U.S. patent application number 11/392892 was filed with the patent office on 2006-10-05 for amnion-derived cell compositions, methods of making and uses thereof.
Invention is credited to Richard A. Banas, Diana L. Clarke, Vivienne S. Marshall, Charlotte A. Smith.
Application Number | 20060222634 11/392892 |
Document ID | / |
Family ID | 37070754 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060222634 |
Kind Code |
A1 |
Clarke; Diana L. ; et
al. |
October 5, 2006 |
Amnion-derived cell compositions, methods of making and uses
thereof
Abstract
The invention is directed to substantially purified
amnion-derived cell populations, compositions comprising the
substantially purified amnion-derived cell populations, and to
methods of creating such substantially purified amnion-derived cell
populations, as well as methods of use. The invention is further
directed to antibodies, in particular, monoclonal antibodies, that
bind to amnion-derived cells or, alternatively, to one or more
amnion-derived cell surface protein markers. The invention is
further directed to methods for producing the antibodies, methods
for using the antibodies, and kits comprising the antibodies.
Inventors: |
Clarke; Diana L.;
(Pittsburgh, PA) ; Smith; Charlotte A.;
(Pittsburgh, PA) ; Banas; Richard A.; (Turtle
Creek, PA) ; Marshall; Vivienne S.; (Glenshaw,
PA) |
Correspondence
Address: |
LINDA O. PALLADINO
45 HONEYSUCKLE COURT
STORMVILLE
NY
12582
US
|
Family ID: |
37070754 |
Appl. No.: |
11/392892 |
Filed: |
March 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60666949 |
Mar 31, 2005 |
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60699257 |
Jul 14, 2005 |
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60742067 |
Dec 2, 2005 |
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Current U.S.
Class: |
424/93.7 ;
435/325 |
Current CPC
Class: |
A61K 8/982 20130101;
C12N 2501/11 20130101; A61K 2800/10 20130101; C12N 5/0629 20130101;
C12N 5/0605 20130101; C12N 2502/02 20130101; A61K 35/50 20130101;
A61K 35/12 20130101; A61Q 19/00 20130101 |
Class at
Publication: |
424/093.7 ;
435/325 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C12N 5/00 20060101 C12N005/00 |
Claims
1. A substantially purified population of amnion-derived cells that
is negative for expression of the protein markers CD90 and
CD117.
2. The substantially purified population of claim 1 that is further
negative for expression of the protein marker CD105.
3. The substantially purified population of claim 1 that is
positive for expression of the protein marker CD29.
4. The substantially purified population of claim 3 that is
negative for expression of the protein marker CD105.
5. The substantially purified population of claim 3 that is further
positive for expression of at least one of the protein markers
selected from the group consisting of CD9, CD10, CD26, CD71, CD166,
CD227, EGF-R, SSEA-4, and HLA-G.
6. The substantially purified population of claim 4 that is further
positive for expression of at least one of the protein markers
selected from the group consisting of CD9, CD10, CD26, CD71, CD166,
CD227, EGF-R, SSEA-4, and HLA-G.
7. The substantially purified population of claim 2 that is further
negative for the expression of at least one of the protein markers
selected from the group consisting of CD140b, telomerase, CD34,
CD44, and CD45.
8. The substantially purified population of claim 4 that is further
negative for the expression of at least one of the protein markers
selected from the group consisting of CD140b, telomerase, CD34,
CD44, and CD45.
9. The substantially purified population of claim 6 that is further
negative for the expression of at least one of the protein markers
selected from the group consisting of CD140b, telomerase, CD34,
CD44, and CD45.
10. The composition of claim 1, which is a pharmaceutical
composition.
11. A method of obtaining the substantially purified population of
amnion-derived cells of claim 1, comprising: a) providing a
population of amnion-derived cells; b) contacting the cells with
(i) one or more antibodies selected from the group consisting of
anti-CD105, anti-CD90, anti-CD117, anti-CD140b, anti-CD34,
anti-CD44, and anti-CD45, antibodies; and (ii) one or more
antibodies selected from the group consisting of anti-CD29,
anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227,
anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies; and c)
separating the cells that do not bind to the antibodies of (i) from
the cells that do bind to the antibody of (i) and separating the
cells that do not bind to the antibodies of (ii) from the cells
that do bind to the antibody of (ii) such that a substantially
purified population of amnion-derived cells that do not bind to the
antibodies of (i) and do bind to the antibody of (ii) is
obtained.
12. A method of obtaining a substantially purified population of
amnion-derived cells, comprising: a) providing a population of
amnion-derived cells; b) contacting the cells with (i) one or more
antibodies selected from the group consisting of anti-CD105,
anti-CD90, anti-CD117, anti-CD140b, anti-CD34, anti-CD44, and
anti-CD45, antibodies; and (ii) one or more antibodies selected
from the group consisting of anti-CD29, anti-CD9, anti-CD10,
anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,
anti-SSEA-4, and anti-HLA-G antibodies; and c) separating the cells
that do not bind to the antibodies of (i) from the cells that do
bind to the antibody of (i) and separating the cells that do not
bind to the antibodies of (ii) from the cells that do bind to the
antibody of (ii) such that a substantially purified population of
amnion-derived cells that do not bind to the antibodies of (i) and
do bind to the antibody of (ii) is obtained.
13. The amnion-derived cells of claim 1, which are an expanded
amnion-derived cell composition.
14. The composition of claim 13 which is animal-free.
15. The composition of claim 13 wherein the amnion-derived cells
form spheroids.
16. A composition comprising conditioned medium obtained from the
expanded amnion-derived cell composition of claim 13.
17. A composition comprising cell lysate obtained from the
amnion-derived cell composition of claim 13.
18. The expanded amnion-derived cell composition of claim 13 having
a concentration of at least 500.times.10.sup.6 amnion-derived
cells/g of starting amnion.
19. A method of creating a hepatocyte comprising differentiating,
in vitro or in vivo, an amnion-derived cell population of claim
1.
20. A hepatocyte created by the method of claim 19.
21. A method of creating a cardiomyocyte comprising
differentiating, in vitro or in vivo, an amnion-derived cell
population of claim 1.
22. A cardiomyocyte created by the method of claim 21.
23. A method for promoting accelerated wound healing in an injured
patient in need thereof comprising administering to the patient one
or more compositions selected from the group consisting of
placental-derived cells, conditioned media derived from
placental-derived cells, placental-derived cell lysates, and
placental-derived cell products.
24. The method according to claim 23 wherein the wound is selected
from the group consisting of mechanical, thermal, acute, chronic,
infected, and sterile wounds.
25. The method according to claim 23 wherein the injured patient is
a human.
26. A cosmetic preparation comprising one or more compositions
selected from the group consisting of placental-derived cells,
conditioned media derived from placental-derived cells,
placental-derived cell lysates, and placental-derived cell
products.
27. A method for treating hearing loss in a patient in need thereof
comprising administering to the patient one or more compositions
selected from the group consisting of placental-derived cells,
conditioned media derived from placental-derived cells,
placental-derived cell lysates, and placental-derived cell
products.
28. The population of claim 1, wherein the cells express a
pancreatic progenitor cell marker protein.
29. The population of claim 28, wherein the progenitor cell marker
is PDX1 protein and wherein the cells further optionally express
any one or more of the protein markers selected from the group
consisting of Foxa2, p48, Hblx9, Neurogenin 3 (Hgn3), NKx2.2,
Nkx6.1, insulin and islet-1.
30. The population of claim 29, wherein the PDX1 protein is
expressed in the nucleus.
31. The population of claim 28, wherein the cells are
differentiated pancreatic progenitor cells.
32. The population of claim 31, wherein the differentiated
progenitor cells express any one or more of the protein markers
selected from the group consisting of PDX1, insulin, C-peptide,
somatostatin, pancreatic polypeptide, and glucagon.
33. The population of claim 31, wherein the differentiated
pancreatic progenitor cells are islet-like cells.
34. The population of claim 33, wherein the islet-like cells are
functional alpha, beta, delta or phi cells.
35. The population of claim 34, wherein functionality of the
islet-like cells is incremental glucose-dependent insulin
secretion.
36. An islet comprising the population of claim 28.
37. A tissue comprising the population of claim 28.
38. The population of claim 28, wherein the cells form
spheroids.
39. The population of claim 38, wherein the spheroids form
buds.
40. The population of claim 39, wherein the buds express PDX1
protein.
41. The population of claim 40, wherein the PDX1 protein is
expressed in the nucleus.
42. The population of claim 28, which comprises one or more
mammalian embryonic islet progenitor cells.
43. The population of claim 42, wherein the mammalian embryonic
islet progenitor cells are human cells.
44. The population of claim 28, wherein the cells express a
heterologous protein.
45. The population of claim 28, wherein the cells have the
identifying characteristics of endoderm, wherein the identifying
characteristics of endoderm are expression of HNF1.alpha.,
HNF1.beta., HNF4.alpha., HNF6, Foxa2 and PDX1 proteins and wherein
the cells further optionally expressing any one or more of the
protein markers Sox17, Cerberus, Hesx1, LeftyA, Otx1 or Otx2.
46. A composition comprising one or more nuclei isolated from
pancreatic progenitor cells of claim 28, wherein the cells express
PDX1 protein in the nucleus and/or express Nkx2.2, Nkx6.1, insulin
and islet-1 protein and/or have the identifying characteristics of
endoderm.
47. The composition of claim 4469, wherein the identifying
characteristics of endoderm are protein expression of HNF1.alpha.,
HNF1.beta., HNF4.alpha., HNF-6, Foxa2 and PDX1 and wherein the
cells, further optionally express any one or more of the protein
markers Sox17, Cerberus, Hesx1, LeftyA, Otx1 or Otx2.
48. A pharmaceutical composition comprising an effective amount of
the population of claim 28 and a carrier.
49. An in vivo method for inducing differentiation of resident
pancreatic cells into islet cells comprising: a) introducing
factors into the pancreas of a subject; and b) allowing the
introduced factors to prime the resident pancreatic cells such that
the cells are induced to differentiate into islet progenitor cells
and/or islet cells.
50. An in vivo method for promoting the generation of islet cells
in a subject comprising: a) transplanting the population of
amnion-derived cells of claim 1 into the pancreas of the subject:
b) introducing factors into the pancreas of the subject; and c)
allowing the introduced factors to promote generation of islet
progenitor cells and or islet cells from the transplanted
amnion-derived cells.
51. An in vivo method for promoting the differentiation of
amnion-derived cells into pancreatic cells comprising (a)
co-culturing the population of amnion-derived cells of claim 1 with
differentiating embryonic pancreatic or non-pancreatic tissue; and
(b) transplanting the co-cultures into the pancreas of a
subject.
52. An in vivo method for promoting the differentiation of
amnion-derived cells into pancreatic cells comprising (a)
co-culturing the population of amnion-derived cells of claim 1 with
differentiating or pre-differentiating non-embryonic heterologous
tissue or autologous tissue; and (b) transplanting the co-cultures
into the pancreas of a subject.
53. An in vivo method for promoting the differentiation of
amnion-derived cells into pancreatic cells comprising (a)
introducing factors to the population of amnion-derived cells of
claim 1 in vitro; and (b) subsequently transplanting the
amnion-derived cells into the pancreas of a subject.
54. A cell culture system comprising a cell culture medium
comprising a SHh antagonist and the population of claim 1 or claim
13.
55. A cell comprising a nucleus isolated from the amnion-derived
cell of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 USC .sctn. 19(e)
to U.S. Provisional Application No. 60/666,949, filed Mar. 31,
2005, U.S. Provisional Application No. 60/699,257, filed Jul. 14,
2005, U.S. Provisional Application No. 60/742,067, filed Dec. 2,
2005, and under 35 USC .sctn.120 to U.S. Utility application Ser.
No. 11/333,849, filed Jan. 18, 2006, the contents of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention is directed to amnion-derived
cell populations, compositions comprising the amnion-derived cell
populations, expanded amnion-derived cell populations, methods of
creating such amnion-derived cell populations, as well as methods
of use. The field is also directed to antibodies, in particular,
monoclonal antibodies, that bind to amnion-derived cells or,
alternatively, to one or more amnion-derived cell surface protein
markers, methods for producing the antibodies, methods for using
the antibodies, and kits comprising the antibodies. The field of
the invention is further directed to novel pancreatic cell
compositions, methods for their production and uses thereof, and to
novel cell culture factor systems.
DESCRIPTION OF RELATED ART
[0003] Preliminary evidence suggests that amnion epithelial cells
isolated and placed in culture exhibit many of the characteristics
necessary to define a stem cell population (Brivanlou, A. H., et
al., Science, 2003. 300(5621): p. 913-6).
[0004] Placental-derived stem cells isolated from placenta have
been shown to exhibit heterogeneous protein expression of the
stage-specific embryonic antigens SSEA-3 and SSEA-4, TRA 1-60, TRA
1-81, c-kit, and Thy-1 (see US2003/0235563 and US2004/0161419).
These cells have also been shown to express the cell surface
proteins Oct-4 and nanog, markers reportedly expressed by
pluripotent stem cells. Under appropriate conditions,
placental-derived stem cells have been shown to differentiate into
cells with characteristics of liver cells (hepatocytes), pancreatic
cells (i.e. alpha and beta cells), central nervous system cells
(neurons and glia), cardiac muscle cells (cardiomyocytes) and
vascular endothelial cells. Placental-derived stem cells are
non-tumorigenic upon transplantation (Miki, T., et al., Stem Cells
2005; 23:1549-1559). In fact, tumors have not been observed in
immuno-compromised mice following transplantation of more than 20
million placental-derived stem cells, conditions under which ES
cells form non-malignant tumors known as teratomas. US2003/0235563
and US2004/0161419 disclose preliminary studies indicating that
placental-derived stem cells cultured in Matrigel supplemented with
10 mM nicotinamide for 14 days express insulin and glucagon as well
as the pancreatic cell markers PDX1 (faint), Pax6 and Nkx2.2.
[0005] Others have transplanted amniotic cells into volunteers and
patients in an attempt to correct lysosomal storage diseases with
no evidence of tumorigenicity (Tylki-Szymanska, A., et al., Journal
of Inherited Metabolic Disease, 1985. 8(3): p. 101-4; Yeager, A.
M., et al.,. American Journal of Medical Genetics, 1985. 22(2): p.
347-55).
[0006] Amniotic membrane is regularly transplanted as a graft for
ocular surface reconstruction without subsequent tumor formation
(John, T., Human amniotic membrane transplantation: past, present,
and future. Opthalmol Clin North Am, 2003. Mar. 16(1): p. 43-65,
vi.). This lack of tumorigenicity is an important distinction
between ES cells and placental-derived stem cells.
[0007] Results of preliminary studies with other cells are
disclosed in WO 2005/017117, WO2005/0042595, US 2005/0019865,
US2005/0032209, US2005/0037491, US2005/0058631, and US2005/0054093.
Results of preliminary studies with these other cells indicate that
they have the potential to differentiate into various cell
types.
[0008] Amniotic membranes have been used clinically as wound
dressing for burn patients for over 100 years to promote
epithelialization, reduce pain, and prevent infection (Bose, B.
(1979) Ann R Coll Surg Engl, 61:444-7; Sawhney, C. P. (1989) Burns,
15:339-42, Thomson, P. D., Parks, D. H. (1981) Ann Plast Surg,
7:354-6). US2003/0235580 describes a method of delivering
therapeutic molecules to skin using amniotic epithelial cells.
US2004/0057938 describes the use of a human amniotic membrane
composition for prophylaxis and treatment of diseases and
conditions of the eye and skin. U.S. Pat. No. 4,361,552 describes a
method of treating a wound or burn, which comprises covering the
surface of the wound or burn with a cross-linked amnion
dressing.
[0009] US2004/0170615 describes the use of compounds expressed in
fetal tissue for use in skin repair and the improvement of skin
appearance.
[0010] Wei, et al, (Wei, J P, et al, (2003) Cell Transplantation
12:545-552) have shown that human amnion-isolated cells can
normalize blood glucose in streptozotocin-induced diabetic
mice.
BACKGROUND OF THE INVENTION
[0011] Stem Cells--Stem cells have the remarkable potential to
develop into many different cell types in the body. Serving as a
repair system for the body, they can theoretically divide without
limit to replenish other cells throughout a person's life. When a
stem cell divides, each new cell has the potential to either remain
a stem cell or become another type of cell with a more specialized
function, such as a muscle cell, a red blood cell, or a brain cell.
Perhaps the most important potential application of human stem
cells is the generation of cells and tissues that could be used for
cell-based therapies. Examples of stem cell studies are provided
(Tylki-Szymanska, A., et al., Journal of Inherited Metabolic
Disease, 1985. 8(3): p. 101-4; Yeager, A. M., et al., American
Journal of Medical Genetics, 1985. 22(2): p. 347-55; John, T.,
2003. 16(1): p. 43-65, vi.).
[0012] Placental tissue is abundantly available as a discarded
source of a type of stem cell called placental-derived stem cells.
Although discarded as part of the placental membranes, lineage
analysis shows that unlike other tissues of the placenta, the
epithelial layer of the amnion, from which the placental-derived
stem cells are isolated, is uniquely descended from the epiblast in
embryonic development (FIG. 1). The epiblast contains the cells
that will ultimately differentiate into the embryo and cells that
will give rise to an extraembryonic tissue, the amnion. Thus far,
only four cell types that have been described in the literature as
being pluripotent. These are the inner cell mass (ICM) of the
pre-implantation embryo, which gives rise to the epiblast, the
epiblast itself, embryonic stem (ES) and embryonic germ cells (EG).
Thus, identification, purification and propagation of a pluripotent
cell population from discarded amnion tissue would provide an
extremely valuable source of stem cells for replacement cell
therapy.
[0013] With an average yield of over 200 million placental-derived
stem cells per placenta, large numbers of cells are available from
this source. If placental-derived stem cells were to become useful
cells for transplantation medicine, they could provide a nearly
inexhaustible supply of starting material in every part of the
world. No other stem cell source provides such a large starting
population of cells, and collection does not require an invasive or
destructive procedure. Furthermore, there are no ethical, religious
or social issues associated with these placental-derived stem cells
as the tissue is derived from the placenta.
[0014] Another important consideration in stem cell therapies is
graft tolerance. In humans, the protein expression of the cell
surface marker HLA-G was originally thought to be restricted to
immune-privileged sites such as placenta, as well as related cells,
including some isolated from amniotic fluid, placental macrophages,
and cord blood, thus implicating its role in maternal-fetal
tolerance (Urosevic, M. and Dummer, R. (2002) ASHI Quarterly; 3rd
Quarter 2002:106-109). Additionally, studies involving heart-graft
acceptance have suggested that the protein expression of HLA-G may
enhance graft tolerance (Lila, N., et al. (2000) Lancet 355:2138;
Lila, N. et al. (2002) Circulation 105:1949-1954). HLA-G protein is
not expressed on the surface of undifferentiated or differentiated
embryonic stem cells (Drukker, M, et al. (2002) PNAS
99(15):9864-9869). Thus, it is desirable that stems cells intended
for cell-based therapies express HLA-G protein.
[0015] Wound Healing--Placental-derived cells have been shown to
secrete many cytokines and growth factors including prostaglandin
E2, PGES, TGF-.beta., EGF, IL-4, IL-8, TNF, interferons, activin A,
noggin, bFGF, some neuroprotective factors, and many angiogenic
factors (Koyano et al., (2002) Develop. Growth Differ. 44:103-112;
Blumenstein et al. (2000) Placenta 21:210-217; Tahara et al. (1995)
J. Clin. Endocrinol. Metabol. 80:138-146; Paradowska et al. (1997)
Placenta 12:441-446; Denison et al. (1998) Hum. Reprod.
13:3560-3565; Keelan et al. (1998) Placenta 19:429-434; Uchida et
al. (2000) J. Neurosci. Res. 62:585-590; Sun et al. (2003) J. Clin.
Endocrinol. Metabol. 88(11):5564-5571; Marvin et al. (2002) Am. J.
Obstet. Gynecol. 187(3):728-734). Many of these cytokines are
associated with wound healing and some have been credited with
contributing to scarless healing in the fetus.
[0016] Approximately 50 million surgical procedures are performed
in the United States each year. An additional 50 million wounds
result from traumatic injuries. Subsequent acute wound healing
failure at any anatomic site results in increased morbidity and
mortality. Non-limiting examples of acute wound failure include
muscle, fascial and skin dehiscence, incisional hernia formation,
gastrointestinal fistulization and vascular anastamotic leaks.
Besides the immediate functional disability, acute wounds that fail
usually go on to form disabling scars.
[0017] Incisional hernias of the abdominal wall provide an
excellent paradigm to study the mechanism and outcome of acute
wound healing failure. Large, prospective, well-controlled series
have shown that 11-20% of over 4 million abdominal wall fascial
closures fail leading to ventral incisional hernia formation. Even
after repair of acute wound failure, recurrence rates remain as
high as 58%. Improvements in suture material, stitch interval,
stitch distance from the margin of the wound, and administration of
prophylactic antibiotics to avoid infection significantly decreased
the rates of clinically obvious acute wound dehiscence, but only
led to small decreases in the rates of ventral hernia formation and
recurrence. The introduction of tissue prostheses, typically
synthetic meshes, to create a tension-free bridge or patch of the
myo-fascial defect reduced first recurrence rates significantly,
supporting the concept that mechanical factors predominate in the
pathogenesis of recurrent hernia.
[0018] Traditional surgical teaching is that laparotomy wound
failure is a rare event, with reported "fascial dehiscence" rates
clustered around 0.1%. One prospective study found that the true
rate of laparotomy wound failure is closer to 11%, and that the
majority of these (94%) go on to form incisional hernias during the
first three years after abdominal operations. This is more in line
with the high incidence of incisional hernia formation. The real
laparotomy wound failure rate is therefore 100 times what most
surgeons think it is. In simplest terms, most incisional hernias
are derived from clinically occult laparotomy wound failures, or
occult fascial dehiscences. The overlying skin wound heals,
concealing the underlying myofascial defect. This mechanism of
early mechanical laparotomy wound failure is more consistent with
modern acute wound healing science. There are no other models of
acute wound healing suggesting that a successfully healed acute
wound goes on to breakdown and mechanically fail at a later date.
This mechanism is also unique in that it assumes that the majority
of abdominal wall laparotomy wound failures occur in hosts with no
clearly identifiable wound healing defect. One model of laparotomy
wound failure that was developed resulted in incisional hernias.
The paramedian skin flap design isolates the skin and myofascial
incisions and allows one to simultaneously study midline laparotomy
wound repair and paramedian dermal repair. Skin and myofascial
repairs can be controlled to achieve 100% intact repairs, or 100%
structural failure and wound dehiscence.
[0019] Cosmetics--Fetal skin has much more effective repair
mechanisms, and, once wounded, it is able to heal without the
formation of scars. This capability does appear to require the
fetal immune system, fetal serum, or amniotic fluid (Bleacher J C,
et al., J Pediatr Surg 28: 1312-4, 1993); Ihara S, Motobayashi Y.,
Development 114: 573-82. 1992). Such abilities of fetal tissue have
led to the suggested use of compounds produced by fetal tissue for
regenerating and/or improving the appearance of skin (see, for
example, US 2004/0170615, which is incorporated by reference in its
entirety herein).
[0020] Diabetes--Traditional insulin therapy prolongs the life of a
patient with Type I diabetes but does not prevent the long-term
systemic complications that arise as the disease progresses. Even
the best injection/infusion regime to monitor and control systemic
glucose levels within an acceptable range inevitably leads to a
deterioration of tissue microvascularization resulting in the
plethora of health-related complications associated with the
disease. These complications can be attributed to the inability of
injectable or orally administered insulin to completely substitute
for the insulin secretion from a normal complement of pancreatic
islets. The failure of insulin as a substitute for the pancreatic
islet beta cell can largely be explained when one examines the
cellular architecture of a pancreatic islet itself. Intensive
inter-cellular regulation of hormone secretion, accomplished by
immediate islet cell proximity, is necessary to prevent the large
temporal fluctuations in blood glucose levels that are responsible
for cellular damage and the ensuing complications of the
disease.
[0021] Presently, transplantation of cadaver pancreas or isolation
and transplantation of cadaver islets are the only alternative
treatments to insulin administration that exist for patients
dependent on insulin to control their diabetes. The scarcity of
donor tissue reserves these alternative therapies for select
patients that are unable to stabilize their blood glucose
adequately using traditional insulin injection/infusion
regimes.
[0022] This conundrum profiles diabetes as a prime candidate for
cell-based therapies. This candidacy is made stronger by the unique
quality of islets to function as self-contained, functional,
glucose-sensing multicellular units
[0023] Studies have also been undertaken to promote differentiation
of stem cells, progenitor cells or their progeny using protein
transduction domains (PTDs) such as that contained in the HIV-1 TAT
protein. The HIV-1 TAT protein has been found to penetrate cells in
a concentration-dependent, receptor-independent fashion. Studies
have been undertaken with TAT PTDs to determine their usefulness in
delivering proteins to cells (see, for example, US 2005/0048629,
Wadia et al., 2004, Nature Medicine 10:310-315 and Krosl et al.,
2003, Nature Medicine 9:1-10). Such proteins may be used to promote
differentiation of stem cells, progenitor cells or their
progeny.
BRIEF SUMMARY OF THE INVENTION
[0024] Although heterogeneous populations of placental-derived stem
cells have been previously characterized using established
embryonic stem cell surface protein markers such as c-kit, SSEA-3,
and SSEA-4, a set of protein markers useful for characterizing and
isolating a preferred substantially purified population of cells is
required. This substantially purified population of cells, termed
amnion-derived cells, could then be fully discriminated from other
cells such as embryonic stem cells, mesenchymal stem cells or
adult-derived stem cells. Therefore, it is an object of this
invention to provide such protein markers capable of characterizing
and isolating amnion-derived cells from placental-derived stem
cells. It is also an object of the invention to use those protein
markers as antigens to make hybridoma cell lines that produce
monoclonal antibodies specific for those protein markers.
[0025] Accordingly, a first aspect of the invention is a
substantially purified population of amnion-derived cells that is
negative for expression of the protein markers CD90 and CD117.
[0026] A second aspect of the invention is a substantially purified
population of the first aspect of the invention that is further
negative for expression of the protein marker CD105.
[0027] A third aspect of the invention is a substantially purified
population of the first aspect of the invention that is positive
for expression of the protein marker CD29.
[0028] A fourth aspect of the invention is a substantially purified
population of the third aspect of the invention that is negative
for expression of the protein marker CD105.
[0029] A fifth aspect of the invention is a substantially purified
population of the third aspect of the invention, that is further
positive for expression of at least one of the protein markers
selected from the group consisting of CD9, CD10, CD26, CD71, CD166,
CD227, EGF-R, SSEA-4, and HLA-G.
[0030] A sixth aspect of the invention is a substantially purified
population of the fourth aspect of the invention, that is further
positive for expression of at least one of the protein markers
selected from the group consisting of CD9, CD10, CD26, CD71, CD166,
CD227, EGF-R, SSEA-4, and HLA-G.
[0031] A seventh aspect of the invention is a substantially
purified population of the second aspect of the invention that is
further negative for the expression of at least one of the protein
markers selected from the group consisting of CD140b, telomerase,
CD34, CD44, and CD45.
[0032] An eighth aspect of the invention is a substantially
purified population of the fourth aspect of the invention that is
further negative for the expression of at least one of the protein
markers selected from the group consisting of CD140b, telomerase,
CD34, CD44, and CD45.
[0033] A ninth aspect of the invention is a substantially purified
population of the sixth aspect of the invention that is further
negative for the expression of at least one of the protein markers
selected from the group consisting of CD140b, telomerase, CD34,
CD44, and CD45.
[0034] A tenth aspect of the invention is a population of
amnion-derived cells of aspects one through nine of the invention,
which is a composition. In a preferred embodiment, the composition
is a pharmaceutical composition.
[0035] An eleventh aspect of the invention is a method of obtaining
the substantially purified population of amnion-derived cells of
the first aspect of the invention, comprising: a) providing a
population of amnion-derived cells; b) contacting the population of
amnion-derived cells with anti-CD90 and anti-CD117 antibodies; and
c) separating the amnion-derived cells that bind to the antibodies
from the cells that do not bind to the antibodies such that the
substantially purified population of amnion-derived cells of the
first aspect of the invention that do not bind to the antibodies is
obtained.
[0036] A twelfth aspect of the invention is a method of obtaining
the substantially purified population of amnion-derived cells of
the second aspect of the invention, comprising: a) providing a
population of amnion-derived cells; b) contacting the cells with
anti-CD90, anti-CD117, and anti-CD105 antibodies; and c) separating
the cells that bind to the antibodies from the cells that do not
bind to the antibodies such that the substantially purified
population of amnion-derived cells of the second aspect of the
invention that do not bind to the antibodies is obtained.
[0037] A thirteenth aspect of the invention is a method of
obtaining the substantially purified population of amnion-derived
cells of the seventh aspect of the invention, comprising: a)
providing a population of amnion-derived cells; b) contacting the
cells with (i) anti-CD90, anti-CD117, and anti-CD105 antibodies and
(ii) with at least one antibody selected from the group consisting
of anti-CD140b, anti-CD34, anti-CD44, and anti-CD45 antibodies; and
c) separating the cells that bind to the antibodies of (i) from the
cells that do not bind to the antibodies of (i) and separating the
cells that bind to the antibodies of (ii) from the cells that do
not bind to the antibodies of (ii); and such that the substantially
purified population of amnion-derived cells of the seventh aspect
of the invention that do not bind to the antibodies of (i) and (ii)
is obtained.
[0038] A fourteenth aspect of the invention is a method of
obtaining the substantially purified population of amnion-derived
cells of the third aspect of the invention, comprising: a)
providing a population of amnion-derived cells; b) contacting the
cells with (i) anti-CD90 and anti-CD117 antibodies and (ii) with an
anti-CD29 antibody; and c) separating the cells that do not bind to
the antibodies of (i) from the cells that do bind to the antibody
of (i) and separating the cells that do not bind to the antibodies
of (ii) from the cells that do bind to the antibody of (ii) such
that the substantially purified population of amnion-derived cells
of the third aspect of the invention that do not bind to the
antibodies of (i) and do bind to the antibody of (ii) is
obtained.
[0039] A fifteenth aspect of the invention is a method of obtaining
the substantially purified population of amnion-derived cells of
the fourth aspect of the invention, comprising: a) providing a
population of amnion-derived cells; b) contacting the cells with
(i) anti-CD90, anti-CD117 and anti-CD105 antibodies and (ii) with
an anti-CD29 antibody; and c) separating the cells that do not bind
to the antibodies of (i) from the cells that do bind to the
antibodies of (i) and separating the cells that do not bind to the
antibody of (ii) from the cells that do bind to the antibody of
(ii) such that the substantially purified population of
amnion-derived cells of the fourth aspect of the invention that do
not bind to the antibodies of (i) and do bind to the antibody of
(ii) is obtained.
[0040] A sixteenth aspect of the invention is a method of obtaining
the substantially purified population of amnion-derived cells of
the fifth aspect of the invention, comprising: a) providing a
population of amnion-derived cells; b) contacting the cells with
(i) anti-CD90, anti-CD117 antibodies and (ii) anti-CD29 antibodies
and (iii) with one or more antibodies selected from the group
consisting of anti-CD9, anti-CD10, anti-CD26, anti-CD71,
anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-G
antibodies; and c) separating the cells that do not bind to the
antibody of (i) from the cells that do bind to the antibodies of
(i) and separating the cells that do not bind to the antibody of
(ii) from the cells that do bind to the antibody of (ii) and
separating the cells that do not bind to the antibodies of (iii)
from the cells that do bind to the antibodies of (iii) such that
the substantially purified population of amnion-derived cells of
the fifth aspect of the invention that do not bind to antibodies of
(i), do bind to antibody of (ii) and do bind to antibodies of (iii)
is obtained.
[0041] A seventeenth aspect of the invention is a method of
obtaining the substantially purified population of amnion-derived
cells of the sixth aspect of the invention, comprising: a)
providing a population of amnion-derived cells; b) contacting the
cells with (i) anti-CD90, anti-CD117, and anti-CD105 antibodies and
(ii) and anti-CD29 antibodies and (iii) with one or more antibodies
selected from the group consisting of anti-CD9, anti-CD10,
anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,
anti-SSEA-4, and anti-HLA-G antibodies; and c) separating the cells
that do not bind to the antibody of (i) from the cells that do bind
to the antibodies of (i) and separating the cells that do not bind
to the antibody of (ii) from the cells that do bind to the antibody
of (ii) and separating the cells that do not bind to the antibodies
of (iii) from the cells that do bind to the antibodies of (iii)
such that the substantially purified population of amnion-derived
cells of the sixth aspect of the invention that do not bind to
antibodies of (i), do bind to antibody of (ii) and do bind to
antibodies of (iii) is obtained.
[0042] An eighteenth aspect of the invention is a method of
obtaining the substantially purified population of amnion-derived
cells of the eighth aspect of the invention, comprising: a)
providing a population of amnion-derived cells; b) contacting the
cells with (i) anti-CD90, anti-CD117, and anti-CD105 antibodies and
(ii) and anti-CD29 antibodies and (iii) with one or more antibodies
selected from the group consisting of anti-CD140b, anti-CD34,
anti-CD44, and anti-CD45 antibodies; and c) separating the cells
that do not bind to the antibody of (i) from the cells that do bind
to the antibodies of (i) and separating the cells that do not bind
to the antibody of (ii) from the cells that do bind to the antibody
of (ii) and separating the cells that do not bind to the antibodies
of (iii) from the cells that do bind to the antibodies of (iii)
such that the substantially purified population of amnion-derived
cells of the eighth aspect of the invention that do not bind to
antibodies of (i), do bind to antibody of (ii) and do not bind to
antibodies of (iii) is obtained.
[0043] A nineteenth aspect of the invention is a method of
obtaining the substantially purified population of amnion-derived
cells of the ninth aspect of the invention, comprising: a)
providing a population of amnion-derived cells; b) contacting the
cells with (i) anti-CD90, anti-CD117, and anti-CD105 antibodies and
(ii) and anti-CD29 antibodies and (iii) one or more antibodies
selected from the group consisting of anti-CD140b, anti-CD34,
anti-CD44, and anti-CD45 antibodies and (iv) one or more antibodies
selected from the group consisting of anti-CD9, anti-CD10,
anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,
anti-SSEA-4, and anti-HLA-G antibodies; and c) separating the cells
that do not bind to the antibody of (i) from the cells that do bind
to the antibodies of (i) and separating the cells that do not bind
to the antibody of (ii) from the cells that do bind to the antibody
of (ii) and separating the cells that do not bind to the antibodies
of (iii) from the cells that do bind to the antibodies of (iii) and
separating the cells from that do bind to the antibody of (iv) from
the cells that do not bind to the antibodies of (iv) such that the
substantially purified population of amnion-derived cells of the
ninth aspect of the invention that do not bind to antibodies of
(i), do bind to antibody of (ii), do not bind to antibodies of
(iii) and do bind to the antibodies of (iv) is obtained.
[0044] A twentieth aspect of the invention is a method of obtaining
a substantially purified population of amnion-derived cells,
comprising: a) providing a population of amnion-derived cells; b)
contacting the cells with one or more antibodies selected from the
group consisting of anti-CD105, anti-CD90, anti-CD117, anti-CD140b,
anti-CD34, anti-CD44, and anti-CD45 antibodies; and one or more
antibodies selected from the group consisting of anti-CD29,
anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227,
anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies; and c)
separating the cells that do not bind to the antibodies of (i) from
the cells that do bind to the antibody of (i) and separating the
cells that do not bind to the antibodies of (ii) from the cells
that do bind to the antibody of (ii) such that a substantially
purified population of amnion-derived cells that do not bind to the
antibodies of (i) and do bind to the antibody of (ii) is
obtained.
[0045] A twenty-first aspect of the invention is the method of
aspects 11 through 20, wherein the cells are separated by FACS
sorting.
[0046] A twenty-second aspect of the invention is one in which the
antibodies of aspects 11 through 20 are monoclonal antibodies,
fully human antibodies, humanized antibodies, chimeric antibodies,
a scfv, or a fragment or derivative of any one of the
aforementioned antibodies.
[0047] In addition to aspects 1 through 22 of the invention,
additional aspects provide for expanded and/or clustered
amnion-derived cells and populations, which provide several
advantages over previously described placental-derived cell
compositions as well as embryonic stem cells compositions.
[0048] Accordingly, a twenty-third aspect of the invention is the
amnion-derived cells of the first aspect of the invention, which
are an expanded amnion-derived cell composition. In a preferred
embodiment, the composition of aspect twenty three is animal-free.
In another preferred embodiment the composition is a clustered
amnion-derived cell composition.
[0049] A twenty-fourth aspect of the invention is a composition
comprising conditioned medium obtained from the expanded
amnion-derived cell composition of the twenty-third aspect of the
invention.
[0050] A twenty-fifth aspect of the invention is a composition
comprising cell lysate obtained from the amnion-derived cell
composition of the twenty-third aspect of the invention.
[0051] A twenty-sixth aspect of the invention is the expanded
amnion-derived cell composition of the twenty-third aspect having a
concentration of at least 500.times.10.sup.6 amnion-derived cells/g
of starting amnion.
[0052] A twenty-seventh aspect of the invention is a method of
creating a hepatocyte comprising differentiating, in vitro or in
vivo, an amnion-derived cell population of the first aspect of the
invention.
[0053] A twenty-eighth aspect of the invention is a hepatocyte
created by the method of the twenty-seventh aspect of the
invention.
[0054] A twenty-ninth aspect of the invention is a liver assist
device comprising an amnion-derived cell composition of the
twenty-seventh aspect of the invention.
[0055] A thirtieth aspect of the invention is a method of creating
a cardiomyocyte comprising differentiating, in vitro or in vivo, an
amnion-derived cell population of the first aspect of the
invention.
[0056] A thirty-first aspect of the invention is a cardiomyocyte
created by the method of the thirtieth aspect of the invention.
[0057] A thirty-second aspect of the invention is a method for
promoting accelerated wound healing in an injured patient in need
thereof comprising administering to the patient one or more
compositions of placental-derived cells. In a preferred embodiment
the composition of placental-derived cells is an expanded
amnion-derived cell composition. In another preferred embodiment of
the method the composition is administered in a scaffold or matrix.
In a specific, preferred embodiment, the scaffold or matrix is
amniotic tissue. In another preferred embodiment, the wound is
selected from the group consisting of mechanical, thermal, acute,
chronic, infected, and sterile wounds. And in yet another preferred
embodiment the injured patient is a human.
[0058] A thirty-third aspect of the invention is a cosmetic
preparation comprising one or more compositions of
placental-derived cells. In a preferred embodiment, the composition
of placental-derived cells is an expanded amnion-derived cell
composition.
[0059] A thirty-fourth aspect of the invention is a method for
treating hearing loss in a patient in need thereof comprising
administering to the patient one or more compositions of
placental-derived cells. In a preferred embodiment, the composition
of placental-derived cells is an expanded amnion-derived cell
composition.
[0060] A thirty-fifth aspect of the invention is a method of
proliferating embryonic stem cells comprising using the
amnion-derived cells of the first aspect as a feeder layer. One
preferred embodiment of this aspect is one which is free of animal
products.
[0061] In addition to aspects 23 through 35 of the invention, the
invention also contemplates compositions comprising differentiated
amnion-derived cell populations, method for identifying such
populations, methods of making such populations, and methods of
using them.
[0062] Accordingly, a thirty-sixth aspect of the invention is the
population of the first aspect of the invention wherein the cells
express a pancreatic progenitor cell marker protein. In a preferred
embodiment, the progenitor cell marker is PDX1 protein. In another
preferred embodiment, the PDX1 protein is expressed in the
nucleus.
[0063] A thirty-seventh aspect of the invention is the population
of cells of the thirty-sixth aspect further optionally expressing
any one or more of the protein markers selected from the group
consisting of Foxa2, p48, Hblx9 and Neurogenin 3 (Ngn3). In a
preferred embodiment, the cells further optionally express any one
or more of the protein markers selected from the group consisting
of NKx2.2, Nkx6.1, insulin and islet-1.
[0064] In a thirty-eighth aspect of the invention is a population
of cells of the thirty-sixth aspect, wherein the cells are
differentiated pancreatic progenitor cells. In a preferred
embodiment, the differentiated progenitor cells express any one or
more of the protein markers selected from the group consisting of
PDX1, insulin, C-peptide, somatostatin, pancreatic polypeptide, and
glucagon. In another preferred embodiment the differentiated
pancreatic progenitor cells are islet-like cells. In a specific,
preferred embodiment the islet-like cells are alpha, beta, delta or
phi cells and in a most preferred embodiment the islet-like cells
are functional islet-like cells. In another preferred embodiment
the functionality of the islet-like cells is incremental
glucose-dependent insulin secretion.
[0065] A thirty-ninth aspect of the invention is an islet
comprising the population of cells of the thirty-sixth and
thirty-eighth aspects.
[0066] A fortieth aspect of the invention is a tissue comprising
the population of the thirty-sixth and thirty-eighth aspects.
[0067] A forty-first aspect of the invention is the population of
the thirty-sixth aspect wherein the cells form spheroids. In a
preferred embodiment the spheroids form buds. In another preferred
embodiment the buds express PDX1 protein and in a most preferred
embodiment the PDX1 protein is expressed in the nucleus.
[0068] A forty-second aspect of the invention is the population of
the thirty-sixth aspect of the invention which comprises one or
more mammalian embryonic islet progenitor cells. In a preferred
embodiment of this aspect, the mammalian embryonic islet progenitor
cells are human cells.
[0069] A forty-third aspect of the invention is the population of
the thirty-sixth aspect wherein the cells express a heterologous
protein. In one embodiment the heterologous protein is a TAT fusion
protein. In specific embodiments the TAT fusion protein is
TAT-PDX1, TAT-Hblx9, TAT-p48, TA-Ngn3 or TAT-Foxa2. In another
preferred embodiment the heterologous protein is a therapeutic
protein of interest.
[0070] A forty-fourth aspect of the invention is the population of
the thirty-sixth aspect wherein the cells have the identifying
characteristics of endoderm. In a preferred embodiment the
identifying characteristics of endoderm are expression of
HNF1.alpha., HNF1.beta., HNF4.alpha., HNF6, Foxa2 and PDX1
proteins. In another preferred embodiment the cells further
optionally express any one or more of the protein markers Sox17,
Cerberus, Hesx1, LeftyA, Otx1 or Otx2.
[0071] A forty-fifth aspect of the invention is a composition
comprising one or more nuclei isolated from pancreatic progenitor
cells of the thirty eighth aspect, wherein the cells express PDX1
protein in the nucleus and/or express Nkx2.2, Nkx6.1, insulin and
islet-1 protein and/or have the identifying characteristics of
endoderm. In a preferred embodiment of this aspect the identifying
characteristics of endoderm are protein expression of HNF1.alpha.,
HNF1.beta., HNF4, HNF6, Foxa2 and PDX1. In another preferred
embodiment the cells further optionally express any one or more of
the protein markers Sox17, Cerberus, Hesx1, LeftyA, Otx1 or
Otx2.
[0072] A forty-sixth aspect of the invention is a pharmaceutical
composition comprising an effective amount of the population of the
first, thirteenth, thirty-six and thirty-eighth aspects of the
invention and a carrier.
[0073] A forty-seventh aspect of the invention is a substantially
purified composition comprising one or more undifferentiated cells
wherein the cells express a pancreatic progenitor cell marker
protein. In a preferred embodiment the cells are embryonic stem
cells. In another preferred embodiment the cells are adult stem
cells. In yet another preferred embodiment the cells are
hematopoietic stem cells and in still another preferred embodiment
the cells are mesenchymal stem cells.
[0074] A forty-eighth aspect of the invention is the composition of
the forty-seventh aspect which is transplanted into a subject. In a
preferred embodiment the subject is a human subject.
[0075] A forty-ninth aspect of the invention is an in vivo method
for inducing differentiation of resident pancreatic cells into
islet cells comprising a) introducing factors into the pancreas of
a subject; and b) allowing the introduced factors to prime the
resident pancreatic cells such that the cells are induced to
differentiate into islet progenitor cells and/or islet cells. In a
preferred embodiment the islet cells are alpha, beta, delta or phi
cells.
[0076] A fiftieth aspect of the invention is an in vivo method for
promoting the generation of islet cells in a subject comprising a)
transplanting amnion-derived cells into the pancreas of the
subject; (b) introducing factors into the pancreas of the subject;
and c) allowing the introduced factors to promote generation of
islet progenitor cells or islet cells from the transplanted
amnion-derived cells. In a preferred embodiment the amnion-derived
cells are undifferentiated amnion-derived cells or partially
differentiated amnion-derived cells. In another embodiment the
cells are transplanted subcutaneously, into liver, mammary gland,
kidney capsule, spleen or any other site in which the cells are
able to engraft.
[0077] A fifty-first aspect of the invention is an in vivo method
for promoting the differentiation of amnion-derived cells into
pancreatic cells comprising (a) co-culturing the amnion-derived
cells with differentiating embryonic pancreatic or non-pancreatic
tissue; and (b) transplanting the co-cultures into the pancreas of
a subject. In a preferred embodiment the non-pancreatic tissue is
selected from the group consisting of epithelium, mesenchyme,
islets, ducts, and exocrine tissue. In another preferred embodiment
the amnion-derived cells are undifferentiated amnion-derived cells
or partially differentiated amnion-derived cells. In another
embodiment the cells are transplanted subcutaneously, into liver,
mammary gland, kidney capsule, spleen or any other site in which
the cells are able to engraft.
[0078] A fifty-second aspect of the invention is an in vivo method
for promoting the differentiation of amnion-derived cells into
pancreatic cells comprising (a) co-culturing the amnion-derived
cells with differentiating or pre-differentiating non-embryonic
heterologous or autologous tissue; and (b) transplanting the
co-cultures into the pancreas of a subject. In a preferred
embodiment the amnion-derived cells are undifferentiated
amnion-derived cells or partially differentiated amnion-derived
cells. In another embodiment the cells are transplanted
subcutaneously, into liver, mammary gland, kidney capsule, spleen
or any other site in which the cells are able to engraft.
[0079] A fifty-third aspect of the invention is an in vivo method
for promoting the differentiation of amnion-derived cells into
pancreatic cells comprising (a) introducing factors to the
amnion-derived cells in vitro; and (b) subsequently transplanting
the amnion-derived cells into the pancreas of a subject. In a
preferred embodiment the amnion-derived cells are undifferentiated
amnion-derived cells or partially differentiated amnion-derived
cells. In another embodiment the cells are transplanted
subcutaneously, into liver, mammary gland, kidney capsule, spleen
or any other site in which the cells are able to engraft.
[0080] A fifty-fourth aspect of the invention is a cell culture
system comprising a cell culture medium comprising a SHh antagonist
and the population of the first aspect or the thirty-sixth aspect
of the invention. In a preferred embodiment the cell culture system
further comprises one or more mammalian embryonic islet progenitor
cells. In another preferred embodiment the SHh antagonist is
cyclopamine or jervine. In a specific preferred embodiment the
cyclopamine is at a concentration of 10 .mu.M. In another preferred
embodiment the cell culture system further comprises a solid
surface. In a specific embodiment the solid is extracellular matrix
and in another specific embodiment the extracellular matrix is
composed of one or more of the substances selected from the group
consisting of Matrigel, fibronectin, superfibronectin, laminin,
collagen, heparin sulfate proteoglycan and naturally occurring
acellular biological substances. In another embodiment the solid
surface forms a scaffold and in a specific embodiment the scaffold
is a fiber, gel, fabric, sponge-like sheet or complex
three-dimensional form containing pores and channels.
[0081] A fifty-fifth aspect of the invention is a cell culture
system comprising a cell culture medium comprising a TAT fusion
peptide and the population of the first aspect or the thirty-sixth
aspect of the invention. In preferred embodiments the TAT fusion
protein is TAT-PDX1, TAT-Hblx9, TAT-Ngn3, TAT-p48, or TAT-Foxa2. In
a preferred embodiment, the cell culture system further comprises a
SHh antagonist. In another preferred embodiment the cell culture
system further comprises one or more mammalian embryonic islet
progenitor cells. In another preferred embodiment the SHh
antagonist is cyclopamine or jervine. In a specific preferred
embodiment the cyclopamine is at a concentration of 10 .mu.M. In
another preferred embodiment the cell culture system further
comprises a solid surface. In a specific embodiment the solid is
extracellular matrix and in another specific embodiment the
extracellular matrix is composed of one or more of the substances
selected from the group consisting of Matrigel, fibronectin,
superfibronectin, laminin, collagen, heparin sulfate proteoglycan
and naturally occurring acellular biological substances. In another
embodiment the solid surface forms a scaffold and in a specific
embodiment the scaffold is a fiber, gel, fabric, sponge-like sheet
or complex three-dimensional form containing pores and
channels.
[0082] A fifty-sixth aspect of the invention is a method for
obtaining a pancreatic progenitor cell comprising culturing an
undifferentiated cell in the culture system of the fifty-fifth
aspect of the invention.
[0083] A fifty-seventh aspect of the invention is a composition
comprising a donor cell comprising a nucleus isolated from the
amnion-derived cell of the first aspect of the invention. In a
preferred embodiment the amnion derived cell is an expanded
amnion-derived cell of the twenty-third aspect of the invention. In
another preferred embodiment amnion-derived cell is a pancreatic
progenitor cell of aspect thirty-six of the invention. In another
preferred embodiment the amnion-derived cell is an alpha, beta,
delta or phi cell. In another preferred embodiment the recipient
cell is a mammalian cell and in a specific embodiment the mammalian
cell is selected from the group consisting of germ cells, oocytes
and sperm.
Definitions
[0084] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the desired function is retained by the polypeptide. NH2 refers to
the free amino group present at the amino terminus of a
polypeptide. COOH refers to the free carboxy group present at the
carboxy terminus of a polypeptide.
[0085] As defined herein "isolated" refers to material removed from
its original environment and is thus altered "by the hand of man"
from its natural state.
[0086] As defined herein, a "gene" is the segment of DNA involved
in producing a polypeptide chain; it includes regions preceding and
following the coding region, as well as intervening sequences
(introns) between individual coding segments (exons).
[0087] As used herein, the term "protein marker" means any protein
molecule characteristic of the plasma membrane of a cell or in some
cases of a specific cell type.
[0088] As used herein, "enriched" means to selectively concentrate
or to increase the amount of one or more materials by elimination
of the unwanted materials or selection and separation of desirable
materials from a mixture (i.e. separate cells with specific cell
markers from a heterogenous cell population in which not all cells
in the population express the marker).
[0089] As used herein, the term "substantially purified" means a
population of cells substantially homogeneous for a particular
marker or combination of markers. By substantially homogeneous is
meant at least 90%, and preferably 95% homogeneous for a particular
marker or combination of markers.
[0090] As used herein, the term "monoclonal antibody library" means
a collection of at least one monoclonal antibody useful for
identifying unique amnion-derived cells protein markers or
generating substantially purified populations of amnion-derived
cells. As defined herein, "specific for" means that the
antibody(ies) specifically bind to amnion-derived cells, but not
embryonic stem cells, mesenchymal stem cells or adult-derived stem
cells.
[0091] The term "placenta" as used herein means both preterm and
term placenta.
[0092] As used herein, the term "totipotent cells" shall have the
following meaning. In mammals, totipotent cells have the potential
to become any cell type in the adult body; any cell type(s) of the
extraembryonic membranes (e.g., placenta). Totipotent cells are the
fertilized egg and approximately the first 4 cells produced by its
cleavage.
[0093] As used herein, the term "pluripotent stem cells" shall have
the following meaning. Pluripotent stem cells are true stem cells
with the potential to make any differentiated cell in the body, but
cannot contribute to making the components of the extraembryonic
membranes which are derived from the trophoblast. The amnion
develops from the epiblast, not the trophoblast. Three types of
pluripotent stem cells have been confirmed to date: Embryonic Stem
(ES) Cells (may also be totipotent in primates), Embryonic Germ
(EG) Cells, and Embryonic Carcinoma (EC) Cells. These EC cells can
be isolated from teratocarcinomas, a tumor that occasionally occurs
in the gonad of a fetus. Unlike the other two, they are usually
aneuploid.
[0094] As used herein, the term "multipotent stem cells" are true
stem cells but can only differentiate into a limited number of
types. For example, the bone marrow contains multipotent stem cells
that give rise to all the cells of the blood but may not be able to
differentiate into other cells types.
[0095] "Amnion-derived cells" are a population of cells that are
derived from the amnion of the placenta. Amnion-derived cells grow
without feeder layers, do not express the protein telomerase and
are non-tumorigenic. Amnion-derived cells do not express the
hematopoietic stem cell marker CD34 protein. The absence of CD34
positive cells in this population indicates the isolates are not
contaminated with hematopoietic stem cells such as umbilical cord
blood or embryonic fibroblasts. Virtually 100% of the cells react
with antibodies to low molecular weight cytokeratins, confirming
their epithelial nature. Freshly isolated amnion-derived cells will
not react with antibodies to the stem/progenitor cell markers c-kit
and Thy-1. Several procedures used to obtain cells from full term
or pre-term placenta are known in the art (see, for example, US
2004/0110287; Anker et al., 2005, Stem Cells 22:1338-1345; Ramkumar
et al., 1995, Am. J. Ob. Gyn. 172:493-500). However, the methods
used herein provide improved compositions and populations of
cells.
[0096] The term "composition of placental-derived cells" as used
herein includes the cells and compositions described in this
application and in US2003/0235563, US2004/0161419, US2005/0124003,
U.S. Provisional Application Nos. 60/666,949, 60/699,257,
60/742,067 and U.S. application Ser. No. 11/333,849, the contents
of which are incorporated herein by reference in their
entirety.
[0097] By the term "animal-free" when referring to compositions,
growth conditions, culture media, etc. described herein, is meant
that no animal-derived materials, such as animal-derived serum,
other than human materials, such as native or recombinantly
produced human proteins, are used in the preparation, growth,
culturing, expansion, or formulation of the composition or
process.
[0098] By the term "expanded", in reference to amnion-derived cell
compositions, means that the amnion-derived cell population
constitutes a significantly higher concentration of multipotent
cells than is obtained using previous methods. The level of
multipotent cells per gram of amniotic tissue in expanded
compositions is at least 50 and up to 150 fold higher than the
number of cells in the primary culture after 5 passages, as
compared to about a 20 fold increase in such cells using previous
methods. Accordingly, an "expanded" population has at least a 2
fold, and up to a 10 fold, improvement in cell numbers per gram of
amniotic tissue over previous methods. The term "expanded" is meant
to cover only those situations in which a person has intervened to
elevate the proportion of the amnion-derived cells. As used herein
"passage" or "passaging" refers to subculturing of cells. For
example, cells isolated from the amnion are referred to as primary
cells. Such cells are expanded in culture by being grown in the
growth medium described herein. When such primary cells are
subcultured, each round of subculturing is referred to as a
passage. As used herein, "primary culture" means the freshly
isolated amnion-derived cell population.
[0099] As used herein a "conditioned medium" is a medium in which a
specific cell or population of cells has been cultured, and then
removed. When cells are cultured in a medium, they may secrete
cellular factors that can provide support to or affect the behavior
of other cells. Such factors include, but are not limited to
hormones, cytokines, extracellular matrix (ECM), proteins,
vesicles, antibodies, and granules. The medium containing the
cellular factors is the conditioned medium. Examples of methods of
preparing conditioned media are described in U.S. Pat. No.
6,372,494 which is incorporated by reference in its entirety
herein. As used herein, conditioned medium also refers to
components, such as proteins, that are recovered and/or purified
from conditioned medium or from amnion-derived cells.
[0100] The term "lysate" as used herein refers to the composition
obtained when the amnion-derived cell are lysed and the cellular
debris (e.g., cellular membranes) is removed. This may be achieved
by mechanical means, by freezing and thawing, by use of detergents,
such as EDTA, or by enzymatic digestion using, for example,
hyaluronidase, dispase, proteases, and nucleases.
[0101] As used herein, the term "substrate" means a defined coating
on a surface that cells attach to, grown on, and/or migrate on. As
used herein, the term "matrix" means a substance that cells grow in
or on that may or may not be defined in its components. The matrix
includes both biological and non-biological substances. As used
herein, the term "scaffold" means a three-dimensional (3D)
structure (substrate and/or matrix) that cells grow in or on. It
may be composed of biological components, synthetic components or a
combination of both. Further, it may be naturally constructed by
cells or artificially constructed. In addition, the scaffold may
contain components that have biological activity under appropriate
conditions.
[0102] The term "transplantation" refers to the administration of a
composition either in an undifferentiated, partially
differentiated, or fully differentiated form into a human or other
animal.
[0103] As used herein, the term "pharmaceutically acceptable" means
that the components, in addition to the therapeutic agent,
comprising the formulation, are suitable for administration to the
patient being treated in accordance with the present invention.
[0104] The term "liver disease" as used herein includes but is not
limited to cirrhosis of the liver, metabolic diseases of the liver,
such as alpha 1-antitrypsin deficiency and ornithine
transcarbamylase (OTC), alcohol-induced hepatitis, chronic
hepatitis, primary sclerosing cholangitis, alpha 1-antitrypsin
deficiency and liver cancer. As used herein, the term "pancreatic
disease" may include but is not limited to pancreatic cancer,
insulin-deficiency disorder such as Insulin-dependent (Type 1)
diabetes mellitus (IDDM) and Non-insulin-dependent (Type 2)
diabetes mellitus (NIDDM), hepatitis C infection, exocrine and
endocrine pancreatic diseases. As used herein, the term
"neurological disease" refers to a disease or condition associated
with any defects in the entire integrated system of nervous tissue
in the body: the cerebral cortex, cerebellum, thalamus,
hypothalamus, midbrain, pons, medulla, brainstem, spinal cord,
basal ganglia and peripheral nervous system. As used herein, the
term "vascular disease" refers to a disease of the human vascular
system. As used herein, the term "cardiac disease" or "cardiac
dysfunction" refers to diseases that result from any impairment in
the heart's pumping function. The term "cardiomyopathy" refers to
any disease or dysfunction of the myocardium (heart muscle) in
which the heart is abnormally enlarged, thickened and/or
stiffened.
[0105] As used herein, the term "hepatocytes" refers to cells that
have characteristics of epithelial cells obtained from liver. As
used herein, the term "pancreatic cell" is used to refer to cells
that produce glucagon, insulin, somatostatin, and/or pancreatic
polypeptide (PP). Preferred pancreatic cells are positive for
pancreatic cell-specific markers, such as homeobox transcription
factor Nkx-2.2, glucagon, paired box gene 6 (Pax6), pancreatic
duodenal homeobox 1 (PDX1), and insulin. As used herein, the term
"vascular endothelial cell" refers to an endothelial cell that
exhibits essential physiological functions characteristic of
vascular endothelial cells including modulation of vasoreactivity
and provision of a semi-permeable barrier to plasma fluid and
protein. As used herein, the term "cardiomyocyte" refers to a
cardiac muscle cell that may spontaneously beat or may exhibit
calcium transients (flux in intracellular calcium concentrations
measurable by calcium imaging). As used herein, the term "neural
cells" refer to cells that exhibit essential functions of neurons,
and glial cells (astrocytes and oligodendrocytes).
[0106] As used herein, the term "tissue" refers to an aggregation
of similarly specialized cells united in the performance of a
particular function.
[0107] As used herein, the term "therapeutic protein" includes a
wide range of biologically active proteins including, but not
limited to, growth factors, enzymes, hormones, cytokines,
inhibitors of cytokines, blood clotting factors, peptide growth and
differentiation factors.
[0108] As used herein, "pancreas" refers generally to a large,
elongated, racemose gland situated transversely behind the stomach,
between the spleen and duodenum. The pancreatic exocrine function,
e.g., external secretion, provides a source of digestive enzymes.
These cells synthesize and secrete digestive enzymes such as
trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease,
deoxyribonuclease, triacylglycerol lipase, phospholipase A2
elastase, and amylase. The endocrine portion of the pancreas
contains the islets of Langerhans. The islets of Langerhans appear
as rounded spheroids of cells embedded within the exocrine
pancreas. Four different types of cells -alpha, beta, delta, and
phi- have been identified in the islets. The alpha cells constitute
about 20% of the cells found in pancreatic islets and produce the
hormone glucagon. Glucagon acts on several tissues to make energy
available in the intervals between feeding. In the liver, glucagon
causes breakdown of glycogen and promotes gluconeogenesis from
amino acid precursors. The delta cells produce somatostatin which
acts in the pancreas to inhibit glucagon release and to decrease
pancreatic exocrine secretion. The hormone pancreatic polypeptide
(PP) is produced in the phi cells. This hormone inhibits pancreatic
exocrine secretion of bicarbonate and enzymes, causes relaxation of
the gallbladder, and decreases bile secretion. The most abundant
cell in the islets, constituting 60-80% of the cells, is the beta
cell, which produces insulin. Insulin is known to cause the storage
of excess nutrients arising during and shortly after feeding. The
major target organs for insulin are the liver, muscle, and
fat-organs specialized for storage of energy. The term "pancreatic
duct" as used herein includes the accessory pancreatic duct, dorsal
pancreatic duct, main pancreatic duct and ventral pancreatic duct,
interlobular pancreatic duct, and interlobular pancreatic duct.
[0109] As used herein, the term "clustered amnion-derived cell
compositions" refers to amnion-derived cell compositions wherein at
least 50% and up to about 95% of the cells form clusters.
[0110] "Pancreatic progenitor cell" as defined herein is a cell
which can differentiate into a cell of pancreatic lineage, e.g., a
cell which can produce a hormone or enzyme normally produced by a
pancreatic cell. For instance, a pancreatic progenitor cell may be
caused to differentiate, at least partially, into alpha, beta,
delta, or phi islet cells, or a cell of exocrine fate. In
accordance with the method of the invention, the pancreatic
progenitor cells of the invention can be cultured prior to
administration to a subject under conditions which promote cell
proliferation and/or differentiation. These conditions include but
are not limited to culturing the cells to allow proliferation in
vitro at which time the cells may form pseudo islet-like spheroids
and secrete insulin, glucagon, and somatostatin. The term
"islet-like cell" as used herein means having some but not
necessarily all of the characteristics of one of the cell types
(.alpha., .beta., .gamma.or .delta.) present in a mature pancreatic
islet. The islet-like cell will express only one of the following
pancreatic endocrine cell hormones: Insulin, glucagon,
Somatostatin, Pancreatic Polypeptide. The term "islet-like
structures" as defined herein are structures containing islet-like
cells. Islet-like structures refers to the spheroids of cells
derived from the methods of the invention which take on both the
appearance of pancreatic alpha, beta, delta or phi cells, as well
as their function. Their coordinated function includes the ability
to respond to glucose.
[0111] As used herein, the term "spheroid" or "spheroids" means
multicellular clusters in suspension cultures. As used herein the
term "bud" or "buds" means the segregation of a subset of cells in
a spheroid into a group on the surface of the spheroid.
[0112] As used herein "germ cells" means embryonic germ cells,
adult germ cells and the cells that they give rise to (i.e. oocyte
and sperm).
[0113] As used herein, "cloning" refers to producing an animal that
develops from the combination of an oocyte and the genetic
information contain within the nucleus or the nucleic acid sequence
of another animal, the animal being cloned. The resulting oocyte
having the donor genome is referred to herein as a "nuclear
transfer cell." The cloned animal has substantially the same or
identical genetic information as that of the animal being cloned.
"Cloning" may also refer to cloning a cell, which includes
producing an oocyte containing genetic information from the nucleus
or the nucleic acid sequence of another animal. Again, the
resulting oocyte having the donor genome is referred to herein as a
"nuclear transfer cell."
[0114] The term "transplantation" as used herein refers to the
administration of a composition comprising cells that are either in
an undifferentiated, partially differentiated, or fully
differentiated form into a human or other animal.
[0115] "Treatment" as used herein covers any treatment of a disease
or condition of a mammal, particularly a human, and includes: (a)
preventing the disease or condition from occurring in a subject
which may be predisposed to the disease or condition but has not
yet been diagnosed as having it; (b) inhibiting the disease or
condition, i.e., arresting its development; or (c) relieving the
disease or condition, i.e., causing regression of the disease or
condition. The population of subjects treated by the methods of the
invention includes subjects suffering from the undesirable
condition or disease, as well as subjects at risk for development
of the condition or disease.
[0116] A "wound" is any disruption, from whatever cause, of normal
anatomy including but not limited to traumatic injuries such as
mechanical, thermal, and incisional injuries; elective injuries
such as surgery and resultant incisional hernias; acute wounds,
chronic wounds, infected wounds, and sterile wounds, as well as
wounds associated with disease states (i.e. ulcers caused by
diabetic neuropathy). A wound is dynamic and the process of healing
is a continuum requiring a series of integrated and interrelated
cellular processes that begin at the time of wounding and proceed
beyond initial wound closure through arrival at a stable scar.
These cellular processes are mediated or modulated by humoral
substances including but not limited to cytokines, lymphokines,
growth factors, and hormones. In accordance with the subject
invention, "wound healing" refers to improving, by some form of
intervention, the natural cellular processes and humoral substances
such that healing is faster, and/or the resulting healed area has
less scaring and/or the wounded area possesses tissue tensile
strength that is closer to that of uninjured tissue.
[0117] Definitions of additional terms are set forth in the table
of abbreviations below. TABLE-US-00001 TABLE 1 Abbreviation
Description Abbreviation Description A1AT Alpha-1 Antitrypsin IE
Islet equivalent CD34 Clustered Differentiation LeftyA Endometrial
bleeding Antigen 34 associated factor preprotein c-Kit Stem Cell
Factor Receptor MBP Myelin basic protein C/EBP.alpha.
CCAAT/enhancer binding Nkx 2.2 NK2 transcription factor
protein-alpha related, locus 2 CNP Natriuretic Peptide C IE Islet
equivalent CYP Cytochrome Oct-4 Octamer binding protein 3/4 ELISA
Enzyme-Linked Pax Paired homeobox gene Immunosorbent Assay EROD
Ethoxyresorufin-o- PCR Polymerase chain reaction deethylase EG
Embryonic Germ PDX1 Pancreatic duodenal homeobox protein-1 ES
Embryonic Stem PP Pancreatic Polypeptide FCS Fetal Calf Serum Rex-1
Reduced expression-1 FGF Fibroblast growth factor RIA Radio
Immuno-Assay FACS Fluorescence Activated Cell Rt-PCR Quantitative
real-time Sorting polymerase chain reaction Foxa2 Forkhead box
protein A2; RT-PCR Reverse Transcription Hepatocyte nuclear factor
3- polymerase chain reaction beta GABA Gamma-amino butyric acid SHh
Sonic Hedgehog GAD Glutamic acid Sox-2 SRY-related HMG-box 2
decarboxylase HB9 Homeobox Protein-HB9 SSEA Stage Specific
Embryonic Antigen HNF4.alpha. Hepatocyte nuclear factor- TDGF-1
Teratocarcinoma-derived 4.alpha. growth factor 1 HNF6 Hepatocyte
nuclear factor-6 Thy-1 Thymus cell antigen-1; CD90 ICC
Immunocytochemistry TRA 1-60 Tumor related antigen-1-60 IHC
Immunohistochemistry TRA 1-81 Tumor related antigen-1-81 ICM Inner
Cell Mass UGT1A1 Uridine diphosphate glucuronosyltransferase
BRIEF DESCRIPTION OF THE FIGURES
[0118] FIG. 1: A schematic representation of human embryological
development.
[0119] FIG. 2--The application of conditioned media overcomes the
inhibition of wound healing caused by bacteria and shifts the
healing trajectory in contaminated wounds to that of near normal
healing.
DETAILED DESCRIPTION
[0120] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook et al, 2001, "Molecular Cloning: A Laboratory Manual";
Ausubel, ed., 1994, "Current Protocols in Molecular Biology"
Volumes I-III; Celis, ed., 1994, "Cell Biology: A Laboratory
Handbook" Volumes 1-111; Coligan, ed., 1994, "Current Protocols in
Immunology" Volumes I-III; Gait ed., 1984, "Oligonucleotide
Synthesis"; Hames & Higgins eds., 1985, "Nucleic Acid
Hybridization"; Hames & Higgins, eds., 1984, "Transcription And
Translation"; Freshney, ed., 1986, "Animal Cell Culture"; IRL
Press, 1986, "Immobilized Cells And Enzymes"; Perbal, 1984, "A
Practical Guide To Molecular Cloning."
[0121] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges may independently be
included in the smaller ranges is also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either both of those included limits are also
included in the invention.
[0122] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now
described.
[0123] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and" and "the" include plural
references unless the context clearly dictates otherwise.
[0124] Production of Amnion-Derived Cell Compositions
[0125] In accordance with the invention, amnion-derived cell
compositions are prepared using the steps of a) recovery of the
amnion from the placenta, b) dissociation of the cells from the
amniotic membrane, c) culturing of the cells in a basal medium with
the addition of a naturally derived or recombinantly produced human
protein; and optionally d) further proliferation of the cells using
additional additives and/or growth factors.
[0126] Recovery of the amnion--The first step in obtaining the
amnion-derived cell compositions of the invention is recovery of
the cells from a placenta. In general, the placenta is processed as
soon as possible after delivery. In preferred embodiments, the
placenta is processed within four hours of delivery. If the
placenta is refrigerated, or the amnion is stripped and
refrigerated, the recovery may be done in up to 36 hours. The
placenta used may be full term or pre-term placenta. Several
procedures used to obtain cells from full term or pre-term placenta
are known in the art (see, for example, US 2004/0110287; Anker et
al., 2005, Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J.
Ob. Gyn. 172:493-500). However, the methods used herein provide
improved compositions and populations of cells.
[0127] Under sterile conditions the amnion is stripped from the
chorion manually, and placed in Hanks Balanced Salt Solution (HBSS)
with no additives. This is preferably done at room temperature. The
membranes are washed at least three times in HBSS, and washed
further if necessary to remove remaining blood clots. Any tissue
still heavily contaminated with blood is cut away and
discarded.
[0128] Dissociation of amniotic membrane cells--The membranes are
incubated with a dissociation reagent. This is done at least once
and as many as ten times. In some embodiments, the dissociation
reagent is a protease. In preferred embodiments, the protease is
Protease XXIII (Sigma; 1 mg/ml). In other embodiments, the
dissociation reagents include, but are not limited to:
trypsin.+-.EDTA, papain, elastase, hyaluronidase, collagenase type
I, II, III, and IV, DNase, Ca+2 and Mg+2-free PBS, EDTA, EGTA,
dispase, collagenase-dispase, Tryple (Gibco), collagenase, and
dispase.
[0129] Characterizing, Identifying Isolating and Creating
Substantially Purified Populations of Amnion-Derived Cells.
[0130] Using commercially available antibodies to known stem cell
markers, freshly isolated amnion-derived cells have been
extensively characterized. As set forth in Example 7, freshly
isolated amnion-derived cells are substantially purified with
respect to CD90 and CD117. In addition, such populations are
essentially negative for protein expression of CD34, CD44, CD45,
CD140b, CD105; essentially positive for protein expression of CD9
and CD29; between about 70-95% positive for protein expression of
SSEA4, CD10, CD166 and CD227; between about 60-95% positive for
protein expression of HLA-G, EGFR and CD26; and between about
10-50% positive for protein expression of CD71.
[0131] In alternative embodiments substantially purified
amnion-derived cell populations can be created using antibodies
against protein markers expressed (positive selection) or not
expressed (negative selection) on the cell surface of the
amnion-derived cells. For instance, Example 8 below demonstrates
how antibodies can be used to create substantially purified
populations. These antibodies may be used to identify,
characterize, isolate or create such substantially purified
populations of amnion-derived cells expressing those protein
markers using a variety of methods. Such procedures may involve a
positive selection, such as passage of sample cells over a column
containing anti-protein marker antibodies or by binding of cells to
magnetic bead-conjugated antibodies to the protein markers or by
panning on plates coated with protein marker antibodies and
collecting the bound cells. Alternatively, a single-cell suspension
may be exposed to one or more fluorescent-labeled antibodies that
immuno-specifically bind to amnion-derived cell protein markers.
Following incubation with the appropriate antibody or antibodies,
the amnion-derived cells are rinsed in buffer to remove any unbound
antibody. Amnion-derived cells expressing the protein marker(s) can
then be sorted by fluorescence-activated cell sorting (FACS) using,
for example, a Becton Dickinson FACStar flow cytometer. To create
substantially purified populations of amnion-derived cells
expressing a desired protein marker(s), the cells may be subjected
to multiple rounds of FACS sorting.
[0132] In addition, protein markers that are not expressed on the
surface of amnion-derived cells may also be used to identify,
isolate or create populations of amnion-derived cells not
expressing those markers. Such procedures may involve a negative
selection method, such as passage of sample cells over a column
containing anti-protein marker antibodies or by binding of cells to
magnetic bead-conjugated antibodies to the protein markers or by
panning on plates coated with protein marker antibodies and
collecting the unbound cells. Alternatively, a single-cell
suspension may be exposed to one or more fluorescent-labeled
antibodies that immuno-specifically bind to the protein markers.
Following incubation with the appropriate antibody or antibodies,
the cells are rinsed in buffer to remove any unbound antibody.
Cells expressing the protein marker(s) can then be sorted by
fluorescence-activated cell sorting (FACS) using, for example, a
Becton Dickinson FACStar flow cytometer and these cells can be
removed. Remaining cells that do not bind to the antibodies can
then be collected. To create substantially purified populations of
amnion-derived cells that do not express a desired protein
marker(s), the cells may be subjected to multiple rounds of FACS
sorting as described above.
[0133] The present invention further contemplates novel antibodies
to amnion-derived cells or to amnion-derived cell protein markers
described herein. The antibodies are useful for detection of the
amnion-derived cell protein markers in, for example, diagnostic
applications. For preparation of monoclonal antibodies directed
toward amnion-derived cells or to amnion-derived cell protein
markers, any technique which provides for the production of
antibody molecules by continuous cell lines in culture may be used.
For example, various protocols for the production of monoclonal
antibodies can be found in Monoclonal Antibody Protocols, Margaret
E. Shelling, Editor, Humana Press; 2nd edition (Mar. 15, 2002). In
addition, the hybridoma technique originally developed by Kohler
and Milstein (1975, Nature 256:495-497), as well as the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
1983, Immunology Today 4:72), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., 1985, in
"Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp.
77-96) and the like are within the scope of the present
invention.
[0134] The monoclonal antibodies may be human monoclonal antibodies
or chimeric human-mouse (or other species) trionoclonal antibodies.
Human monoclonal antibodies may be made by any of numerous
techniques known in the art (e g, Teng et al., 1983, Proc. Natl.
Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology
Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16).
Chimeric antibody molecules may be prepared containing a mouse
antigen-binding domain with human constant regions (Morrison et
al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al.,
1985, Nature 314:452).
[0135] Various procedures known in the art may be used for the
production of polyclonal antibodies to epitopes of the
amnion-derived cells or to the amnion-derived cell protein markers
described herein. For the production of antibody, various host
animals can be immunized by injection with amnion-derived cells or
to amnion-derived cell protein markers, or a fragment or derivative
thereof, including but not limited to rabbits, mice and rats.
Various adjuvants may be used to increase the immunological
response, depending on the host species, and including but not
limited to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanins, dinitrophenol, and potentially useful human
adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium
parvum.
[0136] A molecular clone of an antibody to selected amnion-derived
cells or to amnion-derived cell protein marker epitope(s) can be
prepared by known techniques. Recombinant DNA methodology (Sambrook
et al, 2001, "Molecular Cloning: A Laboratory Manual"; Ausubel,
ed., 1994, "Current Protocols in Molecular Biology" Volumes I-III)
may be used to construct nucleic acid sequences which encode a
monoclonal antibody molecule, or antigen binding region
thereof.
[0137] The present invention provides for antibody molecules as
well as fragments of such antibody molecules. Antibody fragments
which contain the idiotype of the molecule can be generated by
known techniques. For example, such fragments include but are not
limited to: the F(ab')2 fragment which can be produced by pepsin
digestion of the antibody molecule; the Fab' fragments which can be
generated by reducing the disulfide bridges of the F(ab')2
fragment, and the Fab fragments which can be generated by treating
the antibody molecule with papain and a reducing agent. Another
antibody fragment is the single chain Fv (scFv) which is a
truncated Fab having only the V region of a heavy chain linked by a
stretch of synthetic peptide to a V region of a light chain. See,
for example, U.S. Pat. Nos. 5,565,332; 5,733,743; 5,837,242;
5,858,657; and 5,871,907 assigned to Cambridge Antibody Technology
Limited incorporated by reference herein. Antibody molecules may be
purified by known techniques, e.g., immunoabsorption or
immunoaffinity chromatography, chromatographic methods such as HPLC
(high performance liquid chromatography), or a combination
thereof.
[0138] Expanded Populations of Amnion-Derived Cells
[0139] As described herein, Applicants have discovered a novel
method for isolation and propagation of pluripotent, amnion-derived
cells. Such methods result in amnion-derived cell compositions
which are expanded for pluripotent cells, thereby providing, for
the first time, sufficient quantities of cells to enable
therapeutic cell transplantation. Expanded amnion-derived cell
compositions, which are made in accordance with the subject
invention, are compositions in which the level of multipotent cells
per gram of amniotic tissue is at least 50 fold and up to 150 fold
higher after 5 passages, as compared to about 20 fold using
previous methods.
[0140] Additionally, the methods used for cell culture and
proliferation provide a means to culture the cells, as well as
other pluripotent cells, including, but not limited to, embryonic
stem cells, in an animal-free system. Furthermore, the culture
conditions described provide a cell that is less dependent on
attachment to a culture surface for viability, thus allowing for
propagation of the cells using suspension culture for efficient
scale-up.
[0141] The expanded amnion-derived cell compositions described
herein demonstrate extensive proliferative potential, express
certain genes known to be expressed only in undifferentiated cells
(i.e. Nanog and Oct-4) and can differentiate into cell types that
normally arise from all three embryonic germ layers (endoderm,
ectoderm and mesoderm). This differentiation potential suggests
that these expanded amnion-derived cells may be able to contribute
to a variety of cell types. The amnion-derived cell compositions
described herein are also useful as feeder layers for the growth of
a variety of cell types, including but not limited to embryonic
stem cells (ES cells). Amnion-derived cells, including those
described herein, also produce a wide variety of cytokines and
growth factors, thereby making both the cell compositions,
conditioned medium derived from the cells, cell lysates thereform,
extracellular matrices produced by the cells, and combinations
thereof useful to achieve rapid and effective wound healing,
including scarless healing, and also useful in cosmetics, i.e. to
achieve improvement in skin appearance.
[0142] Culturing of the amniotic cells--The cells are cultured in a
basal medium. Such medium includes, but is not limited to, Epilife
(Cascade Biologicals), Opti-pro, VP-SFM, IMDM, Advanced DMEM, K/O
DMEM, 293 SFM II (all made by Gibco; Invitrogen), HPGM, Pro
293S-CDM, Pro 293A-CDM, UltraMDCK, UltraCulture (all made by
Cambrex), Stemline I and Stemline II (both made by Sigma-Aldrich),
DMEM, DMEM/F-12, Ham's F12, M199, and other comparable basal media.
Such media should either contain human protein or be supplemented
with human protein. As used herein a "human protein" is one that is
produced naturally or one that is produced using recombinant
technology. "Human protein" also is meant to include a human fluid
or derivative or preparation thereof, such as human serum or
amniotic fluid, which contains human protein. In preferred
embodiments, the basal media is Stemline I or II, UltraCulture, or
Opti-pro, or combinations thereof and the human protein is human
albumin at a concentration of at least 0.5% and up to 10%. In
particular embodiments, the human albumin concentration is from
about 0.5 to about 2%. The human albumin may come from a liquid or
a dried (powder) form and includes, but is not limited to,
recombinant human albumin, plasbumin and plasmanate.
[0143] In a most preferred embodiment, the cells are cultured using
a system that is free of animal products to avoid
xeno-contamination. In this embodiment, the culture medium is
Stemline I or II, Opti-pro, or DMEM, with human albumin (plasbumin)
added up to concentrations of 10%. Alternatively, UltraCulture may
be used, with substitution of transferrin with human recombinant
transferrin, and replacement of the bovine albumin (BSA) with human
albumin at concentrations of up to 10%. The invention further
contemplates the use of any of the above basal media wherein
animal-derived proteins are replaced with recombinant human
proteins and animal-derived serum, such as BSA, is replaced with
human albumin. In preferred embodiments, the media is serum-free in
addition to being animal-free.
[0144] Further, the culture conditions described herein result in
the formation of three-dimensional clusters of cells called
spheroids, a property that may enhance the likelihood of
differentiation to, for example, pancreatic islet cells, neural
lineages and cardiac cells. Such compositions are prepared as
described above using a basal media selected from the group
consisting of Opti-pro SFM, VP-SFM, Iscove's MDM, HPGM, UltraMDCK,
Stemline II and Stemline I, DMEM, and DMEM:F12 with added human
albumin, plasmanate or plasbumin at levels of up to 10%.
[0145] In alternative embodiments, where the use of non-human serum
is not precluded, such as for in vitro uses, the culture medium may
be supplemented with serum derived from mammals other than humans,
in ranges of up to 40%.
[0146] Additional proliferation--Optionally, other proliferation
factors are used. In one embodiment, epidermal growth factor (EGF),
at a concentration of between 0-1 .mu.g/ml is used. In a preferred
embodiment, the EGF concentration is around 10 ng/ml. Alternative
growth factors which may be used include, but are not limited to,
TGF.alpha. or TGF.beta. (5 ng/ml; range 0.1-100 ng/ml), activin A,
cholera toxin (preferably at a level of about 0.1 .mu.g/ml; range
0-10 .mu.g/ml), transferrin (5 g/ml; range 0.1-100 .mu.g/ml),
fibroblast growth factors (bFGF 40 ng/ml (range 0-200 ng/ml), aFGF,
FGF-4, FGF-8; (all in range 0-200 ng/ml), bone morphogenic proteins
(i.e. BMP-4) or other growth factors known to enhance cell
proliferation.
[0147] Passaging--Cells are initially plated at a density of
25,000/cm.sup.2-1,000,000/cm.sup.2, on tissue culture treated
plates, preferably at a density of about 130,000/cm.sup.2. In one
embodiment, the cells are grown on extracellular matrix treated
plates, such as collagen, laminin, fibronectin, or Matrigel. To
create the expanded amnion-derived cell compositions of the
invention, the cells are passaged at least five (5) times as
described below in Example 1. To create the spheroidal
amnion-derived cell compositions of the invention, only one passage
is required.
[0148] Growth of ES cells--The culture media described above may
also be used to produce expanded or spheroidal preparations of
embryonic stem cells (ES cells). In some embodiments, the culture
medium is free of animal products. In preferred embodiments, the
culture medium is free of animal product and is made without
serum.
[0149] Large scale culture of amnion-derived cells--In further
embodiments, large scale culture is used to produce the
amnion-derived cell compositions, conditioned media therefrom, and
cells for the preparation of cell lysates. The literature
describing large-scale mammalian cell culture has predominantly
related to the culture of cells such as Chinese hamster ovary (CHO)
cells to produce therapeutic proteins (Moreira, J. L. et al. (1995)
Biotechnol Prog, 11:575). In this case, the secretory product of
the cells is the main product of interest. Technologies most often
used for large-scale cell production have been spinner flasks and
roller bottles, although roller bottles are being replaced in some
applications, especially adherent cell culture, by hollow-fibre
culture bioreactors and microcarrier bioreactor systems (Martin,
I., et al. (2004) Trends Biotechnol, 22:80). Hollow fibre
bioreactors combine synthetic fibres with mammalian cells. The
cells are seeded between the fibres and grow in a 3-dimensional
tissue-like formation. The fibres act as conduits for nutritional
factors and oxygen to reach the cells, and also provide an exit for
toxins and cellular by-products which need to be removed from the
proximity of the cells. One of the drawbacks of hollow-fibre
technology is the difficulty in recovery of the cells, although for
some applications, such as extracorporeal hepatic assist devices,
the cells remain in situ to perform their therapeutic purpose
(Gerlach, J. C., (1997) Cell Biol Toxicol, 13:349).
[0150] Large scale cell culture may be used to culture the
amnion-derived cells as a product for some therapeutic purposes,
including both growth of cells for transplantation, as well as for
production of conditioned media. Hematopoietic cells for bone
marrow transplant have been cultured in suspension (Cheshier, S.
H., et al, (1999) Proc Natl Acad Sci USA, 96:3120; Madlambayan, G.
J., et al, (2001) J Hematother Stem Cell Res, 10:481), hepatocytes
for extracorporeal assist devices (Gerlach, J. C., (1997) Cell Biol
Toxicol, 13:349), keratinocytes for artificial skin applications
(Zacchi, V., et al, (1998) J Biomed Mater Res, 40:187; Pellegrini,
G., et al, (1998) Med Biol Eng Comput, 36:778; Waymack, P., et al,
(2000) Burns, 26:609), and neural stem cells for neurodegenerative
diseases (Kallos, M. S., et al. (2003) Med Biol Eng Comput 41:271).
If mammalian cells are anchorage-dependent and cannot be cultured
in suspension, microcarrier beads or microcarriers can be used as a
large surface area to which the cells can attach and grow in the
suspension apparatus. The cell-covered microcarrier beads are
maintained in suspension in the apparati used for cell suspension
cultures, allowing for reductions in media usage and space
requirements. One of the technical difficulties of microcarrier
bead culture is the efficient removal of the cells from the beads
themselves, without compromising viability (Varani, J., et al.
(1986) J Biol Stand, 14:331). The preferred method would be to
culture amnion-derived cells in suspension.
[0151] The scalable production of amnion-derived cells may be
accomplished using systems currently being developed for human
embryonic stem (hES) cells. Most examples of scale-up for hES cells
include partial or complete differentiation during the scale-up
process, for instance Gerecht-Nir and coworkers (Gerecht-Nir, S.,
et al. (2004) Biotechnol Bioeng 86:493) report the scalability of
ES cells as embryoid bodies. Other examples of differentiated
scale-up for ES cells include cardiac cells (Zandstra, P. W., et
al. (2003) Tissue Eng, 9:767) in which embryoid bodies are formed
and treated with retinoic acid, and ES-derived hepatocyte scale-up
in hollow fibre bioreactors (Gerlach, J. C., (1997) Cell Biol
Toxicol, 13:349). Reports of scale-up of undifferentiated human ES
cells are scarce, although mouse ES cells can be proliferated in
hollow fibre bioreactors, with maintenance of their stem cell
surface characteristics.
[0152] In other embodiments, the cells are cultured in suspension
culture conditions including in suspension culture treated plates,
and roller bottles (in roller bottles density range 100,000/ml -5
million/ml; preferred 1 million/ml), or spinner flasks with or
without attachment to microcarrier beads. Examples 2, 3 and 4 sets
forth methods of large scale production that may be used in
accordance with the invention.
[0153] Experiments are performed to determine the medium and
supplements that can be used for optimal cell growth and expression
of differentiated function. The use of multiple spinner flasks
permits the use of 2 or 3 replicates of each condition per
experiment.
[0154] Once cells are grown successfully in suspension, they are
then cultured in sufficient quantities for transplantation. One
skilled in the art will recognize that the number of cells needed
for transplantation will depend upon the specific application.
Cells are harvested as determined in the first set of experiments
above but instead of seeding into T-flasks or spinner flasks they
are put into Wave bags. These are sterile plastic bags (Wave, Inc.)
into which cells and medium are added. The bag and its contents are
placed on a rocker that gently agitates the entire bag. In
addition, CO.sub.2 and air can be added continuously to the bag to
maintain adequate oxygen and pH control. The range of bag sizes is
1 liter to 1000 liters. In one embodiment, 1 liter bags and a
minimum working volume of 125 ml are used. As the cells grow,
additional medium can be added until the 500 ml working volume is
attained.
[0155] Amnion-derived cells are placed both into the Wave bags and
into the normal T-flasks and incubated at 37.degree. C. at 5%
CO.sub.2 in air. Daily samples of cells are withdrawn, in a class
100 biosafety cabinet, from each flask and vessel, and the cells
are stained with trypan blue and counted on a hemacytometer. A
graph of total and viable cell counts per ml is plotted with time
to ensure that amnion-derived cells divide and remain viable with
time in culture.
[0156] Wave bags have two main advantages over alternative culture
vessels. Firstly, they are disposable and therefore cleaning
validation is not necessary for each lot of cells. Secondly,
because the rocking motion creates a wave of liquid in the bags,
the gas exchange in the liquid is much higher than if the cells
were in a stationary flask or spinner flask. As a result, the total
attainable cell number is higher than in either the T-flasks or
spinner flasks.
[0157] At specific intervals, samples are analyzed using reverse
transcriptase and/or real time PCR for gene expression over time.
The samples are examined for amnion-derived cell-specific markers
such as Oct-4, nanog, etc. In addition, samples are measured by
FACS analysis for cell surface markers of undifferentiated cells
(SSEA-3 and 4, Tra-1-60 and Tra-1-81).
[0158] In addition, the ability of amnion-derived cells after
suspension culture and proliferation to undergo differentiation to
all three germ lineages (endoderm, mesoderm, and ectoderm) is
analyzed. Such characterization of differentiative capacity is
assessed by performing differentiation assays and analysis of gene
expression by reverse transcriptase and/or real-time PCR and by low
volume FACS analysis (or IHC).
[0159] At designated time intervals, the cells are removed from the
culture vessels and cultured in differentiation protocols to
determine their ability to differentiate to the three germ lineages
after proliferation.
[0160] Compositions--The compositions of the invention include
substantially purified populations and pharmaceutical compositions
of such. The compositions of the invention can be prepared in a
variety of ways depending on the intended use of the compositions.
For example, a composition useful in practicing the invention may
be a liquid comprising an agent of the invention, i.e. a
substantially purified population of amnion-derived cells, in
solution, in suspension, or both (solution/suspension). The term
"solution/suspension" refers to a liquid composition where a first
portion of the active agent is present in solution and a second
portion of the active agent is present in particulate form, in
suspension in a liquid matrix. A liquid composition also includes a
gel. The liquid composition may be aqueous or in the form of an
ointment, salve, cream, or the like.
[0161] An aqueous suspension or solution/suspension useful for
practicing the methods of the invention may contain one or more
polymers as suspending agents. Useful polymers include
water-soluble polymers such as cellulosic polymers and
water-insoluble polymers such as cross-linked carboxyl-containing
polymers. An aqueous suspension or solution/suspension of the
present invention is preferably viscous or muco-adhesive, or even
more preferably, both viscous and muco-adhesive.
[0162] Pharmaceutical Compositions--The present invention provides
pharmaceutical compositions of substantially purified populations
of amnion-derived cells, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly, in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with
which the composition is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin, and still
others are familiar to skilled artisans.
[0163] The pharmaceutical compositions of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0164] Treatment Kits--The invention also provides for an article
of manufacture comprising packaging material and a pharmaceutical
composition of the invention contained within the packaging
material, wherein the pharmaceutical composition comprises a
substantially purified population of amnion-derived cells, and
wherein the packaging material comprises a label or package insert
which indicates that the substantially purified population of
amnion-derived cells can be used for treating a variety of
disorders including but not limited to diabetes, liver disease,
neural disease, etc.
[0165] Amnion-Derived Cell Nuclei
[0166] In addition to amnion-derived cells themselves and products
derived therefrom, another embodiment of the invention is the
nuclei of amnion-derived cells. Such nuclei may be obtained using
methods known in the art. These include removing the membranes from
cells by either mechanical disruption or chemical means such as
treatment with hyaluronidase or performed by mechanically
extracting the nucleus with a pipet. The nuclei may then
subsequently be transferred into somatic or germ cells, by for
example, intracytoplasmic injection or electrofusion using methods
known in the art as described in for example, US 20030234430 or US
20040268422. As amnion-derived cells are derived from
extraembryonic tissue, specifically the amnion, they are
non-somatic, non-fetal, and non-germ cells, thus unique among the
donor cells typically used in the art.
[0167] Once the nuclei are transferred into another cell, the
resulting cell may be used for any number of applications. One
embodiment relates to methods of therapeutic nuclear cloning or
cloning an animal by combining any enucleated cell with the nuclei
from an amnion-derived cell. This embodiment encompasses the
cloning of a variety of animals. These animals include all mammals
(e.g., human, canines, felines, mice, rats, livestock cattle,
sheep, goats, camels, pigs, horses, llamas). The donor
amnion-derived cell and the oocyte may or may not be from the same
animal.
[0168] The genome of the donor amnion-derived cell can be the
naturally occurring genome, for example, for the production of
cloned animals, or the genome can be genetically altered to
comprise a transgenic sequence, for example, for the production of
transgenic cloned animals.
[0169] The oocytes used in the present invention could be in any
stage of meiotic cell division, including metaphase I, anaphase I,
anaphase II, telophase I, telophase II, and preferably metaphase
II. Oocytes in metaphase II are considered to be in a resting
state. The oocytes can be in the resting stage of metaphase II, and
then be activated, using methods described herein. The stage that
the oocyte is in can be identified by visual inspection of the
oocyte under a sufficient magnification. Methods for identifying
the stage of meiotic cell division are known in the art.
[0170] Oocytes can be activated by physical (e.g. electrical
stimulation, cold shock) and chemical means (e.g. ethanol, acid
tyrode's solution, strontium chloride, calcium ionophore,
puromycin, hyaluronidase and media lacking calcium and magnesium).
Some of these methods activate oocytes by increasing intracellular
calcium levels. Several methods exist that allow for activation of
the oocyte. In particular, a calcium ionophore (e.g., ionomycin) is
an agent that increases the permeability of the oocyte's membrane
and allows calcium to enter into the oocyte. Such methods of
activation are described in U.S. Pat. No. 5,496,720. Ethanol has a
similar affect. Prior to or following enucleation, an oocyte in
metaphase II can be activated with ethanol according to the ethanol
activation treatment as described in Presicce and Yang, Mol.
Reprod. Dev., 37: 61-68 (1994); and Bordignon & Smith, Mol.
Reprod. Dev., 49: 29-36 (1998). Exposure of calcium to the oocyte
also occurs through electrical stimulation. The electrical
stimulation allows increasing levels of calcium to enter the
oocyte. Other known methods of activation can be used with the
present invention to activate the oocyte.
[0171] Oocytes can be obtained from a donor animal during that
animal's reproductive cycle. For example, oocytes can be aspirated
from follicles of ovaries at given times during the reproductive
cycle (exogenous hormone-stimulated i.e. super-ovulation or ovarian
hyperstimulation or non-stimulated). Also at given times following
ovulation, a significant percentage of the oocytes, for example,
are in telophase. Additionally, oocytes can be obtained and then
induced to mature in vitro to an arrested metaphase II stage.
Arrested metaphase II oocytes, produced in vivo or in vitro, can
then be induced in vitro to enter telophase. Thus, oocytes in
telophase can readily be obtained for use in the present invention.
Oocytes can also be recovered surgically by flushing the oocytes
from the oviduct of a female donor. Methods for the collection of
oocytes are known in the art.
[0172] Preferably, the cell stage of the activated oocytes
correlates to the stage of the cell cycle of the donor
amnion-derived cell. This correlation between the meiotic stage of
the oocyte and the mitotic stage of the donor cell is also referred
to herein as "synchronization."
[0173] The present invention utilizes an oocyte that is enucleated.
An enucleated oocyte is one that is devoid of the genome, or one
that is "functionally enucleated." A functionally enucleated oocyte
contains a genome that is non-functional, e.g., cannot replicate or
synthesize DNA. See, for example, Bordignon, V. and L. C. Smith,
Molec. Reprod. Dev., 49: 29-36 (1998). Preferably, the genome of
the oocyte is removed from the oocyte. A genome can be functionally
enucleated from the oocyte by irradiation, by x-ray irradiation, by
laser irradiation, by physically removing the genome, or by
chemical means. Methods of using irradiation are known to those in
the art and are described, for example, in Bradshaw et al.,
Molecul. Reprod. Dev., 41: 503-512 (1995). Methods of chemically
inactivating the DNA are known to those of skilled in the art
(Fulka and Moore, Molecul. Reprod. Dev., 34: 427-430 (1993). Other
known methods of enucleation can be used with the present invention
to enucleate the oocyte.
[0174] To physically remove the genome of an oocyte, one can insert
a micropipette or needle through the zona pellicuda of the oocyte
to remove nuclear material from the oocyte. In one example,
telophase oocytes which have two polar bodies can be enucleated
with a micropipette or needle by removing the second polar body in
surrounding cytoplasm. Specifically, oocytes in telophase stage of
meiosis can be enucleated at any point from the presence of a
protrusion in the plasma membrane from the second polar body up to
the formation of the second polar body itself. Thus, as used
herein, oocytes which demonstrate a protrusion in the plasma
membrane, usually with a spindle abutted to it, up to extrusion of
a second polar body are considered to be oocytes in telophase. In
another example, metaphase II stage oocytes can be enucleated by
puncturing the zona pellucida with a micropipette, abutting the
micropipette to the oocyte nucleus, withdrawing the nucleus and
part of the oolemma (or oocytes membrane) into the pipette. After
withdrawal of the micropipette the oolemma pinches off to leave a
membrane-intact enucleated oocyte. Oocytes can also be pre-treated
with cytochalasin D to aid in this process.
[0175] The present invention includes enucleating the genome of an
oocyte by treating the oocyte with a compound that will induce the
oocyte genome (e.g., nuclear chromatin) to segregate into the polar
bodies during meiotic maturaton thereby leaving the oocyte devoid
of a functional genome, and resulting in the formation of a
recipient cytoplast for use in nuclear transfer procedures.
Examples of agents that will effect such differential segregation
include agents that will disrupt cytoskeletal and metabolism (see,
for example Andreau, J. M. and Timasheff, S. N., Proc. Nat. Acad.
Sci. 79: 6753 (1982), Obrig, T. G., et al, J. Biol. Chem. 246: 174
(1971), Duskin, D. and Mahoney, W. C., J. Biol. Chem. 257: 3105
(1982), Scialli, A. R., et al, Teratogen, Carcinogen, Mutagen 14:
23 (1994), Nishiyama, I and Fujii, T., Exp. Cell Res. 198: 214
(1992), Small, J. V., et al, J. Cell Sci. 89: 21 (1988), Lee, J.
C., et al, Biochem. 19: 6209 (1980). The age of the oocyte and
timing of the events (i.e. enucleation, fusion and activation) are
also very important in successful nuclear transfer and are well
known to those skilled in the art.
[0176] Combination of the activated, enucleated oocyte and the
genome from the amnion-derived cell can occur a variety of ways to
form the nuclear transfer embryo. A genome of an amnion-derived
cell can be injected into the activated oocyte by employing a
microinjector (i.e., micropipette or needle). The nuclear genome of
the amnion-derived cell is extracted using a micropipette or
needle. Once extracted, the amnion-derived cell's nuclear genome
can then be placed into the activated oocyte by inserting the
micropipette, or needle, into the oocyte and releasing the nuclear
genome of the amnion-derived cell. (McGrath, J. and D. Solter,
Science, 226: 1317-1319 (1984)).
[0177] The present invention also includes combining the genome of
an amnion-derived cell with an activated oocyte by fusion e.g.,
electrofusion, viral fusion, liposomal fusion, biochemical reagent
fusion (e.g., phytohemaglutinin (PHA) protein), or chemical fusion
(e.g., polyethylene glycol (PEG) or ethanol). The amnion-derived
cell, an amnion-derived cell karyoplast or the nucleus of the
amnion-derived cell can be deposited within the zona pellucida
which contains the oocyte. The steps of fusing the cell, karyoplast
or nucleus with the oocyte can then be performed by techniques
known in the art. The combination of the genome of the
amnion-derived cell with the activated oocyte results in a nuclear
transfer embryo.
[0178] A nuclear transfer cell of the present invention could then
be transferred into a recipient non-human female animal and allowed
to develop or gestate into a cloned or transgenic animal.
Conditions suitable for gestation are those conditions that allow
for the embryo to develop and mature into a fetus, and eventually
into a live animal. Such conditions are known in the art. The
nuclear transfer cell can be maintained in a culture system until
at least the first cleavage (2-cell stage) up to the blastocyst
stage. Preferably the nuclear transfer cells are transferred at the
2-cell or 4-cell stage. Various culture media for nuclear transfer
cell development are known to those skilled in the art.
[0179] The present invention also relates to methods for generating
transgenic animals by combining an activated oocyte with a
genetically engineered genome from an amnion-derived cell. Such a
combination results in a transgenic nuclear transfer cell. A
transgenic animal is an animal that has been produced from a genome
from a donor cell that has been genetically altered, for example,
to produce a particular protein (a desired protein), or that has
been altered to knock-out a particular gene. Methods for
introducing DNA constructs into the germ line of an animal to make
a transgenic animal are known in the art.
[0180] The present invention is also directed to "therapeutic
cloning", which is the production of ES cells from a cloned embryo.
Previously, Munsie, at al. reported the isolation of mouse ES cells
from blastocysts derived by somatic cell nuclear transfer (Current
Biology 10: 989-992, 2000). Wakayama, et al. obtained mouse ES
cells, which can be induced to various types of specific cells in
vitro, from the cultures of blastocysts derived by somatic cell
nuclear transfer (Science, 292 (5517); 740-743. 2001). The result
of the research done by Wakayama, et al. demonstrates that ES cells
can be isolated from nuclear transfer embryos by somatic cell
nuclear transfer. The nuclear transfer ES cells of somatic cell
origin are pluripotent and can differentiate into any specific cell
types as ES cells derived from the normal zygote.
[0181] In the present invention, therapeutic cloning is
accomplished by the nuclear transfer of an amnion-derived cell
nucleus into an oocyte. The nuclear transfer cells obtained are
further differentiated into a specific cell type needed by, for
example, a patient suffering from a disease (i.e. diabetes, liver
failure, etc.).
[0182] Therapeutic Uses of Amnion-Derived Cells and Differentiated
Cells
[0183] Because these compositions comprise much higher cell numbers
per amniotic tissue than have previously been achieved, they allow
for therapeutic use in situations, such as transplantation, which
require large numbers of cells. These cells have been found to be
multipotent, i.e. capable of differentiating into a variety of
tissue types including but not limited to hematopoietic, liver,
pancreatic, nervous, muscle and endothelial tissues. Such cells are
particularly useful to restore function in diseased tissues via
transplantation therapy or tissue engineering, and to study
metabolism and toxicity of compounds in drug discovery efforts.
[0184] Cell transplantation strategies currently used in the clinic
or in clinical trials have demonstrated promising results, e.g., 1)
Pancreatic islets, isolated from cadaver tissue, are currently
transplanted to restore proper insulin secretion and alleviate the
need for insulin injections in Type I diabetic patients and 2)
Hepatocytes isolated from cadaver livers are transplanted to treat
patients awaiting liver transplant and for treatment of metabolic
disorders. However, the need for clinical grade pancreatic islets
and hepatocytes far exceeds the number of cells that can be
isolated and transplanted from donor tissue. Expanded
amnion-derived cell compositions described herein provide an
abundant cell source that can be differentiated to these cell
types.
[0185] Compositions comprising amnion-derived cell or cells
differentiated therefrom may be administered to a subject to
provide various cellular or tissue functions. As used herein
"subject" may mean either a human or non-human animal.
[0186] Such compositions may be formulated in any conventional
manner using one or more physiologically acceptable carriers
optionally comprising excipients and auxiliaries. Proper
formulation is dependent upon the route of administration chosen.
The compositions may be packaged with written instructions for use
of the cells in tissue regeneration, or restoring a therapeutically
important metabolic function. Amnion-derived cells may also be
administered to the recipient in one or more physiologically
acceptable carriers. Carriers for these cells may include but are
not limited to solutions of phosphate buffered saline (PBS) or
lactated Ringer's solution containing a mixture of salts in
physiologic concentrations.
[0187] One of skill in the art may readily determine the
appropriate concentration of cells for a particular purpose. A
preferred dose is in the range of about 0.25-1.0.times.10.sup.6
cells.
[0188] Amnion-derived cells or cells differentiated therefrom can
be administered by injection into a target site of a subject,
preferably via a delivery device, such as a tube, e.g., catheter.
In a preferred embodiment, the tube additionally contains a needle,
e.g., a syringe, through which the cells can be introduced into the
subject at a desired location. Specific, non-limiting examples of
administering cells to subjects may also include administration by
subcutaneous injection, intramuscular injection, or intravenous
injection. If administration is intravenous, an injectable liquid
suspension of cells can be prepared and administered by a
continuous drip or as a bolus.
[0189] Cells may also be inserted into a delivery device, e.g., a
syringe, in different forms. For example, the cells can be
suspended in a solution contained in such a delivery device. As
used herein, the term "solution" includes a pharmaceutically
acceptable carrier or diluent in which the cells of the invention
remain viable. Pharmaceutically acceptable carriers and diluents
include saline, aqueous buffer solutions, solvents and/or
dispersion media. The use of such carriers and diluents is well
known in the art. The solution is preferably sterile and fluid to
the extent that easy syringability exists. Preferably, the solution
is stable under the conditions of manufacture and storage and
preserved against the contaminating action of microorganisms such
as bacteria and fungi through the use of, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
Solutions of the invention can be prepared by incorporating
amnion-derived cells or differentiated cells as described herein,
in a pharmaceutically acceptable carrier or diluent and, as
required, other ingredients enumerated above, followed by filter
sterilization.
[0190] Undifferentiated, partially differentiated or fully
differentiated amnion-derived cells may be administered
systemically (for example intravenously) or locally (for example
directly into a myocardial defect under echocardiogram guidance or
by direct application under visualization during surgery). For such
injections, the cells may be in an injectable liquid suspension
preparation or in a biocompatible medium which is injectable in
liquid form and becomes semi-solid at the site of damaged tissue. A
conventional intra-cardiac syringe or a controllable endoscopic
delivery device can be used so long as the needle lumen or bore is
of sufficient diameter (e.g. 30 gauge or larger) that shear forces
will not damage the cells being delivered.
[0191] Cells may be administered in a manner that permits them to
graft to the intended tissue site and reconstitute or regenerate
the functionally deficient area. Undifferentiated, partially
differentiated or fully differentiated amnion-derived cells can be
used in therapy by direct administration, or as part of a bioassist
device that provides temporary or permanent organ function. In this
respect, undifferentiated, partially differentiated or fully
differentiated amnion-derived cells may be grown in a bioreactor to
provide extracorporeal organ support for organ relief, such as in
the case of a liver assist device, to provide a plentiful source of
cells for transplantation to restore organ function, or provide a
source of conditioned medium that may be used to stimulate tissue
regeneration. Liver assist devices utilizing primary porcine cells
as well as primary human liver cells have been used successfully
(Sauer, I. M., et al. Xenotransplantation (2003) 10:460-469;
Irgang, M. et al. (2003) 28(2):141-154; Sauer, I. M. et al. (2002)
Int. J. Art. Org. 25(10):1001-1006; Sauer, I. M. et al. (2002) J.
Metabolic Brain Disease 17(4): 477-484, Sauer, I. M. et al. (2003)
J. Hepatology 39(4):649-653). Amnion-derived cell-derived
hepatocytes may be utilized in conjunction with this
technology.
[0192] Alternatively, amnion-derived cells may be transplanted into
the recipient where the cells will proliferate and differentiate to
form new cells and tissues thereby providing the physiological
processes normally provided by that tissue, or may produce factors
that cause the migration and/or differentiation of cells in the
area of the transplant. Tissues are an aggregation of similarly
specialized cells united in the performance of a particular
function. Tissue is intended to encompass all types of biological
tissue including both hard and soft tissue. Soft tissue refers to
tissues that connect, support, or surround other structures and
organs of the body. Soft tissue includes muscles, tendons (bands of
fiber that connect muscles to bones), fibrous tissues, fat, blood
vessels, nerves, and synovial tissues (tissues around joints). Hard
tissue includes connective tissue (e.g., hard forms such as osseous
tissue or bone) as well as other muscular or skeletal tissue.
[0193] Support matrices into which the amnion-derived cells can be
incorporated or embedded include matrices which are
recipient-compatible and which degrade into products which are not
harmful to the recipient. These matrices provide support and
protection for undifferentiated and differentiated amnion-derived
cells in vivo and are, therefore, the preferred form in which such
cells are transplanted into the recipient subjects.
[0194] Natural and/or synthetic biodegradable matrices are examples
of such matrices. Natural biodegradable matrices include plasma
clots, e.g., derived from a mammal, collagen, fibronectin, and
laminin matrices. Suitable synthetic material for a cell
transplantation matrix must be biocompatible to preclude migration
and immunological complications, and should be able to support
extensive cell growth and differentiated cell function. It must
also be resorbable, allowing for a completely natural tissue
replacement. The matrix should be configurable into a variety of
shapes and should have sufficient strength to prevent collapse upon
implantation. Recent studies indicate that the biodegradable
polyester polymers made of polyglycolic acid fulfill all of these
criteria (Vacanti, et al. J. Ped. Surg. 23:3-9 (1988); Cima, et al.
Biotechnol. Bioeng. 38:145 (1991); Vacanti, et al. Plast. Reconstr.
Surg. 88:753-9 (1991)). Other synthetic biodegradable support
matrices include synthetic polymers such as polyanhydrides,
polyorthoesters, and polylactic acid. Further examples of synthetic
polymers and methods of incorporating or embedding cells into these
matrices are also known in the art. See e.g., U.S. Pat. Nos.
4,298,002 and 5,308,701.
[0195] Attachment of the cells to the polymer may be enhanced by
coating the polymers with compounds such as basement membrane
components, agar, agarose, gelatin, gum arabic, collagens types I,
II, III, IV and V, fibronectin, laminin, glycosaminoglycans,
mixtures thereof, and other materials known to those skilled in the
art of cell culture. All polymers for use in the matrix must meet
the mechanical and biochemical parameters necessary to provide
adequate support for the cells with subsequent growth and
proliferation. The polymers can be characterized with respect to
mechanical properties such as tensile strength using an Instron
tester, for polymer molecular weight by gel permeation
chromatography (GPC), glass transition temperature by differential
scanning calorimetry (DSC) and bond structure by infrared (IR)
spectroscopy, with respect to toxicology by initial screening tests
involving Ames assays and in vitro teratogenicity assays, and
implantation studies in animals for immunogenicity, inflammation,
release and degradation studies.
[0196] One of the advantages of a biodegradable polymeric matrix is
that angiogenic and other bioactive compounds can be incorporated
directly into the support matrix so that they are slowly released
as the support matrix degrades in vivo. As the cell-polymer
structure is vascularized and the structure degrades,
amnion-derived cells may differentiate according to their inherent
characteristics. Factors, including nutrients, growth factors,
inducers of differentiation or de-differentiation (i.e., causing
differentiated cells to lose characteristics of differentiation and
acquire characteristics such as proliferation and more general
function), products of secretion, immuno-modulators, inhibitors of
inflammation, regression factors, biologically active compounds
which enhance or allow ingrowth of the lymphatic network or nerve
fibers, hyaluronic acid, and drugs, which are known to those
skilled in the art and commercially available with instructions as
to what constitutes an effective amount, from suppliers such as
Collaborative Research, Sigma Chemical Co., vascular growth factors
such as vascular endothelial growth factor (VEGF), epidermal growth
factor (EGF), and heparin binding epidermal growth factor like
growth factor (HB-EGF), could be incorporated into the matrix or be
provided in conjunction with the matrix. Similarly, polymers
containing peptides such as the attachment peptide RGD
(Arg-Gly-Asp) can be synthesized for use in forming matrices (see
e.g. U.S. Pat. Nos. 4,988,621, 4,792,525, 5,965,997, 4,879,237 and
4,789,734).
[0197] In another example, the undifferentiated, partially
differentiated or fully differentiated amnion-derived cells may be
transplanted in a gel matrix (such as Gelfoam from Upjohn Company)
which polymerizes to form a substrate in which the cells can grow.
A variety of encapsulation technologies have been developed (e.g.
Lacy et al., Science 254:1782-84 (1991); Sullivan et al., Science
252:718-712 (1991); WO 91/10470; WO 91/10425; U.S. Pat. No.
5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538).
During open surgical procedures, involving direct physical access
to the damaged tissue and/or organ, all of the described forms of
undifferentiated, partially differentiated or fully differentiated
amnion-derived cell delivery preparations are available options.
These cells can be repeatedly transplanted at intervals until a
desired therapeutic effect is achieved.
[0198] The present invention also relates to the use of
amnion-derived cells in three dimensional cell and tissue culture
systems to form structures analogous to tissue counterparts in
vivo. The resulting tissue will survive for prolonged periods of
time, and perform tissue-specific functions following
transplantation into the recipient host. Methods for producing such
structures are described in U.S. Pat. Nos. 5,624,840 and 6,428,802,
which are incorporated herein in their entireties.
[0199] The three-dimensional matrices to be used are structural
matrices that provide a scaffold for the cells, to guide the
process of tissue formation. Scaffolds can take forms ranging from
fibers, gels, fabrics, sponge-like sheets, and complex 3-D
structures with pores and channels fabricated using complex Solid
Free Form Fabrication (SFFF) approaches. Cells cultured on a
three-dimensional matrix will grow in multiple layers to develop
organotypic structures occurring in three dimensions such as ducts,
plates, and spaces between plates that resemble sinusoidal areas,
thereby forming new liver tissue. Thus, in preferred aspects, the
present invention provides a scaffold, multi-layer cell and tissue
culture system. As used herein, the term "scaffold" means a
three-dimensional (3D) structure (substrate and/or matrix) that
cells grow in or on. It may be composed of biological components,
synthetic components or a combination of both. Further, it may be
naturally constructed by cells or artificially constructed. In
addition, the scaffold may contain components that have biological
activity under appropriate conditions. The structure of the
scaffold can include a mesh, a sponge or can be formed from a
hydrogel.
[0200] Examples of such scaffolds include a three-dimensional
stromal tissue or living stromal matrix which has been inoculated
with stromal cells that are grown on a three dimensional support.
The extracellular matrix proteins elaborated by the stromal cells
are deposited onto the scaffold, thus forming a living stromal
tissue. The living stromal tissue can support the growth of
amnion-derived cells or differentiated cells later inoculated to
form the three-dimensional cell culture. Examples of other three
dimensional scaffolds are described in U.S. Pat. No. 6,372,494.
[0201] The design and construction of the scaffolding to form a
three-dimensional matrix is of primary importance. The matrix
should be a pliable, non-toxic, injectable porous template for
vascular ingrowth. The pores should allow vascular ingrowth. These
are generally interconnected pores in the range of between
approximately 100 and 300 microns, i.e., having an interstitial
spacing between 100 and 300 microns, although larger openings can
be used. The matrix should be shaped to maximize surface area, to
allow adequate diffusion of nutrients, gases and growth factors to
the cells on the interior of the matrix and to allow the ingrowth
of new blood vessels and connective tissue. At the present time, a
porous structure that is relatively resistant to compression is
preferred, although it has been demonstrated that even if one or
two of the typically six sides of the matrix are compressed, that
the matrix is still effective to yield tissue growth.
[0202] The polymeric matrix may be made flexible or rigid,
depending on the desired final form, structure and function. For
repair of a defect, for example, a flexible fibrous mat is cut to
approximate the entire defect then fitted to the surgically
prepared defect as necessary during implantation. An advantage of
using the fibrous matrices is the ease in reshaping and rearranging
the structures at the time of implantation.
[0203] A sponge-like structure can also be used to create a
three-dimensional framework. The structure should be an open cell
sponge, one containing voids interconnected with the surface of the
structure, to allow adequate surfaces of attachment for sufficient
amnion-derived cells or differentiated cells to form a viable,
functional implant.
[0204] The invention also provides for the delivery of
amnion-derived cells, including amnion-derived cell compositions
described herein, in conjunction with any of the above support
matrices as well as amnion-derived membranes. Such membranes may be
obtained as a by-product of the process described herein for the
recovery of amnion-derived cells, or by other methods, such as are
described, for example, in U.S. Pat. No. 6,326,019 which describes
a method for making, storing and using a surgical graft from human
amniotic membrane, US 2003/0235580 which describes reconstituted
and recombinant amniotic membranes for sustained delivery of
therapeutic molecules, proteins or metabolites, to a site in a
host, U.S. 2004/0181240, which describes an amniotic membrane
covering for a tissue surface which may prevent adhesions, exclude
bacteria or inhibit bacterial activity, or to promote healing or
growth of tissue, and U.S. Pat. No. 4,361,552, which pertains to
the preparation of cross-linked amnion membranes and their use in
methods for treating burns and wounds. In accordance with the
present invention, amnion-derived cells may be grown on such
membranes, added to the membrane in either an undifferentiated,
partially differentiated or fully differentiated form, or
amnion-derived cell conditioned media or cell lysates may be added
to such membranes. Alternatively, amniotic tissue in which
amnion-derived cells have not been stripped away may be used to
deliver amnion-derived cells to a particular site. In all cases,
amnion-derived cells used in conjunction with amniotic tissue or
other matrices can be used in combination with other
therapeutically useful cells and/or cells expressing biologically
active therapeutics such as those described in below.
[0205] Amnion-derived cells and cells differentiated therefrom may
also be used to humanize animal organs. Human amnion-derived cells
may be similarly transplanted into another organ such as pancreas
or brain or heart. The animal organ may or may not be depleted of
its native cells prior to the transplant. "Humanized" organs of an
animal such as a mouse, rat, monkey, pig or dog could be useful for
organ transplants into humans with specific diseases.
[0206] Humanized animal models may also be used for diagnostic or
research purposes relating but not limited to, drug metabolism,
toxicology studies or for the production, study, or replication of
viral or bacterial organisms. Mice transplanted with human
hepatocytes forming chimeric human livers are currently being used
for the study of hepatitis viruses (Dandri et al. Hepatol.
33:981-988 (2001); Mercer et al. Nature Med. 7:927-933 (2001)).
[0207] Amnion-derived cells may be genetically engineered to
produce a particular therapeutic protein. Therapeutic protein
includes a wide range of biologically active proteins including,
but not limited to, growth factors, enzymes, hormones, cytokines,
inhibitors of cytokines, blood clotting factors, peptide growth and
differentiation factors. Particular differentiated cells may be
engineered with a protein that is normally expressed by the
particular cell type. For example, pancreatic cells can be
engineered to produce digestive enzymes. Hepatocytes can be
engineered to produce the enzyme inhibitor, A1AT, or clotting
factors to treat hemophilia. Furthermore, neural cells can be
engineered to produce chemical transmitters.
[0208] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a nucleic acid
encoding the protein of interest linked to appropriate
transcriptional/translational control signals. See, for example,
the techniques described in Sambrook et al, 2001, "Molecular
Cloning: A Laboratory Manual"; Ausubel, ed., 1994, "Current
Protocols in Molecular Biology" Volumes I-III; Celis, ed.,
1994.
[0209] Suitable methods for transferring vector or plasmids into
amnion-derived cells or cells differentiated therefrom include
lipid/DNA complexes, such as those described in U.S. Pat. Nos.
5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202;
and 6,051,429. Suitable reagents include lipofectamine, a 3:1 (w/w)
liposome formulation of the poly-cationic lipid
2,3-dioleyloxy-N-[2(sperminecarbox-amido)ethyl]-N,N-d-imethyl-1-propanami-
nium trifluoroacetate (DOSPA) (Chemical Abstracts Registry name:
N-[2-(2,5-bis[(3-aminopropyl)amino]-1-oxpentyl)amino)ethyl-]-N,N-dimethyl-
-2,3-bis(9-octadecenyloxy)-1-propanamin-trifluoroacetate), and the
neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane
filtered water. Exemplary is the formulation Lipofectamine 2000.TM.
(available from Gibco/Life Technologies # 11668019). Other reagents
include: FuGENE.TM. 6 Transfection Reagent (a blend of lipids in
non-liposomal form and other compounds in 80% ethanol, obtainable
from Roche Diagnostics Corp. # 1814443); and LipoTAXI.TM.
transfection reagent (a lipid formulation from Invitrogen Corp.,
#204110). Transfection of amnion-derived cells can be performed by
electroporation, e.g., as described in Roach and McNeish (Methods
in Mol. Biol. 185:1 (2002)). Suitable viral vector systems for
producing stem cells with stable genetic alterations may be based
on adenoviruses, lentiviruses, retroviruses and other viruses, and
may be prepared using commercially available virus components.
[0210] Amnion-derived cells that are undifferentiated, partially
differentiated, or fully differentiated may be administered or
transplanted to a subject to provide various cellular or tissue
functions specific to the differentiated cell type. For example,
amnion-derived cells differentiated into hepatocytes may be
transplanted into a patient suffering from liver disease. The
progress of the recipient receiving such cells or transplants can
be determined using assays that include blood tests known as liver
function tests. Efficacy of treatment can be determined by
immunocytochemical staining for liver cell markers, microscopic
determination of whether canalicular structures form in growing
tissue, and the ability of the treatment to restore synthesis of
liver-specific proteins. Amnion-derived cell-derived hepatocytes of
the invention can be assessed in animal models for their ability to
repair liver damage. Hepatocytes derived from amnion-derived cells
may be used in assays to detect the activity of specific metabolic
pathways. For detailed examples of the above, see US2003/0235563
and US2004/0161419, which are incorporated herein by reference.
[0211] Pancreatic cells derived from amnion-derived cells can be
used therapeutically for treatment of various diseases associated
with insufficient functioning of the pancreas. Pancreatic diseases
and treatment thereof using the pancreatic cells derived from
amnion-derived cells of the subject invention are described in more
detail below.
[0212] The present invention also provides for administration of
neural cells derived from amnion-derived cells for treatment of
neurological disease. Neurological disease refers to a disease or
condition associated with any defects in the entire integrated
system of nervous tissue in the body: the cerebral cortex,
cerebellum, thalamus, hypothalamus, midbrain, pons, medulla,
brainstem, spinal cord, basal ganglia and peripheral nervous
system. Examples include but are not limited to: Parkinson's
disease, Huntington's disease, Multiple Sclerosis, Alzheimer's
disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's
disease), Muscular dystrophy, choreic syndrome, dystonic syndrome,
stroke, and paralysis.
[0213] The amnion-derived cells may be used in in vitro priming
procedures that result in neural stem cells becoming neurons when
grafted into non-neurogenic or neurogenic areas of the CNS. For
details and examples, see US2003/0235563 and US2004/0161419, both
which are incorporated herein by reference.
[0214] The amnion-derived cells can be used to produce vascular
endothelial cells that may be used in methods for remodeling tissue
or replacing a scar tissue in a subject. Vascular endothelial cells
may also be used to repair vascular damage.
[0215] In an exemplary embodiment, a pharmaceutical composition
comprising an effective amount of the vascular endothelial cells
may be used to treat a subject with a vascular disease. Vascular
disease refers to a disease of the human vascular system. Examples
include peripheral arterial disease, abdominal aortic aneurysm,
carotid disease, and venous disease.
[0216] The present invention also provides for cardiomyocytes
derived from amnion-derived cells, which may be used
therapeutically for treatment of various diseases associated with
cardiac dysfunction. Cardiac disease or cardiac dysfunction as used
herein refers to diseases that result from any impairment in the
heart's pumping function. This includes, for example, impairments
in contractility, impairments in ability to relax (sometimes
referred to as diastolic dysfunction), abnormal or improper
functioning of the heart's valves, diseases of the heart muscle
(sometimes referred to as cardiomyopathy), diseases such as angina
and myocardial ischemia and infarction characterized by inadequate
blood supply to the heart muscle, infiltrative diseases such as
amyloidosis and hemochromatosis, global or regional hypertrophy
(such as may occur in some kinds of cardiomyopathy or systemic
hypertension), and abnormal communications between chambers of the
heart (for example, atrial septal defect). For further discussion,
see Braunwald, Heart Disease: a Textbook of Cardiovascular
Medicine, 5th edition, W B Saunders Company, Philadelphia Pa.
(1997) (hereinafter Braunwald). Cardiomyopathy refers to any
disease or dysfunction of the myocardium (heart muscle) in which
the heart is abnormally enlarged, thickened and/or stiffened. As a
result, the heart muscle's ability to pump blood is usually
weakened. The disease or disorder can be, for example,
inflammatory, metabolic, toxic, infiltrative, fibroblastic,
hematological, genetic, or unknown in origin. There are two general
types of cardiomyopathies: ischemic (resulting from a lack of
oxygen) and nonischemic. Other diseases include congenital heart
disease which is a heart-related problem that is present since
birth and often as the heart is forming even before birth or
diseases that result from myocardial injury which involves damage
to the muscle or the myocardium in the wall of the heart as a
result of disease or trauma. Myocardial injury can be attributed to
many things such as, but not limited to, cardiomyopathy, myocardial
infarction, or congenital heart disease.
[0217] The amnion-derived cells and/or differentiated
cardiomyocytes may be administered and/or transplanted to a subject
suffering from a cardiac disease in any fashion as previously
discussed.
[0218] Methods are also provided for screening agents that affect
cardiomyocyte differentiation or function. For details and examples
see US2003/0235563 and US2004/0161419, which are incorporated
herein by reference.
[0219] In another embodiment, amnion-derived cells, and their
derivatives, can be used to screen various compounds to determine
the effect of the compound on cellular growth, proliferation or
differentiation of the cells. Methods of measuring cell
proliferation are well known in the art and most commonly include
determining DNA synthesis characteristic of cell replication. There
are numerous methods in the art for measuring DNA synthesis, any of
which may be used according to the invention. For example, DNA
synthesis may be determined using a radioactive label
(3H-thymidine) or labeled nucleotide analogues (BrdU) for detection
by immunofluorescence. The efficacy of the compound can be assessed
by generating dose response curves from data obtained using various
concentrations of the compound. A control assay can also be
performed to provide a baseline for comparison. Identification of
the amnion-derived cell population(s) amplified in response to a
given test agent can be carried out according to such phenotyping
as described above.
[0220] In order to assess the effect of a test agent on
amnion-derived cell differentiation or function, the agent may be
contacted with the amnion-derived cells and differentiation
assessed using any means known to one of skill in the art. For
examples and details, see US2003/0235563 and US2004/0161419, which
are incorporated herein by reference.
[0221] In another embodiment, amnion-derived cell compositions
prepared as describe herein are used as feeder layers for the
growth of embryonic stem cells. Such amnion-derived cell
compositions are preferably animal-free. Examples of use of such
cells as feeders layers can be found in Miyamoto, K., et al. Stem
Cells 2004:22:433-440 which is incorporated by reference in its
entirely herein.
[0222] Wound healing--The compositions and methods of the present
invention are effective in accelerating wound healing of wounds
caused by a number of sources, including but not limited to
incisional, compression, thermal, acute, chronic, infected, and
sterile injuries. The instant invention is based upon the discovery
that undifferentiated, partially differentiated or fully
differentiated amnion-derived cells, conditioned medium therefrom,
cell lysates therefrom, extracellular matrices therefrom, alone or
in combination, as well as composition of placental-derived cells
as defined herein can accelerate the wound healing process for all
wound types, particularly when administered topically, i.e. to the
surface of the wound site. Using amnion-derived cells and/or
conditioned medium from such amnion-derived cells, all wound types,
mechanical or thermal, acute or chronic, infected or sterile,
undergo healing more rapidly than similar wounds left to heal
naturally or which are treated with currently available methods. A
"therapeutically effective amount" of a therapeutic agent within
the meaning of the present invention will be determined by a
patient's attending physician or veterinarian. Such amounts are
readily ascertained by one of ordinary skill in the art and will
enable accelerated wound healing when administered in accordance
with the present invention. Factors which influence what a
therapeutically effective amount will be include, the specific
activity of the therapeutic agent being used, the wound type
(mechanical or thermal, full or partial thickness, etc.), the size
of the wound, the wound's depth (if full thickness), the absence or
presence of infection, time elapsed since the injury's infliction,
and the age, physical condition, existence of other disease states,
and nutritional status of the patient. Additionally, other
medication the patient may be receiving will effect the
determination of the therapeutically effective amount of the
therapeutic agent to administer.
[0223] In addition, compositions of the invention may play a role
in more substantial wound healing, such as in the regeneration of
limbs. US2003/0212024, which is incorporated by reference herein,
sets forth methods of testing for such ability by measuring
regeneration in the zebrafish, which is capable of complete
regeneration following amputation of the distal fin. Following
amputation, complete regeneration occurs in several steps,
including formation of a wound epidermis, migration of fibroblasts
and scleroblasts (or osteoblasts) toward the wound epidermis,
formation of a blastema, and outgrowth of the blastema via cell
division and differentiation of the proximal portion of the fin to
form specific structures of the regenerated fin.
[0224] In a preferred embodiment of the present invention,
amnion-derived cells and/or conditioned medium therefrom, and/or
cell lysates thereof should be topically administered to the wound
site to promote accelerated wound healing in the patient. This
topical administration can be as a single dose or as repeated doses
given at multiple designated intervals. It will readily be
appreciated by those skilled in the art that the preferred dosage
regimen will vary with the type and severity of the injury being
treated.
[0225] Formulations suitable for topical administration in
accordance with the present invention comprise therapeutically
effective amounts of the therapeutic agent with one or more
pharmaceutically acceptable carriers and/or adjuvants.
Amnion-derived cells, conditioned media therefrom and cell lysates
thereof may be used in conjunction with a variety of materials
routinely used in the treatment of wounds, such as collagen based
creams, films, microcapsules, or powders; hyaluronic acid or other
glycosaminoglycan-derived preparations; creams, foams, suture
material; and wound dressings. Alternatively, the amnion-derived
cell compositions can be incorporated into a pharmaceutically
acceptable solution designed for topical administration.
[0226] Cosmetic Applications--The same properties that make
undifferentiated, partially differentiated or fully differentiated
amnion-derived cells, conditioned medium therefrom, cell lysates
therefrom, extracellular matrices therefrom, alone or in
combination, as well as composition of placental-derived cells as
defined herein useful for wound healing make them similarly
well-suited for the treatment of cosmetic and/or dermatological
conditions, including aging skin. The dermal layer of skin,
important in maintaining the elasticity and appearance of the skin,
thins with age, leading to sagging and wrinkles.
[0227] As described above, fetal skin has much more effective
repair mechanisms, and, once wounded, it is able to heal without
the formation of scars. This capability does appear to require the
fetal immune system, fetal serum, or amniotic fluid (Bleacher J C,
et al., J Pediatr Surg 28: 1312-4, 1993); Ihara S, Motobayashi Y.,
Development 114: 573-82. 1992). Such abilities of fetal tissue have
led to the suggested use of compounds produced by fetal tissue for
regenerating and/or improving the appearance of skin (see, for
example, US 2004/0170615, which is incorporated by reference in its
entirety herein).
[0228] The present invention contemplates the use of the
amnion-derived cell compositions described herein, as well as
conditioned medium therefrom, and cell lysates thereof, in the use
of novel cosmetic skin care compositions. Such compounds may be
delivered to skin by way of, but not limited to, a solution, a
lotion, an ointment, a cream, a gel, or a skin peelable strip.
[0229] The methods generally include the step of topically applying
a safe and effective amount of the composition to the skin of a
mammal in need thereof. Additional skin care components, as well as
cosmetically acceptable, dermatologically acceptable or
pharmaceutically acceptable carriers may be included in such
compositions.
[0230] Cosmetic compositions usually comprise an aqueous phase that
is gelled, i.e. thickened, using one or more thickener(s) or
gelling agent(s). These may be, for example, lotions which are
aqueous solutions not containing an oily phase, or emulsions which
may be direct oil-in-water emulsions including a fatty phase or
oily phase dispersed in an aqueous continuous phase, or
water-in-oil reverse emulsions including an aqueous phase dispersed
in an oily continuous phase. The term "emulsions" means herein both
the dispersions obtained in the absence of emulsifying surfactants
and the emulsions obtained in the presence of emulsifying
surfactants.
[0231] Oil-in-water emulsions are the emulsions most frequently
sought in cosmetics due to the fact that, when applied to the skin,
they give a softer, less greasy, fresher and lighter feel than
water-in-oil emulsion systems, by virtue of the presence of water
in the continuous outer phase.
[0232] The nature of the compounds used for gelling the aqueous
phase and their content in the composition are chosen as a function
of the desired type of texture, which may range from fluid lotions
to more or less thick emulsions that may constitute milks or
creams. The main thickeners or gelling agents used in cosmetics are
chosen from the following compounds natural polymers such as
xanthan gum and guar gum or cellulose derivatives, starches and
alginates and crosslinked polymeric gelling agents such as the
Carbopols or crosslinked and at least partially neutralized
2-acrylamido-2-methylpropanesulfonic acid polymers.
[0233] Hearing Loss--Undifferentiated, partially differentiated or
fully differentiated amnion-derived cells, conditioned medium
therefrom, cell lysates therefrom, extracellular matrices
therefrom, alone or in combination, as well as composition of
placental-derived cells as defined herein may also be used to treat
hearing loss. Heretofore, hearing loss has been considered
incurable because hair cells, which are the sensory cells of the
cochlea, do not regenerate. Recently, however, both embryonic and
adult stem cells have been shown to be capable of differentiating
into mechanosensory hair cells (Li, H. et al. Nature Med.
9:1293-1299 (2003); Li, H. et al. Proc. Natl. Acad. Sci USA
100:13495-13500). In addition, Atoh1 gene therapy has been used to
promote phenotypic transgeneration, thus promoting the generation
of hair cells from nonsensory cells in deaf mammals (Izumikawa, M.
et al. 2005 Nature Medicine 11 (3):271-276). Further, human
amniotic epithelial cells transplanted into the inner ear of guinea
pigs have been shown to survive for up to three weeks and express
crucial proteins which may maintain homeostasis (Yuge, I. et al.
(2004):77(9) 1452-1471).
[0234] Methods of Differentiating Amnion-Derived Cells and
Differentiated Cell Types
[0235] The amnion-derived cells may be contacted with various
growth factors (termed differentiation factors) that influence
differentiation of such stem cells into particular cell types such
as hepatocytes, pancreatic cells, vascular endothelial cells,
muscle cells, cardiomyocytes and neural cells. For examples, see
US2003/0235563 and US2004/0161419, the contents of which are
incorporated herein by reference).
[0236] The literature is replete with additional differentiation
protocols for embryonic as well as non-embryonic stem or other
multipotent cells, including stem cells. For example, U.S. Pat.
Nos. 6,607,720 and 6,534,052 described methods of improving cardiac
function using embryonic stem cells and genetically altered
embryonic stem cells in which differentiation has been initiated,
for improving cardiac function and repairing heart tissue. U.S.
Pat. No. 6,387,369 provides methods of cardiac tissue and muscle
regeneration using mesenchymal stem cells. Shin, S. et al. have
recently reported the differentiation of embryonic stem cells into
motor neurons using a combination of basic fibroblast growth
factor, sonic hedgehog protein, and retinoic acid. (Human motor
neuron differentiation from human embryonic stem cells. Stem Cells
Dev. 2005 Jun.; 14(3):266-9). All of these references are
incorporated herein in their entirety. One skilled in the art will
recognize that any of these protocols can be applied to the
amnion-derived cell compositions described herein to produce
partially or fully differentiated cells for such uses. Other
exemplary protocols are set forth below:
[0237] Endoderm (pancreatic differentiation). Amnion-derived cell
are exposed to conditions for differentiation of an islet
progenitor cell population expressing PDX1. Briefly, cells are
initially exposed to antagonists of the Sonic Hedgehog (SHh)
signaling pathway to promote endoderm differentiation. Subsequent
differentiation to early islet progenitor cells is accomplished
using a combination of factors and conditions that promote
cessation of cell growth, aggregation of differentiating cells, and
expression of early pancreatic determination genes. Cells are
harvested for RNA and analyzed by reverse transcriptase-PCR
(RT-PCR) for Sox-17 and PDX1.
[0238] Mesoderm (cardiac differentiation). Clusters of cells taken
from the suspension cultures are transferred to gelatin- or
poly-L-lysine coated plates for 8 days in culture medium with serum
(80% KO-DMEM, 1 mM glutamine, 0.1 mM .beta.-mercaptoethanol, 1%
non-essential amino acids, and 20% FBS). For Days 2-4 in this
culture 1 or 10 .mu.M 5-aza-2'-deoxycytidine are added to the
medium (Xu, C. et al. (2002) Circ. Res. 91:501-508). Analysis is
performed on Day 8. Cells are harvested for RNA and analyzed by
reverse transcriptase-PCR (RT-PCR) for GATA-4, Nkx2.5 and MEF-2.
These transcription factors are expressed in precardiac mesoderm
and persist in cardiac development.
[0239] Ectoderm (neural differentiation). Clusters are removed from
the large-scale apparatus and transferred to ultra-low adherence
6-well plates. The differentiation protocol described by Carpenter,
M. K. et al. (2001) Exp Neurol 172:383-397 for human embryonic stem
cells is followed for differentiation as follows. 10 mM all-trans
retinoic acid (RA) will added to the culture medium (80% KO-DMEM, 1
mM glutamine, 0.1 mM .beta.-mercaptoethanol, 1% non-essential amino
acids, and 20% FBS) containing these clusters in suspension. After
4 days in suspension, clusters are plated onto
poly-L-lysine/fibronectin-coated plates in differentiation medium
(DMEM/F-12 with B27 (Gibco), 10 ng/ml human epidermal growth factor
(hEGF), 10 ng/ml human basic fibroblast growth factor (hbFGF)
(Gibco), 1 ng/ml human platelet-derived growth factor-AA (hPDGF-AA)
(R & D Systems), and 1 ng/ml human insulin-like growth factor-1
(hIGF-1) (R & D Systems) for 3 days. After 3 days under these
conditions, the cells are harvested for RNA or fixed. Fixed cells
are immunostained for nestin, polysialylated neural cell adhesion
molecule (PS-NCAM), and A2B5. RNA is analyzed by reverse
transcriptase-PCR (RT-PCR) for nestin, GFAP and MAP-2.
[0240] Differentiated cells derived from amnion-derived cells may
be detected and/or enriched by the detection of tissue-specific
markers by immunological techniques, such as flow
immunocytochemistry for cell-surface markers, immunohistochemistry
(for example, of fixed cells or tissue sections) for intracellular
or cell-surface markers, Western blot analysis of cellular
extracts, and enzyme-linked immunoassay, for cellular extracts or
products secreted into the medium. The expression of
tissue-specific gene products can also be detected at the mRNA
level by Northern blot analysis, dot-blot hybridization analysis,
or by reverse transcriptase initiated polymerase chain reaction
(RT-PCR) using sequence-specific primers in standard amplification
methods.
[0241] Alternatively, differentiated cells may be detected using
selection markers. For example, amnion-derived cells can be stably
transfected with a marker that is under the control of a
tissue-specific regulatory region as an example, such that during
differentiation, the marker is selectively expressed in the
specific cells, thereby allowing selection of the specific cells
relative to the cells that do not express the marker. The marker
can be, e.g., a cell surface protein or other detectable marker, or
a marker that can make cells resistant to conditions in which they
die in the absence of the marker, such as an antibiotic resistance
gene (see e.g., in U.S. Pat. No. 6,015,671).
[0242] Pancreatic Progenitor Cells
[0243] In another embodiment of the invention, cells are treated
such that they differentiate into pancreatic progenitor cells, In
this embodiment, amnion-derived cells, non-insulin producing
embryonic, neonatal or fetal cells are cultured in serum-free
culture medium comprising a SHh antagonist such as cyclopamine or
jervine to obtain pancreatic cells having the identifying
characteristics of endoderm, which include but are not limited to
protein expression of HNF1.alpha., HNF1.beta., HNF4.alpha., HNF6,
Fox2a and PDX1. The cells may be cultured in such medium after
culturing in basal medium.
[0244] Pancreatic progenitor cells of the present invention
expressing PDX1 protein in the nucleus may also be obtained by
culturing cells in medium comprising an SHh antagonist. The cells
may subsequently be cultured in medium comprising TAT-PDX1 fusion
protein. Procedures for obtaining TAT-PDX1 are described infra. In
a preferred embodiment, at least 20% of the cells in the
composition, culture or population of the present invention express
PDX1 protein in the nucleus. In other embodiments, at least 30%,
40%, 50%, 60%, 70%, 80%, or 90% of the cells in the composition,
culture or population of the present invention express PDX1 protein
in the nucleus. In a specific, preferred embodiment, 100% of the
cells express PDX1 protein in the nucleus.
[0245] The composition, culture or population of cells of the
present invention may be cultured in suspension or on a solid
support, such as an adherent matrix or substrate. Details of such
solid supports are described above. In one embodiment, the
composition of cells is cultured on a Matrigel layer. Matrigel
(Collaborative Research, Inc., Bedford, Mass.) is a complex mixture
of matrix and associated materials derived as an extract of murine
basement membrane proteins, consisting predominantly of laminin,
collagen IV, heparin sulfate proteoglycan, and nidogen and
entactin, and was prepared from the EHS tumor (Kleinman et al,
(1986) Biochemistry 25: 312-318). Other such matrices can be
provided, such as Humatrix. Likewise, natural and recombinantly
engineered cells can be provided as feeder layers to the instant
cultures. In another embodiment, the culture vessels are coated
with one or more extra-cellular matrix proteins including, but not
limited to, fibronectin, superfibronectin, laminin, collagen, and
heparin sulfate proteoglycan.
[0246] In another embodiment, the progenitor cells of the present
invention are cultured in the presence of three dimensional
matrices. Examples of such three dimensional matrices are described
in detail above.
[0247] Pancreatic Progenitor Cell Nuclei--The invention is further
directed to the nuclei of the pancreatic progenitor cells of the
present invention. The nuclei of these cells may be obtained using
methods known in the art. These include removing the membranes from
cells by either mechanical disruption or chemical means such as
treatment with hyaluronidase or performed by mechanically
extracting the nucleus with a pipet and inserting it into a
different or similar cell that has had its nucleus removed.
[0248] The nuclei may then subsequently be transferred into somatic
or germ cells by, for example, intracytoplasmic injection, chemical
fusion or electrofusion using methods known in the art as described
in, for example, US 20030234430 or US 20040268422.
[0249] In a particular embodiment, therapeutic cloning may be
undertaken using these nuclei to obtain cells that can be used for
endoderm or other cell differentiation. In a more particular
embodiment, the nuclei may be used to obtain embryonic stem-like
cells. Details concerning therapeutic cloning are described
above.
[0250] In addition to germ cells, the recipient cell may be any
mammalian cell. In one embodiment, the mammalian cell is enucleated
prior to receiving the donor nucleus. In another embodiment, the
mammalian cell in not enucleated prior to receiving the donor
nucleus. In this embodiment, both the recipient and donor nuclei
are present in the recipient cell. The nuclei may fuse or they may
stay separate. Instances in which it is desirable for both
recipient and donor nuclei to be present are ones in which the
object is to confer the tissue-specific functionality of the donor
cell onto the recipient cell while still maintaining the
tissue-specific functionality of the recipient cell. One of skill
in the art will recognize that other combinations are within the
scope of the invention.
[0251] Detection of Pancreatic Cells--The pancreatic cells of the
present invention described hereinabove can be detected in the
composition, culture or population of cells of the present
invention by detecting the presence or absence of various markers,
such as HNF1.alpha., HNF1.beta., HNF4.alpha., HNF6, Foxa2, PDX1,
Nkx2.2, Nkx6.1, Sox17, Cerberus, Hesx1, LeftyA, Otx1 and/or Otx2,
insulin, human C-peptide, somatostatin and islet-1. In one
embodiment, fragments of HNF1.alpha., HNF1.beta., HNF4.alpha.,
HNF6, Foxa2, PDX1, Nkx2.2, Nkx6.1, Sox17, Cerberus, Hesx1, LeftyA,
Otx1 and/or Otx2, insulin, human C-peptide, somatostatin or islet-1
may be used as probes or primers for detecting total or poly A RNA
isolated from the cells with probes and primers between 10-500
nucleotides in length, preferably between 20-200 nucleotides in
length, more preferably between 20-100 nucleotides in length and
most preferably between 20-50 nucleotides in length and subjecting
to agarose gel electrophoresis. Alternatively, these fragments may
be used as RT-PCR primers between about 10-100 nucleotides in
length to amplify the RNA isolated from the cells. Cells suitable
for such an analysis include cells isolated from human tissue.
Methods for performing primer-directed amplification (routine or
long range PCR) are well known in the art (see, for example, PCR
Basics: From Background to Bench, Springer Verlag (2000); Gelfand
et al., (eds.), PCR Strategies, Academic Press (1998)). Such probes
may be between 20-5000 nucleotides in length and may preferably be
between 20-50 nucleotides in length.
[0252] Alternatively, the above-mentioned markers may be detected
using antibodies to the markers in RIA, ELISA, Western Blot or
immunocytochemical techniques. The invention is thus directed to
kits comprising antibodies binding to two or more of the
above-mentioned markers.
[0253] TAT-PDX1 Fusion Protein--The invention also relates to
TAT-PDX1 fusion protein used in the culture medium for the purpose
of obtaining pancreatic progenitor cells comprising PDX1 protein in
the nucleus of the progenitor cells. TAT-PDX1 has the formula:
##STR1##
[0254] Wherein TAT is a TAT peptide having a self cell penetration
property. The TAT peptide is derived from human immunodeficiency
virus type-1 and is capable of passing through a cell membrane to
easily penetrate the cell. This property is thought to be due to
the protein transduction domain in the middle region of the TAT
peptide sequence. R1 may be side chains of glutamine, lysine,
arginine and/or glycine and n is an integer of 4 to 12.
[0255] In a specific embodiment, the TAT peptide may be
Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg;
Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys; or
Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg.
[0256] PDX1 is a homeodomain-containing protein and is thought to
be a key regulator of islet development and insulin gene
transcription in beta cells (Inoue et al., 1996, Diabetes
6:789-794). It has the following amino acid sequence:
TABLE-US-00002 MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPPPPPHPF
PGALGAEQGSPPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFP
EGAEPGVLEENRVQLPFPWMKSTKAHAWKGQWAGGAYAAEPEENKRTRTA
YTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWK
KEEDKKRGGGTAVGGGGVAEPEQDCAVTSGEELLALPPPPPPGGAVPPAA
PVAAREGRLPPGLSASPQPSSVAPRRPQEPR
[0257] and is encoded by the following nucleotide sequence
TABLE-US-00003 gccctgtgtc gcccgcaggc ggcgcctacg ctgcggagcc
ggaggagaac aagcggacgc gcacggccta cacgcgcgca cagctgctag agctggagaa
ggagttccta ttcaacaagt acatctcacg gccgcgccgg gtggagctgg ctgtcatgtt
gaacttgacc gagagacaca tcaagatctg gttccaaaac cgccgcatga agtggaaaaa
ggaggaggac aagaagcgcg gcggcgggac agctgtcggg ggtggcgggg tcgcggagcc
tgagcaggac tgcgccgtga cctccggcga ggagcttctg gcgctgccgc cgccgccgcc
ccccggaggt gctgtgccgc ccgctgcccc cgttgccgcc cgagagggcc gcctgccgcc
tggccttagc gcgtcgccac agccctccag cgtcgcgcct cggcggccgc aggaaccacg
atgagaggca ggagctgctc ctggctgagg ggcttcaacc actcgccgag gaggagcaga
gggcctagga ggaccccggg cgtggaccac ccgccctggc agttgaatgg ggcggcaatt
gcggggccca ccttagaccg aaggggaaaa ccc
[0258] The entire PDX1 sequence may be used in the fusion protein
of the present invention. Alternatively, a fragment of the PDX1
having PDX1 activity (e.g., regulation of insulin transcription,
regulation of PDX1 transcription, regulation of Nkx2.2
transcription) may be used. A non-limiting example of such a
fragment would be a peptide sequence encompassing the homeobox
domain and comprising the sequence: TABLE-US-00004
NKRTRTAYTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQ NRRMKWKKEE
[0259] The PDX1 peptide may contain conservative amino acid
substitutions that do not significantly affect the folding and/or
activity of the protein; small deletions, typically of one to about
30 amino acids; small amino- or carboxyl-terminal extensions, such
as an amino-terminal methionine residue; a small linker peptide of
up to about 20-25 residues; or a small extension that facilitates
purification by changing net charge or another function, such as a
poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of
basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions which
do not generally alter the specific activity are known in the art
and are described, for example, by H. Neurath and R. L. Hill, 1979,
In, The Proteins, Academic Press, New York. The most commonly
occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,
Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,
Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.
Alternatively, the nucleotide sequence encoding PDX1 may contain
alterations which produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are
preferred.
[0260] The fusion protein or peptide may be obtained using
recombinant DNA methods. For example, a nucleic acid sequence
encoding PDX1 or PDX1 peptide may be inserted into a vector
containing nucleic acid sequences encoding the TAT peptide. In a
particular embodiment, the TAT sequence is obtained by PCR and
inserted, for example, into a pET vector, or other protein
expression vector capable of expressing a fusion protein. The
fusion protein is expressed, isolated and purified using procedures
known in the art, such as HPLC and column chromatography.
[0261] Alternatively, the fusion protein may be produced by solid
phase synthesis using an organosynthesizer for peptide synthesis.
The method is Merrifield solid-phase peptide synthesis (J. Am.
Chem. Soc. 85, 2149-2154(1963)). The peptide is synthesized by
sequentially coupling an alpha-amino protected amino acid to an
amino terminal of a peptide chain attached to a solid support resin
after activation. After synthesis, the peptide is cut from the
resin, and the protecting group is removed with a reagent such as
trifluoroacetic acid (TPA). The peptide is separated from the TFA
solution by filtration, centrifugation, or extraction with
diethylether, and it can be purified by high performance liquid
chromatography (HPLC) or other methods.
[0262] In addition, other TAT fusion proteins may be made. For
example, TAT-PDX1, TAT-Hblx9, TAT-Ngn3, TAT-p48, or TAT-Foxa2 may
be used in practicing the methods of the invention. Such fusion
proteins made be made using the methods described above.
[0263] Uses of Pancreatic Progenitor Cells--The pancreatic
progenitor cells of the present invention and compositions thereof
can be used therapeutically for treatment of various diseases
associated with insufficient functioning of the pancreas. As used
herein, the term "pancreatic disease" may include but is not
limited to pancreatic cancer, insulin-deficiency disorder such as
Insulin-dependent (Type 1) diabetes mellitus (IDDM) and
Non-insulin-dependent (Type 2) diabetes mellitus (NIDDM), hepatitis
C infection, exocrine and endocrine pancreatic diseases.
[0264] The progenitor cells of the present invention can be used to
produce populations of differentiated pancreatic cells for repair
subsequent to partial pancreatectomy, e.g., excision of a portion
of the pancreas. Likewise, such cell populations can be used to
regenerate or replace pancreatic tissue loss due to,
pancreatolysis, e.g., destruction of pancreatic tissue, such as
pancreatitis, e.g., a condition due to autolysis of pancreatic
tissue caused by escape of enzymes into the tissue. Pancreatic
cells may be transplanted into the pancreas or to ectopic sites,
such as, but not limited to the liver, portal vein, spleen, mammary
gland, kidney or at or near the intestines. In one embodiment the
cells of the invention may be administered subcutaneously.
[0265] Methods of administration include encapsulating
differentiated beta islet cells producing insulin in implantable
hollow fibers. Such fibers can be pre-spun and subsequently loaded
with the differentiated beta islet cells of the invention (see U.S.
Pat. No. 4,892,538; U.S. Pat. No. 5,106,627; Hoffman et al. Expt.
Neurobiol. 110:39-44 (1990); Jaeger et al. Prog. Brain Res.
82:41-46 (1990); and Aebischer et al. J. Biomech. Eng. 113:178-183
(1991)), or can be co-extruded with a polymer which acts to form a
polymeric coat about the beta islet cells (U.S. Pat. No. 4,391,909;
U.S. Pat. No. 4,353,888; Sugamori et al. Trans. Am. Artif. Intern.
Organs 35:791-799 (1989); Sefton et al. Biotechnol. Bioeng.
29:1135-1143 (1987); and Aebischer et al. Biomaterials 12:50-55
(1991)).
[0266] The cells of the present invention may be genetically
engineered to produce a particular therapeutic protein. As used
herein the term "therapeutic protein" includes a wide range of
biologically active proteins including, but not limited to, growth
factors, enzymes, hormones, cytokines, inhibitors of cytokines,
blood clotting factors, peptide growth and differentiation factors.
Particular differentiated cells may be engineered with a protein
that is normally expressed by the particular cell type. In a
particular embodiment, pancreatic cells can be engineered to
produce digestive enzymes.
[0267] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a nucleic acid
encoding the protein of interest linked to appropriate
transcriptional/translational control signals and are described in
more detail above.
[0268] The pancreatic progenitor cells and compositions,
populations and cultures of the present invention may be used to
determine if a test agent is toxic to a pancreatic cell by
contacting the cells of the present invention with an appropriate
amount of the test agent for a time sufficient for a toxic effect
on the pancreatic cell to be detected and determining whether the
test agent has a toxic effect on the pancreatic cell.
[0269] The pancreatic progenitor cells differentiated therefrom may
also be used to humanize animal organs. Human amnion-derived cells
may be similarly transplanted into another organ such as pancreas
or brain or heart. The animal organ may or may not be depleted of
its native cells prior to the transplant.
EXAMPLES
[0270] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Preparation of Amnion-Derived Cell Compositions
[0271] Recovery of amnion-derived cells--Amnion-derived cells were
dissociated from starting amniotic membrane using the dissociation
agents PXXIII, and trypsin. The average weight range of an amnion
was 18-27 g. The number of cells recovered per g of amnion was
about 10-15.times.10.sup.6 for dissociation with PXXIII and
5-8.times.10.sup.6 for dissociation with trypsin.
[0272] Culture conditions--The primary amnion-derived cells were
cultured for 5 passages in the following media: Stemline II+10%
FBS, Stemline II+10% plasbumin (pb), Ultraculture+10% plasbumin
(pb), and DMEM+10% FBS. Each culture condition was tested using 15
million cells/g amnion, 10 million cells/g amnion, and 5 million
cells/g amnion, depending on the enzyme used for recovery of the
primary cells. For instance, using PXXIII, 15 million cells/g
amnion were obtained, while using trypsin, 10 million cells/g
amnion were obtained, while other enzymes resulted in even lesser
recovery (5 million cells/g amnion).
[0273] Passaging--Cells were passaged 5 times as follows: The cells
were grown attached to a culture flask (on tissue culture treated
plastic). The cells were left to divide and grow. The cells were
removed from the plastic using "tryple" (Gibco), a trypsin-like
product that is animal-free GMP grade. Once unattached, the cells
were centrifuged, and the cell pellet removed and resuspended in
the culture medium with protein and additives (10 ng/ml EGF) and
replated back onto fresh flasks. Cells were grown in a humidified
atmosphere at 37.degree. C. and 5% CO.sub.2.
[0274] Results. The results are show in Table 1 below. The data are
reported as amnion-derived cells.times.10.sup.6/gram of amnion.
TABLE-US-00005 TABLE 1 Starting Isolation Efficiency 5 mill/g 10
mill/g 15 mill/g Stemline + 10% pb 1363 2726 4089 Stemline + 10%
FBS 1024 2048 3072 Ultraculture + 10% pb 575 1151 1726 DMEM + 10%
FBS 128 256 384 DMEM + 10% pb 391 783 1174
[0275] The results indicate that the use of either Stemline or
Ultraculture with added plasbumin (pb) or albumin, the primary
cultures are expanded to a level that is at least 4 fold and as
much as 10 fold higher than is obtained using previous methodology
(DMEM with fetal bovine serum). Even the use of plasbumin (pb) in
the basal media DMEM resulted in an expanded amnion-derived cell
composition, having a 3-fold increase in multipotent cells as
compared to the previous method of using DMEM with fetal bovine
serum.
[0276] Another significant result observed was that cells grown in
medium containing plasbumin displayed a spheroidal phenotype after
passaging. When the amnion-derived cells were removed from the
tissue culture surface with the digestive enzyme and replated,
amnion-derived cells formed small clusters of cells that were not
firmly adhered to the culture surface. Some of the clusters of
cells were completely in suspension. These amnion-derived cell
clusters proliferated until up to 200 cells were present in the
clusters. After a period of 1-5 days, the clusters of cells
reattached and flattened out to form an adherent monolayer. This
clustering phenotype was observed at each passage. Further studies
indicated that such clustering occurs in the following media
containing either recombinant human albumin, plasbumin, or
plasmanate: OptiPRO SFM, VP-SFM, Iscove's MDM, HPGM, UltraMDCK,
Stemline II and Stemline I, DMEM, and DMEM:F12, but not in Advanced
DMEM, Knockout DMEM, 293 SFM II, Pro 293S-CDM, Pro 293A-CDM or
UltracultureVP-SFM.
Example 2
Scale-Up of Amnion-Derived Cells on Microcarrier Beads in Spinner
Flasks
[0277] Methods--One of the most common and oldest techniques for
maintaining cells in suspension culture is by the use of spinner
flasks. The cells can be either attached to microcarrier beads
(adherent) or growing completely without any surface attachment
(non-adherent). In either case, these flasks consist of a sterile
vessel that contains a magnetic stirring mechanism that permits
continuous stirring of the medium and cells under sterile
conditions. This continuous stirring facilitates the diffusion of
nutrients, promotes oxygenation of the medium, and eliminates
concentration gradients. The vessels are stirred in a
temperature-controlled, CO.sub.2 incubator.
[0278] Amnion-derived cells are an epithelial cell type that are
anchorage-dependent which may interfere with or prevent their
adaptation to a pure suspension system. Although amnion-derived
cells may survive in suspension culture, the proliferation of these
cells may not be optimal without any substrate for attachment
and/or many of the cells in suspension may undergo preliminary
differentiation. One method of addressing this while maintaining
the ability to grow the cells in a 3-dimensional system is to grow
the cells on microcarrier beads. Microcarriers are typically small
(30-1000 .mu.m diameter) glass, polystyrene or dextran beads with a
surface treatment to enhance attachment. The microcarriers provide
the advantage of a very large surface area to which the cells
attach allowing for culture at very high densities in a minimal
volume of medium. For example, 1 gram dry weight of typical
microcarriers is equal to 2000 cm.sup.2 of surface area. A small
number of microcarriers in cell culture medium can support the
growth of significant numbers of anchorage-dependent cells.
[0279] Adherent culture: Amnion-derived cells isolated from 3
different placentas were placed into either spinner flasks with
microcarrier beads (adherent cells) or normal T-flasks (control
static adherent cells) and incubated at 37.degree. C. at 5%
CO.sub.2 in air. The cells and beads were seeded into the spinner
flasks at a ratio of 1 g of beads per 66.times.10 6 cells.
10.times. 6 cells were plated into each T-flask. Periodically,
cells were counted, and viability assessed, using a Guava PCA-96
Personal Cell Analyzer (ViaCount package). As little as 20 .mu.l of
sample was used for this analysis. A graph of total and viable cell
counts per ml was blotted with time to ensure that the cells were
able to divide and remain viable over time in culture.
[0280] Results--In all three experiments, amnion-derived cells were
shown to be capable of proliferating to at least 1.5 times seeding
density, thus demonstrating that microcarrier bead spinner flask
culture methods are a feasible alternative to static culture.
Example 3
Scale-Up of Amnion-Derived Cell Compositions in Suspension
[0281] Amnion-derived cells were cultured in ultra-low adherence
tissue culture 6-well plates (Corning) in various mammalian cell
culture media. These culture media were selected on the basis of
their ability to promote proliferation of other mammalian cell
types in suspension culture (i.e. 293S, Ultraculture, Opti-MEM).
Additives to the culture medium in these experiments include a
proprietary source of protein, and EGF (10-20 ng/ml) which
preliminary experiments show is required for proliferation of
amnion-derived cells. Amnion-derived cells were plated at a density
of 1.3.times.10.sup.6 cells/well, and the cultures were maintained
at 37.degree. C., in 5% CO.sub.2 in air. Culture medium was
replaced every two days and cell number was assessed weekly.
Preliminary experiments showed that amnion-derived cells sometimes
form small floating clusters in suspension culture conditions.
These clusters must be dispersed to ensure accurate cell counts and
this was achieved by incubating the cultures in trypsin for 5-10
minutes prior to counting. Cells were counted, and viability
assessed, using a Guava PCA-96 Personal Cell Analyzer (ViaCount
package). As little as 20 PI of sample was used for this analysis.
A graph of total and viable cell counts per ml was plotted with
time to ensure that the cells were able to divide and remain viable
over time in culture. Cells were subcultured at
1.3-1.5.times.10.sup.6 cells per well. The cultures were maintained
until proliferation ceased. Control adherent cultures were
maintained on tissue culture treated 6-well plates to compare rates
of proliferation between adherent and suspension culture. Cultures
were maintained at 37.degree. C., in 5% CO.sub.2 in air. Passage of
adherent cells was performed at confluency. Adherent cultures were
trypsinized and washed before replating at 1.3.times.10.sup.6
cells/well. Cell counts and viability were measured in the adherent
cells at each passage. Culture media was tested on 5 different
donor tissues to account for tissue variability.
[0282] Results: Of the 5 placentas tested, 4 showed at least 2 fold
proliferation at least through day 20 under all conditions tested,
thus demonstrating that non-adherent static culture methods are a
feasible alternative to microcarrier bead or adherent flask
culture.
Example 4
Addition of Growth Factor Additives to Promote More Extensive
Proliferation
[0283] After selection of a culture medium that supports suspension
culture in 6-well plates, various growth factor additives are
tested to promote more extensive proliferation in suspension
culture conditions. These growth factors include EGF, IGF-1,
IGF-II, .alpha.FGF, .alpha.FGF-h, .beta.FGF, FGF-4, FGF-8, KGF,
SCF, Fsk, SHh, Prog, Wnt-1, CT, VPA. These and other factors known
to either promote cell proliferation or decrease apoptosis or
anoikis are tested at various concentrations in the suspension
cultures to determine their effect on proliferation. Additional
testing is performed to ensure that these factors are not promoting
differentiation or changing the secretory profile of the cells.
Example 5
Culturing of Amnion-Derived Cells in Spinner Flasks or Roller
Bottles without Microcarrier Attachment
[0284] After selection of a culture medium that supports
proliferation of amnion-derived cells in suspension, with or
without the addition of growth factors other than EGF, experiments
are performed to assess the proliferation of the cells in a stirred
bioreactor. Amnion-derived cells are placed into the spinner flasks
(suspension cells; 3.times.10.sup.5 cells/ml). Adherent cells
cultured in T-flasks are used as controls (adherent cells;
1.3.times.10.sup.5 cells/cm.sup.2). All cultures are incubated at
37.degree. C. in 5% CO.sub.2 in air. The spinner flasks are treated
with Sigmacote (Sigma-Aldrich) prior to use, to prevent the cells
from attaching to the glass. The flasks are agitated by placing
them on magnetic stir-plate. Daily samples of cells are withdrawn,
in a class II biosafety cabinet, from each spinner flask, and the
number of cells is counted, and viability assessed, using a Guava
PCA-96 Personal Cell Analyzer (ViaCount package). The use of
multiple spinner flasks permits the use of 2 or 3 replicates of
each condition per experiment.
[0285] One of the challenges of a spinner flask system is the
exposure of the cells to sheer forces caused by the rotating
impeller. A gentler alternative to spinner flasks is to culture the
cells in roller bottles. Tissue culture treated roller bottles (1.2
L; Corning) are pre-treated with poly 2-hydroxyethyl methacrylate
(Poly-Hema) to prevent cell attachment. Roller bottles containing
30-40.times.10.sup.6 amnion-derived cells are placed on a roller
bottle apparatus (Integra Biosciences). Cells are sampled on a
biweekly basis and assessed for viability and proliferation, as
indicated above.
Example 6
Generation of Monoclonal Antibodies
[0286] In one embodiment, Balb/c mice are immunized with
amnion-derived cells previously cultured for up to 5 days,
preferably 1-2 days. In this embodiment, both adherent and
non-adherent cells are recovered from the culture and used to
immunize the mice. In another embodiment, after culturing for up to
5 days, preferably 1-2 days, only the adherent cells are recovered
and used for immunization of the mice. In another embodiment, after
culturing for up to 5 days, preferably 1-2 days, only the
non-adherent cells are recovered and used for immunization of the
mice. Four to six weeks after immunization, the spleens are removed
and the spleen cells are fused to a mouse myeloma cell line,
SP2/0-Ag14, using techniques known in the art, resulting in the
generation of viable hybridoma cells. As many as 1000 hybridomas
may be expanded, screened for the expression of monoclonal
antibodies, and further tested for their specific reactivity to
cell-surface protein markers on amnion-derived cells. Antibody
samples are analyzed by flow cytometry and, along with commercially
available antibodies, will be used to identify unique protein
markers on amnion-derived cells. Thus, the invention is directed to
hybridomas producing the monoclonal antibodies of the present
invention as well as the monoclonal antibodies.
Example 7
Antibodies Which React with Amnion-Derived Cell Surface Protein
Markers
[0287] Monoclonal antibodies which react with amnion-derived cell
protein markers on the surface of amnion-derived cells may be used
to separate the cell population into substantially purified
population of amnion-derived cells and will be useful in
characterizing each substantially purified population of
amnion-derived cells for its stem cell characteristics. The
monoclonal antibodies of the present invention may be used to
isolate a stem cell protein marker unique to the substantially
purified population of amnion-derived cells. The newly identified
protein marker may be used to isolate a nucleic acid sequence
encoding the protein. Thus, the invention is directed to unique
markers on amnion-derived cells, the isolated protein marker, the
isolated nucleic acid encoding the protein marker, as well as
expression vectors capable of expression the protein marker when
transfected into mammalian cells such as CHO, COS, etc. The
invention is further directed to bacterial cells carrying the
vector for vector propagation.
[0288] In addition, the invention contemplates using known
antibodies to identify and create substantially purified
populations of amnion-derived cells having unique combinations of
markers useful as identifying characteristics of the substantially
purified populations. This unique combination of markers can be
used to isolate, characterize, purify or create a substantially
purified population of amnion-derived cells having those
characteristics.
[0289] All cell characterizations described herein were done using
freshly isolated amnion-derived cells. One of skill in the art will
recognize that the expression pattern of the markers may vary
depending upon culture conditions and time in culture. For example,
the protein marker expression pattern seen in the expanded
populations of the invention described herein may be different from
that seen in freshly isolated amnion-derived cells. In addition,
one of skill in the art will recognize that the order in which the
cells are contacted with the antibodies is not critical to
obtaining the desired populations of amnion-derived cells. Table 2
shows the results of FACS analysis of amnion-derived cells freshly
isolated from the amnion of a placenta. The antibodies used, alone
or in combination, may be useful to identify, isolate, characterize
or create a substantially purified population of amnion-derived
cells. One preferred embodiment is the use of anti-CD90 and
anti-CD117 antibodies to identify, isolate, characterize or create
a substantially purified population of amnion-derived cells. Other
preferred embodiments for identifying, isolating, characterizing or
creating substantially purified populations of amnion-derived cells
include contacting the cells with anti-CD90, anti-CD117, and
anti-CD105 antibodies; contacting the cells with (i) anti-CD90,
anti-CD117, and anti-CD105 antibodies and (ii) with at least one
antibody selected from the group consisting of anti-CD140b,
anti-CD34, anti-CD44, and anti-CD45 antibodies; contacting the
cells with (i) anti-CD90 and anti-CD117 antibodies and (ii) with an
anti-CD29 antibody; contacting the cells with (i) anti-CD90,
anti-CD117 and anti-CD105 antibodies and (ii) with an anti-CD29
antibody; contacting the cells with (i) anti-CD90, anti-CD117
antibodies and (ii) anti-CD29 antibodies and (iii) with one or more
antibodies selected from the group consisting of anti-CD9,
anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227,
anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies; contacting the
cells with (i) anti-CD90, anti-CD117, and anti-CD105 antibodies and
(ii) and anti-CD29 antibodies and (iii) with one or more antibodies
selected from the group consisting of anti-CD9, anti-CD10,
anti-CD26, anti-CD71, anti-CD166, anti-CD227, anti-EGF-R,
anti-SSEA-4, and anti-HLA-G antibodies; contacting the cells with
(i) anti-CD90, anti-CD117, and anti-CD105 antibodies and (ii) and
anti-CD29 antibodies and (iii) with one or more antibodies selected
from the group consisting of anti-CD140b, anti-CD34, anti-CD44, and
anti-CD45 antibodies; b) contacting the cells with (i) anti-CD90,
anti-CD117, and anti-CD105 antibodies and (ii) and anti-CD29
antibodies and (iii) one or more antibodies selected from the group
consisting of anti-CD140b, anti-CD34, anti-CD44, and anti-CD45
antibodies and (iv) one or more antibodies selected from the group
consisting of anti-CD9, anti-CD10, anti-CD26, anti-CD71,
anti-CD166, anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-G
antibodies; and contacting the cells with one or more antibodies
selected from the group consisting of anti-CD90, anti-CD117,
anti-CD140b, anti-CD34, anti-CD44, and anti-CD45 antibodies; and
one or more antibodies selected from the group consisting of
anti-CD29, anti-CD9, anti-CD10, anti-CD26, anti-CD71, anti-CD166,
anti-CD227, anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies.
TABLE-US-00006 TABLE 2 Population % Designation Surface Marker
.about.95-100% +++ CD9 CD29 .about.70-95% ++ SSEA4 CD10 CD166 CD227
.about.60-95% + HLA-G EGFR CD26 .about.10-50% +/- CD71 <1% -
CD34 CD44 CD45 CD140b CD90 CD105 CD117
[0290] Table 3 shows where antibodies useful for practicing the
methods of the invention can be obtained. TABLE-US-00007 TABLE 3
Antibody Antibody Name Manu. Cat. # Name Manu. Cat. # CD117 PE
BD-Pharm 555714 CD44 APC BD-Pharm 559942 CD44 PE BD-Pharm 555479
CD45 APC BD-Pharm 555485 CD45 PE BD-Pharm 555483 CD140b PE BD-Pharm
558821 EGFR PE BD-Pharm 555997 CD90 Biotin BD-Pharm 555594 CD105
FITC Chemicon CBL418F CD26 BD-Pharm CD117 APC BD-Pharm 550412 CD166
BD-Pharm CD29 APC BD-Pharm 559883 CD10 BD-Pharm CD34 APC BD-Pharm
555824 CD71 BD-Pharm CD227 BD-Pharm CD9 BD-Pharm
Example 8
Generation of Enriched Populations of Amnion-Derived Cells
[0291] Amnion-derived cell protein markers expressed on the cell
surface may be used to enrich for populations of amnion-derived
cells expressing those protein markers using a variety of methods.
Such procedures may involve a positive selection, such as passage
of sample cells over a column containing anti-protein marker
antibodies or by binding of cells to magnetic bead-conjugated
antibodies to the protein markers or by panning on plates coated
with protein marker antibodies and collecting the bound cells.
Alternatively, a single-cell suspension may be exposed to one or
more fluorescent-labeled antibodies that immuno-specifically bind
to amnion-derived cell protein markers. Following incubation with
the appropriate antibody or antibodies, the amnion-derived cells
are rinsed in buffer to remove any unbound antibody. Amnion-derived
cells expressing the protein marker(s) can then be sorted by
fluorescence-activated cell sorting (FACS) using, for example, a
Becton Dickinson FACStar flow cytometer. To enrich for populations
of cells expressing a desired protein marker(s), the cells may be
subjected to multiple rounds of FACS sorting.
[0292] In addition, protein markers that are not expressed on the
surface of amnion-derived cells may also be used to enrich for
populations of amnion-derived cells not expressing those markers.
Such procedures may involve a negative selection method, such as
passage of sample cells over a column containing anti-protein
marker antibodies or by binding of cells to magnetic
bead-conjugated antibodies to the protein markers or by panning on
plates coated with protein marker antibodies and collecting the
unbound cells. Alternatively, a single-cell suspension may be
exposed to one or more fluorescent-labeled antibodies that
immuno-specifically bind to the protein markers. Following
incubation with the appropriate antibody or antibodies, the cells
are rinsed in buffer to remove any unbound antibody. Cells
expressing the protein marker(s) can then be sorted by
fluorescence-activated cell sorting (FACS) using, for example, a
Becton Dickinson FACStar flow cytometer and these cells can be
removed. Remaining cells that do not bind to the antibodies can
then be collected. To enrich for populations of cells that do not
express a desired protein marker(s), the cells may be subjected to
multiple rounds of FACS sorting as described above.
[0293] Non-limiting examples of antibodies that may be useful to
generate such enriched populations of amnion-derived cells include
anti-CD10, anti-CD26, anti-CD71, anti-CD166, anti-CD227,
anti-EGF-R, anti-SSEA-4, and anti-HLA-G antibodies.
[0294] Alternatively, antibodies may be useful to generate enriched
populations of amnion-derived cells by removing undesired cells
(i.e. by conjugating antibodies to beads and adding the beads to a
culture dish containing a heterogeneous population of
amnion-derived cells such that cells in the heterogeneous
population that express the marker to which the antibody is
directed will bind to the beads thus removing them from the
population of cells that do not express the marker). Non-limiting
examples of antibodies that may be useful in this process
anti-CD140b, anti-CD34, anti-CD44, and anti-CD45, anti-CD90,
anti-CD105, and anti-CD117 antibodies.
Example 9
Monoclonal Antibody Library
[0295] To construct a "monoclonal antibody library" a collection of
several monoclonal antibodies may be selected which identifies and
isolates the particular cell population responsible for the
multipotent cell activity characteristic of the population of
amnion-derived cells of the present invention. The panel of
monoclonal antibodies may be reacted with placental tissue, a
placental-derived cell suspension, or a culture of
placental-derived cells. Cells reacting with the collection of
monoclonal antibodies may be identified and isolated using methods
known in the art, e.g., FACS. The invention is therefore directed
to a collection of monoclonal antibodies that are used to form a
monoclonal antibody library.
Example 10
Use of Amnion-Derived Cell Compositions in Wound Healing
[0296] Methods. The keratinocyte cell line isolated from epidermis
(ATCC CRL-1555) was seeded onto 6-well plates at a density of
0.3.times.10.sup.6 cells per well. Cells were left to grow to
confluency then placed into serum-free conditions for 48 hours. In
each well a scrape or wound of the confluent monolayer was made
from the top to the bottom of the well using a 1 ml pipette tip.
Images of the scrape were taken at 0, 24, 30 and 48 hours to
determine cell migration or percent of wound closure in response to
addition of conditioned medium to each well. Conditions tested were
0%, 50%, and 100% of the following: 1) No conditioned media
(control, 0%); 2) Conditioned media from amnion-derived cells
passaged normally at ratio of 1:3; 3) Conditioned media from
amnion-derived cells that were never passaged; 4) Conditioned media
from amnion-derived cells grown in the ATCC cells' media; and 5)
Conditioned media from ATCC cells grown in their own media.
Approximately 6 measurements were taken in microns of each scrape
at each time point using phase microscopy and MetaMorph imaging
software. The percent of healing was calculated by comparing the
width of each wound at 24, 30, and 48 hours to the starting width
of the wound at time zero.
[0297] Results. Conditioned media (CM) from amnion-derived cells
showed a significant increase in cell migration or healing of the
scrape compared to control. CM from other cell types, however, did
not show this increase. Cells that grew in CM from amnion-derived
cells were the only condition that showed complete closure of the
scrape before 24 hours. CM from cells passaged at a ratio of 1:3
and at a concentration of 50% (CM/non-CM) produced the best
results. These results suggest that components of CM from
amnion-derived cells have properties that increase cell migration
or wound healing.
Example 11
Amnion-Derived Cells, Conditioned Media, and Cell Lysates
Accelerate Re-Epithelialization, Collagen Synthesis, and Regain to
Tissue Tensile Strength
[0298] The following experiment was done to assess whether the
application of amnion-derived cells, amnion-derived cell
conditioned media or amnion-derived cell lysates could: 1)
accelerate the rate of re-epithelialization, 2) accelerate collagen
synthesis and deposition in the wound bed and 3) speed up regain to
tissue tensile strength and demonstrate that transplantation of
stem cells may have the same properties. It was also done to assess
whether transplanted amnion-derived cells could incorporate into
epidermal and dermal structures including follicles, glands and
blood vessels.
[0299] Animal model: This initial study utilized a total of 90
rats, distributed into the following groups (5 sacrifice time
points, 3 animals per treatment group, 6 groups per time point),
Table 4. TABLE-US-00008 TABLE 4 Time Points (days) Group #
Treatment 3 5 7 14 21 1 Control (no treatment) 3 3 3 3 3 2
Non-conditioned media + gelfoam 3 3 3 3 3 3 Amnion-derived cell
conditioned 3 3 3 3 3 media + gelfoam 4 Hyaluronic acid vehicle 3 3
3 3 3 5 Amnion-derived cell + hyaluronic 3 3 3 3 3 acid vehicle 6
Amnion-derived cell lysate + 3 3 3 3 3 hyaluronic acid vehicle
[0300] Each animal received 2 dorsal, full-thickness excisional
wounds, for a total of 180 wounds for the entire study, with 6
wounds/group/time point.
[0301] Skin wounding: A pair of wounds was made on each side of the
dorsal midline, using a disposable punch biopsy (6 mm diameter)
These wounds were full-thickness through the epidermis and dermis.
Wounds were treated with: nothing (control), vehicle (10 mm Gelfoam
sponge saturated with non-conditioned media), conditioned media (10
mm Gelfoam sponge saturated with amnion-derived cell conditioned
media), hyaluronic acid vehicle (0.1 ml of Hylan A gel, Genzyme
Corporation), hyaluronic acid+fluorescently (CM-DiI dye, Molecular
Probes, Eugene Oreg.) labeled amnion-derived cells (10.sup.6
cells/wound) or hyaluronic acid+amnion-derived cell lysate (from
10.sup.6 cells/wound), immediately following injury (See above
Table 5). The entire dorsal skin was covered with a sterile
dressing (Tegaderm, 3M, Minneapolis, Minn.) secured with a
biocompatible adhesive (Mastisol, Ferndale Laboratories Inc,
Ferndale, Mich.). Wounds in the first three treatment groups were
re-treated in an identical manner on days 2, 3, 4 and 5 post
wounding. Following the 5th wound treatment, the wounds were left
undisturbed until day 7, at which time the Gelfoam as well as the
sterile dressing wass removed and the wound allowed to heal exposed
to the surrounding environment. Wounds in the last three treatment
groups were left undisturbed until time of sacrifice.
[0302] Imaging and clinical assessment: Two blinded observers
assessed the degree of wound healing for each of the 180 wound
samples at the following days post injury: 1, 2, 3, 4, 5, 7, 14 and
21. The following parameters were ascertained: hemostasis, wound
contraction, re-epithelialization and inflammation. Digital images
were taken of representative wound samples for each treatment group
and stored for later analysis.
[0303] Tissue analysis: Animals were be euthanized according to the
above time table by intracardiac administration of pentobarbital
sodium and phenyloin sodium following heavy sedation with
ketamine/xylazine. Dorsal skin was removed using aseptic technique
and each wound was individually dissected and divided. One half of
each wound was used for tensile strength measurements, with the
other embedded for frozen sectioning and image analysis.
[0304] Tensiometry: Wound samples from the day 7, 14 and 21 groups
were analyzed by tensiometry. For tensile strength measurements the
frozen specimens were trimmed of sub-cutaneous fat and any muscle
that was taken along with the biopsy, and divided into 4-5 samples.
The cross-sectional area of each specimen was measured with
calipers. Then the specimen was clamped in the tensiometer, and
force exerted until the skin teared. Measurements were recorded by
a computer and tensile strength calculated using the formula:
Maximum Tensiometer Reading (converted to g) divided by
Cross-sectional Area (sq-mm)=Tensile strength (g/sq-mm). The
results for individual specimens from one wound were combined to
determine an average TS/wound (tensile strength per wound). This
value was normalized for the TS/skin (tensile strength of uninjured
skin from the opposite side); TS/wound divided by TS/skin=relative
TS/wound. The relative TS/wound was tabulated for each group at
each time point and the mean and standard deviations determined
using Excel database software (Microsoft Office 2000).
[0305] Microscopic analysis: Tissue specimens were embedded in
O.C.T. (Miles, Inc., Elkhart, Ind.) and cryostat-sectioned into
approximately 10 m thick sections, at -23.degree. C. Thin sections,
mounted on glass microscope slides, were stored in moisture-proof
slide boxes at -70.degree. C. Representative slides were processed
for immunohistochemical characterization of the connective tissue
components using standard techniques. Hematoxylin and eosin
staining were used to ascertain the overall histological appearance
of the injured mucosa. Collagen presence in the wound was assayed
using Masson's trichrome stain. Picrosirius-polarization method was
used to analyze collagen fiber organization. Grafting and survival
of fluorescently labeled stem cells in the wound bed was
semi-quantitatively analyzed by measuring the total amount of
fluorescence present in the wound bed. Localization of cells was
recorded and analyzed.
[0306] Effect on the rate of wound re-epithelialization and dermal
collagen deposition and organization was determined. Each of these,
as well as other components of the wound healing process, were
analyzed using specific markers. Transplantation of live
amnion-derived cells into the dermal wound bed was expected to
result in: 1) differentiation and engrafting of stem cells into
various skin compartments and 2) continual regulated release of
various stem cell factors.
[0307] Results--The results of this experiment are set forth in
Table 5 below. TABLE-US-00009 TABLE 5 Summary Amnion-derived cell
conditioned medium Amnion-derived cells Positive Effects 1. Day 5:
CM wounds, >contracted granulation 1. Re-Epithelialization in
early time formation points 2. Day 14: CM wounds appeared smaller,
>contracted, 2. Angiogenesis in early time points >healed 3.
Dynamics of collagen deposition an 3. CM wounds exhibited faster
re- organization epithelialization and angiogenesis. Negative
Effects None No detection of engrafted cells Unaltered 1. Synthesis
and deposition of collagen 1. Clinical observations 2. Regain of
tissue tensile strength 2. Regain of tissue tensile strength
[0308] As shown in Table 5, treatment of wounds with amnion-derived
cell conditioned media showed an increase in contracted granulation
formation by Day 5, and smaller wounds, greater contraction and
healing by Day 14. In addition, the wounds exhibited faster
re-epithelialization and angiogenesis as compared to controls.
Synthesis and deposition of collagen and regain of tissue tensile
strength were unaltered over the course of the experiment.
Treatment of wounds with amnion-derived cells showed
re-epithelialization and angiogenesis at early time points, as well
as evidence of collagen deposition and organization. Engrafted
cells were not detected. No differences based on visual inspection
in clinical observations (redness, swelling, size, etc.) were seen
nor was regain of tissue tensile strength altered over the course
of the experiment.
Example 12
Detection of Cytokines in Conditioned and Unconditioned Media
Samples
[0309] In addition to pluripotency, amnion-derived cells may play a
significant role in the inflammatory response. In the early phases
of wound healing, chemokines and cytokines regulate chemotaxis and
activation of inflammatory cells. Growth factors play dominant
roles in regulating cell proliferation, differentiation, and
synthesis of extracellular matrix. Amnion epithelial cells have
been shown to secrete many cytokines and growth factors. These
factors include prostaglandin E, PDGF, TGF-.alpha., EGF, IL-4,
IL-8, TNF, interferons, activin A, noggin, b-FGF, angiogenic
factors, and other neuroprotective factors (Koyano, S., et al.,
(2002) Dev Growth Differ 44, 103-12; Blumenstein, M., et al.,
(2000) Placenta 21, 210-7; Tahara, M., et al., (1995) J Clin
Endocrinol Metab 80, 138-46; Paradowska, E., et al., (1997)
Placenta 18, 441-6; Denison, F. C., et al., (1998) Hum Reprod 13,
3560-5; Keelan, J. A., (1998) Placenta 19, 429-34; Sun, K., et al.,
(2003) J Clin Endocrinol Metab 88, 5564-71; Uchida, S., et al.,
(2000) J Neurosci Res 62, 585-90).
[0310] Many of these cytokines are associated with wound healing
and some have been credited with contributing to scarless healing
in the fetus (Robson, M. C., et al., (2001) Curr Probl Surg 38,
72-140; Ferguson, M. W. et al., (2004). Philos Trans R Soc Lond B
Biol Sci 359, 839-50).
[0311] To determine which of these cytokines may be secreted by the
amnion-derived cells of the present invention, conditioned media
from amnion-derived cells was isolated from cell cultures that were
seeded onto tissue culture treated flasks at a density of 40,000
cells per cm.sup.2. Cells were cultured in a proprietary serum-free
medium supplemented with 10 ng/ml of EGF. Culture media was
exchanged every 2 days during the growth period. After cells
reached near confluency (1-2 wk after isolation), fresh media was
applied and conditioned media was collected after three days and
stored at -80 C. for subsequent analysis.
[0312] Conditioned media was analyzed for secreted protein content
via antibody arrays for multiple protein detection (RayBiotech,
Norcross, Ga. using RayBio.RTM. Human Cytokine Antibody Arrays V,
VI, and VII). The samples that were analyzed are shown in Table 6
below. TABLE-US-00010 TABLE 6 1. Complete unconditioned media +
plasbumin 2. Complete unconditioned media + EGF (no plasbumin) 3.
Conditioned media from placenta 1 + plasbumin 4. Conditioned media
from placenta 1 (no plasbumin) 5. Conditioned media from placenta 2
+ plasbumin
[0313] Results--Table 7 provides the results of this experiment.
TABLE-US-00011 TABLE 7 Wound Healing Relevant Cytokines Positive in
Conditioned Media Negative Angiopoietin-2, Angiogenin, bFGF, EGF,
FGF-7, TGF-a, TGF-beta 1, FGF-4, IGF-1, IL-1 beta, IL-2, IL-4,
IL-6, IL-8, TGF-beta 2, IL-10, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-
TGF-beta 3 Ra, PDGF-Rb
Example 13
Amnion-Derived Cell/Fibroblast Co-Cultures
[0314] It has been reported in the literature that under certain
conditions when ES cells are co-cultured with fibroblasts, the ES
cells are induced to differentiate into keratinocyte-like cells. To
determine what effect co-culture of amnion-derived cells with
fibroblasts would have on amnion-derived cells, an experiment was
done in which 3.3.times.10 6 amnion-derived cells were co-cultured
with 0.4.times.10 6 fibroblasts on a collagen IV-coated T25 flask
for 3, 5, 10, 15, and 25 days.
[0315] Results--When treated with the trypsin-like enzyme Tryple
(Invitrogen), both amnion-derived cell cultures and fibroblast cell
cultures alone release cells as a single cell suspension. However,
when the amnion-derived cell/fibroblast co-culture was treated with
Tryple, the cells came off the treated culture surface as sheets
rather than as a single cell suspension. Furthermore, the sheets
were very stable and somewhat resistant to enzymatic and mechanical
disruption.
[0316] It is theorized that these sheets may be suitable for use as
wound dressings when it is desirable to have a dermal-type graft.
With demonstrated recent success with mitral resuscitation,
management of inhalation injuries, control of burn wound sepsis,
and understanding of the hypermetabolic response, early excision
and rapid closure of the burn wound with a serviceable integument
becomes a therapeutic imperative. In small surface area burns, this
can be accomplished by autogenous skin grafts. For large surface
area burns, both partial and full-thickness, there is not yet a
totally satisfactory solution. Cutaneous epithelial autografts can
be grown from the patient's skin and massively expanded to cover
the entire body. Unfortunately, the lack of dermis leads to
prolonged fragility and significant scarring, therefore, many
believe that a "dermis" is required along with an epithelium.
[0317] Recent products with a supposed dermal substitute or
neodermis such as Integra, Alloderm, Transcyte, Apligraf, and
Dermagraft have attempted solve the problem. However, all of these
"skin" substitutes have the problem of being expensive and having
lower resistance to infection than autografts. Without a
satisfactory rapid reliable wound closure for burn injuries, the
wound remains in the inflammatory phase of healing for a prolonged
period of time resulting in excessive scarring.
[0318] Robson et.al., (Robson, M. C., and Krizek, T. J. (1973) Ann
Surg 177, 144-9.) reported success in treatment of experimental and
clinical burns (both partial and full thickness) using human
amniotic membranes. It was thought that part of the effect seen
from the treatment with amniotic membranes was due to a humoral
substance or substances stimulating wound healing. These
observations were prior to present knowledge of cytokines and
growth factors. More recently, attempts have been made to use
recombinant growth factors and growth hormones to affect more rapid
healing of the burn wound. Amniotic membranes proved not to be
practical because of the risk of virally transmitted diseases.
However, the observations from those early experiments and coupled
with new knowledge support the possibility that the
pluripotentiality of amnion-derived cells and their now
demonstrated protein secretory profile of cytokines and other
humoral substances stimulatory for wound healing may be useful in
providing rapid early closure for thermal injuries.
Example 14
Effects of Amnion-Derived Cell Conditioned Media in an Animal Model
of Acute Wound Healing
[0319] An animal model of acute excisional granulating wound was
used to evaluate the effect of amnion-derived cell conditioned
media on wound healing.
[0320] Methods: Acute excisional granulating wound model: Twenty
male Sprague-Dawley rats weighing 250-300 g were anesthetized using
ketamine (40 mg/kg), xylazine (10 mg/kg) and acepromazine (0.75
mg/kg). Following anesthesia, the dorsum of each animal was
depilitated and four symmetrical midline areas 1.5.times.1.5 cm
were traced on the skin using a copper template. Four wounds were
then created by excision of the marked areas through the skin and
the panniculus carnonsus muscle. The animals were divided into the
following groups of 5 (Table 8): TABLE-US-00012 TABLE 8 Group No.
Experimental Conditions I Conditioned media, non-infected II
Unconditioned media, non-infected III Conditioned media, infected
IV Unconditioned media, infected
[0321] Analog tracings were made every 72 hours onto acetate sheets
of both open wound areas and of the advancing full-thickness skin
edges of all wounds. To eliminate site-related variability in the
wounds, only the three caudal wounds were measured for statistical
purposes, since the most cephalad wound has been shown to
demonstrate different healing characteristics. Wound area
calculations were performed with the use of digital planimetry
(Sigma Scan; Jandel Scientific, Corte Modera, Calif.). Weekly
quantitative bacterial analyses were performed on a subset of
wounds in each group and are expressed as CFUs/g of tissue.
[0322] After all four wounds of each animal were completely
epithelialized as determined by visual inspection, the animals were
euthanized and the entire dorsum of the rat including the
panniculus carnosus was removed. A 1 cm wide skin strip
perpendicular to each resultant scar, was harvested for breaking
strength analysis. An Instron tensiometer (Model No. 4201; Instron
Corp., Canton, Mass.) with a 5 kg tension load cell and cross head
speed of 10 mm/min was used. Breaking strength is defined as the
force required to rupture the scar and is reported in
kilograms.
[0323] Results--The application of conditioned media overcomes the
inhibition of wound healing caused by bacteria and shifts the
healing trajectory in contaminated wounds to that of near normal
healing (FIG. 2).
Example 15
Effects of Amnion-Derived Cell Conditioned Media in an Animal Model
of Chronic Wound Healing
[0324] Methods: Chronic granulating wound model: Twenty male
Sprague-Dawley rats weighing 300-350 g are anesthetized using
ketamine (40 mg/kg), xylazine (10 mg/kg) and acepromazine (0.75
mg/kg). Following anesthesia, the dorsum of each animal is shaved
and depilitated. A full-thickness dorsal burn measuring 30 cm.sup.3
is created by immersion in boiling water. Animals in the
contaminated group are seeded with 5.times.10 8 Escherichia coli
ATCC #25922 after the rats have been allowed to cool for 15
minutes. Bacteria is obtained from fresh 18 hour broth culture and
inoculum size is confirmed by backplating. The animals are divided
into 8 equal groups of 5 for different treatments after the day 5
escharectomies.
[0325] Animals are individually caged and given food and water ad
libitum. Five days after burning, the eschar is excised from
anesthetized animals, resulting in a chronic granulating wound.
Histological characteristics of this wound with comparison to human
granulating wound have been previously performed. The wounds are
treated with the same experimental groups as described in Table 9
above. Any dried exudates that form are atraumatically removed
prior to wound tracings or biopsies. Every 72 hours the outlines of
the wounds are traced onto acetate sheets and area calculations are
performed using digital planimetry. Care is taken only to record
the advancing full-thickness margin rather than any advancing edge
of epithelium. This avoids the small component of advancement
provided by the smooth, pink, translucent, hairless neoepithelium.
Serial area measurements are plotted against time. For each
animal's data, a Gompertz equation is fitted (typical r2=0.85).
Using this curve the wound half-life is estimated. Comparison
between groups is performed using life table analysis and the
Wilcoxon rank test. The statistical analysis is done using the SAS
(SAS/STAT Guide for Personal Computers, Version 6 Edition, Cary,
N.C., 1987, p. 1028).
Example 16
Effects of Amnion-Derived Cells in Two Animal Models of Wound
Healing
[0326] The two animal models of granulating wounds described above
in Examples 14 and 15 are used to evaluate the effect of
amnion-derived cells on wound healing. The experimental groups are
as follows in Table 9. TABLE-US-00013 TABLE 9 Group No.
Experimental Conditions I Non-contaminated control (PBS only) II
Contaminated control (PBS + bacteria) III Non-contaminated treated
with cells IV Contaminated treated with cells
Example 17
Ability of Amnion-Derived Cells to Promote Complete Regeneration of
Deep Wounds
[0327] Experiments are designed to promote complete regeneration of
deep wounds through re-creating the all of the necessary tissues
including bone, muscle, cartilage, skin, and neural tissue.
Initially, in vitro experiments are designed to determine if
amnion-derived cells can differentiate into all of the cells of
interest. Amnion-derived cells will be cultured as previously
described. Mesenchymal stem cells (Cambrex, Rutherford, N.J.) will
be used as a control for differentiation experiments. MSC's will be
seeded at 5,000-6,000 cells per cm.sup.2 and cultured in
Mesenchymal Stem Cell Growth Medium (MSGM, Cambrex, Rutherford,
N.J.).
[0328] Osteogenic: Once cells are confluent, growth media will be
changed (DMEM, 10% FBS, 1% pen/strep) to osteogenic differentiation
media (Shi, Y. Y., et al., (2005) Plast Reconstr Surg 116,
1686-96.) (DMEM, 10% FBS, 1% pen/strep, 250 uM
ascorbate-2-phosphate, 10 mM beta-glycerophosphate, 2.5 uM retinoic
acid). Osteogenic differentiation media will be changed every 2-3
days. Alkaline phosphatase activity of adipose-derived mesenchymal
cells will be evaluated in duplicate wells after 7 days of culture.
Alkaline phosphatase staining will be performed using the Alkaline
Phosphatase Staining Kit (Sigma) following the manufacturer's
recommendations. Experiments will be performed in triplicate. Von
Kossa staining will be performed in duplicate wells to assess the
ability of cells to mineralize the extracellular matrix and form
bone nodules. Staining will be performed on cells after 21 days of
culture in duplicate wells in differentiation media conditions.
Cells will be fixed in neutral buffered formalin for 30 minutes,
incubated with 1% aqueous silver nitrate for 15 minutes under
ultraviolet light, stained with 5% sodium thiosulfate for 2
minutes, and finally counterstained with 1% Safranin 0 for 10
minutes. In addition, calcium concentration in the extracellular
matrix will be determined via a biochemical colorimetric assay
using the Calcium Reagent Set (Biotron Diagnostics, Hemet, Calif.)
in duplicate wells. Experiments will be performed in
triplicate.
[0329] Adipogenic: Amnion-derived cells and MSC will be cultured in
adipogenic differentiation media (Shi, Y. Y., et al., (2005) Plast
Reconstr Surg 116, 1686-96.) for 3 days (DMEM, 10% FBS, 1%
pen/strep, 10 ug/ml insulin, 1 uM dexamethasone, 0.5 mM
methylxanthine, 200 uM indomethacin), then change to adipocyte
maintenance media for 2 more days (DMEM, 10% FBS, 1% pen/strep, 1
ug/ml insulin). Oil Red 0 staining will be performed to assess for
adipogenic differentiation in duplicate wells (as indicated by the
presence of intracellular lipid-filled droplets) after 5 days of
culture in adipogenic media. Cells will be fixed in 10% neutral
buffered formalin for 30 minutes and then incubated in 60% Oil Red
0 solution for 30 minutes at 37.degree. C. Experiments will be
performed in triplicate.
[0330] Chondrogenic: Amnion-derived cells and MSC will be cultured
in standard non differentiation conditions and then collected and
resuspended at 1.times.10.sup.7 cells/ml concentration. Ten .mu.l
droplets will then be placed onto a culture dish and allowed to
adhere to substratum at 37.degree. C. for 2 hours. Then
chondrogenic media (Malladi, P., et al., (2006) Am J Physiol Cell
Physiol 290, C1139-46.) will be added carefully around cell
aggregates (DMEM, 1% FBS, 1% pen/strep, 37.5 ug/ml
ascorbate-2-phosphate, ITS premix (BD Biosciences), 10 ng/ml TGF-BI
(Research Diagnostics, Inc., Flanders, N.J.). Micromasses will be
fixed in 4% paraformaldehyde with 4% sucrose for 15 minutes,
embedded with Optimal Cutting Temperature (O.C.T.) compound. Ten
.mu.m cryosections will be mounted on slides and stained by
hematoxylin and eosin and alcain blue. Immunohistochemistry will be
performed as follows. Sections will be blocked at room temperature
for 30 minutes and incubated with primary antibody at 4.degree. C.
overnight (anti-collagen II, Santa Cruz Biotechnology, Santa Cruz,
Calif.). Followed by secondary antibody (Vector Labs, Burlingame,
Calif.) incubation, 8 sections will be labeled with ABC reagent
(Vector Labs, Burlingame, Calif.) for 10 minutes at room
temperature. DAB (Vector Labs, Burlingame, Calif.) was applied to
each section and hematoxylin will be used for counterstaining.
[0331] Skeletal myogenic: Amnion-derived cells and MSC will be
cultured as previously described. Skeletal myogenic differentiation
will be induced by culturing cells in myogenic medium (Gang, E. J.,
et al., (2004) Stem Cells 22, 617-24.) (culture medium supplemented
with 5% horse serum, 0.1 .mu.M dexamethasone, and 50 .mu.M
hydrocortisone) for up to 6 weeks. Myogenic differentiation will
analyzed by FACS for MyoD1, myogenin, and myosin heavy chain
(MyHC). For FACS, cells will be detached and stained sequentially
with primary antibodies (human-anti MyoD and anti-myogenin
antibodies; Becton Dickinson) and FITC-conjugated secondary
antibodies (FITC-rat anti-human IgG1; Becton Dickinson). Cells will
be fixed with 2% formaldehyde until analysis with FACS. For
detection of an intracellular protein MyHC, cells were
permeabilized with cold methanol/PBS for 2 minutes at -20.degree.
C. before staining with primary mouse anti-myosin (fast, Sigma) and
FITC-conjugated secondary antibody.
Example 18
Evaluation of Accelerated Wound Strength and Prevention of Acute
Wound Failure
[0332] One object of the invention is to reduce the incidence of
surgical wound failure and to optimize surgical wound outcomes by
treating these acute wounds with conditioned growth media from
amnion-derived cells. The focus is muscle, fascial and skin wound
healing in vivo following surgical injury. Wound fibroblasts are
isolated to measure the effect of soluble mediators derived from
amnion-derived cells on repair fibroblast function in vitro.
[0333] Methods: Male, Sprague-Dawley rats are used for all
experiments. Ventral abdominal wall hair is shaved and the field is
cleansed with alcohol and sterile water. A 6 cm full-thickness skin
incision is placed 2 cm lateral to the ventral midline and a
rectangular skin flap 4 cm in width is subsequently fashioned and
raised through the avascular prefascial plane exposing the linea
alba. In the Sham Operated rats, this skin flap is replaced and
sutured using 4-0 Prolene. In the Experimental group rats, a 5 cm
isolated laparotomy incision is placed through the midline of
muscular layer of the abdominal wall (linea alba). The design of
the ventral abdominal wall skin flap model allows laparotomy
healing to occur isolated from the overlying skin wound. In the
Mechanically Intact Wound group, the laparotomy is repaired with a
running, 3-0 polypropylene suture using 0.3 cm suture bites and 0.5
cm progress between stitches. The suture is tied to itself at the
end of the wound. Experience with this model predicts a 100% intact
wound healing rate. In the Hernia Wound group, the laparotomy
incision is left un-sutured. In both the Disrupted Wound and Hernia
Wound models, the skin flap is sutured in place, acting as a sling
to prevent the evisceration of the abdominal organs. Mortality
using these models has been found to be less than 1%. Following 30
minutes of recovery on a heated pad, the rats are returned to
individual cages. Food and water are provided ad libitum. All rats
are observed daily and weighed weekly. Experience with these models
predicts that by post operative day 28, 100% of the Intact Wound
rats heal the laparotomy incision, and 100% of the Disrupted Wound
rats go on to form incisional hernias.
[0334] Five rats are used at each time point to generate 5 distinct
fibroblast cell lines for each of the four laparotomy wound types.
Necropsies are performed 1, 7, 14, 28 and 60 days after operations.
In the Disrupted Wound group, Day 0 is re-defined at the time of
wound disruption on post-operative day 3. The entire ventral
abdominal wall is excised from each euthanized rat, and the skin
separated from the muscular layer. The peritoneal and subcutaneous
(ventral) surfaces of each abdominal wall is carefully inspected
for the presence of laparotomy wound disruption and herniation. An
incisional hernia is defined as minimum of 2 mm of myo-fascial
separation and/or obvious trans-abdominal wall herniation of
abdominal contents. Biopsies are taken perpendicular to the axis of
the linea alba. One biopsy from each rat is immediately snap frozen
in liquid nitrogen for subsequent RNA isolation and real-time PCR
measurement of collagen and integrin expression. A second adjacent
biopsy from each rat is immediately fixed in 10% buffered formalin
for subsequent paraffin fixation and immunohistological analysis of
wound structure, fibroblast morphology, inflammatory response,
angiogenesis and extracellular matrix formation. The final wound
biopsy is placed in PBS (see Methods below). Primary, first pass
fibroblast cell cultures are used to measure fibroblast
proliferation, collagen synthesis and fibroblast populated collagen
matrix compaction in vivo and following controlled patterns of
mechanical strain in vitro.
[0335] Rapid gain in wound strength is tested in this model as
follows: Animals are randomly assigned into one of 12 Groups (n=10
per group). In Experimental Designs 1 and 2, each of the three
animal models (Sham laparotomy, Healing laparotomy and Hernia) are
treated with four experimental conditions of amnion-derived cell
conditioned media containing the humoral products of amnion-derived
cells. (No treatment, Control amnion-derived cell media (0%
conditioned), 50% amnion-derived cell CM and 100% amnion-derived
cell CM). See Table 10 for Experimental Groups. 100 IU of media is
delivered to the site of the laparotomy myofascial and skin
incisions prior to wounding. This is defined as simple priming and
establishes that this is a reliable and efficient way to deliver
liquid growth media to the site of surgical incisions with a large
experience in this model. Fibroblasts are isolated from healing
wounds over time and assayed for the effect of amnion-derived cell
media on proliferation and collagen matrix compaction in vitro.
TABLE-US-00014 TABLE 10 Table of Experimental Groups Unconditioned
No Treatment media (0% CM) 50% CM 100% CM Sham Sham Sham Sham Wound
Wound Wound Wound Hernia Hernia Hernia Hernia
[0336] Myofascia (laparotomy) and skin incision tensile strength
rats are randomly selected and euthanized at serial timepoints
following laparotomy with an overdose of Nembutal (100 mg/kg i.p.).
The entire ventral abdominal wall is excised and the skin separated
from the musculofascial layer. The wound healing interface is
closely examined for evidence of acute failure (dehiscence) or
primary incisional hernia formation, defined as a fascial defect
greater than 2 mm on or after POD 7. Fascial and skin sutures are
removed. Two myofascial and two skin strips in the shape of the
uppercase letter "I" are taken perpendicular to the wound healing
interface from each abdominal wall. A cutting template is used to
mark the abdominal wall in order to minimize size variability
between specimens. The abdominal wall myofascia and skin strips are
labeled and stored in PBS until tensiometric mechanical analysis is
performed. Biopsies are taken of the myofascial (laparotomy) and
skin wounds and immediately snap frozen in liquid nitrogen for
biochemical analysis or fixed in formalin for histology.
[0337] Mechanical testing of the abdominal wall fascial and skin
strips is performed within 6 hours of necropsy. The sample width
and thickness is measured with Digimatic calipers (Mitutoyo
American Corp., Chicago, Ill.). The samples are each loaded in
tension to failure, during which time the force-extension data are
collected. Force extension curves are generated using an Instron
Tensiometer (model 5542, Instron Corporation, Canton, Mass.)
equipped with a 50 Newton static load cell set at a crosshead speed
of 10 mm per minute. Samples are mounted into the load frame using
pneumatic graspers, preloaded to 0.1 Newtons, and the gauge length
measured between the grips. The load frame applies tensile loads
perpendicular to the suture repaired wounds until mechanical tissue
disruption occurs. The anatomic location of the wound failure is
noted for each specimen. Force and tissue deformation data are
simultaneously recorded and captured on a computer connected to the
load frame via a digital interface card. Data analysis is performed
using the Merlin materials testing software package (Instron
Corporation, Canton, Mass.).
[0338] Data from the stretch loading is used to determine the
following clinically important biomechanical properties: Breaking
strength--the maximum load (F.sub.max) at mechanical failure
(Newtons); Tensile strength--the maximum stress developed in the
specimen per unit area, calculated as F.sub.max/cross sectional
area (N/mm.sup.2); Toughness--the energy absorbed by the specimen
under tension, calculated as the entire area under the
force-extension curve from the origin to mechanical rupture
(Joules); Elongation--the increase in length of the tissue under a
load, defined as the length of the specimen at mechanical
disruption minus the original length (mm); Stiffness--the slope of
the linear elastic region of the force-extension curve (N/mm).
[0339] Histological analyses of provisional matrix structure,
fibroblast migration, inflammatory response and wound angiogenesis
is used to compare the groups using H&E and trichrome staining.
The density of wound collagen formation is measured using
antibodies specific for rat collagen types I and III (Chemicon
International, Inc., Temecula, Calif.). Cellular infiltration into
the wounds at each time-point is measured as the mean cell number
from three high-powered fields by a blinded observer using a
microscope. In addition, histological specimens are digitized using
a UMAX Astra 1200S scanner and analyzed using the computer software
application Adobe PhotoShop version 5.0. Differences in cellularity
and intensity of collagen staining are compared using the Students
t test (SigmaStat, Jandel).
[0340] Samples are collected from laparotomy wounds or incisional
hernias from rats or humans as described previously and are placed
in a sterile 50 mL conical tube (Corning, Corning N.Y.) in cooled
PBS and placed on ice. Each sample is minced into small pieces and
placed in a sterile 6 cm diameter Petri dish (Falcon, Franklin
Lakes N.J.) containing 0.1% collagenase in PBS for 45 minutes at
room temperature. During this time, tissues and cells are
triturated several times using a tissue culture pipette. The
solution is poured into a sterile 50 mL conical tube and
centrifuged at 800 rpm for 6 minutes. The collagenase in PBS is
suctioned off and the remaining cell and macerated tissue pellet is
reconstituted in 15 mL complete growth medium consisting of low
glucose DMEM (GIBCO, Grand Island N.Y.) supplemented with 10%
newborn calf serum (GIBCO, Grand Island N.Y.), 25 .mu.g/mL
gentamicin (GIBCO, Grand Island N.Y.), and 0.375 .mu.g/mL
amphoteracin B (Sigma, St. Louis Mo.). Cells are transferred into a
sterile T75 flask (Corning, Corning N.Y.) and placed into an
incubator at 37.degree. C. with 5% CO.sub.2. Complete growth medium
is changed every two days as soon as cells reached 10-15%
confluence with a minimum of 6 colonies visible using an inverted
microscope with the 5.times. objective. Standard cytokeratin, alpha
smooth muscle actin, vimentin and van Willebrand factor staining is
done to precisely characterize the cells as fibroblasts.
[0341] Once cells reach confluence, they are passaged 1:2. The
medium is removed and the cell layer is washed with 10 mL of HBSS
(GIBCO, Grand Island N.Y.). Cells are trypsinized with 10 mL of
0.05% trypsin with 0.53 mM EDTA (GIBCO, Grand Island N.Y.) for 4-6
minutes at 37.degree. C. The trypsin is inhibited using 10 mL of
complete growth medium. Cells are poured into a sterile 50 mL
conical tube and centrifuged at 600 rpm for 5 minutes. The
supernatant is removed and the cell pellet is resuspended in
complete growth medium. Cells are divided into flasks to give a
final passaging concentration of 1:2. Cells are trypsinized and
centrifuged as above and reconstituted in 4 mL of DMEM with 40%
newborn calf serum. This solution is divided into four 2 mL
cryovials (Corning, Corning N.Y.), and 1 mL of cooled 20% DMSO
(Fischer, Fairlawn N.J.) in DMEM is added to each vial. Vials are
placed in a container with isopropyl alcohol and cooled at
1.degree. C./min in a -80.degree. C. freezer. When completely
frozen, they are transferred to liquid nitrogen for storage.
[0342] One cryovial is removed from liquid nitrogen and quickly
thawed in warm ethanol. The contents are placed in a 50 mL conical
tube with 20 mL warm complete growth medium and centrifuged at 600
rpm for 5 minutes. The supernatant is removed and the cellular
pellet is reconstituted in 15 mL warm complete growth medium. The
cells are plated in a T75 flask. A MIT colorimetric assay is used
to access viability of the fibroblasts by measuring their
mitochondrial activity.
[0343] In vitro protein matrices (FPCL's) fashioned with collagen,
fibrin, and fibronectin are used. Extracellular matrix protein
lattices are prepared as described by the manufacturer (Upstate
Biotechnology, Lake Placid, N.Y.). The gels are incubated for 24
hours at 4.degree. C. The fibroblasts are counted and their cell
number adjusted to 1.times.10.sup.5 cells/ml. One hundred thousand
first passage-cultured fibroblasts are added to each prefabricated
3.5 cm lattice. The lattices are incubated at 37.degree. C. with 5%
CO.sub.2 and the extent of gel contraction is measured every 24
hours for 5 days. The gels are digitally imaged each day and
contraction measurements calculated using Sigma Scan software
(Jandel Scientific, Corte Madera, Calif.). Alternatively, FPCL's
fabricated from rat tail collagen is used for corroborative data.
This assay runs from 30 minutes to several hours and allows
determination of the response of the cells to various functional
inhibitors. Measurement of gel contraction are performed overtime.
Collagen gels are detached from petri dishes, treated with 2.5% FCS
or 2 u/ml thrombin, and the diameter of the gel measured at
perpendicular axis at various times. One function that is evaluated
is the role of MAP kinases on collagen contraction.
[0344] To further characterize the fascial fibroblasts to explain
the effects seen from the animal experiments, a series of tests are
performed on the wound fascial and dermal fibroblast cultures.
These include collagen types I and II gene expression using
quantitative RT-PCR on extracted RNA; measurement of tissue
collagen levels of biopsies of the wound healing interface using
the Sircol collagen assay method (Acurate Chemical and Scientific
Corp., Westburg, N.Y.); measurement of fibroblast, alpha-1 and
beta-1 integrin expression to assure that any reduction in FPCL
contraction was not due to poor migration and reduced fibroblast
function; and immunohistochemistry to evaluate alpha-smooth muscle
action, and Proliferating Cell Nuclear Antigen (PCNA) using
specific monoclonal antibodies for PCNA and alph-SMA (Sant Cruz
Biotechnology, Santa Cruz, Calif.).
[0345] Statistical analyses is as follows: For each experiment, a
factorial design with balanced sample sizes in each group ensures
that the main effect, independent variables in each experiment can
be isolated with appropriate statistical analysis. Outcome
variables are compared using parametric (continuous variables) and
non-parametric (proportions) ANOVA. Nested ANOVA designs are used
to incorporate the main effect variables from each experiment into
a single analysis. If the F ratio for the overall ANOVA is
significant, post hoc comparisons of individual group means are
conducted via t-prime tests of least-squares means for each
comparison. Inherent in this approach is a Bonferonni correction of
the significance level when making multiple pairwise comparisons.
The ANOVA calculations are performed using the general linear
models (GLM) algorithm from the Statistical Analysis System (SAS,
Carey, N.C.), which accounts for unbalanced sample sizes, should
they occur. Post-hoc pairwise testing is conducted using the
lsmeans option with the GLM procedure. The correlations between the
measured variables and if significant covariance is observed
between variables for a given experiment are examined, analysis of
covariance (ANCOVA) is performed as appropriate. A 5% level of
significance is considered statistically significant.
Example 19
Use of Amnion-Derived Cells, Conditioned Media, Cell Lysates, and
Cell Products for Rapid Early Wound Closure of Thermal Injuries
[0346] Outcome and rehabilitation of thermal injuries rely on early
burn wound excision and rapid wound closure. The speed of wound
closure with a serviceable integument or integument substitute is
the key to an improvement in survival. Providing novel approaches
that will facilitate early, rapid wound closure, while minimizing
long-term scarring, is an object of the present invention.
[0347] Established in vitro, animal models, and clinical patients
are used to evaluate the use of amnion-derived cells for early,
rapid wound closure of partial-thickness and full-thickness burns.
In addition to thermal injuries, experiments are done with
established models for chemical, electrical, and cold injuries.
[0348] It is theorized that amnion-derived cells can differentiate
into mesodermal and ectodermal cells. Thus, it may be possible that
use of such cells will provide early and permanent closure of the
burn wound. Since presently, the prolonged time the wound is in the
inflammatory phase is the known variable leading to proliferative
scarring, it is expected that early, permanent closure of the burn
wound would result in decreased scarring and, thus, increased
function.
[0349] Methods: Three animal models of partial-thickness and
full-thickness thermal injuries are used. The three models are
different because the first mimics partial-thickness healing by
epithelialization in approximately three weeks while the second and
third mimic full-thickness healing by contraction and
epithelialization and can remain unhealed for up to eight weeks.
The last two models have been histologically compared to the human
granulating wound. The difference in the last two full-thickness
wounds is the host. One group is a normal rat with an intact immune
system, while the other is an athymic "nude" rat which is devoid of
T-lymphocytes.
[0350] Partial-thickness burn injury: Forty female Hartley strain
guinea pigs weighing 350 to 450 grams are used throughout this part
of the experiment. Under Nembutal anesthesia (35 mg/kg administered
intraperitoneally), the animals' backs are shaved and depilated. A
uniform scald burn over 10% of the body surface is performed at
75.degree. C. for 10 seconds. Guinea pigs are used for this model
because of their lack of an estrus (hair) cycle, and the ability to
develop uniform partial-thickness injuries in them. Animals are
caged individually and fed food and water ad libitum.
[0351] At 24 hours, the animals are reanesthetized and the
partial-thickness eschar gently abraded. The 40 animals are divided
into four groups of 10 animals each. The groups are as follows:
Group I-guinea pigs burned, abraded, and left untreated as
controls; Group II-burned, abraded and treated with nonconditioned
media on day 1 (day of abrasion) and day 7; Group III-burned,
abraded and treated with amnion-derived cell conditioned media on
day 1 and day 7; Group IV-burned, abraded, and treated with a
suspension of amnion-derived cells over the entire burn, and
dressed with Adaptic and bulky dressing. The outer dressing is
gently removed every five days or prn. The animals are premedicated
with buprinorphine (0.1 mg/kg), anesthetized with halothane
inhalation and burn wound biopsies are obtained on a weekly basis
until the time of healing. The biopsy specimens are sectioned and
stained, and the hair follicles are counted microscopically and
expressed as the number per high power field. Additionally,
histological analyses of the healing skin is done. Gross
observations are made and photographically documented for the
quality of healing and hair distribution.
[0352] Full-thickness burn injury (normal rat): Fifty male
Sprague-Dawley rats weighing 300-350 grams are acclimatized for one
week prior to use. Under intraperitoneal Nembutal anesthesia, the
rat dorsum is shaved and depilated. A full-thickness dorsal burn
measuring 30 square cms is created by immersion in actively boiling
water. Seven mL of Ringer's lactate by subcutaneous injection is
given to each rat to prevent dehydration. Animals are individually
caged and given food and water ad libitum. Five days after burning,
the eschar is be excised from anesthetized animals resulting in a
granulating wound. Histological characterization of this wound with
comparison to a human granulating wound has previously been
performed (Robson M C, et al., J Surg Res 16: 299-306, 1974). The
rats are divided into five groups of 10 animals each and treated as
follows: Group I receive no wound treatment and serve as controls;
Group II receives treatment with nonconditioned media on day 0 (day
of escharectomy) and on day 7; Group III receives treatment with
amnion-derived cell conditioned media on day 0 and day 7; Group IV
is treated with a suspension of amnion-derived cells and dressed
with Adaptic and a bulky dressing. The dressing is changed every
five days or prn. Group V is treated with an extracellular matrix
seeded with amnion-derived cells and dressed as in Group IV. Groups
I-III animals' wounds are left exposed. Any dried exudates are
atraumatically removed prior to any wound tracings or biopsies.
Every 72 hours for rats in Groups I-III or whenever dressings
require changing in Groups IV and V the rats are premedicated with
buprinorphine (0.1 mg/kg), anesthetized with halothane inhalation,
and the outlines of their wounds are traced onto acetate sheets.
Area calculations are performed using digital planimetry. Serial
measurements are plotted against time. For each animal's data, a
Gompertz equation is fitted (typical r2=0.85). Using this curve the
wound half-life is estimated. Comparison between groups is
performed using life table analysis and the Wilcoxon rank test. The
statistical analyses is performed using the SAS (SAS/STAT Guide for
Personal Computers, Version 6 Edition, Cary, N.C., 1987, p 1028)
and BMDP (BMDP Statistical Software, Inc. 1988). From the best fit
curves for the individual wounds, the number of days required for
25%, 50%, and 75% healing of the original wounds is calculated.
Randomized wound biopsy sites are obtained from reanesthetized rats
on days 5, 10, 15, 20, and 25 post escharectomy (or days 10, 15,
20, 25, and 30 post burn) and placed in appropriate preservative
solutions for histological studies.
[0353] Full-thickness burn injury (immunologically impaired rat):
Fifty outbred, congenitally athymic "nude" rats are purchased
commercially (Harlan Sprague Dawley, Inc., Indianapolis, Ind.). All
animals are male and weigh between 250 and 300 grams. Because of
their immune defect, the animals are housed in pathogen-free
barrier facilities, in cages with sealed air filters, animal
isolators, laminar flow units, and laminar flow rooms. All supplies
such as food, water, bedding etc. are sterilized to prevent
infection. Procedures recommended in the Guide for the Care and Use
of the Nude Mouse in Biomedical Research (Institute of Laboratory
Animal Resources) are used at all times. Persons handling the rats
wear caps, masks, sterile gowns, sterile gloves, and shoe covers.
All operations on "nude" rats are carried out under intraperitoneal
Nembutal anesthesia, 35 mg/kg body weight, using aseptic surgical
techniques. Operations are performed under a unidirectional airflow
biological hood. Surgical instruments are sterilized by autoclaving
and surgical sites are prepared with povidone iodine solution. The
50 animals are divided into five groups of 10 each. The anesthesia,
analgesia, procedures, wound treatments, and measurements are
identical to the intact rat model described above. Handling of the
tracings, planimetry, and statistics are also the same as
previously described.
[0354] In vitro fibroblast-populated collagen lattice: The
fibroblasts are prepared as previously described by Kuhn, et al
(Kuhn Mass., et al., Internat J Surg Invest 2: 467-474, 2001). The
collagen lattices are prepared from type I rat tail collagen
(acetic acid extracted) as recommended by the manufacturer (Upstate
Biotechnology, Lake Placid, N.Y.) (11). Undiluted collagen (1 ml)
is placed in 35 mm culture dishes (Falcon 1008) and evenly spread.
The dishes are placed in an ammonia vapor chamber for 3 minutes to
solidify. Sterile distilled water (5 ml) is added to the culture
dishes, allowed to stand for one hour, and then aspirated. This is
repeated four times to remove excess ammonia and the collagen
lattices are incubated for 24 hours at 4.degree. C. PBS with 1.0%
serum is added to replace the final aspirate. An 18 gauge needle is
used to detach the collagen gel lattices from the surface of the
culture dishes so that they are loose and suspended in saline. A
total of 30 collagen lattices are prepared to allow quintruplicate
measurements based on 5 treatment groups plus an untreated control.
To form the FPCLs, all saline is aspirated from the 35 mm culture
dishes containing the lattices. Two ml of 2.times.10 5
fibroblasts/ml are placed on the surface of each of the
prefabricated collagen gel lattices. FPCLs are divided into six
groups as follows: Group I is kept as a control with no treatment;
Group II receives nonconditioned media; Group III receives
amnion-derived cell conditioned media; Group IV receives a
suspension of amnion-derived cells; Group V is covered with an
extracellular matrix; and Group VI is covered with an extracellular
matrix seeded with amnion-derived cells. The FPCLs are incubated at
37.degree. C., 5% carbon dioxide. The amount of gel contraction is
measured every 24 hours for 5 days.
[0355] Acetate overlays are used for tracing the area of the gels.
Gels are performed in quintruplicate (5 gels) for the fibroblast
line established and measurements are calculated using digital
planimetry and Sigma Scan software (Jandel Scientific, Corte
Madera, Calif.). Each collagen gel area measurement is converted to
reflect percentage of area remaining over time and subsequently
percentage of gel contraction. A one-way analysis of variance is
used to determine significant differences among groups. When a
difference is identified, a Tukey's Test (all pairwise multiple
comparison test) is used to delineate the differences. Sigma Stat
statistical software is used for data analysis. In addition, the 24
hour FPCLs are examined microscopically and photographed. One of
the gels is evaluated for fibroblast viability on day 5 utilizing
Trypan blue exclusion assay. Another gel is included for cell
number spectrophometrically as a function of mitochondrial activity
using the MTT method. After exposure to test agents, the five day
suspension cultures are reincubated and exposed to
[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or MTT.
Mitochondrial dehydrogenases from viable fibroblasts cleave the
tetrazolium ring, yielding purple formazan crystals. These are
dissolved in an acidified isopropanol resulting in a purple
solution which is spectrophometrically measured. An increase or
decrease in cell number results in a concomitant change in the
amount of formazan formed, indicating any degree of toxicity of the
applied test material.
Example 20
Differentiation of Amnion-Derived Cells to Early Pancreatic
Progenitor Cells
[0356] All four pancreatic endocrine cell types that comprise the
pancreatic islet develop by progressive differentiation from a
common pancreatic progenitor cell. This common pancreatic
progenitor cell expresses the PDX1 gene early in its developmental
ontogeny and later, expression of this gene serves as an early
distinguishable cell marker of pancreatic differentiation. Once
this early pancreatic progenitor cell is generated and/or
propagated in vitro, it may potentially give rise to all four
endocrine cell types comprising the mature pancreatic islet.
[0357] Developmentally, the endocrine and exocrine pancreatic cells
are derived from outgrowths of cuboidal epithelium from the foregut
endoderm. This three-dimensional architecture is thought to be
important in the development and expression of both exocrine and
endocrine cell types. In an attempt to duplicate this environment,
a novel culture condition that preferentially supports and
maintains the spheroids of amnion-derived cell in suspension
culture is used. Briefly, cells are grown as a monolayer, than
treated with proteinase XXIII to obtain single cells or small
clusters of cells. Detection of PDX1 protein expression
(cytoplasmic and peri-nuclear) first occurs in cells present in
buds on the outer-most surface of the amnion-derived cell
spheroids. Immunocytochemical techniques show that these cells also
express CD29 protein. These buds of pancreatic progenitor cells
resemble the cells prominently observed in adult pancreatic duct
cell differentiation (Bonner_Weir, S., et al., Proceedings of the
National Academy of Sciences of the United States of America, 2000.
97(14): p. 7999-8004, Jones, E. M. and N. Sarvetnick, Horm Metab
Res, 1997. 29(6): p. 308-10; Ramiya, V. K., et al., Nature
Medicine, 2000. 6(3): p. 278-82]
[0358] The cells are then plated on a substrate or in a matrix that
maintains the three-dimensional structure of the spheroids but
promotes protein expression of cytoplasmic PDX1. After several
days, the cells are supplemented with factors that promote nuclear
translocation of PDX1 protein in a small proportion of the
differentiating cells. In the presence of factor/cell culture
conditions that promote nuclear localization of PDX1 protein, large
numbers of these buds appear on the majority of the amnion-derived
cell spheroids cultured in suspension. Amnion-derived cells may
also be cultured in the presence of an extracellular matrix in the
presence of the above factors. As a result, cells will form
spheroids on or embedded in the extracellular matrix.
Example 21
Determination of Cells Expressing Islet Cell Proteins
[0359] Cells fixed in 4% buffered PFA and stored in 1.times. PBS
containing 0.02% sodium azide, are rinsed with 1.times.PBS. The
cells are then blocked for nonspecific binding with 5% BSA in
Calcium and Magnesium-Free Phosphate Buffered Saline (CMF-PBS) for
30 minutes and permeabilized with PBS-TX (CMF-PBS/0.3%
Triton-X-100). Staining for nuclear antigens is performed using an
additional high salt treatment (0.3M NaCl, 20 mM Tris-HCl pH 7.2,
0.1% Tween-20, 0.1% Triton-X-100) and overnight incubation with the
primary antibody. All antibodies are diluted in 5% BSA in PBS-TX,
unless otherwise noted. Pancreatic transcription factors are
identified by staining the cells with anti-PDX1 (rabbit polyclonal,
1:2000, C. V. Wright), anti-Nkx2.2 (mouse monoclonal 1:100, T.
Jessell), anti-Nkx6.1 (mouse monoclonal, 1:8000, T. Jessell), and
anti-HB9 (mouse monoclonal, 1:30, T. Jessell). Endocrine cells are
identified by staining with anti-Insulin (1:2000, Linco 4012-01),
anti-Proinsulin, (1:400, Novacastra Peninsula, IHC-7165) and
anti-Somatostatin (1:2000, Peninsula, IHC-8001). Secondary
antibodies used include: Fluorescein isothyocyanate (FITC) (1:200),
Indocarbocyanine (Cy3) (1:1000) and Indodicarbocyanine (Cy5)
(1:400)-conjugated donkey anti-mouse, rabbit and guinea pig IgG
(Jackson ImmunoResearch, ML grade). Cell nuclei are visualized by
DAPI fluorescence as part of the Vectashield mounting medium
(Vector, H1200). Cells are analyzed with a Nikon Eclipse E2000U
fluorescence/DIC inverted microscope equipped with Autoquant 3-D
Imaging software and an Olympus FV300 FluoView Confocal Laser
Scanning Microscope.
Example 22
Transplantation Studies--In Vivo Co-Culture of Amnion-Derived Cell
with Embryonic Islet Progenitor Cells Under the Kidney Capsule
[0360] Three experimental groups are used to determine if the
dorsal pancreatic bud from embryonic day 12.5 (e12.5) mice promotes
the differentiation of PDX1 protein-expressing amnion-derived
cells:
[0361] A. Group 1-12 immuno-compromised mice, 4 months of age, are
transplanted with 10.sup.6 PDX1 protein-expressing cells and two
e12.5 dorsal pancreatic explants under the kidney capsule. Three
mice are sacrificed for analysis every second week (time points: 2,
4, 6, and 8 weeks after transplantation).
[0362] B. Group 2 (control)--12 immuno-compromised mice, 4 months
of age, are transplanted with 10.sup.6 freshly isolated
amnion-derived cells and two e12.5 dorsal pancreatic explants under
the kidney capsule. Three mice are sacrificed for analysis every
second week (time points: 2, 4, 6, and 8 weeks after
transplantation).
[0363] C. Group 3 (control)--12 immuno-compromised mice, 4 months
of age, two e12.5 dorsal pancreatic explants under the kidney
capsule. Three mice are sacrificed for analysis every second week
(time points: 2, 4, 6, and 8 weeks after transplantation).
[0364] Dorsal pancreatic buds are manually dissected from the
foregut of each embryo directly and/or incubated with 0.2 Wunsch
U/ml Liberase Blendzyme-3 (Roche, 11814176) and 0.15 mg/ml DNase I
at 37.degree. C. for 15 min. The enzymatic dissociation is
immediately terminated by the addition of an equal volume of
1.times. PBS containing 10% (w/v) BSA. The dorsal pancreatic buds
are rinsed with HBSS at 4.degree. C. and briefly triturated using a
200 .mu.l pipet tip. The epithelium is stripped manually from the
surrounding mesenchyme and transferred into HBSS at 4.degree. C.
prior to transplantation.
[0365] Mouse and human amnion-derived cells are placed under the
kidney capsule of immuno-compromised mice to prevent
immuno-rejection of the grafted cells over the course of the
experiment. At each time point, three mice are sacrificed, the
transplanted cells isolated and fixed in 4% PFA. The tissue is then
soaked in a series of increasing concentrations of sucrose,
embedded with OCT and sectioned. Cyro-sections (10 .mu.m) are
analyzed for the co-expression of human nuclear antigen protein and
specific islet endocrine cell marker proteins. These markers
include: pro-insulin, C-peptide, glucagon, somatostatin, Nkx2.2,
Pax6, Nkx6.1 and PDX1 proteins.
Example 23
Transplantation Studies--In Vivo Co-Culture of Amnion-Derived Cells
with Embryonic Islet Progenitor Cells in the Mammary Gland
[0366] Three experimental groups are used to determine if the
dorsal pancreatic bud from embryonic day 12.5 (e12.5) mice promotes
the differentiation of PDX1 protein-expressing amnion-derived
cells:
[0367] A. Group 1-12 immuno-compromised mice, 4 months of age, are
transplanted with 10.sup.6 PDX1 protein-expressing cells and two
e12.5 dorsal pancreatic explants in the mammary gland. Three mice
are sacrificed for analysis every second week (time points: 2, 4,
6, and 8 weeks after transplantation).
[0368] B. Group 2 (control)--12 immuno-compromised mice, 4 months
of age, are transplanted with 10.sup.6 freshly isolated
amnion-derived cells and two e12.5 dorsal pancreatic explants in
the mammary gland. Three mice are sacrificed for analysis every
second week (time points: 2, 4, 6, and 8 weeks after
transplantation).
[0369] C. Group 3 (control)--12 immuno-compromised mice, 4 months
of age, two e12.5 dorsal pancreatic explants in the mammary gland.
Three mice are sacrificed for analysis every second week (time
points: 2, 4, 6, and 8 weeks after transplantation).
[0370] Dorsal pancreatic buds are manually dissected from the
foregut of each embryo directly and/or incubated with 0.2 Wunsch
U/ml Liberase Blendzyme-3 (Roche, 11814176) and 0.15 mg/ml DNase I
at 37.degree. C. for 15 min. The enzymatic dissociation is
immediately terminated by the addition of an equal volume of
1.times. PBS containing 10% (w/v) BSA. The dorsal pancreatic buds
are rinsed with HBSS at 4.degree. C. and briefly triturated using a
200 .mu.l pipet tip. The epithelium is stripped manually from the
surrounding mesenchyme and transferred into HBSS at 4.degree. C.
prior to transplantation.
[0371] Mouse and human amnion-derived cells are placed in the
mammary gland of immuno-compromised nude mice to prevent
immuno-rejection of the grafted cells over the course of the
experiment. At each time point, three mice are sacrificed, the
transplanted cells isolated and fixed in 4% PFA. The tissue is then
soaked in a series of increasing concentrations of sucrose,
embedded with OCT and sectioned. Cyro-sections (10 m) are analyzed
for the co-expression of human nuclear antigen protein and specific
islet endocrine cell marker proteins. These markers include:
pro-insulin, C-peptide, glucagon, somatostatin, Nkx2.2, Pax6,
Nkx6.1 and PDX1 proteins.
Example 24
Transplantation of Undifferentiated Amnion-Derived Cells,
Semi-Differentiated Amnion-Derived Cells Expressing Peri-Nuclear
PDX1 and Amnion-Derived Cells Stably Expressing the Nuclear PDX1
Fusion Protein into Immuno-Comprised Mice
[0372] Transplantation of undifferentiated amnion-derived cells,
semi-differentiated amnion-derived cells expressing peri-nuclear
PDX1 and amnion-derived cells stably expressing the nuclear PDX1
fusion protein into non-diabetic immuno-compromised mice was done
to determine if the mammary gland is a permissive transplantation
site that will maintain the morphological characteristics of the
differentiating cells and expression of the PDX1 protein.
[0373] Fifteen immuno-compromised mice (Hilltop Labs) were
transplanted as follows: Group 1: Control Group, Reduced Factor
Matrigel Injected, 5 mice; Group 2: Factor Induced PDX1 expressing
amnion-derived cells; 5 mice; Group 3: amnion-derived cells
infected with Lentiviral PDX1 fusion protein; 5 mice.
[0374] Mice were maintained after transplantation in normal housing
conditions for 31 days. The mammary gland tissue containing the
transplanted cells was subsequently removed, fixed, embedded with
O.C.T. and frozen at -80 C. Additional control samples from the
opposite (non-transplanted) mammary gland were also excised as a
control. Sections will be analyzed for PDX1, Proinsulin and insulin
expression.
Example 25
Further Transplantation Studies
[0375] PDX1 protein-expressing cells are also transplanted into the
portal vein, spleen and mammary gland using non-diabetic
immuno-compromised mice. Differentiating amnion-derived cells as
above are initially transplanted with differentiating mouse e12
dorsal pancreatic buds to determine if they can respond to the same
factors as early embryonic pancreatic epithelial cells and generate
islet-like cells. All tissues transplanted with PDX1
protein-expressing cells are removed two and six weeks following
transplantation, fixed, cryopreserved and sectioned. Tissue
sections are stained with PDX1 anti-sera, Pro-insulin, C-Peptide,
Glucagon and Somatostatin antibodies. Human nuclear antigen
immunostaining will be used to verify the origin of cells
expressing endocrine cell markers in the rat pancreas.
Example 26
Restoration of Normoglycemia in STZ-Induced Diabetic
NOD-Immun-Compromised Mice
[0376] Further experiments are conducted to restore normoglycemia
in STZ-induced diabetic NOD-immuno-compromised mice. Mice
exhibiting initial blood glucose levels over 400 ng/dl are included
in the experiment. Insulin therapy (Linbit) is administered to the
animals after the initial blood glucose determination but prior to
cell transplantation. This allows for the initial engraftment of
amnion-derived cells in a normoglycemic environment. Initial
evidence of human C-peptide expression will be determined using a
human C-Peptide RIA or ELISA assay. Once detection of human
C-peptide is confirmed, insulin therapy is discontinued and the
blood glucose monitored every third day. If the differentiated
cells are able to restore normoglycemia in the STZ-induced diabetic
mice, the transplanted cells will be removed forty to sixty days
after transplantation and the mice evaluated daily for reversion to
the blood glucose levels (>400 ng/dl) previously observed.
Example 27
Factor-Priming Experiments
[0377] In one experiment, the pancreas of a mouse is primed with
factors to promote differentiation of resident pancreatic cells
into functional islets. These factors include any individual or
combination of the following factors: FGF(s), Forskolin,
Follistatin, angiogenic factors, glucocorticoid family members,
Insulin, EGF, EGF-like factors, Heparin, Nicotinamide, SHh
antagonists, HGF, GLP-1 analogs, between 1 and 20 mM Glucose,
divalent cations.
[0378] In another experiment the pancreas of a mouse is primed with
factors to promote the regeneration of transplanted
undifferentiated amnion-derived cells to this site and to allow
differentiation. Undifferentiated amnion-derived cells may be
freshly isolated cells (not cultured) treated with factors to
ensure endoderm differentiation (SHh antagonists, spheroid
growth).
[0379] In another experiment the pancreas of a mouse is primed with
factors to promote the regeneration of transplanted partially
differentiated amnion-derived cells to this site and to allow
further differentiation. Partially differentiated means the
amnion-derived cells have been cultured in vitro in any condition
described herein.
[0380] In another experiment the pancreas of a mouse is primed with
other factors and then the following cells are transplanted:
Co-culture of undifferentiated or partially differentiated
amnion-derived cells with differentiating embryonic pancreatic or
non-pancreatic tissue (epithelium, mesenchyme, islets, ducts,
exocrine cells) or differentiating or pre-differentiated
non-embryonic heterologous (donor) or autologous (self) tissue
(epithelium, mesenchyme, islets, ducts, exocrine cells, etc.).
These cells will provide active factors and/or the biological niche
necessary for the differentiation of undifferentiated or partially
differentiated amnion-derived cells to pancreatic cells. These
factors and/or niche may also promote the molecular organization of
the cells so the cells mature and function as pancreatic islet-like
cells.
[0381] In another experiment the pancreas is primed with
proprietary factor combinations and then transplanted with a
co-culture of undifferentiated or partially differentiated
amnion-derived cells with differentiating embryonic pancreatic or
non-pancreatic tissue (epithelium, mesenchyme, islets, ducts,
exocrine cells) or differentiating or pre-differentiated
non-embryonic heterologous (donor) or autologous (self) tissue
(epithelium, mesenchyme, islets, ducts, exocrine cells, etc.).
These cells will provide active factors and/or the biological niche
necessary for the differentiation of undifferentiated or partially
differentiated amnion-derived cells to pancreatic cells. These
factors and/or niche may also promote the molecular organization of
the cells so the cells mature and function as pancreatic islet-like
cells.
[0382] Another experiment is transplanting undifferentiated or
partially differentiated amnion-derived cells that have been primed
in vitro (not primed at the site of transplantation in vivo)
directly into pancreas. In another experiment, the cells are
transplanted subcutaneously, into liver, mammary gland, kidney
capsule, spleen or any other site in which the cells are able to
engraft.
[0383] Another experiment is transplanting undifferentiated or
partially differentiated amnion-derived cells via intravenous
injection to pancreas that has been injured surgically or
chemically then primed or not primed with the factors listed above.
In this experiment the cells will "home" to the inflammatory site
and integrate with the resident cells.
[0384] Another experiment is the use of undifferentiated, partially
differentiated or functionally differentiated amnion-derived cells
transplanted into the pancreas or any other tissue (i.e.
subcutaneously, into liver, mammary gland, kidney capsule, spleen
or any other site in which the cells are able to engraft) (or
introduced by intravenous injection) to induce immune tolerance in
a patient with an autoimmune disease (for example, diabetes).
Synchronized cell differentiation may occur between the
transplanted amnion-derived cells alone and/or with cells in the
patient (i.e. damaged islets, beta cells, etc). The amnion-derived
cells may provide HLA antigens that will protect cells associated
with them from the immune system.
[0385] Explants will be evaluated for human pancreatic islet
progenitor cells. Undifferentiated and partially differentiated
amnion-derived cells will differentiate into cells expressing islet
cell-specific protein markers of differentiation. Analysis will
include immunocytochemistry and GFP expression of pre-labeled
cells.
[0386] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof. Any equivalent embodiments are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims.
[0387] Throughout the specification various publications have been
referred to. It is intended that each publication be incorporated
by reference in its entirety into this specification.
* * * * *