U.S. patent application number 11/353692 was filed with the patent office on 2006-08-17 for method for expansion of stem cells.
Invention is credited to Neil H. Riordan.
Application Number | 20060182724 11/353692 |
Document ID | / |
Family ID | 36916982 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060182724 |
Kind Code |
A1 |
Riordan; Neil H. |
August 17, 2006 |
Method for expansion of stem cells
Abstract
A method of increasing the growth of stem cells by mixing the
stem cells with a growth medium that has been conditioned by an
incubation with placental tissue. The method increases the
expansion of the stem cell population.
Inventors: |
Riordan; Neil H.; (Weston,
FL) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
36916982 |
Appl. No.: |
11/353692 |
Filed: |
February 14, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60653390 |
Feb 15, 2005 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/368; 435/372 |
Current CPC
Class: |
A61K 35/50 20130101;
A61K 8/982 20130101; A61K 35/51 20130101; A61K 35/50 20130101; A61K
35/51 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 35/44 20130101; A61K 38/1825 20130101; A61K 35/44 20130101;
A61K 38/1825 20130101; C12N 2502/02 20130101; A61K 35/28 20130101;
C12N 5/0018 20130101; A61K 35/28 20130101; A61Q 19/08 20130101;
A61Q 7/00 20130101 |
Class at
Publication: |
424/093.7 ;
435/368; 435/372 |
International
Class: |
A61K 35/14 20060101
A61K035/14; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method for the expansion or growth of stem cells without
substantially inducing differentiation, comprising: incubating at
least a portion of a placenta in a growth medium to condition said
medium, and contacting at least one stem cell with said growth
medium.
2. The method of claim 1, wherein said at least one stem cell is
totipotent, capable of differentiating into cells of all
histological types of the body.
3. The method of claim 1, wherein said at least one stem cell is
pluripotent, capable of differentiating into numerous cells of the
body, but not all.
4. The method of claim 1, wherein said at least one stem cell is a
progenitor cell, capable of differentiating into a restricted
tissue type.
5. The method of claim 2, wherein said totipotent stem cell is
selected from the group consisting of: an embryonic stem cell, an
extra-embryonic stem cell, a cloned stem cell, and a
parthenogenesis derived cell.
6. The method of claim 3, wherein said pluripotent stem cell is
selected from the group consisting of a hematopoietic stem cell, an
adipose stem cell, a mesenchymal stem cell, a cord blood stem cell,
a placental stem cell, an exfoliated tooth derived stem cell, a
hair follicle stem cell and a neural stem cell.
7. The method of claim 4, wherein said progenitor stem cell is
selected from the group consisting of neuronal, hepatic,
nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar,
cardiac, intestinal, and endothelial progenitor cells.
8. The method of claim 5, wherein said embryonic stem cell
expresses at least one marker selected from the group consisting
of: stage-specific embryonic antigens (SSEA) 3, SSEA 4, Tra-1-60
and Tra-1-81, Oct-3/4, Cripto, gastrin-releasing peptide (GRP)
receptor, podocalyxin-like protein (PODXL), and human telomerase
reverse transcriptase (hTERT).
9. The method of claim 6, wherein said hematopoietic stem cell
expresses at least one marker selected from the group consisting
of: CD34, c-kit, and the multidrug resistance transport protein
(ABCG2).
10. The method of claim 6, wherein said adipose-derived stem cell
expresses at least one marker selected from the group consisting
of: CD13, CD29, CD44, CD63, CD73, CD90, CD166, Aldehyde
dehydrogenase (ALDH), and ABCG2.
11. The method of claim 6, wherein said mesenchymal stem cell
expresses at least one marker selected from the group consisting
of: STRO-1, CD105, CD54, CD106, HLA-I markers, vimentin, ASMA,
collagen-1, and fibronectin, but not HLA-DR, CD117, and hemopoietic
cell markers.
12. The method of claim 6, wherein said cord blood stem cell
expresses at least one marker selected from the group consisting
of: CD34, c-kit, and CXCR-4.
13. The method of claim 6, wherein said placental stem cell
expresses at least one marker selected from the group consisting
of: Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3,
SH-4, TRA-1-60, TRA-1-81, SSEA-4 and Sox-2.
14. The method of claim 6, wherein said exfoliated deciduous tooth
stem cell expresses at least one marker s elected from the group
consisting of: STRO-1, CD146 (MUC 18), alkaline phosphatase, MEPE,
and bFGF.
15. The method of claim 7, wherein said neural stem cell is
characterized by expression of RC-2, 3CB2, BLB, Sox-2hh, GLAST, Pax
6, nesting, Muashi-1, and prominin.
16. The method of claim 1, wherein said placenta is derived from a
mammal.
17. The method of claim 1, wherein said placenta is derived from a
human.
18. The method of claim 1, wherein said placenta is derived
preterm.
19. The method of claim 1, wherein said placenta is derived at
term.
20. The method of claim 1, wherein said placenta is perfused for a
period of time with a cell culture media.
21. The method of claim 1, wherein said cell culture media is
supplemented with at least one growth factor.
22. The method of claim 21, wherein said at least one growth factor
is selected from the group consisting of: a WNT signaling agonist,
TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11,
IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG,
angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre,
and a mixture thereof.
23. The method of claim 1 wherein said media is capable of
maintaining viability of a substantial portion of the placental
tissue during the perfusion process.
24. The method of claim 1, wherein said media is selected from the
group consisting of Roswell Park Memorial Institute (RPMI-1640),
Dublecco's Modified Essential Media (DMEM), Eagle's Modified
Essential Media (EMEM), Optimem, and Iscove's Media.
25. The method of claim 1, wherein a source of serum is added to
the media.
26. The method of claim 1, wherein the concentration of serum in
the media is approximately between 0.1% to 25%.
27. The method of claim 1, wherein the concentration of serum in
the media is approximately 10%.
28. The method of claim 1, wherein said serum is selected from the
group consisting of: adult human serum, fetal human serum, fetal
calf serum and umbilical cord blood serum.
29. The method of claim 1, wherein said contacting step occurs
after said incubating step.
30. The method of claim 1, wherein said contacting step occurs
simultaneously with said incubating step.
31. The method of claim 1, wherein said incubating step occurs from
about 1 second to about 3 weeks.
32. The method of claim 1, wherein said incubating step occurs from
about 24 hours to about 10 days.
33. The method of claim 1, wherein said contacting step occurs from
about 1 second to about 3 weeks.
34. The method of claim 1, wherein said contacting step occurs from
about 24 hours to about 10 days.
35. The method of claim 1, wherein said placenta is a hemochorial,
epitheliochorial, or endotheliochorial placenta.
36. The method of claim 1, wherein said placenta is a hemochorial
placenta.
37. The method of claim 1, wherein perfusion is accomplished
through the use of a perfusion apparatus cannulated to blood
vessels connected to the placental body.
38. The method of claim 37, wherein said perfusion apparatus allows
for control of intravasular pressure, oxygen content, carbon
dioxide content, pH, and flow rate of the perfused media flowing
through said placental blood vessels.
39. The method of claim 38, wherein the intravasular pressure of
the perfusate is maintained at 30-80 Hg.
40. The method of claim 39, wherein the intravasular pressure of
the perfusate is maintained at 60 Hg.
41. A stem cell with the preserved ability to proliferate but
having a block in differentiation state induced by culturing in
media conditioned by perfusion through a live placenta.
42. The stem cell of claim 41, wherein said stem cell is selected
from the group consisting of: a totipotent stem cell, a pluripotent
stem cell, and a progenitor stem cell.
43. The stem cell of claim 41, said stem cell is maintained in
contact with the conditioned media for a period of 1 second to 3
weeks.
44. The stem cell of claim 41, wherein said stem cell is maintained
in contact with the conditioned media for a period of 1 second to 3
weeks.
45. The stem cell of claim 41, wherein said stem cell is maintained
in contact with the conditioned media for a period of 2 hours to 72
hours.
46. The stem cell of claim 41, wherein said stem cell is maintained
in contact with the conditioned media for a period of 12 hours to
24 hours.
47. The stem cell of claim 41, wherein said stem cell is maintained
in contact with the conditioned media in a living organism
48. The stem cell of claim 41, wherein said contact between the
conditioned media and the stem cell is prolonged by formulating the
conditioned media in a slow release delivery system.
49. The stem cell of claim 41, wherein said stem cells is initially
cultured in contact with said placentally conditioned media for a
period of time, subsequently to which it is cultured in a second
culture with a different concentration of placentally conditioned
media and an identical or variable mix of cytokines.
50. The stem cell of claim 49, wherein said stem cell is initially
cultured for 48 hours in a concentration of 20-100% placentally
conditioned media, whereas in subsequent cultures it is maintained
in a concentration of 0-50% conditioned media.
51. The stem cell of claim 50, wherein said stem cell is maintained
in a cell culture media that is supplemented with at least one
growth factor selected from the group consisting of: WNT signaling
agonist, TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1,
IL-11, IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG,
angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre,
and a mixture thereof.
52. The stem cell of claim 51, wherein said stem cell is maintained
in a 50% by volume placentally conditioned DMEM media with the
following growth factors also in DMEM media: IL-3 (about 20 ng/ml),
IL-6 (about 250 ng/ml), SCF (about 10 ng/ml), TPO (about 250
ng/ml), flt-3L (about 100 ng/ml).
53. The stem cell of claim 52, wherein said stem cell is maintained
in the presence of an agent selected from one or more of the
following: an inhibitor of GSK-3, an inhibitor of histone
deacetylase activity, and an inhibitor of DNA methyltransferase
activity.
54. The stem cells of claim 52, wherein said stem cell is
rejuvenated by at least one procedure selected from the group
consisting of: fusion with a more primitive stem cell, transfer of
cytoplasm from a more primitive stem cells, and transfer of
karyoplastic extracts from a more primitive stem cell.
55. A method of treating degenerative diseases through
administration of a composition of matter derived from media
conditioned by a live placenta.
56. The method of claim 55, wherein said degenerative disease
effects a tissue selected from the group consisting of: smooth
muscle tissue, striated muscle tissue, cardiac muscle tissue, bone
tissue, bone spongy tissue, nervous system tissue, cartilage
tissue, pancreatic ductal tissue, spleen tissue, thymus tissue,
tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid
tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart
tissue, lung tissue, vascular tissue, endothelial tissue, blood
cells, bladder tissue, kidney tissue, digestive tract tissue,
esophagus tissue, stomach tissue, small intestine tissue, large
intestine tissue, adipose tissue, uterus tissue, eye tissue, lung
tissue, testicular tissue, ovarian tissue, prostate tissue,
connective tissue, endocrine tissue, and mesentery tissue.
57. The method of claim 55, wherein said placenta conditioned media
is administered in combination with an agent capable of inducing
stem cell expansion.
58. The method of claim 55, wherein said placenta conditioned media
is administered in combination with an agent capable of inducing
stem cell differentiation into cells of the tissue in need of
repair.
59. The method of claim 57, wherein said agent capable of inducing
stem cell expansion is selected from the group consisting of: TPO,
SCF, IL-1, IL-3, IL-7, flt-3L, G-CSF, GM-CSF, Epo, FGF-1, FGF-2,
FGF-4, FGF-20, VEGF, activin-A, IGF, EGF, NGF, LIF, PDGF, and a
member of the bone morphogenic protein family.
60. The method of claim 58, wherein said agent capable of inducing
stem cell differentiation is selected from the group consisting of:
HGF, cardiotrophin, BDNF, VEGF, FGF1, FGF2, FGF4, and FGF 20.
61. The method of claim 55, wherein said placental conditioned
media is concentrated to a sufficient extent to allow systemic
administration while retaining biological effects.
62. The method of claim 61, wherein said placental conditioned
media is calibrated for specific Units of Activity based on a
desired biological property.
63. The method of claim 62, wherein said biological activity is the
ability to stimulate proliferation of a defined culture of CD34
stem cells by 50%.
64. The method of claim 61, wherein placentally conditioned media
is administered according to biomarkers of stem cell activity in
the patient in need of treatment.
65. The method of claim 62, wherein said biomarker may be either an
indicator of disease activity, or an indicator of stem cell
regeneration.
66. The method of claim 61, wherein a clinically applicable agent
that possesses stem cell mobilizing activity is administered in
conjunction with the placentally conditioned media and/or the stem
cell proliferation inducing growth factor, and/or the inducer of
stem cell differentation.
67. The method of claim 66, wherein said stem cell mobilizing agent
may be an antibody, a small molecule, or a protein.
68. The method of claim 67, wherein said stem cell mobilizing agent
is an antibody to CXCR-4.
69. The method of claim 67, wherein said stem cell mobilizing agent
is either a small molecular inhibitor of CXCR-4, or a statin.
70. The method of claim 67, wherein said stem cell mobilizing agent
is either a cytotoxic chemotherapy known to mobilize stem cells, or
a growth factor such as G-CSF.
71. The method of claim 61, wherein a dedifferentiation agent is
used for expanding the differentiation potential of said stem
cells.
72. The method of claim 71, wherein said dedifferentiation agent is
either an inhibitor of the enzyme GSK-3, and inhibitor of the
histone deacetylase family of enzymes, or an inhibitor of DNA
methyltransferase activity.
73. The method of claim 72, wherein said dedifferentiation agents
may be trichostatin A, valproic acid, buphenyl, or
5-azacytidine.
74. A method of treating degenerative diseases, comprising
administering a differentiating agent to selectively expand a
population of pluripotent or progenitor cells, while concurrently
administering live placental conditioned media in order to induce
proliferation of the committed stem cell.
75. A method of expanding stem cells that have been therapeutically
reprogrammed, comprising contacting said cells with media that has
been conditioned by a live placenta.
76. The method of claim 75 wherein said therapeutic reprogramming
is accomplished by introduction into the target cell to be
reprogrammed agents capable of acting at the epigenetic level to
modify the cellular transcriptosome into a desired phenotype.
77. The method of claim 76, whereby said target cell is fused with
another cell of a more primitive state of differentiation.
78. The method of claim 75, whereby said cell is temporarily
permeabilized and cytoplasmic and/or karyoplasmic extracts are
introduced into said cell from another cell of a more primitive
state of differentiation.
79. A method of accelerating hematopoietic recovery in a patient in
need thereof, comprising administering placentally conditioned
media.
80. The method of claim 79, wherein said patient has been treated
with chemotherapy, and/or radiotherapy with the scope of either
ablating or diminishing the immune system.
81. The method of claim 79, wherein said patient has been treated
with chemotherapy, and/or radiotherapy with the scope of
eradicating or ameliorating a malignancy.
82. The method claim 79, wherein said patient has been induced into
a state of reduced hematopoiesis as a result of chemical or
radiation poisoning.
83. The method of claim 79, wherein said patient is not
administered a cellular graft to enhance recovery of the
hematopoietic system.
84. The method of claim 79, wherein said patient was administered
either cord blood derived, peripheral blood derived, or bone marrow
derived hematopoietic stem cells or progenitors thereof.
85. The method of claim 79, wherein said patient is administered
placentally conditioned media intravenously at a concentration
sufficient to accelerate recovery of early hematopoietic
progenitors.
86. The method of claim 79, wherein said patient is administered
placentally conditioned media at a concentration of 10-500 Units of
placentally conditioned media per kilogram per day, said Units
based on a logarithmic scale in which 1 Unit is sufficient to
stimulated proliferation of a defined cell culture of CD34+ cells
by 100% compared to control media.
87. The cell culture of claim 86, wherein 1 Unit is defined on a
logarithmic scale as the amount of placentally conditioned media
needed to stimulate proliferation of a 200 .mu.L culture of
5.times.10.sup.3 human cord blood isolated CD34+.
88. The method of claim 79, wherein said patient is treated
intravenously, or through other means, with placental conditioned
media for a period of time needed to obtain a granulocyte count of
500/mm.sup.3.
89. The method of claim 88, wherein said patient is treated
intravenously, or through other means, with placental conditioned
media for a period of time between 7 days to 15 days.
90. The method of claim 79, wherein a growth factor is concurrently
given with the administration of placentally conditioned media.
91. The method of claim 90, wherein said growth factor is selected
from the group consisting of G-CSF, pegylated G-CSF, TPO, IL-11,
GM-CSF, and flt-3L.
92. A method of treating patient with tissue ischemia through
induction of endothelial stem cell expansion using placentally
conditioned media.
93. The method of claim 92, wherein said ischemia is present in at
least one tissue selected from the group consisting of: smooth
muscle tissue, striated muscle tissue, cardiac muscle tissue, bone
tissue, bone spongy tissue, nervous system tissue, cartilage
tissue, pancreatic ductal tissue, spleen tissue, thymus tissue,
tonsil tissue, Peyer's patch tissue, lymph nodes tissue, thyroid
tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart
tissue, lung tissue, vascular tissue, endothelial tissue, blood
cells, bladder tissue, kidney tissue, digestive tract tissue,
esophagus tissue, stomach tissue, small intestine tissue, large
intestine tissue, adipose tissue, uterus tissue, eye tissue, lung
tissue, testicular tissue, ovarian tissue, prostate tissue,
connective tissue, endocrine tissue, and mesentery tissue.
94. The method of claim 93, wherein said ischemia is presenting as
advanced angina.
95. The method of claim 94, wherein placentally conditioned media
is concentrated and administered into the ischemic myocardium using
the minithoracotomy procedure.
96. The method of claim 94, wherein placentally conditioned media
is concentrated and administered into the ischemic myocardium using
the NOGA electromagnetic mapping and injection system.
97. The method of claim 94, wherein placentally conditioned media
is concentrated and administered into the ischemic myocardial area
using a balloon catheter.
98. The method of claim 94, wherein a secondary agent is added that
is capable of inducing proliferation of differentiated and
undifferentiated endothelial cells.
99. The method of claim 98, wherein said secondary agent is either
a nucleic acid, a protein, or a small molecule.
100. The method of claim 99, wherein said secondary agent is
plasmid DNA encoding a polypeptide selected from the group
consisting of: HIF-1, VEGF, FGF1, FGF2, FGF4, FGF20, and
angiopoietin.
101. The method of claim 99, wherein said secondary agent is
selected from the group consisting of: VEGF, FGF1, FGF2, FGF4,
FGF20, and angiopoietin.
102. The method of claim 94, wherein an exogenous source of stem
cells are delivered into the ischemic area.
103. The method of claim 94, wherein an endogenous source of stem
cells are delivered into the ischemic area.
104. The method of claim 102, wherein exogenous stem cells may be
autologous or allogenenic mesenchymal, adipose, endothelial, bone
marrow, mobilized peripheral blood, umbilical, or artificially
reprogrammed stem cells.
105. The method of claim 103, wherein endogenous stem cells are
mobilized with a mobilization agent.
106. The method of claim 92, wherein said patient suffering from
ischemia is a victim of Critical Limb Ischemia.
107. The method of claim 92, wherein said patient is administered a
combination of placentally conditioned media intramuscularly in the
area of ischemia as detected by angiography.
108. The method of claim 107, wherein autologous or allogenenic
mesenchymal, adipose, endothelial, bone marrow, mobilized
peripheral blood, umbilical, or artificially reprogrammed stem
cells are injected with the placentally conditioned media in a
localized environment intramuscularly.
109. The method of claim 108, wherein autologous lymphocytes are
injected with said stem cell source in order to synergize with the
placentally conditioned media and the injected stem cells.
110. The method of claim 92, wherein said patient has suffered from
a cerebral ischemia.
111. The method of claim 110, wherein said patient is treated
immediately after the ischemia episode or in a period of time
subsequently.
112. A method of culturing a placenta in its original 3-dimensional
structure in such a manner to reproduce the in vivo environment in
which it resides in the pregnant women thus retaining capability of
generation and secretion of growth factors and proteins that
maintain the fetal regenerative capacity, said method comprising
the steps of: acquiring a placenta under sterile conditions;
cannulating blood vessels of the placenta in order to allow proper
perfusion in circumstances similar to as if the placenta was
performing its in vivo functions; perfusing said placenta with a
nutrient mix, in a buffer that would mimic physiological
conditions; maintaining a temperature and physical environment
similar to that found in the pregnant woman's body; and imitating
conditions of flow, pH, oxygenation, and pressure similar to that
found in the body.
113. The method of claim 112, wherein perfusion of both the
maternal and fetal circulatory components of the placenta is
performed.
114. The method of claim 112, wherein a nutrient mixture is used
that possesses similar nutrient requirements as the fetal and
maternal circulation, respectively.
115. The method of claim 112, wherein a temperature of 37.degree.
C. is maintained during the perfusion process.
116. The method of claim 112, wherein pH is monitored by the
perfusion apparatus in a real-time basis, and adjusted using
adequate quantities of acids, bases, or buffers.
117. The method of claim 112, wherein oxygen content is maintained
similar to that found in the fetal and maternal circulatory
contribution to the placenta.
118. The method of claim 117, wherein oxygen content may be
increased through the use of adding natural or artificial oxygen
carriers to the perfusion solution.
119. The method of claim 112, wherein an oxygenator may be attached
to the perfusion apparatus, in conjunction with, or separately,
from an oxygen sensor, said combination being used to adjust in
real-time oxygen content.
120. The method of claim 112, wherein osmolality is maintained
through the use of known means such as addition of albumin or
colloids to the perfusion solution.
121. A method of producing a cosmetic for topical use in
rejuvenating aged skin comprising the steps of: concentrating
placentally conditioned media; quantifying and standardizing
biological effect of said media, and formulating said media in a
carrier solution that is suitable for transdermal delivery.
122. The method of claim 121, wherein said media is selected from
the group consisting of: a physiological buffer, a media capable of
maintaining cellular viability, and a media enriched in nutrients
and mimicking the content of the maternal/fetal circulation.
123. The method of claim 122, wherein said media is selected from
the group consisting of DMEM, RPMI, and saline USP.
124. The method of claim 123, wherein said media is DMEM.
125. The method of claim 122, wherein said media contains an
anticoagulant at sufficient quantities to inhibit clotting during
placental perfusion.
126. The method of claim 121, wherein said quantification is based
on ability of placentally conditioned media to induce proliferation
of dermal stem cells.
127. The method of claim 121, wherein a moisturizing agent is added
to the cosmetic preparation.
128. The method of claim 121, wherein said carrier further
comprises at least one nutrient to replenish the skin.
129. The method of claim 121, wherein said carrier contains at
least one anti-oxidant compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. Provisional Application No. 60/653,390, which was filed on
Feb. 15, 2005, the disclosure of which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the field of stem cell technology.
More particularly, the invention describes a new method for
increasing the growth of stem cells by mixing the stem cell culture
with a medium that has been incubated with placental tissue.
BACKGROUND OF THE INVENTION
[0003] Stem cells have the ability to divide for indefinite periods
in culture and to give rise to specialized cells. Typically, stem
cells are divided into two main groups: adult stem cells and
embryonic stem cells. Stem cells may also be generated through
artificial means such as nuclear transfer, cytoplasmic transfer,
cell fusion, parthenogenesis and reprogramming. Isolated stem cells
can give rise to many types of differentiated cells, and can be
used to treat many types of diseases.
[0004] Adult stem cells are undifferentiated but are present in
differentiated tissues, and are capable of differentiation into the
cell types from the tissue that the adult stem cell originated.
Adult stem cells have been derived from various sources, such as
the nervous system (McKay, 1997, Science 276:66-71; Shihabuddin, et
al., 1999, Mol. Med Today 5:474-480); bone marrow (Pittenger, et
al., 1999, Science 284:143-147; Pittenger, M. F. and Marshak, D. R.
(2001) In: Mesenchymal stem cells of human adult bone marrow.
Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds. (Cold Spring
Harbor, N.Y.: Cold Spring Harbor Laboratory Press) 349-374);
adipose tissue (Gronthos, et al., 2001, J. Cell. Physiol.
189:54-63), dermis (Toma, et al., 2001, Nature Cell Biol.
3:778-784); pancreas and liver (Deutsch, et al., 2001, Development
128:871-881). Stem cells have also been isolated from umbilical
cord (Rogers, et al., 2004, Best Pract Res Clin Obstet Gynaecol.
18(6):893-908; Wang et al., 2004, Stem Cells 22(7):1330-1337;
Surbek, et al, 2002, Ther Umsch. 59(11):577-582; and placenta (Yen
et al., 2005, Stem Cells 23(1):3-9), each of which is incorporated
by reference herein in its entirety. It is believed that stem cells
of the adult type are also found in smooth muscle tissue, striated
muscle tissue, cardiac muscle tissue, bone tissue, bone spongy
tissue, cartilage tissue, pancreatic ductal tissue, spleen tissue,
thymus tissue, tonsil tissue, Peyer's patch tissue, lymph nodes
tissue, thyroid tissue, epidermis tissue, dermis tissue,
subcutaneous tissue, heart tissue, lung tissue, vascular tissue,
endothelial tissue, blood cells, bladder tissue, kidney tissue,
digestive tract tissue, esophagus tissue, stomach tissue, small
intestine tissue, large intestine tissue, adipose tissue, uterus
tissue, eye tissue, lung tissue, testicular tissue, ovarian tissue,
prostate tissue, connective tissue, endocrine tissue, and mesentery
tissue.
[0005] Several patents disclose various aspects of adult stem
cells. For example, U.S. Pat. No. 5,556,783 discloses methods of
culturing hair follicle stem cells, while U.S. Pat. No. 5,486,359
discloses methods of isolating human mesenchymal stem cells. U.S.
Pat. Nos. 4,714,680, 5,061,620, and 5,087,570 provide examples of
hematopoietic stem cells. Each of these patents is incorporated by
reference herein in its entirety.
[0006] Embryonic stem cells are undifferentiated cells derived from
the embryo. Typically these cells are extracted from the inner cell
mass of a blastocyte and when cultured under the unique conditions,
either alone or in combination with a variety of feeder cells, the
embryonic stem cells maintain euploid karyotype, do not undergo
senescence, and retain the ability to differentiated into cells of
the endodermal, ectodermal, and mesodermal lineages.
[0007] These cells have the potential to become a wide variety of
specialized cell and tissue types, which can then be used for basic
research, drug discovery, and treatment (or prevention) of many
types of diseases. Patents describing aspects of embryonic stem
cells include U.S. Pat. No. 6,506,574 to Rambhatla, U.S. Pat. No.
6,200,806 to Thomson, U.S. Pat. No. 6,432,711 to Dinsmore, and U.S.
Pat. No. 5,670,372 to Hogan, each of which is incorporated by
reference herein in its entirety. Importantly, murine embryonic
stem cells can be cultured indefinitely under the presence of
leukemia inhibitory factor (LIF), which maintains their
undifferentiated state. In contrast, human embryonic stem cells are
not responsive in the same manner to LIF, thus stimulating the
invention of numerous methodologies to expand them. Unfortunately,
many such methodologies involve the use of either murine feeder
cells or other animal components, hence limiting the therapeutic
potential of these cells. Furthermore, even when established cell
lines, such as the federally approved embryonic stem cells, are
cultured in murine-free conditions, contamination is still present
as recently reported (Martin, et al., 2005, Nat Med 11:228-232,
which is incorporated by reference herein in its entirety).
Accordingly, one object of the invention disclosed is to provide
novel methods of expanding stem cells in absence of animal
components, said invention being applicable to a variety of stem
cells, including embryonic stem cells.
[0008] The importance of technologies associated with expansion of
stem cells, both of adult and/or embryonic derivation is
illustrated by the numerous preclinical and clinical uses of these
cells in treatment of a wide range of diseases.
[0009] One of the earliest clinical uses of stem cells was for
performing bone marrow transplants in patients with hematological
malignancies in which hematopoietic stem cells derived from the
donor bone marrow were administered into the recipient subsequent
to providing said recipient with a sufficient dose of radiation
and/or chemotherapy in order to ablate not only the hematological
malignancy but also non-malignant hematopoiesis. The administration
of, non-malignant hematopoietic stem cells resulted in
donor-specific hematopoiesis and in some patients, cure of the
malignancy. This was first described by Thomas et al in 1957, who
reported that large volumes of donor bone marrow could be safely
infused in patients with acute leukemia following myeloablation and
that donor-specific hematopoiesis was established (Thomas, et al.,
1957, N Engl J Med 257:491-496, which is incorporated by reference
herein in its entirety). The identification of similar
hematopoietic stem cell activity in the peripheral blood led to
development of techniques used to mobilize and harvest peripheral
blood hematopoietic stem cells for use in transplantation settings.
For example, the use of GM-CSF and G-CSF in enhancing the number of
peripheral blood hematopoietic stem cells was reported in the
clinical situation of autologous transplantation subsequent to high
dose chemotherapy (Peters, et al., 1993, Blood 81:1709-1719;
Sheridan, et al., 1992, Lancet 339:640-644, each of which is
incorporated by reference herein in its entirety).
[0010] In addition to treatment of hematological malignancies, stem
cells have been utilized in the context of therapy for solid
tumors. The dose limiting variable in cancer chemotherapy is bone
marrow toxicity. Accordingly, in 1958, Kurnick et al performed an
autologous bone marrow transplant to demonstrate ability of infused
bone marrow to allow use of very high doses of chemotherapy and/or
radiation therapy (Kurnick, et al., 1958, Ann Intern Med
49:973-986, which is incorporated by reference herein in its
entirety). The use of autologous hematopoietic cell transplants
combined with high dose chemo/radiotherapy for solid tumors has
been extensively investigated for breast (Peppercorn, et al., 2005,
Cancer 104:1580-1589; Dillman, et al., 2005, Am J Clin Oncol
28:281-288), colon (Leff, et al., 1986, J Clin Oncol 4:1586-1591;
Franchi, et al., 1994, Eur J Cancer 30A: 1420-1423), lung (Ziske,
et al., 2002, Anticancer Res 22:3723-3726), nasopharyngeal cancer
(Chen, et al., 2003, Jpn J Clin Oncol 33:331-335), and other types
of cancers (Gratwohl, et al., 2004, Ann Oncol 15:653-660), each of
which is incorporated by reference herein in its entirety.
[0011] The identification of the type 1 transmembrane
protein/adhesion molecule, the sialomucin CD34 as a marker of
hematopoietic stem cells led to the use of CD34+ cell selection as
a means of concentrating hematopoietic stem cell activity (Civin,
et al., 1984, J Immunol 133:157-165, which is incorporated by
reference herein in its entirety). Specifically, it was
demonstrated that although bone marrow mononuclear cells contain
approximately 1-4% CD34+ cells, the administration of these cells,
but not bone marrow depleted of CD34+cells, into lethally
irradiated baboons led to hematopoietic reconstitution (Berenson,
et al., 1988, J Clin Invest 81:951-955, which is incorporated by
reference herein in its entirety). Clinical development of purified
CD34+ cells as a source of stem cells was originally sought as a
method of performing bone marrow transplant without contamination
of donor T cells. This would in theory stop development of graft
versus host disease, one of the main causes of allogeneic
transplant associated morbidity and mortality (Ferrara, et al.,
2005, Clin Adv Hematol Oncol 3:415-419, 428, which is incorporated
by reference herein in its entirety). Unfortunately, clinical
evidence demonstrated that patients receiving purified CD34+ stem
cell grafts, although having a lower incidence of graft versus host
disease, also had a higher incidence in leukemic relapse due to an
immunologically mediated graft versus leukemia effect that is
absent when donor bone marrow grafts are depleted of T cells
(Martino, et al., 2000, Haematologica 85:1165-1171; Butt, et al.,
2003, Leuk Lymphoma 44:1509-1513, each of which is incorporated by
reference herein in its entirety). The critical importance of bone
marrow derived T cells in the induction and upkeep of graft versus
leukemia effects was illustrated in studies of leukemic patients
who have relapsed and were subsequently treated by infusion of
donor T cells. This induced a long-term remission in the patients
that had major relapse (Kolb, H. J., 1998, Vox Sang 74 Suppl
2:321-329; Guglielmi, et al., 2002, Blood 100:397-405, each of
which is incorporated by reference herein in its entirety).
Furthermore, it was also observed that under some conditions, bone
marrow derived non-CD34 cells of the osteoblast lineage have a role
in facilitating engraftment in allogeneic settings (Good, R. A.,
2000, World J Surg 24:797-810, which is incorporated by reference
herein in its entirety). Despite these potential drawbacks,
clinical use of CD34+ cells both from mobilized peripheral blood,
as well as bone marrow, during autologous transplantation for high
dose chemo/radiation therapy was considered to be a useful approach
(Korbling, et al., 2001, Blood 98:2900-2908; Pecora, et al., 2001,
Bone Marrow Transplant 27:1245-1253; Pecora, A. L., 1999, Bone
Marrow Transplant 23 Suppl 2:S7-12, each of which is incorporated
by reference herein in its entirety). This is due to the fact that
in this setting, neither facilitator cells are needed, since the
graft is autologous, and the CD34+ selection substantially clears
the marrow of contaminating tumor cells, so that the risk of tumor
relapse is lessened as opposed to using non-purified bone marrow
(Preti, et al., 2001, Cytotherapy 3:85-95; Siena, et al., 2000, J
Clin Oncol 18:1360-1377; Vannucchi, et al., 1998, Br J Haematol
103:610-617, each of which is incorporated by reference herein in
its entirety).
[0012] The use of hematopoietic stem cells has also been described
for "reprogramming" the immune system to induce an antigen-specific
state of non-responsiveness called tolerance. Specifically, this
use can be divided into two main areas: the use of stem cells to
induce donor-specific tolerance during allogeneic or xenogeneic
transplantation, and the use of stem cells to induce tolerance in
situations of autoimmunity. Although common mechanisms of tolerance
maintenance such as generation of T regulatory cells, effector T
cell depletion, and effector T cell anergy have been described in
both types of tolerance, the mechanism of induction seems to be
different; therefore we will describe them individually.
[0013] The possibility of bone marrow hematopoietic stem cells
having the utility of inducing tolerance to a grafted organ was
first elaborated on by Owens in the 1940s. In studies demonstrating
that in utero mixing of blood in the context of shared circulation
between two genetically different cows, he observed bilateral
transplantation tolerance in adulthood. Accordingly, he postulated
that the original sharing of circulation may have contributed to
the state of tolerance which theoretically should not have existed
due to the genetic disparity between the siblings. Furthermore,
definitive roles for using stem cells to induce tolerance came from
Billingham and Medawar in the 1950s in experiments showing
injection of donor bone marrow cells into neonates allowed for
tolerance to the donor antigen when the animal reached adulthood
(Slavin, S., 2002, Int J Hematol 76 Suppl 1:172-175, which is
incorporated by reference herein in its entirety). In animal models
it has been demonstrated that bone marrow cells contribute to
generation of a donor-specific tolerogenic state which is
associated with chimeric hematopoiesis. The combination of
donor-specific bone marrow transplant, with solid organs, has been
used in some clinical situations to induce complete tolerance to
the grafted organ without the need for chronic, continuous immune
suppression (George, et al., 2002, Immunol Res 26:119-129, which is
incorporated by reference herein in its entirety). Unfortunately,
wide spread use of bone marrow induced tolerance is limited by the
fact that bone marrow transplantation is associated with a high
degree of morbidity and mortality during the myeloablative phase.
In addition, the possibility of graft versus host disease is
another pitfall to the full-scale implementation. In order to
overcome this, several methods of inducing partial chimerism, or
mini-chimerism are being investigated through the use of
non-myeloid ablative techniques such as donor-specific transfusions
combined with anti CD154 antibodies (Seung, et al., 2003, J Clin
Invest 112:795-808, which is incorporated by reference herein in
its entirety). Induction of organ tolerance by hematopoietic stem
cells is believed to occur through both thymic dependent (Noris, et
al., 2001, J Am Soc Nephrol 12:2815-2826, which is incorporated by
reference herein in its entirety), and independent (van Pel, et
al., 2003, Transpl Immunol 11:375-384, which is incorporated by
reference herein in its entirety) mechanisms. Specifically, donor
hematopoietic cells generate a variety of both lymphoid and
non-lymphoid cells that express the same antigens found in the
donor organ, but somehow redirect the immune system not to attack
these specific antigens, while maintaining responses against other
antigens not related to the graft. One mechanism that is postulated
to occur is the thymic stromal tissue in the recipient becomes
populated with donor-derived cells. These cells then act at the
level of negative selection in order to induce apoptosis of T cells
reactive to the donor antigen in a similar way to which the immune
system deletes autoreactive T cells during thymic selection
(Shizuru, et al., 2000, Proc Natl Acad Sci U S A 97:9555-9560,
which is incorporated by reference herein in its entirety). Another
mechanism of tolerance involves the persistent presentation of
donor antigen in absence of costimulatory molecules. This was
demonstrated in one situation by the fact that persistence of T
cells from the donor bone marrow is essential in maintaining
tolerance (Xu, et al., 2004, J Immunol 172:1463-1471, which is
incorporated by reference herein in its entirety). The mesenchymal
component of the bone marrow produces a cell population that
consitutively secretes immune inhibitory factors such as IL-10 and
TGF-b while presenting antigens (Liu, et al., 2004, Transplant Proc
36:3272-3275; Togel, et al., 2005, Am J Physiol Renal Physiol
289:F31-42, each of which is incorporated by reference herein in
its entirety). This is believed to further inhibit immunity in an
antigen specific manner. During T cell activation, two general
signals are required for the T cell in order to mount a productive
immune response, the first signal is recognition of antigen, and
the second is recognition of costimulatory or coinhibitory signals.
Mesenchymal cells present antigens to T cells but provide a
coinhibitory signal, thus specifically inhibiting T cells that
recognize them, and other cells expressing similar antigens.
Finally, the fact that CD34+ cells express the T cell killing
molecule FasL has been postulated as another mechanism of
tolerogenesis. Indeed transplantation of bone marrow from mice with
a mutated FasL did not induce tolerogenesis in recipients (George,
et al., 1998, Nat Med 4:333-335, which is incorporated by reference
herein in its entirety).
[0014] The potential of using hematopoietic cell transplantation
for autoimmunity derives from the belief that the immune system can
be deleted and recapitulated, but in such a manner to "reset the
clock" so that autoreactive T cells will not re-appear (Muraro, et
al., Renewing the T cell repertoire to arrest autoimmune
aggression. Trends Immunol., e-published on Jan. 4, 2006, which is
incorporated by reference herein in its entirety). Specifically, it
is known that the process of autoimmunity requires the failure of
several self-tolerance mechanisms before clinical presentation
appears. These include: a) self-reactive T cell deletion in the
thymus; b) anergy/deletion of self reactive T cells in the
periphery; c) failure of the regulatory T cell activity; and d) the
presence of inflammation or antigen release in order to allow
expansion of the autoreactive T cell clone. During autoimmunity the
failure of all of these systems is usually a culmination of
environmental and genetic factors occurring over a protracted
period of time. Accordingly if the immune system could be made to
"start anew" the normal tolerogenic processes would again be
reactivated and the disease would be cured, at least temporarily.
To date clinical use of autologous stem cells has been performed
for a variety of autoimmune indications, including rheumatoid
arthritis (Jantunen, et al., 1999, Scand J Rheumatol 28:69-74,
which is incorporated by reference herein in its entirety),
multiple sclerosis (Karussis, et al., 2004, J Neurol Sci 223:59-64;
Brodsky, et al., 1999, Curr Opin Oncol 11:83-86, each of which is
incorporated by reference herein in its entirety), systemic lupus
erythromatosis (Brunner, et al., 2002, Arthritis Rheum
46:1580-1584; Burt, et al., 2006, Jama 295:527-535, each of which
is incorporated by reference herein in its entirety), and systemic
sclerosis (Viganego, et al., 2000, Curr Rheumatol Rep 2:492-500,
which is incorporated by reference herein in its entirety).
According to a report in 2005, approximately 700 patients in total
have received an autologous stem cells for autoimmune diseases with
a positive benefit/risk ratio that has led to initiation of phase
III prospective randomized controlled trials (Tyndall, et al.,
2005, Clin Exp Immunol 141:1-9, which is incorporated by reference
herein in its entirety).
[0015] Induction of tolerance through hematopoietic stem cell
transplantation, either from bone marrow or peripheral blood
sources possesses the intrinsic danger of bone marrow failure
during ablation of the recipient immune system. Although
non-myeloablative protocols are under development, even these carry
the risk of immune suppression due to the lymphoablation.
Accordingly there is a need in the art to develop novel methods of
either expanding hematopoietic stem cells ex vivo in large enough
quantities to guarantee graft take, as well as methods of in vivo
expanding the stem cells and their progeny so that the period under
which the transplant recipient is immunosuppressed is
minimized.
[0016] Stem cell therapy has also been performed in the context of
administration of mesenchymal stem cells, without the hematopoietic
component, for induction of tolerance. It was demonstrated in a
murine model that flk-1+Sca-1-mesenchymal cell transplantation
leads to permanent donor-specific immunotolerance in allogeneic
host and results in long-term allogeneic skin graft acceptance
(Deng, et al., 2004, Exp Hematol 32:861-867, which is incorporated
by reference herein in its entirety). Other studies have shown that
mesenchymal stem cells are inherently immunosuppressive through
production of PGE-2, interleukin-10 and expression of the
tryptophan catabolizing enzyme indoleamine 2,3,-dioxygenase as well
as Galectin-1 (Kadri, et al., 2005, Stem Cells Dev 14:204-212;
Ryan, et al., 2005, J Inflamm (Lond) 2:8, each of which is
incorporated by reference herein in its entirety). These stem cells
also have the ability to non-specifically modulate the immune
response through the suppression of dendritic cell maturation and
antigen presenting abilities (Beyth, et al., 2005, Blood
105:2214-2219; Aggarwal, et al., 2005, Blood 105:1815-1822, each of
which is incorporated by reference herein in its entirety).
Functional induction of allogeneic T cell apoptosis was also
demonstrated using freshly isolated, irradiated, or long-term
cultured mesenchymal stem cells (Plumas, et al., 2005, Leukemia
19:1597-1604, which is incorporated by reference herein in its
entirety). Others have also demonstrated that mesenchymal stem
cells have the ability to preferentially induce expansion of
antigen specific T regulatory cells with the CD4+ CD25+ phenotype
(Maccario, et al., 2005, Haematologica 90:516-525, which is
incorporated by reference herein in its entirety). Supporting the
potential clinical utility of such cells, it was previously
demonstrated that administration of mesenchymal stem cells inhibits
antigen specific T cell responses in the murine model of multiple
sclerosis, experimental autoimmune encephalomyelitis, leading to
prevention and/or regression of pathology (Zappia, et al., 2005,
Blood 106:1755-1761, which is incorporated by reference herein in
its entirety). Safety of infusing mesenchymal stem cells was
illustrated in studies administering 1-2.2.times.10.sup.6 cells/kg
in order to enhance engraftment of autologous bone marrow cell. No
adverse events were associated with infusion, although level of
engraftment remained to be analyzed in randomized trials (Koc, et
al., 2000, J Clin Oncol 18:307-316, which is incorporated by
reference herein in its entirety). In a matched pair analysis
study, it was demonstrated that in vitro expanded mesenchymal stem
cells reduced both acute and chronic graft versus host disease in
the allogeneic bone marrow transplant setting. Clinical
administration of mesenchymal stem cells was reported in a patient
suffering severe, grade IV graft versus host disease in the liver
and gut subsequent to bone marrow transplant. Administration of
2.times.10.sup.6 cells/kg on day 73 after bone marrow transplant
lead to a long term remission of graft versus host disease, which
was maintained at the time of publication, 1 year subsequent to
administration of the mesenchymal stem cells (Le Blanc, et al.,
2004, Lancet 363:1439-1441, which is incorporated by reference
herein in its entirety). A feasibility study in 46 patients
receiving mesenchymal cells prior to transplant revealed a
favorable safety profile and is encouraging further dose finding
studies (Lazarus, et al., 2005, Biol Blood Marrow Transplant
11:389-398, which is incorporated by reference herein in its
entirety). Unfortunately, mesenchymal cell expansion is relatively
slow and in many situations is not practical for widespread
clinical use. The development of novel methods of expanding stem
cell populations, as for example the methods thought in the present
invention, are likely to increase use of this therapeutically
promising cell population.
[0017] There is evidence that embryonic stem cells are also capable
of inducing immunological tolerance. Indeed, coculture of
alloreactive T cells with embryonic T cells demonstrated an
antigen-specific inhibitory effect (Li, et al., 2004, Stem Cells
22:448-456, which is incorporated by reference herein in its
entirety). Data is still preliminary in this area, and the problem
of embryonic stem cells inducing teratomas currently precludes
their use for this indication. An alternative method of immune
modulation using embryonic stem cells is the generation of defined
immunological cells that can be used directly, or tailored to
possess specific desired properties through modification of culture
conditions or gene manipulation. For example, it was demonstrated
that the murine model of multiple sclerosis, experimental
autoimmune encephalomyelitis can be successfully treated with
dendritic cells generated from embryonic stem cell cultures that
have been manipulated to present the MOG autoantigen in the
presence of TRAIL, a molecule known to induce T cell apoptosis
(Hirata, et al., 2005, J Immunol 174:1888-1897, which is
incorporated by reference herein in its entirety). Generation of
such tailor-made immunological cells would greatly expand the
clinical armamentarium of immunotherapy, however, this is limited
by the currently lack of methodologies for expanding stem cells in
a GMP/GTP compliant and feasible manner.
[0018] One of the main therapeutic uses for stem cells is in the
area of regenerative medicine. The concept of regenerative medicine
is to restore or enhance the ability of tissues to self-organize
and heal themselves following endogenous or exogenous injury.
Although examples of the use of stem cells for tissue regeneration
are almost limitless, several are overviewed below. This should not
be taken as an exhaustive literature review, but rather a general
discussion for example purposes in order to stimulate one skilled
in the art to further investigate this field.
[0019] Bone marrow stem cells have been extensively investigated
for repair of myocardial tissue subsequent to infarction. Early
studies by Orlic demonstrated that administration of GFP c-kit +,
lineage -, bone marrow into ligation induced myocardial infarct
area resulted in regeneration of myocardial and endothelial tissue
by the donor cells (Orlic, et al., 2001, Nature 410:701-705, which
is incorporated by reference herein in its entirety). Subsequent
studies have used mesenchymal bone marrow cells treated with the
DNA methyltransferase inhibitor 5-aza-cytidine to not only
transdifferentiate into myocardial tissue, but also to improve left
ventricular ejection fraction and inhibit cardiac remodeling
(Tomita, et al., 1999. Circulation 100:II247-256, which is
incorporated by reference herein in its entirety). Importantly,
similar experiments were performed in porcine models of infarction,
also indicating improvement in cardiac function (Tomita, et al.,
2002, J Thorac Cardiovasc Surg 123:1132-1140, which is incorporated
by reference herein in its entirety). Accordingly, clinical
experiments were performed administering autologous bone marrow
cells directly into the myocardium during coronary bypass grafting.
In a series of experiments initiated in 1999, 5 patients treated
had no adverse effects, with objective vascularization enhancement
in the area of stem cell administration as detected by nuclear
imaging (Hamano, et al., 2001, Jpn Circ J 65:845-847, which is
incorporated by reference herein in its entirety). A subsequent
study administering AC133 purified bone marrow stem cells into the
infarct area in 12 patient during bypass grafting demonstrated a
marked improvement in left ventricular ejection fraction, a
decreased rate of remodeling, and improved perfusion (Stamm, et
al., 2004, Thorac Cardiovasc Surg 52:152-158, which is incorporated
by reference herein in its entirety). Administration of stem cells
into coronary circulation or directly into the myocardium has also
been performed both in the angina setting, as well as subsequent to
cardiac infarct in order to enhance angiogenesis, and prevent
remodeling, respectively. In patients with end stage angina,
administration of autologous bone marrow cells using the NOGA
catheter system in 14 patients resulted in improved ejection
fraction from a baseline of 20% to 29% (P=0.003) and a reduction in
end-systolic volume (P=0.03) in the treated patients. Furthermore,
electromechanical mapping revealed significant mechanical
improvement of the injected segments (P<0.0005) at 4 months
after treatment (Perin, et al., 2003, Circulation 107:2294-2302,
which is incorporated by reference herein in its entirety).
Improvements were also notably maintained in the same patient
population at 1-year follow-up (Perin, E., 2004, Int J Cardiol 95
Suppl 1:S45-46, which is incorporated by reference herein in its
entirety). Transcoronary administration of bone marrow cells in
patients post-myocardial infarction induced an improvement at 6
months in regional and global LV function, increased thickness of
the infarcted wall, and showed a reduction in myocardial remodeling
as determined by a decrease in the end-systolic volume
(Femandez-Aviles, et al., 2004, Circ Res 95:742-748, which is
incorporated by reference herein in its entirety). In another
study, patients post myocardial infarction were transplanted with
autologous bone marrow cells via a balloon catheter placed into the
infarct-related artery during balloon dilatation (percutaneous
transluminal coronary angioplasty), resulting in decreased infarct
size, improved wall motion score, and a decrease in ventricular
remodeling (Strauer, et al., 2002, Circulation 106:1913-1918, which
is incorporated by reference herein in its entirety). Randomized
trials are currently underway using autologous bone marrow stem
cells for increasing cardiac function post myocardial infarction
although results are still controversial and inconclusive (Assmus,
et al., 2002, Circulation 106:3009-3017; Cleland, et al., 2006, Eur
J Heart Fail 8:105-110, each of which is incorporated by reference
herein in its entirety). In addition to bone marrow hematopoietic
cells, other types of stem cells have been utilized for improvement
in myocardial activity, perfusion, and decreasing ventricular
remodeling. These include mesenchymal stem cells (Chen, et al.,
2004, Chin Med J (Engl) 117:1443-1448, which is incorporated by
reference herein in its entirety), endothelial stem cells (Aoki, et
al., 2005, J Am Coll Cardiol 45:1574-1579, which is incorporated by
reference herein in its entirety), and skeletal myoblasts (Ye, et
al., 2006. Exp Biol Med (Maywood) 231:8-19, which is incorporated
by reference herein in its entirety). A limiting factor in
presently used cellular therapies for myocardial dysfunction is the
lack of ability to induce transdifferentiation of the stem cells
into the desired cardiac tissue in a directed manner. Additionally,
methods do not exist for expanding sufficient numbers of
semi-differentiated progenitor stem cells that possess a high
proclivity for repairing the heart. This drawback is in part due to
lack of proper culture mediums for expansion of such unique cell
populations. The current invention addresses this issue.
[0020] The importance of stem cells inducing regeneration of other
organ systems has been shown in a variety of settings. In a
pathological setting, it was reported that bone marrow derived stem
cells are the precursors of stomach epithelial tissue in
Helicobacter pylori infected mice that progresses to the develop
stomach cancer (Houghton, et al., 2004, Science 306:1568-1571,
which is incorporated by reference herein in its entirety). In a
therapeutic setting, administration of Green Fluorescent Protein
(GFP) bone marrow stem cells into rats with ethanol-induced ulcers
resulted in generation of GFP expressing, cytokeratin-positive
epithelial cells and vimentin-positive interstitial cells,
contributing to a decreased pathology in the stem cell recipients
(Komori, et al., 2005, J Gastroenterol 40:591-599, which is
incorporated by reference herein in its entirety). The human bone
marrow derived Flk1(+)/CD31(-)/CD34(-) cell population was reported
to transdifferentiated into a variety of tissues, including stomach
epithelium when injected into non-obese diabetic, severe combined
immunodeficient (NOD-SCID) mice, thus suggesting human stem cells
also possess such transdifferentiation ability (Fang, et al., 2003,
J Hematother Stem Cell Res 12:603-613, which is incorporated by
reference herein in its entirety). Stomach-homing capacity to
injured tissue of human stem cells was demonstrated human
mesenchymal stem cells infused systemically in NOD-SCID mice that
received radiation to the abdominal area. This resulted in a
specific rise in stem cell engraftment exclusively to the
irradiated areas (Francois, et al., Local irradiation induces not
only homing of human Mesenchymal Stem Cells (hMSC) at exposed sites
but promotes their widespread engraftment to multiple organs: A
study of their quantitative distribution following irradiation
damages. Stem Cells, e-published on Dec. 8, 2005, which is
incorporated by reference herein in its entirety). It is
anticipated that since stem cells can selectively home to the
injured stomach area, addition of factors to allow expansion once
already homed into the injured tissue will increase therapeutic
efficacy of stem cell therapies. The invention teaches methods of
expanding cells that have already homed to an injured tissue.
[0021] The use of stem cells has also been applied to liver
disease. It is known that partial hepatectomy leads to mobilization
of an AC133+stem cell population in clinical situations (Gehling,
et al., 2005, J Hepatol 43:845-853, which is incorporated by
reference herein in its entirety). Furthermore, studies using
carbon tetrachloride induced liver injury have demonstrated a
therapeutic effect of bone marrow flk-1+ cell infusion (Fang, et
al., 2004, Transplantation 78:83-88, which is incorporated by
reference herein in its entirety). It is believed that liver damage
induces expression of several chemokines, including stromal derived
factor-1 (SDF-1) which attracts stem cells into the damaged areas
(Hatch, et al., 2002, Cloning Stem Cells 4:339-351, which is
incorporated by reference herein in its entirety). Therapeutic
mobilization of endogenous stem cells using granulocyte colony
stimulating factor (G-CSF) has also demonstrated protective effects
in liver injury models (Quintana-Bustamante, et al., 2006,
Hepatology 43:108-116, which is incorporated by reference herein in
its entirety). It is anticipated that since stem cells can
selectively home to the injured hepatic area, addition of factors
to allow expansion once already homed into the injured tissue will
increase therapeutic efficacy of stem cell therapies. The invention
teaches methods of expanding cells that have already homed to an
injured tissue.
[0022] Stem cells have also been useful for treatment of
neurological deficiencies in a variety of situations.
Administration of fetal stem cells in the form of mesenchphalic
tissue into the striatal area of Parkinson's disease (PD) patients
have demonstrated that grafted dopaminergic neurons can reinnervate
the striatum, restore regulated dopamine release and
movement-related frontal cortical activation, and result in
observable clinical benefit (Lindvall, et al., 2004, NeuroRx
1:382-393, which is incorporated by reference herein in its
entirety). Patients suffering from stroke have also been treated by
implantation of autologous mesenchymal stem cells into the middle
cerebral arterial territory. Improvements were seen in some
functional indexes such as the Barth's score (Bang, et al., 2005,
Ann Neurol 57:874-882; Rabinovich, et al., 2005, Bull Exp Biol Med
139:126-128, each of which is incorporated by reference herein in
its entirety). A wide variety of neurological indications are
currently under investigation for amenability to stem cell therapy
(Kulbatski, et al., 2005, Curr Drug Targets 6:111-126; Zhu, et al.,
2005, Curr Drug Targets 6:97-110, each of which is incorporated by
reference herein in its entirety). Unfortunately, ethical issues
associated with the use of fetal tissue, as well as inability to
define the activities and functions of neurally injected stem cells
hampers progress in the field. Development of novel culture and
expansion methodologies for stem cell applications is therefore an
important area of issue.
SUMMARY OF THE INVENTION
[0023] In one aspect of the invention, a method of preparing live
placenta conditioned media (LPCM) is disclosed. Said LPCM is
prepared through contacting a media suitable for maintaining
cellular viability in vitro with at least a portion of a placenta
under conditions allowing transfer of molecules from said placenta
into said media.
[0024] In one embodiment of the invention, the placenta may be a
hemochorial, epitheliochorial, or endotheliochorial. In a preferred
embodiment the placenta is hemochorial. The placenta can be
collected subsequent to vaginal delivery or collected pre-term by
cesarean section, depending on biological properties desired. The
placenta can be brought in contact with said media through
immersing said placenta in media, through co-culture of placental
tissue in said media, or through perfusion of said media through
the placenta in a discontinuous or continuous manner. The placenta
can be in its entirety or dissected into individual units or
cellular components. Contact between said placenta and media can be
achieved through a filter apparatus whereby molecules of a specific
size are allowed to permeate through said filter, whereas molecules
of a larger size are excluded.
[0025] In one embodiment of the invention, a full term placenta
obtained from a vaginal delivery is exanguinated and washed in
saline using an anticoagulant. LPCM is produced through perfusing
said placenta in a continuous circuit using a peristaltic pump
preset for a volume of perfusion sufficient to maintain placental
integrity. The peristaltic pump can cause flow of the perfusion
solution in a pulsatile or non-pulsatile manner. In another
embodiment, other means of passaging media through the placenta may
be employed such as a syringe filled with media.
[0026] In some embodiments, LPCM is collected from the perfused
solution at a time-point sufficient to allow transfer of molecules
with desired biological properties from the placenta to the media.
Temperature, pH, intravasular pressure, flow rate, oxygen and
carbon dioxide concentrations, as well as osmolarity of the
perfusion solution may be monitored and adjusting accordingly to
achieve desired properties of the LPCM. Attachment of the perfusion
system to said placenta may be accomplished by perfusion of media
through the umbilical artery(s) and collection through the
umbilical vein and/or through the exterior of the placental
structure through diffusion. Subsections of the placenta may be
perfused individually for example the truncal branch of the
chorionic artery supplying a selected cotyledon and the associated
vein may be perfused on the fetal side, or selective maternal
circulation as described in studies perfusing placenta for
detection of maternal to fetal drug transfer (Forestier, et al.,
2001, Am J Obstet Gynecol 185:178-181, which is incorporated by
reference herein in its entirety). Media chosen for perfusion may
be an isotonic solution, a buffered solution, or a solution capable
of functioning as a growth medium. The growth media can contain, if
desired, a growth factor, combinations of growth factors, or
substantial nutrient content allowing for increased viability of
the placenta to be perfused. Additional agents may be introduced
into the perfusion solution, including agents to prevent clotting,
maintain pH, or to maintain a desired osmolarity or oxygen content.
For example heparin, buffers, zwitterions, or artificial/natural
oxygen carriers can be added. Agents inhibiting apoptosis such as
caspase inhibitors can also be incorporated in order to preserve
certain functions of placental tissue. The contacting step between
the growth media and the placenta can occur, for example, at a
temperature range of from about 32.degree. C. to about 40.degree.
C.
[0027] Another embodiment of the current invention involves
modification of placental conditions through either upregulating or
inhibiting oxygen content in the placenta in order to modify growth
factor release.
[0028] In another aspect of the invention, a method for the
expansion or growth of stem cells is provided, by incubating at
least a portion of a placenta in a growth medium to condition the
medium, and contacting at least one stem cell with the growth
medium hemochorial, epitheliochorial, or endotheliochorial. In a
preferred embodiment the placenta is hemochorial. The placenta may
be collected subsequent to vaginal delivery or collected pre-term
by cesarean section, depending on biological properties desired. In
a preferred embodiment the placenta is hemochorial. The stem cell
can be, for example, a mesenchymal stem cell, or a fetal stem cell.
The stem cells can be derived from an umbilical cord, such as, for
example, from umbilical cord blood. The stem cells can be derived
from an umbilical cord that expresses a CD34.sup.+ cell marker. The
umbilical cord stem cells and said placenta can be derived, for
example, from a mammal, such as a human. The growth medium can also
contain, if desired, a growth factor, combinations of growth
factors, or substantial nutrient content allowing for increased
viability of the stem cells. The incubating step can occur, for
example, at a temperature range of from about 32.degree. C. to
about 40.degree. C. The placenta can be removed from the medium
prior to the contacting step, if desired. The placenta can either
be perfused with the medium or it may be cultured in the medium at
conditions that allow for release of growth factors.
[0029] Further embodiments include a method of optimizing growth
factor production from said placenta conditioned media through the
use of filters that separate compositions based on electrical
charge, size or ability to elute from an adsorbent. Numerous
techniques are known in the art for purification of growth factors
and concentration of said agents. For some particular uses the
placental conditioned medium will be sufficient for use in its
current format and will not require concentration, however numerous
other uses may. In order to identify and standardize placental
conditioned media, one embodiment of the invention is the concept
of "units of activity" for quantification of LPCM activity in which
1 Unit of LPCM is sufficient to stimulate a biological activity
sought to a certain degree. Depending on use, this can be
stimulation of a standardized cell culture to proliferate by a
certain percentage, in other desired uses the Unit may designate
the amount needed to inhibit differentiation a specified culture
condition by a defined percentage.
[0030] The use of placental conditioned media as a combination to
known cocktails is also an embodiment of the invention. In addition
to actual soluble components already used in stem cell culture
medias, LPCM can be used to synergize with plate-bound stimulators,
as well as antibodies and other methods known in the art to induce
cycling in a stem cell. These are well known in the art and include
contact-dependent factors including heparan sulfate-bound cytokines
such as members of the fibroblast growth factor family (de Haan, et
al., 2003, Dev Cell 4:241-251, which is incorporated by reference
herein in its entirety), and ligands of VLA-4 and -5 (Jung, et al.,
2005, Cytokine 32:155-162, which is incorporated by reference
herein in its entirety), as well as antibodies to TGF-.beta.
(Imbert, et al., 1998, Exp Hematol 26:374-381, which is
incorporated by reference herein in its entirety).
[0031] In some embodiments of the invention, a method for the
expansion or growth of stem cells is provided, by incubating at
least a portion of a placenta in a growth medium to condition the
medium, and contacting at least one stem cell with the growth
medium. The stem cell can be a) A totipotent cell such as an
embryonic stem cell, an extra-embryonic stem cell, a cloned stem
cell, a parthenogenesis derived cell; b) A pluripotent cell such as
a hematopoietic stem cell, an adipose derived stem cell, a
mesenchymal stem cell, a cord blood stem cell, a placentally
derived stem cell, an exfoliated tooth derived stem cells, a hair
follicle stem cell or a neural stem cell; or c) A tissue specific
progenitor cell such as a precursor cell for the neuronal, hepatic,
nephrogenic, adipogenic, osteoblastic, osteoclastic, alveolar,
cardiac, intestinal, or endothelial lineage. The incubating step
can occur, for example, at a temperature range of from about
32.degree. C. to about 40.degree. C. The placenta can be removed
from the medium prior to the contacting step, if desired.
[0032] An additional embodiment of the invention teaches addition
of certain factors to the perfusion mixture used to perfuse said
placenta such that the placenta generates endogenous growth
factors, which are capable of either stimulating stem cell
expansion on their own, or having synergy with other growth
factors.
[0033] In additional embodiments of the invention, a method for the
expansion or growth of umbilical cord stem cells is provided, by
contacting at least one stem cell with a liquid that has been
incubated with at least a portion of a placenta. The contacting
step can occur, for example, after the incubating step. The
contacting step can occur simultaneously with the incubating step.
The incubating step can occur, for example from about 1 second to
about 3 weeks. The incubating step can occur, for example, from
about 24 hours to about 10 days. The contacting step can occur, for
example, from about 1 second to about 3 weeks. The contacting step
can occur, for example, from about 24 hours to about 10 days. The
stem cells can be stored, for example, prior to the contacting step
using a freezing process.
[0034] Another embodiment is the use of LPCM alone or in
combination with other approaches expanding cells that have been
generated for a specific phenotype, and are at risk of losing the
phenotype that was artificially endowed upon them. Specifically, it
is known that administration of a certain compounds to stem cells
induces differentiation into certain lineage-specific progenitors.
For example, addition of thrombopoietin alone or in combination
with interleukin 11 to early hematopoietic stem cells will promote
the preferential production of megakaryocytic progenitors. One
embodiment of the current invention is the ability of LPCM, alone
or in combination with other growth factors and/or culture
conditions to maintain and expand the new phenotype of the
differentiated progenitor cell without stimulation of terminal
differentiation. For example, subsequent to increasing the numbers
of megakaryocytic progenitors in a stem cell culture, LPCM may be
added to maintain said progenitors and expand their numbers.
[0035] Another embodiment of the invention relates to generation
and expansion of cells expressing a desired phenotype through
cytoplasmic reprogramming wherein the cytoplasmic extracts of a
cell with a desired property are introduced into the cytoplasm of a
recipient cell with the aim of introducing the properties of the
donor cell into the recipient cell (Hakelien, et al., 2002, Nat
Biotechnol 20:460-466, which is incorporated by reference herein in
its entirety). Such reprogramming can be useful for generating
autologous stem cells from non-stem cells of a patient by
introduction of cytoplasm from the stem cell of an allogeneic
patient. One drawback of this technology has been the limited
ability to expand the reprogrammed cell after introduction of
cytoplasm without differentiation. Accordingly, the invention
teaches the use of LPCM either alone or in combination with other
factors in order to induce expansion of the reprogrammed cell.
[0036] An aspect of the invention is the use of LPCM as an adjuvant
to currently used stem cell feeder-free mixtures that are currently
limited by ability to achieve desired expansion of stem cells of
the phenotype sought.
[0037] Another embodiment of the invention is a stimulator of
proliferation of totipotent stem cells such as such as human
embryonic stem cells characterized by expression of markers such as
SSEA-4, GCTM-2 antigen, TRA 1-60, Cripto, gastrin-releasing peptide
(GRP) receptor, podocalyxin-like protein (PODXL), or human
telomerase reverse transcriptase (hTERT). The LPCM can be used as a
stimulator of proliferation alone or as an additive to media known
to be useful for culturing said cells. An example of such a tissue
culture media is Dulbecco's modified Eagle's medium (DMEM). In an
ideal embodiment LPCM is used in such a manner and under such
conditions so as to alleviate the need for serum or feeder cells in
the culture of human embryonic stem cells.
[0038] Another embodiment of the invention is a stimulator of
proliferation of totipotent stem cells generated by cloning through
the use of nuclear transfer technologies. The LPCM can be used as a
stimulator of proliferation alone or as an additive to media known
to be useful for culturing said cells.
[0039] Another embodiment of the invention is a stimulator of
proliferation of totipotent stem cells such as such as human oocyte
producing stem cells characterized by expression of markers such
Vasa, Oct-4, Dazl, Stella, Fragilis, Nobox, c-Kit and Sca-1. The
LPCM can be used as a stimulator of proliferation alone or as an
additive to media known to be useful for culturing said cells.
[0040] Another embodiment of the invention is a stimulator of
proliferation of totipotent stem cells such as such as
parthenogenetically generated stem cells characterized by
expression of markers such Oct-4, alkaline phosphatase, telomerase,
SSEA-4, TRA 1-60 and TRA 1-81. The LPCM can be used as a stimulator
of proliferation alone or as an additive to media known to be
useful for culturing said cells.
[0041] Another embodiment of the invention is a stimulator of
proliferation of totipotent stem cells such as such as
spermatogonial stem cells reprogrammed to pluripotent germ-line
stem cells characterized by expression of markers such Oct-4,
Nanog, Dppa5 and Rex1. The LPCM can be used as a stimulator of
proliferation alone or as an additive to media known to be useful
for culturing said cells.
[0042] Another embodiment of the invention is the generation of
totipotent stem cells through the steps of: a) treating bone marrow
cells with LPCM in combination with sera from a female in a period
of the menstrual cycle associated with upregulation of oocyte stem
cell markers in the bone marrow; b) addition of a calcium flux
inducing agent to activate said oocyte precursors into the process
of parthenogenesis; c) purifying cells expressing embryonic stem
cell markers such as SSEA-4, TRA 1-60 or TRA 1-81; and d) expanding
said cells in a culture media containing LPCM alone or in
combination with agents and conditions known to induce totipotent
stem cell proliferation.
[0043] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as hematopoietic stem
cells characterized by markers such as Stem Cell Antigen (SCA+),
lineage negative (lin-), c-kit+, CD34+, CD38-, CD33-. The LPCM can
be used as a stimulator of proliferation alone or as an additive to
media known to be useful for culturing said cells.
[0044] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as mesenchymal stem
cells characterized by markers such as LFA-3, ICAM-1, PECAM-1,
P-selectin, L-selectin, CD49b/CD29, CD49c/CD29, CD49d/CD29, CD61,
CD18, CD29, 6-19, thrombomodulin, telomerase, CD10, CD13, STRO-1,
STRO-2, VCAM-1, CD146, THY-1. The LPCM can be used as a stimulator
of proliferation alone or as an additive to media known to be
useful for culturing said cells.
[0045] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as placentally derived
multipotent cells characterized by markers such as Oct-4, Rex-1,
CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3, SH-4, TRA-1-60,
TRA-1-81, SSEA-4 and Sox-2. The LPCM can be used as a stimulator of
proliferation alone or as an additive to media known to be useful
for culturing said cells.
[0046] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as adipose-derived
stem cells characterized by markers such as CD13, CD29, CD44, CD63,
CD73, CD90, CD166, Aldehyde dehydrogenase (ALDH), and ABCG2. The
LPCM can be used as a stimulator of proliferation alone or as an
additive to media known to be useful for culturing said cells.
[0047] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as cord blood stem
cells characterized by markers such as CD34, c-kit, and CXCR-4. The
LPCM can be used as a stimulator of proliferation alone or as an
additive to media known to be useful for culturing said cells.
[0048] Another embodiment of the invention is a stimulator of
proliferation of pluripotent stem cells such as deciduous tooth
stem cells characterized by markers such as STRO-1, CD146 (MUC18),
alkaline phosphatase, MEPE, and bFGF. The LPCM can be used as a
stimulator of proliferation alone or as an additive to media known
to be useful for culturing said cells.
[0049] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as neural stem cells
characterized by markers such as RC-2, 3CB2, BLB, Sox-2hh, GLAST,
Pax 6, nesting, Muashi-1, and prominin. The LPCM can be used as a
stimulator of proliferation alone or as an additive to media known
to be useful for culturing said cells.
[0050] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a stomach epithelial
stem cell characterized by markers such as Musashi-1, c-hairy-1 and
HES-5. The LPCM can be used as a stimulator of proliferation alone
or as an additive to media known to be useful for culturing said
cells.
[0051] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a skeletal muscle
stem cell characterized by markers such as desmin positive, SCA-1+,
CD45- and possessing a side population profile on flow cytometry by
dye exclusion (Challen, et al., 2006, Stem Cells 24:3-12, which is
incorporated by reference herein in its entirety). The LPCM can be
used as a stimulator of proliferation alone or as an additive to
media known to be useful for culturing said cells.
[0052] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a mammary gland stem
cell characterized by markers such as SCA-1 positive, CD45- and
keratin-6. The LPCM can be used as a stimulator of proliferation
alone or as an additive to media known to be useful for culturing
said cells.
[0053] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a dermal stem cell
characterized by markers such as SCA-1 positive, CD34+, CD45- and
positive for alpha6-integrin, beta1-integrin, keratin 14, and
keratin 19. The LPCM can be used as a stimulator of proliferation
alone or as an additive to media known to be useful for culturing
said cells.
[0054] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a myocardial stem
cell characterized by markers such as SCA-1 positive, c-kit
positive, and possessing a side population profile on flow
cytometry by dye exclusion (Challen, supra). The LPCM can be used
as a stimulator of proliferation alone or as an additive to media
known to be useful for culturing said cells.
[0055] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a mesangial stem
cell characterized by markers such as SCA-1 positive, c-kit
positive, and possessing a side population profile on flow
cytometry by dye exclusion (Challen, supra). The LPCM can be used
as a stimulator of proliferation alone or as an additive to media
known to be useful for culturing said cells.
[0056] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a hepatic oval stem
cell characterized by markers such as SCA-1 positive, c-kit
positive, and CD34 positive. The LPCM can be used as a stimulator
of proliferation alone or as an additive to media known to be
useful for culturing said cells.
[0057] Another embodiment of the invention is a stimulator of
proliferation of progenitor stem cells such as a pancreatic stem
cell characterized by markers such as nestin, CK-8, CK-18, Isl-1,
Pdx-1, Pax-4, and Ngn-3. The LPCM can be used as a stimulator of
proliferation alone or as an additive to media known to be useful
for culturing said cells.
[0058] Another embodiment of the invention is the administration of
LPCM into a subject in order to stimulate the proliferation and
expansion of endogenous stem cells that have been activated as part
of the healing process after injury.
[0059] Another embodiment of the invention is the administration of
LPCM into a subject in order to stimulate the proliferation and
expansion of endogenous stem cells that have been mobilized from
the bone marrow to a target organ as a result of injury.
[0060] Another embodiment of the invention a treatment for a
degenerative condition by the application of a combination of LPCM
with known therapies in order to enhance the beneficial effects of
known therapies.
[0061] Another embodiment of the invention an adjuvant to
therapies, interventions, or accidents that destroy or inactivate
stem cells with the goal of accelerating stem cell reconstitution.
Examples of situations where accidental stem cell destruction
occurs would include a nuclear event.
[0062] Within the embodiments of the invention is the use of LPCM,
or extracts thereof, to enhance proliferation of stem cells within
a living organism. Administration of such media can be performed
systemically, or in a localized environment. Clinical situations
where administration of such placentally conditioned media is
desirable can include conditions where an increase in the number of
stem cells is sought due to disease or senescence of endogenous
stem cells. Specific aspects of this include conditions in which a
higher number and/or more rapid recovery of stem cells is needed
after a medical procedure. One such situation would be post bone
marrow transplant where expansion of hematopoietic cells is
desirable in order for the patient not to succumb to bacterial or
viral infections. Specifically, LPCM may be used in conjunction
with a growth factor that stimulates preferential differentiation
of the bone marrow stem cell into the granulocytic and/or monocytic
lineage such as G-CSM or GM-CSF. Such an expansion of granulocytic
and monocytic precursors would be useful in enhancing immunological
defenses subsequent to a bone marrow transplant. If clinically
desirable the number of endogenous dendritic cells can also be
expanded through administration of cytokines such as flt-3L in
combination with LPCM. Accordingly, this invention provides methods
and compositions that can be administered to a patient having
undergone a bone marrow transplant that will enhance proliferation
and bone marrow take.
[0063] Yet another embodiment is supporting the expansion of
endogenous stem cells after a injury has occurred and the
endogenous stem cells are mobilized or begin to differentiated, but
do not do so at high enough levels to stimulate a beneficial
response. Said endogenous stem cells can be present in pancreatic
tissue, liver tissue, smooth muscle tissue, striated muscle tissue,
cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy
tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic
ductal tissue, spleen tissue, thymus tissue, Peyer's patch tissue,
lymph nodes tissue, thyroid tissue, epidermis tissue, dermis
tissue, subcutaneous tissue, heart tissue, lung tissue, vascular
tissue, endothelial tissue, blood cells, bladder tissue, kidney
tissue, digestive tract tissue, esophagus tissue, stomach tissue,
small intestine tissue, large intestine tissue, adipose tissue,
uterus tissue, eye tissue, lung tissue, testicular tissue, ovarian
tissue, prostate tissue, connective tissue, endocrine tissue, and
mesentery tissue.
[0064] Another embodiment is the use of LPCM as a source of
angiogenic/endothelial cell migration/proliferation factor for the
stimulation of angiogenesis in a patient in need thereof. The
amount of angiogenic stimulatory activity can be increased in the
LPCM through perfusing the placenta under conditions of hypoxia or
near hypoxia. LPCM can be used alone or in combination with known
angiogenesis promoters. The angiogenic promoters can include
proteins such as FGF-1, FGF-2, VEGF, transcription factors such as
the Hypoxia Inducible Factor (HIF-1), or small molecule stimulators
of endothelial proliferation such as an artificial agonist of an
angiogenically relevant receptor. Proteins may be administered
locally to the patient through means such as intramuscular
injection, or systemically. Additionally, genes encoding
angiogenically stimulatory growth factors may be delivered in the
form of naked plasmid DNA, adenoviruses, or other means known to
one skilled in the art. Additionally, LPCM can be administered in
combination with stem cells capable of inducing angiogenesis for
augmenting the ability of said stem cells. Said stem cells may be
derived from a variety of tissue, such as adipose, cord blood,
placenta, bone marrow, peripheral blood, or growth
factor/chemotherapy mobilized peripheral blood. In addition, the
cells to be administered can be allogeneic or autologous. For some
purposes, cells can be matched according to the Human Leukocyte
Antigen haplotype.
[0065] Medical conditions amenable to such treatments may include
peripheral limb ischemia, myocardial angina, mesenteric ischemia,
ischemia reperfusion injury and general circulatory disorders.
[0066] Another embodiment of the invention is the use of LPCM alone
or in combination with growth factors to promote healing. In one
aspect, scarless healing is promoted through concentration of
factors such as TGF-b within the LPCM through selective
purification of LCMP fractions containing this TGF-b or similar
proteins involved in tissue repair without promotion of fibrosis.
Alternatively, LPCM can be administered in conjunction with
antibodies or inhibitors to fibrosis promoting cytokines or
factors.
[0067] Another embodiment of the invention is the use of LPCM for
cosmetic purposes in order to enhance skin rejuvenation. LPCM can
be administered in a variety of dermatologically applicable
formulations, either alone or in combination with other factors
capable of restoring certain properties to skin. Additionally, LPCM
can be used in combination with agents capable of
de-differentiating skin such as histone deacetylase inhibitors, DNA
methylase inhibitors, or other epigenetically acting compounds in
order to allow expansion of local dermal precursor cells. Such stem
cell expansion can be tailored to allow formation of skin with
appearances desirable to the common population.
[0068] Another embodiment of the invention is a treatment for
diabetes by administering LPCM in combination with factors capable
of inducing islet regeneration. These factors can be, for example,
soluble proteins, membrane bound proteins or intracellular acting
transcription factors. For example, it is known that administration
into mice combinations of GLP-1, EGF and gastrin leads to
regeneration of islets or islet-like cells that are functionally
effective in models like NOD or streptozocin induced diabetes
(Bonner-Weir, et al., 2005, Nat Biotechnol 23:857-861, which is
incorporated by reference herein in its entirety). When these
experiments were translated to humans, only a marginal therapeutic
effect was seen, and this was observed only in a small subset of
patients (von Herrath, M., 2005, Curr Opin Investig Drugs
6:1037-1042, which is incorporated by reference herein in its
entirety). The invention teaches that use of placental conditioned
media can be added to cultures of differentiating islets in vitro
to expand numbers, but can also be added to a patient in vivo in
order to amplify the relatively minute effect that the hormones are
evoking in terms of differentiation induction.
[0069] Another embodiment of the invention a treatment for multiple
sclerosis utilizing LPCM for expanding neuronal progenitors and
subsequent reintroduction of said progenitors into a host in need
thereof. Alternatively, LPCM can be administered into patients
suffering from multiple sclerosis in combination with agents
capable of inhibiting the autoimmune process. Synergy in
therapeutic effects is anticipated through the concurrent induction
of tissue healing and immune system repair.
[0070] Another embodiment of the invention is the treatment of an
immunological disorder, such as an autoimmune disorder, by
extracting hematopoietic cells from an autologous patient, treating
the cells with LPCM and/or other combinations of stem cell
expanding compounds, ablating the immune system of the patient, and
subsequent reintroduction of stem cells into the host for
reconstitution. LPCM may be subsequently provided to the host in
order to accelerate reconstitution of hematopoiesis. Autoimmune
diseases treatable by these procedures include, but are not limited
to, Type 1 diabetes, multiple sclerosis, rheumatoid arthritis,
systemic sclerosis, Hashimoto's thyroiditis, myasthenia gravis,
scleroderma, systemic lupus erythromatosis, graft versus host
disease, and the like.
[0071] Another embodiment of the invention in relation to
autoimmune diseases, and also transplant rejection, involves the
use of LPCM for expansion of antigen-specific and/or non-specific
immune regulatory cells for use in controlling a pathological
immune response. It is known that cells such as Th2 cells
(Christen, et al., 2004, Immunol Res 30:309-325), Th3 cells
(Prud'homme, et al., 2000, J Autoimmun 14:23-42), TR1 cells
(Bacchetta, et al., 2005, Autoimmun Rev 4:491-496), CD4+CD25+FoxP3+
cells (Bluestone, et al., 2005, Curr Opin Immunol 17:638-642), and
CD3+ double negative cells (Zhang, et al., 2001, J Mol Med
79:419-427), each of which is incorporated by reference herein in
its entirety, are capable of suppressing immune responses in an
antigen specific manner, whereas NKT cells (Van Kaer, L., 2005, Nat
Rev Immunol 5:31-42), myeloid suppressor cells (Serafini, et al.,
2004, Cancer Immunol Immunother 53:64-72), M2 cells (Rauh, et al.,
2004, Biochem Soc Trans 32:785-788), and immature dendritic cells
(Ichim, et al., 2003, Transpl Immunol 11:295-306), each of which is
incorporated by reference herein in its entirety, are capable of
suppressing immune responses in an antigen non-specific manner.
Despite the fact that all of these cells have potential therapeutic
value, their clinical development has been hindered by lack of
methodology for expansion ex vivo and maintenance of function
subsequent to expansion. The invention teaches the use of LPCM for
use in ex vivo culture and expansion of immune regulatory cells
derived from a patient in need thereof. LPCM may be used either
alone or in combination with factors known to be involved in the
development of said cells. Alternatively, immune regulatory cells
can be generated through the use of stem cells through exposure to
factors involved in their development and LPCM being added to
enhance proliferation of said cells without loss of function.
[0072] Another embodiment of the invention is a treatment for
stroke comprising induction of in vitro differentiation of neural
precursor cells using a conditioning regimen, expansion of said
cells using LPCM, and reintroduction of said cells into a patient
in need thereof. Alternatively, in vivo differentiation of
endogenous neural cells can be accomplished by administration of
polypeptides and proteins known in the art. Subsequent or
concurrent with this differentiation, administration of LPCM may be
given in order to expand neural cells.
[0073] Another embodiment is the use of LPCM in storage and
transportation of stem cells in order to maintain viability,
mobility and stem cell function. LPCM may be used alone or added to
a variety of agents known in the art to allow transportation of
stem cells. This is particularly important in situations of bone
marrow stem cell transportation in which cell freezing and thawing
is not performed in numerous situations. The ability to adequately
store stem cells during transportation would allow for tissue
extraction at separate physical locations from the stem cell
processing facility.
[0074] Another embodiment of the invention is the use of LPCM alone
or in combination with other factors for maintaining stem cells in
a totipotent, pluripotent, or progenitor state while allowing
sufficient viability and proliferation ex vivo so as to be useful
for gene therapy. Said gene therapy can be used for introducing new
genes into a host in need thereof. Genes may be introduced by a
wide range of approaches known in the art including adenoviral,
adeno-associated, retroviral, lentiviral, Kunjin virus, or HSV
vectors, as well as electroporation and Sleeping Beauty
transposons. Additionally, gene therapy can include selectively
silencing genes through the use of antisense, ribozyme or RNA
interference technologies.
[0075] In some embodiments of the invention, a method for the
expansion or growth of stem cells without substantially inducing
differentiation is provided, by incubating at least a portion of a
placenta in a growth medium to condition the medium, and contacting
at least one stem cell with the growth medium. The at least one
stem cell can be, for example, totipotent, capable of
differentiating into cells of all histological types of the body.
The totipotent stem cell can be selected, for example, from an
embryonic stem cell, an extra-embryonic stem cell, a cloned stem
cell, a parthenogenesis derived cell. The embryonic stem cell can
express, for example, one or more of the following markers:
stage-specific embryonic antigens (SSEA) 3, SSEA 4, Tra-1-60 and
Tra-1-81, Oct-3/4, Cripto, gastrin-releasing peptide (GRP)
receptor, podocalyxin-like protein (PODXL), or human telomerase
reverse transcriptase (hTERT). The hematopoietic stem cells can
express, for example, one or more of the following markers: CD34,
c-kit, and the multidrug resistance transport protein (ABCG2). The
adipose-derived stem cells can express, for example, one or more of
the following markers: CD13, CD29, CD44, CD63, CD73, CD90, CD166,
Aldehyde dehydrogenase (ALDH), and ABCG2. The mesenchymal stem
cells can express, for example, one or more of the following
markers: STRO-1, CD105, CD54, CD106, HLA-I markers, vimentin, ASMA,
collagen-1, and fibronectin, but not HLA-DR, CD117, and hemopoietic
cell markers. The cord blood stem cells can express, for example,
one or more of the following markers: CD34, c-kit, and CXCR-4. The
placental stem cells can express, for example, one or more of the
following markers: Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166,
CD90, CD105, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4 and Sox-2. The
exfoliated deciduous tooth stem cells can express, for example, one
or more of the following markers: STRO-1, CD146 (MUC18), alkaline
phosphatase, MEPE, and bFGF. The neural stem cell can be
characterized, for example, by expression of RC-2, 3CB2, BLB,
Sox-2hh, GLAST, Pax 6, nesting, Muashi-1, and prominin. The at
least one stem cell can be pluripotent, capable of differentiating
into numerous cells of the body, but not all. The pluripotent stem
cell can be selected from hematopoietic stem cells, adipose stem
cells, mesenchymal stem cells, cord blood stem cells, placental
stem cells, exfoliated teeth derived stem cells, hair follicle stem
cells or neural stem cells. The at least one stem cell can be a
progenitor cell, capable of differentiating into a restricted
tissue type. The progenitor stem cell can be selected from, for
example, neuronal, hepatic, nephrogenic, adipogenic, osteoblastic,
osteoclastic, alveolar, cardiac, intestinal, endothelial progenitor
cells.
[0076] In some embodiments of the present invention, a method for
the expansion or growth of stem cells without substantially
inducing differentiation is provided, by incubating at least a
portion of a placenta in a growth medium to condition the medium,
and contacting at least one stem cell with the growth medium. The
placenta can be derived from a mammal. The placenta can be derived
from a human. The placenta can be derived preterm. The placenta can
be derived at term. The placenta can be perfused for a period of
time with a cell culture media. The cell culture media can be
supplemented, for example, with a single or a plurality of growth
factors. The growth factors can be selected from, for example, a
WNT signaling agonist, TGF-b, bFGF, IL-6, SCF, BMP-2,
thrombopoietin, EPO, IGF-1, IL-11, IL-5, Flt-3/Flk-2 ligand,
fibronectin, LIF, HGF, NFG, angiopoietin-like 2 and 3, G-CSF,
GM-CSF, Tpo, Shh, Wnt-3a, Kirre, or a mixture thereof. The media
can be capable of maintaining viability of a substantial portion of
the placental tissue during the perfusion process. The media can be
selected, for example, from Roswell Park Memorial Institute
(RPMI-1640), Dublecco's Modified Essential Media (DMEM), Eagle's
Modified Essential Media (EMEM), Optimem, and Iscove's Media. The
source of serum can be added to the media. The concentration of
serum in the media can be approximately between 0.1% to 25%. The
concentration of serum in the media can be approximately 10%. The
serum can be selected from adult human serum, fetal human serum,
fetal calf serum and umbilical cord blood serum. The contacting
step can occur after the incubating step. The contacting step can
occur simultaneously with the incubating step. The incubating step
can occur from about 1 second to about 3 weeks. The incubating step
can occur from about 24 hours to about 10 days. The contacting step
can occur from about 1 second to about 3 weeks. The contacting step
can occur from about 24 hours to about 10 days. The placenta can
be, for example, a hemochorial, epitheliochorial, or
endotheliochorial placenta. The perfusion can be accomplished, for
example, through the use of a perfusion apparatus cannulated to
blood vessels connected to the placental body. The perfusion
apparatus can allow for control of intravasular pressure, oxygen
content, carbon dioxide content, pH, and flow rate of the perfused
media flowing through the placental blood vessels. The intravasular
pressure of the perfusate can be maintained, for example, at 30-80
Hg. The intravasular pressure of the perfusate can be maintained at
60 Hg.
[0077] In an additional embodiment of the present invention, a stem
cell with the preserved ability to proliferate, but having a block
in differentiation state is provided, which can be induced by
culturing in media conditioned by perfusion through a live
placenta. The stem cell can be selected, for example, from a
totipotent stem cell, a pluripotent stem cell, and a progenitor
stem cell. The stem cell can be maintained in contact with the
conditioned media, for example, for a period of less than 1 second,
2 hours, 12 hours, 24 hours, 72 hours, or 3 weeks or more. The stem
cell can be maintained in contact with the conditioned media in a
living organism. The contact between the conditioned media and the
stem cell can be prolonged by formulating the conditioned media in
a slow release delivery system. The stem cell can be initially
cultured in contact with the placentally conditioned media for a
period of time, subsequently to which it can be cultured in a
second culture with a different concentration of placentally
conditioned media and an identical or variable mix of cytokines.
The stem cell can be initially cultured for 48 hours in a
concentration of 20-100% placentally conditioned media, whereas in
subsequent cultures it can be maintained in a concentration of
0-50% conditioned media. The stem cell can be maintained in a cell
culture media that can be supplemented with at least one growth
factor selected from the group consisting of WNT signaling agonist,
TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11,
IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, HGF, NFG,
angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre,
and a mixture thereof. The stem cell can be maintained in a 50% by
volume placentally conditioned DMEM media with the following growth
factors also in DMEM media: IL-3 (about 20 ng/ml), IL-6 (about 250
ng/ml), SCF (about 10 ng/ml), TPO (about 250 ng/ml), flt-3L (about
100 ng/ml). The stem cell can be maintained in the presence of an
agent selected from one or more of the following: an inhibitor of
GSK-3, an inhibitor of histone deacetylase activity, and inhibitor
of DNA methyltransferase activity. The stem cell can be rejuvenated
by at fusion with a more primitive stem cell, transfer of cytoplasm
from more primitive stem cells, and/or transfer of karyoplastic
extracts from a more primitive stem cell.
[0078] In an additional embodiment of the present invention, a
method of treating degenerative diseases through administration of
a composition of matter derived from media conditioned by a live
placenta is provided. The degenerative disease effects a tissue
selected from the group consisting of: smooth muscle tissue,
striated muscle tissue, cardiac muscle tissue, bone tissue, bone
spongy tissue, nervous system tissue, cartilage tissue, pancreatic
ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's
patch tissue, lymph nodes tissue, thyroid tissue, epidermal tissue,
dermal tissue, subcutaneous tissue, heart tissue, lung tissue,
vascular tissue, endothelial tissue, blood cells, bladder tissue,
kidney tissue, digestive tract tissue, esophagus tissue, stomach
tissue, small intestine tissue, large intestine tissue, adipose
tissue, uterus tissue, eye tissue, lung tissue, testicular tissue,
ovarian tissue, prostate tissue, connective tissue, endocrine
tissue, and mesentery tissue. The placenta conditioned media can be
administered in combination with an agent capable of inducing stem
cell expansion. The placenta conditioned media can be administered
in combination with an agent capable of inducing stem cell
differentiation into cells of the tissue in need of repair. The
agent capable of inducing stem cell expansion can be selected from
TPO, SCF, IL-1, IL-3, IL-7, flt-3L, G-CSF, GM-CSF, Epo, FGF-1,
FGF-2, FGF-4, FGF-20, VEGF, activin-A, IGF, EGF, NGF, LIF, PDGF,
and a member of the bone morphogenic protein family. The agent
capable of inducing stem cell differentiation can be selected from
HGF, cardiotrophin, BDNF, VEGF, FGF1, FGF2, FGF4, and FGF 20. The
placental conditioned media can be concentrated to a sufficient
extent to allow systemic administration while retaining biological
effects. The placental conditioned media can be calibrated for
specific Units of Activity based on a desired biological property.
The biological activity can be the ability to stimulate
proliferation of a defined culture of CD34 stem cells by 50%. The
placentally conditioned media can be administered according to
biomarkers of stem cell activity in the patient in need of
treatment. The biomarker may be either an indicator of disease
activity, or an indicator of stem cell regeneration. The clinically
applicable agent that possesses stem cell mobilizing activity can
be administered in conjunction with the placentally conditioned
media and/or the stem cell proliferation inducing growth factor,
and/or the inducer of stem cell differentation. The stem cell
mobilizing agent may be an antibody, a small molecule, or a
protein. The stem cell mobilizing agent can be an antibody to
CXCR-4. The stem cell mobilizing agent can be either a small
molecular inhibitor of CXCR-4, or a statin. The stem cell
mobilizing agent can be, for example, a cytotoxic chemotherapy
known to mobilize stem cells, or can be a growth factor such as
G-CSF. The dedifferentiation agent can be used for expanding the
differentiation potential of the stem cells. The dedifferentiation
agent can be, for example, an inhibitor of the enzyme GSK-3, and
inhibitor of the histone deacetylase family of enzymes, or an
inhibitor of DNA methyltransferase activity. The dedifferentiation
agents can be, for example, trichostatin A, valproic acid,
buphenyl, or 5-azacytidine.
[0079] In an additional embodiment of the present invention, a
method of treating degenerative diseases is provided, by
administering a differentiating agent to selectively expand a
population of pluripotent or progenitor cells, while concurrently
administering live placental conditioned media in order to induce
proliferation of the committed stem cell.
[0080] In an additional embodiment of the present invention, a
method of expanding stem cells that have been therapeutically
reprogrammed is provided, by contacting the cells with media that
has been conditioned by a live placenta. The therapeutic
reprogramming can be accomplished by introduction into the target
cell to be reprogrammed agents capable of acting at the epigenetic
level to modify the cellular transcriptosome into a desired
phenotype. The target cell can be fused with another cell of a more
primitive state of differentiation. The cell can be temporarily
permeabilized and cytoplasmic and/or karyoplasmic extracts are
introduced into the cell from another cell of a more primitive
state of differentiation.
[0081] In an additional embodiment of the present invention, a
method of accelerating hematopoietic recovery in a patient in need
thereof is provided, by administering placentally conditioned
media. In some embodiments, the patient has been treated with
chemotherapy, and/or radiotherapy with the scope of either ablating
or diminishing the immune system. The patient can have been
treated, for example, with chemotherapy, and/or radiotherapy with
the scope of eradicating or ameliorating a malignancy. The patient
can have been induced into a state of reduced hematopoiesis as a
result of chemical or radiation poisoning. In some embodiments, the
patient was not administered a cellular graft to enhance recovery
of the hematopoietic system. The patient can have been administered
either cord blood derived, peripheral blood derived, or bone marrow
derived hematopoietic stem cells or progenitors thereof. The
patient can be administered placentally conditioned media
intravenously at a concentration sufficient to accelerate recovery
of early hematopoietic progenitors. The patient can be administered
placentally conditioned media at a concentration of 10-500 Units of
placentally conditioned media per kilogram per day, the Units based
on a logarithmic scale in which 1 Unit can be sufficient to
stimulated proliferation of a defined cell culture of CD34+ cells
by 100% compared to control media. The 1 Unit can be defined on a
logarithmic scale as the amount of placentally conditioned media
needed to stimulate proliferation of a 200 .mu.L culture of
5.times.103 human cord blood isolated CD34+. The patient can be
treated intravenously, or through other means, with placental
conditioned media for a period of time needed to obtain a
granulocyte count of 500/mm.sup.3. The patient can be treated
intravenously, or through other means, with placental conditioned
media for a period of time between 7 days to 15 days. The growth
factor can be concurrently given with the administration of
placentally conditioned media. The growth factor can be selected
from G-CSF, pegylated G-CSF, TPO, IL-11, GM-CSF, or flt-3L.
[0082] In an additional embodiment of the present invention, a
method of treating patient with tissue ischemia through induction
of endothelial stem cell expansion using placentally conditioned
media is provided. The ischemia can be present, for example, in at
least one tissue selected from smooth muscle tissue, striated
muscle tissue, cardiac muscle tissue, bone tissue, bone spongy
tissue, nervous system tissue, cartilage tissue, pancreatic ductal
tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer's patch
tissue, lymph nodes tissue, thyroid tissue, epidermis tissue,
dermis tissue, subcutaneous tissue, heart tissue, lung tissue,
vascular tissue, endothelial tissue, blood cells, bladder tissue,
kidney tissue, digestive tract tissue, esophagus tissue, stomach
tissue, small intestine tissue, large intestine tissue, adipose
tissue, uterus tissue, eye tissue, lung tissue, testicular tissue,
ovarian tissue, prostate tissue, connective tissue, endocrine
tissue, and mesentery tissue. The ischemia can be presenting, for
example, as advanced angina. The placentally conditioned media can
be concentrated and administered into the ischemic myocardium using
the minithoracotomy procedure. The placentally conditioned media
can be concentrated and administered into the ischemic myocardium
using the NOGA electromagnetic mapping and injection system. The
placentally conditioned media can be concentrated and administered
into the ischemic myocardial area using a balloon catheter. A
secondary agent can be added that can be capable of inducing
proliferation of differentiated and undifferentiated endothelial
cells. The secondary agent can be, for example, a nucleic acid, a
protein, or a small molecule. The secondary agent can be, for
example, plasmid DNA encoding a polypeptide selected from HIF-1,
VEGF, FGF1, FGF2, FGF4, FGF20, and angiopoietin. The secondary
agent can be, for example, VEGF, FGF1, FGF2, FGF4, FGF20, or
angiopoietin. An exogenous or endogenous source of stem cells can
be delivered into the ischemic area. The exogenous stem cells can
be autologous or allogenenic mesenchymal, adipose, endothelial,
bone marrow, mobilized peripheral blood, umbilical, or artificially
reprogrammed stem cells. The endogenous stem cells can be mobilized
with a mobilization agent. The patient suffering from ischemia can
be a victim of Critical Limb Ischemia. The patient can be
administered a combination of placentally conditioned media
intramuscularly in the area of ischemia as detected by angiography.
The autologous or allogenenic mesenchymal, adipose, endothelial,
bone marrow, mobilized peripheral blood, umbilical, or artificially
reprogrammed stem cells can be injected with the placentally
conditioned media in a localized environment intramuscularly. The
autologous lymphocytes can be injected with the stem cell source in
order to synergize with the placentally conditioned media and the
injected stem cells. In some embodiments, the patient has suffered
from a cerebral ischemia. The patient can be treated immediately
after the ischemia episode or in a period of time subsequently.
[0083] In an additional embodiment of the present invention, a
method of culturing a placenta in its original 3-dimensional
structure is provided, in such a manner as to reproduce the in vivo
environment in which it resides in the pregnant woman, thus
retaining capability of generation and secretion of growth factors
and proteins that maintain the fetal regenerative capacity. The
method involves acquiring a placenta under sterile conditions,
cannulating blood vessels of the placenta in order to allow proper
perfusion in circumstances similar to as if the placenta was
performing its in vivo functions, perfusing the placenta with a
nutrient mix in a buffer that would mimic physiological conditions,
maintaining a temperature and physical environment similar to that
found in the pregnant woman's body, and imitating conditions of
flow, pH, oxygenation, and pressure similar to that found in the
body. The perfusion of both the maternal and fetal circulatory
components of the placenta can be performed. A nutrient mixture can
be used that possesses similar nutrient requirements as the fetal
and maternal circulation, respectively. A temperature of 37.degree.
C. can be maintained during the perfusion process. The pH can be
monitored, for example, by the perfusion apparatus in a real-time
basis, and adjusted using adequate quantities of acids, bases, or
buffers. The oxygen content can be maintained similar to that found
in the fetal and maternal circulatory contribution to the placenta.
The oxygen content may be increased, for example, through the use
of adding natural or artificial oxygen carriers to the perfusion
solution. An oxygenator may be attached to the perfusion apparatus,
in conjunction with, or separately, from an oxygen sensor, the
combination being used to adjust in real-time oxygen content. The
osmolality can be maintained, for example, through the use of known
means such as addition of albumin or colloids to the perfusion
solution.
[0084] In an additional embodiment of the present invention, a
method of producing a cosmetic for topical use in rejuvenating aged
skin is provided, by concentrating placentally conditioned media,
quantifying and standardizing biological effect of the media, and
formulating the media in a carrier solution that is suitable for
transdermal delivery. The media can be, for example, a
physiological buffer, a media capable of maintaining cellular
viability, or a media enriched in nutrients and mimicking the
content of the maternal/fetal circulation. The media can be
selected from DMEM, RPMI, and saline USP. The media can contain,
for example, an anticoagulant at sufficient quantities to inhibit
clotting during placental perfusion. The quantification can be
based on the ability of placentally conditioned media to induce
proliferation of dermal stem cells. A moisturizing agent can be
added to the cosmetic preparation. The carrier can contain
nutrients replenishing to the skin. The carrier can contain, for
example, a single or a plurality of anti-oxidant compounds.
DETAILED DESCRIPTION OF THE INVENTION
[0085] The present invention provides a method of increasing the
growth of stem cells. The method involves mixing the stem cells
with a growth medium that has been conditioned by an incubation
with placental tissue.
[0086] The invention disclosed herein teaches compositions and
methods relating to using the placenta as a potent source of stem
cell growth factors for the expansion of stem cells either in vitro
or in vivo. Additionally, the ability to almost block cells at a
specific stage of differentiation and allow their expansion through
administration of the compositions described herein, and
extrapolated upon by one skilled in the art will be very important
for the development of stem cell therapeutics as a discipline and
field of medicine.
[0087] Furthermore, there is a need for agents which, in addition
to increasing the rate of stem cell proliferation, also maintain
the stem cells in an undifferentiated state. This becomes
particularly apparent when one considers that, in general, stem
cells reside in unique physiological niches, and while growing
cells within mimics of such niches has been performed, the mimics
of the stem cell niche are often unusable in clinical situations.
An example of this is the fact that early hematopoietic stem cells
require feeder cell lines to be expanded in high quantities, or the
fact that optimal growth of embryonic stem cells is still primarily
achieved using murine feeders. The current invention teaches
methods and compositions for recreating conditions similar to stem
cell niches using approaches that are translatable into the
clinical situation.
General Information Regarding Stem Cell Expansion Techniques
[0088] As shown in the description herein, it becomes apparent that
methods of extracting, expanding and identifying specific
phenotypes of said stem cells is important for clinical
implementation. For example, bone marrow is commonly used as a
source of therapeutic stem cells for myocardial disease, angina,
and hematopoietic cell transplant. However, bone marrow in general
contains a wide number of different stem cells in addition to the
standard, well known, hematopoietic CD34+ stem cell. CD34-
hematopoietic stem cells (Bhatia, et al., 1998, Nat Med
4:1038-1045), oocyte generating stem cells (Johnson, et al., 2005,
Cell 122:303-315), mesenchymal stem cells (Dazzi, et al. The role
of mesenchymal stem cells in haemopoiesis. Blood Rev., e-published
on Dec. 15, 2005), and myogenic precursor cells (Bhagavati, et al.,
2004, Biochem Biophys Res Commun 318:119-124), each of which is
incorporated by reference herein in its entirety, have all been
found in the bone marrow, in addition to T cells, B cells, and
relatively high levels of CD4+ CD25+ T regulatory cells (Zeng, et
al., 2004, Transplantation 77:S9-S11, which is incorporated by
reference herein in its entirety). Given the heterogeneity of bone
marrow as a starting material for stem cell therapy, it is apparent
that understanding of particular cell populations, as well as
ability to isolate and expand them, would substantially advance the
field of stem cell therapeutics.
[0089] Accordingly, whether a stem cell population is derived from
adult or embryonic sources, the stem cells can be grown in a
culture medium to increase the population of a heterogeneous
mixture of cells, or a purified cell population. The cell growth
can be slow, however, and the cells can differentiate to unwanted
cell types during the culture period. Thus, methods of improving
the growth rate of stem cells, in general, and defined stem cell
populations in particular, will be useful for advancing the
clinical use of stem cells. Accordingly, what is needed is novel
methods of increasing the rate of expansion or growth of the stem
cells when grown in culture.
[0090] Several methods of growing stem cells outside of the body
have been developed and are known in the art. Originally, the
majority of work in the area of stem cell growth and expansion was
performed in the hematopoietic system using bone marrow cells. The
ability of either freshly isolated or cultured bone marrow cells to
form colonies on methylcellulose or agar was used as an output.
Colonies of hematopoietic stem cells were typically designated
based on cellular morphology into the broad subsets of colony
forming unit-erythroid (CFU-E), burst forming unit-erythroid
(BFU-E), colony forming unit-granulocytic monocytic (CFU-GM),
colony forming unit-monocytic (CFU-M), colony forming
unit-granulocytic (CFU-G), colony forming unit-granulocytic,
erthrocytic, monocytic, megakarocytic (CFU-E). Identification of
colonies was usually performed under light microscopy and allowed
quantification of relatively mature progenitor cells (Jacobs, et
al., 1979. Exp Hematol 7:177-182, which is incorporated by
reference herein in its entirety). Addition of biologically derived
supernatants to the semi-solid media was performed in order to
search for agents that preferentially enhanced formation of certain
types of colonies. For example, addition of leukocyte or monocyte
supernatant was shown to stimulate preferential growth of CFU-GM
colonies, indicating that this factor possesses similar activity in
vivo (Galbraith, et al., 1979, Can Med Assoc J 121:172-178, which
is incorporated by reference herein in its entirety). In influence
of in vivo situations on the ability of bone marrow to form CFU-E
was used in the identification of erythropoietin biological
activities (Peschle, et al., 1977, Br J Haematol 37:345-352, which
is incorporated by reference herein in its entirety). It is
generally regarded that the longer it takes to form colonies in the
semisolid media, and the larger the size and cellular constitution
of the colonies, the earlier in ontogeny the stem cell that
initiated the colony is. Unfortunately, multilineage stem cells
capable of producing other hematopoietic cells such as lymphocytes
could not be identified using such semisolid culture assays. The
long term culture (LTC) system was developed to detect earlier
progenitor cells then can be detected by semisolid assays. LTC
assays involve the use of pre-established layers of stromal cells
(ie bone marrow fibroblasts or transformed cytokine-secreting cell
lines) that provide signals for viability and proliferation of the
earlier progenitors. It is believed that the cell initiating the
LTC, the LTC initiating cell (LTC-IC) is an early precursor cell
capable of reconstituting a lethally irradiated mouse (Ploemacher,
et al., 1991, Blood 78:2527-2533, which is incorporated by
reference herein in its entirety). The LTC system has been
established as a method of assessing various growth factors and
combinations thereof for ability to expand hematopoietic stem cells
in vitro. The expansion activities of proliferin-2 (Choong, et al.,
2003, FEBS Lett 550:155-162), protease inhibitors (Isgro, et al.,
2005, AIDS Res Hum Retroviruses 21:51-57), PDGF (Su, et al., 2002,
Br J Haematol 117:735-746), each of which is incorporated by
reference herein in its entirety, as well as numerous other factors
was demonstrated using the LTC system. More direct evidence of
hematopoietic stem cell activity is derived from in vivo model
systems. The original identification of the colony forming
unit-spleen (CFU-S) was made by Till and McCulloch in 1964 through
the observation that transfer of bone marrow cells into lethally
irradiated mice gave rise to colonies comprising of multi-lineage
phenotypes (Till, et al., 1964, Proc Natl Acad Sci USA 51:29-36,
which is incorporated by reference herein in its entirety). These
cells demonstrated the ability for serial transplantation and
reconstitution of hematopoiesis, thereby suggesting a stem
cell-like characteristic (Siminovitch, et al., 1964, J Cell Physiol
64:23-31, which is incorporated by reference herein in its
entirety). Modern day implementation of this technique is the SCID
Repopulating Cell (SRC) assay whereby putative human hematopoietic
stem cells are transferred to an irradiated NOD-SCID recipient and
ability to reconstitute full hematopoiesis is observed (Larochelle,
et al., 1996, Nat Med 2:1329-1337, which is incorporated by
reference herein in its entirety). This is currently considered the
standard assay for hematopoietic stem cells, and it is currently
believed that 1 in 3 million bone marrow mononuclear cells have
this ability (Wang, et al., 1997, Blood 89:3919-3924, which is
incorporated by reference herein in its entirety). Assessment of
SRC numbers is routinely performed during evaluation of stem cell
expansion protocols (Kawada, et al., 1999, Exp Hematol 27:904-915;
Yamaguchi, et al., 2001, Exp Hematol 29:174-182, each of which is
incorporated by reference herein in its entirety).
[0091] The development of stem cell expansion techniques began with
work aimed at increasing the number of colonies formed on semisolid
media. Early experiments used a variety of uncharacterized sera and
conditioned media. For example, trophoblast cell line conditioned
media (Ferrero, et al., 1987, Cancer Res 47:6413-6417), xenogeneic
stromal cell conditioned media (Li, et al., 1987, Exp Hematol
15:373-381), supernatants from tumor cells (Tweardy, et al., 1987,
Ann N Y Acad Sci 511:30-38), and healthy lymphocytes were used
(Iscove, et al., 1971, Blood 37:1-5), each of which is incorporated
by reference herein in its entirety. Work was also performed
towards designing serum free systems, using ingredients such as
human transferrin and bovine insulin (Taketazu, et al., 1984,
Cancer Res 44:531-535, which is incorporated by reference herein in
its entirety). The realization that stem cells assessed by the
semisolid CFU assays are already differentiated, led to the use of
stromal feeder cells to allow expansion of LTC-IC as described
above. The specific advantage of the LTC system is that
hematopoietic stem cells could be expanded without concurrent
differentiation. The initial systems of LTC required the use of
murine feeder (stromal) cells since human lines had certain
disadvantages in terms of hematopoietic promoting activity (Petzer,
et al., 1996, Proc Natl Acad Sci USA 93:1470-1474, which is
incorporated by reference herein in its entirety). Numerous
drawbacks existed to the use of murine feeder cell lines to
maintain stem cell viability and proliferative potential. Due to
this, an effort was made to overcome difficulties in growth of
human derived feeder cells, and a variety of such cells have been
developed (Thalmeier, et al., 1994, Blood 83:1799-1807; Kohler, et
al., 1999, Stem Cells 17:19-24; Guo, et al., 2000, Zhongguo Shi Yan
Xue Ye Xue Za Zhi 8:93-96; De Angeli, et al., 2004, Int J Mol Med
13:363-371, each of which is incorporated by reference herein in
its entirety).
[0092] In terms of hematopoiesis, the role of the feeder cells is
to mimic the natural hematopoietic environment in the bone marrow
in which the hematopoietic stem cells reside in an area populated
by fibroblasts and other mesenchymal cells which present growth
factors directly and indirectly to the hematopoietic stem cell. In
this regard, the invention disclosed seeks to mimic the situation
of stem cell generation in general, and hematopoiesis specifically
in some embodiments, by taking advantage of a new method of
utilizing placental tissue. It is known that the placenta contains
endogenous multipotent stem cells characterized by markers such as
SSEA-4, TRA-1-61, TRA-1-80, CD105, endoglin, SH-2, SH-3, and SH-4
(Yen, et al., 2005, Stem Cells 23:3-9), as well as mesenchymal
(Wulf, et al., 2004, Tissue Eng 10:1136-1147) and hematopoietic
stem cells (Gekas, et al., 2005, Dev Cell 8:365-375, Fauza, D.,
2004, Best Pract Res Clin Obstet Gynaecol 18:877-891), each of
which is incorporated by reference herein in its entirety.
Furthermore, the use of cord blood cells from placentas is an
established clinical treatment for a wide variety of diseases and
medical situations (Chao, et al., 2004, Hematology (Am Soc Hematol
Educ Program):354-371, which is incorporated by reference herein in
its entirety). The interest in the placenta is due not only to the
finding of such cells, but the possibility that the presence of
these cells has some biological ramifications. The presence of a
variety of different stem cell tissues in the placenta at high
concentrations suggest that certain factors are present within it
that are hospitable for stem cell growth in a natural milieu.
Indeed, it was recently published that during development, the
placenta acts as a depot for hematopoiesis, much in a similar way
how the liver performs this function during fetal development
(Ottersbach, et al., 2005, Dev Cell 8:377-387; Li, L., 2005, Dev
Cell 8:297-298, each of which is incorporated by reference herein
in its entirety).
[0093] The ease of harvesting placental material, as well as
ability to maintain its viability as a three dimensional structure,
allows for various manipulations and extraction of trophic factors
from it. The placenta is the only stem cell bearing organ that can
be continually perfused while maintaining structural integrity in a
clinically feasible manner. The use of factors derived from
placenta have potent applications not only for ex vivo expansion of
cells for therapeutic purposes, but also for in vivo expansion of
stem cells. It is known in a wide variety of disease conditions
that stem cells are mobilized from various pools and go into the
source of injury (Francois, supra; Wojakowski, et al., 2005, Folia
Histochem Cytobiol 43:229-232; Claps, et al., 2005, Curr Neurovasc
Res 2:319-329; Abbott, et al., 2004, Circulation 110:3300-3305,
each of which is incorporated by reference herein in its entirety).
Unfortunately, unlike the amphibians who are capable of limb
regeneration through production of a high concentration of stem
cells (blastema) (Brockes, et al., 2005, Science 310:1919-1923,
which is incorporated by reference herein in its entirety), human
stem cells only make what is considered to be a relatively
therapeutic "spark" in absence of exogenous support growth factor
support. Furthermore, it is known that higher concentrations of
progenitor cells are associated with scarless healing in certain
mouse strains (Davis, et al., 2005, Blood Cells Mol Dis 34:17-25,
which is incorporated by reference herein in its entirety), and
possibly account for this phenomena in human fetuses (Howell, L.
J., 1994, Nurs Clin North Am 29:681-694, which is incorporated by
reference herein in its entirety). Therefore, many investigators
have attempted to use a variety of growth factors in order to
induce stem cell mobilization and then expansion of the mobilized
stem cells in order to evoke the healing processes, or to transform
the "spark" into an "explosion." Although this was demonstrated by
mobilization of stem cells using G-CSF in patients with myocardial
infarction, the results have accomplished only moderate success
(Powell, et al., 2005, Arterioscler Thromb Vasc Biol 25:296-301;
Belenkov Iu, et al., 2003, Kardiologiia 43:7-12, each of which is
incorporated by reference herein in its entirety). Accordingly,
there is a need for a multifactorial stimulator of stem cells that
is active both in vivo as well as in vitro.
Preparation of Stem Cells to be Expanded
[0094] The term "stem cell" generally refers to any cells that have
the ability to divide for indefinite periods of time and to give
rise to specialized cells. Within the definition of "stem cell" we
include but not limit, to the following: a) totipotent cells such
as an embryonic stem cell, an extra-embryonic stem cell, a cloned
stem cell, a parthenogenesis derived cell, a cell reprogrammed to
possess totipotent properties, or a primordial germ cell; b)
Pluripotent cell such as a hematopoietic stem cell, an adipose
derived stem cell, a mesenchymal stem cell, a cord blood stem cell,
a placentally derived stem cell, an exfoliated tooth derived stem
cells, a hair follicle stem cell or a neural stem cell; and c) A
tissue specific progenitor cell such as a precursor cell for the
neuronal, hepatic, nephrogenic, adipogenic, osteoblastic,
osteoclastic, alveolar, cardiac, intestinal, or endothelial
lineage. The cells can be derived, for example, from tissues such
as pancreatic tissue, liver tissue, smooth muscle tissue, striated
muscle tissue, cardiac muscle tissue, bone tissue, bone marrow
tissue, bone spongy tissue, cartilage tissue, liver tissue,
pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus
tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue,
epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue,
lung tissue, vascular tissue, endothelial tissue, blood cells,
bladder tissue, kidney tissue, digestive tract tissue, esophagus
tissue, stomach tissue, small intestine tissue, large intestine
tissue, adipose tissue, uterus tissue, eye tissue, lung tissue,
testicular tissue, ovarian tissue, prostate tissue, connective
tissue, endocrine tissue, and mesentery tissue.
[0095] The stem cells to be expanded can be isolated from any organ
of any mammalian organism, by any means known to one of skill in
the art. The stem cells can be derived from embryonic or adult
tissue. One of skill of the art can determine how to isolate the
stem cells from the particular organ or tissue of interest, using
methods known in the art. In a preferred embodiment, the stem cells
are isolated from umbilical cord blood. Example 2 describes a
typical method that can be used to isolate stem cells from
umbilical cord blood. In this example, the stem cell marker CD34 is
used to enrich the stem cell population, using antibodies to
CD34.
[0096] The stem cell populations can also be enriched using
antibodies to other stem cell surface markers. Such markers
include, but are not limited to, FLK-1, AC133, CD34, c-kit, CXCR-4,
Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3,
SH-4, TRA-1-60, TRA-1-81, SSEA-4, Sox-2, and the like. One of skill
in the art will be able to determine the specific cell marker
useful for isolating stem cells from the desired tissue.
[0097] One of skill in the art will be able to determine a suitable
growth medium for initial preparation of stem cells. Commonly used
growth media for stem cells includes, but is not limited to,
Iscove's modified Dulbecco's Media (IMDM) media, DMEM, KO-DMEM,
DMEM/F12, RPMI 1640 medium, McCoy's 5A medium, minimum essential
medium alpha medium (.alpha.-MEM), F-12K nutrient mixture medium
(Kaighn's modification, F-12K), X-vivo 20, Stemline, CC100, H2000,
Stemspan, MCDB 131 Medium, Basal Media Eagle (BME), Glasgow Minimum
Essential Media, Modified Eagle Medium (MEM), Opti-MEM I Reduced
Serum Media, Waymouth's MB 752/1 Media, Williams Media E, Medium
NCTC-109, neuroplasma medium, BGJb Medium, Brinster's BMOC-3
Medium, CMRL Medium, CO.sub.2-Independent Medium, Leibovitz's L-15
Media, and the like.
[0098] If desired, other components, such as growth factors, can be
added as desired. Exemplary growth factors and other components
that can be added include but are not limited to thrombopoietin
(TPO), stem cell factor (SCF), IL-1, IL-3, IL-7, flt-3 ligand
(flt-3L), G-CSF, GM-CSF, Epo, FGF-1, FGF-2, FGF-4, FGF-20, IGF,
EGF, NGF, LIF, PDGF, bone morphogenic proteins (BMP), activin-A,
VEGF, forskolin, glucocorticords, and the like. Furthermore, the
media can contain either serum such as fetal calf, horse, or human
serum, or more preferably, serum substitution components. Numerous
agents have been introduced into media to alleviate the need for
serum. For example, serum substitutes have included bovine serum
albumin (BSA), insulin, 2-mercaptoethanol and transferrin (TF).
[0099] The stem cells can then be stored for a desired period of
time, if needed. Stem cell storage methods are known to those of
skill in the art. Typically, the stem cells are treated to a
cryoprotection process, then stored frozen until needed.
Cryopreservation requires attention be paid to three main concepts,
these are: 1) The cryoprotective agent, 2) the control of the
freezing rate, and 3) The temperature at which the cells will be
stored at. Cryoprotective agents are well known to one skilled in
the are and can include but are not limited to dimethyl sulfoxide
(DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol,
albumin, dextran, sucrose, ethylene glycol, i-erythritol,
D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, or
choline chloride as described in U.S. Pat. No. 6,461,645, which is
incorporated by reference herein in its entirety. A method for
cryopreservation of stem cells that is preferred by some skilled
artisans is DMSO at a concentration not being immediately cytotoxic
to cells, under conditions which allow it to freely permeate the
cell whose freezing is desired and to protect intracellular
organelles by combining with water and prevent cellular damage
induced from ice crystal formation. Addition of plasma at
concentrations between 20-25% by volume can augment the protective
effect of DMSO. After addition of DMSO, cells can be kept at
temperatures below 4.degree. C., in order to prevent DMSO mediated
damage. Methods of actually inducing the cells in a state of
suspended animation involve utilization of various cooling
protocols. While cell type, freezing reagent, and concentration of
cells are important variables in determining methods of cooling, it
is generally accepted that a controlled, steady rate of cooling is
optimal. There are numerous devices and apparatuses known in the
field that are capable of reducing temperatures of cells for
optimal cryopreservations. One such apparatus is the Thermo Electro
Cryomed Freezer.TM. manufactured by Thermo Electron Corporation.
Cells can also be frozen in CryoCyte.TM. containers as made by
Baxter. One example of cryopreservation is as follows:
2.times.10.sup.6 CD34 cells/ml are isolated from cord blood using
the Isolex System.TM. as per manufacturer's instructions (Baxter).
Cells can be incubated in DMEM media with 10% DMSO and 20% plasma.
Cooling is generally performed at 1.degree. C./minute from 0 to
-80.degree. C. When cells are needed for use, they can be thawed
rapidly in a water bath maintained at 37.degree. C. water bath and
chilled immediately upon thawing. The cells can then be rapidly
washed, using, for example, either a buffer solution, or a solution
containing a growth factor. Purified cells can then be used for
expansion with LPCM. A database of stored cell information (such as
donor, cell origination types, cell markers, etc.) can also be
prepared, if desired. Further, the stem cells can be obtained, if
desired, from a library of publicly available stored stem cells,
including the National Institute of Health or American Type Culture
Collection.
[0100] The stem cells can be purified prior to contacting the LPCM
by methods known in the art, using, for example, antibody
technology such as panning of cells, through the use of
fluorescence activated cell sorting (FACS) methods, or magnet
activated cell sorting methods such as that MACS apparatus, to
isolate cells having the desired stem cell markers, or to remove
unwanted, contaminating cell types having unwanted cell markers
prior to contacting with LPCM. Other methods of stem cell
purification or concentration can include the use of techniques
such as counterflow centrifugal elutriation, equilibrium density
centrifugation, velocity sedimentation at unit gravity, immune
rosetting and immune adherence, T lymphocyte depletion. Examples of
stem cell markers that can be useful in purification include, but
are not limited to, FLK-1, AC133, CD34, c-kit, CXCR-4, Oct-4,
Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3, SH-4,
TRA-1-60, TRA-1-81, SSEA-4, Sox-2, and the like. Examples of cell
surface markers that can be used as markers of contaminating,
unwanted cell types depends on the stem cell phenotype sought. For
example, if collection of pluripotent hematopoietic cells is
desired, contaminating cells will possess markers of commitment to
the differentiated hematopoietic cells such as CD38 or CD33.
Additionally, non-hematopoietic cell contamination would be
detected by lack of CD45 expression. If selection of stromal
mesenchymal cells is desired, then contaminating cells would be
detected by expression of hematopoietic markers such as CD45.
Additionally, stem cells can be purified based on properties such
as size, density, adherence to certain substrates, or ability to
efflux certain dyes such as Hoechst 33342 or Rhodamine 123.
[0101] The stem cells can be genetically modified at any stage of
the preparation. For example, a gene encoding a selectable marker
or other gene of interest can be introduced to the prepared stem
cells.
[0102] To increase the growth of the stem cells, the stem cells are
mixed with medium that has been incubated with placental tissue. By
"incubation" any type of contact between said media and placental
tissue or cells thereof is implied. The method described herein
provides for a method of expansion of stem cells that in one
embodiment does not require undefined culture medium. Further, the
method described herein does not require a culture medium that
contains animal products with unknown negative effects, such as,
for example, increased antigenicity, or xoonosis.
Preparation and Incubation of the Placental Tissue
[0103] The placental tissue can be derived from fresh sources, or
can have been stored prior to use. Storage can take place with the
placenta as an intact unit, or a deaggrated unit, or as dissociated
placental cells. One of skill in the art can determine the
incubation medium to place the placental tissue in. Any suitable
liquid can be used, such as for example a buffer, water such as
isotonic water, or growth medium. Exemplary media include, but are
not limited to, Iscove's modified Dulbecco's Media (IMDM) media,
DMEM, KO-DMEM, DMEM/F12, RPMI 1640 medium, McCoy's 5A medium,
minimum essential medium alpha medium (.alpha.-MEM), F-12K nutrient
mixture medium (Kaighn's modification, F-12K), X-vivo 20, Stemline,
CC100, H2000, Stemspan, MCDB 131 Medium, Basal Media Eagle (BME),
Glasgow Minimum Essential Media, Modified Eagle Medium (MEM),
Opti-MEM I Reduced Serum Media, Waymouth's MB 752/1 Media, Williams
Media E, Medium NCTC-109, neuroplasma medium, BGJb Medium,
Brinster's BMOC-3 Medium, CMRL Medium, CO2-Independent Medium, and
Leibovitz's L-15 Media or other liquid as determined by one of
skill in the art.
[0104] Any part of the placental tissue can be used for the
incubation process. For example, the whole placenta can be
incubated by submergence in the media which is desired to be
conditioned, alternatively the incubation can occur by perfusion of
the placenta with media which is desired to be conditioned.
Perfusion can be performed through the fetal circulatory system via
the umbilical vein and arteries, or through the maternal side in
distinct placental cotyledons, or in a manner encompassing all of
the maternal circulation. In a preferred embodiment the fetal
circulation is perfused. Furthermore, distinct sections of the
placenta can be used for the incubation. The sections can include
but are not limited to isolated chorionic plate, chorionic villi,
Wharton's Jelly, amniotic membranes, chorionic membranes or
cotyledon units. Furthermore, distinct placentally derived cells
can be isolated and used for incubation. Said cells include but are
not limited to endothelial, epithelial, trophoblastic, macrophages,
and mesenchymal cells. The placental tissue can be rinsed with an
anticoagulation solution prior to incubation, if desired. Exemplary
anticoagulation solutions include but are not limited to saline, a
buffer, or media mixed with heparin, EDTA, antithrombin III, and
the like.
[0105] The placental tissue to be used can be derived from any
mammalian organism. Preferably, the placental tissue is derived
from a human.
[0106] The placental incubation period can be determined by one of
skill in the art. Generally, the placental incubation period can
range from less than about 1 second, 30 seconds, or 60 seconds to
about 2 or 3 weeks or more. Preferably, the placental incubation
period is between about 2, 5, 10, 30, or 45 minutes to about 12,
14, 16, 18, or 20 days. More preferably, the placental incubation
period is between about 1, 3, 5, 8, or 24 hours to about 3, 5, 7,
or 10 days.
[0107] The preferred temperature for the placental incubation
process can be from a range of about 32.degree. C. or less to about
40.degree. C. or more. Preferably, the placental incubation occurs
at a temperature of about 33.degree. C., 34.degree. C., or
35.degree. C. to about 38.degree. C., 39.degree. C., or 40.degree.
C. More preferably, the placental incubation occurs at about
37.degree. C. The incubation medium can be changed regularly, if
desired.
[0108] The placental incubation medium can be mixed, if desired, at
any suitable speed. Any suitable container can be used.
[0109] Antibiotics, antifungals or other contamination preventive
compounds can be added to the incubation medium, if desired.
Exemplary compounds include but are not limited to penicillin,
streptomycin, gentamycin, fungizone or others known in the art.
[0110] The live placenta conditioned medium (LPCM) so produced can
then be filtered, if desired. Preferably, the filtration process
occurs through a sterile 0.2 .mu.m filter. Additionally, other
types of filters can be used depending on the desired sterility of
the LPCM. In some situations filters with nano-sized pores can be
useful in order to prevent viral contamination. Additionally,
methods known in the art for decontamination can be used such as UV
irradiation, X-ray sterilization, ozonation, or hyperthermia in
order to selectively destroy potential contaminants without losing
the desired biological activity of the LPCM. An example of the
placental incubation process is shown in Example 1.
[0111] The LPCM can be stored in a variety of manners prior to use,
this includes lyophilized, frozen, stored under refrigerated
conditions, stored in combination with a preservative agent, or by
other means known to one skilled in the art. It is desired that the
storage step does not effect properties that fresh LPCM would
bestow during incubation with stem cells.
Contacting the Stem Cells with the LPCM
[0112] The prepared stem cells can be contacted with the LPCM. This
can be done, for example, by simply mixing the LPCM with the
culture of stem cell preparations. Mixing can be performed in a
plethora of suitable vessels capable of maintaining viability of
the stem cells. Said vessels can include but are not limited to
tissue culture flasks, conical tubes, culture bags, bioreactors, or
cultures that are continuously mixed. The stem cell/LPCM mixture
can then be allowed to grow as desired. An example of stem cell
growth in the stem cell/LPCM mixture is shown in Example 3. In some
situations it will be desirable to use a combination culture system
in which cells are first grown with one type of culture condition,
then subsequently another culture condition is used. For example,
when rapid expansion of hematopoietic stem cells is needed without
differentiation, cells can be cultured initially in a high
concentration of LPCM for 48 hours, or a time period needed to
induce cycling of the stem cells. Subsequently, media containing
cytokines can be added in the culture for passages after the first
48 hours. Cytokine media can be DMEM supplemented with a
combination of IL-3, IL-6, SCF, TPO, and flt-3L. One skilled in the
art will understand that depending on stem cell type and level of
differentiation desired, different concentrations of LPCM can be
added at the different time points of the culture. For example, in
a particular culture situation, addition of LPCM at the initiation
of culture can not be optimum. In the case that cardiomyocytes are
desired from embryonic stem cells, addition of LPCM to the
embryonic stem cell culture will only increase the number of
undifferentiated stem cells and not allow cardiomyocyte
differentiation, even if differentiation-promoting stimuli are
added. Accordingly, the optimum use of LPCM in this situation is
after differentiation, or partial differentiation along the
cardiomyocyte lineage has occurred. In a practical example, human
embryonic stem cells are cultured on mitotically inactivated
(mitomycin C) murine embryonic feeder layers in culture medium
consisting of 80% knockout DMEM (no-pyruvate, high-glucose
formulation; Life Technologies Inc., Rockville, Md., USA)
supplemented with 20% FBS (HyClone, Logan, Utah, USA), 1 mM
L-glutamine, 0.1 mM mercaptoethanol, and 1% nonessential amino acid
stock (all from Life Technologies Inc). If LPCM is added (at
concentrations ranging from about 2-10 Units/ml, Example 4) to this
culture, there is an accelerated growth of embryonic stem cells but
no differentiation.
[0113] In order to induce cardiomyocyte generation embryonic stem
cells are generally dispersed to small clumps (three to 20 cells)
using collagenase IV (Life Technologies Inc.; at a concentration of
about 1 mg/ml for 20 minutes). Cells can then be transferred to
plastic Petri dishes (Miniplast, Ein Shemer, Israel), at a cell
density of about 5.times.10.sup.6 cells in a 58-mm dish, where they
are cultured in suspension (using same media as above) for 7-10
days. During this stage, the cells aggregated to form embryoid
bodies, which are then plated on 0.1% gelatin-coated culture
dishes, at a density of one to five embryoid in a 1.91-cm.sup.2
well, and observed microscopically for the appearance of
spontaneous contractions. If LPCM (2-10 Units/ml) is added at the
period of day 1-5 of liquid culture, an increased cellularity is
observed but a significant decrease in spontaneously beating
embryoids is observed in the gelatin coated dishes. In contrast, if
LPCM is added at days 6-7 of the liquid culture, then the number of
contracting embryoids on gelatin is generally increased about
4-fold compared to cultures lacking LPCM. This illustrates that
under some culture conditions LPCM is capable of inducing
proliferation of cells at a specific level of differentiation, and
that in some circumstances LPCM can actually inhibit
differentiation even in the presence of conditions that would
normally stimulate it.
[0114] The desired ratio of stem cells to LPCM can be determined by
one of skill in the art. For example, a ratio of less than about
1:1,000, to 1,000:1 or more (stem cell preparation to LPCM) can be
used. For example, a ratio of stem cell preparation to LPCM from
about 1:750, 1:500, 1:250, or 1:100 to about 100:1, 250:1, 500:1,
or 750:1 can be used. This ratio can vary, for example, depending
on temperature, incubation time, number of stem cells, the desired
activity sought in the stem cells, the type of stem cells, the
purity of stem cells, the amount of placental tissue used as a
starting point, and the like. The stem cells can be isolated from
their growth media prior to contacting with the LPCM, or the stem
cells can remain in their growth medium, with the LPCM added.
[0115] The length of the stem cell/LPCM contacting step can be
determined by one of skill in the art. Generally, the contacting
step can range from less than about 1 second, 30 seconds, or 60
seconds to about 2 or 3 weeks or more. Preferably, the contacting
step is between about 2, 5, 10, 30, or 45 minutes to about 12, 14,
16, 18, or 20 days. More preferably, the contacting step is between
about 1, 3, 5, 8, or 24 hours to about 3, 5, 7, or 10 days.
[0116] In some embodiments, other compounds can be added to the
stem cell/LPCM mixture. For example, growth factors can be added to
the mixture. Exemplary factors include but are not limited to
thrombopoietin (TPO), stem cell factor (SCF), IL-1, IL-3, IL-7,
flt-3 ligand (flt-3L), G-CSF, GM-CSF, Epo, FGF-1, FGF-2, FGF-4,
FGF-20, IGF, EGF, NGF, LIF, PDGF, bone morphogenic proteins (BMP),
activin-A, VEGF, forskolin, glucocorticoids, and the like. Specific
concentrations and activities are known to one skilled in the art.
For reference as to applicability to stem cell, the practitioner of
the invention is referred to the following publications: TPO
(Kawada, et al., 1999, Exp Hematol 27:904-915; Wang, et al., 2005,
Ann N Y Acad Sci 1044:29-40; Xie, et al., 2003, Blood
101:1329-1335; Feugier, et al., 2002, J Hematother Stem Cell Res
11:127-138; Won, et al., 2000, J Hematother Stem Cell Res
9:465-473), SCF (Wang, et al., 2005, Cell Biol Int 29:654-661;
Levac, et al., 2005, Haematologica 90:166-172; Peschle, et al.,
1993, Stem Cells 11:356-370), IL-1 (Maurer, et al., 2000, Int J
Hematol 71:203-210; Willems, et al., 2001. Ann Hematol 80:17-25;
Scheding, et al., 2000, Exp Hematol 28:460-470), IL-3 (Ivanovic,
Z., 2004, Eur Cytokine Netw 15:6-13; Inderbitzin, et al., 2005, J
Gastrointest Surg 9:69-74; Bohmer, R. M., 2004, Stem Cells
22:216-224), IL-6 (Quesenberry, et al., 1991, J Cell Biochem
45:273-278; Zhang, et al. Increased myelinating capacity of
embryonic stem cell derived oligodendrocyte precursors after
treatment by interleukin-6/soluble interleukin-6 receptor fusion
protein. Mol Cell Neurosci., e-published on Nov. 30, 2005; Taga, et
al., 2005, Clin Rev Allergy Immunol 28:249-256; Nakamura, et al.,
2005, Clin Rev Allergy Immunol 28:197-204), IL-7 (Ficara, et al.,
2004, Mol Ther 10:1096-1108; Krawczenko, et al., 2005, Arch Immunol
Ther Exp (Warsz) 53:518-525; Andre-Schmutz, et al., 2004, Br J
Haematol 126:844-851; De Waele, et al., 2004, Eur J Haematol
72:193-202), IL-11 (Willems, supra; Lu, et al., 2003, Zhonghua Xue
Ye Xue Za Zhi 24:589-592; Momose, et al., 2002,
Arzneimittelforschung 52:857-861; Van der Meeren, et al., 2002,
Radiat Res 157:642-649), flt-3L (Li, et al., 2005, Eur J Haematol
74:128-135; McGuckin, et al., 2004, Cell Prolif 37:295-306; Lu, et
al., Blood 103:4134-4141; Streeter, et al., 2003, Exp Hematol
31:1119-1125), G-CSF (Aliotta, et al., 2006, Exp Hematol
34:230-241; Jung, et al. Granulocyte colony-stimulating factor
stimulates neurogenesis via vascular endothelial growth factor with
STAT activation. Brain Res., e-published on Jan. 16, 2006; Kogler,
et al., 2005, Exp Hematol 33:573-583), GM-CSF (Quesenberry, supra;
Gangenahalli, et al., 2005, Stem Cells Dev 14:140-152), Epo (Otani,
et al., 2004, Exp Hematol 32:607-613; Yao, et al., 2000, Bone
Marrow Transplant 26:497-503; Mobest, et al., 1998, Biotechnol
Bioeng 60:341-347), FGF-1 (de Haan, supra; Crcareva, et al., 2005,
Exp Hematol 33:1459-1469), FGF-2 (Ratajczak, et al., 1996, Br J
Haematol 93:772-782; Kang, et al., 2005, Stem Cells Dev
14:395-401), FGF-4 (Schwartz, et al., 2005, Stem Cells Dev
14:643-655; Quito, et al., 1996, Blood 87:1282-1291), FGF-20
(Grothe, et al., 2004, Neurobiol Dis 17:163-170), IGF (McDevitt, et
al., 2005, J Mol Cell Cardiol 39:865-873; Musaro, A., 2005, Arch
Ital Biol 143:243-248; Zumkeller, et al., 1999, Blood 94:3653-3657;
Okajima, et al., 1998, J Biol Chem 273:22877-22883), EGF (Miyazaki,
et al., 2004, Cell Transplant 13:385-391; von Ruden, et al., 1988,
Embo J 7:2749-2756), NGF (Bracci-Laudiero, et al., 2003, J
Neuroimmunol 136:130-139; Simone, et al., 1999, Hematol Oncol
17:1-10), LIF (Guo, et al. Murine Embryonic Stem Cells Secrete
Cytokines/Growth Modulators that Enhance Cell
Survival/Anti-Apoptosis and Stimulate Colony Formation of Murine
Hematopoietic Progenitor Cells. Stem Cells, e-published on Dec. 8,
2005; Chodorowska, et al., 2004, Ann Univ Mariae Curie Sklodowska
[Med] 59:189-193), PDGF (Su, et al., 2005, Stem Cells Dev
14:223-230; Lucarelli, et al., 2003, Biomaterials 24:3095-3100;
Yang, et al., 1995, Br J Haematol 91:285-289), BMPs (Ploemacher, et
al., 1999, Leukemia 13:428-437; Zhang, et al., 2005, Dev Biol
284:1-11; Jay, et al., 2004, Cell Res 14:268-282; Chadwick, et al.,
2003, Blood 102:906-915; Dormady, et al.; 2001, J Hematother Stem
Cell Res 10:125-140), activin-A (Shav-Tal, et al., 2002, Stem Cells
20:493-500), VEGF (Cerdan, et al., 2004, Blood 103:2504-2512),
forskolin (Laharrague, et al., 1998, Faseb J 12:747-752; Gaspar
Elsas, et al., 2000, Br J Pharmacol 130:1362-1368), and
glucocorticoids (Grafte-Faure, et al., 1999, Am J Hematol
62:65-73), each of which is incorporated by reference herein in its
entirety.
[0117] Furthermore, conditions promoting certain type of cellular
proliferation or differentiation can be used during the culture.
These conditions include but are not limited to, alteration in
temperature, alternation in oxygen/carbon dioxide content,
alternations in turbidity of said media, or exposure to small
molecules modifiers of cell cultures such as nutrients, inhibitors
of certain enzymes, stimulators of certain enzymes, inhibitors of
histone deacetylase activity such as valproic acid (Bug, et al.,
2005, Cancer Res 65:2537-2541), trichostatin-A (Young, et al.,
2004, Cytotherapy 6:328-336), trapoxin A (Kijima, et al., 1993, J
Biol Chem 268:22429-22435), or Depsipeptide (Gagnon, et al., 2003,
Anticancer Drugs 14:193-202; Fujieda, et al., 2005, Int J Oncol
27:743-748), each of which is incorporated by reference herein in
its entirety, inhibitors of DNA methyltransferase activity such as
5-azacytidine, inhibitors of the enzyme GSK-3 (Trowbridge, et al.,
2006, Nat Med 12:89-98, which is incorporated by reference herein
in its entirety), and the like.
[0118] A variety of factors previously mentioned influence ability
of stem cells to survive, replicate, and differentiate. For
example, in terms of nutrients the amino acid taurine under certain
conditions preferentially inhibits murine bone marrow cells from
forming osteoclasts (Koide, et al., 1999, Arch Oral Biol
44:711-719), the amino acid L-arginine stimulates erythrocyte
differentiation and proliferation of erythroid progenitors (Shima,
et al., 2006, Blood 107:1352-1356), extracellular ATP acting
through P2Y receptors mediates a wide variety of changes to both
hematopoietic and non-hematopoietic stem cells (Lee, et al., 2003,
Genes Dev 17:1592-1604), arginine-glycine-aspartic acid attached to
porous polymer scaffolds increase differentiation and survival of
osteoblast progenitors (Hu, et al., 2003, J Biomed Mater Res A
64:583-590), each of which is incorporated by reference herein in
its entirety. Accordingly, one skilled in the art would know to use
various types of nutrients for inducing differentiation, or
maintaining viability, of certain types of stem cells and/or
progeny thereof.
[0119] The role of oxygen tension in stem cell self-renewal and
viability is also an important issue that is contemplated in the
current invention. It is known that hematopoietic stem cells tend
to reside in hypoxia niches of the bone marrow and that as cells
differentiate into more mature progeny, they progressively migrate
to areas of the bone marrow with higher oxygen tension (Ivanovic,
et al., 2002, Exp Hematol 30:67-73, which is incorporated by
reference herein in its entirety). This important variable in
tissue culture was exploited in studies showing that superior
expansion of human CD34 stem cells capable of full hematopoietic
reconstitution of NOD-SCID mice were obtained in hypoxic conditions
using oxygen tension as low as 1.5%. The potent expansion under
hypoxia, which was 5.8-fold higher as compared to normal oxygen
tension, was attributed to hypoxia induction of HIF-1 dependent
growth factors such as VEGF (Danet, et al., 2003, J Clin Invest
112:126-135, which is incorporated by reference herein in its
entirety). Additionally, other stem cells such as neuronal stem
cells also appear to be localized in hypoxic niches and expand
preferential in low oxygen in vitro conditions as opposed to normal
oxygen tension (Zhu, et al., 2005, Mol Neurobiol 31:231-242, which
is incorporated by reference herein in its entirety). Furthermore,
embryonic stem cells, although grow at similar proliferative rates
between normoxia and hypoxia, they retain superior ability to form
teratomas in vivo and embryoid bodies in vitro when grown under
hypoxic conditions (Ezashi, et al., 2005, Proc Natl Acad Sci USA
102:4783-4788, which is incorporated by reference herein in its
entirety).
[0120] Accordingly, one embodiment of the disclosed invention is
the use of hypoxic conditions for augmenting release of stem cell
proliferating factors in the placenta during production of LPCM.
Hypoxic conditions can be maintained in specialized incubators with
an oxygen tension ranging from 0.1% to 7.5%, preferably 0.5% to 5%,
more preferably 3%-5%. Additionally, another embodiment of the
invention is the use of hypoxic conditions in combination with LPCM
in order to enhance proliferation without differentiation of stem
cells being grown in culture.
[0121] In terms of enhancing the ability of LPCM to stimulate
proliferation of stem cells without differentiation, one adjuvant
approach that is considered an embodiment of the invention is the
use of enzymatic inhibitors in conjunction with LPCM. For example,
histone deacetylases are a class of enzymes involved in
epigenetically opening parts of chromatin to transcription factors,
thus allowing expression of genes that under normal adult
conditions would not be expressed. For example, the telomerase gene
(catalytic subunit hTERT) is involved in the process of cellular
immortalization and is expressed under physiological conditions
only in embryonic stem cells, as well as some bone marrow
hematopoietic cells, abnormally. The functional role of the
telomerase enzyme is to repair the shortened telomeric ends of
chromosomes so that cells can escape replicative senescence.
Pathologically, telomerase is the enzyme responsible for the
ability of cancer cells to proliferate indefinitely in cell
culture. Under normal physiological conditions fibroblasts do not
express telomerase and undergo replicative senescence. A variety of
reports have been published describing that treatment of
fibroblasts with histone deacetylase inhibitors such as
trichostatin A reinduces expression of functional telomerase
(Mukhopadhyay, et al., 2005, J Cell Mol Med 9:662-669; Hou, et al.,
2002, Exp Cell Res 274:25-34; Cong, et al., 2000, J Biol Chem
275:35665-35668, each of which is incorporated by reference herein
in its entirety). This is suggestive that manipulating the histone
deacetylase pathway can be used as a method of de-differentiating
cells or offering the possibility of "rejuvenating" progenitors
that are nearing replicative exhaustion. Indeed, it was
demonstrated that the life extension effect observed due to caloric
restriction is connected to the histone deacetylase pathway
(Howitz, et al., 2003, Nature 425:191-196, which is incorporated by
reference herein in its entirety). The clinical relevance of
manipulating this pathway is illustrated in experiments with
valproic acid, an antidepressant that is in clinical use is a
histone deacetylase inhibitor with similar potency to trichostatin
A in some models. It was demonstrated that treatment of bone marrow
derived hematopoietic stem cells with valproic acid increases both
proliferation and self-renewal through accelerating cell cycle
progression (Bug, supra). Said acceleration was accompanied by a
down-regulation of inhibitor factor p21(cip-1/waf-1). Furthermore,
valproic acid treatment suppressed GSK3 activity and activated the
Wnt signaling pathway, both of which are associated with self
renewal in both hematopoietic (Gotoh, et al., 1997, Cell Growth
Differ 8:721-729; Baba, et al., 2005, Immunity 23:599-609, each of
which is incorporated by reference herein in its entirety), but
also embryonic (Sato, et al., 2004, Nat Med 10:55-63; He, et al.,
2005, Clin Lung Cancer 7:54-60, each of which is incorporated by
reference herein in its entirety) stem cells. The potency of
valproic acid to synergize with known hematopoietic stem cell
stimulatory cytokines such as Flt3L, TPO, SCF and IL-3 was
demonstrated (De Felice, et al., 2005, Cancer Res 65:1505-1513,
which is incorporated by reference herein in its entirety).
[0122] Based on the above discussion, it is apparent to one skilled
in the art that combinations of histone deacetylase inhibitors in
conjunction with LPCM can be useful for expansion of stem cells not
only in vitro, but also in vivo. For example, in an embodiment of
the current invention, the histone deacetylase inhibitor valproic
acid is administered at a concentration ranging from 20 mg/day to
1,500 mg/day on a daily basis in conjunction with 20-500 Units of
LPCM per day in a patient in whom hematopoietic reconstitution is
sought. More preferably, a dose of 150 mg/day to 1,000 mg/day is
given in conjunction with a dose of 100-300 Units of LPCM/day, even
more preferably, a dose of 750 mg/day of valproic acid is given in
conjunction with 250 Units of LPCM. One skilled in the art will
understand to vary the dose based on certain characteristics of the
patient, such as tolerability to valproic acid, as well as amount
and rapidity of hematopoietic reconstitution that is required.
Similar treatments can be used for enhancing the proliferation and
expansion of endogenous stem cells in diseased situations. For
example, patients suffering from a stem cell insufficiency in
smooth muscle tissue, striated muscle tissue, cardiac muscle
tissue, bone tissue, bone spongy tissue, cartilage tissue,
pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil
tissue, Peyer's patch tissue, lymph nodes tissue, thyroid tissue,
epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue,
lung tissue, vascular tissue, endothelial tissue, blood cells,
bladder tissue, kidney tissue, digestive tract tissue, esophagus
tissue, stomach tissue, small intestine tissue, large intestine
tissue, adipose tissue, uterus tissue, eye tissue, lung tissue,
testicular tissue, ovarian tissue, prostate tissue, connective
tissue, endocrine tissue, and mesentery tissue can be treated with
an agent that mobilizes endogenous stem cells, such as G-CSF,
GM-CSF, or antagonist of CXCR-4, in combination with a histone
deacetylase inhibitor and LPCM. Additionally, local concentrations
of LPCM can be added in combination with a histone deacetylase
inhibitor in the tissue in need thereof. Localization can be
achieved through the use of certain delivery polymers known to one
who is skilled in the art. These can include, but are not limited
to, polyvinyl chloride, polylactic acid (PLA), poly-L-lactic acid
(PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid
(PGA), polylactide-co-glycolide (PLGA), polydioxanone,
polygluconate, polylactic acid-polyethylene oxide copolymers,
polyethylene oxide, modified cellulose, collagen,
polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester,
poly(alpha-hydroxy acid), polycaprolactone, polycarbonates,
polyamides, polyanhydrides, polyamino acids, polyorthoesters,
polyacetals, polycyanoacrylates, degradable urethanes, aliphatic
polyester polyacrylates, polymethacrylate, acyl substituted
cellulose acetates, non-degradable polyurethanes, polystyrenes,
polyvinyl flouride, polyvinyl imidazole, chlorosulphonated
polyolifins, polyvinyl alcohol, and the like. Other suitable
polymers can be obtained by reference to The Polymer Handbook, 3rd
edition (Wiley, N.Y., 1989). In addition, specific growth factors
can also be added, guided by the type of stem cell desired and
amount of proliferation/differentiation. Said growth factors can
include can include members of the insulin like growth factor
family, the wingless related factor family, the nerve growth factor
family and the hedgehog factor family. Other specific growth
factors can include brain derived neurotrophic factor,
neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor,
cardiotrophin, members of the transforming growth factor (TGF)/bone
morphogenetic protein/growth and differentiation factor family, the
glial derived neurotrophic factor family including but not limited
to neurturin, neublastin/artemin, and persephin and factors related
to and including hepatocyte growth factor.
[0123] In a preferred embodiment, LPCM is generated using IMDM as a
perfusion solution, however the IMDM is supplemented with a serum
substitute such as commercially available mixtures including, but
not limited to BIT9500 (Stem Cell Technologies, Vancouver Canada).
In other embodiments, the serum substitute is comprised of bovine
serum albumin (BSA), insulin, and transferrin (TF). Alternatively,
human serum albumin USP can be used in cultures intended for
clinical use. The serum substitute can be comprised of, for
example, about 0.1 to about 0.50 g/liter of human serum albumin,
about 0.01 to about 1,000 .mu.g/ml insulin, and about 0.1 to about
1,000 .mu.g/ml transferrin. In another more preferred embodiment
the serum substitute can be comprised of 4 g/liter of human serum
albumin, about 0.71 .mu.g/ml of insulin and about 27 .mu.g/ml of
transferrin. One of skill in the art will understand that depending
on the cells which are intended for culture, or the desired
properties of the LPCM, various concentrations can be
experimentally assessed and tailored according to the biological
response sought.
[0124] In addition, a variety of cytokines can be added to the
perfusing solution during production of LPCM. In one embodiment the
following cytokines are used: TPO, SCF, Flt-3L, IL-3, IL-6, IL-11,
G-CSF and GM-CSF. In a preferred embodiment, a concentration of
about 0.1-500 ng/ml TPO, about 0.1-500 ng/ml SCF, about 0.1-500
ng/ml Flt3L, about 0.1-700 ng/ml IL-3, about 0.1-700 ng/ml IL-6,
about 0.1-500 ng/ml IL-11, about 0.1-500 ng/ml G-CSF, and about
0.1-500 ng/ml GM-CSF is used. In a more preferred embodiment, DMEM
is used as a starting solution for perfusion and is supplemented
with the cytokine cocktail of: about 20 ng/ml IL-3, about 250 ng/ml
IL-6, about 10 ng/ml SCF, about 250 ng/ml TPO, and about 100 ng/ml
flt-3L.
[0125] By treatment with LPCM, the stem cells are able to increase
their growth rate and expand rapidly. When desired, culture
conditions are used that preferentially allow the LPCM to augment
proliferation of stem cells without induction of differentiation.
Any suitable method of determining the growth rate and
differentiation of the stem cells can be used to determine the
growth rate and cell count of the stem cells so produced. For
example, flow cytometry analysis of markers associated with stem
cell retention, LTC-IC and semisolid media assays for
quantification of early and committed progenitors, and in vivo
NOD-SCID Repopulating Activity Assays to quantify the number of in
vivo stem cells with reconstituting activity. Said assays can be
modified and altered in order to allow detection of specific stem
cell subtypes. Assays can also be developed in immune compromised
mice, such as the NOD-SCID strain, by induction of a pathology to
which the human stem cells is anticipated to be therapeutics. For
example, human stem cells have been demonstrated to possess
therapeutic activity in a variety of non-hematopoietic settings in
the NOD-SCID, as well as the NUDE mouse. Other models of
immunodeficiency are known to one skilled in the art and include
the NK deficient beige mouse, the T cell and B cell deficient RAG
knockout mouse, and the common gamma chain knockout mouse.
Furthermore, such immunodeficient mouse strains can be selectively
bred with strains possessing a certain disease pathology so as to
assess the effect of human stem cells expanded by LPCM in them.
[0126] As shown in Example 3, the use of the LPCM method is capable
of greatly increasing the stem cell growth rate. The cell growth
rate can also depend on other factors, such as, for example,
temperature, type of stem cell, contents of the medium, and the
time allowed for the placental incubation step and contacting step.
One of skill in the art will be able to alter these variables to
adjust the growth rate as needed.
[0127] Another embodiment of the invention is a pharmaceutical
preparation comprising LPCM generated in a Good Manufacturing
Practices/Good Tissue Practices environment that will allow it to
be suitable for clinical use. LPCM is generated by perfusing
placentas in an environment that is sterile, using a perfusate
media that does not contain animal proteins or underdefined
components. Such a media can be X-VIVO 10 or other clinically
applicable medias. Subsequent to concentration and quantification
of units of activity, said LPCM can be diluted into an excipient or
carrier. For practical use, will be advantageous to formulate
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for an
individual to be treated; each unit containing a predetermined
quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The dosage unit forms of the invention are dependent upon
the amount of a compound necessary to stimulate proliferation of
the respective stem cells whose proliferation and/or
differentiation is being sought. The amount of a compound necessary
to stimulate proliferation and/or differentiation of the desired
stem cells can be formulated in a single dose, or can be formulated
in multiple dosage units. Treatment can require a one-time dose, or
can require repeated doses.
[0128] Actual formulation of the LPCM will be performed in
agreement with standard practices that are known to one skilled in
the art. These are well known in the art and the one chosen is
based upon the route of administration that will be used, as well
as specific pharmacokinetic properties that are desired. For
example, the preferred embodiment of an LPCM-based therapy is an
injectable, more preferred an injection into the specific area
requiring regeneration of stem cells. However, several embodiments
are possible. For example, routes of administration can include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., ingestion or inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
for formulating an LPCM-based therapeutic can include: sterile
diluent such as water for injection, saline solution (e.g.,
phosphate buffered saline (PBS, UPS)), fixed oils, glycerine, or
other synthetic solvents; antibacterial and antifungal agents such
as parabens, a polyol (for example, glycerol, propylene glycol, and
liquid polyetheylene glycol, and the like), chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The desired fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars, sodium
chloride, polyalcohols such as mannitol or sorbitol, and in the
composition. Prolonged administration of the injectable
compositions can be brought about by including an agent that delays
absorption. Such agents include, for example, aluminum monostearate
and gelatin. The parenteral preparation can be enclosed in ampules,
disposable syringes, or multiple dose vials made of glass or
plastic. It is known in the art, and common practice for oral
compositions to generally include an inert diluent or an edible
carrier. Oral compositions can be liquid, or can be enclosed in
gelatin capsules or compressed into tablets. Tablets, pills,
capsules, troches and the like can contain any of the following
ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or lactose; a disintegrating agent such as alginic
acid, Primogel, or corn starch; a lubricant such as magnesium
stearate or Sterotes; colloidal silicon dioxide. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
EXAMPLES
[0129] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
Incubation of Placenta in Growth Medium
[0130] A fresh human placenta obtained from vaginal delivery was
placed in a sterile plastic container. The placenta was rinsed with
an anticoagulant solution comprising phosphate buffered saline
(Gibco-Invitrogen, Grand Island, N.Y.), containing a 1:1000
concentration of heparin (1% w/w) (American Pharmaceutical
Partners, Schaumburg, Ill.).
[0131] The placenta was then covered with a DMEM media (Gibco) in a
sterile container such that the entirety of the placenta was
submerged in said media, and incubated at 37.degree. C. in a
humidified 5% CO.sub.2 incubator for 24 hours. At the end of the 24
hours, the live placenta conditioned medium (LPCM) was isolated
from the container and sterile-filtered using a commercially
available sterile 0.2 micron filter (VWR).
Example 2
Isolation of CD 34.sup.+ cells from Human Umbilical Cord Blood and
Subsequent Growth of Cells
[0132] Approximately 40 ml of cord blood was collected from a human
umbilical cord via venipuncture and allowed to drop by
gravitational force into a 250 ml sterile bag containing 20 ml
citrate-phosphate-dextrose under sterile conditions. Collected
blood cells were layered onto 50 ml conical tubes containing
Ficoll-Hypaque (density 1.077 gram/ml; Sigma, St Louis, Mo.) and
centrifuged at 400.times.g for 30 minutes. The mononuclear cells in
the interface layer were then collected, washed three times in PBS,
and re-suspended in PBS solution containing 0.5% serum albumin.
CD34+ cells were purified from the mononuclear cell fraction by
immuno-magnetic separation using the Magnetic Activated Cell
Sorting (MACS) CD34+ Progenitor Cell Isolation Kit
(Miltenyi-Biotec, Auburn, Calif.) according to manufacturer's
recommendations. The purity of the CD34+ cells obtained ranged
between 95% and 98%, based on Flow Cytometry evaluation (FACScan
flow cytometer, Becton-Dickinson, Immunofluorometry systems,
Mountain View, Calif.). Cells were plated at a concentration of
10.sup.4 cells/ml in a final volume of 0.5 ml in 24 well culture
plates (Falcon; Becton Dickinson Biosciences) in DMEM. Four
different treatment groups were used. Group 1 consisted of cells in
DMEM with no growth factor supplementation. Group 2 consisted of
cells in DMEM supplemented with the cytokine cocktail of: 20 ng/ml
IL-3, 250 ng/ml IL-6, 10 ng/ml SCF, 250 ng/ml TPO and 100 ng/ml
flt-3L. Group 3 consisted of cells in the cytokine cocktail, with a
50% mixture of LPCM. LPCM was generated as described in Example 1.
Group 4 consisted of cells spun down and resuspended in 100% LPCM.
Cells were cultured for 10 days at 37.degree. C. in a fully
humidified 5% CO.sub.2 incubator. The respective media was added to
each group once every three days at a volume of 0.5 ml. Subsequent
to incubation, cells were collected and numbers of viable CD34+
cells were assessed by flow cytometry using FITC-conjugated
anti-human CD34 antibodies (Beckman Coulter) and the viability dye
propidium iodine (Invitrogen). Cells in Group 1 had a decreased
number of viable CD34+ cells. Of the approximate 5.times.10.sup.3
cells inoculated in the starting culture, output at 10 days was
5-10.times.10.sup.2 viable CD34+ cells. In contrast, Group 2 had an
approximate 50-80 fold increase in the number of viable CD34+ cells
collected at the end of culture as compared with the original input
of 5.times.10.sup.3 cells. Viable CD34+ cells in Group 3 were
synergistically expanded by the combination of growth factors and
LPCM in that the output cell numbers were 300-500 times higher than
the input. The ability of LPCM to induce proliferation of CD34+
cells in absence of growth factor addition was demonstrated in that
the cells of Group 4 were expanded 50-100 fold at the end of
culture. These data demonstrate that LPCM can act as a source of
growth factors on its own, but can also synergize potently with
existing growth factor combinations. Importantly, the fact that no
addition of human proteins was needed for this potent expansion of
CD34+ stem cells is indicative of the utility of LPCM for a variety
of stem cell applications.
Example 3
Expansion of Stem Cells
[0133] At the end of the 24 hour period, the LPCM from Example 1
was added to the wells of the sterile 24 well tissue culture plate
in a volume of 0.25 ml. Umbilical cord mononuclear cells harvested
as described in Example 1 were resuspended in DMEM in a volume of
0.25 ml and added to the wells containing LPCM. The final
concentration of mononuclear cells was 10.times.10.sup.6 cells per
ml. The cultures were subsequently incubated for an additional
seven days at 37.degree. C. in a humidified 5% CO.sub.2 incubator.
The number of CD 34.sup.+ cells and viability was then determined
by flow cytometry as described in Example 2 both at the beginning
of cell culture and subsequently after 7 days of culture. The
number of viable CD34.sup.+ cells had increased 27.4 fold over the
starting number of cells. In contrast, cells that were cultured
with DMEM media alone in absence of LPCM had a decline in viable
CD34+ cell numbers by approximately 7 fold.
Example 4
Generation, Quantification, and Concentration of LPCM
[0134] A fresh human placenta obtained from vaginal delivery was
placed in a sterile plastic container. The placenta was rinsed with
an anticoagulant solution comprising phosphate buffered saline
(Gibco-Invitrogen, Grand Island, N.Y.), containing a 1:1000
concentration of heparin (1% w/w) (American Pharmaceutical
Partners, Schaumburg, Ill.). The umbilical arteries and the
umbilical vein were identified and isolated. Initially, a solution
of heparinized PBS was used to identify patent blood vessels
capable of use for perfusion. The perfusion of vessels was
accomplished by injection of 20 ml of heparinized PBS at a flow
rate of approximately 20 ml per minute into the umbilical arteries.
It was relatively easy to discriminate between the artery and veins
based on the blood filled appearance of the veins. Once patency and
suitability of blood vessels was identified, the vessels were
cannulated using a sterile cannula connected to the tubing of a
pulsitile pumping apparatus. The pump was connected to a collection
flask and arranged as a continous circuit with a total volume of 50
ml of DMEM being perfused through the placenta. The cannulated
placental unit was incubated in a fully humidified environment for
24 hours at 5% CO.sub.2 at 37.degree. C. The perfusion rate was 10
ml per minute at a pressure of 60 Hg.
[0135] Subsequent to incubation, medium was collected used as a
source of LPCM. LPCM was sterilized using 0.2 micron filters (VWR)
and frozen for future use. In order to quantitate biological
activity, dilutions of LPCM in the following ratios by volume 1:1,
1:10, 1:100, 1:1000, were made in DMEM in absence of fetal calf
serum or other serum sources, and the diluted media was added to a
200 .mu.L culture of 5.times.103 human cord blood isolated CD34+
cells per well in 96 well plates in a 48 hour culture condition.
The proliferation of these cells was quantitated by the tritiated
thymidine method. Briefly, 1 .mu.Ci of [.sup.3H]thymidine
(Amersham) was added to each well for the last 12 h of culture. At
the end of the culture period, using an automated cell harvester,
the cells were collected onto glass microfiber filter, and the
radioactive labeling incorporation was measured by a Wallac
Betaplate liquid scintillation counter. 1 Unit of LPCM activity was
designated as the amount of LPCM needed to stimulate proliferation
of cord blood derived CD34+ cells by 100% higher than said cells in
DMEM alone. Calculations are made on a logarithmic curve as
described for other biological agents whose activity is quantitated
in Units (DeKoter, et al., 1997, Cell Immunol 175:120-127, which is
incorporated by reference herein in its entirety).
[0136] In order to concentrate LPCM, a volume of 40 ml of media was
lyophilized under sterile conditions. Lyophilate was subsequently
dialyzed using an exclusion of 5000 Daltons in order to extract
salts and other small molecules in the solution. Reconstitution was
performed in various volumes of USP saline and sterility as well as
activity was quantified. Based on activity as measured using the
CD34+ stimulation assay, various batches of LPCM were manufactured
which are used for some of the experiments described below.
Manufacturing of LPCM in this manner provided a non-toxic substance
in that no maximally tolerated dose was observed when injecting C3H
mice at concentrations ranging up to 1.times.10.sup.6 Units.
Toxicity was evaluated at the histological level after acute and
chronic administration, as well as by enzymatic markers of organ
damage such as creatinine, myocardial kinase, troponin,
transaminases, and albumin secretion. Furthermore, no pyrogenicity
was observed in any of the treated animals. Higher concentrations
were not evaluated since one tenth of this dose is still
substantially above what would be used in a clinical setting.
Example 5
Expansion of Embryonic Stem Cells in Feeder Free Cultures using
LPCM
[0137] The H1 human embryonic stem cell line is obtained from the
Wi-Cell Research Institute (Madison, Wis.) and is propagated and
cultured in mouse embryonic fibroblast (MEF)-conditioned medium
(MEF-CM) containing 4 ng/ml human basic fibroblast growth factor
(bFGF) (Life Technologies, Rockville, Md.) in six-well
(35-mm-diameter) plates precoated with Matrigel (Becton-Dickenson
Labware, Bedford, Mass.) and cultured at 37.degree. C., under 5%
CO.sub.2 in Dulbecco's modified Eagle medium (DMEM)/F12 medium with
20% knockout serum replacement (KOSR), 1 mM L-glutamine, 1%
nonessential amino acids, 0.1 mM 2-mercaptoethanol (2-ME), and 4
ng/ml bFGF (Invitrogen). Cells are harvested from tissue culture
plates by digestion with 200 U/mL collagenase IV for 5 minutes at
37.degree. C. and picking up individual colonies with a 20
microliter pipette tip under a microscope.
[0138] 6-well plates tissue culture plates (Falcon) are coated with
Matrigel.TM. (Becton Dickenson) and 10 colonies per well are added
with 1 ml of either control DMEM media or LPCM. Cells are cultured
for a period of 14 days after initial seeding, subsequently to
which they are deaggregated using collagenase and stained for flow
cytometric analysis of the embryonic stem cell markers SSEA-4. A
two-fold increase in cells expressing SSEA-4 is observed in
cultures treated with LPCM in comparison to DMEM treated cultures.
When cells are maintained under similar conditions for extended
cultures, such as for 120 days, the H-1 cells still possess ability
to form multi-lineage teratomas upon injection of into NUDE mice. A
higher expansion rate of H-1 cells is observed when cells are grown
in LCMP media compared to DMEM on Matrigel.TM. cultures.
Example 6
Expansion of Amnionic Fluid-Derived Multipotent Stem Cells in
Feeder Free Cultures using LPCM
[0139] Following a modification of the methodology described in
U.S. Patent Application No. 2005/0054093, which is incorporated by
reference herein in its entirety, approximately 5 ml of fresh
amniotic fluid is collected during amniocentesis in the second
trimester of pregnancy, mononuclear cells are pelleted by
centrifugation and resuspended in either LPCM or DMEM. Cells are
plated in 24 well plates at a concentration of 5.times.10.sup.3
cells per well. Media is added every two days to the culture. After
a period of 14 days an substantially increased number of cells
positive for SSEA-3, and SSEA-4 as determined by flow cytometry are
found in the cultures with LPCM, in contrast, cultures in DMEM
appear to be populated by cellular debris and fibroblast like cells
lacking stem cell markers (negative for SSEA-1, 3, 4, and
CD34).
Example 7
Expansion of Cord Blood Derived Hematopoietic Cells in Liquid
Culture
[0140] Cord blood CD34 cells are collected as described in Example
2. CD34+ cells are placed into 24-well plates (Falcon) at a
concentration of 2000 cells per well. Each well contained 0.5 ml
IMDM (Gibco) supplemented with BIT9500 (StemCell Technologies,
Vancouver, Canada), instead of serum. Cytokines known to stimulate
hematopoietic cell proliferation are added at the following
concentrations: TPO (50 ng/ml), IL-3 (50 ng/ml), kit-ligand (100
ng/ml) and flt-3L (100 ng/ml). In some cultures 0.2 ml of IMDM is
added, whereas in others LPCM generated using IMDM as a substitute
for DMEM is added. The cultures are incubated at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2. On days 7 and 13,
cells are harvested from the culture and are assayed for the number
of erythroid burst-forming units (BFU-E), granulocyte-macrophage
colony-forming units (CFU-GM) and mixed colony-forming units
(CFU-Mix) are assayed using the methylcellulose semisolid culture
system by the MethoCult7 kit according to the manufacturer's
instructions (StemCell Technologies). Briefly, 1.times.10.sup.4
CD34+ cells expanded in liquid culture (day 7 or 13) are plated
into 35-mm plastic Petri-dishes (Falcon) in culture medium
containing SCF, IL-3, G-CSF, GM-CSF and Epo as colony-stimulating
factors in the presence of the semi-solid MethoCult methylcellulose
base. As a comparison, freshly isolated CD34+ cells are added as an
unstimulated control. Each dish is incubated at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 for 14 d. Colonies consisting
of more than 50 cells are counted under an inverted microscope
(Zeiss) and quantified. The total number of each type of progenitor
cell is calculated from the total number of cells harvested and the
number of each type of colony per well.
[0141] In comparison to day 0 CD34 cells, cells that are grown in
liquid culture for 7 days generate a 150-fold expansion of CFU-GM
when control IMDM media is added. In contrast, day 7 cultures
supplemented with LPCM have a 440-fold expansion of CFU-GM. At 13
days of liquid culture there is a 550-fold expansion of CFU-GM in
the IMDM media, whereas the LPCM supplemented cultures have a
790-fold expansion of CFU-GM. In terms of BFU-E there is a 300-fold
expansion at day 7 of culture in IMDM control in contrast to the
700-fold expansion in the LPCM treated group. Similarly, at day 13,
the control IMDM culture generated a 150-fold expansion of BFU-E in
contrast to the LPCM treated group which had a 550-fold expansion.
When assessing the earlier progenitor colonies, CFU-Mix, a 200-fold
expansion is observed on day 7 in control treated cultures, whereas
a 400-fold increase is observed in the LPCM-treated cultures. More
strikingly is the effect at day 13 of liquid culture where CFU-Mix
actually decreases to a 40-fold expansion in the IMDM control
group, whereas a 500-fold expansion is observed in the LPCM treated
group.
[0142] These results are further confirmed by enumeration of CD34+,
CD38- cells exiting the day 7 and day 13 liquid cultures. In
control cultures, the proportion of CD34+, CD38- cells at day 7 are
40-fold expanded and at day 13 are 23-fold expanded. In contrast,
cultures that are supplemented with LPCM have an increased
expansion rate in that at day 7 a 55-fold expansion is observed,
whereas at day 13 a 150-fold expansion is observed.
[0143] This example illustrates that LPCM has the ability to
synergize with cytokines that are known in the art to act on early
lineage hematopoietic stem cells. In order to demonstrate the
biological activity of expanded precursors, as well as to validate
the possibility that LPCM actually maintains and expands
hematopoietic stem cells with in vivo activity, fresh CD34 cells
isolated from the cord blood, as well as after 7 and 13 days of
culture in the conditions described above, as assessed in the
SCID-repopulating assay. NOD-SCID mice (Jackson Laboratories) are
sublethally irradiated with 350 rads from a Cesium 137 source. CD34
cells from the 3 respective timepoints are administered to 5 groups
of mice, 10 mice per group, through tail vein injection. Group 1
receives 1.times.10.sup.5 freshly isolated CD34 cells, Group 2
receives the same amount of cells that have been cultured for 7
days in liquid culture with IMDM control supplemented media, Group
3 receives the same amount of cells that have been cultured for 7
days in liquid culture with LPCM, Group 4 receives cells from day
13 liquid culture, with IMDM control media, and Group 5 receives
cells from day 13 liquid culture supplemented with LPCM. Assessment
of engraftment is made at 11 weeks by sacrificing the murine
recipients, collecting bone marrow from the femur and tibia, and
detection of the human specific CD45 marker in the murine bone
using flow cytometry. While Group 1 possesses undetectable levels
of human leukocytes. Groups 2 and 4 possess approximately 2% and 4%
human leukocytes, respectively. Groups 3 and 5 possess
approximately 13% and 21% human leukocytes, respectively.
Furthermore, double staining with CD34 and human CD45 suggests that
only Groups 3 and 5 possess detectable levels of human
hematopoietic stem cells. This suggests that LPCM possesses the
important activity of allowing expansion of a human hematopoietic
cell population capable of efficient activity in vitro and in
vivo.
Example 8
Expansion of Tolerogenic Dendritic Cells
[0144] It is known that dendritic cells can act both as immune
stimulators or as immune suppressors. Unfortunately, clinical use
of immune suppressive dendritic cells is hampered by inability to
expand large enough numbers for therapeutic use. This is due to the
fact that numerous dendritic cell-expanding regimens cause
activation, leading to loss of tolerogenic properties. Accordingly
in this experiment we seek to generate expanded numbers of
dendritic cells with a tolerogenic phenotype.
[0145] Bone marrow cells are flushed from the femurs and tibias of
C57/BL6 mice (Jackson Laboratories). Erythrocytes are lysed using
lysis buffer, washed in PBS and cultured at 2.times.10.sup.6
cells/well in 24-well plates (Corning Glass, Corning, N.Y.) in 2 ml
RPMI 1640 (Life Technologies, Ontario, Canada) supplemented with
10% FCS (Life Technologies), 100 U/ml of penicillin, 100 .mu.g/ml
of streptomycin, 50 .mu.M of 2-ME (Life Technologies), 10 ng/ml of
murine rGM-CSF (Peprotech, Rocky Hill, N.J.), 10 ng/ml of IL-4
(Peprotech) and the NF-kappa B inhibitor LF15-0195 (LF) at a
concentration of 5 .mu.g/ml. Cultures in which non-tolerogenic,
conventional DC are desired do not contain LF, and agent previously
demonstrated as a generator of tolerogenic DC (Ichim, supra; Yang,
et al., 2003, J Leukoc Biol 74:438-447, each of which is
incorporated by reference herein in its entirety). Nonadherent
cells are removed after 48 h of culture, and fresh medium is added
every 48 h. In some wells control RPMI is added at a volume of 1:4,
whereas in other wells LPCM generated using RPMI as a base is added
at the same concentration. After 7 days of culture, DC numbers are
quantified by expression of the marker CD11c using flow cytometry.
A 4-fold higher number of CD11c+ cells are extracted from cultures
that were supplemented with LPCM as opposed to control media.
Assessment of tolerogenic function is performed in vitro by
observing upregulation of costimulatory molecules in response to
treatment with 10 ng/ml TNF and 10 ng/ml LPS. While DC generated in
absence of LF were capable of upregulating expression of CD40, CD80
and CD86 after activation, DC generated under the cover of LF, both
with or without LPCM are resistant to upregulation of these
molecules. Functional demonstration that tolerogenic DC raised with
LPCM supplementation actually are tolerogenic is demonstrated by
inability of these cells to stimulate a mixed lymphocyte
reaction.
[0146] Varying numbers of DC are seeded in triplicate in a
flat-bottom 96-well plate (Corning) for use as stimulator cells. DC
are of the C57/BL6 strain and are grown either in the absence of
LF, with LF but no LPCM, or with LF and LPCM. T cells are prepared
from spleens of BALB/c and isolated by T cell enrichment columns
(R&D Systems, Minneapolis Minn.). T cells
(1-5.times.10.sup.5/well) are added to the DC cultures, with the
final MLR taking place in 200 .mu.l of RPMI 1640 (Life
Technologies) supplemented with 10% FCS (Life Technologies), 100
U/ml of penicillin (Life Technologies), and 100 .mu.g/ml of
streptomycin (Life Technologies). Cells are cultured at 37.degree.
C. in a humidified atmosphere of 5% CO.sub.2 for 3 days, and pulsed
with 1 .mu.Ci of [3H]thymidine (Amersham Pharmacia Biotech) for the
last 16 h of culture. Cells were harvested onto glass fiber
filters, and the radioactivity incorporated was quantitated using a
Wallac Betaplate liquid scintillation counter. Results indicate
that while DC raised in absence of LF are potent stimulators of
allogeneic T cell proliferation, DC raised with LF, either in the
presence or absence of LPCM, are non-stimulatory.
[0147] In order to demonstrate functional tolerogenicity in vivo,
control non-LF treated DC, LF-treated DC and LF+LPCM DC of the
C57/BL6 strain are injected at a concentration of 10 million CD11c+
cells into BALB/c recipients 7 days prior to heterotopic cardiac
transplantation with C57/BL6 grafts. Subsequent to grafting
survival of transplanted hearts is 6 days in the non-LF treated DC
recipients, whereas it is 18 days in the LF-treated DC recipients
and 24 days in the LF-treated+LPCM treated recipients.
Example 9
LPMC in Treatment of Stroke
[0148] C57BL/6 (Jackson Laboratory) mice weighing approximately 25
grams each are given free access to food and water before and
during the experiment. Animals are acclimated to the laboratory
environment for 1 week prior to experimentation. Four groups of 10
mice each are treated by intravenous infusion as follows: Group 1
vehicle, Group 2 FGF-1 (10 mg/kg), Group 3 LPCM (100 U/kg), Group 4
FGF-1 (10 mg/kg) and LPCM (100 U/kg). Mice were infused
intravenously, 1 hour after the initiation of ischemia. LPCM is
generated, concentrated, and Units of activity are quantified as
described in Example 4.
[0149] Each mouse is subjected to one hour of cerebral ischemia
followed by 24 hours of reperfusion. At the end of the ischemic
period, animals are treated as described in the above paragraph and
at 14 days are examined for infarct volume. Each mouse is
anesthetized and a thermistor probe is inserted into the rectum to
monitor body temperature, which is maintained at 36-37.degree. C.
by external warming. The left common carotid artery (CCA) is
exposed through a midline incision in the neck. The superior
thyroid and occipital arteries are electrocoagulated and divided. A
microsurgical clip is placed around the origin of the internal
carotid artery (ICA). The distal end of the ECA (external carotid
artery) is ligated with 6-0 silk and transected. A 6-0 silk is tied
loosely around the ECA stump. The clip is removed and the
fire-polished tip of a 5-0 nylon suture (poly-L-lysine coated) is
gently inserted into the ECA stump. The loop of the 6-0 silk is
tightened around the stump and the nylon suture is advanced
approximately 11 mm (adjusted for body weight) into and through the
internal carotid artery (ICA) after removal of the aneurysm clip,
until it rests in the anterior cerebral artery (ACA), thereby
occluding the anterior communicating and middle cerebral arteries.
The animal is returned to home cage after removal from anesthesia.
After the nylon suture is been in place for 1 hour, the animal is
re-anesthetized, rectal temperature is recorded, the suture is
removed and the incision closed.
[0150] Neurological deficits are assessed 14 days after ischemia
based on a scale from 0 (no deficits) to 4 (severe deficits) as
commonly used in the discipline. Neurological scores are as
follows: 0, normal motor function; 1, flexion of torso and
contralateral forelimb when animal is lifted by the tail; 2,
circling to the contralateral side when held by the tail on a flat
surface, but normal posture at rest; 3, leaning to the
contralateral side at rest; 4, no spontaneous activity.
[0151] For infarct volume determination after behavioral testing,
the animals are anesthetized with an intraperitoneal injection of
sodium pentobarbital (50 mg/kg). The brains are removed, sectioned
into 4 2-mm sections through the infracted region and placed in 2%
triphenyltetrazolium chloride (TTC) for 30 minutes at 24 hours.
Subsequently, the sections are placed in 4% paraformaldehyde over
night. The infarct area in each section is determined with a
computer-assisted image analysis system, consisting of a Power
Macintosh computer equipped with a Quick Capture frame grabber
card, Hitachi CCD camera mounted on a camera stand. NIH Image
Analysis Software, v. 1.55 is used for quantification of image
data. The images are captured and the total area of infarct is
determined over the sections. A single operator blinded to
treatment status performs all measurements. Summing the infarct
volumes of the sections calculates the total infarct volume.
[0152] At day 14 after induction of ischemia animals are assessed
by a blinded observer for neurological deficits based on the scale
of 0 to 4 described above. Animals in Group 1 (vehicle control)
have an average score of 3.3 0.335; Animals in Group 2 (FGF-1) have
an average score of 3.0.+-.0.576; Animals in Group 3 (LPCM) have an
average score of 2.2.+-.0.889; Animals in Group 4 (LPCM+FGF-1) have
an average score of 0.4.+-.1.023. This synergistic protective
effect seen between FGF-1 and LPCM, is further supported by
assessment of infarct size. According to present reduction in
infarct size compared to the vehicle control group (Group 1), mice
treated with FGF-1 alone (Group 2) had a reduction of 11%.+-.2.52,
mice treated with the LPCM alone (Group 3) had a reduction of
26%+1.34, and mice treated with the combination of FGF-1 and LPCM
(Group 4) had a reduction in infarct size of 78%.
Example 10
LPMC Augmentation of Cord-Blood Reconstitution after Nuclear
Incident
[0153] A terrorist "dirty bomb" nuclear attack on a populated city
occurs exposing 100 individuals to an estimated 10 Gy Eq of neutron
and gamma irradiation. All 100 patients presented with symptoms of
acute radiation syndrome including severe pancytopenia. Based on
previous experiences (Nagayama, et al., 2002. Int J Hematol
76:157-164, which is incorporated by reference herein in its
entirety), and the lack of sibling related donors or possibility of
autotransplantation, the use of cord blood as a hematopoietic graft
is performed after HLA-matching allowing for only one allele
mismatch. Pretransplantation conditioning consists of antithymocyte
equine 3-globulin alone (2.5 mg/kg for 2 consecutive days), and
GVHD prophylaxis consists of the combined use of cyclosporine A
(CyA) and methylprednisolone (mPSL). Patients are administered
3.times.10.sup.7 nucleated cord blood cells per kilogram through
intravenous infusion. All patients are administered filgrastim
(neupogen) at a concentration of 10 .mu.g/kg/day for 14 days in
order to accelerate leukocytic recovery. Of the 100 patients, 50
receive concurrently with filgrastim, a concentration of 250 Units
of LPCM/kg/day. LPCM is prepared under GMP conditions based on the
description of Example 4. At day 15 after cellular transplantation,
23% of patients treated with filgrastim alone have granulocytic
counts of more than 500/mm.sup.3. In contrast, 100% of the patients
receiving the combination of filgrastim and LPCM have granulocytic
counts of more than 500/mm.sup.3 by day 12 post
transplantation.
[0154] Chimeric hematopoiesis was observed at day 50 in 46% of
patients treated with filgrastim alone, whereas 100% of patients
receiving the combination had achieved this milestone.
Additionally, opportunistic infections are predominantly associated
with the patient group that received filgrastim alone.
[0155] This example suggests the use of LPCM as an adjuvant agent
to standard hematopoiesis stimulating regimens. Additionally,
although GVHD is not observed in any of the patients in the prior
example, most likely due to the low levels of cord blood cells
administered, higher doses of cord blood cells can predispose to
this. Accordingly, LPCM can be used in combination with immune
suppressive cytokines to preferential stimulate expansion of
natural immune regulatory cell subsets.
Example 11
LPMC Augmentation of Endogenous Endothelial Stem Cells for End
Stage Angina
[0156] Twelve patients with advance angina, as defined by the
Seattle Angina Questionnaire and the Canadian Cardiovascular
Society Angina Classification scores III-IV are informed they are
not eligible for surgical or medical intervention. Patients have
either one/two/three vessel disease as determined by angiography as
being greater than or equal to 70% narrowing of a major epicardial
coronary artery such as the right circumflex artery, the left
circumflex artery, or the left anterior descending artery.
Alternatively, some patients have diffuse type of coronary artery
disease as evidenced by the appearance on coronary angiography of
multiple stenoses, multiple atherosclerotic plaques, and/or
peripheral occlusion(s) of coronary vessel(s) with and without a
history of myocardial infarctions. Areas of hypoperfusion are
identified according angiographically. Patients are then subjected
to a mini-thoracotomy procedure similar to the procedure utilized
for transmyocardial revascularization in similar patient subsets
(Lamy, A., 1997, Evid Based Cardiovasc Med 1:77, which is
incorporated by reference herein in its entirety). Specifically,
standard anesthesia with intubation is used according to
institutional guidelines, the left anterior mini-thoracotomy is
performed by a longitudinal incision of the pericardium anterior to
the phrenic nerve with sutures fixing the pericardial edges thus
elevating the heart. Identification of coronary arteries and target
myocardial area of treatment is determined by previous angiography
(either periphery of LAD- or LCX or RCA-branches, depending of
preoperative findings). An intravenous application of beta-blockers
can be used to lower heart rate if mandated according to
institutional procedures and/or the preference of the surgeon.
[0157] According to the procedure pioneered by Stegmann, et al.,
2000, Herz 25:589-599, which is incorporated by reference herein in
its entirety, for intramyocardial administration of growth factors,
a weight-adjusted dose of LPCM of 250 U/kg in saline is then
injected, via a 27 gauge needle, into the myocardium at the target
area. The area of occlusion/lesion, determined from the screening
angiogram, is identified. The injection site is limited to an area
within a 1 cm diameter around the area of occlusion/lesion. The
needle is inserted at a 45.degree. angle directly into the
myocardium at a depth no greater than 1 cm. The axis of the needle
is inserted toward the periphery of the coronary vascular bed. The
surgeon is then to confirm that the LPCM was not injected into the
ventricular cavum by needle aspiration. Pericardial closure with
reabsorbable single sutures is conducted. Insertion of a pleural
drain and closure of thoracic incision is made.
[0158] Patients are subsequently examined at 3 and 6 months using
SPECT radionuclide imaging for perfusion of the treated areas. A
progressive improvement in perfusion area is observed, as well as
restoration of the Canadian Cardiovascular Society Anginal
Classification score from an average of 3.4.+-.0.893 to
1.2.+-.1.052.
Example 12
Generation of Autologous Hematopoietic Stem Cells for Patients
Lacking Donors
[0159] A patient with chronic myeloid leukemia is in need of a bone
marrow transplant in order to destroy the advanced leukemia burden.
Unfortunately a suitable donor is not found and the patient is not
eligible for an autologous bone marrow transplant due to the high
possibility of relapse due to leukemic contamination of the bone
marrow. The novel procedure of cellular reprogramming using
cytoplasmic extracts of undifferentiated embryonic stem cells is
performed, using LPCM as an expansion factor. Specifically, bone
marrow cells are isolated and plated in long term cultures under
standard conditions using imatinib (0.5-1.0 .mu.M for 72 h) and
then mafosfamide (30-90 .mu.g/ml for 30 min) followed by 2 weeks in
culture with cytokines (100 ng/ml each of stem cell factor,
granulocyte colony-stimulating factor and thrombopoietin) as
described (Bhatia, et al., 2004, Hematol Oncol Clin North Am
18:715-732, xi; Yang, et al. A novel triple purge strategy for
eliminating chronic myelogenous leukemia (CML) cells from
autografts. Bone Marrow Transplant, e-published on Jan. 23, 2006,
each of which is incorporated by reference herein in its entirety).
Putatively purged cells are plated under low concentrations in
liquid cultures containing LPCM in order to allow colony formation
and proliferation. Cells are picked from each colony and assessed
for the Philadelphia Chromosome using single cell RT-PCR to amplify
the oncogenic bcr-abl transcript as described (Brail, et al., 1999,
Mutat Res 406:45-54, which is incorporated by reference herein in
its entirety). Cells are picked using a micropipette from colonies
lacking expression of the bcr-abl and are prepared for
reprogramming using embryonic stem cell extracts. The extracts are
prepared according a modification to the method of Collas in U.S.
Patent Application No. 2002/0142397, which is incorporated by
reference herein in its entirety: Interphase cultured embryonic
stem cells of the H--I line are harvested by trypsinization and
washed by centrifugation at 500 g for 10 minutes in a 10 ml conical
tube at 4.degree. C. The supernatant is discarded, and the cell
pellet is resuspended in a total volume of 50 ml of cold PBS. The
cells are centrifuged at 500 g for 10 minutes at 4.degree. C. This
washing step is repeated, and the cell pellet is resuspended in
approximately 20 volumes of ice-cold interphase cell lysis buffer
(20 mM Hepes, pH 8.2, 5 mM MgCl.sub.2, 1 mM DTT, 10 .mu.M
aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M
soybean trypsin inhibitor, 100 .mu.M PMSF, and optionally 20
.mu.g/ml cytochalasin B). The cells are sedimented by
centrifugation at 800 g for 10 minutes at 4.degree. C. The
supernatant is discarded, and the cell pellet is carefully
resuspended in no more than one volume of interphase cell lysis
buffer. The cells are incubated on ice for one hour to allow
swelling of the cells. The cells are lysed by sonication using a
tip sonicator. Cell lysis is performed until at least 90% of the
cells and nuclei are lysed, which is assessed using phase contrast
microscopy. The sonication time required to lyse at least 90% of
the cells and nuclei can vary depending on the type of cell used to
prepare the extract. Accordingly, microscopic evaluation of
cellular morphology is performed to assess degree of sonication
needed. The cell lysate is placed in a 1.5-ml centrifuge tube and
centrifuged at 10,000 to 15,000 g for 15 minutes at 4.degree. C.
using a table top centrifuge. The tubes are removed from the
centrifuge and immediately placed on ice. The supernatant is
carefully collected using a 200 .mu.l pipette tip, and the
supernatant from several tubes is pooled and placed on ice. This
cell extract is then aliquoted into 20 .mu.l volumes of extract per
tube on ice. The tube is then overlayed with mineral oil to the
top. The extract is centrifuged at 200,000 g for three hours at
4.degree. C. to sediment membrane vesicles contained. At the end of
centrifugation, the oil is discarded. The supernatant is carefully
collected, pooled if necessary, and placed in a cold 1.5 ml tube on
ice. This supernatant quantified for protein content and is
referred to as the "cellular extract" that will be used for the
reprogramming of cells.
[0160] CD34+ cells grown in colonies not expressing the bcr-abl
transcript are then permeabilized temporarily. Cells are harvested
by picking the entire colony with a micropipette of 200 .mu.l and
are washed with PBS. Cells are incubated in Streptolysin O solution
(see, for example, Maghazachi et al., 1997, FASEB J. 11(10)765-774,
which is incorporated by reference herein in its entirety) for
15-30 minutes at room temperature. After either incubation, the
cells are washed by centrifugation at 400 g for 10 minutes. This
washing step is repeated twice by resuspension and sedimentation in
PBS. Cells are kept in PBS at room temperature until use. The
permeabilized CD34 stem cells are suspended in the embryonic stem
cell derived reprogramming cytoplasmic extract (generated as
described above) at a concentration of 300 cells per .mu.l. An ATP
generating system (2 mM ATP, 20 mM creatine phosphate, 50 .mu.g/ml
creatine kinase) and 100 .mu.M GTP are added to the extract, and
the reaction is incubated at 30-37.degree. C. for up to two hours
to promote translocation of factors from the extract into the cell
and active nuclear uptake or chromosome-binding of factors. The
reprogrammed cells are centrifuged at 800 g, washed by
resuspension, and centrifugation at 400 g in PBS. The cells are
resuspended in culture medium containing 20-30% fetal calf serum
(FCS) and incubated for 1-3 hours at 37.degree. C. in a regular
cell culture incubator to allow resealing of the cell membrane. The
cells are then washed in regular warm culture medium (10% FCS) and
cultured further using a concentration of 10 U/ml of LPCM in DMEM
media, supplemented with IL-3 (20 ng/ml), IL-6 (250 ng/ml), SCF (10
ng/ml), TPO (250 ng/ml), and flt3-L (100 ng/ml). Media is exchanged
2-3 times per week. After 14 days of culture, cells are assessed by
flow cytometry for expansion of early hematopoietic subtype, and by
RT-PCR for expression of the bcr-abl oncogene. A potent expansion
of CD34+ cells is obtained, and some cells express the embryonic
stem cell marker SSEA-4. The culture is subsequently transferred to
250 ml bag culture and maintained until a concentration of
1.times.10.sup.7 CD34+ cells are generated. Said cells are
subsequently assessed for possible contamination and expression of
bcr-abl. Cells are subsequently used for performing autologous bone
marrow transplant.
Example 13
LPCM for Treatment of Critical Limb Ischemia
[0161] A group of 20 patients are chosen who present with Fountaine
Grade III-IV critical limb ischemia, at risk of amputation. Area of
atherosclerosis and ischemia is identified using angiography.
Doppler scans reveal significant hypoperfusion, and in some areas
almost complete ischemia. LPCM is generated as described in Example
4, in a DMEM base and administered to 10 of the patients, whereas
the other 10 receive the DMEM vehicle without exposure to placenta.
The formulation that is administered to the patients is
reconstituted in saline with 3% human serum albumin to maintain
protein stability. Desalting and batch testing is performed.
Treated patients receive a total of 5 injections per week in areas
identified as ischemic. Untreated control patients receive similar
injections, but with vehicle alone. After 3 months of treatment
initiation a clinical response is observed in 7 out of the 10
patients treated, whereas no responses is seen in the control
group. Clinical response is classified as decrease in Fountaine
Score status over 0.5 percent in combination with improvement in
localized circulation as detected by angiographic examination
performed by an operator blinded to the experiment.
[0162] One skilled in the art will appreciate that these methods
and devices are and can be adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods, procedures, and devices described herein are
presently representative of preferred embodiments and are exemplary
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention and
are defined by the scope of the disclosure.
[0163] It will be apparent to one skilled in the art that varying
substitutions and modifications can be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0164] Those skilled in the art recognize that the aspects and
embodiments of the invention set forth herein can be practiced
separate from each other or in conjunction with each other.
Therefore, combinations of separate embodiments are within the
scope of the invention as disclosed herein.
[0165] All patents and publications mentioned in the specification
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0166] The invention illustratively described herein suitably can
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising,"
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions indicates the exclusion of equivalents of the
features shown and described or portions thereof. It is recognized
that various modifications are possible within the scope of the
invention disclosed. Thus, it should be understood that although
the present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the disclosure.
* * * * *