U.S. patent application number 11/604909 was filed with the patent office on 2007-05-24 for methods of isolation, expansion and differentiation of fetal stem cells from chorionic villus, amniotic fluid, and placenta and therapeutic uses thereof.
This patent application is currently assigned to Children's Medical Center Corporation. Invention is credited to Anthony Atala, Paolo De Coppi.
Application Number | 20070116682 11/604909 |
Document ID | / |
Family ID | 26989930 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070116682 |
Kind Code |
A1 |
Atala; Anthony ; et
al. |
May 24, 2007 |
Methods of isolation, expansion and differentiation of fetal stem
cells from chorionic villus, amniotic fluid, and placenta and
therapeutic uses thereof
Abstract
The present invention is directed to pluripotent fetal stem
cells derived from chorionic villus, amniotic fluid, and placenta
and the methods for isolating, expanding and differentiating these
cells, and their therapeutic uses such as manipulating the fetal
stem cells by gene transfection and other means for therapeutic
applications.
Inventors: |
Atala; Anthony; (Winston
Salem, NC) ; De Coppi; Paolo; (Santa Lucia di Piave,
IT) |
Correspondence
Address: |
DAVID S. RESNICK
100 SUMMER STREET
NIXON PEABODY LLP
BOSTON
MA
02110-2131
US
|
Assignee: |
Children's Medical Center
Corporation
Boston
MA
|
Family ID: |
26989930 |
Appl. No.: |
11/604909 |
Filed: |
November 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10495419 |
Jan 21, 2005 |
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PCT/US02/36966 |
Nov 15, 2002 |
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11604909 |
Nov 28, 2006 |
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60335878 |
Nov 15, 2001 |
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60356295 |
Feb 13, 2002 |
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Current U.S.
Class: |
424/93.7 ;
435/325; 435/366; 435/7.2 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
1/16 20180101; C12N 5/0605 20130101; A61P 25/00 20180101; A61K
35/12 20130101; A61P 19/08 20180101; A61P 25/16 20180101; A61P
43/00 20180101; A61P 21/00 20180101; C12N 5/0607 20130101 |
Class at
Publication: |
424/093.7 ;
435/007.2; 435/366; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; G01N 33/567 20060101 G01N033/567; C12N 5/08 20060101
C12N005/08 |
Claims
1. A method for producing a population of cells enriched for
pluripotent fetal stem cells, comprising selecting c-kit positive
cells from a chorionic villus, amniotic fluid, or placenta
sample.
2. The method of claim 1, wherein the selecting is performed using
an antibody against c-kit.
3. The method of claim 2, wherein the antibody is a monoclonal
antibody.
4. The method of claim 2, wherein the monoclonal antibody is a
mouse monoclonal IgG against an antigenic epitope of human
c-kit.
5. The method of claim 2, wherein the antibody is fluorochrome
conjugated.
6. The method of claim 2, wherein the antibody is conjugated to
magnetic particles.
7. The method of claim 1, wherein the selecting is by flow
cytometry.
8. The method of claim 1, wherein the selecting is by fluorescence
activated cell sorting or high gradient magnetic selection.
9. The method of claim 1, further comprising the step of further
enriching for the pluripotent fetal stem cells by additionally
selecting for cells expressing markers expressed by SSAE and/or
SSAE4 embryonic stem cells.
10. The method of claims 1 or 9, further comprising the steps of
further enriching a population of pluripotent fetal stem cells by
eliminating from the population those cells that express SSAE1
marker.
11. The method of claim 1, wherein the chorionic villus, amniotic
fluid or placenta sample is cryopreserved prior to the selection
step.
12. The method of claim 1, further comprising cyropreserving the
c-kit positive cells.
13. A method of proliferating a population of cells enriched for
pluripotent fetal stem cells comprising: (a) selecting at least one
c-kit positive cell from a chorionic villus, amniotic fluid or
placenta sample; (b) introducing said at least one selected cell to
a culture medium; and (c) proliferating said at least one selected
cell in the culture medium.
14. A method of differentiating c-kit positive pluripotent fetal
stem cells comprising providing a chorionic villus, amniotic fluid
or placenta sample and inducing differentiation of c-kit positive
cells within said sample by exposing the sample to one or more
differentiation-inducing agents.
15. The method of claim 14, wherein the differentiation-inducing
agents result in a cell having a phenotype selected from the group
consisting of osteogenic phenotype, hematopoietic phenotype,
adipogenic phenotype, myogenic phenotype, hepatic phenotype,
neurogenic phenotype, and endothelial phenotype.
16. A method of differentiating c-kit positive pluripotent fetal
stem cells comprising: (a) providing a chorionic villus, amniotic
fluid or placenta sample; (b) obtaining cells from said sample; and
(c) inducing differentiation of c-kit positive cells from step (b)
within said sample by exposing said cells to one or more
differentiation-inducing agents.
17. The method of claim 16, wherein the cells are cultured prior to
exposure to the differentiation-inducing agent.
18. The method of claim 16, wherein the differentiation-inducing
agents result in a cell having a phenotype selected from the group
consisting of osteogenic phenotype, hematopoietic phenotype,
adipogenic phenotype, myogenic phenotype, hepatic phenotype,
neurogenic phenotype and endothelial phenotype.
19. A method for storing pluripotent fetal stem cells comprising
the steps of: (a) obtaining a chorionic villus, amniotic fluid or
placenta sample from a human subject; (b) isolating a substantially
enriched population the c-kit positive pluripotent fetal stem cells
from the sample; and (c) cryopreserving the isolated substantially
enriched population of c-kit positive pluripotent fetal stem
cells.
20. A pluripotent fetal stem cell prepared according to the method
of claims 1 to 11.
21. An isolated pluripotent fetal stem cell which is c-kit
positive.
22. A method of treating disease in a human comprising
administering a substantially enriched population of pluripotent
c-kit positive human fetal stem cells into an individual in need
thereof.
23. A method of transplanting an enriched population of pluripotent
c-kit positive human fetal stem cells into a human in need
thereof.
24. A composition suitable for bonemarrow transplantation
comprising an enriched population of pluripotent c-kit positive
human fetal stem cells.
25. A method of treating disease in a human comprising
administering to a human in need thereof a substantially enriched
population of cells comprising pluripotent c-kit positive human
fetal stem cells which have been differentiated to a lineage
selected from osteogenic, hematopoietic, adipogenic, myogenic,
hepatic, neurogenic and endothelial cell lineage.
26. The method of claims 22 or 25, wherein the disease is
Parkinson's Disease.
27. The method of claim 25, wherein the disease is Parkinson's
Disease and the cell lineage is neurogenic cell lineage.
28. A method of transplanting into a human in need thereof a
substantially enriched population of cells comprising pluripotent
c-kit positive human fetal stem cells which have been
differentiated to a lineage selected from osteogenic,
hematopoietic, adipogenic, myogenic, hepatic, neurogenic and
endothelial phenotype.
29. A composition suitable for bonemarrow transplantation
comprising a substantially enriched population of cells comprising
pluripotent c-kit positive human fetal stem cells which have been
differentiated to a lineage selected from osteogenic,
hematopoietic, adipogenic, myogenic, hepatic, neurogenic and
endothelial phenotype.
30. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising isolating a tissue
specimen from the chorionic villus of a human placenta.
31. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising isolating a tissue
specimen containing said cells from human placenta.
32. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising selecting c-kit positive
cells from a chorionic villus.
33. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising selecting c-kit positive
cells from amniotic fluid.
34. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising selecting c-kit positive
cells from placenta.
35. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising the steps of: (a)
cryopreseverving a tissue specimen from the chorionic villus; (b)
thawing the cryopreserved specimen at a later date; and (c)
selecting c-kit positive cells.
36. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising the steps of: (a)
cryopreseverving a specimen of the amniotic fluid; (b) thawing the
cryopreserved specimen at a later date; and (c) selecting c-kit
positive cells.
37. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising the steps of: (a)
cryopreseverving a tissue specimen from a placenta; (b) thawing the
cryopreserved specimen at a later date; and (c) selecting c-kit
positive cells.
38. A method of producing a population of cells enriched for
pluripotent fetal stem cells comprising, (a) isolating c-kit
positive cells from the chorionic villus; and (b) proliferating
said cells in culture medium.
39. A method of producing differentiated tissue comprising, (a)
providing a tissue specimen from chorionic villus; (b) culturing
the tissue under conditions that cause c-kit positive cells to
proliferate; and (c) upon induction cause the c-kit positive cells
to differentiate.
40. A method of producing differentiated tissue comprising, (a)
providing a tissue specimen from amniotic fluid; (b) culturing the
tissue under conditions that cause c-kit positive cells to
proliferate; and (c) upon induction cause the c-kit positive cells
to differentiate.
41. A method of producing differentiated tissue comprising, (a)
providing a tissue specimen from placenta; (b) culturing the tissue
under conditions that cause c-kit positive cells to proliferate;
and (c) upon induction cause the c-kit positive cells to
differentiate.
42. The method of claim 32, wherein negative selection is used to
enrich c-kit positive cells from the chorionic villus.
43. The method of claim 33, wherein negative selection is used to
enrich c-kit positive cells from the amniotic fluid.
44. The method of claim 34, wherein negative selection is used to
enrich c-kit positive cells from the placenta.
45. The method of claims 30, 31 or 47, wherein the cells are
subsequently cryopreserved.
46. A tissue-engineered construct comprising a biocompatible
polymer scaffold and isolated c-kit positive fetal stem cells.
47. A method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising isolating a tissue
specimen from amniotic fluid.
48. A method of producing a population of cells enriched for
pluripotent fetal stem cells comprising, (a) isolating c-kit
positive cells from the placenta; and (b) proliferating said cells
in culture medium.
49. A method of producing a population of cells enriched for
pluripotent fetal stem cells comprising, (a) isolating c-kit
positive cells from the amniotic fluid; and (b) proliferating said
cells in culture medium.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the isolation, expansion and
differentiation of fetal stem cells from chorionic villus, amniotic
fluid, and placenta and therapeutic uses thereof.
BACKGROUND OF THE INVENTION
[0002] Stem cells are unique cell populations with the ability to
undergo both renewal and differentiation. This fate choice is
highly regulated by intrinsic signals and the external
microenvironment. They can be identified in many adult mammalian
tissues, such as bone marrow, skeletal muscle, skin and adipose
tissue, where they contribute to replenishment of cells lost
through normal cellular senescence or injury. Although stem cells
in adult tissues may be capable of developing into more cell types
than originally thought, they have a limited cellular regeneration
or turnover.
[0003] Stem cells have been reported to exist during embryonic
development and postnatally in bone marrow, skeletal muscle and
skin. Embryonic stem (ES) cells are derived from the inner cell
mass (ICM) at the blastula stage, and have the property of
participating as totipotent cells when placed into host
blastocysts. They are able not only to activate the expression of
genes restricted to each of the three embryonic germ (EG) layers,
but they are also able to express receptors for a number of
different soluble growth factors with established effects on
developmental pathways in vivo.
[0004] Adult stem cells, on the other hand, do not differentiate
spontaneously, but can be induced to differentiate by applying
appropriate growth conditions. Adult stem cells seem to be easier
to maintain in culture than ES cells. Adult stem cells have the
disadvantage of not being immortal, and most of them lose their
pluripotency after a defined number of passages in culture. This
short life-span may be a problem for clinical applications where a
large amount of cells are needed.
[0005] In contrast to adult stem cells, ES cells, derived from
blastocyst-stage early mammalian embryos, have the ability to give
rise to cells that not only proliferate and replace themselves
indefinitely, but that have the potential to form any cell type. ES
cells tend to differentiate spontaneously into various types of
tissues; however, specific growth induction conditions do not
direct differentiation exclusively to specific cell types. Two
reports describing the isolation, long-term culture, and
differentiation of such cells have generated tremendous excitement
in this regard and are herein incorporated by reference (Shamblott,
Michael J., et al., "Derivation of Pluripotent Stem Cells from
Cultured Human Primordial Germ Cells," Proc. Natl. Acad. Sci. USA,
Vol. 95, pp. 13726-31, November 1998; Thomson, James A., et al.,
"Embryonic Stem Cell Lines Derived from Human Blastocysts,"
Science, Vol. 282, pp. 1145-47, Nov. 6, 1998). Although there is a
great scientific interest in ES cell research, the destruction of
embryos in order to harvest and experiment on ES cells still create
unresolved ethical concerns.
[0006] Fetal tissue has been used in the past for autograft and
allograft transplantation and tissue engineering research because
of its pluripotency, proliferative ability and lack of
immunogenicity. Fetal cells maintain a higher capacity to
proliferate than adult cells and may preserve their pluripotency
longer in culture. However, fetal cell transplants are plagued by
problems that are very difficult to overcome. Fetal tissue can be
currently obtained from a biopsy of the fetus itself during
gestation or from cord blood at birth; however, both procedures are
associated with a defined morbidity. Fetal tissue can also be
obtained from aborted embryos, but this resource is limited. Beyond
the ethical concerns regarding the use of cells from aborted
fetuses or living fetuses, there are other issues which remain a
challenge. For example, studies have shown that it generally takes
about six fetuses to provide enough material to treat one patient
with Parkinson's disease.
[0007] Because stem cells, particularly pluripotent stem cells
appear to be an excellent resource for therapeutic applications,
there is a great need for a source of stem cells that is plentiful,
easy to manipulate, and avoids ethical considerations.
SUMMARY OF THE INVENTION
[0008] We have discovered that chorionic villus, amniotic fluid,
and placenta provide an excellent source of pluripotent fetal stem
cells for therapeutic applications. These fetal stem cells have a
better potential for expansion than adult stem cells and avoid the
current controversies associated with the use of human embryonic
stem cells. The c-kit.sup.pos cells isolated from the chorionic
villus, amniotic fluid and placenta samples differentiate into
specific cell lineages, they do not need feeder layers to grow, and
most importantly, the isolation of these cells does not require the
sacrifice of human embryos for their isolation, thus avoiding the
current controversies associated with the use of human embryonic
stem cells.
[0009] Therefore, the present invention is directed to pluripotent
fetal stem cells derived from chorionic villus, amniotic fluid, and
placenta and the methods for isolating, expanding and
differentiating these cells, and their therapeutic uses such as
manipulating the fetal stem cells by gene transfection and other
means for therapeutic applications, including but not limited to
enzyme replacement and gene therapy, tissue regeneration and
replacement, including, for example burn and wound dressings.
[0010] In one aspect, the present invention provides a method for
obtaining pluripotent human fetal stem cells comprising obtaining a
chorionic villus and/or amniotic fluid and/or placenta sample from
a human subject and isolating c-kit positive cells from the sample.
The invention further provides culturing or expanding the c-kit
positive in a culture media before or after isolation. The
chorionic villus, amniotic fluid or placenta sample may be
cryopreserved before isolating or differentiating the c-kit
positive cells. Alternatively, the c-kit positive cells are
isolated from the sample and then cryopreserved. The cells may be
cryopreserved before or after differentiation.
[0011] In yet another aspect, the present invention provides a
method for differentiating the isolated pluripotent human fetal
stem cells derived from chorionic villus and/or amniotic fluid
and/or placenta to cells of different lineages, including, but not
limited to, osteogenic, adipogenic, myogenic, neurogenic,
hematopoitic and endothelial lineages. Differentiation can be
evidenced by, for example, changes in cellular morphology and gene
expression.
[0012] In a further aspect, the present invention provides a method
for differentiating c-kit positive fetal stem cells contained
within a chorionic villus sample, amniotic fluid sample or a
placenta sample to cells of different lineages, including, but not
limited to, osteogenic, adipogenic, myogenic, neurogenic,
hematopoietic, hepatic and endothelial lineages. The method
comprises exposing the sample to one or more
differentiation-inducing agents either in vivo or in vitro. Cells
may be isolated from the sample before differentiation.
[0013] In yet another aspect, the present invention provides a
method for assessing viability, proliferation potential, and
longevity of the pluripotent human fetal stem cells derived from
chorionic villus, amniotic fluid and placenta.
[0014] In another aspect the invention provides a method of
treating disease in a human comprising administering to a human in
need thereof a substantially enriched population of cells
comprising pluripotent c-kit positive human fetal stem cells which
have been differentiated to a lineage selected from osteogenic,
hematopoietic, adipogenic, myogenic, hepatic, neurogenic and
endothelial cell lineage. For example, Parkinson's disease can be
treated with the isolated pluripotent c-kit positive stem cells of
the present invention either directly, or after differentiating
such cells into a neuronal cell lineage capable of producing
dopamine.
[0015] The invention further provides a method of transplanting
into a human in need thereof a substantially enriched population of
cells comprising pluripotent c-kit positive human fetal stem cells
which have been differentiated to a lineage selected from
osteogenic, hematopoietic, adipogenic, myogenic, hepatic,
neurogenic and endothelial phenotype.
[0016] In another aspect, the invention provides a composition
suitable for bonemarrow transplantation comprising a substantially
enriched population of cells comprising pluripotent c-kit positive
human fetal stem cells which have been differentiated to a lineage
selected from osteogenic, hematopoietic, adipogenic, myogenic,
hepatic, neurogenic and endothelial phenotype.
[0017] Further, the invention provides a method of obtaining a
population of cells enriched for pluripotent fetal stem cells,
comprising isolating a tissue specimen from the chorionic villus of
a human placenta.
[0018] The invention also provides a method of obtaining a
population of cells enriched for pluripotent fetal stem cells,
comprising isolating a tissue specimen containing said cells from
human placenta, chorionic villus or amniotic fluid.
[0019] In yet another aspect the invention provides a method of
obtaining a population of cells enriched for pluripotent fetal stem
cells, comprising selecting c-kit positive cells from placenta.
[0020] The invention further provides a method of obtaining a
population of cells enriched for pluripotent fetal stem cells,
comprising the steps of cryopreseverving a tissue specimen from the
chorionic villus, amniotic fluid or placenta, and thawing the
cryopreserved specimen at a later date and selecting c-kit positive
cells.
[0021] In another aspect, the invention provides a method of
producing a population of cells enriched for pluripotent fetal stem
cells comprising isolating c-kit positive cells from the chorionic
villus, placenta or amniotic fluid, and proliferating the cells in
culture medium.
[0022] In another aspect, the invention provides a method of
producing differentiated tissue comprising providing a tissue
specimen from chorionic villus, amniotic fluid or placenta,
culturing the tissue under conditions that cause c-kit positive
cells to proliferate; and upon induction cause the c-kit positive
cells to differentiate.
[0023] The invention also provides a method of the invention
provides a method of obtaining a population of cells enriched for
pluripotent fetal stem cells, comprising isolating a tissue
specimen from the chorionic villus of a human placenta, placenta,
or amniotic fluid further comprising using negative selection to
enrich c-kit positive cells from the chorionic villus.
[0024] In yet another aspect, the invention provides a method of
obtaining a population of cells enriched for pluripotent fetal stem
cells, comprising isolating a tissue specimen containing said cells
from human placenta, chorionic villus or amniotic fluid wherein the
cells are subsequently cryopreserved.
[0025] Finally, the present invention provides therapeutic
applications for the fetal stem cells derived from a chorionic
villus and/or an amniotic fluid and/or a placenta sample including,
but not limited to (a) autologous/heterologous enzyme replacement
therapy; (b) autologous/heterologous transgene carriers in gene
therapy; (c) autologous/heterologous tissue
regeneration/replacement therapy; (d) reconstructive treatment by
surgical implantation; (e) reconstructive treatment of tissues with
products of these cells; and (f) tissue engineering.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1G show results from chorionic villi and amniotic
cell characterization experiments. Between 0.8 and 3% of the
amniotic and chorionic villi cells were c-kit.sup.pos [1A]. The
c-kit.sup.pos cells did not stain with mouse stage specific
embryonic antigen 1 [1B], but stained positively for human stage
specific embryonic antigens 3 and 4 [1C and 1D]. Analyses of late
passage c-kit.sup.pos cells (PD 200) showed a normal karyotype
[1E]. Telomerase activity was evaluated using the Telomerase Repeat
Amplification Protocol (TRAP) assay [1F]. The chorionic villi and
amniotic c-kit.sup.pos cells were telomerase positive (lane1). Upon
differentiation into specific lineages, telomerase activity
diminished to undetectable levels (Lane 2). Lane 3 shows the
positive control. Lane 4 represents negative control cell lysate,
showing no telomerase activity. The telomeric length was evaluated
by terminal restriction fragment (TRF) measurement [1G].
C-kit.sup.pos cells had similar telomere lengths, both at early and
late passages (250 PD) (lane 3 and 4, respectively) as compared
with a high molecular weight marker, approximately 10.2 kbp (lane
2). Lane 1 represents a low molecular weight marker.
[0027] FIGS. 2A-2L demonstrate osteogenic induction of the
c-kit.sup.pos cells isolated from chorionic villi and amniotic
fluid. The shape of chorionic villi and amniotic c-kit.sup.pos
cells treated with osteogenic-inducing medium after 4 days of
induction changed to an osteoblast-like appearance [2A], whereas
cells in the control medium did not lose their spindle-shaped
phenotype [2B]. Alkaline phosphatase activity was quantified in
c-kit.sup.pos cells that were incubated with osteogenic-inducing
and control medium for 32 days [2C]. Numbers represent alkaline
phosphatase production in nMol p-Nitrophenol/min/10.sup.6 cells,
showing a peak of production at day 16 (solid line); whereas
c-kit.sup.pos cells grown in control medium (shaded line) or
c-kit.sup.neg cells grown in osteogenic conditions (dotted line)
did not show any alkaline phosphatase production. C-kit.sup.pos
cells treated with osteogenic-inducing medium and with control
medium stained for alkaline phosphatase after 4, 8, 16, 24 and 32
days [2D]. Strong alkaline phosphatase staining was noted in the
osteogenic-induced cells starting at day 16, and remained high
thereafter. C-kit.sup.pos cells grown in control medium did not
show any alkaline phosphatase staining. When confluent, the cells
formed typical lamellar structures similar to those found in bone
[2F]. C-kit.sup.pos cells in control medium did not form any
lamellar structures [2E]. Mineralization of cells was quantified
using a chemical assay for calcium [2G]. Numbers represent calcium
deposition in mg/dl. Osteogenic-induced ckit.sup.pos cells showed a
significant increase of calcium deposition starting at day 16
(solid line). No calcium deposition was detected in ckit.sup.pos
cells grown in control medium (shaded line) or ckit.sup.pos cells
grown in osteogenic conditions (dotted line). Furthermore cells
treated with control medium or with osteogenic-inducing medium were
analyzed using von Kossa staining after 32 days in culture
(40.times.). The osteogenic-induced cells showed significant
mineralization starting at day 16 [2H]. No mineralization occurred
at any time point in cells grown in control medium [2I]. RNA was
isolated from amniotic c-kit.sup.pos cells grown in control medium
(lanes 1, 2, 3 and 4) and osteogenic-inducing medium (lanes 5, 6, 7
and 8). RT-PCR was performed using primers for alkaline
phosphatase, cbfa1, osteocalcin and .beta.2-microglobulin at days
8, 16, 24 and 32 [2G]. RT-PCR showed upregulation of cbfa1 and
osteocalcin at day 8 and it confirmed the upregulation of alkaine
phosphatase in the osteogenic-induced cells [2J]. C-kit.sup.pos
cells were seeded on hydroxyapatite-collagen scaffolds, induced
into an osteogenic lineage, implanted subcutaneously in athymic
mice, and harvested after 4 and 8 weeks. Bone-like tissue was
evident, surrounded by an extracellular matrix. Toluidine blue
staining confirmed the osteogenic phenotype. Large calcified areas
within the implanted tissue stained positively with von Kossa,
indicating bone formation [2K]. Non seeded scaffold were implanted
and used as control [2L].
[0028] FIGS. 3A-3F demonstrate adipogenic induction of the
c-kit.sup.pos cells isolated from chorionic villi and amniotic
fluid. Clusters of adipocytes appeared at 8 days [3A], and the
percentage of cells increased with time until Oil-O-Red was
uniformly staining the adipogenesis-induced cells at day 16 [3B].
C-kit.sup.pos cells cultured in control medium did not show any
lipid deposits at day 16 [3C]. RNA was isolated from c-kit.sup.pos
cells grown in control (lanes 1 and 2) and adipogenic-inducing
(lanes 3 and 4) medium [3D]. RT-PCR was performed using primers for
PPAR.gamma.2, lipoprotein lipase and .beta.2-microglobulin at days
8 and 16, as indicated. Upregulation of PPAR.gamma.2 and
lipoprotein lipase in cells grown in adipogenic-inducing medium was
noted at days 8 and 16 (lanes 3 and 4). C-kit.sup.pos cells were
seeded on polyglycolic acid polymer scaffolds. Cells were induced
into an adipogenic lineage. The scaffolds were implanted
subcutaneously in athymic mice, harvested after 4 and 8 weeks and
analyzed. The retrieved scaffolds showed the formation of fatty
tissues grossly [3E]. The presence of adipose tissue was confirmed
with Oil-O-Red staining (200.times. magnification) [3F].
[0029] FIGS. 4A-4I demonstrate myogenic induction of the
c-kit.sup.pos cells isolated from chorionic villi and amniotic
fluid. Under myogenic conditions the c-kit.sup.pos cells fused into
multinucleated cells at day 4 [4A] and formed myotube-like
structures after 8 days [4B]. Multinucleated cells stained green
for sarcomeric tropomyosin [4C] and desmin [4D] expression 16 days
after myogenic induction. Cell nuclei were stained blue using DAPI.
Untreated cells did not stain for sarcomeric tropomyosin [4E] or
desmin [4F]. RNA was isolated from c-kit.sup.pos cells grown in
control (lanes 1 and 2) and myogenic-inducing (lanes 3 and 4)
medium [4G]. RT-PCR was performed using primers for MyoD, MRF4
(herculin, Myf6), and desmin at days 8 and 16. Myogenic-induced
cells showed a strong upregulation of desmin expression at day 16
(lane 4). MyoD and MRF4 were induced, with myogenic treatment at
day 8 (lane 1). Specific PCR amplified DNA fragments of MyoD, MRF4
and Desmin could not be detected in the control cells at days 8 and
16 (lanes 1 and 2). C-kit.sup.pos cells were labeled with the
fluorescence marker PKH26 and were induced into a myogenic lineage.
The myogenic cells were injected into the hindlimb musculature of
athymic mice and were retrieved after 4 weeks. The injected
myogenic cells showed the formation of muscle tissue (m) which
expressed desmin [4H] and maintained its fluorescence [4I]. The
native muscle (n) did not express any fluorescence.
[0030] FIGS. 5A-5F demonstrate endothelial induction of the
c-kit.sup.pos cells isolated from chorionic villi and amniotic
fluid. Ckit.sup.pos cells were cultured as monolayers in
PBS-gelatin coated dishes with EBM-2 and bFGF and showed a typical
endothelial appearance in vitro [5A]. The fully differentiated
endothelial cells stained for the endothelial specific markers
FVIII [5B], KDR [5C] and P1H12[5D]. Once cultured in matrigel the
cells were able to form capillary structures over time [5E]. In
order to confirm the phenotypic changes we performed RT-PCR 5[F].
CD31 and VCAM showed a marked increased in the ckit.sup.pos cells
induced in endothelial medium (lane2). Ckit.sup.pos cells cultured
in control medium (lane1) did not show any gene amplification.
[0031] FIGS. 6A-6E demonstrate neurogenic induction of the
c-kit.sup.pos cells isolated from chorionic villi and amniotic
fluid. Ckit.sup.pos cells cultured under neurogenic inducing
conditions changed their morphology within the first 24 hours. The
cell cytoplasm retracted towards the nucleus, forming contracted
multipolar structures, with primary and secondary branches, and
cone-like terminal expansions [6A]. The differentiated cells
stained for specific neurogenic markers .beta. III Tubulin [6B],
Nestin [6C], and glial fibrillary acidic protein (GFAP) [6D]. Only
the C-kit.sup.pos cells cultured under neurogenic conditions showed
the secretion of glutamic acid in the collected medium. Furthermore
the secretion of glutamic acid could be induced (KCl; 20 min in 50
mM KCl buffer) [6E].
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is based upon a discovery that
chorionic villus, amniotic fluid, and placenta cells can be used to
obtain a population of stem cells which are comparable to embryonic
stem cells in their pluripotent differentiation capacity and
therefore are a viable source of stem cells that can be used
therapeutically.
[0033] Chorionic villus sampling and amniocentesis are well
established techniques for the collection of tissue from the human
embryo (10 to 12 weeks) and for the collection of fluid from the
human fetus (12 weeks to term), respectively. Chorionic villus
sampling is performed on pregnant mammal, preferably human, and has
been in use since the 1980s. This procedure involves taking a
sample of the chorion frondosum--that part of the chorionic
membrane containing the villi. The chorionic membrane is the outer
sac which surrounds the developing fetus. Chorionic villi are
microscopic, finger-like projections that emerge from the chorionic
membrane and eventually form the placenta. The cells that make up
the chorionic villi are of fetal origin.
[0034] In humans, chorionic villus sampling is best performed
between 10 and 12 weeks of pregnancy. The procedure is performed
either through the vagina and the cervix (transcervically) or
through the abdomen (transabdominally) depending upon the
preferences of the patient or the doctor. In some cases, the
location of the placenta dictates which method the doctor uses. For
the transcervical procedure, the woman lies on an examining table
on her back with her feet in stirrups. The woman's vaginal area is
thoroughly cleansed with an antiseptic, a sterile speculum is
inserted into her vagina and opened, and the cervix is cleansed
with an antiseptic. Using ultrasound (a device which uses sound
waves to visualize internal organs) as a guide, the doctor inserts
a thin, plastic tube called a catheter through the cervix and into
the uterus. The passage of the catheter through the cervix may
cause cramping. The doctor carefully watches the image produced by
the ultrasound and advances the catheter to the chorionic villi. By
applying suction from the syringe attached to the other end of the
catheter, a small sample of the chorionic villi are obtained. A
cramping or pinching feeling may be felt as the sample is being
taken. The catheter is then easily withdrawn.
[0035] For the transabdominal method, the woman lies on her back on
an examining table. Ultrasound enables the doctor to locate the
placenta. The specific area on the woman's abdomen is cleansed
thoroughly with an antiseptic and a local anesthetic may be
injected to numb the area With ultrasound guidance, a long needle
is inserted through the woman's abdominal wall, through the uterine
wall and to the chorionic villi. The sample is obtained by applying
suction from the syringe. The chorionic villus sample is
immediately placed a into nutrient medium.
[0036] Amniotic fluid is obtained using amniocentesis. The word
amniocentesis literally means "puncture of the amnion," the
thin-walled sac of fluid in which a developing fetus is suspended
during pregnancy. During the sampling procedure, the obstetrician
inserts a very fine needle through the woman's abdomen into the
uterus and amniotic sac and withdraws approximately one ounce of
amniotic fluid.
[0037] The physician uses ultrasound images to guide needle
placement and collect the sample, thereby minimizing the risk of
fetal injury and the need for repeated needle insertions. Once the
sample is collected, the woman can return home after a brief
observation period. She may be instructed to rest for the first 24
hours and to avoid heavy lifting for two days. Consequently, the
fetal cells contained in the fluid are isolated and grown as
explained below.
[0038] These techniques may be used to obtain chorionic villus and
amniotic fluid samples in accordance with the present invention.
Cultured cells from the chorionic villi or amniotic fluid of
pregnancies have been used widely for the prenatal diagnosis of
genetic disorders. The morphologic heterogeneity of these cells is
well known. Numerous cell types from all 3 germ layers are found in
the placenta and the amniotic fluid at different levels of
differentiation (6). Large quantities of chorionic villi and
amniotic fluid are available during pregnancy and at the time of
birth, and cells can be easily obtained from these sources. The
same is true for placenta, which is obtainable after birth.
[0039] A sample of placenta may be obtained using a punch-biopsy, a
scalpel or homogenizing the placenta or a portion thereof using,
for example, a blender. The homogenate may then be used as a source
of cells.
[0040] Stem cell differentiation requires cell-cell contact and
cell-extracellular matrix interactions. While not wishing to be
bound by a particular theory, it is believed that chorionic villus,
amniotic fluid and placenta make a good source of undifferentiated
cells because the cells liberated in the chorionic villus and
amniotic fluid from the fetus during development may not receive
any signal of differentiation, and may be able to maintain their
"pluripotential" state. We have discovered that the preferred cells
are c-kit positive. Thus, the c-kit marker can be used to isolate
these cells. As used herein the terms "pluripotent" or
"pluripotential" cell refers to a cell that has complete
differentiation versatility, i.e., the capacity to differentiate
into at least osteogenic phenotype, hematopoietic phenotype,
adipogenic phenotype, myogenic phenotype, hepatic phenotype and
endothelial phenotype in appropriate inducing conditions,
preferably the pluripotent cell has the capacity to differentiate
to any of the mammalian body's about 260 different cell types.
[0041] The c-kit gene encodes a tyrosine kinase growth factor
receptor for Stem Cell Factor (SCF), also called mast cell growth
factor, that is essential for hematopoiesis, melanogenesis and
fertility. The larger 45 kDa form is processed to generate a 31 kDa
soluble factor while the smaller 32 kDa form gives rise to a 23 kDa
factor. Expression of the two alternatively spliced forms is
somewhat tissue-specific; the 31 kDa form of SCF is expressed in
fibro-blasts and thymus tissue while the 23 kDa factor is found in
spleen, testis, placenta and cerebellum. The c-kit receptor
protein, also known as c-Kit receptor, Steel factor receptor, stem
cell factor receptor and CD117 in standardized terminology of
leukocyte antigens, is constitutively expressed in hematopoietic
stem cells, mast cells, germ cells, melanocytes, certain basal
epithelial cells, luminal epithelium of breast, and the
interstitial cells of Cajal of the gastrointestinal tract. The
c-kit receptor plays a fundamental role during the establishment,
maintenance and function of germ cells. In the embryonal gonad the
c-kit receptor and its ligand SCF are required for the survival and
proliferation of primordial germ cells. In the postnatal animal,
c-kit/SCF are required for production of the mature gametes in
response to gonadotropic hormones, i.e. for the survival and/or
proliferation of the only proliferating germ cells of the testis,
the spermatogonia, and for the growth and maturation of the
oocytes. Experiments have shown that c-kit is a potent growth
factor for primitive hematopoietic cell proliferation in vitro. In
mice, loss of either SCF or c-kit due to mutations in their loci
results in macrocytic anemia, leading to death in utero or within
the first postnatal days.
[0042] Antibodies reactive with the c-kit or portions thereof can
be used to isolate c-kit positive cells. In a preferred embodiment,
the antibodies specifically bind with the c-kit or a portion
thereof. The antibodies can be polyclonal or monoclonal, and the
term antibody is intended to encompass polyclonal and monoclonal
antibodies, and functional fragments thereof. The terms polyclonal
and monoclonal refer to the degree of homogeneity of an antibody
preparation, and are not intended to be limited to particular
methods of production.
[0043] Therefore, examples of antibodies useful according to the
present invention include antibodies recognizing the c-kit. Such
antibodies are herein referred to as "c-kit antibodies." Examples
of commercially available useful c-kit antibodies include, but are
not limited to antibodies in table 1, that can be purchased from
Santa Cruz Biotechnology, Inc. TABLE-US-00001 Antibody Cat.#
Isotype Epitope Applications Species SCF (N-19) sc-1302 goat-IgG
N-terminus WB, IP, IHC human (h) ELISA c-Kit (C-19) sc-168 rabbit
IgG C-terminus WB, IP, IHC, mouse, rat, (h) PAR human c-Kit (M-14)
sc-1494 goat IgG C-terminus WB, IP, IHC mouse, rat, (m) human c-Kit
(Ab 81) sc-13508 mouse IgG.sub.1 FL (h) WB, IP, IHC human FCM c-Kit
(C-14) sc-1493 goat IgG C-terminus WB, IP, IHC human > mouse (h)
c-Kit (104D2) sc-19983 mouse IgG.sub.1 n/a IHC, FCM human c-Kit
(H-300) sc-5535 rabbit IgG 23-322 (h) WB, IP, IHC mouse, rat, human
c-Kit (E-1) sc-17806 mouse IgG.sub.1 23-322 (h) WB, IHC human
[0044] The preferred antibody is c-Kit (E-1), which is a mouse
monoclonal IgG recognizing an epitope corresponding to amni acids
23-322 mapping near the c-kit N-terminys and recognizes both c-Kit
of human origin by both Western blotting and
immunihistochemistry.
[0045] Additional examples of commercially available antibodies
include, but are not limited to YB5.B8 monoclonal antibody,
specific for human CD117 (eBioscience, San Diego, Calif.); an
antibody produced against a human leucaemic cell line UT7
transfected with CD117 cDNA (Chemicon International, Temecula,
Calif.); a polyclonal antibody produced against the C-terminal end
of CD117 (Assay Designs Inc., Ann Arbor, Mich., catalog No. 90572);
clone 28 c-kit monoclonal antibody (catalog no. 612318, from BD
Transduction Laboratories, Franklin Lakes, N.J.); a c-kit tyrosine
kinase receptor antibody ab1462, which is a rabbit polyclonal
anti-human c-kit tyrosine kinase receptor antibody was generated
using a synthetic KLH-conjugated peptide corresponding to the
carboxy-terminus of the CD117; and a monoclonal 13CD anti c-kit
antibody from Zymed Laboratories Inc. (South San Francisco,
Calif.).
[0046] Further, antibodies recognizing c-kit or fragments thereof
may be obtained or prepared as discussed in U.S. Pat. No.
5,454,533, incorporated herein by reference. The c-kit antigen can
be contacted with an antibody, such as various c-kit monoclonal
antibodies, which have specificity for the c-kit antigen. A c-kit
antibody is characterized by binding to the c-kit protein or
fragments thereof under Western blot conditions from reducing
SDS-PAGE gels. For example, the CD117 antigen of c-kit has a
molecular weight, based on commercially available standards, in the
range of about 145 kDa.
[0047] The terms "specific binding" or "specifically binding", as
used herein, refers to the interaction between a c-kit or a
fragment thereof expressed by a cell present in a chorionic villus,
amniotic fluid or placenta sample, and an antibody. The interaction
is dependent upon the presence of a particular structure, i.e., the
antigenic determinant or epitope of c-kit, of the c-kit recognized
by the binding molecule, i.e. the c-kit antibody. For example, if
an antibody is specific for epitope "A" of c-kit, the presence of a
protein containing epitope A (or free, unlabeled A) in a reaction
containing labeled "A" and the antibody will reduce the amount of
labeled A bound to the antibody.
[0048] Additionally, antibodies to c-kit antigen or fragments
thereof can be obtained by immunizing a xenogeneic immunocompetent
mammalian host (including murine, rodentia, lagomorpha, ovine,
porcine, bovine, etc.) with human c-kit or fragments thereof
expressing cells. The choice of a particular host is primarily one
of convenience. A suitable progenitor cell population for
immunization can be obtained, for example by isolating c-kit
positive cells from tissues or cell cultures. Immunizations are
performed in accordance with conventional techniques, where the
cells may be injected subcutaneously, intramuscularly,
intraperitoneally, intravascularly, etc. Normally, from about
10.sup.6 to 10.sup.8 cells will be used, which may be divided up
into one or more injections, usually not more than about 8
injections, over a period of from about one to three weeks. The
injections may be with or without adjuvant, e.g. complete or
incomplete Freund's adjuvant, specol, alum, etc.
[0049] After completion of the immunization schedule, the antiserum
may be harvested in accordance with conventional ways to provide
polygonal antisera specific for the surface membrane proteins of
progenitor cells, including the c-kit antigen or fragments thereof.
Lymphocytes are harvested from the appropriate lymphoid tissue,
e.g. spleen, draining lymph node, etc., and fused with an
appropriate fusion partner, usually a myeloma line, producing a
hybridoma secreting a specific monoclonal antibody. Screening
clones of hybridomas for the antigenic specificity of interest is
performed in accordance with conventional methods.
[0050] Antibodies against c-kit or fragments thereof can be
produced as a single chain, instead of the normal multimeric
structure. Single chain antibodies are described in Jost et al.,
269 J. Biol. Chem. 26267-73 (1994), incorporated herein by
reference, and others. DNA sequences encoding the variable region
of the heavy chain and the variable region of the light chain are
ligated to a spacer encoding at least about 4 amino acids of small
neutral amino acids, including glycine or serine. The protein
encoded by this fusion allows assembly of a functional variable
region that retains the specificity and affinity of the original
antibody.
[0051] Antibodies against c-kit or fragments thereof can be
produced by use of Ig cDNA for construction of chimeric
immunoglobulin genes (Liu et al., 84 Proc. Natl. Acad. Sci. 3439
(1987) and 139 J. Immunol. 3521 (1987), incorporated herein by
reference. mRNA is isolated from a hybridoma or other cell
producing the antibody and used to produce cDNA. The cDNA of
interest may be amplified by the polymerase chain reaction using
specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).
Alternatively, a library is made and screened to isolate the
sequence of interest. The DNA sequence encoding the variable region
of the antibody is then fused to human constant region sequences.
The sequences of human constant regions genes may be found in Kabat
et al., "Sequences of Proteins of Immunological Interest" N.I.H.
publication No. 91-3242 (1991). Human C region genes are readily
available from known clones. The chimeric, humanized antibody is
then expressed by conventional methods.
[0052] Antibodies against c-kit or fragments thereof can also be
produced as antibody fragments, such as Fv, F(ab').sub.2 and Fab.
Antibody fragments may be prepared by cleavage of the intact
protein, e.g. by protease or chemical cleavage. Alternatively, a
truncated gene is designed. For example, a chimeric gene encoding a
portion of the F(ab').sub.2 fragment would include DNA sequences
encoding the CH1 domain and hinge region of the H chain, followed
by a translational stop codon to yield the truncated molecule.
[0053] The c-kit positive cell selection can be by any suitable
means known in the art, including flow cytometry, such as by
fluorescence activated cell sorting using a fluorochrome conjugated
c-kit antibody. The selection can also be by high gradient magnetic
selection using c-kit antibody is conjugated to magnetic particles.
Any other suitable method including attachment to and disattachment
from solid phase, is also contemplated within the scope of the
invention.
[0054] One of skill in the art can derive the population of cells
by immunoselection using an c-kit antibody. The population of cells
should contain at least 30% c-kit positive (c-kit.sup.+ or
c-kit.sup.pos) pluripotent fetal stem cells, preferably at least
50-70% c-kit.sup.+ fetal stem cells, and more preferably greater
than 90% c-kit.sup.+ fetal stem cells. Most preferable would be a
substantially pure population of c-kit.sup.+ fetal stem cells,
comprising at least 95% c-kit.sup.+ fetal stem cells.
[0055] The number of c-kit positive cells in a cell population can
be determined in any well known method known to one skilled in the
art. For example, FACS analysis can be used as shown in FIG. 1A.
Alternatively, magnetic cell sorting technology (MACS) can be used
to separate cells (see, e.g. Miltenyi Biotech, Inc., Auburn,
Calif.). In MACS, the c-kit positive cells can be separated from
the mixture of chorionic villus cells, amniotic fluid, and placenta
cells to very high purity. The c-kit positive cells are
specifically labeled with super-paramagnetic MACS MicroBeads which
can be designed to bind to either the c-kit antigen directly or to
the antibody recognizing c-kit. After magnetic labeling, the cells
are passed through a separation column which is placed in a strong
permanent magnet. The column matrix serves to create a
high-gradient magnetic field. The magnetically labeled cells are
retained in the column while non-labeled cells pass through. After
removal of the column from the magnetic field, the magnetically
retained cells are eluted. Both labeled and non-labeled fractions
can be completely recovered.
[0056] The in vitro cell cultures described herein containing an
enriched population of c-kit positive pluripotent fetal stem cells
are generally characterized in that the cultures stain positive for
c-kit and SSAE3 and SSAE4, produce progeny cells that can
differentiate into at least two, preferably three, most preferably
at least all of the following cell lineages: osteogenic,
adipogenic, neurogenic, myogenic, hematopoietic, hepatic and
endothelial cell lineages in the presence of
differentiation-inducing conditions of which examples are described
in the Example below. Further examples of differentiation-inducing
agenst and combinations thereof for differentiating desired cell
lineages can be found at Stem Cells: Scientific Progress and Future
Research Directions. (Appendix D. Department of Health and Human
Services. June 2001.
http://www.nih.gov/news/stemcell/scireporthtm)
[0057] Immunostaining. Biological samples including the cells
isolated from chrorionic villus samples, amniotic fluid samples or
placenta are assayed for the presence of c-kit.sup.+ fetal stem
cells by any convenient immunoassay method for the presence of
cells expressing the c-kit, bound by the c-kit antibodies. Assays
may be performed on cell lysates, intact cells, frozen sections,
etc.
[0058] Cell Sorting. The use of cell surface antigens to fetal stem
cells, such as c-kit provides a means for the positive
immunoselection of fetal stem cell populations, as well as for the
phenotypic analysis of progenitor cell populations using, for
example, flow cytometry. Cells selected for expression of c-kit
antigen may be further purified by selection for other stem cell
and progenitor cell markers, including, but not limited to SSAE3
and SSAE4 human embryonic stem stage specific markers.
[0059] Alternatively, for the preparation of substantially pure
pluripotent fetal stem cells, a subset of stem cells can be
separated from other cells on the basis of c-kit antibody binding
and the c-kit positive fetal stem cells may be further separated by
binding to other surface markers known in the art.
[0060] Procedures for separation may include magnetic separation,
using antibody-coated magnetic beads, affinity chromatography and
"panning" with antibody attached to a solid matrix, e.g. plate, or
other convenient technique. Techniques providing accurate
separation include fluorescence activated cell sorters, which can
have varying degrees of sophistication, such as multiple color
channels, low angle and obtuse light scattering detecting channels,
impedance channels, etc. Dead cells may be eliminated by selection
with dyes associated with dead cells (propidium iodide (PI), LDS).
Any technique may be employed which is not unduly detrimental to
the viability of the selected cells.
[0061] Conveniently, the antibodies are conjugated with labels to
allow for ease of separation of the particular cell type, e.g.
magnetic beads; biotin, which binds with high affinity to avidin or
streptavidin; fluorochromes, which can be used with a fluorescence
activated cell sorter; haptens; and the like. Multi-color analyses
may be employed with the FACS or in a combination of immunomagnetic
separation and flow cytometry. Multi-color analysis is of interest
for the separation of cells based on multiple surface antigens,
e.g. c-kit.sup.+, and antibodies recognizing SSAE3 and SSAE4 cell
markers. Fluorochromes which find use in a multi-color analysis
include phycobiliproteins, e.g. phycoerythrin and allophycocyanins;
fluorescein and Texas red. A negative designation indicates that
the level of staining is at or below the brightness of an isotype
matched negative control. A dim designation indicates that the
level of staining may be near the level of a negative stain, but
may also be brighter than an isotype matched control.
[0062] In one embodiment, the c-kit antibody is directly or
indirectly conjugated to a magnetic reagent, such as a
superparamagnetic microparticle (microparticle). Direct conjugation
to a magnetic particle is achieved by use of various chemical
linking groups, as known in the art. Antibody can be coupled to the
microparticles through side chain amino or sufhydryl groups and
heterofunctional cross-linking reagents. A large number of
heterofunctional compounds are available for linking to entities. A
preferred linking group is 3-(2-pyridyidithio)propionic acid
N-hydroxysuccinimide ester (SPDP) or
4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid
N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group
on the antibody and a reactive amino group on the magnetic
particle.
[0063] Alternatively, c-kit antibody is indirectly coupled to the
magnetic particles. The antibody is directly conjugated to a
hapten, and hapten-specific, second stage antibodies are conjugated
to the particles. Suitable haptens include digoxin, digoxigenin,
FITC, dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for
conjugation of the hapten to a protein, i.e. are known in the art,
and kits for such conjugations are commercially available.
[0064] To practice the method, the c-kit antibody (Ab) is added to
a cell sample. The amount of c-kit Ab necessary to bind a
particular cell subset is empirically determined by performing a
test separation and analysis. The cells and c-kit antibody are
incubated for a period of time sufficient for complexes to form,
usually at least about 5 min, more usually at least about 10 min,
and usually not more than one hr, more usually not more than about
30 min.
[0065] The cells may additionally be incubated with antibodies or
binding molecules specific for cell surface markers known to be
present or absent on the fetal stem cells. For example, cells
expressing SSAE1 marker can be negatively selected for.
[0066] The labeled cells are separated in accordance with the
specific antibody preparation. Fluorochrome labeled antibodies are
useful for FACS separation, magnetic particles for immunomagnetic
selection, particularly high gradient magnetic selection (HGMS),
etc. Exemplary magnetic separation devices are described in WO
90/07380, PCT/US96/00953, and EP 438,520.
[0067] The purified cell population may be collected in any
appropriate medium. Various media are commercially available and
may be used, including Dulbecco's Modified Eagle Medium (DMEM),
Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered
saline (dPBS), RPMI, Iscove's modified Dulbecco's medium (IMDM),
phosphate buffered saline (PBS) with 5 mM EDTA, etc . . . ,
frequently supplemented with fetal calf serum (FCS), bovine serum
albumin (BSA), human serum albumin (HSA), etc. Preferred culture
media include DMEM, F-12, M1 99, RPMI.
[0068] Populations highly enriched for pluripotent fetal stem cells
are achieved in this manner. The desired cells will be 30% or more
of the cell composition, preferably 50% or more of the cell
population, more preferably 90% or more of the cell population, and
most preferably 95% or more (substantially pure) of the cell
population.
[0069] The use of substantially purified or enriched c-kit positive
pluripotent fetal stem cells of the present invention are useful in
a variety of ways. The c-kit positive cells can be used to
reconstitute a host whose cells have been lost through disease or
injury. Genetic diseases associated with cells may be treated by
genetic modification of autologous or allogeneic stem cells to
correct a genetic defect or treat to protect against disease.
[0070] Alternatively, normal allogeneic fetal stem cells may be
transplanted. Diseases other than those associated with cells may
also be treated, where the disease is related to the lack of a
particular secreted product such as hormone, enzyme, growth factor,
or the like. CNS disorders encompass numerous afflictions such as
neurodegenerative diseases (e.g. Alzheimer's and Parkinson's),
acute brain injury (e.g. stroke, head injury, cerebral palsy) and a
large number of CNS dysfunctions (e.g. depression, epilepsy, and
schizophrenia). In recent years neurodegenerative disease has
become an important concern due to the expanding elderly population
which is at greatest risk for these disorders. These diseases,
which include Alzheimer's Disease, Multiple Sclerosis (MS),
Huntington's Disease, Amyotrophic Lateral Sclerosis, and
Parkinson's Disease, have been linked to the degeneration of neural
cells in particular locations of the CNS, leading to the inability
of these cells or the brain region to carry out their intended
function. By providing for maturation, proliferation and
differentiation into one or more selected lineages through specific
different growth factors the progenitor cells may be used as a
source of committed cells. The pluripotent fetal stem cells
according to the present invention can also be used to produce a
variety of blood cell types, including myeloid and lymphoid cells,
as well as early hematopoietic cells (see, Bjornson et al., 283
Science 534 (1999), incorporated herein by reference).
[0071] A variety of cell differentiation inducing agents can be use
to differentiate the pluripotent fetal stem cells of the present
invention into different phenotypes. To determine the
differentiation status of the stem cells, the phenotypic
characteristic of the cells are observed using conventional methods
such as light microscopy to detect cell morphology (see, e.g.,
FIGS. 2-6), RT-PCT to detect cell lineage specific transcription,
and immunocytochemistry to detect cell surface markers specifically
expressed in a particulate cell lineage. For example, genes
expressed during the osteogenic differentiation serve as markers of
the stem cells differentiating into osteogenic lineage (Long, Blood
Cells Mol Dis 2001 May-June; 27(3):677-90).
[0072] The c-kit positive fetal stem cells may also be used in the
isolation and evaluation of factors associated with the
differentiation and maturation of cells. Thus, the cells may be
used in assays to determine the activity of media, such as
conditioned media, evaluate fluids for growth factor activity,
involvement with dedication of lineages, or the like.
[0073] The isolated c-kit positive fetal stem cells may be
cryopreserved, i.e. frozen at liquid nitrogen temperatures and
stored for long periods of time, being thawed and capable of being
reused. The cells will usually be stored in 5% DMSO and 95% fetal
calf serum. Once thawed, the cells may be expanded by use of growth
factors or stromal cells associated with stem cell proliferation
and differentiation.
[0074] The present invention contemplates also cryopreservation of
the chorionic villus and amniotic fluid samples as well as the
placenta samples, wherein once thawed, c-kit positive cells can be
obtained.
[0075] For illustration purposes, c-kit.sup.pos cells were induced
to different lineages as described in the Example. The ability to
induce specific differentiation was initially evident by
morphological changes, and was confirmed by immunocytochemical and
gene expression analyses. Generally, the c-kit positive fetal stem
cells can be differentiated into different cell lineages according
to methods well known to one skilled in the art (Stem Cells:
Scientific Progress and Future Research Directions. Appendix D.
Department of Health and Human Services. June 2001.
http://www.nih.gov/news/stemcell/scireport.ht).
[0076] Adipogenic specific chemical staining showed that it was
possible to induce lipid accumulation in more than 95% of the
c-kit.sup.pos chorionic villus cells when the cells were cultured
in specific conditions. Adipocyte induction was confirmed with
ppar.gamma.2 and LPL expression at different time points.
[0077] Consistent with bone differentiation, chorionic and amniotic
fetal stem cells showed to be able to produce alkaline phosphatase
and to deposit calcium, and the values of both were higher than
those reached by adult stem cells under the same conditions.
Furthermore, c-kit.sup.pos cells in osteogenic media expressed
specific genes implicated in mammalian bone development. Core
binding factor A1 (Cbfa1) is an osteoblast specific transcription
factor. Cbfa1 regulates the function of genes expressed in
osteoblasts and encodes structural proteins of the bone
extracellular matrix. Forced expression of Cbfa1 in
non-osteoblastic cells leads to osteoblast-specific gene
expression. Cbfa1 deficient mice and deletion or mutation of the
same gene in humans causes cleidocranial dysplasia.
[0078] In postnatal life, growth and repair of skeletal muscle are
mediated by a resident population of mononuclear myogenic
precursors (the "satellite cells"); however their self-renewal
potential is limited and decreases with age. Previous studies have
shown that muscle cells can be derived from mesenchymal stem cells
from bone marrow and peripheral tissue. It has been shown here that
c-kit.sup.pos chorionic villus and amniotic cells can be induced
towards muscle differentiation. The c-kit.sup.pos cells formed
multinucleated cells that were positive for muscle differentiation
markers (Desmin and Sarcomeric Tropomyosin). Furthermore by RT-PCR
analysis, a characteristic pattern of gene expression, reflecting
that seen with embryonic muscle development, was demonstrated.
Previous studies in mouse embryos have shown that Myf6 is expressed
transiently between days 9 and 11. In our study Myf6 was expressed
at day 8 and suppressed at day 16. Myf5 in embryonic mouse
development is expressed early and continues to be expressed until
very late time points. In our study a low expression of Myf5 was
detected in the induced cells throughout the experiment. Also, as
has been shown with ES cells, MyoD expression was detectable at 8
days in the c-kit.sup.pos cells grown under myogenic conditions.
Our findings illustrate that cells derived from chorionic villus,
amniotic fluid and placenta can be induced towards muscle
differentiation.
[0079] Endothelial cells are usually difficult to isolate and
maintain in culture. P1H12, FVIII and KDR are specific markers of
endothelial differentiation. Amniotic c-kit.sup.pos cells cultured
in defined media were able to form fully differentiated endothelial
cells that expressed specific markers.
[0080] In accordance with the present invention, fetal stem cells
are obtained from human chorionic villus, amniotic fluid and
placenta. Large quantities of chorionic villus, amniotic fluid and
placenta cells can be obtained from subjects during pregnancy
and/or at birth depending on which cell source is used. Fetal stem
cells obtained from these sources may be cultured in various media,
such as DMEM, F-12, M1 99, RPMI and combinations thereof,
supplemented with fetal bovine serum (FBS), whole human serum
(WHS), or supplemented with growth factors, cytokines, hormones,
vitamins, antibiotics, or any combination thereof. DMEM media is
preferred.
[0081] The fetal stem cells may also be expanded in the presence of
an agent which suppresses cellular differentiation. Such agents are
well-known in the art (Dushnik-Levinson, M. et al., "Embryogenesis
in vitro: Study of Differentiation of Embryonic Stem Cells," Biol.
Neonate, Vol. 67, 77-83, 1995). Examples of agents which suppress
cellular differentiation include leukemia inhibitory factor (LIF)
and stem cell factor. On the other hand, agents such as
hydrocortisone, Ca.sup.2+, keratinocyte growth factor (KGF), TGF-P,
retinoic acid, insulin, prolactin, sodium butyrate, TPA, DIVISO,
NMF, DMF, collagen, laminin, heparan SO4, androgen, estrogen, and
combinations thereof may be used to induce differentiation (Culture
of Epithelial Cells, (R. Ian Freshney ed., Wiley-Liss 1992)).
[0082] The cells may be assessed for viability, proliferation
potential, and longevity using standard techniques in the art. For
example, a trypanblue exclusion assay, a fluorescein diacetate
uptake assay, a propidium iodide uptake assay, or other techniques
known in the art may be used to assess viability. A thymidine
uptake assay, an MTT cell proliferation assay, or other techniques
known in the art may be used to assess proliferation. Longevity may
be determined by the maximum number of population doublings in
extended cultures or other techniques known in the art
[0083] Additionally, cells of different lineages may be derived by
inducing differentiation of fetal stem cells and as evidenced by
changes in cellular antigens. Various differentiation-inducing
agents are used to accomplish such differentiation, such as growth
factors (for example EGF, aFGF, bFGF, PIDGF, TGF-P), hormones
(including but not limited to insulin, triiodothyronine,
hydrocortisone, and dexamethasone), cytokines (for example
IL-1.alpha. or P, IFN-.gamma., TFN), matrix elements (for example
collagen, laminin, heparan sulfate, Matrigel), retinoic acid,
transferrin, TPA, and DMSO. Such differentiation-inducing agents
are known to those of ordinary skill in the art (Culture of
Epithelial Cells, (R. Ian Freshney ed., Wiley-Liss 1992)). Examples
below describe differentiation of fetal stem cells into osteogenic,
adipogenic, myogenic and endothelial lineages. Identification of
differentiated cells may be accomplished by staining the cells with
tissue-specific antibodies according to techniques known in the
art.
[0084] In contrast to human embryonic stem (ES) cells whose use has
raised ethical concerns, human fetal stem cells of the present
invention are derived from a readily available source (chorionic
villus or amniotic fluid or placenta) which is normally discarded
after birth. Thus, cultured human fetal stem cells are ideal for
use in regenerative and/or reconstructive surgery, as well as for
use in gene therapy. Some specific applications of human fetal stem
cells are described below.
[0085] Fetal stem cells may be used in autologous/heterologous
enzyme replacement therapy in specific conditions including, but
not limited to, lysosomal storage diseases, such as Tay-Sachs,
Niemann-Pick, Fabry's, Gaucher's, Hunter's, Hurler's syndrome, as
well as other gangliosidoses, mucopolysaccharidoses, and
glycogenoses.
[0086] Additionally, the fetal stem cells of the present invention
may be used as autologous/heterologous transgene carriers in gene
therapy to correct inborn errors of metabolism affecting the
cardiovascular, respiratory, gastrointestinal, reproductive, and
nervous systems, or to treat cancer and other pathological
conditions.
[0087] Fetal stem cells of the present invention can be used in
autologous/heterologous tissue regeneration/replacement therapy,
including but not limited to treatment of corneal epithelial
defects, cartilage repair, facial dermabrasion, burn and wound
dressing for traumatic injuries of skin, mucosal membranes,
tympanic membranes, intestinal linings, and neurological
structures. For example, augmentation of myocardial performance can
be achieved by the transplantation of exogenous fetal stem cells
into damaged myocardium, a procedure known as cellular
cardiomyoplasty (CCM) which can be used for enhancing myocardial
performance and treating end-stage cardiac disease. Fetal stem
cells according to the present invention can also be used as a tool
for the repair of a number of CNS disorders as described in a
review by Cao et al. (Stem cell repair of central nervous system
injury, J. Neuroscience Res. 68:501-510, 2002).
[0088] Fetal stem cells of the present invention can also be used
in reconstructive treatment of damaged tissue by surgical
implantation of cell sheets, disaggregated cells, and cells
embedded in carriers for regeneration of tissues for which
differentiated cells have been produced. The cells may also be used
in tissue engineered constructs. Such constructs comprise a
biocompatible polymer formed into a scaffold suitable for cell
growth. The scaffold can be shaped into a heat valve, vessel
(tubular), planar construct or any other suitable shape. Such
constructs are well known in the art (see, e.g., WO02/035992, U.S.
Pat. Nos. 6,479,064, 6,461,628).
[0089] The amniotic fluid, chorionic villus, placenta tissue and
fetal stem cells, before or after differentiation, may be
cryopreserved in a cryoprotective solution comprising a medium or
buffer and a cryoprotective agent. Examples of media are Dulbecco's
Modified Eagle Medium (DMEM), Medium 199 (M199), F-12 Medium, and
RPMI Medium. An example of a buffer is phosphate buffered saline
(PBS). Examples of cryoprotective agents are dimethylsulfoxide
(DMSO) and glycerol. Examples of cryoprotective solutions are:
DMEM/glycerol (1:1), DMEM/7.5% DMSO, M199/7.5% DMSO, and PBS/3.5 M
DMSO. Optionally, the samples may be treated with antibiotics such
as penicillin or streptomycin prior to cryopreservation.
Cryopreservation may be accomplished using a rapid, flash-freeze
method or by more conventional controlled rate-freeze methods.
Rapid freezing of amniotic tissue may be accomplished by placing
sample(s) in a freezing tube containing a cryoprotective solution
and then rapidly immersing the freezing tube in liquid nitrogen.
General slow freezing may be accomplished by placing sample(s) in a
freezing tube containing a cryoprotective solution and then placing
the freezing tube in a -70.degree. C. freezer. Alternatively, the
sample(s) may be subjected to controlled rate freezing using a
standard cryogenic rate controlled system.
[0090] Products of fetal stem cells of the present invention may be
used in reconstructive treatment, either in vivo or ex vivo.
Examples of agents that can be produced using fetal stem cells of
the present invention include growth factors, cytokines, and other
biological response modifiers.
[0091] The references cited herein and throughout the specification
are incorporated by reference in their entirety.
[0092] The invention will be further clarified by the following
examples, which are intended to be purely exemplary of the
invention.
EXAMPLES
[0093] In this example the feasibility of isolating stem cells from
human embryonic and fetal chorionic villi and amniotic fluid was
investigated. Discarded cultures of chorionic villi cells and human
amniotic fluid cells collected for prenatal diagnostic tests were
obtained from more than 300 human pregnant females ranging from 23
to 42 years of age under an approved institutional Investigation
Review Board protocol.
[0094] To establish the cultures, human amniotic fluid was obtained
by transabdominal amniocentesis at 14 to 21 weeks of gestation, and
human embryonic chorionic villus tissue specimens were obtained at
10 to 12 weeks of gestation through a transabdominal approach.
[0095] Amniotic fluid samples were centrifuged and the cell
supernatant was resuspended in culture medium. Approximately
10.sup.4 cells were seeded on 22.times.22 mm cover slips. Cultures
were grown to confluence for about 3 to 4 weeks in 5% CO.sub.2 at
37.degree. C. Fresh medium was applied after five days of culture
and every third day thereafter.
[0096] Chorionic villus cells were isolated from single villus
under light microscopy. The cells were allowed to proliferate in
vitro and were maintained in culture for about 4 weeks. The culture
medium consisted of modified .alpha.MEM (18% Chang Medium B, 2%
Chang C with 15% embryonic stem cell-certified fetal bovine serum,
antibiotics and L-glutamine) (J. H. Priest, Prenatal Chromosomal
Diagnosis and Cell Culture in The ACT Cytogenetics Laboratory
Manual, Margaret J. Barch (Raven Press, New York ed. 2, 1991) cap.5
p. 1.sup.49).
[0097] The cells were sub-cultured using 0.25% trypsin containing 1
mM EDTA for 5 minutes at 37.degree. C. Cells were seeded at 3000
cells/cm.sup.2 in 24 well plates. Cell numbers were determined
after 4, 8, 16, 24 and 32 days in quadruplicate values. For the
first time point (4 days), the medium was removed from the 24 well
plates. The cells were rinsed once with PBS/EDTA, and were
incubated with O.sub.2 ml trypsin/EDTA for 10 minutes at 37.degree.
C. The cells were resuspended with trypsin/EDTA solution several
times to avoid cell clusters, before being transferred to 9.8 ml of
isotonic fluid. Cells were counted as recommended by the
manufacturer's instructions (Coulter Counter). An MTT assay was
performed after 4, 8, 16, 24 and 32 days. 100 .mu.l of MT reagent
(Sigma-Aldrich) was added to 1 ml of medium for 3 hours. The cells
were lysed and color was extracted with isopropanal containing 0.1
M HCl. Extinction was read in a Biorad reader at 570 nm against 655
nm. Results were expressed as a cell count Growth curves from both
cell sources were obtained and the morphology of the cells in
culture was documented.
[0098] Cells from chorionic villi and amniotic fluid underwent
phenotypic analysis. Immunocytochemistry of the amniotic fluid
confirmed that most of the cells were of epithelial origin and
stained positively for cytokeratins. Most of the stromal cells
stained for .alpha.-actin, and only a few cells were positive for
desmin or myosin expression (von Koskull, H., et al., Prenat.
Diagn., 1(4), p. 259 (1981); Medina-Gomez, P. and T. H. Johnston,
Hum. Genet., 60(4), p. 310 (1982)).
[0099] The cells were analyzed using FACS for CD34 (Pharmingen
International, San Diego, Calif.), CD90 (Santa Cruz Biotechnology,
Inc., Santa Cruz, Calif.), CD105 (Pharmingen International), CD133
(Miltenyi Biotec, Bergisch Gladbach, Germany), and c-kit (Santa
Cruz Biotechnology, Inc). For all antibodies, 0.5.times.10.sup.6 of
either chorionic villus or amniotic cells were incubated in 500
.mu.l of primary antibody solution (2% FBS in PBS) at a
concentration of 1:100 on ice for 30 minutes. After incubation with
the primary antibodies, the cells were washed twice with 2 ml of 2%
FBS in PBS, spun down at 1100 RPM for 7 minutes, and either
resuspended in 0.5 ml PBS containing 2% FBS, or incubated in the
dark, on ice for 30 minutes, in 100 .mu.l of FITC labeled secondary
antibody (1:100, Southern Biotechnology Associates Inc.,
Birmingham, Ala.). The cells were washed twice with 2 ml of PBS
containing 2% FBS, spun down, and resuspended in PBS with 2% FBS
for cell analysis. IgG-PE (Pharmingen International) and
IgG1.kappa. (unconjugated, Pharmingen International) were used as
controls. FACS analysis was performed with a FACScalibur (Becton
Dickinson, San Jose, Calif.). Immunocytochemistry was done as
follow: cells, grown on chamber slides Nalge Nunc Int, Naperville,
Ill.), were fixed in 4% formaldehyde and in ice-cold methanol. Cell
layers were washed with PBS. Cell surface gly-colipid- and
glycoprotein-specific mAbs were used at 1:15 to 1:50 dilution.
MC480 (SSEA-1), MC631 (SSEA-3), and MC813-70 (SSEA-4) antibodies
were supplied by the Developmental Studies Hybridoma Bank
(University of Iowa, Iowa City). Antibodies were detected using
biotinylated anti-mouse secondary antibody, strepavi-din-conjugated
horseradish peroxidase, and 3-amino-9-ethylcarbazole chromagen
(BioGenex). Cells prepared for cytogenetic analysis were incubated
in growth media with 0.1 mg/ml of Colcemid for 3-4 hr, trypsinized,
resuspended in 0.075 M KCl, and incubated for 20 min at 37.degree.
C., then fixed in 3:1 methanolyacetic acid.
[0100] FACS analyses of the cells showed that between 18% and 21%
of the cells expressed CD90 and CD105, while much lower percentages
of cells expressed c-kit, CD34 and AC133 (between 0.8% and 3%).
Similar patterns of expression were obtained for the chorionic
villus cells.
[0101] Cells expressing c-kit (c-kit.sup.pos) were successfully
immuno-isolated from chorionic villi and were maintained in culture
in Chang medium. The c-kit.sup.pos cells expressed human embryonic
stage specific markers SSAE3 and SSAE4 and did not express mouse
embryonic stage specific marker SSAE1 (FIG. 1 B-D) (Thomson, J. A.,
et al., Science, 282 (5391), p. 1145 (1998)). The c-kit.sup.pos
cells maintained a round shape when they were cultured in
non-treated culture dishes for almost one week and their
proliferative activity was low. After the first week, the cells
begun to adhere to the plates and changed their morphology,
becoming more elongated, and proliferating more rapidly.
Interestingly and importantly, no feeder layers were required
either for maintenance or expansion.
[0102] In this study c-kit.sup.pos cells, obtained from early to
late passages, were inducible to different cell lineages including
osteogenic, adipogenic, myogenic, neurogenic and endothelial cell
lineages under specific growth factors. The ability to induce
specific differentiation was initially evident by morphological
changes, and was also confirmed immunocytochemically, by gene
expression patterns, and by cell-specific functional analyses.
[0103] Stem cells from bone marrow were purchased (Clonetics) and
used as a positive control. The CD34, CD90, CD105 and AC133
immunoisolated cells, and the remaining non-immunoseparated cells
did not show any pluripotential capacity. Because amniotic fluid
contains both urine and peritoneal fluid, cells isolated from
discarded human neonatal urine and peritoneal fluid were used as
controls. Human urine and peritoneal control fluids did not yield
any c-kit.sup.pos cells, and the c-kit.sup.neg cells did not show
any pluripotential ability.
[0104] It is known that amniotic fluid, in general, contains very
few maternal cells. To determine if any maternal c-kit.sup.pos
cells were present in the chorionic villus or amniotic fluid
samples, studies were performed using cells from male fetuses. All
the caryotyped c-kit.sup.pos cells showed an XY karyotype
indicating that no C-kit.sup.pos maternal cells were present in the
studies samples. C-kit.sup.pos cells from female embryos and
fetuses were used as controls, and they did not show any difference
in their pluripotential ability.
[0105] The c-kit.sup.pos cells derived from chorionic villi and
amniotic fluid showed a high self-renewal capacity with over 250
population doublings, far exceeding Hayflick's limit. The cells
have now been continuously passaged for over 18 months and they
have maintained their undifferentiated state. We have also
demonstrated that late passage c-kit.sup.pos cells maintain their
pluripotential capacity and a normal karyotype after 250 population
doublings (FIG. 1 E).
[0106] Telomerase activity is normally detectable in human germ
cells (Thomson, J. A., et al., Science, 282 (5391), p. 1145
(1998)), most immortalized cell lines, and 80-90% of human tumor
samples, in which the telomere length is preserved. We evaluated
the telomerase activity in the isolated and cultured c-kit.sup.pos
cells using the Telomerase Repeat Amplification Protocol (TRAP)
assay (FIG. 1 F). TRAP analysis (TRAPeze kit, Intergenco
Pharmaceuticals) was performed as described in the manufacturer's
protocol with one modification. CHAP's lysates were subjected to 36
cycles of PCR amplification after the telomerase extension step.
Low telomerase activity was detected with the TRAP-assay in the
amniotic c-kit.sup.pos cells (lane 1) compared to the control
(lanes 3 and 4). However, after differentiation, the c-kit.sup.pos
cells did not show any telomerase activity (lane 2). To confirm
that the measured telomerase activity was of functional relevance
to the isolated cells, the telomere length of the c-kit.sup.pos
cells at early and late passages were determined by terminal
restriction fragment (TRF) analyses. Total cellular DNA was
isolated by the DNeasy Tissue Kit (Qiagen Corp) and 2 .mu.g was
used for Southern Blot analysis of TRF lengths (FeloTAGGG Telomere
Length Assay, Roche Molecular) as described in the manufacturer's
protocol. Briefly, purified genomic DNA was digested with a mixture
of frequently cutting restriction enzymes. The resulting fragments
were agarose gel electrophoresed and transferred to a nylon
membrane by Southern blotting. Hybridization to a digoxigenin
(DIG)-labeled probe specific for the telomeric repeats was followed
by chemiluminescent detection and exposure of the membrane to
autoradiography film. TRF qualitative analysis demonstrated that
the c-kit.sup.pos cells had similar telomere lengths, both at early
(mean TRF length approximately 20 kb) and late (mean TRF length
approximately 20 kb) passages (FIG. 1 G).
[0107] However C-kit.sup.pos cells derived from chorionic villi and
amniotic fluid expanded clonally more than 250 population doublings
while maintaining approximately the same telomere length and had
additionally acquired telomerase activity.
[0108] This phenomenon suggests that the cell population could have
an alternative mechanism for lengthening telomeres (ALT) (16, 17).
One possible answer could be derived from the clonal fluctuations.
Some tested clones could have been overlengthned by the action of
telomere-lengthening by an unknown mechanism. Regulation factors
could therefore influence the activation and inactivation of the
telomerase without influencing the telomere length (Brayan, T. M et
al (1998) Telomere length dynamics in telomerase-positive immortal
human cell population). The explanation of this particular
phenomenon is not clear and the mechanism for the longevity of
these cells in culture is unknown.
[0109] To prove the capacity of the c-kit.sup.pos cells isolated
from amniotic fluid and chorionic villi to differentiate into
various cell lineages we used a method of retroviral marking.
CKit.sup.pos cells were transduced with a puc-CMMP-IC-eGFP
retrovirus and expanded. The infected-cKit.sup.pos were sorted by
FACS-Excalibur and single eGFP.sup.+-cells were plated per well in
a 96 wells-plate and expanded. The derived clones were sorted one
more time by FACScan instrument (Becton Dickinson, San Jose,
Calif.) in-line with a Power Macintosh computer using the CELLQuest
software in order to obtain a subpopulation of clones. The DNA from
the original clones and derived subclones was extracted using a
Dnaesy Tissue Kit (Qiagen) and the concentration was measured with
a Spectrofotometer (Spectronic 601). Three samples of genomic DNA
for each clone and subclone were digest for three hours with a
different mix of restriction enzymes (mix 1. SapI, MfeI, HpaI,
DraIII; mix2. BamHI, NheI, HindIII, XhoI, PacI; mix3. BgIII, AseI).
The fragments were separated by electrophoresis and transfer by
capillarity to a naylon membrane. An eGFP-cDNA probe was
constructed from a plasmid (pEGFP-N 1 . . . ) digesting the plasmid
with AgeI and NotI. The fragment was separated by electrophoresis
and the digested DNA was extract with a Gel Extraction Kit (Qiagen)
and labelled with digoxigenin for detection with alkaline
phosphatase metabolising CDP-Star, a highly sensitive
chemiluminescence substrate (DIG High Prime DNA Labeling and
Detection Starter Kit II, Roche). The blotted DNA fragments were
hybridized to the Dig-labelled eGFP cDNA probe and the retrovirus
insertion was determined by detection exposing the membrane to
X-ray film.
[0110] All experiments were performed with c-kit.sup.pos cells
obtained from twelve clonal cell populations, according to their
gestational age (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21
weeks). Furthermore, all experiments were also performed with five
single c-kit.sup.pos clonal cell populations obtained from a single
fetus (11, 14, 16, 18 and 20 weeks of gestation). Cells from the
different clones showed a similar morphology and growth behavior.
Cells from all clones underwent osteogenic, adipogenic, myogenic,
neurogenic and endothelial differentiation. No statistical
differences were noted in the ability of the 17 clonal cell
populations to differentiate into separate lineages.
[0111] Osteogenic induction. For the induction of osteogenic
differentiation, c-kit.sup.pos cells isolated from amniotic fluid
and chorionic villi were cultured in a defined osteogenic medium.
For the induction of osteogenic differentiation, the cells were
seeded at a density of 3000 cells/cm.sup.2 and were cultured in
DMEM low glucose medium (Gibco/Br1) with 10% fetal bovine serum
(FBS, Gibco/Br1), 1% antibiotics (Gibco/Br1), and osteogenic
supplements [100 nM dexamethasone (Sigma-Aldrich), 10 mM
beta-glycerophosphate (Sigma-Aldrich), and 0.05 mM ascorbic
acid-2-phosphate (Wako Chemicals, Irving, Tex.)]. Jaiswal, N., et
al., J. Cell. Biochem., 64(2), p. 295 (1997).
[0112] Control medium was essentially modified .alpha.MEM. Medium
was changed about every 3 days. Light microscopy analysis of the
cells showed that, within 4 days in the osteogenic medium,
c-kit.sup.pos cells lost their spindle shape phenotype (FIG. 2 A)
and developed an osteoblastic-like appearance with fingerlike
excavations into the cytoplasm (FIG. 2 B). At day 16, the cells
aggregated, showing typical lamellar bone-like structures.
Consistent with bone differentiation, the c-kit.sup.pos cells
cultured under osteogenesis inducing conditions produced alkaline
phosphatase (AP) and showed calcium deposits. Interestingly, both
the amount of AP production and calcium deposition was higher than
those reached by adult osteogenic stem cells cultured under the
same conditions. AP activity was measured using a quantitative
assay for p-Nitrophenol, which is equivalent to AP production.
Alkaline phosphatase enzyme cell activity was measured in
quadruplicate cultures. After rinsing with PBS, the cells were
incubated with 2-amino-2-methyl-1-propanol buffer, pH 10.3
(Sigma-Aldrich #221/26) with 40 mg p-nitrophenyl phosphate
(Sigma-Aldrich #104/40) added, at 37.degree. C. for 3 to 35 min. AP
activity was calculated after measuring the absorbance of
p-nitrophenol products formed at 405 nm on a micro plate reader
(Molecular Devices, Spectra Max Plus). As a standard, p-nitrophenol
standard solution (Sigma-Aldrich #104-1) diluted in
2-amino-2-methyl-1-propanol buffer in concentrations from 0 to 100
nMol p-nitrophenol was used. Enzyme activity was expressed as nMol
p-Nitrophenol/min/10.sup.6 cells.
[0113] Histochemical Analyses. Alkaline phosphatase activity was
determined histologically in cells according to the manufacturer's
instructions (Sigma-Aldrich Kit #85). Briefly, cells were fixed in
a citrate-acetone solution. An alkaline-dye mixture (fast blue RR
solution with naphthol AS-MX phosphate alkaline solution) was added
to the cells in the 35 mm culture dishes. The cell cultures were
protected from direct light. Prior to viewing, the cell cultures
were rinsed with deionized water and air-dried. AP production in
the c-kit.sup.pos cells grown in osteogenic-inducing medium
increased by a factor of 250 compared to c-kit.sup.pos cells grown
in control medium and c-kit.sup.neg cells grown in osteogenic
medium at days 16 and 24 (FIG. 2 C).
[0114] A major feature of osteogenic differentiation is the ability
of the cells to precipitate calcium. Cell associated mineralization
may be analyzed using von Kossa staining and by measuring calcium
content in the cells in culture. Von Kossa staining of cells grown
in the osteogenic medium showed enhanced silver nitrate
precipitation by day 16, indicating high levels of calcium. The
presence of mineralization in cell culture was determined by von
Kossa staining. The cell culture plates were fixed with 10%
formaldehyde for 1 h, incubated with 2% silver nitrate solution for
10 min in the dark, washed thoroughly with deionized water, and
then exposed to UV-light for 15 min. Calcium content continued to
increase exponentially at 24 and 32 days. In contrast, cells in the
control medium did not show any silver nitrate precipitation (FIG.
2 G).
[0115] Calcium deposition by the cells was also measured with a
quantitative chemical assay which measures calcium-cresolophthalein
complexes. Cells undergoing osteogenic induction showed a
significant increase in calcium precipitation after 16 days (up to
4 mg/dl). The precipitation of calcium increased up to 70 mg/dl at
32 days. In contrast, cells grown in the control medium did not
show any increase in calcium precipitation (1.6 mg/dl) by day 32
(FIGS. 2 H and I).
[0116] C-kit.sup.pos cells in osteogenic medium expressed specific
genes implicated in mammalian bone development (AP, core binding
factor A1 (cbfa1), and osteocalcin) (FIG. 2C). RNA was isolated
from cultured cells and cell pellets with RNAzol reagent (Tel-Test
Inc., Friendswood, Tex.) according to the manufacturer's protocol.
RNA (2 .mu.g) was processed for c-DNA synthesis with Superscript II
reverse transcriptase with random hexamers (Life Technologies).
C-DNA was used for each PCR reaction, in a final volume of 30 .mu.l
with 200 nM dNTP, 10 pM of each primer, 0.3U Taq-DNA-polymerase,
reaction buffer, and MgCl2 (Life Technologies), in a PTC-100 cycler
(MJ-Research Inc., Watertown, Mass.). The cycling conditions
consisted of 94.degree. C. for 2 minutes, annealing at 63.degree.
C. for 40 seconds, and elongation at 72.degree. C. for 1 minute.
Cycle numbers varied between 22 and 37 cycles and were chosen in
the exponential phase of the RT-PCR reaction Primer sequences and
fragment sizes are listed in Table 1. All primers were obtained
from Life Technologies. Primers for human core binding factor A1
(cbfa1) primers (sense 5'GGCCTTCCACTCTCAGTAAGA3' (SEQ ID NO:1) and
antisense 5'GATTCATCCATTCTGCCACTA3', (SEQ ID NO:2)28 cycles at
63.degree. C.) amplified a fragment of 474 bp and human osteocalcin
(sense 5'CCCTCACACTCCTCGCCCTAT3' (SEQ ID NO:3) and antisense
5'GGTAGCGCC TGGGTCTCTTCA3', SEQ ID NO:4) amplified a fragment of
144 bp. Human peroxisome proliferator-activated receptor .gamma.2
(ppar.gamma.2) primers (sense 5'TGAACGACCAAGTAACTCTCC3' (SEQ ID
NO:5) and antisense 5' CTCATGTCTGTCTCCGTCTTC3', (SEQ ID NO:6) 29
cycles at 64.degree. C.) yielded a fragment of 460 bp. 533 bp.
Human lipoprotein lipase (lpl) primers (sense
5'CTGGTCGAAGCATTGGAAT3' (SEQ ID NO:7) and antisense
5'TGTAGGGCATCTGAGAACGAG3', (SEQ ID NO:8) 29 cycles at 64.degree.
C.) amplified a fragment of 366 bp. Human Myogenic Regulatory
Factor 4 (MRF4) (sense 5'CGACAGCAGCGGAGAGG3' (SEQ ID NO:9) and
antisense 5'GGAATGATCGGAAACACTTGG3', (SEQ ID NO:10) 37 cycles at
62.degree. C.) was detected as band of 421 bp and human myoD (sense
TCCGCGACGTAGACCTGAC3' (SEQ ID NO:11) and antisense
5'GATATAGCGGATGGCGTTGC3', SEQ ID NO:12) amplified a segment of 449
bp. Human desmin primers (sense 5'CCATCGCGGCTAAGAACATT3'(SEQ ID
NO:13) and antisense 5'TCGGAAGTTGAGGGCAGAGTA3', (SEQ ID NO:14)27
cycles at 62.degree. C.) amplified a fragment of 440 bp, Primers
for human .beta.2-microglobulin (.beta.2-MG) (sense
5'GCTCGCGCTACTCTCTC3' (SEQ ID NO:15) and antisense
5'TTAACTATCTFGGGCTGTGAC3', (SEQ ID NO:16) 23-26 cycles at
62-64.degree. C.) amplified a fragment of 315 bp. Primers for human
CD106 (VCAM) (sense 5'TCCAGCGAGGGTCTACCAG3' (SEQ ID NO:16)
antisense 5'TGTTTGCGTACTCTGCCTTTG3', SEQ ID NO:17) amplified a
segment of 774 bp and human CD31 (PECAM) (sense
5'CCTTCTCTACACCCAAGTTCC3' (SEQ ID NO:18) and antisense
5'GAAATAGGCAAAGTTCCACTG3', SEQ ID NO:19) yielded a fragment of 628
bp.
[0117] C-kit.sup.pos cells grown in osteogenic medium showed an
activation of the AP gene at each time point. No transcription of
the AP gene was detected at 8, 16, 24 and 32 days in the
c-kit.sup.pos cells grown in control medium. Expression of cbfa1, a
transcription factor specifically expressed in osteoblasts and
hypertrophic chondrocytes that regulates gene expression of
structural proteins of the bone extracellular matrix in osteoblasts
(24, 25), was highest in cells grown in osteogenic inducing medium
at day 8 and decreased slightly at days 16, 24 and 32. The
expression of cbfa1 in the controls was significantly lower at each
time point. Osteocalcin was expressed only in the c-kit.sup.pos
cells in osteogenic conditions at 8 days. No expression of
osteocalcin was detected in the c-kit.sup.pos cells in the control
medium and c-kit.sup.neg cells in osteogenic medium at any time
point
[0118] C-kit.sup.pos cells were also seeded on
hydroxyapatite-collagen scaffolds (Collagraft, Neucoll, Zimmer,
Warsaw, Ind.) at a density of 10.times.10.sup.6 cells/cm.sup.2.
Cells were induced into an osteogenic lineage in a bioreactor for
16d. The rods were implanted subcutaneously in athymic mice,
harvested after 4 and 8 weeks, and analyzed. Bone-like tissue was
evident, surrounded by an extracellular matrix which stained blue
with Masson's trichrome. Toluidine blue staining confirmed the
osteogenic phenotype. Small calcified areas within the implanted
tissue stained positively with von Kossa, indicating bone
formation. Unseeded constructs, as controls, showed only a few
infiltrating cells, and no bone-like structures were noted (FIG.
2).
[0119] Adipogenic induction. To promote adipogenic differentiation,
we cultured the c-kit.sup.pos cells in defined adipogenic medium.
For the induction of adipogenic differentiation, the cells were
seeded at a density of 3000 cells/cm.sup.2 and were cultured in
DMEM low glucose medium with 10% FBS, 1% antibiotics, and
adipogenic supplements [1 .mu.M dexamethasone, 1 mM
3-isobutyl-1-methylxanthine (Sigma-Aldrich), 10 .mu.g/ml insulin
(Sigma-Aldrich), and 60 .mu.M indomethacin (Sigma-Aldrich)].
[0120] Control medium consisted of modified .alpha.MEM. Medium
changes were performed every 3 days. C-kit.sup.pos cells cultured
with adipogenic supplements changed their morphology from elongated
to round within 8 days. This coincided with the accumulation of
intracellular triglyceride droplets [FIG. 3 A]. The presence of
adipose elements in cell culture was determined with Oil-O-Red
staining. The 2 well chamber slides were washed in deionized water
and air-dried. The cells were incubated with oil red 0 staining
solution for 15 minutes, rinsed with 50% ethanol 3 times, rinsed
with distilled water, counterstained with Gills hematoxilin for 30
sec to 1 min, and rinsed in deionized water 3 to 4 times. After 16
days in culture, more than 95% of the cells had their cytoplasm
filled with lipid-rich vacuoles, which stained with Oil-O-Red (FIG.
3 B).
[0121] Chamber slides were mounted with water-based mounting media.
The c-kit.sup.pos cells cultured in control medium and the
c-kit.sup.neg cells cultured in adipogenic medium did not show any
phenotypic changes consistent with adipogenic differentiation and
did not stain with Oil-O-Red (FIG. 3 C).
[0122] Adipogenic differentiation was confirmed by RT-PCR analysis.
We analyzed the expression of peroxisome proliferation-activated
receptor .gamma.2 (ppar.gamma.2) (28, 29) a transcription factor
that regulates adipogenesis, and of lipoprotein lipase. Expression
of these genes was upregulated in the c-kit.sup.pos cells under
adipogenic conditions. C-kit.sup.pos cells cultured under control
conditions and c-kit.sup.neg cells cultured under adipogenic
conditions did not express either gene at any time point [FIG. 3
D].
[0123] C-kit.sup.pos cells were seeded on polyglycolic acid (PGA)
polymer scaffolds at a density of 10.times.10.sup.6 cells/cm.sup.2.
Cells were induced into an adipogenic lineage in a bioreactor for
16d. The scaffolds were implanted subcutaneously in athymic mice,
harvested after 4 and 8 weeks, and analyzed. The retrieved
scaffolds showed the formation of fatty tissues grossly. The
presence of adipose tissue was confirmed with Oil-O-Red staining
[FIG. 3].
[0124] Myogenic induction. In postnatal life, growth and repair of
skeletal muscle are mediated by a resident population of
mononuclear myogenic precursors (satellite cells); however their
self-renewal potential is limited and decreases with age. Previous
studies have shown that muscle cells can be derived from
mesenchymal stem cells from bone marrow and peripheral tissue (30).
In this study, c-kit.sup.pos cells were induced towards muscle
differentiation. We seeded c-kit.sup.pos cells in 35 mm plates
precoated with Matrigel in a defined medium. The defined myoblast
growth medium consisted of DMEM low glucose containing 10% horse
serum (GIBCO, BRL), 0.5% chick embryo extract (GIBCO, BRL) and 1%
penicillin/streptomycin (GIBCO, BRL) (Reddel, R. R. et al., (1997).
Immortalized cells with no detectable telomerase activity.
Biochemistry 62, 1254-1262). Matrigel (Collaborative Biomedical
Products, Universal Biologicals Ltd.) was diluted in DMEM to 1
mg/ml, plated and incubated for 1 h at 37.degree. C., before the
cells were seeded. Rosenblatt, J. D., et al., In Vitro Cell Dev.
Biol. Anim., 31 (10), p. 773 (1995). Defined medium containing
5-azacytidine was added after 12 hours and replaced 24 hours later
with 5-azacytidine-free defined medium. As a control,
undifferentiated cells were grown in 35 mm plates with modified
.alpha.MEM. Medium changes were performed every 3 days.
[0125] Induction with 5-azacytidine for 24 hours induced the
formation of multinucleated cells after a 24 to 48 hour period
[FIG. 4 A]. The multinucleated cells expressed the muscle
differentiation markers desmin and sarcomeric tropomyosin. [FIGS. 4
D and F]. C-kit.sup.pos cells grown in control medium and
c-kit.sup.neg cells grown in myogenic conditions did not lead to
cell fusion or multinucleated cells.
[0126] We analyzed the expression of MyoD, Myf6 (MRF4) and Desmin
in cells undergoing myogenic differentiation, using RT-PCR. A
characteristic pattern of gene expression, reflecting that seen
with embryonic muscle development, was demonstrated [FIG. 4 G] (32,
33). Previous studies in mouse embryos have shown that Myf6 is
expressed transiently between days 9 and 11 (34, 35). In our study
Myf6 was expressed at day 8 and suppressed at day 16. As has been
shown with ES cells, MyoD expression was detectable at 8 days and
suppressed at 16 days in the c-kit.sup.pos cells grown under
myogenic conditions. Desmin expression was induced at 8 days and
increased by 16 days in the c-kit.sup.pos cells cultured in
myogenic medium. In contrast, there was no activation of Myf6, MyoD
or Desmin in the control cells at 8 and 16 days.
[0127] C-kit.sup.pos cells were labeled with a fluorescence marker
(PKH26 Green Fluorescent Cell Linker, Sigma-Aldrich) and were
induced into a myogenic lineage. The myogenic cells were
resuspended in rat tail collagen containing 17% Matrigel (BD
Biosciences), were injected into the hindlimb musculature of
athymic mice, and were retrieved after 4 weeks. The injected
myogenic cells showed the formation of muscle tissue which express
desmin (A) and maintained its fluorescence (B) [FIG. 4].
[0128] Endothelial induction. To induce endothelial
differentiation, we plated the cells in dishes precoated with
PBS-gelatin. The cells were maintained in culture for 1 month in
endothelial-defined medium. To induce endothelial differentiation,
the cells were plated at a density of 3000 cells/cm.sup.2 in 35 mm
dishes precoated with PBS-gelatin. The cells were maintained in
culture for 1 month in endothelial basal medium-2 (EBM-2 Clonetics
BioWittaker inc., Walkersville, Md.) supplemented with 10% FBS
(GIBCO/BRL, Grand Island, N.Y.), 1% antibiotics (GIBCO/BRL, Grand
Island, N.Y.) and 1% L-glutamine (GIBCO/BRL, Grand Island, N.Y.).
Basic fibroblast growth factor (bFGF) was added every other day.
After 1 week in culture the c-kit.sup.pos cells changed their
morphology, and by the second week, the cells were mostly tubular
[FIG. 5 A]. Human-specific endothelial cell For hepatic
differentiation, c-kit.sup.pos cells surface markers (P H12),
factor VIII (FVIII) and KDR are specific for differentiated
endothelial cells. The differentiated cells stained positively for
FVIII, KDR and P1H12 [FIG. 5 B-D]. C-kit.sup.pos cells cultured in
Chang medium for the same period of time were not able to form
tubular structures and did not stain for endothelial specific
markers. Endothelial cells are usually difficult to isolate and
maintain in culture. In our study the endothelial cells, once
differentiated, were able to grow in culture and formed
capillary-like structures in vitro [FIG. 5 E]. In order to confirm
the phenotypic changes we performed RT-PCR. Platelet endothelial
cell adhesion molecule 1 (PECAM-1 or CD31) and vascular cell
adhesion molecule (VCAM) were markedly increased in the
ckit.sup.pos cells induced in endothelial media but were not
amplified in the ckit.sup.pos cells cultured in control media [FIG.
5 F].
[0129] Hepatic induction. For hepatic differentiation,
c-kit.sup.pos cells from amniotic fluid and chorionic villi, seeded
in Matrigel coated dishes, were cultured in hepatic condition for 9
days. The c-kit.sup.pos cells seeded in Matrigel (Collaborative
Biomedical Products, Universal Biologicals Ltd.) using a modified
manufacturer thin gel method using 100 ul/cm.sup.2 surface. The
cells seeded in 24-well plates at a density of 25,000
cells/cm.sup.2 were allowed to establish themselves in this culture
in Chang medium for 3 days to achieve a semi-confluent density.
Differentiation was induced in three steps. The base medium
consisted of low glucose Dulbecco's medium (Gibco/Br1) containing
300 uM monothioglycerol (Sigma-Aldrich), 100 U/ml penicillin, and
100 U/ml streptomycin (Gibco/Br1) with 15% fetal bovine serum FBS,
(Gibco/Br1). Cells were grown initially for 3 days in the presence
of 100 ng/ml acidic fibroblastic growth factor ( ) . This step was
followed by exposure to 20 ng/ml hepatocyte growth factor ( ) for 3
days and concluded with 20 ng/ml hepatocyte growth factor, 10 ng/ml
oncostatin M ( ), 10-7 M dexamethasone (Sigma-Aldrich).sup.1. Cells
were maintained in the same media used for end stage
differentiation. Control cell populations were seeded in the same
manner as differentiated cells, but were simply maintained in
control medium. After the differentiation process the cells were
maintained in culture for 30 days.
[0130] In order to evaluate the hepatic differentiation, the
expression of albumin was evaluated and the urea production was
measured using a standard urea nitrogen essay in the differentiated
cells and in the control cells. Cells suspended in matrigel were
trypsanized for 10 minutes with light mechanical assistance and
cytospin onto slides at a density of 1000 cells/slide. Cells were
probed for albumin with goat anti-human albumin ( ) using standard
immunocytochemistry protocol with DAPI nuclear counterstain. Urea
production was measured using a colorometric urea nitrogen assay
(Sigma-Aldrich). Differentiated or control cell populations were
placed in ammonium chloride at a supraphysiological level of 20 mM
NH4Cl to examine maximum rate of urea production of each of these
cell types. The medium was then collected after 30 minutes of
exposure and tested per manufacturer instructions with and without
urease to obtain true levels of urea. After 7 days of the
differentiation process, cells tended to show morphological changes
from elongated fibroblastic cells to more epitheliod cobblestone
appearances. Cells showed positive staining for at day 12 post
differentiation. The maximum rate of urea production for hepatic
differentiation induced cells was 4.7.times.10.sup.-4 .mu.g
urea/hour/cell as opposed to 2.36.times.10.sup.-4 .mu.g
urea/hour/cell for control cell populations.
[0131] Neurogenic induction. For neurogenic induction, we cultured
amniotic and chorionic villi ckit.sup.pos cells in defined
neurogenic medium (40, 41). For neurogenic induction, the amniotic
cells were seeded at a concentration of 3000 cells/cm.sup.2 in 100
mm plates and were cultured in DMEM low glucose medium (GIBCO/BRL,
Grand Island, N.Y.), 1% antibiotics (GIBCO/BRL, Grand Island,
N.Y.), 2% DMSO and 200 .mu.M butylated hydroxyanisole (BHA,
Sigma-Aldrich, St.Louis, Mo.). Neuron growth factor (NGF) (8
.mu.l/ml) was added to the culture every 2 days. After 2 days the
medium was changed to control medium and the same amount of NGF was
continuously supplemented. Cells were fixed for immunocytochemistry
at 4 and 8 days.
[0132] After 2 days the medium was changed to control medium and
the same amount of NGF was continuously supplemented. Cells were
fixed for immunocytochemistry at 4 and 8 days. Control medium
consisted of modified .alpha.MEM. Medium changes were performed
every 3 days.
[0133] C-kit.sup.pos cells cultured in neurogenic conditions
changed their morphology within the first 24 hours. Two different
cell populations were apparent, morphologically large flat cells
and small bipolar cells. The bipolar cell cytoplasm retracted
towards the nucleus, forming contracted multipolar structures. Over
the subsequent hours, the cells displayed primary and secondary
branches and cone-like terminal expansions [FIG. 6 A]. Induced
C-kit.sup.pos cells showed a characteristic sequence of expression
of neural-specific proteins. At an early stage the intermediate
filament protein nestin (BD Bioscience), which is specifically
expressed in neuroepithelial stem cells, was highly expressed [FIG.
6 B]. The expressions of .beta. III tubulin [FIG. 6 C] and glial
fibrillary acidic protein (GFAP) (Santa Cruz) [FIG. 6 D], markers
of neuron and glial differentiation (42), respectively, increased
over time and seemed to reach a plateau at about 6 days.
C-kit.sup.pos cells cultured in Chang medium and c-kit.sup.neg
cells cultured in neurogenic medium for the same period did not
stain for neurogenic specific markers. Furthermore we analyzed the
functional behavior of the neuronal cells. The C-kit.sup.pos cells
cultured under neurogenic conditions showed the presence of the
neurotransmitter glutamic acid in the collected medium. Glutamic
acid is usually secreted by fully differentiated neurons in culture
(43). Non-induced cells, heat inactivated cells and control
urothelial cells did not secrete any glutamic acid [FIG. 6 E].
[0134] Hematopoietic differentiation. For the hematopoietic
differentiation we used a liquid media (StemSpan by STEM CELL
TECHNOLOGIES, see www.stemcell.com). The following growth factors
were added to the cell culture medium: stem cell factor, GM-CSF,
IL6, IL3, G-CSF according to the manufacturer's instructions (STEM
CELL TECHNOLOGIES). The hematopoietic differentiation was assessed
by analyzing the cell morphology.
[0135] Murine chorionic villi amniotic fluids and were collected
from 12 days pregnant female C57BL/6 mice aging from 6 to 9 weeks
of age (protocol approved Animal Care and Use Committee, Children's
Hospital, Boston) under light microscopy. The samples were
proceeded as previously described. Briefly placentas were dissected
under light microscope and the chorionic villi were explanted.
Chorionic villi and amniotic fluid derived cells were cultured in
the same conditions used for human cells with addition of LIF (10
ng/ml) (Sigma-Aldrich). The ckit.sup.pos cells were transduced with
a puc-CMMP-IC-eGFP retrovirus and expanded. The
infected-cKit.sup.pos were sorted by FACS-Excalibur and a single
eGFP.sup.+-cells was plated per well in a 96 wells-plate and
expanded.
[0136] In order to assess the ability of these cells to contribute
to different tissue 10-12 ckit.sup.pos infected cells were
microinjected into 4-day-old blastocysts of
C57BL/6-TgN(lacZpl)60Vij. The blastocysts were transferred to
foster mothers and mice were allowed to develop until 16 days of
gestation.
[0137] The fetuses were collected, embedded in OCT and 10 .mu.m
whole-body sections were prepared. Tissue sections were stained for
.beta.-galactosidase enzyme activity and observed under fluorescent
microscope in order to identify the c-kit.sup.pos cells carrying
the gene for the green protein.
[0138] We also collected multiple organs, they were embedded in OCT
and 5 .mu.m sections were prepared as described. The sections were
stained for .beta.-galactosidase enzyme activity and observed.
[0139] Frozen section were cut at 10 .mu.m and fixed with 2%
formaldhyde, 0.2% glutaraldehyde, 0.02% NP-40 and 0.01% sodium
deoxycolate in PBS pH7.8 for 30 min at RT and then wash 3 times
with PBS. Samples were incubated in LacZ staining solution (2 mM
MgCl.sub.2, 0.02% NP-40, 0.01% sodium deoxycolate, 5 mM
K-ferricyanide, 5 mM K-ferrocyanide and 0.1% X-gal in PBS pH7.8) at
37C for 8 to 16 hours in dark. Images were acquired using IX-70
microscope with Magna Fire Digital Imaging Camera System (Olympus)
and processed using Adobe Photoshop 5.0.
[0140] Discussion. Stem cells have been reported to exist during
embryonic development and postnatally, in bone marrow, skeletal
muscle and skin (for a recent review discussion of stem cells see,
J. Pathol, Vol 197, Issue 4, 2002). Embryonic stem (ES) cells are
derived from the inner cell mass (ICM) at the blastula stage. ES
cells tend to differentiate spontaneously into various types of
tissues. However, isolation of these cells, particularly from human
embryos, has resulted in heated debate about ethical concerns of
this procedure which results in destruction of the embryo.
[0141] Adult stem cells do not differentiate spontaneously, but can
be induced to differentiate by applying appropriate growth
conditions. Adult stem cells seem to be easier to maintain in
culture than ES cells. However, adult stem cells have the
disadvantage of not being immortal, and most of them lose their
pluripotency after a defined number of passages in culture. This
short life-span is a significant obstacle in clinical applications
where a large amount of cells are needed.
[0142] Fetal tissue has been used in the past for transplantation
and tissue engineering research because of its pluripotency and
proliferative ability. Fetal cells have a higher proliferative
capacity than adult cells and may preserve their pluripotency
longer in culture. However, there are several issues concerning the
availability of fetal cell transplants. Beyond the ethical concerns
regarding the use of cells from aborted fetuses or living fetuses,
there are other issues, which remain a challenge. For example,
previous studies have shown that up to six fetuses are required to
provide enough material to treat one patient with Parkinson's
disease (45).
[0143] This invention is based upon a finding that chorionic villi
and amniotic fluid cells, which have been used for decades for
prenatal diagnosis, represent a viable source of human fetal stem
cells from both embryonic and fetal sources and can be used
therapeutically. It is well known that both chorionic villi tissue
specimens and amniotic fluid contain a large variety of cells. The
vast majority of the cells collected from chorionic villi and
amniotic fluid are already differentiated, and therefore have a
limited proliferative ability (46). We have here identified and
isolated cells that maintained both their pluripotential and
proliferative ability.
[0144] Many efforts in the past were aimed at trying to identify
antibodies that bind cell surface markers on undifferentiated
cells. C-kit, CD105, CD34 and CD90 have been identified as
potential stem cell markers. We found that less than 1% of the
embryonic and fetal cells isolated from chorionic villi and
amniotic fluid were c-kit.sup.pos and that only the isolated
c-kit.sup.pos cells had the pluripotent phenotype. The c-kit gene
encodes for a tyrosine kinase growth factor receptor for Stem Cell
Factor (SCF), also called mast cell growth factor that is essential
for hematopoesis, melanogenesis and fertility (46). The Kit protein
(CD117) is constitutively expressed in hematopoetic stem cells,
mast cells, germ cells, melanocytes, certain basal epithelial
cells, luminal epithelium of breast, and the interstitial cells of
Cajal of the gastrointestinal tract (47). The c-kit gene plays a
fundamental role during the establishment, maintenance and function
of germ cells (48). In the embryonal gonad, the c-kit receptor and
its ligand SCF are required for the survival and proliferation of
primordial germ cells. Furthermore recent studies have shown that
c-kit is expressed in placental tissue during pregnancy. C-kit and
SCF may have an important role in embryonic development as
evidenced by expression and localization at the feto-maternal
interface (49). In the postnatal animal, c-kit/SCF are required for
production of the mature gametes in response to gonadotropic
hormones, i.e. for the survival and/or proliferation of the only
proliferating germ cells of the testis, the spermatogonia, and for
the growth and maturation of the oocytes. Experiments in vitro have
shown that c-kit is a potent mitogen for primitive hematopoetic
cells. In mice, loss of either SCF or c-kit results in macrocytic
anemia, leading to death in-utero or within the first postnatal
days.
[0145] Adult stem cells have a limited capacity to proliferate and
they undergo senescence when the Hayflick limit is reached.
Furthermore, the adult stem cells are not able to preserve their
ability to differentiate into multiple lineages after a few
passages. Contrary to adult stem cells, embryonic stem (ES) cells
have an unlimited capacity to proliferate and they are able to
maintain their potential for differentiation in culture. We found
that the c-kit.sup.pos cells derived from human embryonic and fetal
chorionic villi and amniotic fluid were pluripotent and were able
to differentiate into osteogenic, adipogenic, myogenic, neurogenic,
hepatic and endothelial phenotypes. The possibility of forming
different types of tissues was confirmed in vivo. The cells were
telomerase positive, highly clonogenic, and the cloned fetal stem
cell lines were able to undergo more than 250 cell divisions,
exceeding Hayflick's limit. The stem cell lines maintained their
telomere length and differentiation potential in culture, even
after 250 population doublings. In addition, the c-kit.sup.pos
cells did not require a feeder layer for growth The c-kit positive
human fetal stem cells also expressed markers known to be
associated with human embryonic stem cells (SSAE3 and SSAE4).
[0146] In conclusion, we describe the isolation, expansion and
differentiation of stem cells from human embryonic and fetal
chorionic villi and amniotic fluid. These cells provide an
excellent source for both research and therapeutic applications.
Embryonic and fetal stem cells have a better potential for
expansion than adult stem cells and for this reason they represent
a significantly better source for therapeutic applications where
large numbers of cells are needed.
[0147] Further, the ability to isolate stem cells during gestation
may also be advantageous for treatment of fetuses with congenital
malformations in utero. When compared with ES cells, c-kit.sup.pos
fetal stem cells isolated from chorionic villi and amniotic fluid
have many similarities: they can differentiate into all three germ
layers, they express common markers and show telomerase activity.
However c-kit.sup.pos cells isolated from the chorionic villi and
amniotic fluid have considerable advantages over ES cells. The
c-kit.sup.pos cells isolated from the chorionic villi and amniotic
fluid easily differentiate into specific cell lineages, they do not
need feeder layers to grow, and most importantly, the isolation of
these cells does not require the sacrifice of human embryos for
their isolation, thus avoiding the current controversies associated
with the use of human embryonic stem cells.
[0148] The references cited herein and throughout the specification
are incorporated by reference in their entirety.
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Sequence CWU 1
1
20 1 21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 1 ggccttccac tctcagtaag a 21 2 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 2
gattcatcca ttctgccact a 21 3 21 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 3 ccctcacact cctcgcccta t
21 4 21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 4 ggtagcgcct gggtctcttc a 21 5 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 5
tgaacgacca agtaactctc c 21 6 21 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 6 ctcatgtctg tctccgtctt c
21 7 19 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 7 ctggtcgaag cattggaat 19 8 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 8
tgtagggcat ctgagaacga g 21 9 17 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 9 cgacagcagc ggagagg 17 10
21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 10 ggaatgatcg gaaacacttg g 21 11 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 11
tccgcgacgt agacctgac 19 12 20 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 12 gatatagcgg atggcgttgc 20
13 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 13 ccatcgcggc taagaacatt 20 14 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 14
tcggaagttg agggcagagt a 21 15 17 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 15 gctcgcgcta
ctctctc 17 16 21 DNA Artificial Sequence Description of Artificial
Sequence Synthetic primer 16 ttaactatct tgggctgtga c 21 17 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
primer 17 tgtttgcgta ctctgccttt g 21 18 21 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 18 ccttctctac
acccaagttc c 21 19 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 19 gaaataggca aagttccact g 21
20 19 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 20 tccagcgagg gtctaccag 19
* * * * *
References