U.S. patent application number 17/006257 was filed with the patent office on 2020-12-17 for isolation, cultivation and uses of stem/progenitor cells.
This patent application is currently assigned to CELLRESEARCH CORPORATION PTE. LTD.. The applicant listed for this patent is CELLRESEARCH CORPORATION PTE. LTD.. Invention is credited to Ivor Jiun LIM, Toan Thang PHAN.
Application Number | 20200390820 17/006257 |
Document ID | / |
Family ID | 1000005059620 |
Filed Date | 2020-12-17 |
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United States Patent
Application |
20200390820 |
Kind Code |
A1 |
PHAN; Toan Thang ; et
al. |
December 17, 2020 |
ISOLATION, CULTIVATION AND USES OF STEM/PROGENITOR CELLS
Abstract
The present invention relates to a method of cultivating
mammalian cells. This method comprises cultivating the mammalian
cells on a feeder layer, wherein the feeder layer comprises a
stem/progenitor cell population isolated from the amniotic membrane
of the umbilical cord
Inventors: |
PHAN; Toan Thang;
(Singapore, SG) ; LIM; Ivor Jiun; (Singapore,
SG) |
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Applicant: |
Name |
City |
State |
Country |
Type |
CELLRESEARCH CORPORATION PTE. LTD. |
Singapore |
|
SG |
|
|
Assignee: |
CELLRESEARCH CORPORATION PTE.
LTD.
Singapore
SG
|
Family ID: |
1000005059620 |
Appl. No.: |
17/006257 |
Filed: |
August 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16425735 |
May 29, 2019 |
10780130 |
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17006257 |
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11205248 |
Aug 15, 2005 |
10363275 |
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16425735 |
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60602208 |
Aug 16, 2004 |
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60632209 |
Dec 1, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/51 20130101;
A61K 35/36 20130101; C12N 5/0605 20130101; C12N 2506/025 20130101;
C12N 5/0629 20130101; C12N 2506/1392 20130101; C12N 5/0606
20130101; A61K 9/06 20130101; C12N 5/0668 20130101; C12N 5/0603
20130101; A61K 35/28 20130101; C12N 5/063 20130101; A61K 9/0014
20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C12N 5/073 20060101 C12N005/073; C12N 5/071 20060101
C12N005/071; C12N 5/0775 20060101 C12N005/0775; C12N 5/0735
20060101 C12N005/0735; A61K 9/00 20060101 A61K009/00; A61K 9/06
20060101 A61K009/06; A61K 35/36 20060101 A61K035/36; A61K 35/51
20060101 A61K035/51 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2005 |
SG |
PCT/SG05/00174 |
Claims
1. A method of cultivating mammalian cells comprising cultivating
said mammalian cells on a feeder layer, wherein the feeder layer
comprises a stem/progenitor cell population isolated from the
amniotic membrane of the umbilical cord.
2. The method of claim 1, wherein the stem/progenitor cell
population isolated from the amniotic membrane of the umbilical
cord is an epithelial or mesenchymal stem/progenitor cell
population that express the following genes: POU5f1, Bmi-1,
leukemia inhibitory factor (LIF), and secrete Activin A and
Follistatin.
3. The method of claim 2, wherein the epithelial or mesenchymal
stem/progenitor cell population express the following growth
factors: connective tissue growth factor (CTGF), vascular
endothelial growth factor (VEGF), placenta-like growth factor PLGF,
STAT3, stem cell factor (SCF), Hepatoma-derived Growth Factor
(HDGF), Fibroblast Growth Factor-2 (FGF-2), Platelet-derived Growth
Factor (PDGF), alpha-Smooth Muscle Actin (.alpha.-SMA),
Fibronectin, Decorin, and Syndecan-1,2,3,4.
4. The method of claim 1, wherein the stem/progenitor cell
population is isolated from the amniotic membrane of the umbilical
cord by a method comprising: (a) separating the amniotic membrane
from the other components of the umbilical cord in vitro to obtain
amniotic membrane tissue; (b) culturing the amniotic membrane
tissue obtained in step (a) under conditions allowing cell
proliferation; and (c) isolating the stem/progenitor cells, and
wherein the stem/progenitor cells are epithelial or mesenchymal
stem/progenitor cells.
5. The method of claim 1, wherein the feeder layer essentially
consists of a stem/progenitor cell population isolated from the
amniotic membrane of the umbilical cord.
6. The method of claim 1, wherein the stem/progenitor cell
population isolated from the amniotic membrane of the umbilical
cord is a human stem/progenitor cell population.
7. The method of claim 1, wherein the mammalian cells are stem
cells.
8. The method of claim 7, wherein the stem cells are embryonic stem
cells.
9. The method of claim 1, wherein the mammalian cells are cultured
under serum free conditions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/425,735 filed May 29, 2019 which is
a continuation application of U.S. patent application Ser. No.
11/205,248 filed Aug. 15, 2005, now granted U.S. Pat. No.
10,363,275 issued Jul. 19, 2019, which claims the benefit of
priority to U.S. Provisional Application No. 60/602,208, filed Aug.
16, 2004, and to U.S. Provisional Application No. 60/632,209, filed
Dec. 1, 2004, the contents of each being hereby incorporated by
reference it its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for isolating
stem/progenitor cells from the amniotic membrane of umbilical cord,
wherein the method comprises separating the amniotic membrane from
the other components of the umbilical cord in vitro, culturing the
amniotic membrane tissue under conditions allowing cell
proliferation, and isolating the stem/progenitor cells from the
tissue cultures. In particular, the invention relates to the
isolation and cultivation of stem cells having embryonic properties
such as epithelial and/or mesenchymal stem/progenitor cells under
conditions allowing the cells to undergo mitotic expansion.
Furthermore, the invention is directed to a method for the
differentiation of the isolated stem/progenitor cells into
epithelial and/or mesenchymal cells and therapeutic uses of these
stem/progenitor cells.
BACKGROUND OF THE INVENTION
[0003] Stem cells are a cell population possessing the capacities
to self-renew indefinitely and to differentiate in multiple cell or
tissue types. Embryonic stem cells (from approximately days 3 to 5
after fertilisation) proliferate indefinitely and can differentiate
spontaneously into all tissue types: they are thus termed
pluripotent stem cells (reviewed, for example, in Smith, A. G.
(2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462). Adult stem cells,
however, are more tissue-specific and may have less replicative
capacity: they are thus termed multipotent stem cells (reviewed,
for example, in Paul, G. et al. (2002) Drug Discov. Today 7,
295-302). The "plasticity" of embryonic and adult stem cells relies
on their ability to trans-differentiate into tissues different from
their origin and, perhaps, across embryonic germ layers.
[0004] The ability of stem cells to self-renew is critical to their
function as reservoir of primitive undifferentiated cells. In
contrast, most somatic cells have a limited capacity for
self-renewal due to telomere shortening (reviewed, for example, in
Dice, J. F. (1993) Physiol. Rev. 73, 149-159). Stem cell-based
therapies thus have the potential to be useful for the treatment of
a multitude of human and animal diseases.
[0005] Stem cells as well as stem/progenitor cells can be derived
from different sources. The "multi-lineage" potential of embryonic
and adult stem cells has been extensively characterized. Even
though the potential of embryonic stem cells is enormous, their use
implies many ethical problems. Therefore, non-embryonic stem cells
derived from the bone marrow stroma, fat tissue, dermis and
umbilical cord blood have been proposed as alternative sources.
These cells can differentiate inter alia into chondrocytes,
adipocytes, osteoblasts, myoblasts, cardiomyocytes, astrocytes, and
tenocytes in vitro and undergo differentiation in vivo, making
these stem cells--in general referred to as mesenchymal stem
cells--promising candidates for mesodermal defect repair and
disease management.
[0006] In clinical use, however, harvesting of such mesenchymal
stem cells causes several problems. The collection of the cells is
a mental and physical burden to the patient as a surgical procedure
is required to obtain the cells (for example, the collection of
bone marrow is an invasive technique performed with a biopsy needle
that requires local or even general anesthesia). Furthermore, in
many cases the number of stem cells extracted is rather low. More
importantly, no epithelial cells are derived or differentiated from
these cells. This prompted the search for other possible sources of
stem cells.
[0007] Umbilical cord blood has been identified as a rich source of
haematopoetic stem/progenitor cells. However, the existence of
mesenchymal stem/progenitor cells is discussed controversially. On
the one hand, such cells could not be isolated or successfully
cultured from term umbilical cord blood (Mareschi, K. et al. (2001)
Haematologica 86, 1099-1100). At the same time, results obtained by
Campagnoli, C. et al. (Blood (2001) 98, 2396-2402) as well as
Erices, A. et al. (Br. J. Haematol. (2000) 109, 235-242) suggest
that mesenchymal stem cells are present in several fetal organs and
circulate in the blood of pre-term fetuses simultaneously with
hematopoietic precursors. Accordingly, International Patent
Application WO 03/070922 discloses isolation and culture-expansion
methods of mesenchymal stem/progenitor cells from umbilical cord
blood and a differentiation method of such cells into various
mesenchymal tissues. Isolation efficiencies of about 60% have been
reported (Bieback, K. et al. (2004) Stem Cells 22, 625-634). In the
same study, both the time period from collection of the umbilical
cord blood to isolation of the cells and the volume of the blood
sample used have been determined as crucial parameters for
achieving such a yield. However, it is still a matter of debate
whether these stem/progenitor cells are indeed derived of umbilical
cord tissue.
[0008] Recently, mesenchymal stem/progenitor cells have been
successfully isolated from umbilical cord tissue, namely from
Wharton's jelly, the matrix of umbilical cord, (Mitchell, K. E. et
al. (2003) Stem Cells 21, 50-60; U.S. Pat. No. 5,919,702; US Patent
Application 2004/0136967). These cells have been shown to have the
capacity to differentiate, for example, into a neuronal phenotype
and into cartilage tissue, respectively. Furthermore, mesenchymal
stem/progenitor cells have also been isolated from the endothelium
and the subendothelial layer of the umbilical cord vein, one of the
three vessels (two arteries, one vein) found within the umbilical
cord (Romanov, Y. A. et al. (2003) Stem Cells 21, 105-110; Covas,
D. T. et al. (2003) Braz. J. Med. Biol. Res. 36, 1179-1183).
[0009] However, none of these approaches employed thus far has
resulted in the isolation or cultivation of epithelial
stem/progenitor cells as a source for epithelial cell-based
therapies such as skin resurfacing, liver repair, bladder tissue
engineering and other engineered surface tissues. Thus, there is
still a need for methods and reliable sources useful for the
isolation and cultivation of epithelial stem/progenitor cells.
Furthermore, rapid and efficient methods which are ethically
acceptable and do not pose a biomedical burden on the patient for
the isolation of epithelial and mesenchymal stem/progenitor cells
are still required in order to provide such cells in a sufficient
amount for various applications in regenerative medicine and tissue
engineering.
SUMMARY OF THE INVENTION
[0010] The invention provides a method for isolating
stem/progenitor cells from the amniotic membrane of umbilical cord,
the method comprising:
[0011] (a) separating the amniotic membrane from the other
components of the umbilical cord in vitro;
[0012] (b) culturing the amniotic membrane tissue obtained in step
(a) under conditions allowing cell proliferation; and
[0013] (c) isolating the stem/progenitor cells.
[0014] In one embodiment, the invention provides a method, further
comprising:
[0015] (a'') separating the cells from the amniotic membrane tissue
before cultivation by a technique selected from the group
consisting of enzymatic digestion and direct tissue explant.
[0016] In one preferred embodiment, the invention provides a method
for isolating stem/progenitor cells that have embryonic stem
cell-like properties.
[0017] In another preferred embodiment, the invention provides a
method for isolating epithelial and/or mesenchymal stem/progenitor
cells.
[0018] In another embodiment, the invention provides a method
further comprising:
[0019] (d) culturing the stem/progenitor cells under conditions
allowing the cells to undergo clonal expansion.
[0020] In yet another embodiment, the invention provides a method
further comprising:
[0021] (e) culturing the stem/progenitor cells under conditions
allowing the differentiation of said cells into epithelial cells
and/or mesenchymal cells; and
[0022] (f) isolating the differentiated cells.
[0023] In yet another embodiment, the invention provides a method,
further comprising:
[0024] (g) preserving the isolated stem/progenitor cells for
further use.
[0025] In yet a further embodiment, the invention comprising a
method of cultivating stem/progenitors cells of the invention,
comprising:
[0026] Obtaining a tissue explant from the amniotic membrane of
umbilical cord;
[0027] Cultivating the tissue explant in suitable cultivation media
and cultivation conditions over a suitable period of time.
[0028] In yet other embodiments, the invention is directed to
therapeutic uses of the stem/progenitor cells or cells
differentiated therefrom or cellular secretions or extracts
thereof. One of these embodiments provide a method of treating a
subject having a disorder comprising administering to the subject
an effective amount of a stem/progenitor cell isolated by the
inventive method of explained above. Another embodiment comprises
administering to the subject an effective amount of a cell
differentiated from a stem/progenitor cell of the invention. Other
embodiments provide a corresponding pharmaceutical composition,
i.e. a pharmaceutical composition comprising a stem progenitor cell
or a cell differentiated therefrom, as well as cellular secretions
into the cell medium and extracts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the drawings, in which:
[0030] FIG. 1A depicts epithelial cell outgrowth from umbilical
cord amniotic membrane by the method of direct tissue explant
(40.times. magnification) at day 2 of tissue culture. FIG. 1B
depicts epithelial cell outgrowth from umbilical cord amniotic
membrane by the method of direct tissue explant (40.times.
magnification) at day 5 of tissue culture. FIG. 1C also depicts
epithelial cell outgrowth from umbilical cord amniotic membrane by
the method of direct tissue explant (40.times. magnification) at
day 5 of tissue culture. Cell culture plastic surfaces were coated
with collagen 1/collagen 4 mixtures (1:2; Becton Dickinson) before
placing the amniotic membrane on the surface. The amniotic membrane
specimens were submerged in 5 ml EpiLife medium or Medium 171 (both
from Cascade Biologics). Medium was changed every 2 or 3 days and
cell outgrowth by explant was monitored under light microscopy.
Microphotographs were taken at different time intervals as stated
above. The observed polyhedral cell morphology is typical of
epithelial cells.
[0031] FIG. 2A depicts enzymatic digestion of the umbilical cord
segments yielding similar epithelial (40.times. magnification)
cells at day 2. FIG. 2C also depicts enzymatic digestion of the
umbilical cord segments yielding similar epithelial (40.times.
magnification) cells at day 2. FIG. 2B depicts enzymatic digestion
of the umbilical cord segments yielding similar epithelial
(40.times. magnification) cells at day 5. FIG. 2D also depicts
enzymatic digestion of the umbilical cord segments yielding similar
epithelial (40.times. magnification) cells at day 5. Umbilical cord
amniotic membrane was divided into small pieces of 0.5 cm.times.0.5
cm and digested in 0.1% (w/v) collagenase type 1 solution (Roche
Diagnostics) at 37.degree. C. for 8 hours. The samples were
vortexed every 30 min for 3 min. Cells were harvested by
centrifugation at 4000 rpm for 30 min. Cell pellets were
resuspended in EpiLife medium or Medium 171 (both from Cascade
Biologics) supplemented with 50 .mu.g/ml insulin-like growth
factor-1 (IGF-1), 50 .mu.g/ml platelet-derived growth factor-BB
(PDGF-BB), 5 .mu.g/ml transforming growth factor-.beta.1
(TGF-.beta.1) and 5 .mu.g/ml insulin (all obtained from R&D
Systems), counted and seeded on 10 cm tissue culture dishes
pre-coated with collagen 1/collagen 4 mixtures (1:2; Becton
Dickinson) at density of 1.times.10.sup.6 cells/dish. After 24
hours, attached cells were washed with warm phosphate buffered
saline (PBS) and the culture medium was replaced with EpiLife
medium or Medium 171 (both from Cascade Biologics). The medium was
changed every 2 or 3 days, and cell outgrowth was monitored under
light microscopy. Microphotographs were taken at different time
intervals as stated above. Once again the cells demonstrated
typical epithelial cell polyhedral morphology.
[0032] FIGS. 3A to 3D depict outgrowing mesenchymal cells explanted
from umbilical cord amniotic membrane. FIG. 3A shows the outgrowth
of mesenchymal cells explanted from umbilical cord amniotic
membrane observed as early as 48 hours after placement in tissue
culture dishes using DMEM supplemented with 10% fetal calf serum
(FCS) as culture medium (40.times. magnification). FIG. 3C also
shows the outgrowth of mesenchymal cells explanted from umbilical
cord amniotic membrane observed as early as 48 hours after
placement in tissue culture dishes using DMEM supplemented with 10%
fetal calf serum (FCS) as culture medium (40.times. magnification).
The explants were submerged in 5 ml DMEM (Invitrogen) supplemented
with 10% fetal bovine serum (Hyclone) (DMEM/10% FBS). Medium was
changed every 2 or 3 days. Cell outgrowth was monitored under light
microscopy. Microphotographs were taken at different time
intervals. FIG. 3B shows cells characterized by their spindle
shaped morphology, which migrate and expand both easily and quickly
in vitro, closely resembling fibroblasts (40.times. magnification).
FIG. 3D also shows cells characterized by their spindle shaped
morphology, which migrate and expand both easily and quickly in
vitro, closely resembling fibroblasts (40.times.
magnification).
[0033] FIG. 4A (40.times. magnification) depicts mesenchymal cells
from umbilical cord amniotic membrane cells isolated by collagenase
enzymatic digestion, showing mesenchymal cells isolated from
umbilical cord amniotic membrane at day 2. FIG. 4B (40.times.
magnification) depicts mesenchymal cells from umbilical cord
amniotic membrane cells isolated by collagenase enzymatic
digestion, showing cell proliferation observed at day 5. Umbilical
cord amniotic membrane was divided into small pieces of 0.5
cm.times.0.5 cm and digested in 0.1% (w/v) collagenase type1
solution (Roche Diagnostics) at 37.degree. C. for 6 hours. The
samples were vortexed every 15 min for 2 min. Cells were harvested
by centrifugation at 4000 rpm for 30 min. Cell pellets were
resuspended in DMEM/10% FBS, counted and seeded on 10 cm tissue
culture dish at density of 1.times.10.sup.6 cells/dish. Medium was
changed every 2 or 3 days. Cell outgrowing was monitored under
light microscopy. Microphotographs were taken at different time
intervals. Once again, cells demonstrated spindle shaped morphology
typical of mesenchymal cells as fibroblasts.
[0034] FIG. 5A (40.times. magnification) depicts the morphology in
serum culture condition (DMEM/10% FCS) of normal dermal fibroblasts
(NF109 cells). FIG. 5B (40.times. magnification) depicts the
morphology in serum-free culture condition (DMEM) of normal dermal
fibroblasts (NF109 cells). FIG. 5C (40.times. magnification)
depicts the morphology in serum culture condition (DMEM/10% FCS) of
adipose-derived mesenchymal cells (ADMC). FIG. 5D (40.times.
magnification) depicts the morphology in serum-free culture
condition (DMEM) of adipose-derived mesenchymal cells (ADMC). FIG.
5E (40.times. magnification) depicts the morphology in serum
culture condition (DMEM/10% FCS) of umbilical cord amniotic
membrane mesenchymal cells (UCMC) isolated according to the method
of the invention. FIG. 5G (40.times. magnification) also depicts
the morphology in serum culture condition (DMEM/10% FCS) of
umbilical cord amniotic membrane mesenchymal cells (UCMC) isolated
according to the method of the invention. FIG. 5F (40.times.
magnification) depicts the morphology in serum-free culture
condition (DMEM) of umbilical cord amniotic membrane mesenchymal
cells (UCMC) isolated according to the method of the invention.
FIG. 5H (40.times. magnification) also depict the morphology in
serum-free culture condition (DMEM) of umbilical cord amniotic
membrane mesenchymal cells (UCMC) isolated according to the method
of the invention. Morphology of NF and ADMC cultured in serum
starvation conditions (DMEM only) is reflected by flatter cells and
less dense cytoplasm as compared with serum rich conditions
(DMEM/10% FCS) where cells are more rounded with a dense cytoplasm.
No change in morphology was observed in both UCMC groups cultured
under identical conditions of serum-free vs. serum rich media,
indicating a difference in behavior and physiology of these latter
mesenchymal cells.
[0035] FIG. 6 (40.times. magnification) depicts UCMC isolated
according to the invention cultured in DMEM/10% FCS at days 3 and 7
without a 3T3 feeder layer. The cells are seen to be growing well,
and are forming a colony (vertical growth) instead of exhibiting
radial spread. Once again, this indicates a difference in behavior
of these mesenchymal cells as compared to their more differentiated
counterparts.
[0036] FIG. 7 (40.times. magnification) depicts colony formation of
umbilical cord epithelial cells (UCEC) cultured on a 3T3 feeder
layer at days 3 and 7. This appearance is similar to that of normal
skin derived epithelial keratinocyte stem cells. In the latter, the
3T3 feeder layer maintains stemness of the cells.
[0037] FIG. 8A (40.times. magnification) depicts obvious colony
formation of umbilical cord mesenchymal cells (UCMC) isolated
according to the invention cultured on a 3T3 feeder layer at days 3
and 7. The 3T3 feeder layer normally suppresses the growth of
differentiated mesenchymal cells as human dermal fibroblasts. Once
again, this indicates a difference in behavior of these mesenchymal
cells as compared to their more differentiated counterparts. FIG.
8B shows the colony forming efficiency assay of the umbilical cord
mesenchymal cells.
[0038] FIG. 9-1 shows Western blot analysis by which the expression
of OCT-4 in UCEC and UCMC isolated according to the invention, was
compared to the expression of these markers in human dermal
fibroblasts (NF), in bone marrow mesenchymal cells (BMSC) and
adipose-derived mesenchymal cells (ADMC). FIG. 9-2 shows Western
blot analysis by which the expression of STAT3 in UCEC and UCMC
isolated according to the invention, was compared to the expression
of these markers in human dermal fibroblasts (NF), in bone marrow
mesenchymal cells (BMSC) and adipose-derived mesenchymal cells
(ADMC). FIG. 9-3 shows Western blot analysis by which the
expression of STAT3 in UCEC and UCMC isolated according to the
invention, was compared to the expression of these markers in human
dermal fibroblasts (NF), in bone marrow mesenchymal cells (BMSC)
and adipose-derived mesenchymal cells (ADMC). FIG. 9-4 shows
Western blot analysis by which the expression of PLGF in UCEC and
UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-5 shows Western blot analysis by
which the expression of PLGF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-6 shows
Western blot analysis by which the expression of CTGF in UCEC and
UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-7 shows Western blot analysis by
which the expression of CTGF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-8 shows
Western blot analysis by which the expression of PDGF in UCEC and
UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-9 shows Western blot analysis by
which the expression of PDGF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-10
shows Western blot analysis by which the expression of VEGF in UCEC
and UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-11 shows Western blot analysis by
which the expression of VEGF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-12
shows Western blot analysis by which the expression of FGF-2 in
UCEC and UCMC isolated according to the invention, was compared to
the expression of these markers in human dermal fibroblasts (NF),
in bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-13 shows Western blot analysis by
which the expression of FGF-2 in UCEC and UCMC isolated according
to the invention, was compared to the expression of these markers
in human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-14
shows Western blot analysis by which the expression of HDGF in UCEC
and UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-15 shows Western blot analysis by
which the expression of HDGF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-16
shows Western blot analysis by which the expression of SCF in UCEC
and UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-17 shows Western blot analysis by
which the expression of .alpha.-SMA in UCEC and UCMC isolated
according to the invention, was compared to the expression of these
markers in human dermal fibroblasts (NF), in bone marrow
mesenchymal cells (BMSC) and adipose-derived mesenchymal cells
(ADMC). FIG. 9-18 shows Western blot analysis by which the
expression of fibronectin in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-19
shows Western blot analysis by which the expression of fibronectin
in UCEC and UCMC isolated according to the invention, was compared
to the expression of these markers in human dermal fibroblasts
(NF), in bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-20 shows Western blot analysis by
which the expression of decorin in UCEC and UCMC isolated according
to the invention, was compared to the expression of these markers
in human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-21
shows Western blot analysis by which the expression of syndecan-1
in UCEC and UCMC isolated according to the invention, was compared
to the expression of these markers in human dermal fibroblasts
(NF), in bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-22 shows Western blot analysis by
which the expression of syndecan-2 in UCEC and UCMC isolated
according to the invention, was compared to the expression of these
markers in human dermal fibroblasts (NF), in bone marrow
mesenchymal cells (BMSC) and adipose-derived mesenchymal cells
(ADMC). FIG. 9-23 shows Western blot analysis by which the
expression of syndecan-2 in UCEC and UCMC isolated according to the
invention, was compared to the expression of these markers in human
dermal fibroblasts (NF), in bone marrow mesenchymal cells (BMSC)
and adipose-derived mesenchymal cells (ADMC). FIG. 9-24 shows
Western blot analysis by which the expression of syndecan-3 in UCEC
and UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-25 shows Western blot analysis by
which the expression of syndecan-3 in UCEC and UCMC isolated
according to the invention, was compared to the expression of these
markers in human dermal fibroblasts (NF), in bone marrow
mesenchymal cells (BMSC) and adipose-derived mesenchymal cells
(ADMC). FIG. 9-26 shows Western blot analysis by which the
expression of syndecan-4 in UCEC and UCMC isolated according to the
invention, was compared to the expression of these markers in human
dermal fibroblasts (NF), in bone marrow mesenchymal cells (BMSC)
and adipose-derived mesenchymal cells (ADMC). FIG. 9-27 shows
Western blot analysis by which the expression of Bmi-1 in UCEC and
UCMC isolated according to the invention, was compared to the
expression of these markers in human dermal fibroblasts (NF), in
bone marrow mesenchymal cells (BMSC) and adipose-derived
mesenchymal cells (ADMC). FIG. 9-28 shows Western blot analysis by
which the expression of LIF in UCEC and UCMC isolated according to
the invention, was compared to the expression of these markers in
human dermal fibroblasts (NF), in bone marrow mesenchymal cells
(BMSC) and adipose-derived mesenchymal cells (ADMC). FIG. 9-29
shows secretion of Leukemia inhibitory factor detected by Western
blot analysis in supernatants of umbilical cord mesenchymal and
epithelial stem cell culture in comparison with bone marrow,
adipose derived stem cells, human dermal fibroblasts and epidermal
keratinocytes. FIG. 9-30 shows secretion of highly secreted
ActivinA and Follistatin detected by ELISA assay in supernatants of
umbilical cord mesenchymal and epithelial stem cell culture in
comparison with bone marrow, adipose derived stem cells, human
dermal fibroblasts and epidermal keratinocytes.
[0039] FIG. 10-1 shows indirect immunofluorescent analysis of
markers of epithelial cells expressed in umbilical cord epithelial
stem cells: cytokeratins (CK)-general, CK17, CK6, CK10, CK19, CK18,
CK16, CK15. FIG. 10-2 shows indirect immunofluorescent analysis of
markers of epithelial cells expressed in umbilical cord epithelial
stem cells: Hemidesmosome components-integrin alpha6, integrin
beta4; Desmosome components. FIG. 10-3 shows indirect
immunofluorescent analysis of markers of epithelial cells expressed
in umbilical cord epithelial stem cells: Basement membrane
components-laminin1, laminin5, collagen IV, collagen VII. FIG. 10-4
shows indirect immunofluorescent analysis of markers of epithelial
cells expressed in umbilical cord epithelial stem cells:
extracellular matrix components integrin-beta1 and fibronectin.
[0040] FIGS. 11-1 to 11-4 show cytokine array analysis of secreted
cytokines and growth factors by umbilical cord mesenchymal stem
cells (UCMC) in comparison with human bone-marrow mesenchymal stem
cells. In more detail, FIG. 11-1 shows an expression profile of
secreted cytokines and growth factors by umbilical cord mesenchymal
cells, FIG. 11-2 also shows an expression profile of secreted
cytokines and growth factors by umbilical cord mesenchymal cells,
FIG. 11-3 further shows an expression profile of secreted cytokines
and growth factors by umbilical cord mesenchymal cells, and also
FIG. 11-4 shows an expression profile of secreted cytokines and
growth factors by umbilical cord mesenchymal cells.
[0041] FIGS. 12-1 to 12-7 show cytokine array analysis of secreted
cytokines and growth factors by umbilical cord epithelial stem
cells (UCEC) in comparison with human epidermal keratinocytes. In
more detail, FIG. 12-1 shows an expression profile of secreted
cytokines and growth factors by umbilical cord epithelial cells,
FIG. 12-2 also shows an expression profile of secreted cytokines
and growth factors by umbilical cord epithelial cells, FIG. 12-3
shows an expression profile of secreted cytokines and growth
factors by human epidermal keratinocytes, FIG. 12-4 also shows an
expression profile of secreted cytokines and growth factors by
human epidermal keratinocytes, FIG. 12-5 shows an expression
profile of secreted cytokines and growth factors by umbilical cord
epithelial cells, FIG. 12-6 shows a spotting of a chip used for
cytokine array, and FIG. 12-7 also shows a spotting of a chip used
for cytokine array.
[0042] FIG. 13-1 shows UCMC cells cultured in DMEM supplemented
with 10% fetal calf serum (FCS); FIG. 13-2 shows UCMC cells
cultured in serum-free media PTT-1; FIG. 13-3 shows UCMC cells
cultured in serum-free media PTT-2; FIG. 13-4 also shows UCMC cells
cultured in serum-free media PTT-2; and FIG. 13-5 shows UCMC cells
cultured in serum-free media PTT-3. FIG. 13-6 shows the growth of
adipose derived stromal cells in serum free medium PTT-3 and FIG.
13-7 shows bone marrow derived stromal cells in serum free medium
PTT-3.
[0043] FIG. 14 shows global gene expression in umbilical cord
epithelial and mesenchymal stem cells analyzed by DNA microarray.
UCEC expressed a total of 28055 genes and UCMC expressed a total of
34407 genes. There are 27308 overlapping genes expressing in both
cell types. 747 genes expressed were unique to UCEC and 7099 genes
expressed were unique to UCMC. The selected genes of interest are
presented in this Figure. Both stem cell types expressed 140 genes
related to embryonic stem cells and embryonic development.
[0044] FIG. 15 shows a schematic illustration of expansion of
umbilical cord epithelial and mesenchymal stem cells using
repetitive explants of umbilical cord lining membrane tissues.
[0045] FIG. 16 depicts a cross section of an umbilical cord
demonstrating the umbilical cord amniotic lining membrane (LM), the
contained Wharton's jelly (WJ), as well as two umbilical arteries
(UA) and one umbilical vein (UV) supported within this jelly.
[0046] FIG. 17A depicts direct (in-vitro) differentiation of
epithelial cells isolated from the amniotic membrane of umbilical
cord (UCEC) into skin epidermal keratinocytes. FIG. 17B depicts
direct in-vitro differentiation of mesenchymal cells isolated from
the amniotic membrane of umbilical cord (UCMC) into
osteoblasts.
DETAILED DESCRIPTION
[0047] The invention is based on the surprising finding that the
amniotic membrane of umbilical cord represents a source, from which
stem/progenitor cells such as mesenchymal and epithelial
stem/progenitor cells can be successfully isolated and expanded
under in vitro conditions. Even more surprising is the finding that
these cells show embryonic stem cell-like characteristics. The
amniotic membrane (also called amniotic lining membrane), i.e. thin
innermost membranous sac enclosing the placenta and developing
embryo of mammals, has recently been used as a natural substrate in
ocular surface reconstruction and as a biological substrate for
expanding limbal epithelial stem cells (cf., e.g., Anderson, D. F.
et al. (2001) Br. J. Ophthalmol. 85, 567-575; Gruterich, M. et al.
(2003) Surv. Ophthalmol. 48, 631-646). However, no methods have
been described thus far for the isolation of stem/progenitor cells
from the amniotic membrane, at least for humans, nor has the
amniotic membrane covering the umbilical cord been reported as a
source for stem cells.
[0048] The invention provides a method for isolating
stem/progenitor cells from the amniotic membrane of umbilical cord,
the method comprising:
[0049] (a) separating the amniotic membrane from the other
components of the umbilical cord in vitro;
[0050] (b) culturing the amniotic membrane tissue obtained in step
(a) under conditions allowing cell proliferation; and
[0051] (c) isolating the stem/progenitor cells.
[0052] For isolation of the cells of the invention from umbilical
cord, the umbilical cord or a part thereof is usually collected
immediately after birth (of a child in the case of humans) and for
transport to the laboratory transferred in a medium that is
suitable for handling of mammalian tissue. Examples of such media
include, but are not limited to Leibovitz media which are
commercially available from suppliers such as Sigma Aldrich, Saint
Louis, USA or HyClone, Logan, Utah, USA. The umbilical cord is then
typically processed under sterile conditions. Processing of the
cord typically includes removing the blood that has remained on the
surface or within the blood vessels of the umbilical cord by
washing with a suitable buffer such as phosphate buffered saline.
The umbilical cord is then typically reduced to smaller pieces, for
example by cutting, and washed again before separating the amniotic
membrane from the other components. In this conjunction, it is
noted that it is not necessary to process the umbilical cord of a
mammalian donor immediately after birth but it is also possible, to
collect the umbilical cord and, optionally after washing under
sterile conditions and reducing it into smaller pieces, to preserve
the umbilical cord or parts thereof by cryo-preservation and to
store the so obtained specimen, for example in liquid nitrogen, for
later isolation of the cells of the invention from the umbilical
cord. Accordingly, an (intact) umbilical cord or a portion of an
intact umbilical cord that is treated by cryo-preservation is also
encompassed in the present invention. In addition, the umbilical
cord amniotic membrane that has been separated from the other
components of the umbilical cord and is then treated by
cryo-preservation is also encompassed in the present invention.
[0053] The term "cryo-preservation" is used herein in its regular
meaning to describe a process where cells or whole tissues are
preserved by cooling to low sub-zero temperatures, such as
(typically) -80.degree. C. or -196.degree. C. (the boiling point of
liquid nitrogen). Cryo-preservation can be carried out as known to
the person skilled in the art and can include the use of
cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol, which
slow down the formation of ice-crystals in the cells of the
umbilical cord.
[0054] The term "stem/progenitor cell" as used herein refers to any
cell derived of umbilical cord having the capacities to self-renew
indefinitely and to differentiate in multiple cell or tissue types
such as endothelial cells, epithelial cells, fibroblasts, myocytes
or neurons. Furthermore, the cells may be derived of any mammalian
species, such as mouse, rat, guinea pig, rabbit, goat, dog, cat,
sheep, monkey or human, with cells of human origin being preferred
in one embodiment.
[0055] The term "embryonic stem cell-like properties" refers to the
ability of the cells derived of umbilical cord that they
can--almost like or exactly like embryonic stem
cells--differentiate spontaneously into all tissue types, meaning
that they are pluripotent stem cells.
[0056] The term "amniotic membrane" as used herein refers to the
thin innermost membranous sac enclosing the developing embryo of
mammals. During pregnancy, the fetus is surrounded and cushioned by
a liquid called amniotic fluid. This fluid, along with the fetus
and the placenta, is enclosed within a sac called the amniotic
membrane, which also covers the umbilical cord. The amniotic fluid
is important for several reasons. It cushions and protects the
fetus, allowing the fetus to move freely. The amniotic fluid also
allows the umbilical cord to float, preventing it from being
compressed and cutting off the fetus' supply of oxygen and
nutrients derived from the circulating blood within the placental
blood vessels. The amniotic sac contains the amniotic fluid which
maintains a homeostatic environment protecting the fetal
environment from the outside world. This barrier additionally
protects the fetus from organisms (like bacteria or viruses) that
could travel up the vagina and potentially cause infection.
[0057] Media and reagents for tissue culture are well known in the
art (cf., for example, Pollard, J. W. and Walker, J. M. (1997)
Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa,
N.J.; Freshney, R. I. (2000) Culture of Animal Cells, Fourth
Edition, Wiley-Liss, Hoboken, N.J.). Examples of suitable media for
incubating/transporting umbilical cord tissue samples include, but
are not limited to, Dulbecco's Modified Eagle Medium (DMEM), RPMI
media, Hanks' Balanced Salt Solution (HBSS) phosphate buffered
saline (PBS), and L-15 medium, with the latter one being preferred
in some embodiments. Examples of appropriate media for culturing
stem/progenitor cells according to the invention include, but are
not limited to, Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12,
RPMI media, EpiLlfe medium, and Medium 171, with the latter being
preferred in some embodiments. The media may be supplemented with
fetal calf serum (FCS) or fetal bovine serum (FBS) as well as
antibiotics, growth factors, amino acids, inhibitors or the like,
which is well within the general knowledge of the skilled
artisan.
[0058] In one embodiment, the invention provides a method, further
comprising:
[0059] (a'') separating these stem/progenitor cells from the
amniotic membrane tissue by a enzymatic digestion and/or direct
tissue explant technique before cultivation. The term "enzymatic
digestion technique" as used herein means that enzymes are added to
cleave the cells from the main tissue mass (here the amniotic
membrane of the umbilical cord). The separated cells are
subsequently collected. The term "direct tissue explant technique"
as used herein means that the tissue is first placed in media
without enzymes. Then under careful conditions the cells separate
from the main tissue mass by itself- and the cells are then
harvested for collection.
[0060] Methods for separating cells of a particular tissue or organ
by treatment with enzymes or by direct tissue explant are well
known in the art (cf., for example, Pollard, J. W. and Walker, J.
M. (1997) Basic Cell Culture Protocols, Second Edition, Humana
Press, Totowa, N.J.; Freshney, R. I. (2000) Culture of Animal
Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Any enzyme
catalyzing tissue dissociation may be used for performing the
methods of the present invention. In some embodiments, collagenase
is used for that purpose. The enzyme may be used as a crude
preparation or in purified form. It may be purified from any
prokaryotic or eukaryotic organism (with Clostridium histolyticum
being most preferred) or produced recombinantly by means of gene
technology. Any type of collagenase may be employed, i.e. type 1,
type 2, type 3, type 4, or any combination thereof. In some
embodiments the use of collagenase type 1 is being preferred.
[0061] In one embodiment, the invention provides a method for
isolating stem/progenitor cells that have embryonic stem cell-like
properties. These cells can ultimately be differentiated into, but
not limited to, by morphology, epithelial or mesenchymal cells.
[0062] Accordingly, in another embodiment, the invention provides a
method for isolating epithelial and/or mesenchymal stem/progenitor
cells, wherein in accordance with the above disclosure these cells
may have embryonic stem cell-like properties.
[0063] Epithelial stem/progenitor cells include any cells
exhibiting a epithelial cell like morphology (i.e. a polyhedral
shape) that can be differentiated into any type of epithelial cell
such as, but not limited to, skin epithelial cells, hair follicular
cells, cornea epithelial cells, conjunctival epithelial cells,
retinal epithelial cells, liver epithelial cells, kidney epithelial
cells, pancreatic epithelial cells, oesophageal epithelial cells,
small intestinal epithelial cells, large intestinal epithelial
cells, lung and airway epithelial cells, bladder epithelial cells
or uterine epithelial cells.
[0064] Mesenchymal stem/progenitor cells include any cells
exhibiting a mesenchymal cell like morphology (i.e. a spindle-like
shape) that can be differentiated into any type of mesenchymal cell
such as, but not limited to, skin fibroblasts, chondrocytes,
osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes,
smooth muscle cells, skeletal muscle cells, adipocytes, cells
derived from endocrine glands, and all varieties and derivatives of
neurectodermal cells.
[0065] In another embodiment, the invention provides a method
further comprising:
[0066] (d) culturing the stem/progenitor cells under conditions
allowing the cells to undergo clonal expansion.
[0067] The term "clonal expansion" (sometimes also referred to as
"mitotic clonal expansion") relates to a process that occurs early
in the differentiation program of a cell, by which stem/progenitor
cells become committed to a particular lineage and then undergo
terminal differentiation. It is well known in the art that the
conditions to induce clonal expansion of progenitor cells may vary
significantly between different cell types. Without being limited
to a particular method, the induction of clonal expansion is
generally achieved by cultivating the stem/progenitor cells in a
medium that has been optimized for cell proliferation. Such media
are commercially available from many providers. Non-limiting
examples of such media are KGM.RTM.-Keratinocyte Medium (Cambrex),
MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife medium
(Cascade Biologics) or Medium 171 (Cascade Biologics).
Alternatively, a culture medium may be supplemented with reagents
inducing cell proliferation such as growth factors. Such reagents
may be admixed in a single solution such as the Human Keratinocyte
Growth Supplement Kit (Cascade Biologics), to name one example, or
may be supplemented individually. Such reagents include, but are
not limited to, growth factors (such as epidermal growth factor,
insulin-like growth factor-1, platelet-derived growth factor-BB,
transforming growth factor-.beta.1, insulin, for example), hormones
(such as a bovine pituitary extract), hydrocortisone, transferrin
and the like in any suitable combination to induce clonal expansion
of a given cell type. The term "clonal expansion" also includes
cultivation of the cell in vivo, for example, by injection of the
cells into mammals such as humans, mice, rats, monkeys, apes to
name only a few.
[0068] In yet another embodiment, the invention provides a method
further comprising:
[0069] (e) culturing the stem/progenitor cells under conditions
allowing the differentiation of said cells into epithelial cells
and/or mesenchymal cells; and
[0070] (f) isolating the differentiated cells.
[0071] Thus, the invention also provides for a method of
differentiating a stem/progenitor cell into a differentiated
cell.
[0072] In yet another embodiment, the invention provides a method,
further comprising:
[0073] (g) preserving the isolated stem/progenitor cells for
further use.
[0074] Methods and protocols for preserving and storing of
eukaryotic cells, and in particular mammalian cells, are well known
in the art (cf., for example, Pollard, J. W. and Walker, J. M.
(1997) Basic Cell Culture Protocols, Second Edition, Humana Press,
Totowa, N.J.; Freshney, R. I. (2000) Culture of Animal Cells,
Fourth Edition, Wiley-Liss, Hoboken, N.J.). Any method maintaining
the biological activity of the isolated stem/progenitor cells such
as epithelial or mesenchymal stem/progenitor cells may be utilized
in connection with the present invention. In one preferred
embodiment, the stem/progenitor cells are maintained and stored by
using cryo-preservation.
[0075] Accordingly, the invention is also directed to a
progenitor/stem cell derived from the amniotic membrane of
umbilical cord by means of the above methods and to a cell
differentiated from the progenitor/stem cell. In addition, the
invention is also directed to a cell bank comprising or consisting
of one or more progenitor/stem cells that have been isolated as
described here. This cell bank of progenitor/stem cells may be
autologous to an individual or pooled (the latter for subsequent
allogeneic transplantation, for example), and subsequently can be
employed by further differentiation for regenerative medicine,
tissue repair and regeneration, for example.
[0076] In accordance with the above, the invention is also directed
to a pharmaceutical composition comprising a stem/progenitor cell
isolated from the amniotic membrane of umbilical cord by the above
inventive method. The pharmaceutical composition can also include a
cell differentiated from the stem/progenitor cell. The
pharmaceutical composition can be of any kind, and usually
comprises the stem/progenitor cells, a cell differentiated
therefrom or a cellular secretion or cellular extract thereof
together with a suitable therapeutically acceptable
carrier/excipient. In case of a cellular secretion, the desired
compound(s) can be used in some embodiments in the form of the
supernatant into which the compound(s) is/are secreted. In other
embodiment, the supernatant might be processed, for example, by
purification and concentration prior to be included in a
pharmaceutical composition. In some embodiments, the pharmaceutical
composition is adapted for systemic or topical application.
[0077] A pharmaceutical composition adapted for topical application
may be in liquid or viscous form. Examples thereof include an
ointment, a cream, and a lotion and the like. Examples for
pharmaceutical compositions that are suitable for systemic use are
liquid compositions, wherein the stem/progenitor cells or the
cellular extract are dissolved in a buffer that is acceptable for
injection or infusion, for example. The preparation of such
pharmaceutical compositions is within the knowledge of the person
skilled in the art and described in Gennaro, A. L. and Gennaro, A.
R. (2000) Remington: The Science and Practice of Pharmacy, 20th
Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., for
example.
[0078] Accordingly, the invention also relates to a method of
treating a subject having a disorder. This method comprises
administering to the subject an effective amount either of a
stem/progenitor cell isolated as explained herein or of a cellular
extract derived from such a cell.
[0079] In principle, any condition or disorder which is suitable
for being treated by means of stem cells/progenitor cells can be
treated with a cell or a cellular extract of present invention. It
is also possible to differentiate cells of the invention into a
desired type of cell, for example, but not limited to, a skin cell,
a bone cell, an hormone producing cell such as a beta islet insulin
producing cell, and use the differentiated cell therapeutically. In
some embodiments, the disorder is selected from the group
consisting of neoplastic disease, accelerated skin aging and skin
disorders, tissue disorders, visceral endocrine deficiencies, and
neural disorders.
[0080] The tissue disorder to be treated can be a congenital or an
acquired tissue deficiency. Examples of visceral endocrine
deficiency that can be treated with a cell of the invention
include, but are not limited to, Diabetes mellitus associated with
insulin deficiency, testosterone deficiency, anemia, hypoglycemia,
hyperglycemia, pancreatic deficiency, adrenal deficiency, and
thyroid deficiencies.
[0081] Examples of neural disorders that can be treated include,
but are not limited to, Alzheimer's disease, Parkinson's disease,
Jacob Kreutzfeld's disease, Lou Gehrig's disease, Huntington's
disease and neural neoplastic conditions.
[0082] An example of a skin disease is a wound or a damaged part of
the skin, for example, sun burned skin. Also aging of the skin is
considered to be a skin disease herein. Topical or similar delivery
of stem/progenitor cells of the invention or cellular extracts
thereof, for example, as a constituent in lotions or creams or any
other suitable vehicle may thus be used for repair of sun damaged
skin and in addition may slow also down the aging process of skin
(anti-aging properties) by replenishing, and thus fortifying,
deficient growth factors and related peptide elements, without
which skin aging would be accelerated. The stem/progenitor cells
may also migrate to injured regions of the body such as surface
wounds to form the necessary required cellular elements necessary
for the local reparative processes (cf. The Journal of Immunology,
2001, 166: 7556-7562; or International Journal of Biochemical and
Cell Biology 2004; 36: 598-606.
[0083] The neoplastic disease may be cancer, in particular as
recent studies have demonstrated that stem cells may selectively
target neoplastic tumor tissue (Journal of the National Cancer
Institute 2004; 96 (21): 1593-1603) allowing for directed delivery
of antineoplastic agents such as interferon to neoplastic foci. The
cancer can be any kind of cancer, including those cancers that are
able to form solid tumors, ranging from skin cancer to cancer of
the internal organs. Examples of cancers to be treaded include,
squamous cell carcinoma, breast ductal and lobular carcinoma,
hepatocellular carcinoma, nasopharyngeal carcinoma, lung cancer,
bone cancer, pancreatic cancer, skin cancer, cancer of the head or
neck, cutaneous or intraocular malignant melanoma, uterine cancer,
ovarian cancer, rectal cancer, cancer of the anal region, stomach
cancer, colon cancer, breast cancer, testicular cancer, uterine
cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma,
cancer of the esophagus, cancer of the small intestine, cancer of
the endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft
tissue, cancer of the urethra, cancer of the penis, prostate
cancer, chronic or acute leukemias, solid tumors of childhood,
lymphocytic lymphoma, cancer of the bladder, cancer of the kidney
or ureter, renal cell carcinoma, carcinoma of the renal pelvis,
neoplasm of the central nervous system (CNS), primary CNS lymphoma,
tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary
adenoma, Kaposi's sarcoma, epidermoid cancer or any combination of
such cancers, including disseminated (metastasising) forms thereof.
In case of treatment of a neoplastic disease the umbilical cord
amnion derived stem cells and/or their cellular extracts disclosed
herein can be administered systemically both as a direct treatment
and/or as a carrier vehicle. In the latter case of anti-neoplastic
tumor therapy, the cells comprise an anti-neoplastic agent.
[0084] In another pharmaceutical use, stem/progenitor cells of the
present invention can be used for gene therapy. For this purpose,
the cells can be transformed with a nucleic acid encoding the
protein that is to be produced in the cells. The nucleic acid can
be introduced into a cells of the invention using any of the
various methods that are well known to the skilled person, for
example, using a viral vector and/or a lipid containing
transfection composition such as as IBAfect (IBA GmbH, Gottingen,
Germany), Fugene (Roche), GenePorter (Gene Therapy Systems),
Lipofectamine (Invitrogen), Superfect (Qiagen), Metafecten
(Biontex) or those ones described in the PCT application WO
01/015755). In a related embodiment, the cells of the invention,
after being transformed with a nucleic acid encoding a polypeptide
of choice, can be used of recombinantly producing this
polypeptide.
[0085] As mentioned above, stem cell extracts are rich in a variety
of growth factors and peptides that are relevant for normal tissue
physiology. Such growth factors and/or peptides may be deficient in
exposed parts of the body, such as the skin, which is the surface
layer of all human beings protecting the body from external
elements for the maintenance of internal homeostasis. Therefore in
a further embodiment, stem/progenitor cells of the invention or
cellular extracts thereof are suitable for the treatment and/or
maintenance of internal homeostasis.
[0086] In a further embodiment and in line with the above
disclosure, the stem/progenitor cells of the invention can be used
for the production of any biological molecule. The biological
molecule can be, for instance, any molecule that is naturally
produced in the cells or a molecule the coding nucleic acid of
which has been introduced into the cells via recombinant DNA
technology. Examples of molecules that can be produced by the cells
of the invention include, but are not limited to, a protein such as
a cytokine, a growth factor such as insulin-like growth factor
(IGF), epidermal growth factor (EGF), transforming growth factor
beta (TGF-beta), Activin A, a bone morphogenetic protein (BMP),
PDGF or a hormone as insulin or erythropoietin or a transporter
protein such transferrin, a peptide such a growth factor or hormone
(e.g. luteinic hormone (LSH), follicle stimulating hormone (FSH)),
a small organic molecule such as a steroid hormone, an oligo- or
polysaccharide, for example, heparin or heparan sulfate (cf.,
example WO 96/23003, or WO 96/02259 in this regard), a
proteoglycan, a glycoprotein such as collagen or laminin, or a
lipid, to name only a few.
[0087] In a further aspect and in accordance with recent approaches
(see, for example, Amit, M et al., Human feeder layers for human
embryonic stell cells, Biol Reprod 2003; 68: 2150-2156), the
stem/progenitor cells described here can be used as feeder layer
for the cultivation of other embryonic stem cells, in particular
human embryonic stem cells. In one of these embodiments the cells
of the present invention are preferably of human origin, since
using human cells as feeder layer minimizes the risk of
contaminating the cell culture with animal-derived components such
as animal pathogens or immunogens. In this respect, it is to be
noted that the cells of the invention can be cultivated under serum
free conditions. Accordingly, employing the cells as feeder layer
and cultivating the cell culture under with serum free media as the
one described herein later, or in Draper et al. (Culture and
characterization of human embryonic stem cell lines, Stem Cells Dev
2004, 13:325-336) or in the International patent application WO
98/30679, for example.
[0088] In this connection, it is noted that in transplantation
surgery and cell-based therapy high quantities of low passage cells
with a minimal proportion of senescent cells (i.e., large
proportion of high quality cells) are crucial and are required to
be derived within the shortest possible time during cell expansion.
For example, mesenchymal stem cells from bone marrow and cord blood
are low in quantity and therefore require expansion over many
passages for a long period of time in order to achieve the
sufficient number of cells required for cell transplant. The high
passage cells however tend to deteriorate in quality and may lead
to cell senescence or cancerous transformation. It has been found
here that high quantities of cells of the present invention can be
obtained by low passage numbers using a repetitive explantation
technique. The present invention thus also relates to a method of
cultivating stem/progenitors cells of the invention, wherein this
method comprises:
[0089] Obtaining a tissue explant from the amniotic membrane of
umbilical cord;
[0090] Cultivating the tissue explant in suitable cultivation media
and cultivation conditions over a suitable period of time,
[0091] Optionally exposing the tissue explant to fresh cultivation
media and continuing the cultivation under suitable conditions over
a suitable period of time (cf., FIG. 15).
[0092] The cultivation can be carried out in for as many cycles
(passages) as wanted and be stopped once the desired number of
cells has been obtained. Exposing the tissue explant to fresh
cultivation can be carried out by removing the used cell
cultivation medium from the vessel used for growing the cells and
adding fresh media to that vessel. Instead of replacing the media
in the used vessel, exposing to fresh cultivation media can be
achieved by transferring the tissue explant to a new vessel which
is filled with cultivation media. The tissue explant used for
cultivation/propagation of the cells can be obtained by any
suitable method, for example by the "direct tissue explant
technique" as explained above (in which the tissue is first placed
in media without enzymes, and then under careful conditions the
cells separate from the main tissue mass by itself- and the cells
are then harvested for collection).
[0093] The cultivation of the tissue explants can be carried out in
any media that is suitable for cultivation of mammalian cells.
Examples include the conventional and commercially available media
that are given above with respect to the cultivation or the clonal
expansion of the cells of the invention such as, but not limited
to, KGM.RTM.-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial
Cell Medium (Cambrex) EpiLife medium (Cascade Biologics), Medium
171 (Cascade Biologics), DMEM, DMEM-F12 or RPMI media. The
cultivation is typically carried out at conditions (temperature,
atmosphere) that are normally used for cultivation of cells of the
species of which the cells are derived, for example, at 37.degree.
C. in air atmosphere with 5% CO.sub.2. In one embodiment, the
cultivation is carried out using serum free, in particular bovine
serum free media. The cultivation (in one passage) is performed for
any suitable time the cells need for growth, typically, but by no
means limited to, for about 1 to several days, for example to about
7 or about 8 days.
[0094] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including", "containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0095] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0096] Other embodiments are within the following claims and
non-limiting examples. In addition, where features or aspects of
the invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
EXAMPLES
Example 1: Collection of Umbilical Cord Tissue
[0097] Umbilical cord tissue is collected immediately after
delivery of the child. The specimen is rinsed clean and immediately
transferred into a 500 ml sterile glass bottle containing culture
transport medium (L-15 medium supplemented with 50 IU/ml
penicillin, 50 .mu.g/ml streptomycin, 250 .mu.g/ml fungizone, 50
.mu.g/ml gentamicin; all reagents purchased from Invitrogen) prior
to transport to the laboratory. In the laboratory, stem cell
extraction is conducted in a laminar flow hood under sterile
conditions. The specimen is first transferred to a sterile
stainless steel tray. All remaining blood in the cord vessels is
removed by multiple syringing washes using warm phosphate-buffered
saline (PBS) supplemented with 5 IU/ml heparin (from Sigma). Plain
PBS without heparin is used in the final washes. The umbilical cord
tissue specimen is then cut into pieces 2 cm in length and
transferred into 10 cm diameter cell culture dishes, where further
washing and disinfection is performed with 70% ethanol followed by
multiple washes using PBS containing an antibiotic mixture (50
IU/ml penicillin, 50 .mu.g/ml streptomycin, 250 .mu.g/ml fungizone,
50 .mu.g/ml gentamicin; all purchased from Invitrogen) until the
solution becomes clear.
Example 2: Cell Separation/Cultivation
[0098] Dissection of umbilical cord tissue is first performed to
separate the umbilical cord amniotic membrane from Wharton's jelly
(i.e. the matrix of umbilical cord) and other internal components.
The isolated amniotic membrane is then cut into small pieces (0.5
cm.times.0.5 cm) for cell isolation. Explant is performed by
placing the pieces of umbilical cord amniotic membrane on tissue
culture dishes at different cell culture conditions for isolation
of either epithelial or mesenchymal stem cells.
[0099] For mesenchymal cell separation/cultivation, the explants
were submerged in 5 ml DMEM (Invitrogen) supplemented with 10%
fetal bovine serum (Hyclone) (DMEM/10% FBS) and maintained in a
CO.sub.2 cell culture incubator at 37.degree. C. The medium was
changed every 2 or 3 days. Cell outgrowth was monitored under light
microscopy. Outgrowing cells were harvested by trypsinization
(0.125% trypsin/0.05% EDTA) for further expansion and
cryo-preservation using DMEM/10% FBS.
[0100] For epithelial cell separation/cultivation, cell culture
plastic surfaces were coated with collagen 1/collagen 4 mixtures
(1:2) before placing the tissue samples on the surface. The tissue
samples were submerged in 5 ml EpiLife medium or Medium 171 (both
from Cascade Biologics). The medium was changed every 2 or 3 days.
Cell outgrowth from tissue culture explants was monitored under
light microscopy. Outgrowing cells were harvested by trypsinization
(0.125% trypsin/0.05% EDTA) using EpiLife medium or Medium 171.
[0101] For the enzymatic extraction method of cells, umbilical cord
amniotic membrane was divided into small pieces of 0.5 cm.times.0.5
cm and digested in 0.1% (w/v) collagenase type1 solution (Roche
Diagnostics) at 37.degree. C. for 6 hours. The samples were
vortexed every 15 min for 2 min. Cells were harvested by
centrifugation at 4000 rpm for 30 min. Two different approaches
were employed to isolate either epithelial or mesenchymal stem
cells.
[0102] For isolation of epithelial stem cells, cell pellets were
resuspended in EpiLife medium or Medium 171 (both from Cascade
Biologics) supplemented with 50 .mu.g/ml insulin-like growth
factor-1 (IGF-1), 50 .mu.g/ml platelet-derived growth factor-BB
(PDGF-BB), 5 .mu.g/ml transforming growth factor-.beta.1
(TGF-.beta.1), and 5 .mu.g/ml insulin (all obtained from R&D
Systems), counted and seeded on 10 cm tissue culture dishes
pre-coated with collagen 1/collagen 4 mixtures (1:2; Becton
Dickinson) at density of 1.times.10.sup.6 cells/dish. After 24
hours, attached cells were washed with warm PBS and medium was
replaced with supplement-added EpiLife medium or Medium 171. The
medium was changed every 2 or 3 days. Cell growth and expanding
clonal formation was monitored under light microscopy. At a
confluence of about 70%, cells were sub-cultured by trypsinization
(0.125% trypsin/0.05% EDTA) for further expansion and
cryo-preservation.
[0103] For isolation of mesenchymal stem cells, cell pellets were
resuspended in DMEM/10% FBS, counted and seeded on 10 cm tissue
culture dishes at density of 1.times.10.sup.6 cells/dish. The
culture medium was changed every 2 or 3 days. Cell growth and
expansion was monitored under light microscopy. At a confluence of
about 90%, cells were sub-cultured as outlined above.
[0104] For cultivation of epithelial and mesenchymal stem cells on
feeder layer, umbilical cord lining membrane was digested by
collagenase treatment, counted and seeded on 10 cm tissue culture
dishes coated with lethally irradiated or Mitomycin C treated 3T3
fibroblasts (feeder layer) in Green's medium. The culture medium
was changed every 2 or 3 days. Colony formation was monitored under
light microscopy and photographed.
Example 3: Identification of Stem/Progenitor Cells
[0105] Epithelial cells: FIGS. 1A-1C show pictures of outgrowing
epithelial cells from umbilical cord amniotic membrane prepared by
the method using tissue explant (40.times. magnification). Pictures
were taken at day 2 (FIG. 1A) and day 5 (FIGS. 1B, 1C) of tissue
culture. Cell morphology analysis demonstrated polyhedral shaped
epithelial-like cells. Enzymatic digestion of the umbilical cord
segments yielded similar (FIGS. 2A-2C), epithelial cells at day 2
(FIGS. 2A, 2C) and day 5 (FIGS. 2B, 2D) (40.times. magnification).
FIG. 7 shows pictures of colony formation of epithelial stem cells
from umbilical cord amniotic membrane cultured on feeder layer
using Green's method (40.times. magnification). A colony of
polyhedral shaped epithelial-like cells expanded rapidly from day 3
to day 7.
[0106] Mesenchymal cells: Outgrowth of mesenchymal cells explanted
from umbilical cord amniotic membrane was observed as early as 48
hours after placement in tissue culture dishes using DMEM
supplemented with 10% fetal calf serum (FCS) as culture medium
(FIGS. 3A, 3C) (40.times. magnification). The cells were
characterized by their spindle shaped morphology, and migrated and
expanded both easily and quickly in vitro, closely resembling
fibroblasts (FIGS. 3B, 3D) (40.times. magnification). Similar
observations were noted in the cell group isolated by collagenase
enzymatic digestion (FIGS. 4A-4B). FIG. 4A shows mesenchymal cells
isolated from umbilical cord amniotic membrane at day 2. Cell
proliferation was observed at day 5 (FIG. 4B) (40.times.
magnification). FIGS. 6 and 8-1 show pictures of colony formation
of mesenchymal stem cells from umbilical cord amniotic membrane
cultured on non-feeder layer (FIG. 6) and feeder layer condition
(FIG. 8-1, using a 3T3 feeder layer) in DMEM/10% FCS (40.times.
magnification). The colonies of elongated shaped fibroblastic-like
cells expanded rapidly from day 3 to day 7. It is noted in this
respect, that the 3T3 feeder layer normally suppresses the growth
of mesenchymal cells as human dermal fibroblasts. Once again, this
indicates a difference in the behavior of the mesenchymal cells of
the invention as compared to more differentiated counterparts.
[0107] In further experiments the colony forming ability of the
mesenchymal cells of the invention (UCMC) was studied. For colony
forming efficiency assay, 100-200 single cells were seeded in 100
mm tissue culture dishes or T75 flasks without feeder layers. Cells
were maintained in DMEM/10% FCS for 12 days. Single colony
formation was monitored under the inverted light microscope
(experiment was carried out in duplicate, experiments termed
UCMC-16 and UCMC-17 in FIG. 8-2). Microphotographs were
sequentially taken. At day 12, colonies were fixed and stained with
Rhodamine. UCMC colony forming units were seen (FIG. 8-2). The
multiple large colonies observed, indicated self-renewal of UMCM
in-vitro (FIG. 8-2).
[0108] Western blot analysis (FIGS. 9-1 to 9-30) shows that
mesenchymal stem cells from umbilical cord amniotic membrane (UCMC)
and umbilical cord epithelial cells (UCEC) isolated in accordance
with the invention expressed the POU5f1 gene which encodes the
transcription factor Octamer-4 (Oct-4) a specific marker of
embryonic stem cells (cf. Niwa, H., Miyazaki, J., and Smith, A. G.
(2000). Nat. Genet. 24, 372-376) (FIG. 9-1). Thus, this analysis
indicates the embryonic-like properties of these stem cells. These
mesenchymal and epithelial cells also expressed Bmi-1, a marker
that is required for the self-renewal of adult stem cells (cf.,
Park et al., J. Clin. Invest. 113, 175-179 (2004) (FIG. 9-27) as
well as leukemia inhibitory factor (LIF) (FIG. 9-28) that is
considered to maintain the pluripotency of stem cells and embryonic
cells and has thus, for example been used for isolation and
expansion of human neural stem cells. These cells also highly
expressed the other growth factors such as connective tissue growth
factor (CTGF) (FIGS. 9-6, 9-7), vascular endothelial growth factor
(VEGF) (FIGS. 9-10, 9-11), placenta-like growth factor PLGF (FIGS.
9-4, 9-5), STAT3 (FIGS. 9-2, 9-3), stem cell factor (SCF) (FIG.
9-16), Hepatoma-derived Growth Factor (HDGF) (FIGS. 9-14, 9-15),
Fibroblast Growth Factor-2 (FGF-2) (FIGS. 9-12, 9-13),
Platelet-derived Growth Factor (PDGF) (FIGS. 9-8, 9-9),
alpha-Smooth Muscle Actin (.alpha.-SMA) (FIG. 9-17), Fibronectin
(FIGS. 9-18, 9-19), Decorin (FIG. 9-20), Syndecan-1,2,3,4 (FIGS.
9-21 to 9-26). In FIG. 9, the expression of these genes is compared
to human dermal fibroblasts, bone marrow mesenchymal cells (BMSC)
and adipose-derived mesenchymal cells (ADMC). FIG. 9-29 shows
Western blot data of the secretion of leukemia inhibitory factor
(LIF) by both UCEC and UCMC. FIG. 9-30 shows highly secreted
Activin A and Follistatin (both of which proteins are well known to
promote tissue repair and regeneration, enhanced angiogenesis, and
maintain embryonic stem cell culture, so that expression of the
respective genes is a sign for the embryonic properties and ability
of the cells to differentiate) detected ELISA assay (FIG. 9-30) in
supernatants of umbilical cord mesenchymal and epithelial stem cell
culture in comparison with bone marrow, adipose derived stem cells,
human dermal fibroblasts and epidermal keratinocytes. Also these
results indicate that the cells of the invention are promising
candidates in therapeutic application of these cells areas such as
regenerative medicine, aging medicine, tissue repair and tissue
engineering. In addition, FIGS. 9-29 and 9-30 show the capability
of the cells to secret an expression product into the culture
medium.
[0109] Mesenchymal cells were further characterized by analysis of
secreted cytokines and growth factors in comparison with human
bone-marrow mesenchymal stem cells. The umbilical cord epithelial
stem cells (UCEC) were analysed in comparison with human epidermal
keratinocytes. This analysis was carried out as follows: Briefly,
UCMC, UCEC, dermal fibroblasts, bone-marrow mesenchymal cells,
epidermal keratinocytes were cultured in growth media until 100%
confluence (37.degree. C., 5% CO.sub.2) and then synchronized in
starvation medium (serum-free DMEM) for 48 hours. The next day, the
medium was replaced the next against fresh serum-free DMEM and the
cells then were cultivated for another 48 hours. Conditioned media
were collected, concentrated and analyzed using a Cytokine Array
(RayBiotech, Inc, GA, USA).
[0110] The results of this analysis show that UCMC secrete
Interleukin-6 (IL-6); (MCP1); hepatocyte growth factor (HGF);
Interleukin-8 (IL8); sTNFR1; GRO; TIMP1; TIMP2; TRAILR3; uPAR;
ICAM1; IGFBP3; IGFBP6 (FIG. 11), whereas UCEC secrete IGFBP-4;
PARC; EGF; IGFBP-2; IL-6; Angiogenin; GCP-2; IL1R.alpha.; MCP-1;
RANTES; SCF; TNF.beta.; HGF; IL8; sTNFR; GRO; GRO-.alpha.;
Amphiregulin; IL-1R4/ST2; TIMP1; TIMP2; uPAR; VEGF (FIG. 12).
[0111] Accordingly, this shows that both cells types secrete large
amounts of cytokines and growth factors that play important roles
in developmental biology, tissue homeostasis, tissue repair and
regeneration and angiogenesis. This further demonstrates the
versatility of the cells of the invention for use in the respective
therapeutic applications.
[0112] In addition, the cells of the invention were further
examined with respect to their safety profile using mouse teratoma
formation assay as an indicator. Six SCID mice were used in these
experiments. A suspension of more than 2 million UCMC was injected
with a sterile 25G needle into the thigh muscle of each SCID mouse.
Animals were kept up to 6 months and tumor formation was assessed.
No tumor formation was observed in these mice (data not shown).
This indicates that the cells of the invention are safe and do not
have any capability to form tumors, benign or otherwise.
[0113] The UCMC were also analysed for their expression of human
leukocyte antigen (HLA) molecules. When testing on major
histocompatibility complex (MHC) class I molecules, this analysis
showed that HLA-A molecules were present in high number (test
result in arbitrary unit: 3201), meaning that the cells are HLA-A
positive whereas expression of HLA-B molecules was insignificant
(test result in arbitrary units: 35), meaning the cells are HLA-B
negative. As HLA-B is mainly responsible for rejection reaction in
transplantation, this result indicates that the cells of the
invention are not only suitable for autologous transplantation but
also for allogeneic transplantation. The cells were tested positive
for Class II MHC molecule HLA-DR52 and tested negative for Class II
MHC molecule HLA-DRB4. HLA-DRB1 was also found to be present
(0301/05/20/22.
Example 4: Cultivation of Stem/Progenitor Cells in Serum Free
Media
[0114] UCMC cells were cultured in DMEM containing 10 FCS and in
serum-free media, PTT-1, PTT-2 and PTT-3. The three media PTT-1,
PTT-2 and PTT-3 were prepared by one of the present inventors, Dr
Phan. In brief, these 3 media do not contain fetal bovine or human
serum, but contain different cytokines and growth factors such as
IGF, EGF, TGF-beta, Activin A, BMPs, PDGF, transferrin, and
insulin. The growth factor components vary between media to assess
differential growth characteristics. The cultivation was carried
out as follows: Different proportions of growth factors and
cytokines were added in basal media. UCMC were thawed and
maintained in these media for 10 days. Cell proliferation was
monitored under light microscopy.
[0115] FIGS. 13-1 to 13-7 show good UCMC growth in the 4 different
media groups (FIG. 13-1 to FIG. 13-5), wherein the morphology of
UCMC cells is different depending on the ratio or proportion of
cytokines or growth factors present in the respective media. In
contrast, bone marrow and adipose-derived mesenchymal cells did not
grow well in these serum-free media (FIG. 13-6 and FIG. 13-7).
Accordingly, the good growth of the UCMC demonstrates the
robustness of the cells of the invention and their high viability,
indicating that their growth characteristics are superior to
conventional sources of mesenchymal stem cells as bone marrow
derived and adipose-derived mesenchymal cells. In this respect, it
is worth to note that (bovine) serum free medium was used in these
experiments and that the majority of human mesenchymal cells do not
grow well in serum-free medium systems. Thus, using the cells of
the invention in connection with defined serum-free media
technologies is a big advantage in cell therapy as the risks of
using fetal bovine serum for cell culture and expansion are
removed. (Although use of bovine serum has been practiced for a
long time and typically optimizes cell growth, concerns of its used
have been raised as to the transmission of zoonoses as Bovine
Spongiform Encephalopathy (Mad Cow Disease)).
Example 5: Characterization of the Gene Expression Profile of
Umbilical Cord Epithelial and Mesenchymal Stem Cells
[0116] The gene expression profile of umbilical cord epithelial and
mesenchymal stem cells was analyzed using a DNA microarray. For
this purpose, UCMC and UCEC were cultured in growth media at
37.degree. C., 5% CO.sub.2 until 100% confluence. Cells were
synchronized in basal media for 48 hours then replaced with fresh
basal media for another 48 hours. Total RNA was harvested and sent
to Silicon Genetics Microarray Service. Data analysis was performed
using GeneSpring 7.2). FIG. 14 summarizes the global gene
expression. UCEC expressed a total of 28055 genes and UCMC
expressed a total of 34407 genes. There are 27308 overlapping genes
expressing in both cell types. 747 genes expressed were unique to
UCEC and 7099 genes expressed were unique to UCMC. The selected
genes of interest are presented in FIG. 14.
[0117] Both stem cell types expressed 140 genes related to
embryonic stem cells and embryonic development, further supporting
that the cells of the invention have embryonic stem cell-like
properties: Nanog; Alpha-fetal protein; Pre-B-cell leukemia
transcription factor 3; Laminin alpha 5; Carcinoembryonic
antigen-like 1; abhydrolase domain containing 2; Delta-like 3
(Drosophila); Muscleblind-like (Drosophila); GNAS complex locus;
Carcinoembryonic antigen-related cell adhesion molecule 3;
Palmitoyl-protein thioesterase 2; Pregnancy specific
beta-1-glycoprotein 2; Carcinoembryonic antigen-like 1; Embryonic
ectoderm development; Maternal embryonic leucine zipper kinase;
Chorionic somatomammotropin hormone 2; Forkhead box D3; radical
fringe homolog (Drosophila); Kinesin family member 1B; Myosin,
heavy polypeptide 3, skeletal muscle, embryonic; Split hand/foot
malformation (ectrodactyly) type 3; TEA domain family member 3;
Laminin, alpha 1; Chorionic somatomammotropin hormone 1; placental
lactogen; Corticotropin releasing hormone receptor 1; thyrotrophic
embryonic factor; Aryl-hydrocarbon receptor nuclear translocator 2;
Membrane frizzled-related protein; Neuregulin 1'Collagen, type XVI,
alpha 1; Neuregulin 1; Chorionic somatomammotropin hormone 1
(placental lactogen); CUG triplet repeat, RNA binding protein 1;
Chorionic somatomammotropin hormone 1 (placental lactogen)
Bystin-like; MyoD family inhibitor; Retinoic acid induced 2; GNAS
complex locus; Pre-B-cell leukemia transcription factor 4; Laminin,
alpha 2 (merosin, congenital muscular dystrophy); SMAD, mothers
against DPP homolog 1 (Drosophila); Homo sapiens transcribed
sequence with moderate similarity to protein pir:D28928 (H.
sapiens) D28928 pregnancy-specific beta-1 glycoprotein IB,
abortive-human (fragment); Kinesin family member 1B; Bruno-like 4,
RNA binding protein (Drosophila); Embryo brain specific protein;
Pregnancy-induced growth inhibitor; SMAD, mothers against DPP
homolog 5 (Drosophila); Chorionic somatomammotropin hormone 2;
Adenylate cyclase activating polypeptide 1 (pituitary);
Carcinoembryonic antigen-related cell adhesion molecule; Laminin,
alpha 3; Protein 0-fucosyltransferase 1; Jagged 1 (Alagille
syndrome); Twisted gastrulation homolog 1 (Drosophila); ELAV
(embryonic lethal, abnormal vision, Drosophila)-like 3 (Hu antigen
C); Thyrotrophic embryonic factor; Solute carrier family 43, member
3; Inversin; nephronophthisis 2 (infantile); inversion of embryonic
turning; Homo sapiens inversin (INVS), transcript variant 2, mRNA;
Homo sapiens transcribed sequences; Homeo box D8; Embryonal
Fyn-associated substrate; ELAV (embryonic lethal, abnormal vision,
Drosophila)-like 1 (Hu antigen R); Basic helix-loop-helix domain
containing, class B, 2; Oxytocin receptor; Teratocarcinoma-derived
growth factor 1; Fms-related tyrosine kinase 1 (vascular
endothelial growth factor/vascular permeability factor receptor);
Adrenomedullin; Nuclear receptor coactivator 6-CUG triplet repeat,
RNA binding protein 1; Twisted gastrulation homolog 1 (Drosophila);
Carcinoembryonic antigen-related cell adhesion molecule 4; Protein
tyrosine phosphatase, receptor type, R; Acrg embryonic lethality
(mouse) minimal region ortholog; EPH receptor A3; Delta-like 1
(Drosophila); Nasal embryonic LHRH factor; Transcription factor
CP2-like 1; Split hand/foot malformation (ectrodactyly) type 3;
Jagged 2; Homo sapiens transcribed sequence; Neuregulin 1; Split
hand/foot malformation (ectrodactyly) type 1; Solute carrier family
43, member 3; Hydroxyacyl-Coenzyme A
dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A
hydratase (trifunctional protein), alpha subunit;
Fucosyltransferase 10 (alpha (1,3) fucosyltransferase); Acrg
embryonic lethality (mouse) minimal region ortholog;
Carcinoembryonic antigen-related cell adhesion molecule 7;
Nucleophosmin/nucleoplasmin, 2; Fc fragment of IgG, receptor,
transporter, alpha; Twisted gastrulation homolog 1 (Drosophila);
Homo sapiens similar to vacuolar protein sorting 35;
maternal-embryonic 3 (LOC146485), mRNA; abhydrolase domain
containing 2; T, brachyury homolog (mouse); A disintegrin and
metalloproteinase domain 10; Ribosomal protein L29; Endothelin
converting enzyme 2; ELAV (embryonic lethal, abnormal vision,
Drosophila)-like 1 (Hu antigen R); Trophinin; Homeo box B6;
Laminin, alpha 4; Homeo box B6; hypothetical protein FLJ13456;
NACHT, leucine rich repeat and PYD containing 5; ELAV (embryonic
lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R);
Undifferentiated embryonic cell transcription factor 1;
Pregnancy-associated plasma protein A, pappalysin 1; Secretoglobin,
family 1A, member 1 (uteroglobin); Parathyroid hormone-like
hormone; Carcinoembryonic antigen-related cell adhesion molecule 1
(biliary glycoprotein); Laminin, alpha 1.
[0118] Both stem cell types also expressed thousands of genes
related to developmental biology, cell growth and differentiation,
cell homeostasis, cell and tissue repair and regeneration. Examples
of such growth factors and their receptors is as follows: (G-CSF,
FGFs, IGFs, KGF, NGF, VEGFs, PIGF, Angiopoietin, CTGF, PDGFs, HGF,
EGF, HDGF, TGF-beta, Activins and Inhibins, Follistatin, BMPs,
SCF/c-Kit, LIF, WNTs, SDFs, OncostatinM, Interleukins, Chemokines
and many others); MMPs, TIMPs extracellular matrices (collagens,
laminins, fibronectins, vitronectins, tenascins, intergrins,
syndecans, decorin, fibromoludin, proteoglycans, sparc/osteonectin,
mucin, netrin, glypican, cartilage associated protein, matrilin,
hyaluronan, fibulin, ADAMTS, biglycan, discoidin, desmosome
components, ICAMs, cadherins, catenins and many others);
cytokeratins.
[0119] There are groups of genes present only in UCMC. These genes
are related to the following: Normal Physiological Processes
(Insulin-like growth factor 1 (somatomedin C); Insulin-like 4
(placenta); Relaxin 1; Plasminogen; Insulin-like growth factor 1
(somatomedin C); Insulin-like 5; Insulin-like growth factor 1
(somatomedin C); Insulin-like growth factor 2 (somatomedin A),
Homeostasis (Radial spokehead-like 1; Hemochromatosis; Chemokine
(C-C motif) ligand 5; Interleukin 31 receptor A; Chemokine (C-X-C
motif) ligand 12 (stromal cell-derived factor 1); Nuclear receptor
subfamily 3, group C, member 2; Hemochromatosis; Chemokine (C-C
motif) ligand 23; Chemokine (C-C motif) ligand 23; Ferritin
mitochondrial; Peroxisome proliferative activated receptor, gamma,
coactivator 1, alpha; Surfactant, pulmonary-associated protein D;
Chemokine (C-C motif) ligand 11; Chemokine (C-C motif) ligand 3;
Egl nine homolog 2 (C. elegans); Peroxisome proliferative activated
receptor, gamma, coactivator 1, beta; Chemokine (C-C motif) ligand
1; Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor
1); ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide; Chemokine
(C motif) ligand 2; Hemopexin; Ryanodine receptor 3), Morphogenesis
(Spectrin, alpha, erythrocytic 1 (elliptocytosis 2); Homeo box D3;
Eyes absent homolog 1 (Drosophila); Ras homolog gene family, member
J; Leukocyte specific transcript 1; Ectodysplasin A2 receptor;
Glypican 3; Paired box gene 7; Corin, serine protease; Dishevelled,
dsh homolog 1 (Drosophila); Ras homolog gene family, member J;
T-box 3 (ulnar mammary syndrome); Chondroitin beta1,4
N-acetylgalactosaminyltransferase; Chondroitin beta1,4
N-acetylgalactosaminyltransferase; SRY (sex determining region
Y)-box 10; Myosin, heavy polypeptide 9, non-muscle; Luteinizing
hormone/choriogonadotropin receptor; radical fringe homolog
(Drosophila); Secreted frizzled-related protein 5; Wingless-type
MMTV integration site family, member 11; Eyes absent homolog 2
(Drosophila); Muscleblind-like (Drosophila); T-box 5; Mab-21-like 1
(C. elegans); Growth arrest-specific 2; Sex comb on midleg homolog
1 (Drosophila); T-box 6; Filamin-binding LIM protein-1; Melanoma
cell adhesion molecule; Twist homolog 1 (acrocephalosyndactyly 3;
Saethre-Chotzen syndrome) (Drosophila); Homeo box A11; Keratocan;
Fibroblast growth factor 1 (acidic); Carboxypeptidase M; CDCl42
effector protein (Rho GTPase binding) 4; LIM homeobox transcription
factor 1, beta; Engrailed homolog 1; Carboxypeptidase M; Fibroblast
growth factor 8 (androgen-induced); Fibroblast growth factor 18;
Leukocyte specific transcript 1; Endothelin 3; Paired-like
homeodomain transcription factor 1), Embryonic Development
(Pregnancy specific beta-1-glycoprotein 3; ELAV (embryonic lethal,
abnormal vision, Drosophila)-like 4 (Hu antigen D); G
protein-coupled receptor 10; Ectodysplasin A2 receptor; ATP-binding
cassette, sub-family B (MDR/TAP), member 4; Pregnancy specific
beta-1-glycoprotein 11; Nasal embryonic LHRH factor; Relaxin 1;
Notch homolog 4 (Drosophila); Pregnancy specific
beta-1-glycoprotein 6; pih-2P; Homo sapiens pregnancy-induced
hypertension syndrome-related protein (PIH2); Oviductal
glycoprotein 1, 120 kDa (mucin 9, oviductin);
Progestagen-associated endometrial protein; Myosin, light
polypeptide 4, alkali; atrial, embryonic; Prolactin; Notch homolog
4 (Drosophila); Pre-B-cell leukemia transcription factor 1; radical
fringe homolog (Drosophila); Corticotropin releasing hormone;
Nuclear receptor subfamily 3, group C, member 2; Neuregulin 2;
Muscleblind-like (Drosophila); Myosin, light polypeptide 4, alkali;
atrial, embryonic; Homo sapiens cDNA FLJ27401 fis, clone WMC03071;
Extraembryonic, spermatogenesis, homeobox 1-like; Insulin-like 4
(placenta); Human processed pseudo-pregnancy-specific glycoprotein
(PSG12) gene, exon B2C containing 3' untranslated regions of 2
alternative splice sites C1 and C2; Fms-related tyrosine kinase 1
(vascular endothelial growth factor/vascular permeability factor
receptor); Pre-B-cell leukemia transcription factor 1; Pregnancy
specific beta-1-glycoprotein 3; carcinoembryonic antigen-related
cell adhesion molecule 1 (biliary glycoprotein); Steroid sulfatase
(microsomal), arylsulfatase C, isozyme S; Homeo box B6; Protein
0-fucosyltransferase 1; LIM homeobox transcription factor 1, beta;
Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
glycoprotein); Follicle stimulating hormone, beta polypeptide;
Angiotensinogen (serine (or cysteine) proteinase inhibitor, Glade A
(alpha-1 antiproteinase, antitrypsin), member 8); Carcinoembryonic
antigen-related cell adhesion molecule 6 (non-specific cross
reacting antigen); Protein kinase C, alpha binding protein;
Collectin sub-family member 10 (C-type lectin); Laminin, alpha 1),
the Extracellular Space (Carboxylesterase 1 (monocyte/macrophage
serine esterase 1); Fibroblast growth factor 5; Progastricsin
(pepsinogen C); Sperm associated antigen 11; Proprotein convertase
subtilisin/kexin type 2; Hyaluronan binding protein 2; Sema domain,
immunoglobulin domain (Ig), short basic domain, secreted,
(semaphorin) 3F; Interleukin 2; Chymotrypsin-like; Norrie disease
(pseudoglioma); mucin 5, subtypes A and C,
tracheobronchial/gastric; Carboxypeptidase B2 (plasma,
carboxypeptidase U); radical fringe homolog (Drosophila); Pregnancy
specific beta-1-glycoprotein 11; Meprin A, alpha (PABA peptide
hydrolase); Tachykinin, precursor 1 (substance K, substance P,
neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha,
neuropeptide K, neuropeptide gamma); Fibroblast growth factor 8
(androgen-induced); Fibroblast growth factor 13; Hemopexin; Breast
cancer 2, early onset; Fibroblast growth factor 14; Retinoschisis
(X-linked, juvenile) 1; Chitinase 3-like 1 (cartilage
glycoprotein-39); Dystonin; Secretoglobin, family 1 D, member 2;
Noggin; WAP four-disulfide core domain 2; CD5 antigen-like
(scavenger receptor cysteine rich family); Scrapie responsive
protein 1; Gremlin 1 homolog, cysteine knot superfamily (Xenopus
laevis); Interleukin 16 (lymphocyte chemoattractant factor);
Chemokine (C-C motif) ligand 26; Nucleobindin 1; Fibroblast growth
factor 18; Insulin-like growth factor binding protein 1;
Surfactant, pulmonary-associated protein A1; Delta-like 1 homolog
(Drosophila); Cocaine- and amphetamine-regulated transcript; Meprin
A, beta; Interleukin 17F; Complement factor H; Cysteine-rich
secretory protein 2; Dystonin; WAP four-disulfide core domain 1;
Prolactin; Surfactant, pulmonary-associated protein B; Fibroblast
growth factor 5; Dickkopf homolog 2 (Xenopus laevis); Sperm
associated antigen 11; Chemokine (C-C motif) ligand 11; Meprin A,
alpha (PABA peptide hydrolase); Chitinase 3-like 2; C-fos induced
growth factor (vascular endothelial growth factor D); Chemokine
(C-C motif) ligand 4; Poliovirus receptor; Hyaluronoglucosaminidase
1; Oviductal glycoprotein 1, 120 kDa (mucin 9, oviductin);
Chemokine (C-X-C motif) ligand 9; Secreted frizzled-related protein
5; Amelogenin (amelogenesis imperfecta 1, X-linked); Relaxin 1;
Sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican); Chemokine (C-C motif) ligand 26; Fibroblast growth
factor 1 (acidic); Angiopoietin-like 2; Fms-related tyrosine kinase
1 (vascular endothelial growth factor/vascular permeability factor
receptor); Dystonin; Insulin-like 4 (placenta); Transcobalamin II;
macrocytic anemia; Chemokine (C-C motif) ligand 1; Insulin-like
growth factor binding protein, acid labile subunit; Complement
factor H; Pregnancy specific beta-1-glycoprotein 6; Silver homolog
(mouse); Proteoglycan 4; Fibroblast growth factor 16; Cytokine-like
protein C17; Granulysin; Angiopoietin 2; Chromogranin B
(secretogranin 1); Sema domain, immunoglobulin domain (Ig), and GPI
membrane anchor, (semaphorin) 7A; Pleiotrophin (heparin binding
growth factor 8, neurite growth-promoting factor 1); Chloride
channel, calcium activated, family member 3; Secretoglobin, family
1 D, member 1; Fibulin 1; Phospholipase A2 receptor 1, 180 kDa),
and the Extracellular Matrix (ADAMTS-like 1; Periostin, osteoblast
specific factor; Glypican 5; Leucine rich repeat neuronal 3;
Transglutaminase 2 (C polypeptide,
protein-glutamine-gamma-glutamyltransferase); A disintegrin-like
and metalloprotease (reprolysin type) with thrombospondin type 1
motif, 2; Microfibrillar-associated protein 4; Glypican 3;
Collagen, type V, alpha 3; Tissue inhibitor of metalloproteinase 2;
Keratocan; Cartilage oligomeric matrix protein; Lumican; Hyaluronan
and proteoglycan link protein 3; Statherin; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
3; Spondin 1, extracellular matrix protein; Chitinase 3-like
(cartilage glycoprotein-39); Collagen, type IV, alpha 3
(Goodpasture antigen); Wingless-type MMTV integration site family,
member 7B; Collagen, type VI, alpha 2; Lipocalin 7; Hyaluronan and
proteoglycan link protein 4; A disintegrin-like and metalloprotease
(reprolysin type) with thrombospondin type 1 motif, 5
(aggrecanase-2); Fibronectin 1; Matrilin 1, cartilage matrix
protein; Hypothetical protein FLJ13710; Chondroitin beta1,4
N-acetylgalactosaminyltransferase; Matrix metalloproteinase 16
(membrane-inserted); Von Willebrand factor; Collagen, type VI,
alpha 2; Transmembrane protease, serine 6; Matrix metalloproteinase
23B; Matrix metalloproteinase 14 (membrane-inserted); Leucine rich
repeat neuronal 3; SPARC-like (mast9, hevin); Sparc/osteonectin,
cwcv and kazal-like domains proteoglycan (testican) 3;
Dermatopontin; collagen, type XIV, alpha 1 (undulin); Amelogenin,
Y-linked; Nidogen (enactin); ADAMTS-like 2; Hyaluronan and
proteoglycan link protein 2; Collagen, type XV, alpha 1; Glypican
6; Matrix metalloproteinase 12 (macrophage elastase); Amelogenin
(amelogenesis imperfecta 1, X-linked); A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
15; Transmembrane protease, serine 6; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
16; Sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican); A disintegrin-like and metalloprotease (reprolysin
type) with thrombospondin type 1 motif, 20; Collagen, type XI,
alpha 1; Hyaluronan and proteoglycan link protein 1; Chondroitin
beta1,4 N-acetylgalactosaminyltransferase; Asporin (LRR class 1);
Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV,
autosomal dominant); Secreted phosphoprotein 1 (osteopontin, bone
sialoprotein I, early T-lymphocyte activation 1); Matrix Gla
protein; Fibulin 5; collagen, type XIV, alpha (undulin); Tissue
inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy,
pseudoinflammatory); Collagen, type XXV, alpha 1; Cartilage
oligomeric matrix protein; Collagen, type VI, alpha 1;
Chondroadherin; Collagen, type XV, alpha 1; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
16; Collagen, type IV, alpha 4; Dentin matrix acidic
phosphoprotein; Collagen, type IV, alpha 1; Thrombospondin repeat
containing 1; Matrix metalloproteinase 16 (membrane-inserted);
Collagen, type I, alpha 2; Fibulin 1; Tectorin beta;
Glycosylphosphatidylinositol specific phospholipase D1; Upregulated
in colorectal cancer gene 1). Cytoskeleton: (Filamin B, beta (actin
binding protein 278); Centrin, EF-hand protein, 1; FERM domain
containing 3; Bridging integrator 3; Parvin, gamma; Rho guanine
nucleotide exchange factor (GEF) 11; Tyrosine kinase 2; Kelch-like
4 (Drosophila); Spectrin, beta, erythrocytic (includes
spherocytosis, clinical type I); Arg/Abl-interacting protein
ArgBP2; Advillin; Spectrin repeat containing, nuclear envelope 1;
Catenin (cadherin-associated protein), delta 1; Erythrocyte
membrane protein band 4.1 like 5; Catenin (cadherin-associated
protein), alpha 2; Chemokine (C-C motif) ligand 3; Sarcoglycan,
gamma (35 kDa dystrophin-associated glycoprotein); Nebulin;
Thymosin, beta, identified in neuroblastoma cells;
3-phosphoinositide dependent protein kinase-1; Wiskott-Aldrich
syndrome protein interacting protein; Dystonin; Huntingtin
interacting protein 1; KIAA0316 gene product; Tropomodulin 4
(muscle); Deleted in liver cancer 1; Villin-like; Syntrophin, beta
1 (dystrophin-associated protein A1, 59 kDa, basic component 1);
Protein kinase, cGMP-dependent, type I; Homo sapiens similar to
keratin 8; cytokeratin 8; keratin, type II cytoskeletal 8
(LOC345751), mRNA; Adducin 1 (alpha); Protein kinase C and casein
kinase substrate in neurons 3; Dystonin; Kell blood group; Filamin
A interacting protein 1; Growth arrest-specific 2; Chromosome 1
open reading frame 1; Stathmin-like 2; Spectrin, alpha,
erythrocytic 1 (elliptocytosis 2); FKSG44 gene; Kinesin family
member 1C; Tensin; Kaptin (actin binding protein); Neurofibromin 2
(bilateral acoustic neuroma); Pleckstrin homology, Sec7 and
coiled-coil domains 2 (cytohesin-2); Actin-related protein T1;
Wiskott-Aldrich syndrome-like; Kelch-like 4 (Drosophila); Fascin
homolog 1, actin-bundling protein (Strongylocentrotus purpuratus);
Amphiphysin (Stiff-Man syndrome with breast cancer 128 kDa
autoantigen); Polycystic kidney disease 2-like 1; Ankyrin 2,
neuronal; CDCl42 binding protein kinase alpha (DMPK-like);
Hypothetical protein FLJ36144; Arg/Abl-interacting protein ArgBP2;
Formin-like 3; Catenin (cadherin-associated protein), beta 1, 88
kDa; Profilin 2; Synaptopodin 2-like; Syntrophin, gamma 2;
Phospholipase D2; Engulfment and cell motility 2 (ced-12 homolog,
C. elegans); Neurofilament, light polypeptide 68 kDa; Dystonin;
Actin-like 7B; Kinesin family member 1C; PDZ and LIM domain 3;
Adducin 2 (beta); obscurin, cytoskeletal calmodulin and
titin-interacting RhoGEF; Tubulin, beta polypeptide paralog;
Filamin A interacting protein 1; Talin 1; Homo sapiens similar to
[Segment 1 of 2] Piccolo protein (Aczonin) (LOC375597); CDCl42
effector protein (Rho GTPase binding) 4; Syndecan 1; Filamin A,
alpha (actin binding protein 280); Profilin 2; Tensin like Cl
domain containing phosphatase; Hypothetical protein MGC33407; Rho
family GTPase 1; Flavoprotein oxidoreductase MICAL2; Ca2+-dependent
secretion activator; Rabphilin 3A-like (without C2 domains); Myosin
XVA; Protein kinase, cGMP-dependent, type I; Myosin regulatory
light chain interacting protein; Kinesin family member 13B; Muscle
RAS oncogene homolog; Spectrin, beta, non-erythrocytic 1; TAO
kinase 2; Filamin B, beta (actin binding protein 278);
Neurofibromin 2 (bilateral acoustic neuroma); Catenin
(cadherin-associated protein), alpha 3; obscurin, cytoskeletal
calmodulin and titin-interacting RhoGEF; Coronin, actin binding
protein, 1A; Erythrocyte membrane protein band 4.1-like 1;
Spectrin, beta, non-erythrocytic 4; Thymosin, beta 4, Y-linked;
Tektin 2 (testicular); Ras homolog gene family, member J;
Serine/threonine kinase with Dbl- and pleckstrin homology domains;
Dystrobrevin, beta; Actin, gamma 2, smooth muscle, enteric;
Tara-like protein; Caspase 8, apoptosis-related cysteine protease;
Kelch repeat and BTB (POZ) domain containing 10; Mucin 1,
transmembrane; Microtubule-associated protein tau; Tensin; Ras
homolog gene family, member F (in filopodia); Adducin 1 (alpha);
Actinin, alpha 4; Erythrocyte membrane protein band 4.1
(elliptocytosis 1, RH-linked); Bicaudal D homolog 2 (
Drosophila); Ankyrin 3, node of Ranvier (ankyrin G); Myosin VIIA
(Usher syndrome 1B (autosomal recessive, severe)); Catenin
(cadherin-associated protein), alpha 2; Homo sapiens similar to
keratin 8, type II cytoskeletal--human (LOC285233); Fascin homolog
3, actin-bundling protein, testicular; Ras homolog gene family,
member J; Beaded filament structural protein 2, phakinin; Desmin;
Myosin X; Signal-induced proliferation-associated gene 1;
Scinderin; Coactosin-like 1 (Dictyostelium); Engulfment and cell
motility 2 (ced-12 homolog, C. elegans); Tubulin, beta 4;
Ca.sup.2+-dependent secretion activator; FERM domain containing 4A;
Actin, alpha 1, skeletal muscle; Talin 1; Caldesmon 1;
Filamin-binding LIM protein-1; Microtubule-associated protein tau;
Syntrophin, alpha 1 (dystrophin-associated protein A1, 59 kDa,
acidic component); Adducin 2 (beta); Filamin A interacting protein
1; PDZ and LIM domain 3; Erythrocyte membrane protein band 4.1 like
4B; FYN binding protein (FYB-120/130); Bridging integrator 3).
Extracellular: (A disintegrin-like and metalloprotease (reprolysin
type) with thrombospondin type 1 motif, 20; SPARC-like 1 (mast9,
hevin); Serine (or cysteine) proteinase inhibitor, Glade G (C1
inhibitor), member 1, (angioedema, hereditary); Urocortin;
Chymotrypsin-like; Platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v-sis) oncogene homolog); BMP-binding
endothelial regulator precursor protein; Complement factor H;
Chorionic somatomammotropin hormone-like 1; Chemokine (C-C motif)
ligand 18 (pulmonary and activation-regulated); Fibronectin 1;
Pregnancy specific beta-1-glycoprotein 3; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
3; CocoaCrisp; Insulin-like 4 (placenta); Wingless-type MMTV
integration site family, member 11; Cartilage oligomeric matrix
protein; Transmembrane protease, serine 6; C-fos induced growth
factor (vascular endothelial growth factor D); Family with sequence
similarity 12, member B (epididymal); Protein phosphatase 1,
regulatory subunit 9B, spinophilin; Transcobalamin II; macrocytic
anemia; Coagulation factor V (proaccelerin, labile factor);
Phospholipase A2, group IID; Tumor necrosis factor, alpha-induced
protein 6; Collagen, type XV, alpha 1; Hyaluronan and proteoglycan
link protein 3; collagen, type XIV, alpha 1 (undulin); Interleukin
19; Protease inhibitor 15; Cholinergic receptor, nicotinic, beta
polypeptide 1 (muscle); Lysyl oxidase-like 3; Insulin-like growth
factor binding protein 5; Growth hormone 1; Casein beta; NEL-like 2
(chicken); I factor (complement); Chemokine (C-C motif) ligand 23;
Interferon, alpha 2; Matrix metalloproteinase 16
(membrane-inserted); Matrix metalloproteinase 12 (macrophage
elastase); Glypican 5; Pregnancy specific beta-1-glycoprotein 3;
Fibroblast growth factor 6; Gremlin 1 homolog, cysteine knot
superfamily (Xenopus laevis); Protein S (alpha); Chondroitin
beta1,4 N-acetylgalactosaminyltransferase;
Glycosylphosphatidylinositol specific phospholipase D1; Fibroblast
growth factor 1 (acidic); Spondin 1, extracellular matrix protein;
Bone morphogenetic protein 1; Surfactant, pulmonary-associated
protein B; Dentin matrix acidic phosphoprotein; Lipoprotein, Lp(a);
Mucin 1, transmembrane; Mannan-binding lectin serine protease 1
(C4/C2 activating component of Ra-reactive factor); Meprin A, beta;
Secretoglobin, family 1D, member 1; Asporin (LRR class 1);
Chemokine (C-C motif) ligand 25; Cytokine-like protein C17;
Insulin-like 5; Meprin A, alpha (PABA peptide hydrolase); Scrapie
responsive protein 1; Fibroblast growth factor 18; Chemokine (C-X-C
motif) ligand 9; Inhibin, beta B (activin AB beta polypeptide);
Fibroblast growth factor 8 (androgen-induced); Granulysin; Cocaine-
and amphetamine-regulated transcript; Collagen, type I, alpha 2;
Chemokine (C-C motif) ligand 17; Chemokine (C-C motif) ligand 23;
Sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican) 3; Gamma-aminobutyric acid (GABA) A receptor, beta 3;
Defensin, alpha 4, corticostatin; Leucine rich repeat neuronal 3;
Glypican 6; Mitogen-activated protein kinase kinase 2; Coagulation
factor XI (plasma thromboplastin antecedent); Chemokine (C-C motif)
ligand 5; Dystonin; Frizzled-related protein; Coagulation factor
XIII, A1 polypeptide; Insulin-like growth factor 1 (somatomedin C);
Hypothetical protein MGC45438; Sperm associated antigen 11;
Insulin-like growth factor 1 (somatomedin C); Periostin, osteoblast
specific factor; Alpha-2-macroglobulin; Gamma-aminobutyric acid
(GABA) A receptor, alpha 5; Serine (or cysteine) proteinase
inhibitor, Glade A (alpha-1 antiproteinase, antitrypsin), member 3;
Silver homolog (mouse); Frizzled-related protein; Chondroadherin;
Chondroitin beta1,4 N-acetylgalactosaminyltransferase;
5-hydroxytryptamine (serotonin) receptor 3, family member C;
Collagen, type VI, alpha 2; Toll-like receptor 9; Amelogenin,
Y-linked; Vascular endothelial growth factor B; Radial
spokehead-like 1; Fms-related tyrosine kinase 1 (vascular
endothelial growth factor/vascular permeability factor receptor);
Protease inhibitor 16; Interleukin 2; Clusterin (complement lysis
inhibitor, SP-40,40, sulfated glycoprotein 2,
testosterone-repressed prostate message 2, apolipoprotein J);
Follicle stimulating hormone, beta polypeptide; A disintegrin-like
and metalloprotease (reprolysin type) with thrombospondin type 1
motif, 16; Lysozyme (renal amyloidosis); radical fringe homolog
(Drosophila); Insulin-like growth factor binding protein 5;
Taxilin; Apolipoprotein A-V; Platelet derived growth factor C;
Chemokine (C-C motif) ligand 3-like 1; Fibroblast growth factor 16;
Collagen, type VI, alpha 2; Serine (or cysteine) proteinase
inhibitor, Glade C (antithrombin), member 1; Chemokine (C-C motif)
ligand 11; Collagen, type IV, alpha 4; Bruton agammaglobulinemia
tyrosine kinase; Insulin-like growth factor 2 (somatomedin A);
Kazal-type serine protease inhibitor domain 1; Fibrinogen, A alpha
polypeptide; Chemokine (C-C motif) ligand 1; Inhibin, beta E; Sex
hormone-binding globulin; Collagen, type IV, alpha 1;
Lecithin-cholesterol acyltransferase; Cysteine-rich secretory
protein 2; Hyaluronan and proteoglycan link protein 1; Natriuretic
peptide precursor C; Ribonuclease, RNase A family, k6; Fibroblast
growth factor 14; ADAMTS-like 2; Collagen, type IV, alpha 3
(Goodpasture antigen); Angiopoietin 2; Apolipoprotein L, 3;
Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);
Hyaluronan binding protein 2; Coagulation factor VII (serum
prothrombin conversion accelerator); collagen, type XIV, alpha 1
(undulin); Oviductal glycoprotein 1, 120 kDa (mucin 9, oviductin);
Matrilin 1, cartilage matrix protein; mucin 5, subtypes A and C,
tracheobronchial/gastric; Tumor necrosis factor receptor
superfamily, member 11b (osteoprotegerin); Transglutaminase 2 (C
polypeptide, protein-glutamine-gamma-glutamyltransferase);
Keratocan; Collagen, type V, alpha 3; WAP four-disulfide core
domain 2; Chemokine (C-X3-C motif) ligand 1; Serine (or cysteine)
proteinase inhibitor, Glade D (heparin cofactor), member 1;
Secretory protein LOC348174; Coagulation factor X; Interleukin 16
(lymphocyte chemoattractant factor); Pancreatic lipase-related
protein 2; HtrA serine peptidase 3; Glycine receptor, alpha 3; CD5
antigen-like (scavenger receptor cysteine rich family);
Hypothetical protein MGC39497; Coagulation factor VIII,
procoagulant component (hemophilia A); Dermatopontin; Noggin;
Secreted LY6/PLAUR domain containing 1; ADAMTS-like 1; Alpha-1-B
glycoprotein; Chromosome 20 open reading frame 175; Wingless-type
MMTV integration site family, member 8B; Fibulin 1; Fibulin 5;
Cathepsin S; Nidogen (enactin); Chemokine (C-C motif) ligand 26;
Endothelial cell-specific molecule 1; Chitinase 3-like 1 (cartilage
glycoprotein-39); Gamma-aminobutyric acid (GABA) A receptor, beta
1; Secretoglobin, family 1 D, member 2; Mannan-binding lectin
serine protease 1 (C4/C2 activating component of Ra-reactive
factor); ADAMTS-like 1; Sema domain, immunoglobulin domain (Ig),
and GPI membrane anchor, (semaphorin) 7A; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
15; Proprotein convertase subtilisin/kexin type 2; Insulin-like
growth factor 1 (somatomedin C); Retinoschisis (X-linked, juvenile)
1; A disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 16; Chemokine (C motif) ligand 2;
Fibroblast growth factor 5; Sperm associated antigen 11;
Microfibrillar-associated protein 4; Poliovirus receptor;
Extracellular signal-regulated kinase 8; Transmembrane protease,
serine 6; Protein kinase C, alpha; Chitinase 3-like 2; Interleukin
9; Apolipoprotein L, 6; Surfactant, pulmonary-associated protein
A1; Collagen, type VI, alpha 1; Apolipoprotein L, 6; Hypothetical
protein FLJ13710; Carboxypeptidase B2 (plasma, carboxypeptidase U);
Bactericidal/permeability-increasing protein-like 2; Fibroblast
growth factor 5; Secreted phosphoprotein 1 (osteopontin, bone
sialoprotein I, early T-lymphocyte activation 1); HtrA serine
peptidase 3; Deleted in liver cancer 1; Endothelial cell-specific
molecule 1; Von Willebrand factor; A disintegrin-like and
metalloprotease (reprolysin type) with thrombospondin type 1 motif,
5 (aggrecanase-2); Sema domain, immunoglobulin domain (Ig), short
basic domain, secreted, (semaphorin) 3A; Chemokine (C-X-C motif)
ligand 12 (stromal cell-derived factor 1); Statherin; Extracellular
signal-regulated kinase 8; Tissue inhibitor of metalloproteinase 3
(Sorsby fundus dystrophy, pseudoinflammatory); Platelet factor 4
(chemokine (C-X-C motif) ligand 4); Surfactant,
pulmonary-associated protein D; Complement factor H; Delta-like 1
homolog (Drosophila); WAP four-disulfide core domain 1;
Insulin-like growth factor binding protein, acid labile subunit;
Breast cancer 2, early onset; Pre-B lymphocyte gene 1;
Corticotropin releasing hormone; Hypothetical protein DKFZp434B044;
Prolactin-induced protein; RAS guanyl releasing protein 4;
Progastricsin (pepsinogen C); Sema domain, immunoglobulin domain
(Ig), short basic domain, secreted, (semaphorin) 3F; Upregulated in
colorectal cancer gene 1; Proteoglycan 4; Cholinergic receptor,
nicotinic, delta polypeptide; Cartilage oligomeric matrix protein;
ABO blood group (transferase A, alpha
1-3-N-acetylgalactosaminyltransferase; transferase B, alpha
1-3-galactosyltransferase); Interleukin 12A (natural killer cell
stimulatory factor 1, cytotoxic lymphocyte maturation factor 1,
p35); Fibroblast growth factor 7 (keratinocyte growth factor); Kin
of IRRE like 3 (Drosophila); Cholinergic receptor, nicotinic, alpha
polypeptide 2 (neuronal); Palate, lung and nasal epithelium
carcinoma associated; Collagen, type XV, alpha 1; Pleiotrophin
(heparin binding growth factor 8, neurite growth-promoting factor
1); Angiopoietin-like 2; Norrie disease (pseudoglioma); Chemokine
(C-C motif) ligand 3; Chitinase 3-like 1 (cartilage
glycoprotein-39); Inter-alpha (globulin) inhibitor H3; Amelogenin
(amelogenesis imperfecta 1, X-linked); Epidermal growth factor
(beta-urogastrone); Fibroblast growth factor 13; Wingless-type MMTV
integration site family, member 7B; Cholinergic receptor,
nicotinic, gamma polypeptide; Pregnancy specific
beta-1-glycoprotein 6; Matrix metalloproteinase 14
(membrane-inserted); Chemokine (C-C motif) ligand 26; Interferon,
alpha 6; Tachykinin, precursor 1 (substance K, substance P,
neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha,
neuropeptide K, neuropeptide gamma); Secreted frizzled-related
protein 5; Hyaluronan and proteoglycan link protein 4; Complement
component 4B; Matrix metalloproteinase 16 (membrane-inserted);
Fibroblast growth factor 7 (keratinocyte growth factor);
Apolipoprotein C-II; Chloride channel, calcium activated, family
member 3; Tetranectin (plasminogen binding protein); Collagen, type
III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant);
KIAA0556 protein; Chemokine (C-C motif) ligand 4; Hemopexin;
Inter-alpha (globulin) inhibitor H1; Relaxin 1; Matrix Gla protein;
A disintegrin-like and metalloprotease (reprolysin type) with
thrombospondin type 1 motif, 2; Interferon (alpha, beta and omega)
receptor 2; Acid phosphatase, prostate; Guanine nucleotide binding
protein (G protein), gamma 8; Matrix metalloproteinase 23B; Meprin
A, alpha (PABA peptide hydrolase); Hyaluronoglucosaminidase 1;
Angiotensinogen (serine (or cysteine) proteinase inhibitor, Glade A
(alpha-1 antiproteinase, antitrypsin), member 8); Cartilage
intermediate layer protein, nucleotide pyrophosphohydrolase;
Purinergic receptor P2X, ligand-gated ion channel, 7; Glypican 3;
Tectorin beta; Interferon, alpha 5; Lipocalin 7; Platelet factor 4
variant 1; Nucleobindin 1; Collagen, type XI, alpha 1; Gastric
inhibitory polypeptide; Thrombospondin repeat containing 1;
5-hydroxytryptamine (serotonin) receptor 3 family member D;
Collagen, type XXV, alpha 1; Growth differentiation factor 9;
Hypothetical protein DKFZp434B044; Endothelin 3; Chemokine (C
motif) ligand 2; Prokineticin 2; Tumor necrosis factor receptor
superfamily, member 11b (osteoprotegerin); Tissue inhibitor of
metalloproteinase 2; Dystonin; Chromogranin B (secretogranin 1);
Hyaluronan and proteoglycan link protein 2; Leucine rich repeat
neuronal 3; Lumican; Matrilin 1, cartilage matrix protein;
Phospholipase A2, group IIA (platelets, synovial fluid);
Carboxylesterase 1 (monocyte/macrophage serine esterase 1);
Sparc/osteonectin, cwcv and kazal-like domains proteoglycan
(testican); Dickkopf homolog 2 (Xenopus laevis); Gamma-aminobutyric
acid (GABA) A receptor, alpha 3; Pregnancy specific
beta-1-glycoprotein 11; Insulin-like growth factor binding protein
1; Defensin, beta 106; Interleukin 17F; Ligand-gated ion channel
subunit; Phospholipase A2 receptor 1, 180 kDa; I factor
(complement); Dystonin; LAG1 longevity assurance homolog 1 (S.
cerevisiae); Prolactin; Testis expressed sequence 264; Sema domain,
immunoglobulin domain (Ig), short basic domain, secreted,
(semaphorin) 3D; secreted frizzled-related protein 2; secreted
frizzled-related protein 4).
[0120] There are groups of genes present only in UCEC. These genes
are related to the following: Homeostasis (Albumin; Calcium-sensing
receptor; Aquaporin 9; Lactotransferrin. Morphogenesis: Homeo box
HB9; Epithelial V-like antigen 1). Embryonic Development (Relaxin
2; Carcinoembryonic antigen-related cell adhesion molecule 8;
Indoleamine-pyrrole 2,3 dioxygenase; EPH receptor A3; Thyrotrophic
embryonic factor; Pregnancy specific beta-1-glycoprotein 1;
Laminin, alpha 3), the Extracellular Space (Surfactant,
pulmonary-associated protein A1; Pregnancy specific
beta-1-glycoprotein 1; Lactotransferrin; TGF-alpha; Albumin;
FGF-23; S100 calcium binding protein A9 (calgranulin B)), the
Extracellular Matrix (Laminin, beta 4; Laminin, alpha 3; Zona
pellucida glycoprotein 4. Structural Molecule Activity: Chromosome
21 open reading frame 29; Laminin, alpha 3; Microtubule-associated
protein 2; Laminin, beta 4; Keratin 6B; Ladinin 1; Keratin 6A;
Occludin; Loricrin; Erythrocyte membrane protein band 4.1
(elliptocytosis 1, RH-linked); Crystallin, beta A2; eye lens
structural protein; Contactin associated protein-like 4; Claudin
19; Hypothetical protein LOC144501; Keratin 6E; Keratin 6L; Lens
intrinsic membrane protein 2, 19 kDa), the Cytoskeleton
(Microtubule-associated protein 2; Erythrocyte membrane protein
band 4.1 like 5; Homo sapiens trichohyalin (THH); Keratin 6B;
Keratin 6A; Epithelial V-like antigen 1; Hook homolog 1
(Drosophila); Loricrin; Erythrocyte membrane protein band 4.1
(elliptocytosis 1, RH-linked); Tropomodulin 1; MAP/microtubule
affinity-regulating kinase 1; Keratin 6E; Actin binding LIM protein
family, member 2), Cell Adhesion Molecules (Cadherin 19, type 2;
Myeloid/lymphoid or mixed-lineage leukemia; Chromosome 21 open
reading frame 29; Kin of IRRE like 2; Laminin, alpha 3;
Sialoadhesin; CD84 antigen (leukocyte antigen); Lectin,
galactoside-binding, soluble, 2 (galectin 2); Epithelial V-like
antigen 1; CD96 antigen; Tubulointerstitial nephritis antigen;
Carcinoembryonic antigen-related cell adhesion molecule 8; IL-18;
Immunoglobulin superfamily, member 1; Integrin, beta 8; Ornithine
arbamoyltransferase; Integrin, beta 6; Contactin associated
protein-like 4; Collagen, type XVII, alpha 1; Cadherin-like 26;
Mucin and cadherin-like), Cell Differentiation proteins (Protein
tyrosine phosphatase, receptor-type, Z polypeptide 1; Laminin,
alpha 3; CD84 antigen (leukocyte antigen); EDRF2; Homo sapiens
erythroid differentiation-related factor 2; Tumor protein p73-like;
NB4 apoptosis/differentiation related protein; Homo sapiens
PNAS-133; Similar to seven in absentia 2; Interleukin 24; Keratin
6B; Keratin 6A; Dehydrogenase/reductase (SDR family) member 9; Gap
junction protein, beta 5 (connexin 31.1); Iroquois homeobox protein
4; Ventral anterior homeobox 2; Chemokine (C-X-C motif) ligand 10;
Tumor necrosis factor receptor superfamily, member 17; Calcium
channel, voltage-dependent, beta 2 subunit; Parkinson disease
(autosomal recessive, juvenile) 2, parkin; Kallikrein 7
(chymotryptic, stratum corneum); Glial cells missing homolog 2;
AP-2 alpha; Protein tyrosine phosphatase, receptor-type, Z
polypeptide 1; Troponin T1; Sciellin; Glucosaminyl (N-acetyl)
transferase 2, I-branching enzyme; Collagen, type XVII, alpha 1;
Suppressor of cytokine signaling 2; Distal-less homeo box 1; Zygote
arrest 1; Interleukin 20; Growth differentiation factor 3; FGF-23;
Wingless-type MMTV integration site family, member 8A.
Extracellular: Chromosome 21 open reading frame 29; Laminin, alpha
3; Laminin, beta 4; Interleukin 24; Pregnancy specific
beta-1-glycoprotein 1; Chemokine (C-X-C motif) ligand 11;
Surfactant, pulmonary-associated protein A1; Prepronociceptin;
5-hydroxytryptamine (serotonin) receptor 3B; Carcinoembryonic
antigen-related cell adhesion molecule 8; Chemokine (C-X-C motif)
ligand 10; IL-18 (interferon-gamma-inducing factor);
Lactotransferrin; Albumin; Fas ligand (TNF superfamily, member 6);
Cholinergic receptor, nicotinic, beta polypeptide 4; Cathelicidin
antimicrobial peptide; Airway trypsin-like protease; S100 calcium
binding protein A9 (calgranulin B); TGF-alpha; Kallikrein 10;
Serine protease inhibitor, Kunitz type 1; WNT1 inducible signaling
pathway protein 3; Relaxin 2; Interferon, kappa; Defensin, beta
103A; IL-20; Zona pellucida glycoprotein 4; Growth differentiation
factor 3; FGF-23; Wingless-type MMTV integration site family,
member 8A; Complement factor H-related 5), Developmental proteins
(EPH receptor A3; NIMA (never in mitosis gene a)-related kinase 2;
Zinc finger protein 282; TANK-binding kinase 1; MRE11 meiotic
recombination 11 homolog A; E2F transcription factor 2; Protein
tyrosine phosphatase, receptor-type, Z polypeptide 1; Homo sapiens
clone 161455 breast expressed mRNA from chromosome X; Laminin,
alpha 3; v-myb myeloblastosis viral oncogene homolog (avian)-like
1; Regulator of G-protein signalling 11; Microtubule-associated
protein 2; Transmembrane protein 16A; Adenomatosis polyposis coli
2; Homeo box HB9; Centromere protein F, 350/400 ka (mitosin); CD84
antigen (leukocyte antigen); EDRF2; Homo sapiens erythroid
differentiation-related factor 2; Tumor protein p73-like; NB4
apoptosis/differentiation related protein; Homo sapiens PNAS-133;
Forkhead box P2; Homo sapiens gastric-associated
differentially-expressed protein YA61P (YA61); Tenascin N;
Chromosome 6 open reading frame 49; Zinc finger protein 462; Zinc
finger protein 71 (Cos26); SRY (sex determining region Y)-box 7;
Triggering receptor expressed on myeloid cells-like 4; Interleukin
24; Pregnancy specific beta-1-glycoprotein 1; Chondroitin sulfate
proteoglycan 5 (neuroglycan C); Keratin 6B; Keratin 6A;
Dehydrogenase/reductase (SDR family) member 9; Epithelial V-like
antigen 1; Gap junction protein, beta 5 (connexin 31.1); G
protein-coupled receptor 51; Interferon regulatory factor 6;
Neurotrophin 5 (neurotrophin 4/5); CD96 antigen; Iroquois homeobox
protein 4; Interleukin 1 receptor-like 1; G-2 and S-phase expressed
1; Nuclear receptor subfamily 2, group E, member 3; Ventral
anterior homeobox 2; Zinc finger protein 215; DNA segment on
chromosome 4 (unique) 234 expressed sequence; Carcinoembryonic
antigen-related cell adhesion molecule 8; Chemokine (C-X-C motif)
ligand 10; IL-18; Indoleamine-pyrrole 2,3 dioxygenase; Albumin;
Calcium-sensing receptor (hypocalciuric hypercalcemia 1, severe
neonatal hyperparathyroidism); Fas ligand (TNF superfamily, member
6); TNFR superfamily, member 17; Calcium channel,
voltage-dependent, beta 2 subunit; Parkinson disease (autosomal
recessive, juvenile) 2, parkin; Kallikrein 7 (chymotryptic, stratum
corneum); Glial cells missing homolog 2; TGF-alpha; Thyrotrophic
embryonic factor; AP-2 alpha (activating enhancer binding protein 2
alpha); Kallikrein 10; Regulator of G-protein signalling 7; Protein
tyrosine phosphatase, receptor-type, Z polypeptide 1; Serine
protease inhibitor, Kunitz type 1; WNT1 inducible signaling pathway
protein 3; Zic family member 3 heterotaxy 1 (odd-paired homolog,
Drosophila); TTK protein kinase; Troponin T1, skeletal, slow;
Sciellin; TGFB-induced factor 2-like, X-linked; Kallikrein 8
(neuropsin/ovasin); Glucosaminyl (N-acetyl) transferase 2,
I-branching enzyme; Ankyrin repeat domain 30A; Relaxin 2; Collagen,
type XVII, alpha 1; Gene differentially expressed in prostate;
Phosphatase and actin regulator 3; Suppressor of cytokine signaling
2; Nuclear receptor subfamily 4, group A, member 3; Angiotensin I
converting enzyme (peptidyl-dipeptidase A) 1; Hypothetical protein
MGC17986; Distal-less homeo box 1; LAG1 longevity assurance homolog
3 (S. cerevisiae); Zygote arrest 1; Interferon, kappa; IL-20;
ICEBERG caspase-1 inhibitor; Growth differentiation factor 3;
FGF-23; Testis expressed sequence 15; Wingless-type MMTV
integration site family, member 8A; SRY (sex determining region
Y)-box 7; Carnitine deficiency-associated, expressed in ventricle
1; Prokineticin 1; CAMP responsive element binding protein 3-like
3; Caspase recruitment domain family, member 15; FLJ23311
protein).
Example 6: Direct Differentiation of Umbilical Cord Epithelial Stem
Cells (UCEC) into Skin Epidermal Keratinocytes
[0121] For differentiation into skin epidermal keratinocytes,
umbilical cord epithelial stem cells, UCEC cells, were cultured
according to a standard protocol for the cultivation of
keratinocytes. Cell isolation techniques were as described above.
UCEC were then cultured in serum-free keratinocyte growth media,
KGM, KGM-2 (Cambrex), EpiLife (Cascade Biologics) or in Green's
medium in the presence of irradiated or Mytomycin-C treated 3T3
mouse embryonic feeder layer at 37.degree. C., 5% CO.sub.2). UCEC
cell morphology thus differentiated resembled human epidermal
keratinocytes. Epithelial cells have similar morphology under light
microscope and can be easily turned into fibroblasts using
conventional and commercially available media (cf., FIGS.
2A-2D).
[0122] Immunofluorescent analysis shows that the cultivated UCEC
also express epidermal keratinocyte molecular markers such as
keratins, desmosome, hemidesmosome and basement membrane components
(see also FIGS. 10-1 to 10-4 that show that UCEC are qualified to
be epithelial cells in general by expressing a variety of these
epithelial cell markers). Accordingly, these results show that
umbilical cord epithelial progenitor/stem cells of the present
invention can be differentiated into skin cells such as epidermal
keratinocytes which can be used for wound healing and have great
potential for the development of cultured skin equivalents.
Example 7: Expansion of Umbilical Cord Epithelial and Mesenchymal
Stem Cells Using Repetitive Tissue Explants of Umbilical Cord
Lining Membrane Tissues
[0123] Umbilical cord epithelial and mesenchymal stem cells of the
invention were expanded using repetitive explants of umbilical cord
amniotic membrane tissue as follows. Briefly, at day 1 of process,
tissue explants were plated onto tissue culture dishes in growth
media (DMEM/10% FCS, EpiLife, KGM, KGM-2 or M171) at 37.degree. C.,
5% CO.sub.2; media was changed every 2 or 3 days. Cell outgrowths
started and continued migrating from the explants for 7 days. After
that, tissue explants were transferred to other dishes to allow
further cell outgrowth. This process was continued until the
explants had diminished in size, preventing further explantation.
In this connection it is noted that the explants progressively
shrink in size until they are too small for further tissue explant
since during the process of cells outgrowing and migrating from
tissue explants, the cells produce proteases to digest and break
down tissue. FIG. 16 schematically illustrates the rapid and robust
expansion process of umbilical cord epithelial and mesenchymal stem
cells achieved using this protocol. Thus, this study demonstrates
the high yield of UCMC and UMEC cells can be obtained from this
source, further reflecting the high viability and pro-growth
characteristics oft these cells in comparison with other sources of
cells as bone-marrow or adipose-derived stem cells. In addition,
being a solid tissue, the successful repetitive explant technique
used herein demonstrates that the cells of the invention can be
uniformly extracted from the entire tissue instead of only certain
portions. This allows the maximum number of cells that can be
derived at a low passage instead of passing the cells through many
generations causing deterioration of cells.
Example 8: Direct Differentiation of Umbilical Cord Mesenchymal
Cells (UCMC) into Skin Dermal Fibroblasts
[0124] For differentiation into skin dermal fibroblasts, umbilical
cord mesenchymal stem cells, UCMC cells were cultured according to
a standard protocol for the cultivation of fibroblasts. Cell
isolation techniques were as described above in Example 6. UCMC
were then cultured in DMEM or commercially available fibroblast
growth media (FGM). UCMC cell morphology thus differentiated
resembled human dermal fibroblasts. Mesenchymal cells have similar
morphology under light microscope and can be easily turned into
fibroblasts using conventional and commercially available media
(cf., FIGS. 3A-3D).
Example 9: Direct Differentiation of Epithelial Stem/Progenitor
Cells into Skin Epidermal Keratinocytes
[0125] In an approach similar to Example 6, epithelial
stem/progenitor cells of the amniotic membrane of the umbilical
cord (UCEC) were isolated as described in Example 2. For
differentiation of UCEC into epidermal keratinocytes, the cells
were cultured in keratinocyte media (EpiLife or KGM) until 100%
(cultivation after 5 days shown in FIG. 17-A) confluent before
changing the media to DMEM/10% FCS for 3 days to form epidermal
cell sheets. As shown in FIG. 17-A (in which photographs of two
experiments termed "UCEC-10" and UCEC-17 are depicted), after
cultivation in DMEM/10% FCS, UCEC, had differentiated into
epidermal keratinocytes that formed cell sheets (photograph of FIG.
17-A taken after 10 days). These results thus provide further
evidence for the pluripotency of the cells of the present
invention.
Example 10: Direct Differentiation of Mesenchymal Stem/Progenitor
Cells into Osteoblasts
[0126] Mesenchymal stem/progenitor cells of the amniotic membrane
of the umbilical cord (UCMC) were isolated as described in Example
2. For differentiation of UCMC into osteoblasts, cells were
cultured in DMEM/10% FCS until 100% confluent, and then in
starvation medium of serum-free DMEM for another 48 hours. UCMC
were subjected to osteogenic induction media for 4 weeks before
subjecting the cells to von Kossa staining (bone cell staining).
The osteogenic induction medium contained DMEM/10% FCS; 1%
antibiotic (streptomycin and penicillin)/antimycotic (fungizone);
0.01 .mu.M 1,25-dihydroxyvitamin D3, 50 .mu.M
ascorbate-2-phosphate, 10 mM .beta.-glycerophosphate, 1% antibiotic
(streptomycin and penicillin)/antimycotic (fungizone).
[0127] As shown in FIG. 17B, von Kossa staining of UCMC cells that
were cultivated in the osteogenic induction medium indicated bone
nodule formation in the UCMC and thus differentiation of the UCMC
into osteoblasts whereas no such differentiation was indicated in
untreated UCMC which were cultured in DMEM/10% FCS without
induction under otherwise same conditions as negative control. As a
further negative control, dermal fibroblasts from an 8 months old
donor and keloid fibroblasts from an 20 year old donor were
cultivated under the same conditions as the induced or un-induced
UCMC. Both cell types did not yield a positive result using von
Kossa staining, which is a further evidence for the pluripotency of
UCMC of the present invention and thus to differentiate, for
example, also into osteoblasts.
Example 11: Direct Differentiation of Mesenchymal Stem/Progenitor
Cells into Adipocytes
[0128] Mesenchymal stem/progenitor cells from the amniotic membrane
of the umbilical cord (UCMC) were isolated as described in Example
2. For differentiation of UCMC into adipocytes, cells were cultured
in DMEM/10% FCS until 100% confluent, and then in starvation medium
of serum-free DMEM for another 48 hours. UCMC were subjected to
adipogenic induction media for 4 weeks before subjecting the cells
to Oil-Red-O staining. The adipogenic induction medium contained
DMEM/10% FCS; 1% antibiotic (streptomycin and
penicillin)/antimycotic (fungizone)); 0.5 mM
isobutyl-methylxanthine (IBMX), 1 .mu.M dexamethasone, 10 .mu.M
insulin, and 200 .mu.M indomethacin.
[0129] Oil-Red-O staining of UCMC cells that were cultivated in the
adipogenic induction medium indicated fat accumulation in the UCMC
and thus differentiation of the UCMC into adipocytes whereas no
such differentiation was indicated in untreated UCMC which were
cultured in DMEM/10% FCS without induction under otherwise same
conditions as negative control. As a further negative control,
dermal fibroblasts from an 8 month old donor and keloid fibroblasts
from a 20 year old donor were cultivated under the same conditions
as the induced or un-induced UCMC. Both cell types did not yield a
positive result in the staining with Oil-Red-O, which is a further
evidence for the pluripotency of UCMC of the present invention and
to differentiate, for example, also into adipocytes.
* * * * *