United States Patent: (10) Patent No.: US 9,085,755 B2

US009085755B2
(12) United States Patent (10) Patent No.: US 9,085,755 B2

Phan et al. (45) Date of Patent: *Jul. 21, 2015
(54) ISOLATION, CULTIVATION AND USES OF 5,861,315 A 1/1999 Nakahata
STEMAPROGENITOR CELLS 5,919,702 A 7/1999 Purchio et al.
5,919,808 A 7/1999 Petrie et al.
5,962,325 A 10/1999 Naughton et al.
(71) Applicant: CELLRESEARCH CORPORATION 6,152,142 A 1 1/2000 Tseng
PTE LTD 6,231,879 B1 5, 2001 Li et al.
6,326,019 B1 12/2001 Tseng
(72) Inventors: Toan-Thang Phan, Singapore (SG); 6,497,875 B1 12/2002 Sorrell et al.
Ivor Jiun Lim, Singapore (SG) 2003/0044977 A1 3/2003 Sakuragawa et al.
2003/0096400 A1 5/2003 Kinstler
2004/0048372 A1 3/2004 Hariri
(73) Assignee: CellResearch Corporation Pte Ltd., 2004/O136967 A1 7/2004 Weiss et al.
Singapore (SG) 2004/O247573 A1 12/2004 Kim et al.
2005.0054098 A1 3/2005 Mistry et al.
(*) Notice: Subject to any disclaimer, the term of this 2005, 0118714 A1 6/2005 Ha et al.
patent is extended or adjusted under 35 2005, 0124003 A1 6/2005 Atala et al.
U.S.C. 154(b) by 206 days. FOREIGN PATENT DOCUMENTS
This patent is Subject to a terminal dis
claimer. CN 1127638 A T 1996
EP 1288293 A1 3, 2003
JP 2003154000 A 5, 2003
(21) Appl. No.: 13/781,135 JP 2003231639 A 8, 2003
JP 2004121419 A 4/2004
(22) Filed: Feb. 28, 2013
(Continued)
(65) Prior Publication Data
OTHER PUBLICATIONS
US 2013/0337563 A1 Dec. 19, 2013
Abe et al., Peripheral blood fibrocytes: differentiation pathway and
Related U.S. Application Data migration to wound sites. J Immunol. Jun. 15, 2001:166(12):7556
7562.
(62) Division of application No. 11/205.248, filed on Aug. Amitet al., Human Feeder Layers for Human Embryonic StemCells.
15, 2005. Biol Reprod. Jun. 2003:68(6):2150-2156.
(60) Provisional application No. 60/602,208, filed on Aug. Anderson et al., Amniotic membrane transplantation for partial
16, 2004, provisional application No. 60/632,209, limbal stem cell deficiency. BrJ Ophthalmol. May 2001:85(5):567
filed on Dec. 1, 2004. 575.
Bailo et al., Engraftment Potential of Human Amnion and Chorion
(30) Foreign Application Priority Data Cells Derived from Term Placenta. Transplantation Nov. 27.
2004;78(10): 1439-1448.
Jun. 3, 2005 (WO)................ PCTFSG2005/000174 (Continued)
(51) Int. Cl.
CI2N 5/073 (2010.01)
CI2N 5/0735 (2010.01) Primary Examiner — Taeyoon Kim
CI2N 5/071 (2010.01) (74) Attorney, Agent, or Firm — Michael A. Whittaker;
CI2N 5/0775 (2010.01) Acuity Law Group PC
(52) U.S. Cl.
CPC .............. CI2N5/0606 (2013.01); CI2N5/063 (57) ABSTRACT
(2013.01); C12N5/0605 (2013.01); C12N
5/0629 (2013.01); C12N5/0668 (2013.01); The present invention relates to a method for isolating stem/
CI2N2506/025 (2013.01); C12N 2506/1392 progenitor cells from the amniotic membrane of umbilical
(2013.01) cord, wherein the method comprises separating the amniotic
(58) Field of Classification Search membrane from the other components of the umbilical cord
CPC. A61K 35/51; C12N5/0668; C12N 5/0662; in vitro, culturing the amniotic membrane tissue under con
C12N 2506/025; C12N 2506/1392: C12N ditions allowing cell proliferation, and isolating the stem/
5/0606; C12N5/0605; C12N 5/0629; C12N progenitor cells from the tissue cultures. The isolated stem
5/063 cell cells can have embryonic stem cell-like properties and
USPC .................. 435/325,377, 371; 424/93.7, 583 can be used for various therapeutic purposes. In one embodi
See application file for complete search history. ment, the invention relates to the isolation and cultivation of
stem cells Such as epithelial and/or mesenchymal stem/pro
(56) References Cited genitor cells under conditions allowing the cells to undergo
U.S. PATENT DOCUMENTS mitotic expansion. Furthermore, the invention is directed to a
method for the differentiation of the isolated stem/progenitor
5,612,028 A 3, 1997 Sackier et al. cells into epithelial and/or mesenchymal cells.
5,665,557 A 9/1997 Murray et al.
5,744,347 A 4/1998 Wagner et al.
5,858,390 A 1/1999 Boss, Jr. 12 Claims, 67 Drawing Sheets
US 9,085,755 B2
Page 2
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WO 2005.001081 A1 1, 2005 Medicine. Shinsyu Medical Journal, Feb. 2004:52(1):7-14.
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WO 2006O19357 A1 2, 2006 Medicine. Shinsyu Medical Journal, Feb. 2004:52(1):7-14 (Partial
WO 2007046775 A1 4/2007 English translation).
OTHER PUBLICATIONS Niwa et al., Quantitative expression of Oct-3/4 defines differentia
Bieback et al., Critical Parameters for the Isolation of Mesenchymal tion, dedifferentiation or self-renewal of ES cells. Nat Genet. Apr.
StemCells from Umbilical Cord Blood. StemCells 2004:22(4):625 2000:24(4):372-376.
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tion in adults. Proc Nat Acad Sci USA. May 1, 1992; 89(9):4109 Quan et al., Circulating fibrocytes: collagen-secreting cells of the
4113. peripheral blood. IntJ Biochem Cell Biol. Apr. 2004:36(4):598-606.
Campagnoli et al., Identification of mesenchymal stem/progenitor Romanov et al., Searching for Alternative Sources of Postnatal
cells in human first-trimester fetal blood, liver, and bone marrow. Human Mesenchymal Stem Cells: Candidate MSC-like Cells from
Blood Oct. 15, 2001:98(8):2396-2402. Umbilical Cord. Stem Cells 2003:21(1):105-110.
Chen et al., Chondrogenic differentiation of human mesenchymal Sakuragawa et al., Human Amnion Mesenchyme Cells Express Phe
stem cells cultured in a cobweb-like biodegradable scaffold. notypes of Neuroglial Progenitor Cells. J Neurosci Res. Oct. 15,
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Office in 10-2007-7005989 on Jan. 14, 2013. Cell Dev Biol. 2001:17:435-462.
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Office in 10-2007-700.5989 on Jan. 14, 2013 (English translation). Tumor Stroma and Targeted-Delivery Vehicles for Anticancer
Office Action issued May 22, 2012, in JP 2007-527147. Agents. J Natl Cancer Inst. Nov. 3, 2004:96(21): 1593-1603.
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translation). stem cell line derived from human amniotic membranes and initiation
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dated Feb. 29, 2012. only).
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stem cells. Braz J Med Biol Res. Sep. 2003:36(9): 1179-1183. from second-trimester amniotic fluid using a novel two-stage culture
Erices et al., Mesenchymal progenitor cells in human umbilical cord protocol. Hum Reprod. Jun. 2004; 19(6): 1450-1456.
blood. BrJ Haematol. Apr. 2000; 109(1):235-242. Zangrossi et al., Oct-4 Expression in Adult Human Differentiated
Gershengornet al., Epithelial-to-Mesenchymal Transition Generates Cells Challenges. Its Role as a Pure StemCell Marker. StemCells Jul.
Proliferative Human Islet Precursor Cells. Science Dec. 2007:25(7): 1675-1680.
2004:306(5705)2261-2264. Zhao et al., Human Amniotic Mesenchymal Cells Have Some Char
Grueterich et al., ExVivo Expansion of Limbal Epithelial StemCells: acteristics of Cardiomyocytes. Transplantation Mar. 15,
Amniotic Membrane Serving as a Stem Cell Niche. Surv 2005:79(5):528-535 (abstract only).
Ophthalmol. Nov.-Dec. 2003:48(6):631-646. Office Action and Search Report issued by the Austrian Patent Office
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Maternal Origin from Human Placenta. StemCells 2004:22(7): 1338 surface reconstruction. Graefe's Arch Clin Exp Ophthalmol Jan.
1345. 2000:238(1):68-75.
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US 9,085,755 B2
1. 2
ISOLATION, CULTIVATION AND USES OF cells—in general referred to as mesenchymal stem cells—
STEMAPROGENITOR CELLS promising candidates for mesodermal defect repair and dis
ease management.
CROSS-REFERENCE TO RELATED In clinical use, however, harvesting of Such mesenchymal
APPLICATIONS stem cells causes several problems. The collection of the cells
is a mental and physical burden to the patient as a Surgical
This application is a divisional of application Ser. No. procedure is required to obtain the cells (for example, the
11/205.248 filed Aug. 15, 2005, which claims the benefit of collection of bone marrow is an invasive technique performed
priority of U.S. Provisional Application No. 60/602,208, filed with a biopsy needle that requires local or even general anes
Aug. 16, 2004, U.S. Provisional Application No. 60/632,209, 10 thesia). Furthermore, in many cases the number of stem cells
extracted is rather low. More importantly, no epithelial cells
filed Dec. 1, 2004 and International Application No. PCT/ are derived or differentiated from these cells. This prompted
SG2005/000174, filed Jun. 3, 2005, the contents of each the search for other possible sources of stem cells.
being hereby incorporated by reference it its entirety for all Umbilical cord blood has been identified as a rich source of
purposes.
15
haematopoetic stem/progenitor cells. However, the existence
of mesenchymal stem/progenitor cells is discussed contro
FIELD OF THE INVENTION versially. On the one hand, such cells could not be isolated or
successfully cultured from term umbilical cord blood (Mare
The present invention relates to a method for isolating schi, K. et al. (2001) Haematologica 86, 1099-1100). At the
stem/progenitor cells from the amniotic membrane of umbili same time, results obtained by Campagnoli, C. et al. (Blood
cal cord, wherein the method comprises separating the amni (2001) 98, 2396-2402) as well as Erices, A. et al. (Br. J.
otic membrane from the other components of the umbilical Haematol. (2000) 109, 235-242) suggest that mesenchymal
cord in vitro, culturing the amniotic membrane tissue under stem cells are present in several fetal organs and circulate in
conditions allowing cell proliferation, and isolating the stem/ the blood of pre-term fetuses simultaneously with hemato
progenitor cells from the tissue cultures. In particular, the poietic precursors. Accordingly, International Patent Appli
invention relates to the isolation and cultivation of stem cells 25 cation WO 03/070922 discloses isolation and culture-expan
having embryonic properties such as epithelial and/or mes sion methods of mesenchymal stem/progenitor cells from
umbilical cord blood and a differentiation method of such
enchymal stem/progenitor cells under conditions allowing cells into various mesenchymal tissues. Isolation efficiencies
the cells to undergo mitotic expansion. Furthermore, the of about 60% have been reported (Bieback, K. et al. (2004)
invention is directed to a method for the differentiation of the
isolated stem/progenitor cells into epithelial and/or mesen 30 Stem Cells 22, 625-634). In the same study, both the time
chymal cells and therapeutic uses of these stem/progenitor period from collection of the umbilical cord blood to isolation
cells.
of the cells and the volume of the blood sample used have
been determined as crucial parameters for achieving Such a
BACKGROUND OF THE INVENTION
yield. However, it is still a matter of debate whether these
35
stem/progenitor cells are indeed derived of umbilical cord
tissue.
Stem cells area cell population possessing the capacities to Recently, mesenchymal stem/progenitor cells have been
self-renew indefinitely and to differentiate in multiple cell or Successfully isolated from umbilical cord tissue, namely from
tissue types. Embryonic stem cells (from approximately days Wharton's jelly, the matrix ofumbilical cord, (Mitchell, K. E.
3 to 5 after fertilisation) proliferate indefinitely and can dif et al. (2003) Stem Cells 21, 50-60; U.S. Pat. No. 5,919,702:
ferentiate spontaneously into all tissue types: they are thus 40 US Patent Application 2004/0136967). These cells have been
termed pluripotent stem cells (reviewed, for example, in shown to have the capacity to differentiate, for example, into
Smith, A. G. (2001) Annu. Rev. Cell. Dev. Biol. 17, 435-462). a neuronal phenotype and into cartilage tissue, respectively.
Adult stem cells, however, are more tissue-specific and may Furthermore, mesenchymal stem/progenitor cells have also
have less replicative capacity: they are thus termed multipo been isolated from the endothelium and the subendothelial
tent stem cells (reviewed, for example, in Paul, G. etal. (2002) 45 layer of the umbilical cord vein, one of the three vessels (two
Drug Discov. Today 7, 295-302). The “plasticity” of embry arteries, one vein) found within the umbilical cord (Romanov,
onic and adult stem cells relies on their ability to trans-differ Y.A. et al. (2003) Stem Cells 21, 105-110; Covas, D.T. et al.
entiate into tissues different from their origin and, perhaps, (2003) Braz. J. Med. Biol. Res. 36, 1179-1183).
across embryonic germ layers. However, none of these approaches employed thus far has
The ability of stem cells to self-renew is critical to their 50 resulted in the isolation or cultivation of epithelial stem/pro
function as reservoir of primitive undifferentiated cells. In genitor cells as a source for epithelial cell-based therapies
contrast, most Somatic cells have a limited capacity for self Such as skin resurfacing, liver repair, bladder tissue engineer
renewal due to telomere shortening (reviewed, for example, ing and other engineered Surface tissues. Thus, there is still a
in Dice, J. F. (1993) Physiol. Rev. 73, 149-159). Stem cell need for methods and reliable sources useful for the isolation
based therapies thus have the potential to be useful for the 55 and cultivation of epithelial stem/progenitor cells. Further
treatment of a multitude of human and animal diseases. more, rapid and efficient methods which are ethically accept
Stem cells as well as stem/progenitor cells can be derived able and do not pose a biomedical burden on the patient for the
from different sources. The “multi-lineage' potential of isolation of epithelial and mesenchymal stem/progenitor cells
embryonic and adult stem cells has been extensively charac are still required in order to provide such cells in a sufficient
terized. Even though the potential of embryonic stem cells is 60 amount for various applications in regenerative medicine and
enormous, their use implies many ethical problems. There tissue engineering.
fore, non-embryonic stem cells derived from the bone mar
row stroma, fat tissue, dermis and umbilical cord blood have SUMMARY OF THE INVENTION
been proposed as alternative sources. These cells can differ
entiate inter alia into chondrocytes, adipocytes, osteoblasts, 65 The invention provides a method for isolating stem/pro
myoblasts, cardiomyocytes, astrocytes, and tenocytes in vitro genitor cells from the amniotic membrane of umbilical cord,
and undergo differentiation in vivo, making these stem the method comprising:
US 9,085,755 B2
3 4
(a) separating the amniotic membrane from the other com outgrowth by explant was monitored under light microscopy.
ponents of the umbilical cord in vitro: Microphotographs were taken at different time intervals as
(b) culturing the amniotic membrane tissue obtained in stated above. The observed polyhedral cell morphology is
step (a) under conditions allowing cell proliferation; and typical of epithelial cells.
(c) isolating the stem/progenitor cells. FIG. 2 depicts enzymatic digestion of the umbilical cord
In one embodiment, the invention provides a method, fur segments yielding similar epithelial (40xmagnification) cells
ther comprising: at day 2 (FIG. A. C) and day 5 (FIG. B., D). Umbilical cord
(a") separating the cells from the amniotic membrane tis amniotic membrane was divided into small pieces of 0.5
Sue before cultivation by a technique selected from the cmx0.5 cm and digested in 0.1% (w/v) collagenase type 1
group consisting of enzymatic digestion and direct tis 10 solution (Roche Diagnostics) at 37° C. for 8 hours. The
Sue explant. samples were vortexed every 30 min for 3 min. Cells were
In one preferred embodiment, the invention provides a harvested by centrifugation at 4000 rpm for 30 min. Cell
method for isolating stem/progenitor cells that have embry pellets were resuspended in EpiLife medium or Medium 171
onic stem cell-like properties. (both from Cascade Biologics) supplemented with 50 g/ml
In another preferred embodiment, the invention provides a 15 insulin-like growth factor-1 (IGF-1), 50 lug/ml platelet-de
method for isolating epithelial and/or mesenchymal stem/ rived growth factor-BB (PDGF-BB), 5 g/ml transforming
progenitor cells. growth factor-B1 (TGF-31) and 5ug/ml insulin (all obtained
In another embodiment, the invention provides a method from R&D Systems), counted and seeded on 10 cm tissue
further comprising: culture dishes pre-coated with collagen 1/collagen 4 mixtures
(d) culturing the stem/progenitor cells under conditions (1:2. Becton Dickinson) at density of 1x10° cells/dish. After
allowing the cells to undergo clonal expansion. 24 hours, attached cells were washed with warm phosphate
In yet another embodiment, the invention provides a buffered saline (PBS) and the culture medium was replaced
method further comprising: with EpiLife medium or Medium 171 (both from Cascade
(e) culturing the stem/progenitor cells under conditions Biologics). The medium was changed every 2 or 3 days, and
allowing the differentiation of said cells into epithelial 25 cell outgrowth was monitored underlight microscopy. Micro
cells and/or mesenchymal cells; and photographs were taken at different time intervals as stated
(f) isolating the differentiated cells. above. Once again the cells demonstrated typical epithelial
In yet another embodiment, the invention provides a cell polyhedral morphology.
method, further comprising: FIG. 3 depicts outgrowing mesenchymal cells explanted
(g) preserving the isolated stem/progenitor cells for further 30 from umbilical cord amniotic membrane. Cellular outgrowth
SC. was observed as early as 48 hours after placement in tissue
In yet a further embodiment, the invention comprising a culture dishes using DMEM supplemented with 10% fetal
method of cultivating stem/progenitors cells of the invention, calf serum (FCS) as culture medium (40xmagnification)
comprising: (FIG.3A, C). The explants were submerged in 5 ml DMEM
Obtaining a tissue explant from the amniotic membrane of 35 (Invitrogen) supplemented with 10% fetal bovine serum (Hy
umbilical cord; clone) (DMEM/10% FBS). Medium was changed every 2 or
Cultivating the tissue explant in Suitable cultivation media 3 days. Cell outgrowth was monitored under light micros
and cultivation conditions over a suitable period of time. copy. Microphotographs were taken at different time inter
In yet other embodiments, the invention is directed to thera vals. The cells were characterized by their spindle shaped
peutic uses of the stem/progenitor cells or cells differentiated 40 morphology, and migrated and expanded both easily and
therefrom or cellular secretions or extracts thereof. One of quickly in vitro, closely resembling fibroblasts (FIG.3B, D).
these embodiments provide a method of treating a subject FIG. 4 (40xmagnification) depicts mesenchymal cells
having a disorder comprising administering to the Subject an from umbilical cord amniotic membrane cells isolated by
effective amount of a stem/progenitor cell isolated by the collagenase enzymatic digestion. FIG. 4A shows mesenchy
inventive method of explained above. Another embodiment 45 mal cells isolated from umbilical cord amniotic membrane at
comprises administering to the Subject an effective amount of day 2. Cell proliferation was observed at day 5 (FIG. 4B).
a cell differentiated from a stem/progenitor cell of the inven Umbilical cord amniotic membrane was divided into small
tion. Other embodiments provide a corresponding pharma pieces of 0.5 cmx0.5 cm and digested in 0.1% (w/v) collage
ceutical composition, i.e. a pharmaceutical composition com nase type 1 solution (Roche Diagnostics) at 37°C. for 6 hours.
prising a stem progenitor cell or a cell differentiated 50 The samples were vortexed every 15 min for 2 min. Cells
therefrom, as well as cellular secretions into the cell medium were harvested by centrifugation at 4000 rpm for 30 min. Cell
and extracts. pellets were resuspended in DMEM/10% FBS, counted and
seeded on 10 cm tissue culture dish at density of 1x10'
BRIEF DESCRIPTION OF THE DRAWINGS cells/dish. Medium was changed every 2 or 3 days. Cell
55 outgrowing was monitored under light microscopy. Micro
The invention will be better understood with reference to photographs were taken at different time intervals. Once
the detailed description when considered in conjunction with again, cells demonstrated spindle shaped morphology typical
the non-limiting examples and the drawings, in which: of mesenchymal cells as fibroblasts.
FIG. 1 depicts epithelial cell outgrowth from umbilical FIG. 5 (40xmagnification) depicts the morphology in
cord amniotic membrane by the method of direct tissue 60 serum-free culture condition (DMEM) and serum culture
explant (40xmagnification) at day 2 (FIG. 1A) and day 5 condition (DMEM/10% FCS) of umbilical cord amniotic
(FIG. 1B, C) of tissue culture. Cell culture plastic surfaces membrane mesenchymal cells (UCMC, FIG. 5E, F, G, H)
were coated with collagen 1/collagen 4 mixtures (1:2; Becton isolated according to the method of the invention, normal
Dickinson) before placing the amniotic membrane on the dermal fibroblasts (NF109 cells, FIG. 5A, B) and adipose
Surface. The amniotic membrane specimens were Submerged 65 derived mesenchymal cells (ADMC, FIG. 5C, D). FIG. 5
in 5 ml EpiLife medium or Medium 171 (both from Cascade shows changes in cell morphology of NF and ADMC cultured
Biologics). Medium was changed every 2 or 3 days and cell in serum starvation conditions (DMEM only) reflected by
US 9,085,755 B2
5 6
flatter cells and less dense cytoplasm as compared with serum FIG. 14 shows global gene expression in umbilical cord
rich conditions (DMEM/10%FCS) where cells are more epithelial and mesenchymal stem cells analyzed by DNA
rounded with a dense cytoplasm (FIG. 5A, B, C, D). No microarray. UCEC expressed a total of 28055 genes and
change in morphology was observed in both UCMC groups UCMC expressed a total of 34407 genes. There are 27308
cultured under identical conditions of serum-free vs. serum 5 overlapping genes expressing in both cell types. 747 genes
rich media (FIG. 5E, F, G, H), indicating a difference in expressed were unique to UCEC and 7099 genes expressed
behavior and physiology of these latter mesenchymal cells. were unique to UCMC. The selected genes of interest are
FIG. 6 (40xmagnification) depicts UCMC isolated accord presented in this Figure. Both stem cell types expressed 140
ing to the invention cultured in DMEM/10% FCS at days 3 genes related to embryonic stem cells and embryonic devel
and 7 without a 3T3 feeder layer. The cells are seen to be 10 opment.
growing well, and are forming a colony (vertical growth) FIG. 15 shows a schematic illustration of expansion of
instead of exhibiting radial spread. Once again, this indicates umbilical cord epithelial and mesenchymal stem cells using
a difference in behavior of these mesenchymal cells as com repetitive explants of umbilical cordlining membrane tissues.
pared to their more differentiated counterparts. FIG. 16 depicts a cross section of an umbilical cord dem
FIG. 7 (40xmagnification) depicts colony formation of 15 onstrating the umbilical cord amniotic lining membrane
umbilical cord epithelial cells (UCEC) cultured on a 3T3 (LM), the contained Wharton's jelly (WJ), as well as two
feeder layer at days 3 and 7. This appearance is similar to that umbilical arteries (UA) and one umbilical vein (UV) sup
of normal skin derived epithelial keratinocyte stem cells. In ported within this jelly.
the latter, the 3T3 feederlayer maintains stemness of the cells. FIG. 17 depicts direct (in-vitro) differentiation of epithelial
FIG. 8 (40xmagnification) depicts obvious colony forma- 20 cells isolated from the amniotic membrane of umbilical cord
tion of umbilical cord mesenchymal cells (UCMC) isolated (UCEC) into skin epidermal keratinocytes (FIG. 17A), and
according to the invention cultured on a 3T3 feeder layer at in-vitro differentiation of mesenchymal cells isolated from
days 3 and 7(FIG. 8-1). The 3T3 feeder layer normally sup the amniotic membrane of umbilical cord (UCMC) into
presses the growth of differentiated mesenchymal cells as osteoblasts (FIG. 17B).
human dermal fibroblasts. Once again, this indicates a differ- 25
ence in behavior of these mesenchymal cells as compared to DETAILED DESCRIPTION
their more differentiated counterparts. FIG. 8-2 shows the
colony forming efficiency assay of the umbilical cord mes The invention is based on the Surprising finding that the
enchymal cells. amniotic membrane of umbilical cord represents a source,
FIG. 9-1 to FIG.9-28 show Western blot analysis by which 30 from which stem/progenitor cells such as mesenchymal and
the expression of several embryonic stem cell markers in epithelial stem/progenitor cells can be successfully isolated
UCEC and UCMC isolated according to the invention, was and expanded under invitro conditions. Even more surprising
compared to the expression of these markers in human dermal is the finding that these cells show embryonic stem cell-like
fibroblasts (NF), in bone marrow mesenchymal cells (BMSC) characteristics. The amniotic membrane (also called amniotic
and adipose-derived mesenchymal cells (ADMC). FIGS. 35 lining membrane), i.e. thin innermost membranous sac
9-29 and 9-30 show secretion of Leukemia inhibitory factor enclosing the placenta and developing embryo of mammals,
detected by Western blot analysis (FIG. 9-29) and highly has recently been used as a natural Substrate in ocular surface
secreted ActivinA and Follistatin detected by ELISA assay reconstruction and as a biological Substrate for expanding
(FIG. 9-30), respectively in supernatants of umbilical cord limbal epithelial stem cells (cf., e.g., Anderson, D. F. et al.
mesenchymal and epithelial stem cell culture in comparison 40 (2001) Br. J. Ophthalmol. 85,567-575: Griterich, M. et al.
with bone marrow, adipose derived stem cells, human dermal (2003) Surv. Ophthalmol. 48, 631-646). However, no meth
fibroblasts and epidermal keratinocytes. ods have been described thus far for the isolation of stem/
FIG. 10 shows indirect immunofluorescent analysis of progenitor cells from the amniotic membrane, at least for
markers of epithelial cells expressed in umbilical cord epi humans, nor has the amniotic membrane covering the umbili
thelial stem cells such as cytokeratins (CK)-general, CK17, 45 cal cord been reported as a source for stem cells.
CK6, CK10, CK19, CK18, CK16, CK15 (FIG. 10-1): The invention provides a method for isolating stem/pro
Hemidesmosome components-integrin alpha6, integrin genitor cells from the amniotic membrane of umbilical cord,
beta4: Desmosome components (FIG. 10-2); Basement the method comprising:
membrane components-laminin1, laminin5, collagen IV, col (a) separating the amniotic membrane from the other com
lagen VII (FIG. 10-3) and other important extracellular 50 ponents of the umbilical cord in vitro:
matrix components as integrin-betal and fibronectin (FIG. (b) culturing the amniotic membrane tissue obtained in
10-4). step (a) under conditions allowing cell proliferation; and
FIG. 11-1 to FIG. 11-4 shows cytokine array analysis of (c) isolating the stem/progenitor cells.
secreted cytokines and growth factors by umbilical cord mes For isolation of the cells of the invention from umbilical
enchymal stem cells (UCMC) in comparison with human 55 cord, the umbilical cord or a part thereof is usually collected
bone-marrow mesenchymal stem cells. immediately after birth (of a child in the case of humans) and
FIG. 12-1 to FIG. 12-7 shows cytokine array analysis of for transport to the laboratory transferred in a medium that is
secreted cytokines and growth factors by umbilical cord epi Suitable for handling of mammalian tissue. Examples of Such
thelial stem cells (UCEC) in comparison with human epider media include, but are not limited to Leibovitz, media which
mal keratinocytes. 60 are commercially available from Suppliers such as Sigma
FIG. 13 shows UCMC cells cultured in DMEM supple Aldrich, Saint Louis, USA or HyClone, Logan, Utah, USA.
mented with 10% fetal calfserum (FCS) (FIG. 13-1), serum The umbilical cord is then typically processed under sterile
free media PTT-1 (FIG. 13-2), in serum-free media PTT-2 conditions. Processing of the cord typically includes remov
(FIG. 13-3, FIG. 13-4) and in serum-free media PTT-3 (FIG. ing the blood that has remained on the surface or within the
13-5). FIG. 13 also shows the growth of adipose derived 65 blood vessels of the umbilical cord by washing with a suitable
stromal cells (FIG. 13-6) and bone marrow derived stromal buffer such as phosphate buffered saline. The umbilical cord
cells (FIG. 13-7) in serum free medium PTT-3. is then typically reduced to Smaller pieces, for example by
US 9,085,755 B2
7 8
cutting, and washed again before separating the amniotic are not limited to, Dulbecco's Modified Eagle Medium
membrane from the other components. In this conjunction, it (DMEM), DMEM-F12, RPMI media, EpiLlfe medium, and
is noted that it is not necessary to process the umbilical cord Medium 171, with the latter being preferred in some embodi
of a mammalian donor immediately after birth but it is also ments. The media may be supplemented with fetal calf serum
possible, to collect the umbilical cord and, optionally after (FCS) or fetal bovine serum (FBS) as well as antibiotics,
washing under sterile conditions and reducing it into Smaller growth factors, amino acids, inhibitors or the like, which is
pieces, to preserve the umbilical cord or parts thereof by well within the general knowledge of the skilled artisan.
cryo-preservation and to store the so obtained specimen, for In one embodiment, the invention provides a method, fur
example in liquid nitrogen, for later isolation of the cells of ther comprising:
the invention from the umbilical cord. Accordingly, an (in 10 (a") separating these stem/progenitor cells from the amni
tact) umbilical cord or a portion of an intact umbilical cord otic membrane tissue by a enzymatic digestion and/or
that is treated by cryo-preservation is also encompassed in the direct tissue explant technique before cultivation. The
present invention. In addition, the umbilical cord amniotic term “enzymatic digestion technique' as used herein
membrane that has been separated from the other components means that enzymes are added to cleave the cells from
of the umbilical cord and is then treated by cryo-preservation 15 the maintissue mass (here the amniotic membrane of the
is also encompassed in the present invention. umbilical cord). The separated cells are Subsequently
The term "cryo-preservation' is used herein in its regular collected. The term “direct tissue explant technique' as
meaning to describe a process where cells or whole tissues are used herein means that the tissue is first placed in media
preserved by cooling to low Sub-Zero temperatures, such as without enzymes. Then under careful conditions the
(typically) -80° C. or -196° C. (the boiling point of liquid cells separate from the main tissue mass by itself and
nitrogen). Cryo-preservation can be carried out as known to the cells are then harvested for collection.
the person skilled in the art and can include the use of cryo Methods for separating cells of a particular tissue or organ
protectors such as dimethylsulfoxide (DMSO) or glycerol, by treatment with enzymes or by direct tissue explant are well
which slow down the formation of ice-crystals in the cells of known in the art (cf., for example, Pollard, J. W. and Walker,
the umbilical cord. 25 J. M. (1997) Basic Cell Culture Protocols, Second Edition,
The term “stem/progenitor cell as used herein refers to Humana Press, Totowa, N.J.; Freshney, R.I. (2000) Culture of
any cell derived of umbilical cord having the capacities to Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.).
self-renew indefinitely and to differentiate in multiple cell or Any enzyme catalyzing tissue dissociation may be used for
tissue types such as endothelial cells, epithelial cells, fibro performing the methods of the present invention. In some
blasts, myocytes or neurons. Furthermore, the cells may be 30 embodiments, collagenase is used for that purpose. The
derived of any mammalian species, such as mouse, rat, guinea enzyme may be used as a crude preparation or in purified
pig, rabbit, goat, dog, cat, sheep, monkey or human, with cells form. It may be purified from any prokaryotic or eukaryotic
of human origin being preferred in one embodiment. organism (with Clostridium histolyticum being most pre
The term "embryonic stem cell-like properties’ refers to ferred) or produced recombinantly by means of gene technol
the ability of the cells derived of umbilical cord that they 35 ogy. Any type of collagenase may be employed, i.e. type 1,
can—almost like or exactly like embryonic stem cells—dif type 2, type 3, type 4, or any combination thereof. In some
ferentiate spontaneously into all tissue types, meaning that embodiments the use of collagenase type 1 is being preferred.
they are pluripotent stem cells. In one embodiment, the invention provides a method for
The term "amniotic membrane' as used herein refers to the isolating stem/progenitor cells that have embryonic stem cell
thin innermost membranous sac enclosing the developing 40 like properties. These cells can ultimately be differentiated
embryo of mammals. During pregnancy, the fetus is Sur into, but not limited to, by morphology, epithelial or mesen
rounded and cushioned by a liquid called amniotic fluid. This chymal cells.
fluid, along with the fetus and the placenta, is enclosed within Accordingly, in another embodiment, the invention pro
a sac called the amniotic membrane, which also covers the vides a method for isolating epithelial and/or mesenchymal
umbilical cord. The amniotic fluid is important for several 45 stem/progenitor cells, wherein in accordance with the above
reasons. It cushions and protects the fetus, allowing the fetus disclosure these cells may have embryonic stem cell-like
to move freely. The amniotic fluid also allows the umbilical properties.
cord to float, preventing it from being compressed and cutting Epithelial stem/progenitor cells include any cells exhibit
off the fetus' supply of oxygen and nutrients derived from the ing a epithelial cell like morphology (i.e. a polyhedral shape)
circulating blood within the placental blood vessels. The 50 that can be differentiated into any type of epithelial cell such
amniotic sac contains the amniotic fluid which maintains a as, but not limited to, skin epithelial cells, hair follicular cells,
homeostatic environment protecting the fetal environment cornea epithelial cells, conjunctival epithelial cells, retinal
from the outside world. This barrier additionally protects the epithelial cells, liver epithelial cells, kidney epithelial cells,
fetus from organisms (like bacteria or viruses) that could pancreatic epithelial cells, oesophageal epithelial cells, Small
travel up the vagina and potentially cause infection. 55 intestinal epithelial cells, large intestinal epithelial cells, lung
Media and reagents for tissue culture are well known in the and airway epithelial cells, bladder epithelial cells or uterine
art (cf., for example, Pollard, J. W. and Walker, J. M. (1997) epithelial cells.
Basic Cell Culture Protocols, Second Edition, Humana Press, Mesenchymal stem/progenitor cells include any cells
Totowa, N.J.; Freshney, R.I. (2000) Culture of Animal Cells, exhibiting a mesenchymal cell like morphology (i.e. a
Fourth Edition, Wiley-Liss, Hoboken, N.J.). Examples of 60 spindle-like shape) that can be differentiated into any type of
Suitable media for incubating/transporting umbilical cord tis mesenchymal cell Such as, but not limited to, skin fibroblasts,
sue samples include, but are not limited to, Dulbecco's Modi chondrocytes, osteoblasts, tenocytes, ligament fibroblasts,
fied Eagle Medium (DMEM), RPMI media, Hanks’ Balanced cardiomyocytes, Smooth muscle cells, skeletal muscle cells,
Salt Solution (HBSS) phosphate buffered saline (PBS), and adipocytes, cells derived from endocrine glands, and all vari
L-15 medium, with the latter one being preferred in some 65 eties and derivatives of neurectodermal cells.
embodiments. Examples of appropriate media for culturing In another embodiment, the invention provides a method
stem/progenitor cells according to the invention include, but further comprising:
US 9,085,755 B2
10
(d) culturing the stem/progenitor cells under conditions In accordance with the above, the invention is also directed
allowing the cells to undergo clonal expansion. to a pharmaceutical composition comprising a stem/progeni
The term “clonal expansion' (sometimes also referred to as tor cell isolated from the amniotic membrane of umbilical
“mitotic clonal expansion”) relates to a process that occurs cord by the above inventive method. The pharmaceutical
early in the differentiation program of a cell, by which stem/ 5 composition can also include a cell differentiated from the
progenitor cells become committed to aparticular lineage and stem/progenitor cell. The pharmaceutical composition can be
then undergo terminal differentiation. It is well known in the of any kind, and usually comprises the stem/progenitor cells,
art that the conditions to induce clonal expansion of progeni a cell differentiated therefrom or a cellular secretion or cel
tor cells may vary significantly between different cell types. lular extract thereof together with a suitable therapeutically
Without being limited to a particular method, the induction of 10 acceptable carrier/excipient. In case of a cellular secretion,
clonal expansion is generally achieved by cultivating the the desired compound(s) can be used in Some embodiments in
stem/progenitor cells in a medium that has been optimized for the form of the Supernatant into which the compound(s) is/are
cell proliferation. Such media are commercially available secreted. In other embodiment, the Supernatant might be pro
from many providers. Non-limiting examples of Such media cessed, for example, by purification and concentration prior
are KGMR)-Keratinocyte Medium (Cambrex), MEGM- 15 to be included in a pharmaceutical composition. In some
Mammary Epithelial Cell Medium (Cambrex), EpiLife embodiments, the pharmaceutical composition is adapted for
medium (Cascade Biologics) or Medium 171 (Cascade Bio systemic or topical application.
logics). Alternatively, a culture medium may be supple A pharmaceutical composition adapted for topical appli
mented with reagents inducing cell proliferation Such as cation may be in liquid or viscous form. Examples thereof
growth factors. Such reagents may be admixed in a single 20 include an ointment, a cream, and a lotion and the like.
solution such as the Human Keratinocyte Growth Supple Examples for pharmaceutical compositions that are Suitable
ment Kit (Cascade Biologics), to name one example, or may for systemic use are liquid compositions, wherein the stem/
be supplemented individually. Such reagents include, but are progenitor cells or the cellular extract are dissolved in a buffer
not limited to, growth factors (such as epidermal growth that is acceptable for injection or infusion, for example. The
factor, insulin-like growth factor-1, platelet-derived growth 25 preparation of Such pharmaceutical compositions is within
factor-BB, transforming growth factor-f1, insulin, for the knowledge of the person skilled in the art and described in
example), hormones (such as a bovine pituitary extract), Gennaro, A. L. and Gennaro, A. R. (2000) Remington. The
hydrocortisone, transferrin and the like in any suitable com Science and Practice of Pharmacy, 20th Ed., Lippincott Wil
bination to induce clonal expansion of a given cell type. The liams & Wilkins, Philadelphia, Pa., for example.
term “clonal expansion' also includes cultivation of the cell in 30 Accordingly, the invention also relates to a method of treat
Vivo, for example, by injection of the cells into mammals such ing a Subject having a disorder. This method comprises
as humans, mice, rats, monkeys, apes to name only a few. administering to the subject an effective amount either of a
In yet another embodiment, the invention provides a stem/progenitor cell isolated as explained herein or of a cel
method further comprising: lular extract derived from such a cell.
(e) culturing the stem/progenitor cells under conditions 35 In principle, any condition or disorder which is suitable for
allowing the differentiation of said cells into epithelial being treated by means of stem cells/progenitor cells can be
cells and/or mesenchymal cells; and treated with a cell or a cellular extract of present invention. It
(f) isolating the differentiated cells. is also possible to differentiate cells of the invention into a
Thus, the invention also provides for a method of differenti desired type of cell, for example, but not limited to, a skin cell,
ating a stem/progenitor cell into a differentiated cell. 40 a bone cell, an hormone producing cell Such as a beta islet
In yet another embodiment, the invention provides a insulin producing cell, and use the differentiated cell thera
method, further comprising: peutically. In some embodiments, the disorder is selected
(g) preserving the isolated stem/progenitor cells for further from the group consisting of neoplastic disease, accelerated
SC. skin aging and skin disorders, tissue disorders, visceral endo
Methods and protocols for preserving and storing of 45 crine deficiencies, and neural disorders.
eukaryotic cells, and in particular mammalian cells, are well The tissue disorder to be treated can be a congenital or an
known in the art (cf., for example, Pollard, J. W. and Walker, acquired tissue deficiency. Examples of visceral endocrine
J. M. (1997) Basic Cell Culture Protocols, Second Edition, deficiency that can be treated with a cell of the invention
Humana Press, Totowa, N.J.; Freshney, R.I. (2000) Culture of include, but are not limited to, Diabetes mellitus associated
Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). 50 with insulin deficiency, testosterone deficiency, anemia,
Any method maintaining the biological activity of the iso hypoglycemia, hyperglycemia, pancreatic deficiency, adre
lated stem/progenitor cells such as epithelial or mesenchymal nal deficiency, and thyroid deficiencies.
stem/progenitor cells may be utilized in connection with the Examples of neural disorders that can be treated include,
present invention. In one preferred embodiment, the stem/ but are not limited to, Alzheimer's disease, Parkinson's dis
progenitor cells are maintained and stored by using cryo- 55 ease, Jacob Kreutzfeld's disease, Lou Gehrig's disease, Hun
preservation. tington's disease and neural neoplastic conditions.
Accordingly, the invention is also directed to a progenitor/ An example of a skin disease is a wound or a damaged part
stem cell derived from the amniotic membrane of umbilical of the skin, for example, Sun burned skin. Also aging of the
cord by means of the above methods and to a cell differenti skin is considered to be a skin disease herein. Topical or
ated from the progenitor/stem cell. In addition, the invention 60 similar delivery of stem/progenitor cells of the invention or
is also directed to a cell bank comprising or consisting of one cellular extracts thereof, for example, as a constituent in
or more progenitor/stem cells that have been isolated as lotions or creams or any other suitable vehicle may thus be
described here. This cell bank of progenitor/stem cells may be used for repair of Sundamaged skin and in addition may slow
autologous to an individual or pooled (the latter for Subse also down the aging process of skin (anti-aging properties) by
quent allogeneic transplantation, for example), and Subse- 65 replenishing, and thus fortifying, deficient growth factors and
quently can be employed by further differentiation for regen related peptide elements, without which skin aging would be
erative medicine, tissue repair and regeneration, for example. accelerated. The stem/progenitor cells may also migrate to
US 9,085,755 B2
11 12
injured regions of the body Such as Surface wounds to form In a further embodiment and in line with the above disclo
the necessary required cellular elements necessary for the Sure, the stem/progenitor cells of the invention can be used for
local reparative processes (cf. The Journal of Immunology, the production of any biological molecule. The biological
2001, 166: 7556-7562; or International Journal of Biochemi molecule can be, for instance, any molecule that is naturally
cal and Cell Biology 2004:36:598-606. produced in the cells or a molecule the coding nucleic acid of
The neoplastic disease may be cancer, in particular as which has been introduced into the cells via recombinant
recent studies have demonstrated that stem cells may selec DNA technology. Examples of molecules that can be pro
tively target neoplastic tumor tissue (Journal of the National duced by the cells of the invention include, but are not limited
Cancer Institute 2004: 96 (21): 1593-1603) allowing for to, a protein Such as a cytokine, a growth factor Such as
directed delivery of antineoplastic agents such as interferonto 10 insulin-like growth factor (IGF), epidermal growth factor
neoplastic foci. The cancer can be any kind of cancer, includ (EGF), transforming growth factor beta (TGF-beta), Activin
ing those cancers that are able to form solid tumors, ranging A, a bone morphogenetic protein (BMP), PDGF or a hormone
as insulin or erythropoietin or a transporter protein Such trans
from skin cancerto cancer of the internal organs. Examples of ferrin, a peptide Sucha growth factor or hormone (e.g. luteinic
cancers to be treaded include, squamous cell carcinoma, 15 hormone (LSH), folliclestimulating hormone (FSH)), a small
breast ductal and lobular carcinoma, hepatocellular carci organic molecule Such as a steroid hormone, an oligo- or
noma, nasopharyngeal carcinoma, lung cancer, bone cancer, polysaccharide, for example, heparin or heparan Sulfate (cf.
pancreatic cancer, skin cancer, cancer of the head or neck, example WO 96/23003, or WO 96/02259 in this regard), a
cutaneous or intraocular malignant melanoma, uterine can proteoglycan, a glycoprotein Such as collagen or laminin, or a
cer, ovarian cancer, rectal cancer, cancer of the anal region, lipid, to name only a few.
stomach cancer, colon cancer, breast cancer, testicular cancer, In a further aspect and in accordance with recent
uterine cancer, carcinoma of the fallopian tubes, carcinoma of approaches (see, for example, Amit, Met al., Human feeder
the endometrium, carcinoma of the cervix, carcinoma of the layers for human embryonic stell cells, Biol Reprod 2003; 68:
vagina, carcinoma of the Vulva, Hodgkin’s Disease, non 2150-2156), the stem/progenitor cells described here can be
Hodgkin’s lymphoma, cancer of the esophagus, cancer of the 25 used as feeder layer for the cultivation of other embryonic
Small intestine, cancer of the endocrine system, cancer of the stem cells, in particular human embryonic stem cells. In one
thyroid gland, cancer of the parathyroid gland, cancer of the of these embodiments the cells of the present invention are
adrenal gland, sarcoma of Soft tissue, cancer of the urethra, preferably of human origin, since using human cells as feeder
cancer of the penis, prostate cancer, chronic or acute leuke layer minimizes the risk of contaminating the cell culture
mias, Solid tumors of childhood, lymphocytic lymphoma, 30 with animal-derived components such as animal pathogens or
cancer of the bladder, cancer of the kidney or ureter, renal cell immunogens. In this respect, it is to be noted that the cells of
carcinoma, carcinoma of the renal pelvis, neoplasm of the the invention can be cultivated under serum free conditions.
central nervous system (CNS), primary CNS lymphoma, Accordingly, employing the cells as feederlayer and cultivat
tumor angiogenesis, spinal axis tumor, brain stem glioma, ing the cell culture under with serum free media as the one
pituitary adenoma, Kaposi's sarcoma, epidermoid cancer or 35 described herein later, or in Draper et al. (Culture and char
any combination of Such cancers, including disseminated acterization of human embryonic stem cell lines, StemCells
(metastasising) forms thereof. In case of treatment of a neo Dev 2004, 13:325-336) or in the International patent applica
plastic disease the umbilical cord amnion derived stem cells tion WO 98/30679, for example.
and/or their cellular extracts disclosed herein can be admin In this connection, it is noted that in transplantation Surgery
istered systemically both as a direct treatment and/or as a 40 and cell-based therapy high quantities of low passage cells
carrier vehicle. In the latter case of anti-neoplastic tumor with a minimal proportion of senescent cells (i.e., large pro
therapy, the cells comprise an anti-neoplastic agent. portion of high quality cells) are crucial and are required to be
In another pharmaceutical use, stem/progenitor cells of the derived within the shortest possible time during cell expan
present invention can be used for gene therapy. For this pur Sion. For example, mesenchymal stem cells from bone mar
pose, the cells can be transformed with a nucleic acid encod 45 row and cord blood are low in quantity and therefore require
ing the protein that is to be produced in the cells. The nucleic expansion over many passages for a long period of time in
acid can be introduced into a cells of the invention using any order to achieve the sufficient number of cells required for cell
of the various methods that are well known to the skilled transplant. The high passage cells howevertend to deteriorate
person, for example, using a viral vector and/or a lipid con in quality and may lead to cell senescence or cancerous trans
taining transfection composition Such as as IBAfect (IBA 50 formation. It has been found here that high quantities of cells
GmbH, Göttingen, Germany), Fugene (Roche), GenePorter of the present invention can be obtained by low passage
(Gene Therapy Systems), Lipofectamine (Invitrogen), Super numbers using a repetitive explantation technique. The
fect (Qiagen), Metafecten (Biontex) or those ones described present invention thus also relates to a method of cultivating
in the PCT application WO 01/015755). In a related embodi stem/progenitors cells of the invention, wherein this method
ment, the cells of the invention, after being transformed with 55 comprises:
a nucleic acid encoding a polypeptide of choice, can be used Obtaining a tissue explant from the amniotic membrane of
of recombinantly producing this polypeptide. umbilical cord;
As mentioned above, stem cell extracts are rich in a variety Cultivating the tissue explant in Suitable cultivation media
of growth factors and peptides that are relevant for normal and cultivation conditions over a Suitable period of time,
tissue physiology. Such growth factors and/or peptides may 60 Optionally exposing the tissue explant to fresh cultivation
be deficient in exposed parts of the body, such as the skin, media and continuing the cultivation under Suitable con
which is the Surface layer of all human beings protecting the ditions over a suitable period of time (cf., FIG. 15).
body from external elements for the maintenance of internal The cultivation can be carried out in for as many cycles
homeostasis. Therefore in a further embodiment, stem/pro (passages) as wanted and be stopped once the desired number
genitor cells of the invention or cellular extracts thereof are 65 of cells has been obtained. Exposing the tissue explant to
suitable for the treatment and/or maintenance of internal fresh cultivation can be carried out by removing the used cell
homeostasis. cultivation medium from the vessel used for growing the cells
US 9,085,755 B2
13 14
and adding fresh media to that vessel. Instead of replacing the ately transferred into a 500 ml sterile glass bottle containing
media in the used vessel, exposing to fresh cultivation media culture transport medium (L-15 medium Supplemented with
can be achieved by transferring the tissue explant to a new 50 IU/ml penicillin, 50 g/ml streptomycin, 250 ug/ml fun
vessel which is filled with cultivation media. The tissue giZone, 50 ug/ml gentamicin; all reagents purchased from
explant used for cultivation/propagation of the cells can be Invitrogen) prior to transport to the laboratory. In the labora
obtained by any suitable method, for example by the “direct tory, stem cell extraction is conducted in a laminar flow hood
tissue explant technique' as explained above (in which the understerile conditions. The specimen is first transferred to a
tissue is first placed in media without enzymes, and then sterile stainless steel tray. All remaining blood in the cord
under careful conditions the cells separate from the main vessels is removed by multiple syringing washes using warm
tissue mass by itself and the cells are then harvested for 10 phosphate-buffered saline (PBS) supplemented with 5 IU/ml
collection). heparin (from Sigma). Plain PBS without heparin is used in
The cultivation of the tissue explants can be carried out in
any media that is suitable for cultivation of mammalian cells. the final washes. The umbilical cord tissue specimen is then
Examples include the conventional and commercially avail cut into pieces 2 cm in length and transferred into 10 cm
able media that are given above with respect to the cultivation diameter cell culture dishes, where further washing and dis
or the clonal expansion of the cells of the invention such as, 15 infection is performed with 70% ethanol followed by multiple
but not limited to, KGMR)-Keratinocyte Medium (Cambrex), washes using PBS containing an antibiotic mixture (50 IU/ml
MEGM Mammary Epithelial Cell Medium (Cambrex) penicillin, 50 g/ml streptomycin, 250 ug/ml fungizone, 50
EpiLife medium (Cascade Biologics), Medium 171 (Cascade ug/ml gentamicin; all purchased from Invitrogen) until the
Biologics), DMEM, DMEM-F12 or RPMI media. The culti Solution becomes clear.
Vation is typically carried out at conditions (temperature,
atmosphere) that are normally used for cultivation of cells of Example 2
the species of which the cells are derived, for example, at 37°
C. in air atmosphere with 5% CO. In one embodiment, the Cell Separation/Cultivation
cultivation is carried out using serum free, in particular
bovine serum free media. The cultivation (in one passage) is 25 Dissection of umbilical cord tissue is first performed to
performed for any suitable time the cells need for growth, separate the umbilical cord amniotic membrane from Whar
typically, but by no means limited to, for about 1 to several ton's jelly (i.e. the matrix ofumbilical cord) and other internal
days, for example to about 7 or about 8 days. components. The isolated amniotic membrane is then cut into
The inventions illustratively described herein may suitably small pieces (0.5 cmx0.5 cm) for cell isolation. Explant is
be practiced in the absence of any element or elements, limi 30 performed by placing the pieces of umbilical cord amniotic
tation or limitations, not specifically disclosed herein. Thus, membrane on tissue culture dishes at different cell culture
for example, the terms "comprising”, “including”, “contain
ing, etc. shall be read expansively and without limitation. conditions for isolation of either epithelial or mesenchymal
Additionally, the terms and expressions employed herein stem cells.
have been used as terms of description and not of limitation, For mesenchymal cell separation/cultivation, the explants
and there is no intention in the use of such terms and expres 35 were submerged in 5 ml DMEM (Invitrogen) supplemented
sions of excluding any equivalents of the features shown and with 10% fetal bovine serum (Hyclone) (DMEM/10% FBS)
described orportions thereof, but it is recognized that various and maintained in a CO cell culture incubator at 37°C. The
modifications are possible within the scope of the invention medium was changed every 2 or 3 days. Cell outgrowth was
claimed. Thus, it should be understood that although the monitored under light microscopy. Outgrowing cells were
present invention has been specifically disclosed by preferred 40 harvested by trypsinization (0.125% trypsin/0.05% EDTA)
embodiments and optional features, modification and varia for further expansion and cryo-preservation using DMEM/
tion of the inventions embodied therein herein disclosed may 10% FBS.
be resorted to by those skilled in the art, and that such modi For epithelial cell separation/cultivation, cell culture plas
fications and variations are considered to be within the scope tic Surfaces were coated with collagen 1/collagen 4 mixtures
of this invention. 45 (1:2) before placing the tissue samples on the Surface. The
The invention has been described broadly and generically tissue samples were submerged in 5 ml EpiLife medium or
herein. Each of the narrower species and Subgeneric group Medium 171 (both from Cascade Biologics). The medium
ings falling within the generic disclosure also form part of the was changed every 2 or 3 days. Cell outgrowth from tissue
invention. This includes the generic description of the inven culture explants was monitored under light microscopy. Out
tion with a proviso or negative limitation removing any Sub 50 growing cells were harvested by trypsinization (0.125%
ject matter from the genus, regardless of whether or not the trypsin/0.05% EDTA) using EpiLife medium or Medium
excised material is specifically recited herein. 171.
Other embodiments are within the following claims and For the enzymatic extraction method of cells, umbilical
non-limiting examples. In addition, where features or aspects cord amniotic membrane was divided into small pieces of 0.5
of the invention are described in terms of Markush groups, 55 cmx0.5 cm and digested in 0.1% (w/v) collagenase typel
those skilled in the art will recognize that the invention is also solution (Roche Diagnostics) at 37° C. for 6 hours. The
thereby described in terms of any individual member or sub samples were vortexed every 15 min for 2 min. Cells were
group of members of the Markush group. harvested by centrifugation at 4000 rpm for 30 min. Two
different approaches were employed to isolate either epithe
EXAMPLES 60 lial or mesenchymal stem cells.
For isolation of epithelial stem cells, cell pellets were
Example 1 resuspended in EpiLife medium or Medium 171 (both from
Cascade Biologics) supplemented with 50 g/ml insulin-like
Collection of Umbilical Cord Tissue growth factor-1 (IGF-1), 50 lug/ml platelet-derived growth
65 factor-BB (PDGF-BB), 5 g/ml transforming growth factor
Umbilical cord tissue is collected immediately after deliv B1 (TGF-B1), and 5 g/ml insulin (all obtained from R&D
ery of the child. The specimen is rinsed clean and immedi Systems), counted and seeded on 10 cm tissue culture dishes
US 9,085,755 B2
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pre-coated with collagen 1/collagen 4 mixtures (1:2; Becton In further experiments the colony forming ability of the
Dickinson) at density of 1x10° cells/dish. After 24 hours, mesenchymal cells of the invention (UCMC) was studied. For
attached cells were washed with warm PBS and medium was colony forming efficiency assay, 100-200 single cells were
replaced with supplement-added EpiLife medium or Medium seeded in 100 mm tissue culture dishes or T75 flasks without
171. The medium was changed every 2 or 3 days. Cell growth 5 feederlayers. Cells were maintained in DMEM/10% FCS for
and expanding clonal formation was monitored under light 12 days. Single colony formation was monitored under the
microscopy. At a confluence of about 70%, cells were sub inverted light microscope (experiment was carried out in
cultured by trypsinization (0.125% trypsin/0.05% EDTA) for duplicate, experiments termed UCMC-16 and UCMC-17 in
further expansion and cryo-preservation. FIG. 8-2). Microphotographs were sequentially taken. At day
For isolation of mesenchymal stem cells, cell pellets were 10 12, colonies were fixed and stained with Rhodamine. UCMC
resuspended in DMEM/10% FBS, counted and seeded on 10 colony forming units were seen (FIG. 8-2). The multiple large
cm tissue culture dishes at density of 1x10° cells/dish. The colonies observed, indicated self-renewal of UMCM in-vitro
culture medium was changed every 2 or 3 days. Cell growth (FIG. 8-2).
Western blot analysis (FIG. 9) shows that mesenchymal
and expansion was monitored under light microscopy. At a 15 stem cells from umbilical cord amniotic membrane (UCMC)
confluence of about 90%, cells were sub-cultured as outlined and umbilical cord epithelial cells (UCEC) isolated in accor
above. dance with the invention expressed the POU5fl gene which
For cultivation of epithelial and mesenchymal stem cells encodes the transcription factor Octamer-4 (Oct-4) a specific
on feederlayer, umbilical cord lining membrane was digested marker of embryonic stem cells (cf. Niwa, H., Miyazaki, J.,
by collagenase treatment, counted and seeded on 10 cm tissue and Smith, A. G. (2000). Nat. Genet. 24, 372-376) (FIG.9-1).
culture dishes coated with lethally irradiated or Mitomycin C Thus, this analysis indicates the embryonic-like properties of
treated 3T3 fibroblasts (feeder layer) in Green's medium. The these stem cells. These mesenchymal and epithelial cells also
culture medium was changed every 2 or 3 days. Colony expressed Bmi-1, a marker that is required for the self-re
formation was monitored under light microscopy and photo newal of adult stem cells (cf., Park et al., J. Clin. Invest.
graphed. 25 113,175-179 (2004) (FIG. 9-27) as well as leukemia inhibi
tory factor (LIF) (FIG.9-28) that is considered to maintain the
Example 3 pluripotency of stem cells and embryonic cells and has thus,
for example been used for isolation and expansion of human
Identification of Stem/Progenitor Cells neural stem cells. These cells also highly expressed the other
30 growth factors such as connective tissue growth factor
Epithelial cells: FIG. 1 shows pictures of outgrowing epi (CTGF) (FIGS. 9-6, 9-7), vascular endothelial growth factor
thelial cells from umbilical cord amniotic membrane pre (VEGF) (FIGS. 9-10, 9-11), placenta-like growth factor
pared by the method using tissue explant (40xmagnification). PLGF (FIGS. 9-4, 9-5), STAT3 (FIGS. 9-2, 9-3), stem cell
Pictures were taken at day 2 (FIG. 1A) and day 5 (FIG. 1B, C) factor (SCF) (FIG. 9-16), Hepatoma-derived Growth Factor
of tissue culture. Cell morphology analysis demonstrated 35 (HDGF) (FIGS. 9-14, 9-15), Fibroblast Growth Factor-2
polyhedral shaped epithelial-like cells. Enzymatic digestion (FGF-2) (FIGS. 9-12, 9-13), Platelet-derived Growth Factor
of the umbilical cord segments yielded similar (FIG. 2), epi (PDGF) (FIGS. 9-8, 9–9), alpha-Smooth Muscle Actin
thelial cells at day 2 (FIG. A. C) and day 5 (FIG. B., D) (C-SMA) (FIG.9-17), Fibronectin (FIGS. 9-18,9-19), Deco
(40xmagnification). FIG. 7 shows pictures of colony forma rin (FIG. 9-20), Syndecan-1,2,3,4 (FIGS. 9-21 to 9-26). In
tion of epithelial stem cells from umbilical cord amniotic 40 FIG. 9, the expression of these genes is compared to human
membrane cultured on feeder layer using Green's method dermal fibroblasts, bone marrow mesenchymal cells (BMSC)
(40xmagnification). A colony of polyhedral shaped epithe and adipose-derived mesenchymal cells (ADMC). FIG. 9-29
lial-like cells expanded rapidly from day 3 to day 7. shows Western blot data of the secretion of leukemia inhibi
Mesenchymal cells: Outgrowth of mesenchymal cells tory factor (LIF) by both UCEC and UCMC. FIG.9-30 shows
explanted from umbilical cord amniotic membrane was 45 highly secreted Activin A and Follistatin (both of which pro
observed as early as 48 hours after placement in tissue culture teins are well known to promote tissue repair and regenera
dishes using DMEM supplemented with 10% fetal calf serum tion, enhanced angiogenesis, and maintain embryonic stem
(FCS) as culture medium (FIG. 3A, C) (40xmagnification). cell culture, so that expression of the respective genes is a sign
The cells were characterized by their spindle shaped mor for the embryonic properties and ability of the cells to differ
phology, and migrated and expanded both easily and quickly 50 entiate) detected ELISA assay (FIG.9-30) in supernatants of
in vitro, closely resembling fibroblasts (FIG. 3B, D) (40x umbilical cord mesenchymal and epithelial stem cell culture
magnification). Similar observations were noted in the cell in comparison with bone marrow, adipose derived stem cells,
group isolated by collagenase enzymatic digestion (FIG. 4). human dermal fibroblasts and epidermal keratinocytes. Also
FIG. 4A shows mesenchymal cells isolated from umbilical these results indicate that the cells of the invention are prom
cord amniotic membrane at day 2. Cell proliferation was 55 ising candidates in therapeutic application of these cells areas
observed at day 5 (FIG. 4B) (40xmagnification). FIGS. 6 and Such as regenerative medicine, aging medicine, tissue repair
8-1 show pictures of colony formation of mesenchymal stem and tissue engineering. In addition, FIGS. 9-29 and 9-30
cells from umbilical cord amniotic membrane cultured on show the capability of the cells to secret an expression prod
non-feeder layer (FIG. 6) and feeder layer condition (FIG. uct into the culture medium.
8-1, using a 3T3 feeder layer) in DMEM/10% FCS (40 x 60 Mesenchymal cells were further characterized by analysis
magnification). The colonies of elongated shaped fibroblas of secreted cytokines and growth factors in comparison with
tic-like cells expanded rapidly from day 3 to day 7. It is noted human bone-marrow mesenchymal stem cells. The umbilical
in this respect, that the 3T3 feeder layer normally suppresses cord epithelial stem cells (UCEC) were analysed in compari
the growth of mesenchymal cells as human dermal fibro son with human epidermal keratinocytes. This analysis was
blasts. Once again, this indicates a difference in the behavior 65 carried out as follows: Briefly, UCMC, UCEC, dermal fibro
of the mesenchymal cells of the invention as compared to blasts, bone-marrow mesenchymal cells, epidermal kerati
more differentiated counterparts. nocytes were cultured in growth media until 100% confluence
US 9,085,755 B2
17 18
(37° C., 5% CO) and then synchronized in starvation UCMC cells is different depending on the ratio or proportion
medium (serum-free DMEM) for 48 hours. The next day, the of cytokines or growth factors present in the respective media.
medium was replaced the next against fresh serum-free In contrast, bone marrow and adipose-derived mesenchymal
DMEM and the cells then were cultivated for another 48 cells did not grow well in these serum-free media (FIG. 13-6
hours. Conditioned media were collected, concentrated and 5 and FIG. 13-7). Accordingly, the good growth of the UCMC
analyzed using a Cytokine Array (RayBiotech, Inc., GA, demonstrates the robustness of the cells of the invention and
USA). their high viability, indicating that their growth characteris
The results of this analysis show that UCMC secrete Inter tics are Superior to conventional sources of mesenchymal
leukin-6 (IL-6); (MCP1); hepatocyte growth factor (HGF); stem cells as bone marrow derived and adipose-derived mes
Interleukin-8 (IL8); sTNFR1; GRO, TIMP1: TIMP2: 10 enchymal cells. In this respect, it is worth to note that (bovine)
TRAILR3; uPAR: ICAM1; IGFBP3; IGFBP6 (FIG. 11), serum free medium was used in these experiments and that
whereas UCEC secrete IGFBP-4: PARC: EGF: IGFBP-2: the majority of human mesenchymal cells do not grow well in
IL-6: Angiogenin; GCP-2: IL1RC.: MCP-1; RANTES: SCF; serum-free medium systems. Thus, using the cells of the
TNFB; HGF; 1L8; STNFR: GRO; GRO-C: Amphiregulin; invention in connection with defined serum-free media tech
IL-1R4/ST2: TIMP1: TIMP2; uPAR, VEGF (FIG. 12). 15 nologies is a big advantage in cell therapy as the risks of using
Accordingly, this shows that both cells types secrete large fetal bovine serum for cell culture and expansion are
amounts of cytokines and growth factors that play important removed. (Although use of bovine serum has been practiced
roles in developmental biology, tissue homeostasis, tissue for a long time and typically optimizes cell growth, concerns
repair and regeneration and angiogenesis. This further dem of its used have been raised as to the transmission of Zoonoses
onstrates the versatility of the cells of the invention for use in as Bovine Spongiform Encephalopathy (Mad Cow Disease)).
the respective therapeutic applications.
In addition, the cells of the invention were further exam Example 5
ined with respect to their safety profile using mouse teratoma
formation assay as an indicator. Six SCID mice were used in Characterization of the Gene Expression Profile of
these experiments. A Suspension of more than 2 million 25 Umbilical Cord Epithelial and Mesenchymal Stem
UCMC was injected with a sterile 25G needle into the thigh Cells
muscle of each SCID mouse. Animals were kept up to 6
months and tumor formation was assessed. No tumor forma The gene expression profile of umbilical cord epithelial
tion was observed in these mice (data not shown). This indi and mesenchymal stem cells was analyzed using a DNA
cates that the cells of the invention are safe and do not have 30 microarray. For this purpose, UCMC and UCEC were cul
any capability to form tumors, benign or otherwise. tured in growth media at 37° C., 5% CO, until 100% conflu
The UCMC were also analysed for their expression of human ence. Cells were synchronized in basal media for 48 hours
leukocyte antigen (HLA) molecules. When testing on major then replaced with fresh basal media for another 48 hours.
histocompatibility complex (MHC) class I molecules, this Total RNA was harvested and sent to Silicon Genetics
analysis showed that HLA-A molecules were present in high 35 Microarray Service. Data analysis was performed using
number (test result in arbitrary unit: 3201), meaning that the GeneSpring 7.2). FIG. 14 Summarizes the global gene expres
cells are HLA-A positive whereas expression of HLA-B mol sion. UCEC expressed a total of 28055 genes and UCMC
ecules was insignificant (test result in arbitrary units: 35), expressed a total of 34407 genes. There are 27308 overlap
meaning the cells are HLA-B negative. As HLA-B is mainly ping genes expressing in both cell types. 747 genes expressed
responsible for rejection reaction in transplantation, this 40 were unique to UCEC and 7099 genes expressed were unique
result indicates that the cells of the invention are not only to UCMC. The selected genes of interest are presented in FIG.
Suitable for autologous transplantation but also for allogeneic 14.
transplantation. The cells were tested positive for Class II Both stem cell types expressed 140 genes related to embry
MHC molecule HLA-DR52 and tested negative for Class II onic stem cells and embryonic development, further Support
MHC molecule HLA-DRB4. HLA-DRB1 was also found to 45 ing that the cells of the invention have embryonic stem cell
be present (0301/05/20/22, like properties: Nanog: Alpha-fetal protein; Pre-B-cell
leukemia transcription factor 3; Laminin alpha 5; Carcino
Example 4 embryonic antigen-like 1; abhydrolase domain containing 2;
Delta-like 3 (Drosophila); Muscleblind-like (Drosophila);
Cultivation of Stem/Progenitor Cells in Serum Free 50 GNAS complex locus; Carcinoembryonic antigen-related
Media cell adhesion molecule 3: Palmitoyl-protein thioesterase 2:
Pregnancy specific beta-1-glycoprotein 2; Carcinoembryonic
UCMC cells were cultured in DMEM containing 10 FCS antigen-like 1; Embryonic ectoderm development; Maternal
and in serum-free media, PTT-1, PTT-2 and PTT-3. The three embryonic leucine Zipper kinase; Chorionic Somatomam
media PTT-1, PTT-2 and PTT-3 were prepared by one of the 55 motropin hormone 2: Forkhead box D3; radical fringe
present inventors, Dr Phan. In brief, these 3 media do not homolog (Drosophila); Kinesin family member 1B: Myosin,
contain fetal bovine or human serum, but contain different heavy polypeptide 3, skeletal muscle, embryonic; Split hand/
cytokines and growth factors such as IGF. EGF, TGF-beta, foot malformation (ectrodactyly) type 3: TEA domain family
Activin A, BMPs, PDGF, transferrin, and insulin. The growth member 3; Laminin, alpha 1: Chorionic Somatomammotro
factor components vary between media to assess differential 60 pin hormone 1; placental lactogen; Corticotropin releasing
growth characteristics. The cultivation was carried out as hormone receptor 1; thyrotrophic embryonic factor; Aryl
follows: Different proportions of growth factors and cytok hydrocarbon receptor nuclear translocator 2: Membrane
ines were added in basal media. UCMC were thawed and frizzled-related protein; Neuregulin 1'Collagen, type XVI,
maintained in these media for 10 days. Cell proliferation was alpha 1: Neuregulin 1: Chorionic Somatomammotropin hor
monitored under light microscopy. 65 mone 1 (placental lactogen); CUG triplet repeat, RNA bind
FIG.13 shows good UCMC growth in the 4 different media ing protein 1: Chorionic Somatomammotropin hormone 1
groups (FIG. 13-1 to FIG. 13-5), wherein the morphology of (placental lactogen) Bystin-like: MyoD family inhibitor; Ret
US 9,085,755 B2
19 20
inoic acid induced 2: GNAS complex locus; Pre-B-cell leu WNTs, SDFs, OncostatinM, Interleukins, Chemokines and
kemia transcription factor 4: Laminin, alpha 2 (merosin, con many others); MMPs, TIMPs extracellular matrices (col
genital muscular dystrophy); SMAD, mothers against DPP lagens, laminins, fibronectins, vitronectins, tenascins, inter
homolog 1 (Drosophila); Homo sapiens transcribed sequence grins, Syndecans, decorin, fibromolludin, proteoglycans,
with moderate similarity to protein pir:D28928 (H. sapiens) sparcfosteonectin, mucin, netrin, glypican, cartilage associ
D28928 pregnancy-specific beta-1 glycoprotein IB, abor ated protein, matrilin, hyaluronan, fibulin, ADAMTS, bigly
tive human (fragment); Kinesin family member 1B: Bruno can, discoidin, desmosome components, ICAMs, cadherins,
like 4, RNA binding protein (Drosophila); Embryo brain catenins and many others); cytokeratins.
specific protein; Pregnancy-induced growth inhibitor; There are groups of genes present only in UCMC. These
SMAD, mothers against DPP homolog 5 (Drosophila); 10 genes are related to the following: Normal Physiological Pro
Chorionic Somatomammotropin hormone 2: Adenylate cesses (Insulin-like growth factor 1 (Somatomedin C); Insu
cyclase activating polypeptide 1 (pituitary); Carcinoembry lin-like 4 (placenta); Relaxin 1: Plasminogen; Insulin-like
onic antigen-related cell adhesion molecule; Laminin, alpha growth factor 1 (somatomedin C); Insulin-like 5: Insulin-like
3: Protein O-fucosyltransferase 1: Jagged 1 (Alagile syn growth factor 1 (somatomedin C); Insulin-like growth factor
drome); Twisted gastrulation homolog 1 (Drosophila); ELAV 15 2 (Somatomedin A). Homeostasis (Radial spokehead-like 1;
(embryonic lethal, abnormal vision, Drosophila)-like 3 (Hu Hemochromatosis; Chemokine (C-C motif) ligand 5; Inter
antigen C); Thyrotrophic embryonic factor; Solute carrier leukin 31 receptor A: Chemokine (C-X-C motif) ligand 12
family 43, member 3: Inversin; nephronophthisis 2 (infan (stromal cell-derived factor 1); Nuclear receptor subfamily 3,
tile); inversion of embryonic turning; Homo sapiens inversin group C, member 2: Hemochromatosis: Chemokine (C-C
(INVS), transcript variant 2, mRNA: Homo sapiens tran motif) ligand 23; Chemokine (C-C motif) ligand 23; Ferritin
scribed sequences; Homeo box D8; Embryonal Fyn-associ mitochondrial; Peroxisome proliferative activated receptor,
ated substrate; ELAV (embryonic lethal, abnormal vision, gamma, coactivator 1, alpha; Surfactant, pulmonary-associ
Drosophila)-like 1 (Hu antigen R); Basic helix-loop-helix ated protein D; Chemokine (C-C motif) ligand 11; Chemok
domain containing, class B, 2: Oxytocin receptor, Teratocar ine (C-C motif) ligand 3; Egl nine homolog 2 (C. elegans);
cinoma-derived growth factor 1: Fms-related tyrosine kinase 25 Peroxisome proliferative activated receptor, gamma, coacti
1 (vascular endothelial growth factor/vascular permeability vator 1, beta; Chemokine (C-C motif) ligand 1: Chemokine
factor receptor); Adrenomedullin: Nuclear receptor coactiva (C-X-C motif) ligand 12 (stromal cell-derived factor 1);
tor 6-CUG triplet repeat, RNA binding protein 1; Twisted ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide;
gastrulation homolog 1 (Drosophila); Carcinoembryonic Chemokine (C motif) ligand 2: Hemopexin; Ryanodine
antigen-related cell adhesion molecule 4:Protein tyrosine 30 receptor 3), Morphogenesis (Spectrin, alpha, erythrocytic 1
phosphatase, receptor type, R: Acrg embryonic lethality (elliptocytosis 2); Homeo box D3; Eyes absent homolog 1
(mouse) minimal region ortholog; EPH receptor A3:Delta (Drosophila); Ras homolog gene family, member J. Leuko
like 1 (Drosophila); Nasal embryonic LHRH factor; Tran cyte specific transcript 1; Ectodysplasin A2 receptor, Glypi
scription factor CP2-like 1: Split hand/foot malformation can 3: Paired box gene7: Corin, serine protease; Dishevelled,
(ectrodactyly) type 3: Jagged 2; Homo sapiens transcribed 35 dish homolog 1 (Drosophila); Ras homolog gene family,
sequence; Neuregulin 1: Split hand/foot malformation (ectro member J; T-box 3 (ulnar mammary syndrome); Chondroitin
dactyly) type 1: Solute carrier family 43, member 3: beta 1.4 N-acetylgalactosaminyltransferase; Chondroitin
Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Co beta 1.4 N-acetylgalactosaminyltransferase: SRY (sex deter
enzyme A thiolase/enoyl-Coenzyme A hydratase (trifunc mining regionY)-box 10, Myosin, heavy polypeptide 9, non
tional protein), alpha Subunit, Fucosyltransferase 10 (alpha 40 muscle; Luteinizing hormone/choriogonadotropin receptor;
(1.3) fucosyltransferase); Acrg embryonic lethality (mouse) radical fringe homolog (Drosophila); Secreted frizzled-re
minimal region ortholog: Carcinoembryonic antigen-related lated protein 5: Wingless-type MMTV integration site family,
cell adhesion molecule 7: Nucleophosmin/nucleoplasmin, 2. member 11; Eyes absent homolog 2 (Drosophila); Mus
Fc fragment of IgG, receptor, transporter, alpha; Twisted gas cleblind-like (Drosophila); T-box 5: Mab-21-like 1 (C.
trulation homolog 1 (Drosophila); Homo sapiens similar to 45 elegans); Growth arrest-specific 2: Sex comb on midleg
vacuolar protein sorting 35, maternal-embryonic 3 homolog 1 (Drosophila); T-box 6: Filamin-binding LIM pro
(LOC146485), mRNA:abhydrolase domain containing 2: T. tein-1; Melanoma cell adhesion molecule; Twist homolog 1
brachyury homolog (mouse); A disintegrin and metallopro (acrocephalosyndactyly 3: Saethre-Chotzen syndrome)
teinase domain 10: Ribosomal protein L29; Endothelin con (Drosophila); Homeobox A11; Keratocan; Fibroblast growth
verting enzyme 2: ELAV (embryonic lethal, abnormal vision, 50 factor 1 (acidic); Carboxypeptidase M. CDC42 effector pro
Drosophila)-like 1 (Huantigen R); Trophinin; Homeo box tein (Rho GTPase binding) 4: LIM homeobox transcription
B6; Laminin, alpha 4: Homeo box B6; hypothetical protein factor 1, beta; Engrailed homolog 1; Carboxypeptidase M:
FLJ13456; NACHT, leucine rich repeat and PYD containing Fibroblast growth factor 8 (androgen-induced); Fibroblast
5; ELAV (embryonic lethal, abnormal vision, Drosophila)- growth factor 18; Leukocyte specific transcript 1; Endothelin
like 1 (Huantigen R); Undifferentiated embryonic cell tran 55 3: Paired-like homeodomain transcription factor 1), Embry
Scription factor 1; Pregnancy-associated plasma protein A, onic Development (Pregnancy specific beta-1 -glycoprotein
pappalysin 1: Secretoglobin, family 1A, member 1 (uteroglo 3: ELAV (embryonic lethal, abnormal vision, Drosophila)-
bin); Parathyroid hormone-like hormone; Carcinoembryonic like 4 (Huantigen D); G protein-coupled receptor 10; Ecto
antigen-related cell adhesion molecule 1 (biliary glycopro dysplasin A2 receptor; ATP-binding cassette, sub-family B
tein); Laminin, alpha 1. 60 (MDR/TAP), member 4: Pregnancy specific beta-1 -glyco
Both stem cell types also expressed thousands of genes protein 11; Nasal embryonic LHRH factor; Relaxin 1: Notch
related to developmental biology, cell growth and differen homolog 4 (Drosophila); Pregnancy specific beta-1-glyco
tiation, cell homeostasis, cell and tissue repair and regenera protein 6: pih-2P; Homo sapiens pregnancy-induced hyper
tion. Examples of such growth factors and their receptors is as tension syndrome-related protein (PIH2); Oviductal glyco
follows: (G-CSF, FGFs, IGFs, KGF, NGF, VEGFs, PIGF, 65 protein 1, 120 kDa (mucin 9, oviductin); Progestagen
Angiopoietin, CTGF, PDGFs, HGF, EGF, HDGF, TGF-beta, associated endometrial protein; Myosin, light polypeptide 4.
Activins and Inhibins, Follistatin, BMPs, SCF/c-Kit, LIF, alkali; atrial, embryonic; Prolactin: Notch homolog 4 (Droso
US 9,085,755 B2
21 22
phila); Pre-B-cell leukemia transcription factor 1; radical (vascular endothelial growth factor/vascular permeability
fringe homolog (Drosophila); Corticotropin releasing hor factor receptor); Dystonin; Insulin-like 4 (placenta); Transco
mone; Nuclear receptor Subfamily 3, group C, member 2; balamin II; macrocytic anemia; Chemokine (C-C motif)
Neuregulin 2: Muscleblind-like (Drosophila); Myosin, light ligand 1; Insulin-like growth factor binding protein, acid
polypeptide 4, alkali; atrial, embryonic; Homo sapiens cDNA labile subunit; Complement factor H; Pregnancy specific
FLJ27401 fis, clone WMCO3071; Extraembryonic, sper beta-1-glycoprotein 6: Silver homolog (mouse); Proteogly
matogenesis, homeobox 1-like; Insulin-like 4 (placenta); can 4: Fibroblast growth factor 16: Cytokine-like protein
Human processed pseudo-pregnancy-specific glycoprotein C17: Granulysin; Angiopoietin 2; Chromogramin B (secre
(PSG12) gene, exon B2C containing 3' untranslated regions togranin 1); Sema domain, immunoglobulin domain (Ig), and
of 2 alternative splice sites C1 and C2: Fms-related tyrosine 10 GPI membrane anchor, (semaphorin) 7A: Pleiotrophin (hep
kinase 1 (vascular endothelial growth factor/vascular perme arin binding growth factor 8, neurite growth-promoting factor
ability factor receptor); Pre-B-cell leukemia transcription 1); Chloride channel, calcium activated, family member 3:
factor 1; Pregnancy specific beta-1-glycoprotein 3; carcino Secretoglobin, family 1 D, member 1: Fibulin 1: Phospholi
embryonic antigen-related cell adhesion molecule 1 (biliary pase A2 receptor 1, 180 kDa), and the Extracellular Matrix
glycoprotein); Steroid Sulfatase (microsomal), arylsulfatase 15 (ADAMTS-like 1: Periostin, osteoblast specific factor;
C, isozyme S; Homeobox B6; Protein O-fucosyltransferase Glypican 5; Leucine rich repeat neuronal 3; Transglutami
1: LIM homeobox transcription factor 1, beta; Carcinoem nase 2 (C polypeptide, protein-glutamine-gamma-glutamyl
bryonic antigen-related cell adhesion molecule 1 (biliary gly transferase); A disintegrin-like and metalloprotease (reprol
coprotein); Follicle stimulating hormone, beta polypeptide; ysin type) with thrombospondin type 1 motif. 2;
Angiotensinogen (serine (or cysteine) proteinase inhibitor, Microfibrillar-associated protein 4: Glypican 3: Collagen,
clade A (alpha-1 antiproteinase, antitrypsin), member 8); type V, alpha 3: Tissue inhibitor of metalloproteinase 2: Kera
Carcinoembryonic antigen-related cell adhesion molecule 6 tocan; Cartilage oligomeric matrix protein; Lumican; Hyalu
(non-specific cross reacting antigen); Protein kinase C, alpha roman and proteoglycan link protein 3: Statherin; A disinte
binding protein; Collectin sub-family member 10 (C-type grin-like and metalloprotease (reprolysin type) with
lectin); Laminin, alpha 1), the Extracellular Space (Carboxy 25 thrombospondin type 1 motif, 3: Spondin 1, extracellular
lesterase 1 (monocyte/macrophage serine esterase 1); Fibro matrix protein; Chitinase 3-like 1 (cartilage glycoprotein-39);
blast growth factor 5: Progastricsin (pepsinogen C); Sperm Collagen, type IV, alpha 3 (Goodpasture antigen); Wingless
associated antigen 11; Proprotein convertase Subtilisin/kexin type MMTV integration site family, member 7B; Collagen,
type 2: Hyaluronan binding protein 2; Sema domain, immu type VI, alpha 2: Lipocalin 7: Hyaluronan and proteoglycan
noglobulin domain (Ig), short basic domain, secreted. (Sema 30 link protein 4: A disintegrin-like and metalloprotease (reprol
phorin)3F: Interleukin 2: Chymotrypsin-like: Norrie disease ysin type) with thrombospondin type 1 motif, 5 (aggrecanase
(pseudoglioma); mucin 5, subtypes A and C, tracheobron 2); Fibronectin 1: Matrilin 1, cartilage matrix protein: Hypo
chial/gastric; Carboxypeptidase B2 (plasma, carboxypepti thetical protein FLJ13710; Chondroitin beta 1.4
dase U); radical fringe homolog (Drosophila); Pregnancy N-acetylgalactosaminyltransferase; Matrix metalloprotein
specific beta-1-glycoprotein 11; MeprinA, alpha (PABA pep 35 ase 16 (membrane-inserted); Von Willebrand factor; Col
tide hydrolase); Tachykinin, precursor 1 (Substance K. Sub lagen, type VI, alpha 2: Transmembrane protease, serine 6:
stance P. neurokinin 1, neurokinin 2, neuromedin L, neuro Matrix metalloproteinase 23B; Matrix metalloproteinase 14
kinin alpha, neuropeptide K, neuropeptide gamma); (membrane-inserted); Leucine rich repeat neuronal 3:
Fibroblast growth factor 8 (androgen-induced); Fibroblast SPARC-like 1 (mast9, hevin); Sparcfosteonectin, cwcv and
growth factor 13, Hemopexin; Breast cancer 2, early onset; 40 kazal-like domains proteoglycan (testican) 3: Dermatopon
Fibroblast growth factor 14: Retinoschisis (X-linked, juve tin; collagen, type XIV, alpha 1 (undulin); Amelogenin,
nile) 1: Chitinase 3-like 1 (cartilage glycoprotein-39); Dys Y-linked; Nidogen (enactin); ADAMTS-like 2: Hyaluronan
tonin; Secretoglobin, family 1D, member 2: Noggin: WAP and proteoglycan link protein 2; Collagen, type XV, alpha 1:
four-disulfide core domain 2; CD5 antigen-like (Scavenger Glypican 6: Matrix metalloproteinase 12 (macrophage
receptor cysteine rich family); Scrapie responsive protein 1: 45 elastase); Amelogenin (amelogenesis imperfecta 1.
Gremlin 1 homolog, cysteine knot Superfamily (Xenopus lae X-linked); A disintegrin-like and metalloprotease (reprolysin
vis); Interleukin 16 (lymphocyte chemoattractant factor); type) with thrombospondin type 1 motif, 15: Transmembrane
Chemokine (C-C motif) ligand 26; Nucleobindin 1: Fibro protease, serine 6: A disintegrin-like and metalloprotease (re
blast growth factor 18: Insulin-like growth factor binding prolysin type) with thrombospondin type 1 motif, 16; Sparc/
protein 1: Surfactant, pulmonary-associated protein A1, 50 osteonectin, cwcV and kazal-like domains proteoglycan (tes
Delta-like 1 homolog (Drosophila); Cocaine- and amphet tican); A disintegrin-like and metalloprotease (reprolysin
amine-regulated transcript; Meprin A, beta; Interleukin 17F: type) with thrombospondin type 1 motif, 20; Collagen, type
Complement factor H. Cysteine-rich secretory protein 2: XI, alpha 1: Hyaluronan and proteoglycan link protein 1:
Dystonin; WAP four-disulfide core domain 1: Prolactin; Sur Chondroitin beta 1.4 N-acetylgalactosaminyltransferase;
factant, pulmonary-associated protein B; Fibroblast growth 55 Asporin (LRR class 1); Collagen, type III, alpha 1 (Ehlers
factor 5; Dickkopfhomolog 2 (Xenopus laevis); Sperm asso Danlos syndrome type IV, autosomal dominant); Secreted
ciated antigen 11; Chemokine (C-C motif) ligand 11; Meprin phosphoprotein 1 (osteopontin, bone sialoprotein I, early
A, alpha (PABA peptide hydrolase); Chitinase 3-like 2: C-fos T-lymphocyte activation 1); Matrix Gla protein; Fibulin 5:
induced growth factor (vascular endothelial growth factor D): collagen, type XIV, alpha 1 (undulin); Tissue inhibitor of
Chemokine (C-C motif) ligand 4: Poliovirus receptor; Hyalu 60 metalloproteinase 3 (Sorsby fundus dystrophy, pseudoin
ronoglucosaminidase 1: Oviductal glycoprotein 1, 120 kDa flammatory); Collagen, type XXV, alpha 1: Cartilage oligo
(mucin 9, oviductin); Chemokine (C-X-C motif) ligand 9: meric matrix protein; Collagen, type VI, alpha 1: Chondroad
Secreted frizzled-related protein 5: Amelogenin (amelogen herin; Collagen, type XV, alpha 1: A disintegrin-like and
esis imperfecta 1, X-linked); Relaxin 1: Sparcfosteonectin, metalloprotease (reprolysin type) with thrombospondin type
cwcV and kazal-like domains proteoglycan (testican); 65 1 motif, 16: Collagen, type IV, alpha 4: Dentin matrix acidic
Chemokine (C-C motif) ligand 26; Fibroblast growth factor 1 phosphoprotein; Collagen, type IV, alpha 1: Thrombospondin
(acidic); Angiopoietin-like 2; Fms-related tyrosine kinase 1 repeat containing 1; Matrix metalloproteinase 16 (mem
US 9,085,755 B2
23 24
brane-inserted); Collagen, type I, alpha 2: Fibulin 1; Tectorin lated cysteine protease; Kelch repeat and BTB (POZ) domain
beta; Glycosylphosphatidylinositol specific phospholipase containing 10; Mucin 1, transmembrane; Microtubule-asso
D1; Upregulated in colorectal cancer gene 1). Cytoskeleton: ciated protein tau; Tensin; Rashomologgene family, member
(Filamin B, beta (actin binding protein 278); Centrin, EF F (in filopodia); Adducin 1 (alpha); Actinin, alpha 4: Eryth
hand protein, 1: FERM domain containing 3: Bridging inte rocyte membrane protein band 4.1 (elliptocytosis 1.
grator 3; Parvin, gamma; Rho guanine nucleotide exchange RH-linked); Bicaudal D homolog 2 (Drosophila); Ankyrin 3,
factor (GEF) 11: Tyrosine kinase 2: Kelch-like 4 (Droso node of Ranvier (ankyrin G); Myosin VIIA (Usher syndrome
phila); Spectrin, beta, erythrocytic (includes spherocytosis, 1B (autosomal recessive, severe)); Catenin (cadherin-associ
clinical type I); Arg/Abl-interacting protein ArgBP2: Advil ated protein), alpha2; Homo sapiens similar to keratin 8, type
lin; Spectrin repeat containing, nuclear envelope 1; Catenin 10 II cytoskeletal-human (LOC285233); Fascin homolog 3.
(cadherin-associated protein), delta 1; Erythrocyte mem actin-bundling protein, testicular, Ras homolog gene family,
brane protein band 4.1 like 5: Catenin (cadherin-associated member J. Beaded filament structural protein 2, phakinin;
protein), alpha2; Chemokine (C-C motif) ligand 3; Sarcogly Desmin, Myosin X; Signal-induced proliferation-associated
can, gamma (35kDa dystrophin-associated glycoprotein); gene 1: Scinderin; Coactosin-like 1 (Dictyostelium); Engulf
Nebulin; Thymosin, beta, identified in neuroblastoma cells; 15 ment and cell motility 2 (ced-12 homolog, C. elegans); Tubu
3-phosphoinositide dependent protein kinase-1; Wiskott-Al lin, beta 4: Ca"-dependent secretion activator, FERM
drich syndrome protein interacting protein; Dystonin; Hun domain containing 4A, Actin, alpha 1, skeletal muscle; Talin
tingtin interacting protein 1; KIAA0316 gene product; Tro 1; Caldesmon 1: Filamin-binding LIM protein-1; Microtu
pomodulin 4 (muscle); Deleted in liver cancer 1; Villin-like: bule-associated protein tau; Syntrophin, alpha 1 (dystrophin
Syntrophin, beta 1 (dystrophin-associated protein A1, 59 associated protein A1, 59 kDa, acidic component); Adducin2
kDa, basic component 1); Protein kinase, c0MP-dependent, (beta); Filamin A interacting protein 1; PDZ and LIM domain
type I: Homo sapiens similar to keratin 8: cytokeratin 8: 3: Erythrocyte membrane protein band 4.1 like 4B; FYN
keratin, type II cytoskeletal 8 (LOC345751), mRNA: Addu binding protein (FYB-120/130); Bridging integrator 3).
cin 1 (alpha); Protein kinase C and casein kinase Substrate in Extracellular. (A disintegrin-like and metalloprotease (re
neurons 3: Dystonin; Kellblood group; FilaminA interacting 25 prolysin type) with thrombospondin type 1 motif, 20;
protein 1: Growth arrest-specific 2: Chromosome 1 open SPARC-like 1 (mast9, hevin); Serine (or cysteine) proteinase
reading frame 1: Stathmin-like 2: Spectrin, alpha, erythro inhibitor, clade G (C1 inhibitor), member 1, (angioedema,
cytic 1 (elliptocytosis 2); FKSG44 gene; Kinesin family hereditary); Urocortin; Chymotrypsin-like: Platelet-derived
member 1C: Tensin; Kaptin (actin binding protein); Neurofi growth factor beta polypeptide (simian sarcoma viral (v-sis)
bromin 2 (bilateral acoustic neuroma); Pleckstrin homology, 30 oncogene homolog); BMP-binding endothelial regulator pre
Sec7 and coiled-coil domains 2 (cytohesin-2); Actin-related cursor protein; Complement factor H. Chorionic
protein T1: Wiskott-Aldrich syndrome-like: Kelch-like 4 somatomammotropin hormone-like 1; Chemokine (C-C
(Drosophila); Fascin homolog 1, actin-bundling protein motif) ligand 18 (pulmonary and activation-regulated);
(Strongylocentrotus purpuratus); Amphiphysin (Stiff-Man Fibronectin 1: Pregnancy specific beta-1-glycoprotein 3: A
syndrome with breast cancer 128kDa autoantigen); Polycys 35 disintegrin-like and metalloprotease (reprolysin type) with
tic kidney disease 2-like 1; Ankyrin 2, neuronal; CDC42 thrombospondin type 1 motif, 3: CocoaCrisp. Insulin-like 4
binding protein kinase alpha (DMPK-like); Hypothetical pro (placenta); Wingless-type MMTV integration site family,
tein FLJ36144: Arg/Abl-interacting protein ArgBP2: member 11, Cartilage oligomeric matrix protein; Transmem
Formin-like 3: Catenin (cadherin-associated protein), beta 1, brane protease, serine 6: C-fos induced growth factor (vascu
88 kDa. Profilin 2: Synaptopodin 2-like: Syntrophin, gamma 40 lar endothelial growth factor D); Family with sequence simi
2: Phospholipase D2: Engulfment and cell motility 2 (ced-12 larity 12, member B (epididymal); Protein phosphatase 1,
homolog, C. elegans); Neurofilament, light polypeptide 68 regulatory subunit 9B, spinophilin; Transcobalamin II; mac
kDa; Dystonin; Actin-like 7B; Kinesin family member 1C: rocytic anemia; Coagulation factor V (proaccelerin, labile
PDZ and LIM domain 3: Adducin 2 (beta); obscurin, cytosk factor); Phospholipase A2, group IID; Tumor necrosis factor,
eletal calmodulin and titin-interacting RhoGEF; Tubulin, 45 alpha-induced protein 6: Collagen, type XV, alpha 1; Hyalu
beta polypeptide paralog: Filamin A interacting protein 1: roman and proteoglycan link protein 3; collagen, type XIV,
Talin 1; Homo sapiens similar to Segment 1 of 2 Piccolo alpha 1 (undulin) ; Interleukin 19; Protease inhibitor 15;
protein (Aczonin) (LOC375597); CDC42 effector protein Cholinergic receptor, nicotinic, beta polypeptide 1 (muscle);
(Rho GTPase binding) 4: Syndecan 1: FilaminA, alpha (actin Lysyl oxidase-like 3: Insulin-like growth factor binding pro
binding protein 280); Profilin 2: Tensin like C1 domain con 50 tein 5: Growth hormone 1: Casein beta; NEL-like 2 (chicken):
taining phosphatase: Hypothetical protein MGC33407; Rho I factor (complement); Chemokine (C-C motif) ligand 23;
family GTPase 1: Flavoprotein oxidoreductase MICAL2: Interferon, alpha 2: Matrix metalloproteinase 16 (membrane
Ca2+-dependent secretion activator; Rabphilin 3A-like inserted); Matrix metalloproteinase 12 (macrophage
(without C2 domains); Myosin XVA; Protein kinase, c0MP elastase); Glypican 5: Pregnancy specific beta-1-glycopro
dependent, type I: Myosin regulatory light chain interacting 55 tein 3: Fibroblast growth factor 6: Gremlin 1 homolog, cys
protein; Kinesin family member 13B; Muscle RAS oncogene teine knot Superfamily (Xenopus laevis); Protein S (alpha);
homolog: Spectrin, beta, non-erythrocytic 1: TAOkinase 2; Chondroitin beta 1.4 N-acetylgalactosam inyltransferase;
Filamin B, beta (actin binding protein 278); Neurofibromin 2 Glycosylphosphatidylinositol specific phospholipase D1;
(bilateral acoustic neuroma); Catenin (cadherin-associated Fibroblast growth factor 1 (acidic); Spondin 1, extracellular
protein), alpha 3: obscurin, cytoskeletal calmodulin and titlin 60 matrix protein; Bone morphogenetic protein 1: Surfactant,
interacting RhoGEF: Coronin, actin binding protein, 1A: pulmonary-associated protein B; Dentin matrix acidic phos
Erythrocyte membrane protein band 4.1-like 1: Spectrin, phoprotein; Lipoprotein, Lp(a); Mucin 1, transmembrane;
beta, non-erythrocytic 4: Thymosin, beta4, Y-linked: Tektin2 Mannan-binding lectin serine protease 1 (C4/C2 activating
(testicular); Ras homolog gene family, member J. Serine/ component of Ra-reactive factor); Meprin A, beta; Secreto
threonine kinase with Dbl- and pleckstrin homology 65 globin, family 1D, member 1: Asporin (LRR class 1);
domains; Dystrobrevin, beta; Actin, gamma 2, Smooth Chemokine (C-C motif) ligand 25; Cytokine-like protein
muscle, enteric: Tara-like protein; Caspase 8, apoptosis-re C17: Insulin-like 5: Meprin A, alpha (PABA peptide hydro
US 9,085,755 B2
25 26
lase); Scrapie responsive protein 1: Fibroblast growth factor nent (hemophilia A); Dermatopontin; Noggin; Secreted LY6/
18; Chemokine (C-X-C motif) ligand 9; Inhibin, beta B (ac PLAUR domain containing 1: ADAMTS-like 1: Alpha-1-B
tivin AB beta polypeptide); Fibroblast growth factor 8 (an glycoprotein; Chromosome 20 open reading frame 175;
drogen-induced); Granulysin; Cocaine- and amphetamine Wingless-type MMTV integration site family, member 8B;
regulated transcript; Collagen, type I, alpha 2: Chemokine Fibulin 1: Fibulin 5: Cathepsin S: Nidogen (enactin):
(C-C motif) ligand 17: Chemokine (C-C motif) ligand 23; Chemokine (C-C motif) ligand 26; Endothelial cell-specific
Sparcfosteonectin, cwcV and kazal-like domains proteogly molecule 1: Chitinase 3-like 1 (cartilage glycoprotein-39);
can (testican) 3; Gamma-aminobutyric acid (GABA) A Gamma-aminobutyric acid (GABA) A receptor, beta 1:
receptor, beta3; Defensin, alpha 4, corticostatin: Leucine rich Secretoglobin, family 1D, member 2: Mannan-binding lectin
repeat neuronal 3; Glypican 6: Mitogen-activated protein 10 serine protease 1 (C4/C2 activating component of Ra-reactive
kinase kinase 2; Coagulation factor XI (plasma thromboplas factor); ADAMTS-like 1: Sema domain, immunoglobulin
tin antecedent); Chemokine (C-C motif) ligand 5; Dystonin; domain (Ig), and GPI membrane anchor, (Semaphorin) 7A. A
Frizzled-related protein; Coagulation factor XIII, A1 disintegrin-like and metalloprotease (reprolysin type) with
polypeptide; Insulin-like growth factor 1 (Somatomedin C); thrombospondin type 1 motif, 15: Proprotein convertase sub
Hypothetical protein MGC45438: Sperm associated antigen 15 tilisin/kexin type 2: Insulin-like growth factor 1 (Somatome
11; Insulin-like growth factor 1 (somatomedin C); Periostin, din C); Retinoschisis (X-linked, juvenile) 1: A disintegrin
osteoblast specific factor; Alpha-2-macroglobulin; Gamma like and metalloprotease (reprolysin type) with
aminobutyric acid (GABA) A receptor, alpha 5: Serine (or thrombospondin type 1 motif, 16; Chemokine (C motif)
cysteine) proteinase inhibitor, clade A (alpha-1 antiprotein ligand 2: Fibroblast growth factor 5: Sperm associated anti
ase, antitrypsin), member 3; Silver homolog (mouse); gen 11; Microfibrillar-associated protein 4: Poliovirus recep
Frizzled-related protein; Chondroadherin; Chondroitin tor; Extracellular signal-regulated kinase 8: Transmembrane
beta1,4 N-acetylgalactosam inyltransferase; 5-hydrox protease, serine 6: Protein kinase C, alpha; Chitinase 3-like 2:
ytryptamine (serotonin) receptor 3, family member C. Col Interleukin 9; Apollipoprotein L. 6; Surfactant, pulmonary
lagen, type VI, alpha 2: Toll-like receptor 9; Amelogenin, associated protein A1, Collagen, type VI, alpha 1: Apollipo
Y-linked; Vascular endothelial growth factor B: Radial spoke 25 protein L. 6: Hypothetical protein FLJ13710; Carboxypepti
head-like 1: Fms-related tyrosine kinase 1 (vascular endothe dase B2 (plasma, carboxypeptidase U) ; Bactericidal/
lial growth factor/vascular permeability factor receptor); Pro permeability-increasing protein-like 2: Fibroblast growth
tease inhibitor 16; Interleukin 2: Clusterin (complement lysis factor 5: Secreted phosphoprotein 1 (osteopontin, bonesia
inhibitor, SP-40.40, sulfated glycoprotein 2, testosterone-re loprotein I, early T-lymphocyte activation 1): Htra serine
pressed prostate message 2, apolipoprotein J); Follicle stimu 30 peptidase 3: Deleted in liver cancer 1; Endothelial cell-spe
lating hormone, beta polypeptide; A disintegrin-like and met cific molecule 1; Von Willebrand factor; A disintegrin-like
alloprotease (reprolysin type) with thrombospondin type 1 and metalloprotease (reprolysin type) with thrombospondin
motif, 16; Lysozyme (renal amyloidosis); radical fringe type 1 motif, 5 (aggrecanase-2); Sema domain, immunoglo
homolog (Drosophila); Insulin-like growth factor binding bulin domain (Ig), short basic domain, secreted, (semaphorin)
protein 5: Taxilin; Apolipoprotein A-V: Platelet derived 35 3A; Chemokine (C-X-C motif) ligand 12 (stromal cell-de
growth factor C; Chemokine (C-C motif) ligand 3-like 1: rived factor 1); Statherin; Extracellular signal-regulated
Fibroblast growth factor 16; Collagen, type VI, alpha 2: kinase 8: Tissue inhibitor of metalloproteinase 3 (Sorsby
Serine (or cysteine) proteinase inhibitor, clade C (antithrom fundus dystrophy, pseudoinflammatory); Platelet factor 4
bin), member 1: Chemokine (C-C motif) ligand 11; Collagen, (chemokine (C-X-C motif) ligand 4); Surfactant, pulmonary
type IV, alpha 4; Bruton agammaglobulinemia tyrosine 40 associated protein D; Complement factor H; Delta-like 1
kinase; Insulin-like growth factor 2 (Somatomedin A); Kazal homolog (Drosophila); WAP four-disulfide core domain 1:
type serine protease inhibitor domain 1: Fibrinogen, Aalpha Insulin-like growth factor binding protein, acid labile sub
polypeptide; Chemokine (C-C motif) ligand 1; Inhibin, beta unit; Breast cancer 2, early onset; Pre-B lymphocyte gene 1:
E.; Sex hormone-binding globulin; Collagen, type IV, alpha 1: Corticotropin releasing hormone: Hypothetical protein
Lecithin-cholesterol acyltransferase: Cysteine-rich secretory 45 DKFZp434B044; Prolactin-induced protein: RAS guanyl
protein 2; Hyaluronan and proteoglycan link protein 1; Natri releasing protein 4: Progastricsin (pepsinogen C); Sema
uretic peptide precursor C; Ribonuclease, RNase A family, domain, immunoglobulin domain (Ig), short basic domain,
k6: Fibroblast growth factor 14; ADAMTS-like 2: Collagen, secreted, (semaphorin)3F: Upregulated in colorectal cancer
type IV, alpha 3 (Goodpasture antigen); Angiopoietin 2: Apo gene 1: Proteoglycan 4; Cholinergic receptor, nicotinic, delta
lipoprotein L, 3; Chemokine (C-X-C motif) ligand 12 (stro 50 polypeptide; Cartilage oligomeric matrix protein; ABO blood
mal cell-derived factor 1); Hyaluronan binding protein 2: group (transferase A, alpha 1-3-N-acetylgalactosaminyl
Coagulation factor VII (serum prothrombin conversion accel transferase; transferase B, alpha 1-3-galactosyltransferase);
erator); collagen, type XIV, alpha 1 (undulin); Oviductal gly Interleukin 12A (natural killer cell stimulatory factor 1, cyto
coprotein 1, 120 kDa (mucin 9, oviductin); Matrilin 1, carti toxic lymphocyte maturation factor 1, p35); Fibroblast
lage matrix protein; mucin 5. Subtypes A and C. 55 growth factor 7 (keratinocyte growth factor); Kin of IRRE
tracheobronchial/gastric; Tumor necrosis factor receptor like 3 (Drosophila); Cholinergic receptor, nicotinic, alpha
Superfamily, member 11b (osteoprotegerin); Transglutami polypeptide 2 (neuronal); Palate, lung and nasal epithelium
nase 2 (C polypeptide, protein-glutamine-gamma-glutamyl carcinoma associated; Collagen, type XV, alpha 1: Pleiotro
transferase); Keratocan; Collagen, type V, alpha 3: WAP four phin (heparin binding growth factor 8, neurite growth-pro
disulfide core domain 2: Chemokine (C-X3-C motif) ligand 60 moting factor 1); Angiopoietin-like 2: Norrie disease
1: Serine (or cysteine) proteinase inhibitor, clade D (heparin (pseudoglioma); Chemokine (C-C motif) ligand 3; Chitinase
cofactor), member 1: Secretory protein LOC348174; Coagul 3-like 1 (cartilage glycoprotein-39); Inter-alpha (globulin)
lation factor X; Interleukin 16 (lymphocyte chemoattractant inhibitor H3; Amelogenin (amelogenesis imperfecta 1.
factor); Pancreatic lipase-related protein 2: Htra serine pep X-linked); Epidermal growth factor (beta-urogastrone);
tidase 3: Glycine receptor, alpha 3: CD5 antigen-like (scav 65 Fibroblast growth factor 13; Wingless-type MMTV integra
enger receptor cysteine rich family); Hypothetical protein tion site family, member 7B: Cholinergic receptor, nicotinic,
MGC39497; Coagulation factor VIII, procoagulant compo gamma polypeptide; Pregnancy specific beta-1-glycoprotein
US 9,085,755 B2
27 28
6; Matrix metalloproteinase 14 (membrane-inserted); Crystallin, beta A2; eye lens structural protein; Contactin
Chemokine (C-C motif) ligand 26: Interferon, alpha 6; Tachy associated protein-like 4: Claudin 19; Hypothetical protein
kinin, precursor 1 (Substance K. Substance P. neurokinin 1, LOC144501; Keratin 6E; Keratin 6L; Lens intrinsic mem
neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide brane protein 2, 19 kDa), the Cytoskeleton (Microtubule
K, neuropeptide gamma); Secreted frizzled-related protein 5: associated protein 2: Erythrocyte membrane protein band 4.1
Hyaluronan and proteoglycan link protein 4; Complement like 5: Homo sapiens trichohyalin (THFH); Keratin 6B. Kera
component 4B; Matrix metalloproteinase 16 (membrane-in tin 6A: Epithelial V-like antigen 1: Hook homolog 1 (Droso
serted); Fibroblast growth factor 7 (keratinocyte growth fac phila); Loricrin; Erythrocyte membrane protein band 4.1 (el
tor): Apollipoprotein C-II; Chloride channel, calcium acti liptocytosis 1, RH-linked); Tropomodulin 1: MAP/
vated, family member 3: Tetranectin (plasminogen binding 10 microtubule affinity-regulating kinase 1; Keratin 6E: Actin
protein); Collagen, type III, alpha 1 (Ehlers-Danlos Syndrome binding LIM protein family, member 2), Cell Adhesion Mol
type IV, autosomal dominant); KIAA0556 protein; Chemok ecules (Cadherin 19, type 2: Myeloid/lymphoid or mixed
ine (C-C motif) ligand 4: Hemopexin; Inter-alpha (globulin) lineage leukemia; Chromosome 21 open reading frame 29;
inhibitor H1; Relaxin 1; Matrix Gla protein: A disintegrin Kin of IRRE like 2: Laminin, alpha 3: Sialoadhesin: CD84
like and metalloprotease (reprolysin type) with thrombospon 15 antigen (leukocyte antigen); Lectin, galactoside-binding,
din type 1 motif. 2: Interferon (alpha, beta and omega) recep soluble, 2 (galectin 2); Epithelial V-like antigen 1: CD96
tor 2: Acid phosphatase, prostate; Guanine nucleotide antigen; Tubulointerstitial nephritis antigen; Carcinoembry
binding protein (G protein), gamma 8, Matrix metallopro onic antigen-related cell adhesion molecule 8: IL-18; Immu
teinase 23B; Meprin A, alpha (PABA peptide hydrolase); noglobulin Superfamily, member 1; Integrin, beta 8: Orni
Hyaluronoglucosaminidase 1: Angiotensinogen (serine (or thine arbamoyltransferase; Integrin, beta 6: Contactin
cysteine) proteinase inhibitor, clade A (alpha-1 antiprotein associated protein-like 4: Collagen, type XVII, alpha 1: Cad
ase, antitrypsin), member 8); Cartilage intermediate layer herin-like 26; Mucin and cadherin-like), Cell Differentiation
protein, nucleotide pyrophosphohydrolase; Purinergic recep proteins (Protein tyrosine phosphatase, receptor-type, Z
tor P2X, ligand-gated ion channel. 7: Glypican 3: Tectorin polypeptide 1: Laminin, alpha 3: CD84 antigen (leukocyte
beta; Interferon, alpha 5; Lipocalin 7: Platelet factor 4 variant 25 antigen); EDRF2; Homo sapiens erythroid differentiation
1: Nucleobindin 1: Collagen, type XI, alpha 1: Gastric inhibi related factor 2; Tumor protein p73-like; NB4 apoptosis/
tory polypeptide; Thrombospondin repeat containing 1,5-hy differentiation related protein; Homo sapiens PNAS-133;
droxytryptamine (serotonin) receptor 3 family member D; Similar to seven in absentia 2: Interleukin 24; Keratin 6B;
Collagen, type XXV, alpha 1: Growth differentiation factor 9: Keratin 6A: Dehydrogenase/reductase (SDR family) member
Hypothetical protein DKFZp434B044: Endothelin 3: 30 9; Gap junction protein, beta 5 (connexin 31.1); Iroquois
Chemokine (C motif) ligand 2: Prokineticin 2; Tumor necro homeobox protein 4; Ventral anterior homeobox 2: Chemok
sis factor receptor superfamily, member 11b (osteoprote ine (C-X-C motif) ligand 10; Tumor necrosis factor receptor
gerin); Tissue inhibitor of metalloproteinase 2; Dystonin; Superfamily, member 17; Calcium channel, Voltage-depen
Chromogramin B (Secretogranin 1); Hyaluronan and pro dent, beta 2 subunit; Parkinson disease (autosomal recessive,
teoglycan link protein 2: Leucine rich repeat neuronal 3: 35 juvenile) 2, parkin; Kallikrein 7 (chymotryptic, stratum cor
Lumican; Matrilin 1, cartilage matrix protein: Phospholipase neum); Glial cells missing homolog 2; AP-2 alpha; Protein
A2, group IIA (platelets, synovial fluid); Carboxylesterase 1 tyrosine phosphatase, receptor-type, Z polypeptide 1: Tropo
(monocyte/macrophage serine esterase 1); Sparcfosteonec nin T1: Sciellin; Glucosaminyl (N-acetyl) transferase 2.
tin, cwcV and kazal-like domains proteoglycan (testican); I-branching enzyme: Collagen, type XVII, alpha 1: Suppres
Dickkopf homolog 2 (Xenopus laevis); Gamma-aminobu 40 sor of cytokine signaling 2: Distal-less homeo box 1, Zygote
tyric acid (GABA) A receptor, alpha 3: Pregnancy specific arrest 1; Interleukin 20; Growth differentiation factor 3; FGF
beta-1 -glycoprotein 11; Insulin-like growth factor binding 23: Wingless-type MMTV integration site family, member
protein 1: Defensin, beta 106; Interleukin 17F: Ligand-gated 8A. Extracellular: Chromosome 21 open reading frame 29;
ion channel subunit; Phospholipase A2 receptor 1, 180 kDa; Laminin, alpha 3; Laminin, beta 4: Interleukin 24; Pregnancy
I factor (complement); Dystonin; LAG 1 longevity assurance 45 specific beta-1 -glycoprotein 1: Chemokine (C-X-C motif)
homolog 1 (S. cerevisiae); Prolactin; Testis expressed ligand 11; Surfactant, pulmonary-associated protein A1; Pre
sequence 264, Sema domain, immunoglobulin domain (Ig), pronociceptin; 5-hydroxytryptamine (serotonin) receptor 3B;
short basic domain, secreted, (semaphorin) 3D: Secreted Carcinoembryonic antigen-related cell adhesion molecule 8:
frizzled-related protein 2; secreted frizzled-related protein 4). Chemokine (C-X-C motif) ligand 10; IL-18 (interferon
There are groups of genes present only in UCEC. These 50 gamma-inducing factor); Lactotransferrin; Albumin; Fas
genes are related to the following: Homeostasis (Albumin; ligand (TNF superfamily, member 6); Cholinergic receptor,
Calcium-sensing receptor, Aquaporin 9; Lactotransferrin. nicotinic, beta polypeptide 4: Cathelicidin antimicrobial pep
Morphogenesis: Homeo box HB9: Epithelial V-like antigen tide; Airway trypsin-like protease:S100 calcium binding pro
1). Embryonic Development (Relaxin 2: Carcinoembryonic tein A9 (calgranulin B); TGF-alpha; Kallikrein 10; Serine
antigen-related cell adhesion molecule 8: Indoleamine-pyr 55 protease inhibitor, Kunitz type 1; WNT1 inducible signaling
role 2.3 dioxygenase; EPH receptor A3; Thyrotrophic embry pathway protein 3: Relaxin 2: Interferon, kappa; Defensin,
onic factor; Pregnancy specific beta-1-glycoprotein 1; Lami beta 103A, IL-20; Zona pellucida glycoprotein 4: Growth
nin, alpha 3), the ExtracellularSpace (Surfactant, pulmonary differentiation factor 3; FGF-23; Wingless-type MMTV inte
associated protein A1; Pregnancy specific beta-1 gration site family, member 8A: Complement factor H-re
-glycoprotein 1: Lactotransferrin; TGF-alpha; Albumin; 60 lated 5), Developmental proteins (EPH receptor A3: NIMA
FGF-23; S100 calcium binding protein A9 (calgranulin B)), (never in mitosis genea)-related kinase 2; Zinc finger protein
the Extracellular Matrix (Laminin, beta 4: Laminin, alpha 3: 282; TANK-binding kinase 1: MRE 11 meiotic recombina
Zona pellucida glycoprotein 4. Structural Molecule Activity: tion 11 homolog A: E2F transcription factor 2: Protein
Chromosome 21 open reading frame 29; Laminin, alpha 3; tyrosine phosphatase, receptor-type, Z polypeptide 1; Homo
Microtubule-associated protein 2: Laminin, beta 4: Keratin 65 sapiens clone 161455 breast expressed mRNA from chromo
6B; Ladinin 1; Keratin 6A: Occludin; Loricrin; Erythrocyte Some X; Laminin, alpha 3; V-myb myeloblastosis viral onco
membrane protein band 4.1 (elliptocytosis 1, RH-linked): gene homolog (avian)-like 1; Regulator of G-protein signal
US 9,085,755 B2
29 30
ling 11; Microtubule-associated protein 2: Transmembrane growth media, KGM, KGM-2 (Cambrex), EpiLife (Cascade
protein 16A: Adenomatosis polyposis coli 2; Homeo box Biologics) or in Green's medium in the presence of irradiated
HB9; Centromere protein F, 350/400 ka (mitosin); CD84 or Mytomycin-C treated 3T3 mouse embryonic feeder layer
antigen (leukocyte antigen); EDRF2; Homo sapiens eryth at 37° C., 5% CO). UCEC cell morphology thus differenti
roid differentiation-related factor 2; Tumor protein p73-like: ated resembled human epidermal keratinocytes. Epithelial
NB4 apoptosis/differentiation related protein; Homo sapiens cells have similar morphology under light microscope and
PNAS-133; Forkhead box P2: Homo sapiens gastric-associ can be easily turned into fibroblasts using conventional and
ated differentially-expressed protein YA61P (YA61); Tenas commercially available media (cf., FIG. 2).
cin N. Chromosome 6 open reading frame 49; Zinc finger Immunofluorescent analysis shows that the cultivated
protein 462; Zinc finger protein 71 (Cos26); SRY (sex deter 10 UCEC also express epidermal keratinocyte molecular mark
mining region Y)-box 7; Triggering receptor expressed on erS Such as keratins, desmosome, hemidesmosome and base
myeloid cells-like 4: Interleukin 24; Pregnancy specific beta ment membrane components (see also FIG. 10 that shows that
1-glycoprotein 1: Chondroitin Sulfate proteoglycan 5 (neuro UCEC are qualified to be epithelial cells in general by
glycan C); Keratin 6B. Keratin 6A: Dehydrogenase/reductase expressing a variety of these epithelial cell markers). Accord
(SDR family) member 9: Epithelial V-like antigen 1: Gap 15 ingly, these results show that umbilical cord epithelial pro
junction protein, beta 5 (connexin 31.1); G protein-coupled genitor/stem cells of the present invention can be differenti
receptor 51; Interferon regulatory factor 6: Neurotrophin 5 ated into skin cells such as epidermal keratinocytes which can
(neurotrophin 4/5); CD96 antigen; Iroquois homeobox pro be used for wound healing and have great potential for the
tein 4: Interleukin 1 receptor-like 1: G-2 and S-phase development of cultured skin equivalents.
expressed 1: Nuclear receptor Subfamily 2, group E, member
3:Ventral anterior homeobox2; Zinc finger protein 215; DNA Example 7
segment on chromosome 4 (unique) 234 expressed sequence;
Carcinoembryonic antigen-related cell adhesion molecule 8: Expansion of Umbilical Cord Epithelial and
Chemokine (C-X-C motif) ligand 10; IL-18; Indoleamine Mesenchymal StemCells Using Repetitive Tissue
pyrrole 2.3 dioxygenase; Albumin; Calcium-sensing receptor 25 Explants of Umbilical Cord Lining Membrane
(hypocalciuric hypercalcemia 1, severe neonatal hyperpar Tissues
athyroidism); Fas ligand (TNF superfamily, member 6);
TNFR superfamily, member 17: Calcium channel, voltage Umbilical cord epithelial and mesenchymal stem cells of
dependent, beta 2 subunit; Parkinson disease (autosomal the invention were expanded using repetitive explants of
recessive, juvenile) 2, parkin; Kallikrein 7 (chymotryptic, 30 umbilical cord amniotic membrane tissue as follows. Briefly,
stratum corneum); Glial cells missing homolog 2; TGF-al at day 1 of process, tissue explants were plated onto tissue
pha; Thyrotrophic embryonic factor; AP-2 alpha (activating culture dishes in growth media (DMEM/10% FCS, EpiLife,
enhancer binding protein 2 alpha); Kallikrein 10; Regulator KGM, KGM-2 or M171) at 37° C., 5% CO; media was
of G-protein signalling 7: Protein tyrosine phosphatase, changed every 2 or 3 days. Cell outgrowths started and con
receptor-type, Z polypeptide 1: Serine protease inhibitor, 35 tinued migrating from the explants for 7 days. After that,
Kunitz type 1; WNT1 inducible signaling pathway protein 3: tissue explants were transferred to other dishes to allow fur
Zic family member 3 heterotaxy 1 (odd-paired homolog, ther cell outgrowth. This process was continued until the
Drosophila); TTK protein kinase; Troponin T1, skeletal, explants had diminished in size, preventing further explanta
slow; Sciellin; TGFB-induced factor 2-like, X-linked; Kal tion. In this connection it is noted that the explants progres
likrein 8 (neuropsin/ovasin); Glucosaminyl (N-acetyl) trans 40 sively shrink in size until they are too small for further tissue
ferase 2, I-branching enzyme; Ankyrin repeat domain 30A; explant since during the process of cells outgrowing and
Relaxin 2: Collagen, type XVII, alpha 1: Gene differentially migrating from tissue explants, the cells produce proteases to
expressed in prostate; Phosphatase and actin regulator 3: Sup digest and break down tissue. FIG. 16 schematically illus
pressor of cytokine signaling 2; Nuclear receptor Subfamily 4, trates the rapid and robust expansion process of umbilical
group A, member 3; Angiotensin I converting enzyme (pep 45 cord epithelial and mesenchymal stem cells achieved using
tidyl-dipeptidase A) 1; Hypothetical protein MGC 17986: this protocol. Thus, this study demonstrates the high yield of
Distal-less homeobox 1, LAG 1 longevity assurance homolog UCMC and UMEC cells can be obtained from this source,
3 (S. cerevisiae): Zygote arrest 1: Interferon, kappa, IL-20; further reflecting the high viability and pro-growth character
ICEBERG caspase-1 inhibitor; Growth differentiation factor istics oft these cells in comparison with other sources of cells
3; FGF-23; Testis expressed sequence 15: Wingless-type 50 as bone-marrow or adipose-derived stem cells. In addition,
MMTV integration site family, member 8A: SRY (sex deter being a solid tissue, the Successful repetitive explant tech
mining region Y)-box 7; Carnitine deficiency-associated, nique used herein demonstrates that the cells of the invention
expressed in ventricle 1: Prokineticin 1: CAMP responsive can be uniformly extracted from the entire tissue instead of
element binding protein 3-like 3: Caspase recruitment only certain portions. This allows the maximum number of
domain family, member 15; FLJ23311 protein). 55 cells that can be derived at a low passage instead of passing
the cells through many generations causing deterioration of
Example 6 cells.
Direct Differentiation of Umbilical Cord Epithelial Example 8

Stem Cells (UCEC) into Skin Epidermal 60
Keratinocytes Direct Differentiation of Umbilical Cord
Mesenchymal Cells (UCMC) into Skin Dermal
For differentiation into skin epidermal keratinocytes, Fibroblasts
umbilical cord epithelial stem cells, UCEC cells, were cul
tured according to a standard protocol for the cultivation of 65 For differentiation into skin dermal fibroblasts, umbilical
keratinocytes. Cell isolation techniques were as described cord mesenchymal stem cells, UCMC cells were cultured
above. UCEC were then cultured in serum-free keratinocyte according to a standard protocol for the cultivation of fibro
US 9,085,755 B2
31 32
blasts. Cell isolation techniques were as described above in described in Example 2. For differentiation of UCMC into
Example 6. UCMC were then cultured in DMEM or commer adipocytes, cells were cultured in DMEM/10% FCS until
cially available fibroblast growth media (FGM). UCMC cell 100% confluent, and then in starvation medium of serum-free
morphology thus differentiated resembled human dermal DMEM for another 48 hours. UCMC were subjected to adi
fibroblasts. Mesenchymal cells have similar morphology pogenic induction media for 4 weeks before Subjecting the
under light microscope and can be easily turned into fibro cells to Oil-Red-O staining. The adipogenic induction
blasts using conventional and commercially available media
(cf., FIG. 3). medium contained DMEM/10%FCS: 1% antibiotic (strepto
mycin and penicillin)/antimycotic (fungizone)): 0.5 mM
Example 9 10
isobutyl-methylxanthine (IBMX), 1 uM dexamethasone, 10
uM insulin, and 200 uMindomethacin.
Direct Differentiation of Epithelial Stem/Progenitor Oil-Red-O staining of UCMC cells that were cultivated in
Cells into Skin Epidermal Keratinocytes the adipogenic induction medium indicated fat accumulation
in the UCMC and thus differentiation of the UCMC into
In an approach similar to Example 6, epithelial stem/pro adipocytes whereas no such differentiation was indicated in
genitor cells of the amniotic membrane of the umbilical cord 15
untreated UCMC which were cultured in DMEM/10% FCS
(UCEC) were isolated as described in Example 2. For differ without induction under otherwise same conditions as nega
entiation of UCEC into epidermal keratinocytes, the cells
were cultured in keratinocyte media (EpiLife or KGM) until tive control. As a further negative control, dermal fibroblasts
100% (cultivation after 5 days shown in FIG. 17-A) confluent from an 8 month old donor and keloid fibroblasts from a 20
before changing the media to DMEM/10% FCS for 3 days to year old donor were cultivated under the same conditions as
form epidermal cell sheets. As shown in FIG. 17-A (in which the induced or un-induced UCMC. Both cell types did not
photographs of two experiments termed “UCEC-10 and yield a positive result in the staining with Oil-Red-O, which is
UCEC-17 are depicted), after cultivation in DMEM/10% a further evidence for the pluripotency of UCMC of the
FCS, UCEC, had differentiated into epidermal keratinocytes present invention and to differentiate, for example, also into
that formed cell sheets (photograph of FIG. 17-A taken after 25 adipocytes.
10 days). These results thus provide further evidence for the
pluripotency of the cells of the present invention. What is claimed is:
Example 10 1. A method for isolating stem/progenitor cells from the
amniotic membrane of an umbilical cord, the method com
Direct Differentiation of Mesenchymal 30 prising:
Stem/Progenitor Cells into Osteoblasts (a) separating the amniotic membrane from the other com
ponents of the umbilical cord in vitro to obtain an amni
Mesenchymal stem/progenitor cells of the amniotic mem otic membrane tissue;
brane of the umbilical cord (UCMC) were isolated as (b) culturing the amniotic membrane tissue obtained in
described in Example 2. For differentiation of UCMC into 35 step (a) under conditions allowing cell proliferation; and
osteoblasts, cells were cultured in DMEM/10% FCS until (c) isolating the stem/progenitor cells, wherein the stem/
100% confluent, and then in starvation medium of serum-free progenitor cells are epithelial or mensenchymal stem/
DMEM for another 48 hours. UCMC were subjected to osteo progenitor cells expressing the following genes:
genic induction media for 4 weeks before Subjecting the cells POU5fl. Bmi-1, leukemia inhibitory factor (LIF), and
to von Kossa staining (bone cell staining). The osteogenic 40 Secreting Activin A and follistatin.
induction medium contained DMEM/10% FCS: 1% antibi 2. The method of claim 1, further comprising:
otic (streptomycin and penicillin)fantimycotic (fungizone); (a") separating the cells of the amniotic membrane tissue
0.01 uM 1,25-dihydroxyvitamin D3, 50 uMascorbate-2- before cultivation by a method selected from the group
phosphate, 10 mM f-glycerophosphate, 1% antibiotic (strep consisting of enzymatic separation and direct tissue
tomycin and penicillin)/antimycotic (fungizone). 45 explant.
As shown in FIG. 17B. von Kossa staining of UCMC cells 3. The method of claim 1, further comprising:
that were cultivated in the osteogenic induction medium indi (d) culturing the stem/progenitor cells under conditions
cated bone nodule formation in the UCMC and thus differ allowing the cells to undergo clonal expansion.
entiation of the UCMC into osteoblasts whereas no such 4. The method of claim 3, further comprising:
differentiation was indicated inuntreated UCMC which were 50 (e) culturing the stem/progenitor cells under conditions
cultured in DMEM/10% FCS without induction under other allowing the differentiation of said cells into epithelial
wise same conditions as negative control. As a further nega cells or mesenchymal cells; and
tive control, dermal fibroblasts from an 8 months old donor (f) isolating the differentiated cells.
and keloid fibroblasts from an 20 year old donor were culti 5. The method of claim 4, wherein the epithelial cells are
vated under the same conditions as the induced or un-induced 55 skin epidermal keratinocytes.
UCMC. Both cell types did not yield a positive result using 6. The method of claim 4, wherein the mesenchymal cells
Von Kossa staining, which is a further evidence for the pluri are selected from the group consisting of skin fibroblasts,
potency of UCMC of the present invention and thus to differ osteoblasts and adipocytes.
entiate, for example, also into osteoblasts. 7. The method of claim 1, further comprising
60 (g) preserving the isolated stem/progenitor cells for further
Example 11 SC.
8. The method of claim 7, wherein preserving is carried out
Direct Differentiation of Mesenchymal by using cryo-preservation.
Stem/Progenitor Cells into Adipocytes 9. A method of differentiating a stem/progenitor cell iso
65 lated by the method as defined in claim 1, comprising cultur
Mesenchymal stem/progenitor cells from the amniotic ing the stem/progenitor cells under conditions allowing the
membrane of the umbilical cord (UCMC) were isolated as cells to undergo clonal expansion.
US 9,085,755 B2
33 34
10. The method of claim 9, further comprising culturing the
stem/progenitor cells under conditions allowing the differen
tiation of said cells into epithelial cells and/or mesenchymal
cells.
11. The method of claim 10, wherein the epithelial cells are 5
skin epidermal keratinocytes.
12. The method of claim 10, wherein the mesenchymal
cells are selected from the group consisting of skin fibro
blasts, osteoblasts, and adipocytes.
k k k k k
10

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(12) United States Patent (10) Patent No.: US 9,085,755 B2

Direct Differentiation of Umbilical Cord Epithelial Example 8

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