U.S. patent application number 14/595934 was filed with the patent office on 2015-07-16 for method of deriving mesenchymal stem cells.
This patent application is currently assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH. The applicant listed for this patent is Sai Kiang LIM, Elias LYE. Invention is credited to Sai Kiang LIM, Elias LYE.
Application Number | 20150197725 14/595934 |
Document ID | / |
Family ID | 37075090 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197725 |
Kind Code |
A1 |
LIM; Sai Kiang ; et
al. |
July 16, 2015 |
Method of Deriving Mesenchymal Stem Cells
Abstract
We describe a method of obtaining a cell culture, the method
comprising providing a cell obtained by dispersing a human
embryonic stem cell (hESC) colony, or a descendent thereof, and
propagating the cell in the absence of a feeder cell layer in a
serum free medium comprising FGF2 and optionally PDGF AB.
Preferably, the human embryonic stem cell (hESC) colony is
dispersed with a dispersing agent which is trypsin.
Inventors: |
LIM; Sai Kiang; (Singapore,
SG) ; LYE; Elias; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIM; Sai Kiang
LYE; Elias |
Singapore
Singapore |
|
SG
SG |
|
|
Assignee: |
AGENCY FOR SCIENCE, TECHNOLOGY AND
RESEARCH
Singapore
SG
|
Family ID: |
37075090 |
Appl. No.: |
14/595934 |
Filed: |
January 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12065551 |
Mar 3, 2008 |
8962318 |
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PCT/SG2006/000232 |
Aug 15, 2006 |
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14595934 |
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60713992 |
Sep 2, 2005 |
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Current U.S.
Class: |
435/366 ;
435/405 |
Current CPC
Class: |
A61P 17/00 20180101;
C12N 2501/115 20130101; C12N 5/0603 20130101; C12Q 1/6809 20130101;
C12N 2506/02 20130101; A61P 35/00 20180101; A61P 9/00 20180101;
A61P 17/02 20180101; C12N 2501/734 20130101; G01N 33/5044 20130101;
A61P 3/10 20180101; C12N 2501/135 20130101; C12N 5/0662 20130101;
A61P 25/28 20180101; C12N 2502/1352 20130101; A61P 35/02 20180101;
A61P 25/16 20180101 |
International
Class: |
C12N 5/073 20060101
C12N005/073 |
Claims
1-54. (canceled)
55. A cell line obtained by providing a cell obtained by dispersing
an embryonic cell colony or a descendent thereof and propagating
the cell in the absence of co-culture in a serum free medium
comprising FGF2.
56. A method of conditioning a cell culture medium, the method
comprising obtaining a cell by dispersing an embryonic cell colony
or a descendent thereof, propagating the cell in the absence of
co-culture in a serum free medium comprising FGF2, and culturing a
mesenchymal stem cell, mesenchymal stem cell line or differentiated
mesenchymal stem cell in a cell culture medium.
57. A conditioned medium obtained by obtaining a cell by dispersing
an embryonic cell colony or a descendent thereof, propagating the
cell in the absence of co-culture in a serum free medium comprising
FGF2, and culturing a mesenchymal stem cell, mesenchymal stem cell
line or differentiated mesenchymal stem cell in a cell culture
medium.
58. The conditioned medium according to claim 57, wherein said
medium is used to treat a disease in an individual.
59. The cell line according to claim 55, wherein said embryonic
cell colony is a human embryonic cell colony.
60. The cell line according to claim 55, wherein said embryonic
cell colony is dispersed with trypsin.
61. The cell line according to claim 55, having a cell surface
marker selected from the group consisting of CD105 and CD73.
62. A cell line obtained by providing a cell obtained by dispersing
an embryonic cell colony or a descendent thereof, propagating the
cell in the absence of co-culture in a serum free medium containing
FGF2, and selecting a mesenchymal stem cell from the propagated
cells based on expression of a cell surface marker, and deriving a
cell line from the cell selected therefrom.
63. The cell line according to claim 62, wherein said embryonic
cell colony is a human embryonic cell colony.
64. The cell line according to claim 62, wherein said embryonic
cell colony is dispersed with trypsin.
65. The cell line according to claim 62, having a cell surface
marker selected from the group consisting of CD105 and CD73.
66. The method according to claim 56, further comprising the step
of isolating the cell culture medium.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 12/065,551, filed on Mar. 3, 2008, which is the national stage
of international application no. PCT/SG2006/000232, filed on Aug.
15, 2006, which claims priority to US provisional application
serial no. U.S. 60/713,992, filed on Sep. 2, 2005.
[0002] The foregoing application, and each document cited or
referenced in each of the present and foregoing applications,
including during the prosecution of each of the foregoing
application ("application and article cited documents"), and any
manufacturer's instructions or catalogues for any products cited or
mentioned in each of the foregoing application and articles and in
any of the application and article cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, and all documents cited or reference in documents
cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text or in
any document hereby incorporated into this text, are hereby
incorporated herein by reference. Documents incorporated by
reference into this text or any teachings therein may be used in
the practice of this invention. Documents incorporated by reference
into this text are not admitted to be prior art.
FIELD
[0003] The present invention relates to the fields of development,
cell biology, molecular biology and genetics. More particularly,
the invention relates to a method of deriving mesenchymal stem
cells from embryonic stem cells.
BACKGROUND
[0004] Stem cells, unlike differentiated cells have the capacity to
divide and either self-renew or differentiate into phenotypically
and functionally different daughter cells (Keller, Genes Dev. 2005;
19:1129-1155; Wobus and Boheler, Physiol Rev. 2005; 85:635-678;
Wiles, Methods in Enzymology. 1993; 225:900-918; Choi et al,
Methods Mol Med. 2005; 105:359-368).
[0005] The pluripotency of mouse embryonic stem cells (ESCs) and
their ability to differentiate into cells from all three germ
layers makes embryonic stem cells an ideal source of cells for
regenerative therapy for many diseases and tissue injuries (Keller,
Genes Dev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev.
2005; 85:635-678). However, this very property of embryonic stem
cells also poses a unique challenge, i.e. generating the
appropriate cell types for the treatment of a specific diseased or
injured tissue in sufficient quantity and homogeneity to ensure
therapeutic efficacy, and inhibiting the generation of other cell
types that may have a deleterious effect on the tissue repair and
regeneration. At present, protocols that either enhance
differentiation of embryonic stem cells towards specific lineages
and/or enrich for specific tissue cell types are too inefficient
and generally yield heterogeneous cell populations that might be
tumorigenic (Keller, Genes Dev. 2005; 19:1129-1155; Wobus and
Boheler, Physiol Rev. 2005; 85:635-678).
[0006] Mesenchymal stem cells (MSCs) are multipotent stem cells
that have documented evidence of therapeutic efficacy in treating
musculoskeletal injuries, improving cardiiac function in
cardiovascular disease and ameliorating the severity of GVHD (Le
Blanc and Pittenger, 2005). Being lineage restricted, they have
limited but robust potential to differentiate into mesenchymal cell
types, e.g adipocytes, chondrocytes and osteocytes, and have
negligible risk of teratoma formation. Host immune rejection of
transplanted MSCs is routinely circumvented through autologous or
allogeneic transplantation. MSCs can be isolated from several adult
tissues including bone marrow (BM), adipose tissues (ad), cord
blood and expanded ex vivo. However, availability of tissues for
their isolation remains limiting and requires risky invasive
procedures, and ex vivo expansion of MSCs while significant, is
nonetheless finite.
[0007] An alternative source of MSCs is the unlimited supply of
infinitely expandable and pluripotent human embryonic stem cells
(hESCs) that will also eliminate the need for potentially risky
invasive techniques. Host immune rejection of hESC-derived MSCs
(hESC-MSC) could potentially be circumvented by either autologous
hESCs generated by therapeutic cloning or immune compatible
allogeneic hESCs when banks of hESC lines become sufficiently
large.
[0008] The isolation of MSC or MSC-like cells from hESC has been
previously described. Barberi, et al. (2005) PLoS Med 2, e161
describes a protocol which involves co-culturing hESCs with mouse
OP9 cells in the presence of serum for 40 days before sorting for
CD73+ cells that constitute about 5% of the total cell population.
Xu, C. et al. (2004) Stem Cells 22, 972-80 describes a protocol
which involves infecting hESC-derived embryoid bodies with a
retrovirus expressing hTERT (Xu et al). However, the critical
components of these protocols i.e. viral infection of exogenous
DNA, exposure to mouse cells and use of serum introduce
unacceptable risks of tumorigenicity and xenozootic infection, and
preclude the use of these MSCs for clinical applications.
[0009] This invention seeks to solve this and other problems with
methods in the art.
SUMMARY
[0010] We provide, according to the invention, mesenchymal stem
cells (MSC), mesenchymal stem cell lines, differentiated
mesenchymal stem cells, methods of obtaining these and uses of
these, as set out in the claims. Preferrred embodiments are set out
in the claims and are described in the description.
[0011] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe,
J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; J. M. Polak and James
O'D. McGee, 1990, Oligonucleotide Synthesis: A Practical Approach,
Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of
Enzymology: DNA Structure Part A: Synthesis and Physical Analysis
of DNA Methods in Enzymology, Academic Press; Using Antibodies: A
Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David
Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN
0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow
(Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory
Press, ISBN 0-87969-314-2), 1855; and Lab Ref: A Handbook of
Recipes, Reagents, and Other Reference Tools for Use at the Bench,
Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor
Laboratory, ISBN 0-87969-630-3. Each of these general texts is
herein incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1G. Characterisation of hESC-MSC cultures.
[0013] FIG. 1A. Cellular morphology under phase contrast.
[0014] FIG. 1B. Expression of pluripotency-associated genes in
hESC-MSC. Transcript levels are measured by Taqman-based
quantitative RT-PCR and normalized to that of hESC. The transcript
level in hESC is derived from the average of HuES9 and H1 hESC
lines.
[0015] FIG. 1C. Western blot analysis for pluripotency-associated
genes in HuES9 and H1 hESC lines, HuES9.E1 HuES9.E3 and H1.E2
hESC-MSC cultures and E14 mouse ESC line.
[0016] FIG. 1D. Renal subcapsular transplantation of HuES9 and
HuES9.E1. Paraffin-embedded, H&E stained cross sections of
kidney four months after transplantation with either HuES9.E1 (top)
or HuES9 (bottom).
[0017] FIG. 1E. Alkaline phosphatase activity in human HuES9 ESC
line, mouse E14 ESC line, mouse embryonic fibroblast (MEF) feeder
and HuES9.E1.
[0018] FIG. 1F. Genomic DNA analysis by PCR for the presence of
human Alu and mouse c-mos repeat sequences.
[0019] FIG. 1G. Chromosomal analysis of HuES9.E1 by G-banding (top
panel) and Spectral Karyotyping (SKY) (middle panel). Inversion of
chromosome 9 shown in bottom panel.
[0020] FIGS. 2A-2B. Surface antigen profiling by FACS analysis.
[0021] FIG. 2A. HuES9.E1 HuES9.E3 and H1.E2 hESC-MSCs, HuES9 hESCs,
and murine embryonic fibroblast feeder cells are stained and
analyzed on a Cyan LX (Dako North America, Inc., Carpinteria,
Calif.) instrument using WinMDI software. Nonspecific fluorescence
is determined by incubation of similar cell aliquots with
isotype-matched mouse monoclonal antibodies.
[0022] FIG. 2B. HuES9.E1 and HuES9.E3 hESC-MSCs are passaged twice
in serum-containing BM-MSC media before being analyzed in parallel
with BM-MSCs by FACS analysis.
[0023] FIGS. 3A-3C. Differentiation of HuES9.E1. HuES9.E1 cells are
induced to undergo adipogenesis, chondrogenesis and osteogenesis
using standard protocols.
[0024] FIG. 3A. Adipogenesis. i) Day 14 after inducing
adipogenesis, cells are stained for oil droplets by oil red; ii)
PPAR.gamma. mRNA at day 7 and day 14 are measured by Taqman
quantitative RT-PCR. All values are normalized to that of HuES9.E1;
iii) Relative PPAR.gamma. mRNA levels in HuES9 and HI hESCs, their
derivative MSC cell cultures (HuES9.E1, HuES9.E3 and H1.E2) and
adult tissue-derived MSCs (BM-MSC and ad-MSC) as measured by Taqman
quantitative RT-PCR. All values are normalized to that of
HuES9.E1.
[0025] FIG. 3B. Chondrogenesis. i) Day 21 after inducing
chondrogenesis, cells are stained for proteoglycans by alcian blue
(left) and immunoreactivity to collagen type II using a HRP-based
visualization assay; ii) Aggrecan and PPAR.gamma. mRNA at day 7 and
day 14 are measured by Taqman quantitative RT-PCR. All values are
normalized to that of HuES9.E1; iii) Relative PPAR.gamma. mRNA
levels in HuES9 and HI hESCs, their derivative MSC cell cultures
(HuES9.E1, HuES9.E3 and H1.E2) and adult tissue-derived MSCs
(BM-MSC and ad-MSC) as measured by Taqman quantitative RTPCR. All
values are normalized to that of HuES9.E1.
[0026] FIG. 3C. Osteogenesis i) Day 21 after inducing
chondrogenesis, cells are stained for mineralization by von Kossa
stain, ii, iii) Bone-specific alkaline phosphatase (ALP) and bone
sialoprotein (BSP) mRNA at day 7 and day 14 are measured by Taqman
quantitative RT-PCR. All values are normalized to that of
HuES9.E1
[0027] FIGS. 4A-4E. Gene expression analysis.
[0028] FIG. 4A. Hierarchical clustering of expressed genes in three
hESC-MSC cultures consisting of HuES9.E1, HuES9.E3, and H1.E2,
three BMMSC samples, three ad-MSC samples and three hESC lines
consisting of HuES9, H1 and Hes3.
[0029] FIG. 4B. Pairwise comparison of gene expression between
hESC-MSCs and BMMSCs (left) and between hESC-MSCs and hESCs
(right).
[0030] FIG. 4C. Analysis of commonly expressed genes (<2 fold
difference) in hESC-MSCs and BM-MSCs. The genes are classified into
biological processes using the Panther classification system. Each
biological process is determined if it is significantly over- or
under-represented (p<0.01) by comparing the observed frequency
of genes to the expected frequency of genes in the NCBI: H. sapiens
gene database of 23481 genes for each biological process.
Significantly over- or under-represented processes are grouped and
graphically presented.
[0031] FIGS. 4D, 4E. Analysis of differentially expressed genes
(>2 fold difference) in hESC-MSCs and BM-MSCs. Biological
processes that are significantly over- or under-represented
(p<0.01) by genes highly expressed in hESC-MSCs or BM-MSCs are
grouped and graphically presented.
[0032] FIGS. 5A-5E. Positive and negative sorting for generation of
hESC-MSC.
[0033] FIG. 5A. FACS analysis HuES9.E1 HuES9.E3 and H1.E2
hESC-MSCs, HuES9 hESCs, and murine embryonic fibroblast feeder
cells are stained and analyzed for the presence of CD24 on a Cyan
LX (Dako North America, Inc., Carpinteria, Calif.) instrument using
WinMDI software. Nonspecific fluorescence is determined by
incubation of similar cell aliquots with isotype-matched mouse
monoclonal antibodies.
[0034] FIG. 5B. Sorting for CD105+, CD24- cells from HuES9 cells
that have been trypsinized and propagated without feeder in media
supplemented with PDGF and FGF2 for one week. CD105+, CD24- cells
represented in Q4 are selected for culture.
[0035] FIG. 5C. Pairwise comparison of gene expression between Q4.1
and each of the other Q4 cultures, namely Q4.2 to Q4.5.
[0036] FIG. 5D. Pairwise comparison of gene expression between all
Q4 cultures and hESC-MSCs consisting of HuES9.E1, HuES9.E3, and
H1.E2, and between all Q4 cultures and BM-MSCs.
[0037] FIG. 5E. SKY analysis of Q4.3.
[0038] FIG. 6. Protein analysis of media conditioned by HuES9.E1
MSC culture. 80% confluent HuES9.E1 cell cultures are washed 3
times with PBS, cultured overnight in a chemically defined media
consisting of DMEM media without phenol red and supplemented with
ITS, 5 ng/ml FGF2, 5 ng/ml PDGF AB
glutamine-penicillin-streptomycin and .beta.-mercaptoethanol. The
cultures are then rinsed three times with PBS and then replaced
with fresh defined media. An aliquot of media are removed at 0, 24,
48 and 72 hours, centrifuged at 500.times.g and the supernatant is
0.2.mu. filtered. 10 .mu.l of the media is separated on a 4-12%
SDS-PAGE and silver-stained.
[0039] FIG. 7. Antibody array. One ml of conditioned (CM) or
non-conditoned media (NCM) is incubated with a RayBio.RTM. Cytokine
Antibody Arrays according to manufacturer's instruction (RayBio
Norcross, Ga.). The antibody map for each array is listed in the
Examples. Binding of ligands to specific antibody is visualized
using HRP-based chemiluminescence assay. Different exposures of
each membrane is analysed. An antigen is scored as present if a
signal is present on the membrane hybridized with CM but absent on
that hybridized with NCM. Data from analysis of 4 independent
batches of CM and NCM is summarized in the Examples.
[0040] FIG. 8. Distribution of 201 gene products into biological
processes. The 201 genes are classified into different biological
processes in the GO classification system. 58 biological processes
that are over-represented by the frequency of genes in the
secretory proteome relative to the frequency of the genes in a
database collated from Unigene, Entrez and GenBank with a p-value
of <0.05 are grouped into three major groups: metabolism,
defense response and tissue differentiation.
[0041] FIG. 9. Distribution of 201 gene products into pathways. The
201 genes are classified into different pathways in the GO
classification system. 30 biological processes that are
over-represented by the frequency of genes in the secretory
proteome relative to the frequency of the genes in a database
collated from Unigene, Entrez and GenBank with a p-value of
<0.05 are categorised into several major categories: receptor
binding, signal transduction, cell-cell interaction, cell
migration, immune response and metabolism.
DETAILED DESCRIPTION
Obtaining Mesenchymal Stem Cells (MSC)
[0042] The prior art methods of obtaining mesenchymal stem cells
(MSC) or MSC-like cells from hESCs involve either transfection of a
human telomerase reverse transcriptase (hTERT) gene into
differentiating hESCs (Xu et al., 2004) or coculture with mouse OP9
cell line (Barberi et al., 2005). The use of exogenous genetic
material and mouse cells in these derivation protocols introduces
unacceptable risks of tumorigenicity or infection of xenozootic
infectious agents.
[0043] In contrast, our method provides for a clinically relevant
and reproducible protocol for isolating similar or identical
(preferably homogenous) MSC populations from differentiating hESCs.
In general, our method comprises dispersing a embryonic stem (ES)
cell colony into cells. The cells are then plated out and
propagated. The cells are propagated in the absence of co-culture
in a serum free medium comprising fibroblast growth factor 2
(FGF2), in order to obtain mesenchymal stem cells (MSCs).
[0044] Thus, our protocol does not require serum, use of mouse
cells or genetic manipulations and requires less manipulations and
time, and is therefore highly scalable. The Examples describe the
isolation of MSCs from two different hESC lines, HuES9 and H-1 and
also a third one, Hes-3.sup.23, and demonstrates the robustness of
the protocol. Human ES cell derived MSCs (hESC-MSCs) obtained by
the methods and compositions described here are remarkably similar
to bone-marrow derived MSCs (BM-MSCs).
[0045] In preferred embodiments, the embryonic stem cell culture
comprises a human embryonic stem cell (hESC) culture.
[0046] In a one embodiment, a method of generating mesenchymal stem
cells (MSC) comprises trypsinizing and propagating hESCs without
feeder support in media supplemented with FGF2 and optionally PDGF
AB before sorting for CD105+CD24-cells.
[0047] In a preferred embodiment, the method comprises sorting for
CD105+, CD24- cells from trypsinized hESCs one week after
feeder-free propagation in a media supplemented with FGF2 and
optionally PDGF AB will generate to generate a hESC-MSC cell
culture in which at least some, preferably substantially all, more
preferably all cells are similar or identical (preferably
homogenous) to each other.
[0048] The methods described here for generating clinically
relevant hESCMSC cultures that are physically, biologically and
functionally similar to BM-MSCs could potentially alleviate the
limiting supply of BM for isolation of BM-MSCs that have
demonstrated therapeutic efficacy in many clinical and preclinical
animal studies.sup.10,25. This will also remove the need for risky
invasive BM aspiration procedure, reduce the waiting time and cost
of preparing BM-MSCs on a per-patient basis, and reduce batch to
batch variations. Furthermore, the robust derivation of MSCs from a
defined cell type such as hESC provides a useful model to study and
better understand the derivation and biology of MSC that has remain
an enigma despite its present widespread clinical and preclinical
applications.sup.9.
[0049] Further uses of the mesenchymal stem cells generated by the
process include replacement of adult tissue-derived MSCs in
clinical or therapeutic applications; replacement of adult
tissue-derived MSCs as feeders for propagation of other cell types
such as human ESCs, expansion of cord blood or bone marrow stem
cell populations; and preparation of MSC-conditioned media for
treatment of cardiovascular disease.
Disaggregating Embryonic Stem Cell Colonies
[0050] Our methods of producing mesenchymal stem cells comprise
dispersing or disaggregating an embryonic stem cell colony into
cells.
[0051] The embryonic stem cell colony may comprise a huES9 colony
(Cowan C A, Klimanskaya I, McMahon J, Atienza J, Witmyer J, et al.
(2004) Derivation of embryonic stem-cell lines from human
blastocysts. N Engl J Med 350: 1353-1356) or a H1 ESC colony
(Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A,
Swiergiel J J, et al. (1998) Embryonic Stem Cell Lines Derived from
Human Blastocysts. Science 282: 1145-1147.).
[0052] Preferably, the cells in the colony are disaggregated or
dispersed to a substantial extent, i.e., at least into clumps. More
preferably, the colony is disaggregated or dispersed to the extent
that all the cells in the colony are single, i.e., the colony is
completely disaggregated.
[0053] The disaggregation may be achieved with a dispersing
agent.
[0054] The dispersing agent may be anything that is capable of
detaching at least some embryonic stem cells in a colony from each
other. The dispersing agent may preferably comprise a reagent which
disrupts the adhesion between cells in a colony, or between cells
and a substrate, or both. Preferably, the dispersing agent may
comprise a protease.
[0055] In preferred embodiments, the dispersing agent comprises
trypsin. The treatment with trypsin may last for example for 3
minutes or thereabouts at 37 degrees C. The cells may then be
neutralised, centrifuged and resuspended in medium before plating
out.
[0056] In preferred embodiments, the method comprises dispersing a
confluent plate of human embryonic stem cells with trypsin and
plating the cells out.
[0057] The disaggregation may comprise at least some of the
following sequence of steps: aspiration, rinsing, trypsinization,
incubation, dislodging, quenching, re-seeding and aliquoting. The
following protocol is adapted from the Hedrick Lab, UC San
Diego.
[0058] In the aspiration step, the media is aspirated or generally
removed from the vessel, such as a flask. In the rinsing step, the
cells are rinsed with a volume, for example 5-10 mls, of a buffered
medium, which is preferably free from Ca.sup.2+ and Mg.sup.2+. For
example, the cells may be rinsed with calcium and magnesium free
PBS. In the trypsinization step, an amount of dispersing agent in
buffer is added to the vessel, and the vessel rolled to coat the
growing surface with the dispersing agent solution. For example, 1
ml of trypsin in Hank's BSS may be added to a flask.
[0059] In the incubation step, the cells are left for some time at
a maintained temperature. For example, the cells may be left at
37.degree. C. for a few minutes (e.g., 2 to 5 minutes). In the
dislodging step, the cells may be dislodged by mechanical action,
for example by scraping or by whacking the side of the vessel with
a hand. The cells should come off in sheets and slide down the
surface.
[0060] In the quenching step, a volume of medium is added to the
flask. The medium preferably contains a neutralising agent to stop
the action of the dispersing agent. For example, if the dispersing
agent is a protease such as trypsin, the medium may contain a
protein, such as a serum protein, which will mop up the activity of
the protease. In a particular example, 3 ml of serum containing
cell culture medium is added to the flask to make up a total of 4
mls. The cells may be pipetted to dislodge or disperse the
cells.
[0061] In the re-seeding step, the cells are re-seeded into fresh
culture vessels and fresh medium added. A number of re-seedings may
be made at different split ratios. For example, the cells may be
reseeded at 1/15 dilution and 1/5 dilution. In a particular
example, the cells may be re-seeded by adding 1 drop of cells into
a 25 cm.sup.2 flask and 3 drops into another to re-seed the
culture, and 7-8 mls media is then added to each to provide for
1/15 dilution and 1/5 dilution from for example a 75 cm.sup.2
flask. In the aliquoting step, the cells may be aliquoted into new
dishes or whatever split ratio is desired, and media added.
Maintenance as Cell Culture
[0062] The disaggregated cells are plated and maintained as a cell
culture.
[0063] The cells may be plated onto a culture vessel or substrate
such as a gelatinized plate. Crucially, the cells are grown and
propagated without the presence of co-culture, e.g., in the absence
of feeder cells.
[0064] The cells in the cell culture are grown in a serum-free
medium which is supplemented by one or more growth factors such as
fibroblast growth factor 2 (FGF2) and optionally platelet-derived
growth factor AB (PDGF AB), at for example 5 ng/ml. The cells in
the cell culture are preferably split or subcultured 1:4 when
confluent, by treatment with trypsin, washing and replating.
Absence of Co-Culture
[0065] In highly preferred embodiments, our methods involve
culturing cells in the absence of co-culture. The term "co-culture"
refers to a mixture of two or more different kinds of cells that
are grown together, for example, stromal feeder cells.
[0066] Thus, in typical ES cell culture, the inner surface of the
culture dish is usually coated with a feeder layer of mouse
embryonic skin cells that have been treated so they will not
divide. The feeder layer provides an adherent surface to enable the
ES cells to attach and grow. In addition, the feeder cells release
nutrients into the culture medium which are required for ES cell
growth. In the methods and compositions described here, the ES and
MSC cells are cultured in the absence of such co-culture.
[0067] Preferably, the cells are cultured as a monolayer or in the
absence of feeder cells. According to preferred embodiments of the
methods described here, the embryonic stem cells are cultured in
the absence of feeder cells to establish mesenchymal stem cells
(MSC).
[0068] In preferred embodiments, the dissociated or disaggregated
embryonic stem cells are plated directly onto a culture substrate.
The culture substrate may comprise a tissue culture vessel, such as
a Petri dish. The vessel may be pre-treated. In preferred
embodiments, the cells are plated onto, and grow on, a gelatinised
tissue culture plate.
[0069] An example protocol for the gelatin coating of dishes
follows. A solution of 0.1% gelatin in distilled water is made and
autoclaved. This may be stored at room temp. The bottom of a tissue
culture dish is covered with the gelatin solution and incubated for
5-15 min. Remove gelatin and plates are ready to use. Medium should
be added before adding cells to prevent hypotonic lysis.
Serum Free Media
[0070] The dissociated or disaggregated embryonic stem cells are
cultured in a medium which is preferably a serum-free medium.
[0071] The term "serum-free media" may comprise cell culture media
which is free of serum proteins, e.g., fetal calf serum. Serum-free
media are known in the art, and are described for example in U.S.
Pat. Nos. 5,631,159 and 5,661,034. Serum-free media are
commercially available from, for example, Gibco-BRL
(Invitrogen).
[0072] The serum-free media may be protein free, in that it may
lack proteins, hydrolysates, and components of unknown composition.
The serum-free media may comprise chemically-defined media in which
all components have a known chemical structure. Chemically-defined
serum-free media is advantageous as it provides a completely
defined system which eliminates variability allows for improved
reproducibility and more consistent performance, and decreases
possibility of contamination by adventitious agents.
[0073] In a preferred embodiment, the serum-free media comprises
Knockout DMEM media (Invitrogen-Gibco, Grand Island, N.Y.).
[0074] The serum-free media may be supplemented with one or more
components, such as serum replacement media, at a concentration of
for example, 5%, 10%, 15%, etc. The serum-free media is preferably
supplemented with 10% serum replacement media from Invitrogen-Gibco
(Grand Island, N.Y.).
Growth Factor
[0075] The serum-free medium in which the dissociated or
disaggregated embryonic stem cells are cultured preferably
comprises one or more growth factors. A number of growth factors
are known in the art, including PDGF, EGF, TGF-a, FGF, NGF,
Erythropoietin, TGF-b, IGF-I and IGF-II.
[0076] In a preferred embodiment, the growth factor comprises
fibroblast growth factor 2 (FGF2). The medium may also contain
other growth factors such as platelet-derived growth factor AB
(PDGF AB). Both of these growth factors are known in the art. In a
highly preferred embodiment, the method comprises culturing cells
in a medium comprising both FGF2 and PDGF AB.
[0077] FGF2 is a wide-spectrum mitogenic, angiogenic, and
neurotrophic factor that is expressed at low levels in many tissues
and cell types and reaches high concentrations in brain and
pituitary. FGF2 has been implicated in a multitude of physiologic
and pathologic processes, including limb development, angiogenesis,
wound healing, and tumor growth. FGF2 may be obtained commercially,
for example from Invitrogen-Gibco (Grand Island, N.Y.).
[0078] Platelet Derived Growth Factor (PDGF) is a potent mitogen
for a wide range of cell types including fibroblasts, smooth muscle
and connective tissue. PDGF, which is composed of a dimer of two
chains termed the A chain and B chain, can be present as AA or BB
homodimers or as an AB heterodimer. Human PDGF-AB is a 25.5 kDa
homodimer protein consisting of 13.3 kDa A chain and 12.2 B chain.
PDGF AB may be obtained commercially, for example from Peprotech
(Rocky Hill, N.J.).
[0079] The growth factor(s), preferably FGF2 and optionally PDGF
AB, are preferably present in the medium at concentrations of about
100 pg/ml, preferably about 500 pg/ml, preferably about 1 ng/ml,
preferably about 2 ng/ml, preferably about 3 ng/ml, preferably
about 4 ng/ml, most preferably about 5 ng/ml. In preferred
embodiments, the medium contains FGF2 at about 5 ng/ml. The medium
may also contain PDGF AB, preferably at about 5 ng/ml.
Splitting Cells
[0080] Cells in culture will generally continue growing until
confluence, when contact inhibition causes cessation of cell
division and growth. Such cells may then be dissociated from the
substrate or flask, and "split", subcultured or passaged, by
dilution into tissue culture medium and replating.
[0081] The methods and compositions described here may therefore
comprise passaging, or splitting during culture. Preferably, the
cells in the cell culture are split at a ratio of 1:2 or more,
preferably 1:3, more preferably 1:4, 1:5 or more. The term
"passage" designates the process consisting in taking an aliquot of
a confluent culture of a cell line, in inoculating into fresh
medium, and in culturing the line until confluence or saturation is
obtained.
Selection, Screening or Sorting Step
[0082] In highly preferred embodiments, the method further
comprises a selection or sorting step, to further isolate or select
for mesenchymal stem cells.
[0083] The selection or sorting step may comprise selecting
mesenchymal stem cells (MSC) from the cell culture by means of one
or more surface antigen markers. The use of a selection or sorting
step further enhances the stringency of sorting and selection
specificity for MSCs and furthermore potentially reduces possible
contamination from embryonic stem cells such as hESCs and other
hESC-derivatives from the starting material. This would then
further reduce the risk of teratoma formation and further increase
the clinical relevance of the protocol we describe.
[0084] A number of methods are known for selection or sorting based
on antigen expression, and any of these may be used in the
selection or sorting step described here. In particularly preferred
embodiments, the selection or sorting is achieved by means of
fluorescence activated cell sorting (FACS). Thus, as known in the
art, FACS involves exposing cells to a reporter, such as a labelled
antibody, which binds to and labels antigens expressed by the cell.
Methods of production of antibodies and labelling thereof to form
reporters are known in the art, and described for example in Harlow
and Lane. The cells are then passed through a FACS machine, which
sorts the cells from each other based on the labelling.
[0085] We have realised that while a number of candidate surface
antigens known to be associated with MSCs e.g. CD105, CD73, ANPEP,
ITGA4 (CD49d), PDGFRA, some of the MSC associated surface antigens
e.g. CD29 and CD49e are also highly expressed in ES cells such as
hESCs and their expression are verified by FACS analysis. The
association of a surface antigen with MSCs may not be sufficient to
qualify the antigen as a selectable marker for isolating MSCs from
ES cells such as hESC. Accordingly, the selection or sorting step
preferably employs antigens which are differentially expressed
between MSCs and ES cells.
[0086] The selection or sorting step of our method may positively
select for mesenchymal stem cells based on the expression of
antigens. Such antigens may be identified by, for example,
comparing the gene expression profiles of hESCs and hESCMSCs as
described in the Examples. In particular embodiments, the selection
or sorting may specifically make use of any of the antigens shown
in Table E1A and E1B below.
[0087] In preferred embodiments, the selection or sorting step of
our method may positively select for mesenchymal stem cells based
on the expression of antigens which are identified as expressed on
MSCs, but not expressed on ES cells such as hESCs.
[0088] Thus, as shown in the Examples, we demonstrate that CD73 is
highly expressed on MSCs.sup.3, while being not highly expressed on
hESCs (FIG. 4A). Furthermore, as the Examples demonstrate that both
CD73 and CD105 are highly expressed surface antigens in MSCs and
are among the top 20 highly expressed surface antigens in hESC-MSCs
relative to hESC (FIG. 3, table), the use of either CD73 or CD105
(or both) as selectable marker for putative MSCs will be equally
effective in sorting for putative MSCs generated by differentiating
hESCs.
[0089] Alternatively, or in addition, the selection or sorting step
may negatively select against antigens based on surface antigens
that are highly expressed as surface antigen on embryonic stem
cells (ES cells) such as hESCs, and not mesenchymal stem cells
e.g., hESC-MSC. Selection or sorting may be based on known or
previously identified hESC-specific surface antigens such as MIBP,
ITGB1BP3 and PODXL.sup.22, and CD24.
[0090] The Examples show that FACS analysis confirms the expression
of CD24 on hESC but not hESC-MSCs. Therefore, CD24 may be used as a
negative selection or sorting marker either on its own, or in
conjunction with CD105 as a positive selectable marker for
isolating putative MSCs from differentiating hESC cultures.
[0091] Importantly, the mesenchymal stem cells are able to maintain
self-renewal without the requirement for transformation. Thus, for
example, known transformation treatments such as fusion with
immortalised cells such as tumour cells or tumour cell lines, viral
infection of a cell line with tranforming viruses such as SV40,
EBV, HBV or HTLV-1, transfection with specially adapted vectors,
such as the SV40 vector comprising a sequence of the large T
antigen (R. D. Berry et al., Br. J. Cancer, 57, 287-289, 1988),
telomerase (Bodnar-A-G. et. al., Science (1998) 279: p. 349-52) or
a vector comprising DNA sequences of the human papillomavirus (U.S.
Pat. No. 5,376,542), introduction of a dominant oncogene, or by
mutation are therefore not required in the methods described here
for making mesenchymal stem cells.
[0092] In preferred embodiments, the mesenchymal stem cells and
cell lines (or the differentiated cells derived from them) do not
display one or more characteristics of embryonic stem cells.
Preferred such characteristics include expression of the OCT4 gene
and alkaline phosphatase activity. Preferably, the mesenchymal stem
cell exhibits reduced expression of one or more characteristic
markers of pluripotency. Such pluripotency markers are described in
further detail below, but include Nanog, BMP4, FGF5, Oct4, Sox-2
and Utf1.
[0093] Mesenchymal stem cells made by the methods described here
are preferably non-tumorigenic. Preferably, the mesenchymal stem
cells when implanted into an immune compromised or immunodeficient
host animal do not result in tumours, compared to implantation of
parental embryonic stem cells which results in tumour formation.
Preferably, the immune compromised or immunodeficient host animal
is a SCID mouse or a Rag1-/- mouse. Preferably, the mesenchymal
stem cells do not form tumours after prolonged periods of
implantation, preferably greater than 2 weeks, more preferably
greater than 2 months, most preferably greater than 9 months.
Detailed protocols for tumourigenicity testing are set out in the
Examples.
[0094] Mesenchymal stem cells made by the methods described here
are also preferably display one or more of the following
characteristics. They have a substantially stable karyotype as
assessed by chromosome number, preferably when maintained in cell
culture for at least 10 generations. They also preferably display a
substantially stable gene expression pattern from generation to
generation. By this we mean that the expression levels one or more,
preferably substantially all, of a chosen set of genes does not
vary significantly between a mesenchymal stem cells in one
generation and mesenchymal stem cells in the next generation.
[0095] Preferably, the set of genes comprises one or more, a
subset, or all of, the following: cerberus (GenBank Accession nos:
NM.sub.--009887, AF031896, AF035579), FABP (GenBank Accession nos:
NM.sub.--007980, M65034, AY523818, AY523819), Foxa2 (GenBank
Accession nos: NM.sub.--010446, X74937, L10409), Gata-1 (GenBank
Accession nos: NM.sub.--008089, X15763, BCO52653), Gata-4 (GenBank
Accession nos: NM.sub.--008092, AF179424, U85046, M98339,
AB075549), Hesx1 (GenBank Accession nos: NM.sub.--010420, X80040,
U40720, AK082831), HNF4a (GenBank Accession nos: NM.sub.--008261,
D29015, BCO39220), c-kit (GenBank Accession nos: NM.sub.--021099,
Y00864, AY536430, BC075716, AK047010, BCO26713, BCO52457,
AK046795), PDGFR.alpha. (NM.sub.--011058, M57683, M84607,
BCO53036), Oct4 (GenBank Accession nos: NM.sub.--013633, X52437,
M34381, BC068268), Runx1 (GenBank Accession nos: NM.sub.--009821,
D26532, BC069929, AK051758), Sox17 (GenBank Accession nos:
NM.sub.--011441, D49474, L29085, AK004781), Sox2 (GenBank Accession
nos: NM.sub.--011443, U31967, AB108673), Brachyury
(NM.sub.--009309, X51683), TDGF1 (GenBank Accession nos:
NM.sub.--011562, M87321) and Tie-2 (GenBank Accession nos:
NM.sub.--013690, X67553, X71426, D13738, BC050824).
[0096] The methods described here enable the production of
mesenchymal stem cells as well as differentiated cells, which
comprise clonal descendants of mesenchymal stem cells. The term
"clonal descendant" of a cell refers to descendants of the cells
which have not undergone substantially any transforming treatment
or genetic alteration. Such clonal descendants have not undergone
substantial genomic changes are substantially genetically identical
to the parent cell, or an ancestor, preferably, the embryonic stem
cell (save with reduced potency). The term "mesenchymal stem cells"
should also preferably be taken to include cell lines derived from
mesenchymal stem cells, i.e., mesenchymal stem cell lines, and vice
versa.
Derivation of Progenitor Cells
[0097] In preferred embodiments, the methods described here may
employ further steps to select or screen for mesenchymal stem
cells.
[0098] Such further steps may take place prior to the steps
described above, in between such steps, or after these steps. The
further steps may be in fact be conducted independently, and may
specifically comprise deriving one or more progenitor cells or cell
lines from the ES cells.
[0099] We therefore disclose an alternative method of deriving
mesenchymal stem cells. The method comprises: (a) providing an
embryonic stem (ES) cell; and (b) establishing a progenitor cell
line from the embryonic stem cell; in which the progenitor cell
line is selected based on its ability to self-renew.
[0100] However, preferably, the methods are conducted together.
Thus, for example, our methods may comprise deriving progenitor
cells or progenitor cell lines from the embryonic stem cell prior
to, during, or after the dispersal step or the propagation step.
Thus, the method may for example comprise obtaining a mesenchymal
stem cell (MSC) by providing a cell obtained by dispersing a
embryonic stem (ES) cell colony, or a descendent thereof, deriving
one or more progenitor cells or progenitor cell lines from the
embryonic stem cell and propagating the cell in the absence of
co-culture in a serum free medium comprising FGF2.
[0101] Preferably, the progenitor cell line is selected based on
its ability to self-renew, or the method may select against somatic
cells based on their inability to self-renew, or both.
[0102] In a preferred embodiment, the progenitor cell line is
derived or established in the absence of co-culture, preferably in
the absence of feeder cells. Preferably, the absence of co-culture
selects against embryonic stem cells.
[0103] In preferred embodiments, the progenitor cell line is
established without transformation. The progenitor cell line may be
established by exposing embryonic stem cells or their descendants
to conditions which promote self-renewal of putative progenitor
cells. Preferably, the self-renewal-promoting conditions discourage
the propagation of embryonic stem cells.
[0104] The self-renewal-promoting conditions may comprise growth in
rich media. More preferably, the self-renewal-promoting conditions
comprise growing cells in the absence of LIF. Preferably, the
self-renewal-promoting conditions comprise serial passages.
Preferably, the self-renewal promoting conditions comprise at least
12 serial passages.
[0105] In preferred embodiments, the progenitor cell line has
reduced potential compared to the embryonic stem cell. Preferably,
the progenitor cell line is lineage restricted, preferably
non-pluripotent. Preferably, the progenitor cell line is
non-tumorigenic.
[0106] Preferably, the step of deriving the progenitor cell line
comprises a step of exposing the embryonic stem cell to conditions
that enhance differentiation to a specific lineage. Preferably, the
differentiation enhancing-conditions comprises generating an
embryoid body from the embryonic stem cell. Preferably, the cells
are removed from differentiation enhancing-conditions after
pluripotency is lost.
[0107] Preferably, the removing of the cells from lineage
restriction-promoting conditions comprises disaggregating an
embryoid body. Preferably, the method comprises disaggregating
embryoid bodies which have been grown from between about 3 to 6
days.
[0108] In preferred embodiments, the progenitor cell line displays
reduced expression of or does not substantially express either or
both of OCT4 and alkaline phosphatase activity.
[0109] Preferably, the progenitor cell line displays reduced
expression of a pluripotency marker compared to an embryonic stem
cell from which it is derived, the pluripotency marker preferably
selected from the group consisting of: Nanog, BMP4, FGF5, Oct4,
Sox-2 and Utf1.
[0110] In preferred embodiments, the progenitor cell lines display
one or more of the following characteristics: (a) are maintainable
in cell culture for greater than 40 generations; (b) have a
substantially stable karyotype or chromosome number when maintained
in cell culture for at least 10 generations; (c) have a
substantially stable gene expression pattern from generation to
generation.
[0111] Preferably, the progenitor cell line does not substantially
induce formation of teratoma when transplanted to a recipient
animal, preferably an immune compromised recipient animal,
preferably after 3 weeks, more preferably after 2 to 9 months.
[0112] Preferably, the embryonic stem cell or progenitor cell line
is a mammalian, preferably mouse or human, embryonic stem cell or
progenitor cell line.
[0113] Preferably, the progenitor cell line comprises an
endothelial progenitor cell line, preferably a E-RoSH cell line.
Alternatively, or in addition, the progenitor cell line may
comprise a mesenchymal progenitor cell line, preferably a huES9.E1
cell line.
[0114] In some embodiments, the method further comprises the step
of (d) deriving a differentiated cell from the progenitor cell
line.
[0115] Preferably, the progenitor cell line is propagated for at
least 5 generations prior to differentiation.
[0116] We provide a method of generating a differentiated cell from
an embryonic stem (ES) cell, the method comprising: (a) deriving a
progenitor cell line from the embryonic stem cell; (b) propagating
the progenitor cell line; and (c) deriving a differentiated cell
from the progenitor cell line.
[0117] There is provided a method comprising: (a) providing an
embryonic stem (ES) cell; (b) deriving a progenitor cell from the
embryonic stem cell; and (c) establishing a progenitor cell line
from the progenitor cell, in which progenitor cells are selected
based on their ability to self-renew.
[0118] The method may specifically be used for generating a
differentiated cell from an embryonic stem (ES) cell. Preferably,
the differentiated cell is an endothelial cell or a mesenchymal
cell. More preferably, the differentiated cell is an adipocyte or
an osteocyte.
[0119] We provide a progenitor cell line produced by a method
according to any preceding aspect of the invention.
[0120] The methods and compositions described here may also further
comprise further steps which employ other factors or
characteristics of such MSCs for selection or screening or
both.
[0121] Biasing Differentiation
[0122] In preferred embodiments, the method for generating
embryonic stem cell-derived progenitor cell lines of specific
lineages preferably further comprises a first step of biasing
differentiation of embryonic stem cells towards a specific desired
lineage or lineage of interest. Our methods may also comprise a
second step of encouraging self-renewal of putative progenitor
cells and discouraging the propagation of embryonic stem cells.
[0123] The first step may comprise promoting the growth or
propagation of a specific lineage of interest. Different progenitor
cell lines of specific lineages of interest may be made by exposing
the cells to conditions that promote the differentiation of those
lineages of interest. For example, the embryonic stem cells may be
exposed to growth factors or small molecules such as ligands that
promote or enable differentiation.
[0124] Thus, the methods described here for establishing embryonic
stem cell-derived cell lines of specific lineages preferably
include a step of enhancing differentiation of embryonic stem cells
towards that specific lineage. Preferably, the
differentiation-enhancing step is carried out for a predetermined
period of time. Thus, preferably, the embryonic stem cells or their
descendants are transiently exposed to differentiation-enhancing
environment.
[0125] The choice of the method of enhancing or biasing
differentiation will depend on the specific cell lineage of
interest for which it is desired to produce progenitor cells. The
person skilled in the art will be aware of the various methods
which may be used for different cells.
[0126] Endodermal Progenitor Cells
[0127] Where it is desired to bias differentiation of embryonic
stem cells towards endodermal types of tissues, for example,
embryoid bodies may be formed and disaggregated (see later). The
disaggregated embryoid bodies may be exposed to growth factors or
drugs or combinations thereof that induce endodermal
differentiation. Examples of such growth factors and drugs include
activin A, FGF4, dexamethasone and retinoic acid.
[0128] Hematopoietic and Endothelial Progenitor Cells
[0129] On the other hand, where it is desired to bias
differentiation of embryonic stem cells towards hematopoietic or
endothelial lineages, the disaggregated embryoid bodies may be
exposed to growth factors or drugs or combinations thereof that
induce hematopoietic or endothelial differentiation. Examples of
such growth factors and drugs include GM-CSF, G-CSF, SCF, PDGF,
IL-3, erythropoietin, thrombopoeittin, TNF.alpha. and
rapamycin.
[0130] Cardiac Mesoderm and Skeletal Myoblast Progenitor Cells
[0131] On the other hand, where it is desired to bias
differentiation of embryonic stem cells towards cardiac mesoderm or
skeletal myoblast lineages, the disaggregated embryoid bodies may
be exposed to growth factors or drugs or combinations thereof that
induce cardiac mesoderm or skeletal myoblast differentiation.
Examples of such growth factors and drugs include dexamethasone,
inhibitors of PPAR.gamma. and testosterone or its analogs.
[0132] The second step may comprise plating the differentiating
cells in a rich media. In such embodiments, continued propagation
will selectively enrich for progenitor cells which can then be
cloned.
[0133] Formation of Embryoid Bodies
[0134] In some embodiments, the differentiation-enhancing step
comprises formation of embryoid bodies from embryonic stem cells.
Embryoid bodies, and methods for making them, are known in the art.
The term "embryoid body" refers to spheroid colonies seen in
culture produced by the growth of embryonic stem cells in
suspension. Embryoid bodies are of mixed cell types, and the
distribution and timing of the appearance of specific cell types
corresponds to that observed within the embryo. Preferably, the
embryoid bodies are generated by plating out embryonic stem cells
onto semi-solid media, preferably methylcellulose media as
described in Lim et al, Blood. 1997; 90:1291-1299. Preferably, the
embryoid bodies are between 3 to 6 days old.
[0135] In such embodiments, the embryoid body is disaggregated,
i.e., separating the component cells from each other, e.g., by
collagenase or trypsin treatment, in order to remove the cells from
lineage restriction-promoting conditions.
[0136] The method in preferred embodiments comprises a step of
choosing a putative progenitor cell for the desired specific
lineage. The choosing may be conducted based on morphology of the
cell, or by expression or markers, etc. Gene expression profiling
or antigen profiling may also be used to choose specific progenitor
cells which are of a desired lineage. The chosen putative
progenitor cell for the desired specific lineage may then be
cultured, or further choosing steps conducted thereon.
[0137] In preferred embodiments, the differentiation-enhancing step
is followed by exposing differentiating cells to conditions which
encourage self-renewal of putative progenitor cells and discourage
the propagation of embryonic stem cells. Such conditions may
preferably comprise culture in the absence of co-culture or feeder
cells (see above).
[0138] Rich Media
[0139] Alternatively, or in addition, such conditions comprise
plating in rich media. The term "rich media" as used in this
document is intended to refer to media which is nutrient rich.
Preferably, such media comprises essential nutrients required for
growth of the relevant cell. Preferably, the rich media contain
serum. More preferably, it comprises substantially all the
nutrients required for such growth. Most preferably, the rich
medium supports, promotes and encourages growth of the relevant
cells. in highly preferred embodiments, the relevant cell is a
progenitor cell or a putative progenitor cell of interest. An
example of a rich medium is DMEM with 4500 mg/l D-glucose,
supplemented with 20% fetal calf serum, non essential amino acids,
L-glutamine and .beta.-mercaptoethanol.
[0140] In preferred embodiments, such rich media does not comprise
additional growth regulators or hormones that allow, promote or
encourage growth of embryonic stem cells, such as Leukemia
Inhibitory Factor (LIF).
[0141] According to such embodiments, continued propagation will
selectively enrich for progenitor cells which can then be
cloned.
[0142] Long-Term Maintenance in Culture
[0143] Preferably, the methods described here involve culturing the
embryonic stem cells or their descendants for more than one
generation. Preferably, the cells are cultured for more than 5,
more than 10, more than 15, more than 20, more than 25, more than
50, more than 40, more than 45, more than 50, more than 100, more
than 200, more than 500 or more than 800 generations. In
particular, the cell lines may be maintained for 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75,
100, 200, 500 or more generations.
[0144] Cells in culture will generally continue growing until
confluence, when contact inhibition causes cessation of cell
division and growth. Such cells may then be dissociated from the
substrate or flask, and "split" or passaged, by dilution into
tissue culture medium and replating. The progenitor cells may
therefore be passaged, or split during culture; preferably they are
split at a ratio of 1:2 or more, preferably 1:3, more preferably
1:4, 1:5 or more. The term "passage" designates the process
consisting in taking an aliquot of a confluent culture of a cell
line, in inoculating into fresh medium, and in culturing the line
until confluence or saturation is obtained.
[0145] The progenitor cells derived according to the methods
described here may however be maintained for a large number of
generations, based on their capacity to self-renew. On the other
hand, it has been established that "normal" (i.e., untransformed
somatic) cells derived directly from an organism are not immortal.
In other words, such somatic cells have a limited life span in
culture (they are mortal). They will not continue growing
indefinitely, but will ultimately lose the ability to proliferate
or divide after a certain number of generations. On reaching a
"crisis phase" such cells die after about 50 generations. Thus,
such somatic cells may only be passaged a limited number of
times.
[0146] Importantly, the progenitor cells are able to maintain
self-renewal without the requirement for transformation. Thus, for
example, known transformation treatments such as fusion with
immortalised cells such as tumour cells or tumour cell lines, viral
infection of a cell line with tranforming viruses such as SV40,
EBV, HBV or HTLV-1, transfection with specially adapted vectors,
such as the SV40 vector comprising a sequence of the large T
antigen (R. D. Berry et al., Br. J. Cancer, 57, 287-289, 1988),
telomerase (Bodnar-A-G. et. al., Science (1998) 279: p. 349-52) or
a vector comprising DNA sequences of the human papillomavirus (U.S.
Pat. No. 5,376,542), introduction of a dominant oncogene, or by
mutation are therefore not required in the methods described here
for making progenitor cell lines.
[0147] According to preferred embodiments of the methods described
here, progenitor cells may be propagated without transformation for
more than 50 generations. In preferred embodiments, the progenitor
cells may be propagated indefinitely and without transformation as
progenitor cell lines. The progenitor cells and progenitor cell
lines are preferably lineage restricted compared to their parental
embryonic stem cells. In particular, they are not capable of giving
rise to all three germ layers. In highly preferred embodiments, the
progenitor cell lines are preferably non-pluripotent.
[0148] Characteristics of Progenitor Cells
[0149] In preferred embodiments, the progenitor cells and cell
lines (or the differentiated cells derived from them) do not
display one or more characteristics of embryonic stem cells.
Preferred such characteristics include expression of the OCT4 gene
and alkaline phosphatase activity. Preferably, the progenitor cell
line exhibits reduced expression of one or more characteristic
markers of pluripotency. Such pluripotency markers are described in
further detail below, but include Nanog, BMP4, FGF5, Oct4, Sox-2
and Utf1.
[0150] Progenitor cells made by the methods described here are
preferably non-tumorigenic. Preferably, the progenitor cells when
implanted into an immune compromised or immunodeficient host animal
do not result in tumours, compared to implantation of parental
embryonic stem cells which results in tumour formation. Preferably,
the immune compromised or immunodeficient host animal is a SCID
mouse or a Rag1-/- mouse. Preferably, the progenitor cells do not
form tumours after prolonged periods of implantation, preferably
greater than 2 weeks, more preferably greater than 2 months, most
preferably greater than 9 months. Detailed protocols for
tumourigenicity testing are set out in the Examples.
[0151] Progenitor cells made by the methods described here are also
preferably display one or more of the following characteristics.
They have a substantially stable karyotype as assessed by
chromosome number, preferably when maintained in cell culture for
at least 10 generations. They also preferably display a
substantially stable gene expression pattern from generation to
generation. By this we mean that the expression levels one or more,
preferably substantially all, of a chosen set of genes does not
vary significantly between a progenitor cell in one generation and
a progenitor cell in the next generation.
[0152] Preferably, the set of genes comprises one or more, a
subset, or all of, the following: cerberus (GenBank Accession nos:
NM.sub.--009887, AF031896, AF035579), FABP (GenBank Accession nos:
NM.sub.--007980, M65034, AY523818, AY523819), Foxa2 (GenBank
Accession nos: NM.sub.--010446, X74937, L10409), Gata-1 (GenBank
Accession nos: NM.sub.--008089, X15763, BCO52653), Gata-4 (GenBank
Accession nos: NM.sub.--008092, AF179424, U85046, M98339,
AB075549), Hesx1 (GenBank Accession nos: NM.sub.--010420, X80040,
U40720, AK082831), HNF4a (GenBank Accession nos: NM.sub.--008261,
D29015, BCO39220), c-kit (GenBank Accession nos: NM.sub.--021099,
Y00864, AY536430, BC075716, AK047010, BCO26713, BCO52457,
AK046795), PDGFR.alpha. (NM.sub.--011058, M57683, M84607,
BCO53036), Oct4 (GenBank Accession nos: NM.sub.--013633, X52437,
M34381, BC068268), Runx1 (GenBank Accession nos: NM.sub.--009821,
D26532, BC069929, AK051758), Sox17 (GenBank Accession nos:
NM.sub.--011441, D49474, L29085, AK004781), Sox2 (GenBank Accession
nos: NM.sub.--011443, U31967, AB108673), Brachyury
(NM.sub.--009309, X51683), TDGF1 (GenBank Accession nos:
NM.sub.--011562, M87321) and Tie-2 (GenBank Accession nos:
NM.sub.--013690, X67553, X71426, D13738, BC050824).
[0153] The methods described here enable the production of
progenitor cells and progenitor cell lines as well as
differentiated cells, which comprise clonal descendants of
progenitor cells. The term "clonal descendant" of a cell refers to
descendants of the cells which have not undergone substantially any
transforming treatment or genetic alteration. Such clonal
descendants have not undergone substantial genomic changes are
substantially genetically identical to the parent cell, or an
ancestor, preferably, the embryonic stem cell (save with reduced
potency). The term "progenitor cell" should also preferably be
taken to include cell lines derived from progenitor cells, i.e.,
progenitor cell lines, and vice versa.
[0154] Regulators of Self-Renewal and Differentiation
[0155] Our methods may also be used to identify putative regulators
of self-renewal or differentiation. The methods involve conducting
the methods described for production of progenitor cell lines or
differentiated cells in the presence and absence of a candidate
molecule, and identifying if the presence of the molecule has any
effect on the process. For example, a molecule which accelerates
the production of progenitor cells or differentiated cells may be
used as a positive regulator of differentiation (or alternatively
as an inhibitor of self-renewal). Conversely, a molecule which
retards the process can be considered an inhibitor of
differentiation or a promoter of self-renewal.
[0156] In preferred embodiments, we also provide a cell, preferably
a progenitor, of a selected lineage, obtainable according to the
method. Hitherto, preparations of progenitors were too impure for
certainty as to whether any chosen cell was a progenitor cell. With
culture according to the invention that can give rise to
substantially 100% pure preparations of progenitors, isolation of a
single progenitor is achieved.
[0157] We further provide in preferred embodiments a composition
comprising a plurality of cells, wherein a majority of the cells
are progenitor cells of a selected lineage. Preferably, at least
60% of the cells are progenitor cells of the selected lineage. More
preferably, at least 60% of the cells are progenitor cells. In
addition, the invention provides an isolated progenitor cell. The
term cell line preferably refers to cells that can be maintained
and grown in culture and display an immortal or indefinite life
span.
[0158] The methods described here may be combined with decreasing
the activity of mTOR to promote differentiation, as described in
U.S. 60/609,216, herein incorporated by reference.
[0159] Uses of Progenitor Cells
[0160] Our methods are capable of producing of progenitor cells and
cell lines of various types.
[0161] For example, we disclose a method of making peripheral blood
progenitor cells (PBPC), neuronal progenitor cells, haematopoeitic
progenitor cells, myeloid progenitor cells, epithelial progenitor
cells, bone marrow stromal cells, skeletal muscle progenitor cells,
pancreatic islet progenitor cells, mesenchymal progenitor cells,
cardiac mesodermal stem cells, lung epithelial progenitor cells,
liver progenitors, and endodermal progenitor cells.
[0162] Progenitor cells made according to the methods described
here can be used for a variety of commercially important research,
diagnostic, and therapeutic purposes. These uses are generally well
known in the art, but will be described briefly here.
[0163] For example, stem cells may be used to generate progenitor
cell populations for regenerative therapy. Progenitor cells may be
made by ex vivo expansion or directly administered into a patient.
They may also be used for the re-population of damaged tissue
following trauma.
[0164] Thus, hematopoietic progenitor cells may be used for bone
marrow replacement, while cardiac progenitor cells may be used for
cardiac failure patients. Skin progenitor cells may be employed for
growing skin grafts for patients and endothelial progenitor cells
for endothelization of artificial prosthetics such as stents or
artificial hearts.
[0165] Embryonic stem cells and their tissue stem cell derivatives
may be used as sources of progenitor cells for the treatment of
degenerative diseases such as diabetes, Alzheimer's disease,
Parkinson's disease, etc. Stem cells, for example may be used as
sources of progenitors for NK or dendritic cells for immunotherapy
for cancer, which progenitors may be made by the methods and
compositions described here.
[0166] It will be evident that the methods and compositions
described here enable the production of progenitor cells, which may
of course be made to differentiate using methods known in the art.
Thus, any uses of differentiated cells will equally attach to those
progenitor cells for which they are sources.
[0167] Progenitor cells produced by the methods and compositions
described here may be used for, or for the preparation of a
pharmaceutical composition for, the treatment of a disease. Such
disease may comprise a disease treatable by regenerative therapy,
including cardiac failure, bone marrow disease, skin disease,
burns, degenerative disease such as diabetes, Alzheimer's disease,
Parkinson's disease, etc and cancer.
[0168] We therefore describe a method of treatment of a disease
comprising: (a) providing an embryonic stem (ES) cell; (b)
establishing a progenitor cell line from the embryonic stem cell in
which the progenitor cell line is selected based on its ability to
self-renew; (d) optionally deriving a differentiated cell from the
progenitor cell line; and (e) administering the progenitor cell
line or the differentiated cell into a patient.
[0169] Characteristics of Obtained MSCs
[0170] The MSCs obtained by the methods and compositions described
here preferably satisfy the morphologic, phenotypic and functional
criteria commonly used to identify MSCs.sup.9, as known in the art.
For ease of reference, the mesenchymal stem cells obtained by the
methods and compositions described here, particularly as derived
from human embroynic stem cells, may be referred to as
"hESC-MSC"s.
[0171] Thus, the MSCs obtained by the methods and compositions
described here may preferably exhibit one or more morphological
characteristics of mesenchymal stem cells. For example, the MSCs
obtained may form an adherent monolayer with a fibroblastic
phenotype.
[0172] Furthermore, the MSCs obtained may preferably display a
surface antigen profile which is similar or identical to
mesenchymal stem cells. Thus, the surface antigen profile of the
MSCs obtained may include one or more, preferably all, of CD29+,
CD44+, CD49a and e+, CD105+, CD166+ and CD34-, CD45-.sup.9-11.
[0173] The MSCs obtained may be differentiated into any mesenchymal
lineage, using methods known in the art and described below. Thus,
the MSCs obtained by the methods and compositions described here
may display a differentiation potential that include adipogenesis,
chondrogenesis and osteogenesis.sup.9.
[0174] The mesenchymal stem cells obtained as described, e.g.,
hESC-MSCs, can have a substantial proliferative capacity in vitro.
In some embodiments, the mesenchymal stem cells obtained may
undergo at least 10 population doublings while maintaining a normal
diploid karyotype. Preferably, however, the MSCs are capable of
undergoing at least 20-30 population doublings while maintaining a
normal diploid karyotype. In preferred embodiments, the MSCs
display a stable gene expression and surface antigen profile
throughout this time.
[0175] Preferably, the MSCs obtained do not display any defects,
such as chromosomal aberrations and/or alterations in gene
expression. In preferred embodiments, such defects are not evident
until after 10 passages, preferably after 13 passages, more
preferably after 15 passages.
hESC-MSC Proteome
[0176] The Examples describe experiments to analyse the proteome of
human ESC-derived MSCs (hESC-MSCs).
[0177] As shown in Examples 7 to 15, the hESC-MSCs have an
expression profile which is similar to that of adult bone marrow
derived MSCs (BM-MSCs). These results demonstrate that the MSCs
derived by our methods have significant biological similarities to
their bone marrow derived counterparts, e.g., in their ability to
secrete paracrine factors. Accordingly, the hESC-MSCs may be used
for any purpose for which BM-MSCs are suitable.
[0178] We further provide a medium which is conditioned by culture
of hESC-MSCs. Such a conditioned medium comprises molecules
secreted by the hESC-MSC, including 201 unique gene products. Such
a conditioned medium, and combinations of any of the molecules
comprised therein, including in particular proteins or
polypeptides, may be used to supplement the activity of, or in
place of, the hESC-MSCs, for the purpose of for example treating or
preventing a disease.
[0179] Analysis of the proteome of the hESC-MSCs shows that the
proteins expressed are involved in three biological processes:
metabolism, defense response, and tissue differentiation including
vascularization, hematopoiesis and skeletal development.
Accordingly, the hESC-MSCs may be used to treat diseases which
these functions may have a role in, or whose repair or treatment
involves any one or more of these biological processes. Similarly,
the proteins expressed by the hESC-MSCs, singly or in combination,
preferably in the form of a conditioned medium, may be used to
supplement the activity of, or in place of, the hESC-MSCs, for the
purpose of for example treating or preventing such diseases.
[0180] The 201 gene products expressed by the hESC-MSCs are shown
to activate important signalling pathways in cardiovascular
biology, bone development and hematopoiesis such as Jak-STAT, MAPK,
Toll-like receptor, TGF-beta signalling and mTOR signaling
pathways. Accordingly, the hESC-MSCs, proteins expressed by them,
etc, may be used to prevent or treat a disease in which any of
these signalling pathways is involved, or whose aetiology involves
one or more defects in any one or more of these signalling
pathways.
[0181] Furthermore, any one or more proteins secreted from the MSCs
described here, including in the form of conditioned media, may be
used for the same purposes as the BM-MSCs described herein.
[0182] The hESC-MSCs may also be used as sources for any of the
proteins secreted or expressed by them, as listed in the in
Examples 10 to 14, particularly the tables therein. We therefore
provide for a method of producing a polypeptide as shown in any of
Examples 10 to 14, the method comprising obtaining a mesenchymal
stem cell as described, culturing the mesenchymal stem cell and
isolating the polypeptide from the mesenchymal stem cell,
preferably from a medium in which the mesenchymal stem cell is
growing.
Homogeneity
[0183] In a preferred embodiment, the mesenchymal stem cells
produced by the method described here are similar or identical
(preferably homogenous) in nature. That is to say, mesenchymal stem
cell (MSC) clones isolated by the protocol show a high degree of
similarity or identity with each other, whether phenotypically or
otherwise.
[0184] Similarity or identity may be gauged by a number of ways and
measured by one or more characteristics. In a preferred embodiment,
the clones are similar or identical in gene expression. Preferably,
the method is such that any two or more mesenchymal stem cells
selected by the method exhibit substantially identical or similar
gene expression profiles, that is to say, a combination of the
identity of genes expressed and the level to which they are
expressed. Preferably, substantially all of the mesenchymal stem
cells isolated exhibit substantially identical or similar gene
expression profiles.
[0185] Homogeneity of gene expression may be measured by a number
of methods. Preferably, genome-wide gene profiling is conducted
using, for example, array hybridisation of extracted RNA as
described in the Examples. Total RNA may be extracted and converted
into cDNA, which is hybridised to an array chip comprising a
plurality of gene sequences from a relevant genome. Preferably, the
array comprises NCBI Reference Sequence (RefSeq) genes, which are
well characterised genes validated, annotated and curated on an
ongoing basis by National Center for Biotechnology Information
(NCBI) staff and collaborators.
[0186] Gene expression between samples is then compared using
analysis software. In a preferred embodiment, the similarity or
identity of gene expression expressed as a "correlation
coefficient". In such measures, a high correlation coefficient
between two samples indicates a high degree of similarity between
the pattern of gene expression in the two samples. Conversely, a
low correlation coefficient between two samples indicates a low
degree of similarity between the pattern of gene expression in the
two samples. Normalisation may be conducted to remove systematic
variations or bias (including intensity bias, spatial bias, plate
bias and background bias) prior to data analysis.
[0187] Correlation tests are known in the art and include a T-test
and Pearson's test, as described in for example Hill, T. &
Lewicki, P. (2006). Statistics: Methods and Applications. StatSoft,
Tulsa, Okla., ISBN: 1884233597 (also StatSoft, Inc. (2006).
Electronic Statistics Textbook. Tulsa, Okla.: StatSoft. WEB).
Reference is made to Khojasteh et al., 2005, A stepwise framework
for the normalization of array CGH data, BMC Bioinformatics 2005,
6:274. An Intra-class correlation coefficient (ICC) may also be
conducted, as described in Khojasteh et al, supra.
[0188] In preferred embodiments, a Pearson's test is conducted to
generate a Pearson's correlation coefficient. A correlation
coefficient of 1.0 indicates an identical gene expression
pattern.
[0189] In a preferred embodiment, the cDNA is hybridised to a
Sentrix HumanRef-8 Expression BeadChip and scanned using a Illumina
BeadStation 500.times.. Preferably, the data is extracted,
normalised and analysed using Illumina BeadStudio (Illumina, Inc,
San Diego, Calif., USA). It will be clear to the reader however
that any suitable chip and scanning hardware and software (which
outputs a correlation measurement) may be used to assay similarity
of gene expression profile.
[0190] Preferably, the gene expression correlation coefficient
between any two isolates as preferably measured by the above means
is greater than 0.65, preferably greater than 0.70, more preferably
greater than 0.80, more preferably greater than 0.85, more
preferably greater than 0.90, most preferably more than 0.95.
[0191] In some embodiments, the method described here generates
mesenchymal stem cells whose gene expression correlation
coefficient between any two or more isolates of mesenchymal stem
cells so obtained is in the same order as, or slightly less than,
the correlation coefficient between technical replicates of the
same RNA sample, performed a period of time apart such as 1 month
apart. In other embodiments, the gene expression correlation
coefficient between any two or more isolates of mesenchymal stem
cells is greater than 0.90, preferably greater than 0.95.
[0192] Preferably, the gene expression correlation coefficients are
in such ranges for cells which have undergone the selection or
sorting procedure described above. Preferably, the gene expression
correlation coefficient between the majority of isolates,
preferably all isolates, is in such ranges.
[0193] Thus, as shown in the Examples, the correlation coefficient
shows a high degree of similarity between five mesenchymal stem
cell cultures obtained, with a correlation coefficient between four
of the lines of 0.96 and the one with 0.90; in contrast, the
correlation coefficient between technical replicates of a RNA
sample analysed one month apart is between 0.97 and 0.98.
[0194] Accordingly, we provide for a method of generating
mesenchymal stem cells which are substantially similar or identical
(preferably homogenous) with each other. The isolates preferably
display a near identical gene expression profile.
[0195] As well as the "internal" homogeneity described above (i.e.,
homogeneity between the isolates of MSCs from the method),
homogeneity may also be assessed between such isolates and other
cells or cell types. In particular, comparisons may be made with
mesenchymal stem cells derived by other methods, such as
bone-marrow derived mesenchymal stem cells (BM-MSCs). In preferred
embodiments, the MSCs obtained by the methods and compositions
described here display a gene expression profile which is similar
to, homogenous with, or identical with a BM-MSC. Thus, the MSCs
obtained may show a correlation coefficient of gene expression of
greater than 0.5, preferably greater than 0.6, preferably greater
than 0.7, with BM-MSCs.
[0196] Thus, as shown in the Examples, pairwise comparision of gene
expression between three independently derived hESC-MSC populations
and three individual BM-MSC samples are found to be similar with a
correlation coefficient of 0.72.
Regulators of Mesenchymal Stem Cell Formation
[0197] Our methods may also be used to identify putative regulators
of mesenchymal stem cell formation from embryonic stem cells. The
methods involve conducting the methods described for production of
mesenchymal stem cells in the presence and absence of a candidate
molecule, and identifying if the presence of the molecule has any
effect on the process. For example, a molecule which accelerates
the production of mesenchymal stem cells may be used as a positive
regulator of mesenchymal stem cell formation. Conversely, a
molecule which retards the process can be considered an inhibitor
of mesenchymal stem cell formation.
[0198] In preferred embodiments, we also provide a cell, preferably
a mesenchymal stem cell, obtainable according to the method.
Hitherto, preparations of mesenchymal stem cells were either too
impure, or not substantially phenotypically similar or identical
(e.g., with respect to gene expression), or were not suitable for
clinical purposes as they are produced by methods involving
co-culture or presence of serum. With culture according to the
invention, this can give rise to substantially 100% pure
preparations of mesenchymal stem cells which are similar or
identical (preferably homogenous) to each other.
[0199] In addition, we describe a process for producing
differentiated cells, the method comprising obtaining a mesenchymal
stem cell by a method as described herein, and differentiating the
mesenchymal stem cell. For example, we provide for methods of
differentiating to adipocytes, chondrocytes and osteocytes, etc. We
further provide differentiated cells obtainable by such methods.
Cell lines made from such mesenchymal stem cells and differentiated
cells are also provided. The term cell line preferably refers to
cells that can be maintained and grown in culture and display an
immortal or indefinite life span.
[0200] The methods described here may be combined with decreasing
the activity of mTOR to promote differentiation, as described in
U.S. 60/609,216, herein incorporated by reference.
Uses
[0201] The methods and compositions described here may be used for
up-regulating expression of mesenchymal or endothelial markers of a
cell. They may also or instead be used for down-regulating
expression of stem cell or pluripotency markers of a cell. The
methods and compositions described here may be used to identify an
agent capable of promoting or retarding self-renewal or
differentiation of a stem cell. Such a method comprises performing
a method according to any preceding claim in the presence of a
candidate molecule, and determining an effect thereon.
[0202] The methods and compositions described here may also be used
for the production of a progenitor cell line or a differentiated
cell for the treatment of, or the preparation of a pharmaceutical
composition for the treatment of, any one of the following: a
disease treatable by regenerative therapy, cardiac failure, bone
marrow disease, skin disease, burns, degenerative disease such as
diabetes, Alzheimer's disease, Parkinson's disease and cancer.
[0203] Mesenchymal stem cells and differentiated cells made
according to the methods described here can be used for a variety
of commercially important research, diagnostic, and therapeutic
purposes. These uses are generally well known in the art, but will
be described briefly here.
[0204] For example, stem cells may be used to generate mesenchymal
stem cells and differentiated cell populations for regenerative
therapy. Mesenchymal stem cells and differentiated cells may be
made by ex vivo expansion or directly administered into a patient.
They may also be used for the re-population of damaged tissue
following trauma.
[0205] Thus, adipocytes or fat tissues may therefrom may be used to
fill up cavities or depressions during reconstructive or plastic
surgery. Chondrocytes may be used for cartilage repair while
osteocytes may be used for bone repair. The mesenchymal stem cells
made by the methods and compositions described here may be
differentiated into any of these cell types and used for the
purposes described.
[0206] Embryonic stem cells and their tissue stem cell derivatives
may be used as sources of mesenchymal stem cells and differentiated
cells for the treatment of degenerative diseases such as diabetes,
Alzheimer's disease, Parkinson's disease, etc. Stem cells, for
example may be used as sources of mesenchymal stem cells and
differentiated cells for NK or dendritic cells for immunotherapy
for cancer, which mesenchymal stem cells and differentiated cells
may be made by the methods and compositions described here.
[0207] It will be evident that the methods and compositions
described here enable the production of mesenchymal stem cells,
which may of course be made to differentiate using methods known in
the art. Thus, any uses of differentiated cells will equally attach
to those mesenchymal stem cells for which they are sources.
[0208] Mesenchymal stem cells and differentiated cells produced by
the methods and compositions described here may be used for, or for
the preparation of a pharmaceutical composition for, the treatment
of a disease. Such disease may comprise a disease treatable by
regenerative therapy, including cardiac failure, bone marrow
disease, skin disease, burns, degenerative disease such as
diabetes, Alzheimer's disease, Parkinson's disease, etc and
cancer.
[0209] We therefore describe a method of treatment of a disease
comprising: (a) providing an embryonic stem (ES) cell; (b)
establishing a mesenchymal stem cell line from the embryonic stem
cell in which the mesenchymal stem cell is obtained by dispersing a
embryonic stem (ES) cell colony, or a descendent thereof, and
propagating the cell in the absence of co-culture in a serum free
medium comprising FGF2; (d) optionally deriving a differentiated
cell from the mesenchymal stem cell line; and (e) administering the
mesenchymal stem cell line or the differentiated cell into a
patient. The medium may optionally contain PDGF AB.
[0210] Our methods may generally be used for up-regulating
expression of mesenchymal or endothelial markers of a cell.
Alternatively, or in addition, they may be used for down-regulating
expression of stem cell or pluripotency markers of a cell.
[0211] We further provide a method according to any preceding
aspect of the invention for the production of a progenitor cell
line or a differentiated cell for the treatment of, or the
preparation of a pharmaceutical composition for the treatment of,
any one of the following: a disease treatable by regenerative
therapy, cardiac failure, bone marrow disease, skin disease, burns,
degenerative disease such as diabetes, Alzheimer's disease,
Parkinson's disease and cancer.
[0212] We also provide a differentiated cell produced by a method
according to any preceding aspect of the invention.
Differentiated Cells
[0213] Differentiated cells, such as terminally differentiated
cells, may be derived from the mesenchymal stem cells or cell lines
made according to the methods described. We therefore disclose
methods for generating differentiated cells, the methods comprising
generating mesenchymal stem cells or cell lines as described, and
deriving differentiated cells from these. The mesenchymal stem
cells made by the methods and compositions described here may be
differentiated into any of these cell types and used for the
purposes described.
[0214] Differentiated cells which may be made according to the
methods described here may include any or all of the following:
[0215] i) adipocyte: the functional cell type of fat, or adipose
tissue, that is found throughout the body, particularly under the
skin. Adipocytes store and synthesize fat for energy, thermal
regulation and cushioning against mechanical shock
[0216] ii) cardiomyocytes: the functional muscle cell type of the
heart that allows it to beat continuously and rhythmically
[0217] iii) chondrocyte: the functional cell type that makes
cartilage for joints, ear canals, trachea, epiglottis, larynx, the
discs between vertebrae and the ends of ribs
[0218] iv) fibroblast: a connective or support cell found within
most tissues of the body. Fibroblasts provide an instructive
support scaffold to help the functional cell types of a specific
organ perform correctly.
[0219] v) hepatocyte: the functional cell type of the liver that
makes enzymes for detoxifying metabolic waste, destroying red blood
cells and reclaiming their constituents, and the synthesis of
proteins for the blood plasma
[0220] vi) hematopoietic cell: the functional cell type that makes
blood. Hematopoietic cells are found within the bone marrow of
adults. In the fetus, hematopoietic cells are found within the
liver, spleen, bone marrow and support tissues surrounding the
fetus in the womb.
[0221] vii) myocyte: the functional cell type of muscles
[0222] viii) neuron: the functional cell type of the brain that is
specialized in conducting impulses
[0223] ix) osteoblast: the functional cell type responsible for
making bone
[0224] x) islet cell: the functional cell of the pancreas that is
responsible for secreting insulin, glucogon, gastrin and
somatostatin. Together, these molecules regulate a number of
processes including carbohydrate and fat metabolism, blood glucose
levels and acid secretions into the stomach.
Uses of Mesenchymal Stem Cells and Differentiated Cells
[0225] Mesenchymal stem cells and differentiated cells made
according to the methods and compositions described here may be
used for a variety of commercially important research, diagnostic,
and therapeutic purposes.
[0226] For example, populations of differentiated cells may be used
to prepare antibodies and cDNA libraries that are specific for the
differentiated phenotype. General techniques used in raising,
purifying and modifying antibodies, and their use in immunoassays
and immunoisolation methods are described in Handbook of
Experimental Immunology (Weir & Blackwell, eds.); Current
Protocols in Immunology (Coligan et al., eds.); and Methods of
Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH
Verlags GmbH). General techniques involved in preparation of mRNA
and cDNA libraries are described in RNA Methodologies: A Laboratory
Guide for Isolation and Characterization (R. E. Farrell, Academic
Press, 1998); cDNA Library Protocols (Cowell & Austin, eds.,
Humana Press); and Functional Genomics (Hunt & Livesey, eds.,
2000). Relatively homogeneous cell populations are particularly
suited for use in drug screening and therapeutic applications.
[0227] These and other uses of mesenchymal stem cells and
differentiated cells are described in further detail below, and
elsewhere in this document. The mesenchymal stem cells and
differentiated cells may in particular be used for the preparation
of a pharmaceutical composition for the treatment of disease. Such
disease may comprise a disease treatable by regenerative therapy,
including cardiac failure, bone marrow disease, skin disease,
burns, degenerative disease such as diabetes, Alzheimer's disease,
Parkinson's disease, etc and cancer.
[0228] As shown in the Examples, the mesenchymal stem cells made by
the methods and compositions described here have similar or
identical properties to bone marrow derived mesenchymal stem cells
(BM-MSCs). Therefore, the mesenchymal stem cells, and any
differentiated cells made from these, may be used in any of the
applications for which BM-MSCs are known to be used, or in which it
is possible for them to be used.
Drug Screening
[0229] Mesenchymal stem cells and differentiated cells made
according to the methods and compositions described here may also
be used to screen for factors (such as solvents, small molecule
drugs, peptides, polynucleotides, and the like) or environmental
conditions (such as culture conditions or manipulation) that affect
the characteristics of mesenchymal stem cells or differentiated
cells.
[0230] In some applications, mesenchymal stem cells and
differentiated cells are used to screen factors that promote
maturation, or promote proliferation and maintenance of such cells
in long-term culture. For example, candidate maturation factors or
growth factors are tested by adding them to mesenchymal stem cells
or differentiated cells in different wells, and then determining
any phenotypic change that results, according to desirable criteria
for further culture and use of the cells.
[0231] Furthermore, gene expression profiling of mesenchymal stem
cells and differentiated cells may be used to identify receptors,
transcription factors, and signaling molecules that are unique or
highly expressed in these cells. Specific ligands, small molecule
inhibitors or activators for the receptors, transcription factors
and signaling molecules may be used to modulate differentiation and
properties of mesenchymal stem cells and differentiated cells.
[0232] Particular screening applications relate to the testing of
pharmaceutical compounds in drug research. The reader is referred
generally to the standard textbook "In vitro Methods in
Pharmaceutical Research", Academic Press, 1997, and U.S. Pat. No.
5,030,015), as well as the general description of drug screens
elsewhere in this document. Assessment of the activity of candidate
pharmaceutical compounds generally involves combining the
differentiated cells with the candidate compound, determining any
change in the morphology, marker phenotype, or metabolic activity
of the cells that is attributable to the compound (compared with
untreated cells or cells treated with an inert compound), and then
correlating the effect of the compound with the observed
change.
[0233] The screening may be done, for example, either because the
compound is designed to have a pharmacological effect on certain
cell types, or because a compound designed to have effects
elsewhere may have unintended side effects. Two or more drugs can
be tested in combination (by combining with the cells either
simultaneously or sequentially), to detect possible drug-drug
interaction effects. In some applications, compounds are screened
initially for potential toxicity (Castell et al., pp. 375-410 in
"In vitro Methods in Pharmaceutical Research," Academic Press,
1997). Cytotoxicity can be determined in the first instance by the
effect on cell viability, survival, morphology, and expression or
release of certain markers, receptors or enzymes. Effects of a drug
on chromosomal DNA can be determined by measuring DNA synthesis or
repair. [.sup.3H]thymidine or BrdU incorporation, especially at
unscheduled times in the cell cycle, or above the level required
for cell replication, is consistent with a drug effect. Unwanted
effects can also include unusual rates of sister chromatid
exchange, determined by metaphase spread. The reader is referred to
A. Vickers (PP 375-410 in "In vitro Methods in Pharmaceutical
Research," Academic Press, 1997) for further elaboration.
Tissue Regeneration
[0234] Mesenchymal stem cells and differentiated cells made
according to the methods and compositions described here may also
be used for tissue reconstitution or regeneration in a human
patient in need thereof. The cells are administered in a manner
that permits them to graft to the intended tissue site and
reconstitute or regenerate the functionally deficient area.
[0235] For example, the methods and compositions described here may
be used to modulate the differentiation of stem cells. Mesenchymal
stem cells and differentiated cells may be used for tissue
engineering, such as for the growing of skin grafts. Modulation of
stem cell differentiation may be used for the bioengineering of
artificial organs or tissues, or for prosthetics, such as
stents.
Cancer
[0236] Mesenchymal stem cells and differentiated cells made by the
methods and compositions described here may be used for the
treatment of cancer.
[0237] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and
leukemia.
[0238] More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
gastric cancer, pancreatic cancer, glial cell tumors such as
glioblastoma and neurofibromatosis, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial carcinoma, salivary
gland carcinoma, kidney cancer, renal cancer, prostate cancer,
vulval cancer, thyroid cancer, hepatic carcinoma and various types
of head and neck cancer. Further examples are solid tumor cancer
including colon cancer, breast cancer, lung cancer and prostrate
cancer, hematopoietic malignancies including leukemias and
lymphomas, Hodgkin's disease, aplastic anemia, skin cancer and
familiar adenomatous polyposis. Further examples include brain
neoplasms, colorectal neoplasms, breast neoplasms, cervix
neoplasms, eye neoplasms, liver neoplasms, lung neoplasms,
pancreatic neoplasms, ovarian neoplasms, prostatic neoplasms, skin
neoplasms, testicular neoplasms, neoplasms, bone neoplasms,
trophoblastic neoplasms, fallopian tube neoplasms, rectal
neoplasms, colonic neoplasms, kidney neoplasms, stomach neoplasms,
and parathyroid neoplasms. Breast cancer, prostate cancer,
pancreatic cancer, colorectal cancer, lung cancer, malignant
melanoma, leukaemia, lympyhoma, ovarian cancer, cervical cancer and
biliary tract carcinoma are also included.
[0239] The mesenchymal stem cells and differentiated cells made
according to the methods and compositions described here may also
be used in combination with anticancer agents such as endostatin
and angiostatin or cytotoxic agents or chemotherapeutic agent. For
example, drugs such as such as adriamycin, daunomycin,
cis-platinum, etoposide, taxol, taxotere and alkaloids, such as
vincristine, and antimetabolites such as methotrexate. The term
"cytotoxic agent" as used herein refers to a substance that
inhibits or prevents the function of cells and/or causes
destruction of cells. The term is intended to include radioactive
isotopes (e.g. I, Y, Pr), chemotherapeutic agents, and toxins such
as enzymatically active toxins of bacterial, fungal, plant or
animal origin, or fragments thereof.
[0240] Also, the term includes oncogene product/tyrosine kinase
inhibitors, such as the bicyclic ansamycins disclosed in WO
94/22867; 1,2-bis(arylamino)benzoic acid derivatives disclosed in
EP 600832; 6,7-diamino-phthalazin-1-one derivatives disclosed in EP
600831; 4,5-bis(arylamino)-phthalimide derivatives as disclosed in
EP 516598; or peptides which inhibit binding of a tyrosine kinase
to a SH2-containing substrate protein (see WO 94/07913, for
example). A "chemotherapeutic agent" is a chemical compound useful
in the treatment of cancer. Examples of chemotherapeutic agents
include Adriamycin, Doxorubicin, 5-Fluorouracil (5-FU), Cytosine
arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,
Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,
Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine,
VP-16, Vinorelbine, Carboplatin, Teniposide, Daunomycin,
Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Nicotinamide,
Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other
related nitrogen mustards, and endocrine therapies (such as
diethylstilbestrol (DES), Tamoxifen, LHRH antagonizing drugs,
progestins, anti-progestins etc).
Stem Cells
[0241] As used in this document, the term "stem cell" refers to a
cell that on division faces two developmental options: the daughter
cells can be identical to the original cell (self-renewal) or they
may be the progenitors of more specialised cell types
(differentiation). The stem cell is therefore capable of adopting
one or other pathway (a further pathway exists in which one of each
cell type can be formed). Stem cells are therefore cells which are
not terminally differentiated and are able to produce cells of
other types.
[0242] Stem cells as referred to in this document may include
totipotent stem cells, pluripotent stem cells, and multipotent stem
cells.
[0243] Totipotent Stem Cells
[0244] The term "totipotent" cell refers to a cell which has the
potential to become any cell type in the adult body, or any cell of
the extraembryonic membranes (e.g., placenta). Thus, the only
totipotent cells are the fertilized egg and the first 4 or so cells
produced by its cleavage.
[0245] Pluripotent Stem Cells
[0246] "Pluripotent stem cells" are true stem cells, with the
potential to make any differentiated cell in the body. However,
they cannot contribute to making the extraembryonic membranes which
are derived from the trophoblast. Several types of pluripotent stem
cells have been found.
[0247] Embryonic Stem Cells
[0248] Embryonic Stem (ES) cells may be isolated from the inner
cell mass (ICM) of the blastocyst, which is the stage of embryonic
development when implantation occurs.
[0249] Embryonic Germ Cells
[0250] Embryonic Germ (EG) cells may be isolated from the precursor
to the gonads in aborted fetuses.
[0251] Embryonic Carcinoma Cells
[0252] Embryonic Carcinoma (EC) cells may be isolated from
teratocarcinomas, a tumor that occasionally occurs in a gonad of a
fetus. Unlike the first two, they are usually aneuploid. All three
of these types of pluripotent stem cells can only be isolated from
embryonic or fetal tissue and can be grown in culture. Methods are
known in the art which prevent these pluripotent cells from
differentiating.
[0253] Adult Stem Cells
[0254] Adult stem cells comprise a wide variety of types including
neuronal, skin and the blood forming stem cells which are the
active component in bone marrow transplantation. These latter stem
cell types are also the principal feature of umbilical cord-derived
stem cells. Adult stem cells can mature both in the laboratory and
in the body into functional, more specialised cell types although
the exact number of cell types is limited by the type of stem cell
chosen.
[0255] Multipotent Stem Cells
[0256] Multipotent stem cells are true stem cells but can only
differentiate into a limited number of types. For example, the bone
marrow contains multipotent stem cells that give rise to all the
cells of the blood but not to other types of cells. Multipotent
stem cells are found in adult animals. It is thought that every
organ in the body (brain, liver) contains them where they can
replace dead or damaged cells.
[0257] Methods of characterising stem cells are known in the art,
and include the use of standard assay methods such as clonal assay,
flow cytometry, long-term culture and molecular biological
techniques e.g. PCR, RT-PCR and Southern blotting.
[0258] In addition to morphological differences, human and murine
pluripotent stem cells differ in their expression of a number of
cell surface antigens (stem cell markers). Antibodies for the
identification of stem cell markers including the Stage-Specific
Embryonic Antigens 1 and 4 (SSEA-1 and SSEA-4) and Tumor Rejection
Antigen 1-60 and 1-81 (TRA-1-60, TRA-1-81) may be obtained
commercially, for example from Chemicon International, Inc
(Temecula, Calif., USA). The immunological detection of these
antigens using monoclonal antibodies has been widely used to
characterize pluripotent stem cells (Shamblott M. J. et. al. (1998)
PNAS 95: 13726-13731; Schuldiner M. et. al. (2000). PNAS 97:
11307-11312; Thomson J. A. et. al. (1998). Science 282: 1145-1147;
Reubinoff B. E. et. al. (2000). Nature Biotechnology 18: 399-404;
Henderson J. K. et. al. (2002). Stem Cells 20: 329-337; Pera M. et.
al. (2000). J. Cell Science 113: 5-10.).
Sources of Stem Cells
[0259] Stem cells of various types, which may include the following
non-limiting examples, may be used in the methods and compositions
described here for producing mesenchymal stem cells and
differentiated cells.
[0260] U.S. Pat. No. 5,851,832 reports multipotent neural stem
cells obtained from brain tissue. U.S. Pat. No. 5,766,948 reports
producing neuroblasts from newborn cerebral hemispheres. U.S. Pat.
Nos. 5,654,183 and 5,849,553 report the use of mammalian neural
crest stem cells. U.S. Pat. No. 6,040,180 reports in vitro
generation of differentiated neurons from cultures of mammalian
multipotential CNS stem cells. WO 98/50526 and WO 99/01159 report
generation and isolation of neuroepithelial stem cells,
oligodendrocyte-astrocyte precursors, and lineage-restricted
neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem
cells obtained from embryonic forebrain and cultured with a medium
comprising glucose, transferrin, insulin, selenium, progesterone,
and several other growth factors.
[0261] Primary liver cell cultures can be obtained from human
biopsy or surgically excised tissue by perfusion with an
appropriate combination of collagenase and hyaluronidase.
Alternatively, EP 0 953 633 A1 reports isolating liver cells by
preparing minced human liver tissue, resuspending concentrated
tissue cells in a growth medium and expanding the cells in culture.
The growth medium comprises glucose, insulin, transferrin, T.sub.3,
FCS, and various tissue extracts that allow the hepatocytes to grow
without malignant transformation. The cells in the liver are
thought to contain specialized cells including liver parenchymal
cells, Kupffer cells, sinusoidal endothelium, and bile duct
epithelium, and also precursor cells (referred to as "hepatoblasts"
or "oval cells") that have the capacity to differentiate into both
mature hepatocytes or biliary epithelial cells (L. E. Rogler, Am.
J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol.
10:710, 1998; Lazaro et al., Cancer Res. 58:514, 1998).
[0262] U.S. Pat. No. 5,192,553 reports methods for isolating human
neonatal or fetal hematopoietic stem or progenitor cells. U.S. Pat.
No. 5,716,827 reports human hematopoietic cells that are Thy-1
positive progenitors, and appropriate growth media to regenerate
them in vitro. U.S. Pat. No. 5,635,387 reports a method and device
for culturing human hematopoietic cells and their precursors. U.S.
Pat. No. 6,015,554 describes a method of reconstituting human
lymphoid and dendritic cells.
[0263] U.S. Pat. No. 5,486,359 reports homogeneous populations of
human mesenchymal stem cells that can differentiate into cells of
more than one connective tissue type, such as bone, cartilage,
tendon, ligament, and dermis. They are obtained from bone marrow or
periosteum. Also reported are culture conditions used to expand
mesenchymal stem cells. WO 99/01145 reports human mesenchymal stem
cells isolated from peripheral blood of individuals treated with
growth factors such as G-CSF or GM-CSF. WO 00/53795 reports
adipose-derived stem cells and lattices, substantially free of
adipocytes and red cells. These cells reportedly can be expanded
and cultured to produce hormones and conditioned culture media.
[0264] Stem cells of any vertebrate species can be used. Included
are stem cells from humans; as well as non-human primates, domestic
animals, livestock, and other non-human mammals such as rodents,
mice, rats, etc.
[0265] Amongst the stem cells suitable for use in this invention
are primate pluripotent stem (pPS) cells derived from tissue formed
after gestation, such as a blastocyst, or fetal or embryonic tissue
taken any time during gestation. Non-limiting examples are primary
cultures or established lines of embryonic stem cells.
Media and Feeder Cells
[0266] Media for isolating and propagating pluripotent stem cells
can have any of several different formulas, as long as the cells
obtained have the desired characteristics, and can be propagated
further.
[0267] Suitable sources are as follows: Dulbecco's modified Eagles
medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagles
medium (KO DMEM), Gibco#10829-018; 200 mM L-glutamine,
Gibco#15039-027; non-essential amino acid solution, Gibco
11140-050; beta-mercaptoethanol, Sigma#M7522; human recombinant
basic fibroblast growth factor (bFGF), Gibco#13256-029. Exemplary
serum-containing embryonic stem (ES) medium is made with 80% DMEM
(typically KO DMEM), 20% defined fetal bovine serum (FBS) not heat
inactivated, 0.1 mM non-essential amino acids, 1 mM L-glutamine,
and 0.1 mM beta-mercaptoethanol. The medium is filtered and stored
at 4 degrees C. for no longer than 2 weeks. Serum-free embryonic
stem (ES) medium is made with 80% KO DMEM, 20% serum replacement,
0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM
beta-mercaptoethanol. An effective serum replacement is
Gibco#10828-028. The medium is filtered and stored at 4 degrees C.
for no longer than 2 weeks. Just before use, human bFGF is added to
a final concentration of 4 ng/mL (Bodnar et al., Geron Corp,
International Patent Publication WO 99/20741).
[0268] In a preferred embodiment, the media comprises Knockout DMEM
media (Invitrogen-Gibco, Grand Island, N.Y.), supplemented with 10%
serum replacement media (Invitrogen-Gibco, Grand Island, N.Y.), 5
ng/ml FGF2 (Invitrogen-Gibco, Grand Island, N.Y.) and 5 ng/ml PDGF
AB (Peprotech, Rocky Hill, N.J.).
[0269] Feeder cells (where used) are propagated in mEF medium,
containing 90% DMEM (Gibco#11965-092), 10% FBS (Hyclone#30071-03),
and 2 mM glutamine. mEFs are propagated in T150 flasks
(Coming#430825), splitting the cells 1:2 every other day with
trypsin, keeping the cells subconfluent. To prepare the feeder cell
layer, cells are irradiated at a dose to inhibit proliferation but
permit synthesis of important factors that support human embryonic
stem cells (.about.4000 rads gamma irradiation). Six-well culture
plates (such as Falcon#304) are coated by incubation at 37 degrees
C. with 1 mL 0.5% gelatin per well overnight, and plated with
375,000 irradiated mEFs per well. Feeder cell layers are typically
used 5 h to 4 days after plating. The medium is replaced with fresh
human embryonic stem (hES) medium just before seeding pPS
cells.
[0270] Conditions for culturing other stem cells are known, and can
be optimized appropriately according to the cell type. Media and
culture techniques for particular cell types referred to in the
previous section are provided in the references cited.
Embryonic Stem Cells
[0271] Embryonic stem cells can be isolated from blastocysts of
members of the primate species (Thomson et al., Proc. Natl. Acad.
Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be
prepared from human blastocyst cells using the techniques described
by Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;
Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al, Nature
Biotech. 18:399, 2000.
[0272] Briefly, human blastocysts are obtained from human in vivo
preimplantation embryos. Alternatively, in vitro fertilized (IVF)
embryos can be used, or one cell human embryos can be expanded to
the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989).
Human embryos are cultured to the blastocyst stage in G1.2 and G2.2
medium (Gardner et al., Fertil. Steril. 69:84, 1998). Blastocysts
that develop are selected for embryonic stem cell isolation. The
zona pellucida is removed from blastocysts by brief exposure to
pronase (Sigma). The inner cell masses are isolated by
immunosurgery, in which blastocysts are exposed to a 1:50 dilution
of rabbit anti-human spleen cell antiserum for 30 minutes, then
washed for 5 minutes three times in DMEM, and exposed to a 1:5
dilution of Guinea pig complement (Gibco) for 3 minutes (see Solter
et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two
further washes in DMEM, lysed trophectoderm cells are removed from
the intact inner cell mass (ICM) by gentle pipetting, and the ICM
plated on mEF feeder layers.
[0273] After 9 to 15 days, inner cell mass-derived outgrowths are
dissociated into clumps either by exposure to calcium and
magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by
exposure to dispase or trypsin, or by mechanical dissociation with
a micropipette; and then replated on mEF in fresh medium.
Dissociated cells are replated on mEF feeder layers in fresh
embryonic stem (ES) medium, and observed for colony formation.
Colonies demonstrating undifferentiated morphology are individually
selected by micropipette, mechanically dissociated into clumps, and
replated. embryonic stem cell-like morphology is characterized as
compact colonies with apparently high nucleus to cytoplasm ratio
and prominent nucleoli. Resulting embryonic stem cells are then
routinely split every 1-2 weeks by brief trypsinization, exposure
to Dulbecco's PBS (without calcium or magnesium and with 2 mM
EDTA), exposure to type IV collagenase (.about.200 U/mL; Gibco) or
by selection of individual colonies by micropipette. Clump sizes of
about 50 to 100 cells are optimal.
Embryonic Germ Cells
[0274] Human Embryonic Germ (hEG) cells can be prepared from
primordial germ cells present in human fetal material taken about
8-11 weeks after the last menstrual period. Suitable preparation
methods are described in Shamblott et al., Proc. Natl. Acad. Sci.
USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.
[0275] Briefly, genital ridges are rinsed with isotonic buffer,
then placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution
(BRL) and cut into <1 mm.sup.3 chunks. The tissue is then
pipetted through a 100/.mu.L tip to further disaggregate the cells.
It is incubated at 37 degrees C. for about 5 min, then about 3.5 mL
EG growth medium is added. EG growth medium is DMEM, 4500 mg/L
D-glucose, 2200 mg/L mM sodium bicarbonate; 15% embryonic stem (ES)
qualified fetal calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium
pyruvate (BRL); 1000-2000 U/mL human recombinant leukemia
inhibitory factor (LIF, Genzyme); 1-2 ng/ml human recombinant basic
fibroblast growth factor (bFGF, Genzyme); and 10 .mu.M forskolin
(in 10% DMSO). In an alternative approach, EG cells are isolated
using hyaluronidase/collagenase/DNAse. Gonadal anlagen or genital
ridges with mesenteries are dissected from fetal material, the
genital ridges are rinsed in PBS, then placed in 0.1 ml HCD
digestion solution (0.01% hyaluronidase type V, 0.002% DNAse I,
0.1% collagenase type IV, all from Sigma prepared in EG growth
medium). Tissue is minced and incubated 1 h or overnight at 37
degrees C., resuspended in 1-3 mL of EG growth medium, and plated
onto a feeder layer.
[0276] Ninety-six well tissue culture plates are prepared with a
sub-confluent layer of feeder cells cultured for 3 days in modified
EG growth medium free of LIF, bFGF or forskolin, inactivated with
5000 rad y-irradiation. Suitable feeders are STO cells (ATCC
Accession No. CRL 1503). 0.2 mL of primary germ cell (PGC)
suspension is added to each of the wells. The first passage is
conducted after 7-10 days in EG growth medium, transferring each
well to one well of a 24-well culture dish previously prepared with
irradiated STO mouse fibroblasts. The cells are cultured with daily
replacement of medium until cell morphology consistent with EG
cells are observed, typically after 7-30 days or 1-4 passages.
Self-Renewal and Differentiation
Self-Renewal
[0277] Stem cells which are self-renewing may be identified by
various means known in the art, for example, morphology,
immunohistochemistry, molecular biology, etc.
[0278] Such stem cells preferably display increased expression of
Oct4 and/or SSEA-1. Preferably, expression of any one or more of
Flk-1, Tie-2 and c-kit is decreased. Stem cells which are
self-renewing preferably display a shortened cell cycle compared to
stem cells which are not self-renewing.
[0279] For example, in the two dimensions of a standard microscopic
image, human embryonic stem cells display high nuclear/cytoplasmic
ratios in the plane of the image, prominent nucleoli, and compact
colony formation with poorly discemable cell junctions. Cell lines
can be karyotyped using a standard G-banding technique (available
at many clinical diagnostics labs that provides routine karyotyping
services, such as the Cytogenetics Lab at Oakland Calif.) and
compared to published human karyotypes.
[0280] Human embryonic stem and human embryonic germ cells may also
be characterized by expressed cell markers. In general, the
tissue-specific markers discussed in this disclosure can be
detected using a suitable immunological technique--such as flow
cytometry for membrane-bound markers, immunohistochemistry for
intracellular markers, and enzyme-linked immunoassay, for markers
secreted into the medium. The expression of protein markers can
also be detected at the mRNA level by reverse transcriptase-PCR
using marker-specific primers. See U.S. Pat. No. 5,843,780 for
further details.
[0281] Stage-specific embryonic antigens (SSEA) are characteristic
of certain embryonic cell types. Antibodies for SSEA markers are
available from the Developmental Studies Hybridoma Bank (Bethesda
Md.). Other useful markers are detectable using antibodies
designated Tra-1-60 and Tra-1-81 (Andrews et al., Cell Linesfrom
Human Gern Cell Tumors, in E. J. Robertson, 1987, supra). Human
embryonic stem cells are typically SSEA-1 negative and SSEA-4
positive. hEG cells are typically SSEA-1 positive. Differentiation
of pPS cells in vitro results in the loss of SSEA-4, Tra-1-60, and
Tra-1-81 expression and increased expression of SSEA-1. pPS cells
can also be characterized by the presence of alkaline phosphatase
activity, which can be detected by fixing the cells with 4%
paraformaldehyde, and then developing with Vector Red as a
substrate, as described by the manufacturer (Vector Laboratories,
Burlingame Calif.).
[0282] Embryonic stem cells are also typically telomerase positive
and OCT-4 positive. Telomerase activity can be determined using
TRAP activity assay (Kim et al., Science 266:2011, 1997), using a
commercially available kit (TRAPeze.RTM. XK Telomerase Detection
Kit, Cat. s7707; Intergen Co., Purchase N.Y.; or TeloTAGGG.TM.
Telomerase PCR ELISA plus, Cat. 2,013,89; Roche Diagnostics,
Indianapolis). hTERT expression can also be evaluated at the mRNA
level by RT-PCR. The LightCycler TeloTAGGG.TM.hTERT quantification
kit (Cat. 3,012,344; Roche Diagnostics) is available commercially
for research purposes.
Differentiation
[0283] Differentiating cells, including mesenchymal stem cells and
differentiated cells derived from these, preferably display
enhanced dephosphorylation of 4E-BP1 and/or S6K1. They preferably
display decreased expression of Oct4 and/or SSEA-1. Preferably,
expression of any one or more of Flk-1, Tie-2 and c-kit is
increased. Preferably, expression of any one or more of Brachyury,
AFP, nestin and nurr1 expression increased. Stem cells which are
self-renewing preferably display a lengthened cell cycle compared
to stem cells which are not self-renewing.
[0284] Differentiating stem cells, i.e., cells which have started
to, or are committed to a pathway of differentiation can be
characterized according to a number of phenotypic criteria,
including in particular transcript changes. The criteria include
but are not limited to characterization of morphological features,
detection or quantitation of expressed cell markers and enzymatic
activity, gene expression and determination of the functional
properties of the cells in vivo. In general, differentiating stem
cells will have one or more features of the cell type which is the
final product of the differentiation process, i.e., the
differentiated cell. For example, if the target cell type is a
muscle cell, a stem cell which is in the process of differentiating
to such a cell will have for example a feature of myosin
expression.
[0285] In many respects, therefore, the criteria will depend on the
fate of the differentiating stem cell, and a general description of
various cell types is provided below.
[0286] Markers of interest for differentiated or differentiating
neural cells include beta-tubulin EIII or neurofilament,
characteristic of neurons; glial fibrillary acidic protein (GFAP),
present in astrocytes; galactocerebroside (GaIC) or myelin basic
protein (MBP); characteristic of oligodendrocytes; OCT-4,
characteristic of undifferentiated human embryonic stem cells;
nestin, characteristic of neural precursors and other cells. A2B5
and NCAM are characteristic of glial progenitors and neural
progenitors, respectively. Cells can also be tested for secretion
of characteristic biologically active substances. For example,
GABA-secreting neurons can be identified by production of glutamic
acid decarboxylase or GABA. Dopaminergic neurons can be identified
by production of dopa decarboxylase, dopamine, or tyrosine
hydroxylase.
[0287] Markers of interest for differentiated or differentiating
liver cells include alpha-fetoprotein (liver progenitors); albumin,
.alpha..sub.1-antitrypsin, glucose-6-phosphatase, cytochrome p450
activity, transferrin, asialoglycoprotein receptor, and glycogen
storage (hepatocytes); CK7, CK19, and gamma-glutamyl transferase
(bile epithelium). It has been reported that hepatocyte
differentiation requires the transcription factor BNF-4 alpha (Li
et al., Genes Dev. 14:464, 2000). Markers independent of HNF-4
alpha expression include alpha.sub.1-antitrypsin,
alpha-fetoprotein, apoE, glucokinase, insulin growth factors 1 and
2, IGF-1 receptor, insulin receptor, and leptin. Markers dependent
on HNF-4 alpha expression include albumin, apoAl, apoAII, apoB,
apoCIII, apoCII, aldolase B, phenylalanine hydroxylase, L-type
fatty acid binding protein, transferrin, retinol binding protein,
and erythropoietin (EPO).
[0288] Cell types in mixed cell populations derived from pPS cells
can be recognized by characteristic morphology and the markers they
express. For skeletal muscle: myoD, myogenin, and myf-5. For
endothelial cells: PECAM (platelet endothelial cell adhesion
molecule), Flk-1, tie-i, tie-2, vascular endothelial (VE) cadherin,
MECA-32, and MEC-14.7. For smooth muscle cells: specific myosin
heavy chain. For cardiomyocytes: GATA-4, Nkx2.5, cardiac troponin
I, alpha-myosin heavy chain, and ANF. For pancreatic cells, pdx and
insulin secretion. For hematopoietic cells and their progenitors:
GATA-1, CD34, AC133, .beta.-major globulin, and .beta.-major
globulin like gene PH1.
[0289] Certain tissue-specific markers listed in this disclosure or
known in the art can be detected by immunological techniques--such
as flow immunocytochemistry for cell-surface markers,
immunohistochemistry (for example, of fixed cells or tissue
sections) for intracellular or cell-surface markers, Western blot
analysis of cellular extracts, and enzyme-linked immunoassay, for
cellular extracts or products secreted into the medium. The
expression of tissue-specific gene products can also be detected at
the mRNA level by Northern blot analysis, dot-blot hybridization
analysis, or by reverse transcriptase initiated polymerase chain
reaction (RT-PCR) using sequence-specific primers in standard
amplification methods. Sequence data for the particular markers
listed in this disclosure can be obtained from public databases
such as GenBank.
Examples
Example 1
Methods
[0290] Derivation of hESC-MSC (Mesenchymal Stem Cells)
[0291] Hues9 and H1 hESCs are grown as previously
described.sup.6,7.
[0292] To differentiate hESCs, a confluent 6 cm plate of hESCs is
trypsinized for 3 mins, 37.degree. C., neutralized, centrifuged and
resuspended in Knockout DMEM media (Invitrogen-Gibco, Grand Island,
N.Y.), supplemented with 10% serum replacement media
(Invitrogen-Gibco, Grand Island, N.Y.), 5 ng/ml FGF2
(Invitrogen-Gibco, Grand Island, N.Y.), and 5 ng/ml PDGF AB
(Peprotech, Rocky Hill, N.J.) on a gelatinized 10 cm plate. The
cells are trypsinized when confluent and split 1:4.
[0293] Sorting for CD105+ and CD24- is performed one week after
hESCs have been trypsinized. The differentiating hESCs are
trypsinized for 3 mins, neutralized, centrifuged, resuspended in
the culture media and then plated on bacterial culture dish. After
2 hours at 37.degree. C. in CO.sup.2 incubator, the cells are
harvested, washed with PBS and incubated with CD24-PE and
CD105-FITC (PharMingen, San Diego, Calif.) for 90 mins at room
temperature.
[0294] The cells are then washed with PBS and sorted on a FACS Aria
using FACS Diva software (BD Biosciences Pharmingen, San Diego,
Calif.). BM MSCs are prepared as previously described.sup.26. The
cells are cultured in DMEM supplemented with
penicillin-streptomycin-glutamine, non-essential amino acids and
10% fetal calf serum (Invitrogen-Gibco, Grand Island, N.Y.)
[0295] Differentiation into adipocytes, chondrocytes and osteocytes
is performed as previously described.sup.3. Oil red, alcian blue
and von Kossa staining is performed using standard techniques
Immunoreactivity for collagen type II is performed on
paraformaldehyde fixed, paraffin-embedded sections using a goat
anti-collagen a1 Type II and donkey anti-goat IgG antibody
conjugated with HRP (Santa Cruz, Santa Cruz, Calif.).
[0296] Karyotyping
[0297] Cells received at about 80% confluence in Petri dish. Cells
are treated with colcemid for mitotic arrest and harvested by
standard hypotonic treatment and methanol:acetic acid (3:1)
fixation. Slides are prepared by standard air drying method and
hybridized with SKY paint probe (ASI). Post hybridization washes
are performed according to the protocols provided by the
manufacturer and established in our laboratory. 20-30 metaphase
cells per culture were are. The karyotype of each culture is
representative of >80% metaphase cells.
[0298] Transplantation Studies
[0299] 2.times.10.sup.6 cells are resuspended in 30 .mu.l of saline
and transferred into the renal subcapsular space as previously
described' After four months, the mice are sacrificed and the
kidneys are removed, fixed in 4% paraformaldehyde,
paraffin-embedded, sectioned at 4 .mu.M and stained with
H&E.
[0300] Western Blot Analysis
[0301] Standard procedures are used.sup.28. Briefly, cells are
lysed in RIPA buffer and centrifuged at 14,000 rpm for 15 minutes
at 4.degree. C. 20 ng supernatant is denatured, separated on 10
SDS-polyacrylamide gel, electro-blotted onto a nitrocellulose
membrane and membrane is incubated sequentially with a primary
antibody, then either a HRP conjugated-secondary antibody or a
biotinylated secondary antibody followed by neutroavidin-HRP, and
finally, a HRP enhanced chemiluminescent substrate, ECS (Pierce,
Rockford, Ill.).
[0302] Primary antibodies used are 1:200 dilution of anti-OCT4,
anti-SOX-2 and .beta.-actin (Santa Cruz Biotechnology, CA),
Secondary antibodies are HRP-conjugated goat anti-rabbit, rabbit
anti-goat and rabbit antimouse.
[0303] PCR
[0304] Genomic PCR for mouse- and human-specific repeat sequences
are performed as previously described. Genomic PCR for mouse- and
human-specific repeat sequences are performed as previously
described. Real time RT-PCR is performed by reverse transcribing 1
.mu.g of total RNA using a High Capacity cDNA Archive Kit (Applied
Biosystems, Foster City, Calif.). The cDNA is diluted 10.times. in
water and amplified by Taqman primers (Applied Biosystems, Foster
City, Calif.) for 40 cycles.
[0305] Surface Antigen Analysis
[0306] Cell surface antigens on hESC-MSCs, and hESCs are analyzed
using FACS. The cells are tryspinized for 5 minutes, centrifuged,
resuspended in culture media and incubated in a bacterial culture
dish for 2-3 hours in a 37.degree. C., 5% CO.sup.2 incubator. Cell
surface antigens on hESC-MSCs and hESCs are analyzed by FACS.
[0307] The cells are trypsinized for 1 minute, centrifuged, washed
with PBS, fixed in 4% paraformaldehyde for 0.5 hour at room
temperature, washed and blocked in 2% FCS for 0.5 hour at room
temperature with agitation. 1.5.times.10.sup.5 cells are then
incubated with each of the following conjugated monoclonal
antibodies: CD24-PE, CD29-PE, CD44-FITC, CD49a-PE, CD49e-PE,
CD105-FITC, CD166-PE, CD34-FITC, CD45-FITC (PharMingen, San Diego,
Calif.) for 90 mins at room temperature.
[0308] After incubation, cells are washed and resuspended in PBS.
Nonspecific fluorescence is determined by incubation of similar
cell aliquots with isotype-matched mouse monoclonal antibodies
(PharMingen, San Diego, Calif.). Data are analyzed by collecting
20,000 events on a Cyan LX (Dako North America, Inc., Carpinteria,
Calif.) instrument using WinMDI software. Nonspecific fluorescence
is determined by incubation of similar cell aliquots with
isotype-matched mouse monoclonal antibodies (PharMingen, San Diego,
Calif.), or with secondary antibody alone.
[0309] Illumina Gene Chip Analysis
[0310] Total RNA (2 .mu.g) from 3 samples each of primary BM and
adipose-derived MSCs, from two biological replicates of HuES9.E1,
HuES9.E3, HuES9.E1 and three undifferentiated hESC lines, H1, Hes3
and HuES9 are cnverted to biotinylated cRNA using the Illumina RNA
Amplification Kit (Ambion, Inc., Austin, Tex.) according to the
manufacturer's directions.
[0311] Samples are purified using the RNeasy kit (Qiagen, Valencia,
Calif.). Hybridization to the Sentrix HumanRef-8 Expression
BeadChip (Illumina, Inc., San Diego, Calif.), washing and scanning
are performed according to the Illumina BeadStation 500.times.
manual. The data are extracted, normalized and analyzed using
Illumina BeadStudio provided by the manufacturer. Transcript
signals that are below the limit of detection (LOD) at 99%
confidence are eliminated as genes not expressed.
Example 2
Generating MSC Cultures from Human ES Cell Lines
[0312] When hESC colonies are dispersed by trypsin and then
passaged on gelatinized tissue culture plates in the absence of
feeder, and in serum-free media that is supplemented with serum
replacement media, FGF2 and optionally PDGF AB, a culture of
fibroblast-like cells which is similar or identical (preferably
homogenous) to each other is generated within two weeks.
[0313] The cultures have a fibroblastic cellular morphology that
resembled BM-MSCs (FIG. 1A). Dispersing hESC colonies by
collagenase is not efficient in generating these fibroblast-like
cells.
[0314] Two polyclonal cultures, huES9.E1 and huES9.E3, are
independently generated from huES9 ESC line, while the third, H1.E2
is generated from H1 ESC line. Expression of several
pluripotency-associated genes is generally reduced. For example,
transcript levels of HESX1, POUFL5, SOX-2, UTF-1 and ZFP42 are
>10.sup.1-5 fold below that in the hESCs (FIG. 1B). Protein
levels of OCT4, NANOG and SOX2 are also reduced (FIG. 1C). As
typified by huES9.E1, these cells did not have detectable alkaline
phosphatase activity (FIG. 1D).
[0315] Unlike its parental HuES9 cells, renal subcapsular
transplantation of 1.times.10.sup.6 HuES9.E1 cells in immune
compromised SCID mice did not induce the formation of a teratoma
during a four-month observation period (FIG. 1E). To assess the
possibility that these cells are contaminated or fused with mouse
feeder cells.sup.8, these cultures are tested and shown to be
negative for mouse-specific c-mos repeat sequences but positive for
human specific alu repeat sequences (FIG. 1F).
[0316] The average population doubling time of HuES9.E1, HuES9.E3
and H1.E2 are 72, 72 and 120 hours respectively. Population
doubling time is highly dependent on cell density and is most
optimal at between 30-80% confluency. After about 80 population
doublings, HuES9.E1 have a 46 XX inv(9)(p13q12 karyotype. The
chromosome 9 inversion originated from the parental huES9 hESC
line.sup.6. HuES9.E3 also have 46 XX inv(9)(p13q12 karyotype while
H1.E1 has a 46, XY karyotype after 36 and 32 population doubling
time. We observed that the cells began to manifest abnormal,
nonclonal chromosomal aberrations in .about.10-30% of cells after
about 35 population doublings (data not shown) and cells are not
used after 35 population doublings.
Example 3
Surface Antigen Profile
[0317] Surface antigen profiling of HuES9.E1, HuES9.E3 and H1.E2 by
FACS analysis revealed a surface antigen profile that is
qualitatively similar to that defined for BMMSCs i.e. CD29+, CD44+,
CD49a and e+, CD105+, CD166+ and CD34-, CD45-.sup.9-11 (FIG. 2A)
The intensity of fluorescent labelling and distribution of labelled
cells varied with each of the hESC-MSC cultures (FIG. 2A).
[0318] To compare the surface antigen profile of these cells to
that of BM-MSCs, HuES9.E1, HuES9.E3 and H1.E1 are grown in the same
BM-MSC culture media supplemented with 10% fetal calf serum for two
passages. Despite the change in culture condition, HuES9.E1,
HuES9.E3 and H1.E1 continued to be CD29+, CD44+, CD49a+, CD105+,
CD166+ and CD34-, CD45- (FIG. 2B; data not shown for H1.E1) and are
largely similar to that of BM-MSCs. An exception is CD49a which had
a much lower expression in BM-MSCs. These data indicated that the
hESC-MSCs exhibited characteristic BM-MSC surface antigen profiles
that are stable and are not significantly influenced by changes in
their microenvironment.
Example 4
Differentiation Potential of hESC-MSC
Adipogenesis, Chondrogenesis and Osteogenesis
[0319] As all of the surface antigens associated with MSCs are also
expressed on many other cell types and the expression of these
surface antigens are variable, identification of presumptive MSCs
have traditionally relies on functional parameters.sup.9. It is
reported that the default differentiation pathway of MSCs in
culture is osteogenesis with varying degrees of adipogenesis and
chondrogenesis.sup.9.
[0320] Differentiation potential of HuES9.E1 cells is therefore
tested using standard differentiation conditions for adipogenesis,
chondrogenesis and osteogenesis using published
protocols.sup.3.
[0321] Adipocytic differentiation is highly efficient with oil
droplets observed in >99% of the cells (FIG. 3A). Consistent
with its role as important transcription factor in adipogenesis',
PPAR.gamma. mRNA in the hESC derived MSCs which is about 10-100
fold higher than that in their respective parental ESC lines
increased by a further 2 fold (FIG. 3A).
[0322] Chondrogeneiss or the formation of cartilage is also
efficient with >90% of cells producing proteoglycan in
extracellular matrix as detected by alcian blue staining (FIG. 3B)
and .about.20% of the cells being immunoreactive for collagen II.
Transcript level of aggrecan, an cartilage-specific extracellular
matrix protein is also increased.sup.13 (FIG. 3B). However,
transcript level of collagen II, another cartilage-specific
extracellular matrix protein is decreased despite the presence of
collagen II immunoreactivity in the matrix. The reason is not known
but some mRNAS particularly those with AU-rich elements are known
to be destabilized when translated.sup.14,15.
[0323] When HuES9.E1 cells are induced to undergo osteogenesis or
the formation of bone, expression of bone-specific alkaline
phosphatase (ALP) and bone sialoprotein (BSP).sup.16 is upregulated
by 2-3 fold (FIG. 3C). However, mineralization, a more advanced
stage of bone formation.sup.17 as determined by von Kossa staining
is poor (FIG. 3C). There is <1% positive staining in the
differentiated culture.
Example 5
Gene Expression Profile
[0324] Gene expression profiling of the hESC-MSCs are performed to
1) assess the relatedness of hESC-MSC cultures with adult
tissue-derived MSCs using BM-MSCs and adipose derived (ad)-MSCs
from three different individuals, and three human ESC lines; 2)
assess the relatedness between each of the three hESC-MSC cultures;
3) compare the similarity and differences between MSCs derived from
hESC and those derived from BM.
[0325] Labeled cDNA prepared from total RNA RNA are hybridized to
Illumina BeadArray containing about 24,000 unique features.
Hierarchical clustering of expressed genes in three hESC-MSC
cultures i.e. HuES9.E1, HuES9.E3 and H1.E2, three BM-MSC samples
and three adipose derived (ad)-MSC samples revealed that the gene
expression profile of hESC-MSCs is more closely related to that of
adult tissue-derived MSCs, namely BM-MSC and ad-MSC than to their
parent hESCs (FIG. 4A).
[0326] Interestingly, MSCs clustered according to their tissue of
origin, and this can be further demarcated into adult versus
embryonic tissue as suggested by the clustering of ad-MSCs and
BMMSCs as a distinct group from hESC-MSCs. Pairwise comparison of
gene expression between hESC-MSCs and BM-MSCs revealed a
correlation coefficient of 0.72 suggesting that while there is
significant conservation of gene expression in both hESC-MSCs and
BM-MSCs, there are also significant differences (FIG. 4B). Pairwise
comparison between hESC-MSCs and hESCs confirmed the distinction of
hESC-MSCs from hESCs with a low correlation coefficient of 0.65
(FIG. 4C).
[0327] To assess the relatedness between each of the three hESC-MSC
cultures, HuES9.E1, H1.E2 and HuES9.E3 are each compared to the
same reference consisting of HuES9.E1, H1.E2 and HuES9.E3. The
correlation coefficients of HuES9.E1, H1.E2 and HuES9.E3 to the
same reference are virtually identical i.e. 0.93, 0.95 and 0.93,
respectively suggesting that HuES9.E1, H1.E2 and HuES9.E3 are
highly similar (FIG. 4C).
[0328] Of 8699 and 8505 genes that are expressed above the limit of
detection at 99% confidence level in hESC-MSC and BM-MSC,
respectively, 6376 genes are expressed in both hESC-MSCs and
BM-MSCs at <2.0 fold difference. As these genes are likely to
provide insights into the fundamental biology of MSCs, we examine
the biological processes that are driven by these genes. Of the
6376 commonly expressed genes, 4,064 are found in the Panther
classified gene list (March 2006).
[0329] Classification of these genes into different biological
processes revealed that the frequency of genes in some of the
biological processes are significantly over or under represented
(p<0.01) when compared to the reference list consisting of 23481
genes in NCBI: H. sapiens gene database. For example, there are an
overrepresentation of genes in metabolic processes that are likely
to be important for growth and self-renewal of putative stem cells.
These processes include basal metabolic processes for catabolic and
anabolic activities, biosynthesis of secretory products that
require extensive post-translational modifications e.g.
glycosylation, and cellular proliferation (FIG. 4C).
[0330] Consistent with their mesenchymal potential, there is also
an under-representation of genes involved in ectoderm
differentiation particularly neural development. The gene
expression analysis also suggested that MAPKKK signalling is
prominent in both BM-MSC and hESC-MSCs. MAPKKK signalling which
consists of at least three subfamilies, namely the classical MAPK
(also known as ERK), stress-activated protein kinase/c-Jun
N-terminal kinase (JNK) and p38 kinase, are associated with
proliferation, differentiation, development, regulation of
responses to cellular stresses, cell cycle, death and
survival.sup.18-21.
[0331] Further analysis of the gene expression profiles of hESC-MSC
and BM-MSC revealed that 1142 and 1134 genes are expressed at
>2.0 fold in hESC-MSC and BM-MSC, respectively. Of these, 738
and 880 genes respectively, are located in Panther classified gene
list (March 2006) and classified into biological processes.
Biological processes that are significantly over- or
under-represented (p<0.01) when compared to the reference list
of 23481 genes in NCBI: H. sapiens gene database are selected.
[0332] Genes that are preferentially expressed in BM-MSCs are
clustered in biological processes that are involved in metabolic
processes, cell structure, differentiation and signalling while
preferentially expressed hESC-MSC genes are clustered in those
processes involved in proliferation, differentiation, immunity and
signal transduction (FIG. 3C). The over-representation of genes in
biological processes associated with proliferation is consistent
with the higher proliferative capacity of hESC-MSC over BM-MSC.
[0333] Although highly expressed genes in either hESC-MSC or BM-MSC
are over-represented in the general categories of differentiation
and signalling, the specific biological processes within each
category are differently represented in hESC-MSC and BM-MSC. For
example, differentiation processes that are associated with early
embryonic development such as embryogenesis and segmentation are
over-represented in hESC-MSC while those associated with late
embryonic development e.g. skeletal development and muscle
development are overrepresented in BM-MSC. Similarily,
extracellular matrix protein-mediated signalling and MAPKKK cascade
are overrepresented in BM-MSC. Together, these observations suggest
that differentiation potential and signalling pathway utilisation
in hESC-MSC and BM-MSC may not be identical.
Example 5
Distinguishing Surface Markers for hESCs and hESC-Derived MSCs for
Isolating of Single Cell-Derived MSC Population
[0334] The genome-wide gene expression is queried for highly
expressed genes in either hESC-MSC or hESC that encode for membrane
proteins to facilitate the isolation of MSCs from differentiating
hESCs. From a list of top 20 highly expressed genes encoding for
putative membrane proteins in either hESC-MSCs or hESCs, candidate
genes are selected for which antibodies against their gene product
is commercially available (Table E1A and E1B below). Among those
candidate genes that are highly expressed in hESC derived
TABLE-US-00001 TABLE E1A Highly expressed membrane proteins in
hESC-derived MSC over hESC Fold Symbol change Accession Synonyms
ANPEP 715.50 NM_001150.1 CD13; LAP1; PEPN; gp150 ENG 479.50
NM_000118.1 END; ORW; HHT1; ORW1; CD105 SCN9A 251.20 NM_002977.1
PN1; NE-NA TRPV2 187.90 NM_016113.3 VRL; VRL1; VRL-1; MGC12549
RAMP1 182.30 NM_005855.1 F2RL2 152.03 NM_004101.2 PAR3 NTSR1 141.15
NM_002531.1 NTR GABRA2 122.05 NM_000807.1 SLC16A4 106.60
NM_004696.1 MCT4 ITGA4 103.60 NM_000885.2 CD49D NCAM2 93.31
NM_004540.2 NCAM21; MGC51008 IL1R1 86.80 NM_000877.2 P80; IL1R;
IL1RA; CD121A; D2S1473; IL-1R-alpha PDGFRA 80.25 NM_006206.2
CD140A; PDGFR2 VCAM1 71.30 NM_080682.1 INCAM-100 SSFA2 69.74
NM_006751.3 CS1; CS-1; KRAP; SPAG13; KIAA1927 TRHDE 58.63
NM_013381.1 PAP-II EDG2 55.82 NM_001401.3 LPA1; edg-2; vzg-1;
Gpcr26; Mrec1.3; rec.1.3 NT5E 48.15 NM_002526.1 eN; NT5; NTE; eNT;
CD73; E5NT FLRT2 46.51 NM_013231.2 KIAA0405 FAP 44.43 NM_004460.2
FAPA; DPPIV; SEPRASE
TABLE-US-00002 TABLE E1B Highly expressed membrane proteins in hESC
over hESC-derived MSC Fold Symbol change Accession Synonyms
ITGB1BP3 2642.33 NM_014446.1 MIBP PTPRZ1 2126.50 NM_002851.1 PTPZ;
HPTPZ; PTP18; PTPRZ; RPTPB CNTN1 430.00 NM_175038.1 F3; GP135 PCDH1
342.08 NM_002587.3 PC42; PCDH42; MGC45991 PODXL 303.06 NM_005397.2
PCLP; Gp200 GPR64 217.67 NM_005756.1 HE6; TM7LN2 PROM1 209.04
NM_006017.1 AC133; CD133; PROML1 GPRC5C 205.00 NM_022036.2 RAIG3;
RAIG-3 CD24 166.98 NM_013230.1 CD24A CLDN3 166.42 NM_001306.2 RVP1;
HRVP1; CPE-R2; CPETR2 TACSTD1 163.81 NM_002354.1 EGP; KSA; M4S1;
MK-1; EGP40; MIC18; TROP1; Ep-CAM; hEGP-2; CO17-1A; GA733-2 HTR3A
140.00 NM_000869.1 HTR3 FGFR4 139.96 NM_022963.1 TKF; JTK2 ADCY1
127.44 NM_021116.1 FGFR3 123.27 NM_022965.1 ACH; CEK2; JTK4;
HSFGFR3EX IL17RB 91.70 NM_018725.2 CRL4; EVI27; IL17BR; IL17RH1;
MGC5245 SORL1 66.79 NM_003105.3 LR11; LRP9; SORLA; gp250; SorLA-1
GPM6B 60.70 NM_005278.2 M6B KCNS3 35.26 NM_002252.3 KV9.3; MGC9481
FZD3 33.94 NM_017412.2 Fz-3; hFz3
[0335] Tables E1A and E1B above show highly expressed surface
antigen encoding genes in hESC-derived MSCs (Table E1A) or their
parental hESCs (Table E1B). Based on gene expression analysis by
microarray hybridization, the top twenty genes that were highly
expressed in hESC-derived MSCs (HuES9.E1, HuES9.E3 and H1.E2) vs
hESCs (HuES9, H1 and Hes3 hESCs) (Table E1A) and vice versa (Table
E1B).
[0336] MSCs are ENG (CD105), ITGA4 (CD49d), PDGFRA, NTSE (CD73)
that are characteristic surface markers of MSCs derived from adult
tissues.sup.9-11 and among those candidate genes that are highly
expressed in hESC are previously identified as highly expressed
hESC-specific genes, ITGB1BP3 and PODXL.sup.22 and CD 24 whose
expression has not been associated with hESCs. We confirmed that
CD24 is highly expressed in hESC vs hESC-MSC (FIG. 5A).
Example 6
Deriving a Homogenous hESC-MSC Population
[0337] We next tested the utility of these markers to enhance the
homogeneity of hESC-MSCs. One week after trypsinization and culture
in media supplemented with serum replacement media, FGF2 and
optionally PDGF AB, the cells in the culture is sorted by FACS for
CD105 and against CD24.
[0338] CD105+ and CD24- cells constituted .about.5% of the culture
(FIG. 5B). Sorted cells are plated onto 10.times.96 well plates at
1 cell/well, 1.times.24 well plate at 100 cells/well and 3.times.6
well plates at 1000 cells/well. Of these, only five of the eighteen
1000 cells/wells generated MSC-like cultures suggesting that these
cultures are likely to be generated from a single cell.
[0339] Genome-wide gene expression profiling of these five
cultures, Q4.1 to Q4.5 using the Illumina BeadArray containing
about 24,000 unique features revealed a high degree of similarity
among the five cultures with four of the lines having a correlation
coefficient of 0.96 and the remaining one with 0.90 (FIG. 5C). In
our hands, the correlation coefficient between technical replicates
performed at least one month apart using the same RNA sample is
routinely in the range of 0.97 to 0.98. Q4.1 to Q4.5 are also
highly similar to the hESC-MSCs consisting of huES9.E1, H1.E2 and
huES9.E3, and BM-MSCs with a correlation coefficient of 0.87 and
0.81, respectively (FIG. 5D).
[0340] In contrast, the correlation coefficient of Q4.1 to Q4.5 to
their parental HuES9.E1 hESC line is a low 0.55 (FIG. 5D).
Chromosomal analysis using G banding and SKY is performed on
randomly selected Q4.3 culture. Q4.3 has a normal karyotype with a
chromosome 9 inversion that originated from its parental HuES9 hESC
line (FIG. 5E).sup.6. Together these observations show that highly
homogenous MSC cultures can be generated by sorting for CD105+ and
CD24- cells from trypsinized hESC culture after propagation in
media supplemented with bFGF2 and optionally PDGF BB for one
week.
[0341] The incorporation of positive and negative selectable
markers into the derivation protocol resulted in the derivation of
five monoclonal isolates with a genome-wide expression profile that
is almost identical to each other and confirmed the specificity of
the selection or sorting criteria. Global pairwise gene expression
comparison between the five isolates reveal a near identical gene
expression profile that is comparable to that observed for
technical replicates using the same RNA samples.
[0342] Examples 7 to 15 describe experiments to analyse the
proteome of human ESC-derived MSCs (hESC-MSCs). In these
experiments, a chemically defined serum-free culture media is
conditioned by human ESC-derived MSCs (hESC-MSCs) is analysed using
a clinically compliant protocol. The conditioned media is analyzed
by multidimensional protein identification technology (MuDPIT) and
cytokine antibody array analysis, and reveals the presence of 201
unique gene products.
[0343] 86-88% of these gene products have detectable transcript
levels by microarray or qRT-PCR assays. Computational analysis
predicts that these gene products will significantly drive three
major groups of biological processes: metabolism, defense response,
and tissue differentiation including vascularization, hematopoiesis
and skeletal development. It also predicts that the 201 gene
products activate important signalling pathways in cardiovascular
biology, bone development and hematopoiesis such as Jak-STAT, MAPK,
Toll-like receptor, TGF-beta signalling and mTOR signaling
pathways.
Example 7
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
Materials and Methods
[0344] Preparation of Conditioned Media
[0345] HuES9.E1 cells are cultured as described above. 80%
confluent HuES9.E1 cell cultures are washed 3 times with PBS,
cultured overnight in a chemically defined media consisting of DMEM
media without phenol red (Catalog 31053; Invitrogen-Gibco, Grand
Island, N.Y.) and supplemented with ITS (Invitrogen-Gibco, Grand
Island, N.Y.), 5 ng/ml FGF2 (Invitrogen-Gibco, Grand Island, N.Y.),
5 ng/ml PDGF AB (Peprotech, Rocky Hill, N.J.)
glutamine-penicillin-streptomycin and .beta.-mercaptoethanol. The
cultures are then rinsed three times with PBS and then replaced
with fresh defined media. After three days, the media are
collected, centrifuged at 500.times.g and the supernatant is
0.2.mu. filtered. For LC MS/MS analysis, the conditioned media is
placed in dialysis cassettes with MW cutoff of 3500 (Pierce
Biotechnology, Rockford, Ill.), dialyzed against 3 changes of 10
vol. 0.9% NaCl, then concentrated 20 times using Slide-A-Lyzer
Concentrating Solution, then dialysing against 10 changes of 100
vol. 0.9% NaCl before filtering with a 0.2.mu. filter. A same
volume of non-conditioned media is dialyzed and concentrated in
parallel with the conditioned media.
[0346] Cytokine Antibody Blot Assays
[0347] One ml of conditioned or non-conditioned media is assayed
for the presence of cytokines and other proteins using RayBio.RTM.
Cytokine Antibody Arrays according to manufacturer's instruction
(RayBio Norcross, Ga.).
[0348] LC MS/MS Analysis
[0349] Proteins in two ml of dialyzed conditioned (CM) or
non-conditioned media (NCM) are reduced, alkylated, and tryptic
digested as described {Washburn, 2001 #2997}. The samples are then
desalted by passing the digested mixture through a conditioned
Sep-Pak C-18 SPE cartridge (Waters, Milford, Mass., USA), washed
twice with a 3% acetonitrile (ACN) (JT Baker, Phillipsburg, N.J.)
and 0.1% formic acid (FA) buffer, and eluted with a 70% ACN and
0.1% FA buffer. The eluted samples are then dehydrated by speedvac
to about 10% of their initial volumes and adjusted to 50 .mu.l with
0.1% formic acid. The samples are kept at 4.degree. C. prior to
LC-MS/MS analysis.
[0350] The desalted peptide mixture is analyzed by MudPIT with a
LC-MS/MS system (LTQ, ThermoFinnign, San Jose, Calif., USA). The
sample is loaded into a strong cation exchange (SCX) column
(Biobasic SCX, Sum, Thermo Electron, San Jose, USA) and fractioned
by 6 salt steps with 50 ul of buffers (0, 2, 5, 10, 100, and 1000
mM of ammonium chloride in a 5% ACN and 0.1% FA) in first
dimension. The peptides eluted from SCX column are concentrated and
desalted in a Zorbax peptide trap (Agilent, Pola Alto, Calif.,
USA). The second dimensional chromatographic separation is carried
out with a home-packed nanobored C18 column (75 um i.d.times.10 cm,
5 .mu.m particles) directly into a pico-frit nanospray tip (New
Objective, Wubrun, Mass., USA), operating at a flow rate of 200
nL/min with a 120 min gradient.
[0351] The LTQ is operated in a data-dependent mode by performing
MS/MS scans for the 3 of the most intense peaks from each MS scan.
For each experiment, ms/ms (dta) spectra of the 6 salt steps are
combined into a single mascot generic file by a home-written
program. Protein identification is achieved by searching the
combined data against the IPI human protein database via an
in-house Mascot server. The search parameters are: a maximum of 2
missed cleavages using trypsin; fixed modification is
carbaminomethylation and variable modifications are oxidation of
methionine and protein N-terminal acetylation. The mass tolerances
are set to 2.0 and 0.8 Da for peptide precursor and fragment ions
respectively. Protein identifications are accepted as true positive
if two different peptides are found with score 50 or above. Since
many growth factors and cytockines are small proteins/peptides and
secreted in small amount, the corresponding MS/MS spectra will be
weak and only one peptide per protein will be identified. For those
peptides with Mascot score of between 20 and 50, manual validation
of the MS/MS spectra is performed.
[0352] Bioinformatics
[0353] The validated proteins are collated by removing the
background proteins identified in the non-conditioned medium. The
IPI identifier of each protein is then converted to gene symbol by
using the protein cross-reference table. Gene products are
classified into the different biological processes or pathways of
the GO classification system Gene Ontology (GO) classification on
GeneSpring GX7.3 Expression Analysis (Agilent Technologies, Palo
Alto, Calif.). and then comparing the frequency of genes in each
process or pathway to that in the Genbank human genome database,
those processes of pathways with significantly higher gene
frequency (p<0.05) are assumed to be significantly modulated by
the secretion of MSC.
[0354] qRT-PCR
[0355] Total RNA is extracted from HuES9.E1 cells with Trizol
Reagent (Gibco-BRL) and purified over a spin column (Nucleospin RNA
II System, Macherey-Nagel GmbH &Co., Duren, Germany) according
to the manufacturers' protocol. 1 .mu.g total RNA is converted to
cDNA with random primers in a 50 .mu.l reaction volume using a High
Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif.,
USA). The cDNA is diluted with distilled water to a volume of 100
.mu.l. 1 .mu.l is used for each primer set in a pathway-specific
RT.sup.2 Profiler PCR Arrays (SuperArray, Frederick, USA) according
to the manufacturer's protocol. The plates used for the analysis
are: Chemokines & Receptors PCR Array (cat. no. APH-022), NFkB
Signaling Pathway PCR Array (cat. no. APH-025), Inflammatory
Cytokines & Receptors PCR Array (cat. no. APH-011), Common
Cytokine PCR Array (cat. no. APH-021), JAK/STAT Signaling Pathway
PCR Array (cat. no. APH-039).
Example 8
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
Preparation of Conditioned Media (CM) and Non-Conditioned Media
(NCM)
[0356] To ensure that there is minimal contamination of conditioned
media by media supplements such as serum replacement media,
HuES9.E1 MSCs are grown to about 80% confluency, washed three times
with PBS, incubated overnight in a chemically defined media
consisting of DMEM supplemented with ITS (insulin, transferrin and
selenoprotein), 5 ng/ml FGF2, 5 ng/ml PDGF AB,
glutamine-penicillin-streptomycin and .beta.-mercaptoethanol.
HuES9.E1 MSCs can be propagated in this minimal media for at least
a week. The next day, the cell culture is again washed three times
with PBS, and incubated with the fresh defined media.
[0357] The media is collected after three days of conditioning. The
conditioned media (CM) is always analyzed or processed in paralled
with an equivalent volume of non-condtioned media (NCM). For LC
MS/MS analysis, the media is concentrated .about.10.times. before
extensive dialysis against 0.9% saline as described in the
materials and methods. The average protein concentration of
concentrated CM and NCM are 98.0.+-.17.9 .mu.g/ml and 41.6.+-.1.2
.mu.g/ml (n=3), respectively. The conditioning of media by MSCs is
monitored by running aliquots of the media on protein gels. Protein
composition of the media increased in complexity with time (FIG.
6). The CM had a more complex protein composition than NCM.
Example 9
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
Analysis of MSC Conditioned Media by LC MS/MS and Antibody
Array
[0358] LC MS/MS analysis identified 250 proteins that are present
in two independently prepared batches of CM, but not in a similarly
processed NCM. These are:
[0359] 1. IPI00021428 Actin, alpha skeletal muscle; 2. IPI00414057
Actin alpha 1 skeletal muscle protein; 3. IPI00008603 Actin, aortic
smooth muscle; 4. IPI00021439 Actin, cytoplasmic 1; 5. IPI00023006
Actin, alpha cardiac; 6. IPI00021440 Actin, cytoplasmic 2; 7.
IPI00025416 Actin, gamma-enteric smooth muscle; 8. IPI00479925
agrin; 9. IPI00015102 CD166 antigen precursor; 10. IPI00007423
Acidic leucine-rich nuclear phosphoprotein 32 family member B; 11.
IPI00413331 36 kDa protein; 12. IPI00412577 34 kDa protein; 13.
IPI00413506 33 kDa protein; 14. IPI00418169 Hypothetical protein
DKFZp686P03159; 15. IPI00003815 Rho GDP-dissociation inhibitor 1;
16. IPI00004656 Beta-2-microglobulin precursor; 17. IPI00218042
Splice Isoform BMP1-5 of Bone morphogenetic protein 1 precursor;
18. IPI00009054 Splice Isoform BMP1-3 of Bone morphogenetic protein
1 precursor; 19. IPI00014021 Splice Isoform BMP1-1 of Bone
morphogenetic protein 1 precursor; 20. IPI00218040 Splice Isoform
BMP1-4 of Bone morphogenetic protein 1 precursor; 21. IPI00006980
Protein C14orf166; 22. IPI00296165 Complement C1r subcomponent
precursor; 23. IPI00152540 OTTHUMP00000016748; 24. IPI00305064
Splice Isoform CD44 of CD44 antigen precursor; 25. IPI00297160
Hypothetical protein DKFZp451K1918; 26. IPI00293539 Splice Isoform
2 of Cadherin-11 precursor; 27. IPI00304227 Splice Isoform 1 of
Cadherin-11 precursor; 28. IPI00386476 Cadherin 11, type 2, isoform
1 preproprotein; 29. IPI00024046 Cadherin-13 precursor;
[0360] 30. IPI00290085 Neural-cadherin precursor; 31. IPI00029739
Splice Isoform 1 of Complement factor H precursor; 32. IPI00012011
Cofilin, non-muscle isoform; 33. IPI00007257 calsyntenin 1 isoform
2; 34. IPI00218539 Splice Isoform B of Collagen alpha-1(XI) chain
precursor; 35. IPI00477350 Collagen, type XI, alpha 1; 36.
IPI00329573 Splice Isoform Long of Collagen alpha-1(XII) chain
precursor; 37. IPI00221384 Splice Isoform Short of Collagen
alpha-1(XII) chain precursor; 38. IPI00400935 Collagen alpha-1(XVI)
chain precursor; 39. IPI00297646 Collagen alpha-1(I) chain
precursor; 40. IPI00164755 Prepro-alpha2(I) collagen precursor; 41.
IPI00304962 Collagen alpha-2(I) chain precursor; 42. IPI00021033
Collagen alpha-1(III) chain precursor; 43. IPI00167087 COL3A1
protein; 44. IPI00021034 Collagen alpha-1(IV) chain precursor; 45.
IPI00479324 alpha 2 type W collagen preproprotein; 46. IPI00306322
Collagen alpha-2(IV) chain precursor; 47. IPI00303313 Collagen
alpha-1(V) chain precursor; 48. IPI00477611 184 kDa protein; 49.
IPI00293881 COL5A2 protein; 50. IPI00018279 Collagen alpha-3(V)
chain precursor; 51. IPI00291136 Collagen alpha-1(VI) chain
precursor; 52. IPI00304840 Splice Isoform 2C2 of Collagen
alpha-2(VI) chain precursor; 53. IPI00220613 Splice Isoform 2C2A of
Collagen alpha-2(VI) chain precursor; 54. IPI00022200 alpha 3 type
VI collagen isoform 1 precursor; 55. IPI00072918 alpha 3 type VI
collagen isoform 4 precursor; 56. IPI00220701 Splice Isoform 2 of
Collagen alpha-3(VI) chain precursor; 57. IPI00072917 alpha 3 type
VI collagen isoform 3 precursor; 58. IPI00021828 Cystatin B; 59.
IPI00007778 Di-N-acetylchitobiase precursor; 60. IPI00295741
Cathepsin B precursor;
[0361] 61. IPI00299219 Protein CYR61 precursor; 62. IPI00514900 42
kDa protein; 63. IPI00333770 Similar to Dedicator of cytokinesis
protein 10; 64. IPI00478332 Similar to Dedicator of cytokinesis
protein 9; 65. IPI00000875 Elongation factor 1-gamma; 66.
IPI00465248 Alpha-enolase; 67. IPI00013769 Alpha-enolase, lung
specific; 68. IPI00216171 Gamma-enolase; 69. IPI00218803 Splice
Isoform B of Fibulin-1 precursor; 70. IPI00296537 Splice Isoform C
of Fibulin-1 precursor; 71. IPI00328113 Fibrillin-1 precursor; 72.
IPI00019439 fibrillin 2 precursor; 73. IPI00385645 Splice Isoform 2
of Fibroblast growth factor 17 precursor; 74. IPI00216602 Splice
Isoform 5 of Fibroblast growth factor receptor 2 precursor; 75.
IPI00216604 Splice Isoform 8 of Fibroblast growth factor receptor 2
precursor; 76. IPI00034099 Hypothetical protein FLJ21918; 77.
IPI00333541 Filamin-A; 78. IPI00302592 Filamin A, alpha; 79.
IPI00339227 Hypothetical protein DKFZp686O1166; 80. IPI00414283
Fibronectin precursor (FN) (Cold-insoluble globulin) (CIG). Splice
isoform 3; 81. IPI00339225 Splice Isoform 5 of Fibronectin
precursor; 82. IPI00339319 Splice Isoform 11 of Fibronectin
precursor; 83. IPI00556632 Splice Isoform 12 of Fibronectin
precursor; 84. IPI00411462 Hypothetical protein DKFZp686B18150; 85.
IPI00029723 Follistatin-related protein 1 precursor; 86.
IPI00005401 Polypeptide N-acetylgalactosaminyltransferase 5; 87.
IPI00219025 Glutaredoxin-1; 88. IPI00171411 Golgi phosphoprotein 2;
89. IPI00026314 Gelsolin precursor;
[0362] 90. IPI00219757 Glutathione S-transferase P; 91. IPI00027569
Heterogeneous nuclear ribonucleoprotein C-like 1; 92. IPI00003881
HNRPF protein; 93. IPI00442294 Splice Isoform 1 of Neurotrimin
precursor; 94. IPI00003865 Splice Isoform 1 of Heat shock cognate
71 kDa protein; 95. IPI00037070 Splice Isoform 2 of Heat shock
cognate 71 kDa protein; 96. IPI00220362 10 kDa heat shock protein,
mitochondrial; 97. IPI00024284 Basement membrane-specific heparan
sulfate proteoglycan core protein precursor; 98. IPI00297284
Insulin-like growth factor binding protein 2 precursor; 99.
IPI00297284 Insulin-like growth factor binding protein 2 precursor;
100. IPI00029236 Insulin-like growth factor binding protein 5
precursor; 101. IPI00029236 Insulin-like growth factor binding
protein 5 precursor; 102. IPI00029235 Insulin-like growth factor
binding protein 6 precursor; 103. IPI00029235 Insulin-like growth
factor binding protein 6 precursor; 104. IPI00016915 Insulin-like
growth factor binding protein 7 precursor; 105. IPI00016915
Insulin-like growth factor binding protein 7 precursor; 106.
IPI00328163 K-ALPHA-1 protein; 107. IPI00021396 Vascular
endothelial growth factor receptor 2 precursor; 108. IPI00298281
Laminin gamma-1 chain precursor; 109. IPI00219219 Galectin-1; 110.
IPI00023673 Galectin-3 binding protein precursor; 111. IPI00021405
Splice Isoform A of Lamin-A/C; 112. IPI00216953 Splice Isoform
ADelta10 of Lamin-A/C; 113. IPI00180173 PREDICTED: similar to
tropomyosin 4; 114. IPI00401614 PREDICTED: similar to FKSG30; 115.
IPI00374397 PREDICTED: similar to tropomyosin 4; 116. IPI00374732
PREDICTED: similar to PPIA protein; 117. IPI00402104 PREDICTED:
similar to peptidylprolyl isomerase A isoform 1; cyclophilin A;
peptidyl-pro; 118. IPI00455415 PREDICTED: similar to Heterogeneous
nuclear ribonucleoprotein C-like dJ845O24.4; 119. IPI00454722
PREDICTED: similar to Phosphatidylethanolamine-binding protein;
120. IPI00454852 PREDICTED: similar to Teratocarcinoma-derived
growth factor 1;
[0363] 121. IPI00002802 Protein-lysine 6-oxidase precursor; 122.
IPI00410152 latent transforming growth factor beta binding protein
1 isoform LTBP-1L; 123. IPI00220249 Latent transforming growth
factor beta-binding protein, isoform 1L precursor; 124. IPI00220249
Latent transforming growth factor beta-binding protein, isoform 1L
precursor"; 125. IPI00410152 latent transforming growth factor beta
binding protein 1 isoform LTBP-1L; 126. IPI00020986 Lumican
precursor; 127. IPI00291006 Malate dehydrogenase, mitochondrial
precursor; 128. IPI00005707 Macrophage mannose receptor 2
precursor; 129. IPI00020501 Myosin-11; 130. IPI00019502 Myosin-9;
131. IPI00604620 Nucleolin; 132. IPI00220740 Splice Isoform 2 of
Nucleophosmin; 133. IPI00219446 Phosphatidylethanolamine-binding
protein; 134. IPI00299738 Procollagen C-endopeptidase enhancer 1
precursor; 135. IPI00015902 Beta platelet-derived growth factor
receptor precursor; 136. IPI00216691 Profilin-1; 137. IPI00169383
Phosphoglycerate kinase 1; 138. IPI00219568 Phosphoglycerate
kinase, testis specific; 139. IPI00296180 Urokinase-type
plasminogen activator precursor; 140. IPI00215943 Splice Isoform 3
of Plectin 1; 141. IPI00215942 Splice Isoform 2 of Plectin 1; 142.
IPI00014898 Splice Isoform 1 of Plectin 1; 143. IPI00398777 plectin
1 isoform 8; 144. IPI00398776 plectin 1 isoform 7; 145. IPI00186711
plectin 1 isoform 6; 146. IPI00420096 plectin 1 isoform 3; 147.
IPI00398779 plectin 1 isoform 11; 148. IPI00398778 plectin 1
isoform 10; 149. IPI00398002 plectin 1 isoform 1; 150. IPI00419585
Peptidyl-prolyl cis-trans isomerase A;
[0364] 151. IPI00472718 peptidylprolyl isomerase A isoform 2; 152.
IPI00000874 Peroxiredoxin-1; 153. IPI00024915 Peroxiredoxin-5,
mitochondrial precursor; 154. IPI00375306 peroxiredoxin 5
precursor, isoform b; 155. IPI00012503 Splice Isoform Sap-mu-0 of
Proactivator polypeptide precursor; 156. IPI00374179 proteasome
activator subunit 1 isoform 2; 157. IPI00030154 Proteasome
activator complex subunit 1; 158. IPI00168812 PTK7 protein tyrosine
kinase 7 isoform d precursor; 159. IPI00419941 PTK7 protein
tyrosine kinase 7 isoform a precursor; 160. IPI00003590 Quiescin
Q6, isoform a; 161. IPI00015916 Bone-derived growth factor
(Fragment); 162. IPI00015916 Bone-derived growth factor; 163.
IPI00298289 Splice Isoform 2 of Reticulon-4; 164. IPI00021766
Splice Isoform 1 of Reticulon-4; 165. IPI00013895 Calgizzarin; 166.
IPI00010402 Hypothetical protein; 167. IPI00218733 Superoxide
dismutase; 168. IPI00014572 SPARC precursor; 169. IPI00005614
Splice Isoform Long of Spectrin beta chain, brain 1; 170.
IPI00008780 Stanniocalcin-2 precursor; 171. IPI00301288 SEL-OB
protein; 172. IPI00216138 Transgelin; 173. IPI00018219 Transforming
growth factor-beta-induced protein ig-h3 precursor; 174.
IPI00304865 transforming growth factor, beta receptor III''; 175.
IPI00296099 Thrombospondin-1 precursor; 176. IPI00032292
Metalloproteinase inhibitor 1 precursor; 177. IPI00027166
Metalloproteinase inhibitor 2 precursor; 178. IPI00220828 Thymosin
beta-4; 179. IPI00180240 thymosin-like 3;
[0365] 180. IPI00299633 OTTHUMP00000031270 (Fragment); 181.
IPI00465028 Triosephosphate isomerase 1 variant (Fragment); 182.
IPI00451401 Splice Isoform 2 of Triosephosphate isomerase; 183.
IPI00010779 Tropomyosin 4; 184. IPI00216975 Splice Isoform 2 of
Tropomyosin alpha-4 chain; 185. IPI00180675 Tubulin alpha-3 chain;
186. IPI00218343 Tubulin alpha-6 chain; 187. IPI00216298
Thioredoxin; 188. IPI00472175 CDNA FLJ46672 fis, clone
TRACH3009008, highly similar to Thioredoxin reductase; 189.
IPI00450472 Ubiquitin-conjugating enzyme E2I; 190. IPI00018352
Ubiquitin carboxyl-terminal hydrolase isozyme L1; 191. IPI00010207
Ubiquitin-fold modifier 1 precursor; 192. IPI00260630 URB; 193.
IPI00021263 14-3-3 protein zeta/delta; 194. IPI00642991
Hypothetical protein DKFZp686F10164; 195. IPI00470919 Hypothetical
protein DKFZp686K08164; 196. IPI00719088 collagen, type VI, alpha 1
precursor; 197. IPI00654685 Similar to SPARC precursor; 198.
IPI00641961 Collagen, type XII, alpha 1; 199. IPI00645849
Extracellular matrix protein 1; 200. IPI00554786 Thioredoxin
reductase 1; 201. IPI00645018 Plasminogen activator, urokinase;
202. IPI00552339 Tissue inhibitor of metalloproteinase 1; 203.
IPI00642997 Actin, cytoplasmic 2; 204. IPI00719778 Similar to
Annexin A2; 205. IPI00647915 Transgelin 2; 206. IPI00552815
Collagen, type V, alpha 1; 207. IPI00552981 CDNA PSECO266 fis,
clone NT2RP3003649, highly similar to Homo sapiens fibulin-1D mRNA;
208. IPI00180776 29 kDa protein; 209. IPI00552416 Filamin A,
alpha;
[0366] 210. IPI00640698 Actin, gamma-enteric smooth muscle; 211.
IPI00514530 Actin, alpha 1, skeletal muscle; 212. IPI00556442
Insulin-like growth factor binding protein 2 variant (Fragment);
213. IPI00513782 Gelsolin; 214. IPI00478731 29 kDa protein; 215.
IPI00396479 24 kDa protein; 216. IPI00334627 39 kDa protein; 217.
IPI00555762 PTK7 protein tyrosine kinase 7 isoform a variant
(Fragment); 218. IPI00658202 97 kDa protein; 219. IPI00006273 CYR61
protein; 220. IPI00719405 TMSL6 protein; 221. IPI00658096 Thymosin
beta-4; 222. IPI00376163 5 kDa protein; 223. IPI00556217 Fibrillin
1 variant (Fragment); 224. IPI00514817 Similar to Lamin A/C; 225.
IPI00644087 Progerin; 226. IPI00655812 Rhabdomyosarcoma antigen
MU-RMS-40.12; 227. IPI00604517 Similar to Nucleolin; 228.
IPI00444262 CDNA FLJ45706 fis, clone FEBRA2028457, highly similar
to Nucleolin; 229. IPI00412473 Protein; 230. IPI00414489 Protein;
231. IPI00411463 Protein; 232. IPI00556415 Transgelin variant
(Fragment); 233. IPI00718825 Calmodulin; 234. IPI00478156 17 kDa
protein; 235. IPI00386621 CALM3 protein; 236. IPI00647001 Acidic;
237. IPI00642650 Similar to Stanniocalcin 2 precursor; 238.
IPI00641471 Collagen-like protein; 239. IPI00514669 SH3 domain
binding glutamic acid-rich protein like 3; 240. IPI00719422
Triosephosphate isomerase (Fragment); 241. IPI00003734 Putative
5100 calcium-binding protein H_NH0456N16.1; 242. IPI00029574 11 kDa
protein; 243. IPI00641047 Gelsolin; 244. IPI00647556 Gelsolin; 245.
IPI00654821 hypothetical protein L0054845 isoform 1; 246.
IPI00647572 Dickkopf related protein-3 precursor; 247. IPI00639879
Similar to Cytokinesis protein sepA; 248. IPI00657746 Similar to
Dedicator of cytokinesis protein 8; 249. IPI00555993 Vascular
endothelial growth factor receptor 3 variant; 250. IPI00552545
Dedicator of cytokinesis protein 8.
[0367] Together, these 250 proteins are encoded by 132 unique known
genes: ACTA1; COL5A2; HSPA8; PSAP; ACTA2; COL5A3; HSPE1; PSME1;
ACTB; COL6A1; HSPG2; PTK7; ACTC; COL6A2; IGFBP2; QSCN6; ACTG1;
COL6A3; IGFBP5; RTN4; ACTG2; CSTB; IGFBP6; S100A11; AGRN; CTBS;
IGFBP7; SH3BGRL3; ALCAM; CTSB; K-ALPHA-1; SOD1; ANP32B; CYR61; KDR;
SPARC; ANXA2; DOCK10; LAMC1; SPTBN1; ARHGDIA; DOCK8; LGALS1; STC2;
B2M; ECM1; LGALS3BP; SVEP1; BMP1; EEF1G; LMNA; TAGLN; C14orf166;
ENO1; LOX; TAGLN2; C1R; ENO1B; LTBP1; TGFBI; CALM1; ENO2; LUM;
TGFBR3; CD109; FBLN1; MDH2; THBS1; CD44; FBN1; MRC2; TIMP1; CDH11;
FBN2; MYH11; TIMP2; CDH13; FGF17; MYH9; TMSB4X; CDH2; FGFR2; NCL;
TMSL3; CFH/HF1; FLJ21918; NPM1; TMSL6; CFL1; FLNA; PBP; TPI1;
CLSTN1; FN1; PCOLCE; TPM4; COL11A1; FSTL1; PDGFRB; TUBA3; COL12A1;
GALNT5; PFN1; TUBA6; COL16A1; GLRX; PGK1; TXN; COL1A1; GOLPH2;
PGK2; TXNRD1; COL1A2; GSN; PLAU; UBE2I; COL3A1; GSTP1; PLEC1;
UCHL1; COL4A1; HNRPCL1; PPIA; UFM1; COL4A2; HNRPF; PRDX1; URB;
COL5A1; HNT; PRDXS; YWHAZ.
[0368] There are 32 unknown proteins: IPI00642991 Hypothetical
protein DKFZp686F10164; IPI00470919 Hypothetical protein
DKFZp686K08164; IPI00654685 Similar to SPARC precursor; IPI00719778
Similar to Annexin A2; IPI00552981 CDNA PSECO266 fis, clone
NT2RP3003649, highly similar to Homo sapiens fibulin-1D mRNA;
IPI00180776 29 kDa protein; IPI00478731 29 kDa protein; IPI00396479
24 kDa protein; IPI00334627 39 kDa protein; IPI00658202 97 kDa
protein; IPI00376163 5 kDa protein; IPI00514817 Similar to Lamin
A/C; IPI00644087 Progerin; IPI00655812 Rhabdomyosarcoma antigen
MU-RMS-40.12; IPI00604517 Similar to Nucleolin; IPI00444262 CDNA
FLJ45706 fis, clone FEBRA2028457, highly similar to Nucleolin;
IPI00412473 Protein; IPI00414489 Protein; IPI00411463 Protein;
IPI00478156 17 kDa protein; IPI00386621 CALM3 protein; IPI00647001
Acidic; IPI00642650 Similar to Stanniocalcin 2 precursor;
IPI00641471 Collagen-like protein; IPI00514669 SH3 domain binding
glutamic acid-rich protein like 3; IPI00003734 Putative 5100
calcium-binding protein HNH0456N16.1; IPI00029574 11 kDa protein;
IPI00654821 hypothetical protein L0054845 isoform 1; IPI00647572
Dickkopf related protein-3 precursor; IPI00639879 Similar to
Cytokinesis protein sepA; IPI00657746 Similar to Dedicator of
cytokinesis protein 8; IPI00555993 Vascular endothelial growth
factor receptor 3 variant.
[0369] The MSCs described in this document can therefore be used as
sources of any or all of these proteins, or any proteins or other
molecules which are secreted or expressed by them.
[0370] MSCs have been shown to secrete a broad spectrum of
cytokines and growth factors that affect cells on their
vicinity.sup.8. Many of these factors are small molecules that are
not easily detectable during shot-gun LC MS/MS analysis. Therefore,
the CM and NCM are also analyzed by hybridization to 5 different
antibody arrays that together carried antibodies against 101
cytokines/growth factors (See also FIG. 7).
[0371] Layout of Antibody Arrays
TABLE-US-00003 RayBio .RTM. Angiogenesis array I A B C D E F G H 1
POS POS NEG NEG Angiogenin EGF ENA-78 b FGF 2 POS POS NEG NEG
Angiogenin EGF ENA-78 b FGF 3 GRO IFN-.quadrature..quadrature.
IGF-I IL-6 IL-8 LEPTIN MCP-1 PDGF-BB 4 GRO
IFN-.quadrature..quadrature. IGF-I IL-6 IL-8 LEPTIN MCP-1 PDGF-BB 5
PIGF RANTES TGF-.quadrature..quadrature. TIMP-1 TIMP-2 Thrombo-
VEGF VEGF-D poietin 6 PIGF RANTES
TGF-.quadrature..quadrature..quadrature. TIMP-1 TIMP-2 Thrombo-
VEGF VEGF-D poietin 7 BLANK BLANK BLANK BLANK BLANK BLANK Neg POS 8
BLANK BLANK BLANK BLANK BLANK BLANK Neg POS
TABLE-US-00004 RayBio .RTM. Human Chemokine Antibody Array I A B C
D E F G H I J K L 1 POS POS NEG NEG BLC CCL28 Ck.beta.8-1 CTACK
CXCL16 ENA-78 Eotaxin Eotaxin-2 2 POS POS NEG NEG BLC CCL28
Ck.beta.8-1 CTACK CXCL16 ENA-78 Eotaxin Eotaxin-2 3 Eotaxin-3
Fractalkine GCP-2 GRO GRO.alpha. HCC-4 I-309 I-TAC IL-8 IP-10
Lymphotactin MCP-1 4 Eotaxin-3 Fractalkine GCP-2 GRO GRO.alpha.
HCC-4 I-309 I-TAC IL-8 IP-10 Lymphotactin MCP-1 5 MCP-2 MCP-3 MCP-4
MDC MIG MIP-1.alpha. MIP-1.beta. MIP-1.delta. MIP-3.alpha.
MIP-3.beta. MPIF-1 NAP 2 6 MCP-2 MCP-3 MCP-4 MDC MIG MIP-1.alpha.
MIP-1.beta. MIP-1.delta. MIP-3.alpha. MIP-3.beta. MPIF-1 NAP 2 7
PARC RANTES SDF-1 .alpha. SDF-1 .beta. TARC TECK BLANK BLANK BLANK
BLAND BLANK POS 8 PARC RANTES SDF-1 .alpha. SDF-1 .beta. TARC TECK
BLANK BLANK BLANK BLAND BLANK POS
TABLE-US-00005 RayBio .RTM. Matrix Metalloproteinases Antibody
Array I A B C D E F G H 1 POS POS NEG NEG MMP-1 MMP-2 MMP-3 MMP-8 2
POS POS NEG NEG MMP-1 MMP-2 MMP-3 MMP-8 3 MMP-9 MMP-10 MMP-13
TIMP-1 TIMP-2 TIMP-3 TIMP-4 POS 4 MMP-9 MMP-10 MMP-13 TIMP-1 TIMP-2
TIMP-3 TIMP-4 POS
TABLE-US-00006 RayBio .RTM. Human Cytokine Antibody Array I A B C D
E F G H 1 POS POS NEG NEG GCSF GM-CSF GRO GRO.alpha. 2 POS POS NEG
NEG GCSF GM-CSF GRO GRO.alpha. 3 IL-1 .alpha. IL-2 IL-3 IL-5 IL-6
IL-7 IL-8 IL-10 4 IL-1 .alpha. IL-2 IL-3 IL-5 IL-6 IL-7 IL-8 IL-10
5 IL-13 IL-15 IFN-.gamma. MCP-1 MCP-2 MCP-3 MIG RANTES 6 IL-13
IL-15 IFN-.gamma. MCP-1 MCP-2 MCP-3 MIG RANTES 7 TGF-.beta.1
TNF-.alpha. TNF-.beta. BLANK BLANK BLANK BLANK POS 8 TGF-.beta.1
TNF-.alpha. TNF-.beta. BLANK BLANK BLANK BLANK POS
TABLE-US-00007 RayBio .RTM. Human Cytokine Antibody Array V A B C D
E F G H I J K L 1 POS POS POS POS NEG NEG ENA-78 GCSF CM-CSF GRO
GRO- POS 2 I-309 IL-1 IL-1.beta. IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8
IL-10 I-309 3 IL-12 IL-13 IL-15 IFN- .gamma. MCP-1 MCP-2 MCP-3 MCSF
MDC MIG MIP-1.beta. IL-12 p40p70 p40p70 4 MIP-1 .delta. RANTES SCF
SDF-1 TARC TGF-.beta..quadrature. TNF- TNF-.beta. EGF IGF-1
Angiogenin MIP-1 .delta. 5 Oncostatin Thrombo- VEGF PDGF-BB LEPTIN
BDNF BLC Ck.beta.8-.quadrature..quadrature. Eotaxin Eotaxin-2
Eotaxin-3 Oncostatin M poietin M 6 FGF-4 FGF-6 FGF-7 FGF-9 Flt-3
Fractalkine GCP-2 GDNF HGF IGFBP-1 IGFBP-2 FGF-4 Ligand 7 IGFBP-3
IGFBP-4 IL-16 IP-10 LIF LIGHT MCP-4 MIF MIP-3.alpha. NAP-2 NT-3
IGFBP-3 8 NT-4 Osteo- PARC PIGF TGF-.beta.2 TGF-.beta.3 TIMP-1
TIMP-2 NEG POS POS NT-4 protegerin
[0372] 72 of the cytokines/growth factors are found to be
reproducibly secreted by HuES9.E1 MSCs in at least three of four
independently prepared CM and not in NCM. 72 proteins identified by
antibody array (Name GeneSymbol GeneID): 1. MDC ADAM11
GI.sub.--11496997-A; 2. Angiogenin ANG GI.sub.--42716312-S; 3. BDNF
BDNF GI.sub.--34106709-A; 4. I-309 CCL1 GI.sub.--4506832-S; 5.
Eotaxin CCL11 GI.sub.--22538399; 6. MIP-1.delta. CCL15
GI.sub.--34335180-A; 7. HCC-4 CCL16 GI.sub.--22538800; 8. MCP-1
CCL2 GI.sub.--22538812-S; 9. Ck.beta.8-1 CCL23 GI.sub.--22538807-A;
10. Eotaxin-2 CCL24 GI.sub.--22165426-S; 11. Eotaxin-3 CCL26
GI.sub.--22547151-S; 12. RANTES CCL5 GI.sub.--22538813; 13. MCP-3
CCL7 GI.sub.--13435401-S; 14. MCP-2 CCL8 GI.sub.--22538815-S; 15.
MCSF CSF1 GI.sub.--27262666-I; 16. GM-CSF CSF2 GI.sub.--27437029-S;
17. GCSF CSF3 GI.sub.--27437048; 18. Fractalkine CX3CL1
GI.sub.--4506856-S; 19. GRO-a CXCL1 GI.sub.--4504152-S; 20. I-TAC
CXCL11 GI.sub.--14790145-S; 21. SDF-1 CXCL12 GI.sub.--40316923; 22.
BLC CXCL13 GI.sub.--5453576-S; 23. ENA-78 CXCLS
GI.sub.--41872613-S; 24. IP-10 CXCR3 GI.sub.--4504098-S; 25. EGF
EGF GI.sub.--6031163-S; 26. FGF-4 FGF4 GI.sub.--4503700; 27. FGF-6
FGF6 GI.sub.--10337586; 28. FGF-7 FGF7 GI.sub.--15147344; 29. FGF-9
FGF9 GI.sub.--4503706-S; 30. Fit 3 Ligand FLT3LG
GI.sub.--38455415-S; 31. GDNF GDNF GI.sub.--40549410; 32. GCP-2
GOLGA4 GI.sub.--45359854-S; 33. HGF HGF GI.sub.--33859834-S; 34.
IFN-.gamma. IFNG GI.sub.--10835170-S; 35. IGF-I IGF1
GI.sub.--19923111-S; 36. IGFBP-1 IGFBP1 GI.sub.--4504614-S; 37.
IGFBP-2 IGFBP2 GI.sub.--10835156-S; 38. IGFBP-4 IGFBP4
GI.sub.--10835020-S; 39. IL-10 IL10 GI.sub.--24430216-S; 40.
IL-12p40p70 IL12B GI.sub.--24497437-S; 41. IL-13 IL13
GI.sub.--26787977-S; 42. IL-16 IL16 GI.sub.--27262654-A; 43. IL-1a
IL1A GI.sub.--27894329-S; 44. IL-1.beta. IL1B GI.sub.--27894305-S;
45. IL-2 IL2 GI.sub.--28178860-S; 46. IL-3 IL3 GI.sub.--28416914-S;
47. IL-6 IL6 GI.sub.--10834983-S; 48. IL-7 IL7 GI.sub.--28610152-S;
49. IL-8 IL8 GI.sub.--28610153-S; 50. SCF KITLG GI.sub.--4580419-A;
51. LEPTIN LEP GI.sub.--4557714-S; 52. LIF LIF GI.sub.--6006018-S;
53. MIF MIF GI.sub.--4505184-S; 54. MMP-1 MMP1 GI.sub.--13027798-S;
55. MMP-10 MMP10 GI.sub.--4505204-S; 56. MMP-13 MMP13
GI.sub.--13027796-S; 57. MMP 3 MMP3 GI.sub.--13027803-S; 58. MMP-9
MMP9 GI.sub.--4826835-S; 59. PARC PARC GI.sub.--24307990; 60.
PDGF-BB PDGFB GI.sub.--15451785-A; 61. PIGF PIGF
GI.sub.--27894289-A; 62. TGF-.beta.1 TGFB1 GI.sub.--10863872; 63.
TGF-.beta.2 TGFB2 GI.sub.--4507462-S; 64. Thrombopoietin THPO
GI.sub.--40805871-S; 65. TIMP-1 TIMP1 GI.sub.--4507508-S; 66.
TIMP-2 TIMP2 GI.sub.--9257247-S; 67. TIMP-3 TIMP3
GI.sub.--21536431-S; 68. TIMP-4 TIMP4 GI.sub.--4507514-S; 69.
TNF-.alpha. TNF GI.sub.--25952110; 70. Osteoprotegerin TNFRSF11B
GI.sub.--22547122-S; 71. VEGF VEGF GI.sub.--30172563-S; 72.
Lymphotactin XCL1 GI.sub.--4506852-S.
[0373] 29 proteins not detected by antibody array (Name GeneSymbol
GeneID): b FGF CCL23 GI.sub.--22538807-A; MCP-4 CCL13
GI.sub.--22538799-S; TARC CCL17 GI.sub.--22538801-S; MIP-3b CCL19
GI.sub.--22165424-S; MIP-3a CCL20 GI.sub.--4759075-S; MPIF-1 CCL23
GI.sub.--22538807-A; TECK CCL25 GI.sub.--22538797-A; CTACK CCL27
GI.sub.--22165428-S; CCL28 CCL28 GI.sub.--22538810-A; MIP-1b CCL4
GI.sub.--4506844-S; SDF-1a CXCL12 GI.sub.--403169234; SDF-1b CXCL12
GI.sub.--403169234; CXCL16 CXCL16 GI.sub.--11545764-S; MIG CXCL9
GI.sub.--4505186-S; MIP-1a CXCR6 GI.sub.--5730105-S; IGFBP-3 IGFBP3
GI.sub.--19923110-S; IL-15 IL15 GI.sub.--26787979-A; IL-41L4
GI.sub.--27477091-A; IL-5 IL5 GI.sub.--28559032-S; TNF-b LTA
GI.sub.--6806892-S; MMP-2 MMP2 GI.sub.--11342665-S; MMP-8 MMP8
GI.sub.--4505220-S; NAP-2 NAP1L4 GI.sub.--21327711-S; NT-3 NTF3
GI.sub.--45359869-S; NT-4 NTFS GI.sub.--34328933-S; Oncostatin M
OSM GI.sub.--28178862-S; TGF-b1 TGFB1 GI.sub.--10863872-S; LIGHT
TNFSF14 GI.sub.--25952146-A; VEGF-D VEGFD GI.sub.--19924297-S.
[0374] However, only 3 gene products, namely IGFBP2, TIMP1 and
TIMP2 are also detected by LC MS/MS analysis, possibly because many
of cytokines and growth factors are small molecules and are not
detectable during conventional LC MS/MS analysis.
[0375] The lists of 132 and 72 gene products identified by LC MS/MS
and antibody array analysis respectively have 3 common genes,
namely IGFBP2, TIMP1 and TIMP2. Therefore in the final tally, a
total of 201 unique gene products are identified (Table E2
below).
TABLE-US-00008 ACTA1 CD44 CXCL5* GLRX IL16* PFN1 TIMP2 ACTA2 CD109
CXCL11* GOLGA4 K-ALPHA-1 PGK1 TIMP3 ACTB CDH2 CXCL12* GOLPH2 KDR
PGK2 TIMP4 ACTC CDH11 CXCL13.dagger. GSN KITLG PIGF TMSB4X ACTG1
CDH13 CXCR3* GSTP1 LAMC1 PLAU TMSL3 ACTG2 CFH/HF1 CYR61 HGF LEP
PLEC1 TMSL6 ADAM11 CFL1 DOCK8 HNRPCL1 LGALS1 PPIA TNF* AGRN CLSTN1
DOCK10 HNRPF LGALS3BP PRDX1 TNFRSF11B* ALCAM COL1A1 ECM1 HNT LIF
PRDX5 TPI1 ANG COL1A2 EEF1G HSPA8 LMNA PSAP TPM4 ANP32B COL3A1 EGF
HSPE1 LOX PSME1 TUBA3 ANXA2 COL4A1 ENO1 HSPG2 LTBP1 PTK7 TUBA6
ARHGDIA COL4A2 ENO1B IFNG.dagger. LUM QSCN6 TXN B2M COL5A1 ENO2
IGF1 MDH2 RTN4 TXNRD1 BDNF* COL5A2 FBLN1 IGFBP1 MIF* S100A11 UBE2I
BMP1 COL5A3 FBN1 IGFBP2 MMP1 SH3BGRL3 UCHL1 C14orf166 COL6A1 FBN2
IGFBP4 MMP3* SOD1 UFM1 C1R COL6A2 FGF4 IGFBP5 MMP9 SPARC URB CALM1
COL6A3 FGF6 IGFBP6 MMP10 SPTBN1 VEGF.dagger. CCL1 COL11A1 FGF7
IGFBP7 MMP13 STC2 XCL1 CCL2 COL12A1 FGF9 IL1A* MRC2 SVEP1 YWHAZ
CCL5 COL16A1 FGF17 IL1B* MYH11 TAGLN CCL7 CSF1* FGFR2 IL2* MYH9
TAGLN2 CCL8 CSF2* FLJ21918 IL3* NCL TGFB1 CCL11 CSF3* FLNA IL6*
NPM1 TGFB2* CCL15 CSTB FLT3LG IL7* PARC TGFBI* CCL16 CTBS FN1 IL8*
PBP TGFBR3 CCL23 CTSB FSTL1 IL10* PCOLCE THBS1 CCL24 CX3CL1* GALNT5
IL12B* PDGFB THPO CCL26 CXCL1* GDNF IL13* PDGFRB TIMP1
[0376] Table E2. Alphabetical list of 201 unique gene products
identified by LC-MS/MS and antibody array. The proteins identified
by LC-MS/MS and antibody array are combined and represented by
their gene symbol. Transcript level for each gene is assessed using
a high throughput Illumina BeadArray.
[0377] 29 of these gene products namely, ENA-78, FGF-4, FGF-7,
FGF-9, GCP-2, G-CSF, GM-CSF, GRO-a, HCC-4, HGF, IGFBP-1, IGFBP-2,
IGFBP-4, IL-1.beta., IL-6, IL-8, IP-10, LIF, MCP-1, MCSF, MIF,
Osteoprotegerin, PARC, PIGF, SCF, TGF-.beta.2, TIMP-1, TIMP-2, VEGF
have been previously reported to be secreted by adult tissue
derived MSCs (13,16,19-21). Four other proteins that are reported
to be secreted by adult tissue derived MSCs, namely IGFBP-3,
MIP-3a, Oncostatin M and TGF-.beta.3 (see above) are not present in
our list of 201 gene products.
Example 10
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
Verification by Genome Wide-Gene Expression Analysis
[0378] Comparison of the 201 gene products to a genome-wide gene
expression profile of the hESC-MSCs generated by hybridizing total
RNA to a Illumina BeadArray revealed that 134 or 67% of the gene
products had gene transcript levels that are present at above the
limit of detection (LOD) with a 99% confidence (Table E2). While
115 or 88% of the 132 gene products identified by LC MS/MS had
detectable transcript levels (Table E2), 27 or 38% of the 72 gene
products identified by antibody array had detectable transcript
levels and 45 or 62% had no detectable transcript level (Table E2).
Probes for two of the gene products, ENO1B and SVEP1 are not
present on the Illumina BeadArray. It is possible that transcript
levels for most of the 72 gene may be too low in abundance for
detection by Illumina BeadArray as mRNAs encoding for
cytokines/chemokines are known to contain AU-rich elements that
caused rapid degradation of the mRNA during translation
(22,23).
[0379] More sensitive qRT-PCR assays are therefore performed. 42 of
the 72 gene products are randomly selected and tested. 36 or 86% of
the 42 gene products have detectable transcript levels as defined
as having a normalized C.sub.t value of <35 (Table E3 below).
This frequency is similar to 88% frequency observed for gene
products identified by LC MS/MS and whose gene transcripts are
detectable by Illumina BeadArray. In addition, all 15 that have
detectable transcript levels by Illumina BeadArray, also have
detectable transcript levels by qRT-PCR (Table E3). 21 of 27 (78%)
gene products that did not have detectable transcript levels by
Illumina BeadArray have transcript level detectable by qRT-PCR
(Table E3).
TABLE-US-00009 Symbol Illumina Bead Array Normalized C.sub.t 1 BDNF
>LOD 13.38 2. CCL2 >LOD 11.28 3. CCL7 >LOD 17.35 4. CCL8
>LOD 30.28 5. CXCL1 >LOD 14.09 6. CXCL12 >LOD 16.83 7.
CXCL5 >LOD 19.93 8. IL1A >LOD 16.6 9. IL1B >LOD 11.44 10.
IL6 >LOD 18.92 11. IL8 >LOD 8.53 12. MIF >LOD 16.23 13.
MMP3 >LOD 15.96 14. TGFB2 >LOD 15.16 15. TNFRSF11B >LOD
12.28 16. CCL1 <LOD 34.67 17. CCL11 <LOD 26.13 18. CCL23
<LOD 37.33 19. CCL24 <LOD 10.82 20. CCL26 <LOD 30.38 21.
CCL5 <LOD 27.97 22. CSF1 <LOD 14.35 23. CSF2 <LOD 23.92
24. CSF3 <LOD 32.47 25. CX3CL1 <LOD 25.04 26. CXCL11 <LOD
22.38 27. CXCR3 <LOD 28.41 28. IL10 <LOD 27.4 29. IL12B
<LOD 22.17 30. IL13 <LOD 14.96 31. IL16 <LOD 32.98 32. IL2
<LOD 31.87 33. IL3 <LOD 32.24 34. IL7 <LOD 22.3 35. TGFB1
<LOD 10.63 36. TNF <LOD 31.72 37. CCL15 <LOD >35 38.
CCL16 <LOD >35 39. CXCL13 <LOD >35 40. IFNG <LOD
>35 41. VEGF <LOD >35 42. XCL1 <LOD >35
[0380] Table E3. Quantitative RT-PCR assay for the presence of
transcripts. 42 of the 72 gene products identified by antibody
array are randomly selected for qRT-PCR analysis. 12 gene products
have transcript levels that are above the limit of detection (LOD)
at 99% confidence on the Illumina BeadArray, a high throughput
genome-wide gene expression assay. The C.sub.t value for each gene
is normalized against .beta.-actin.
Example 11
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
Biological Processes that are Modulated by the Secreted
Proteins
[0381] To investigate if the secreted products have the potential
to repair the injured tissues or organs, gene products are first
classified according to their biological processes and pathways
according to the Gene Ontology (GO). The frequency of unique genes
in the secreted MSC proteome associated with each process or
pathway is then compared to the gene-frequency for the respective
pathway or process in a database collated from Unigene, Entrez and
GenBank. Significantly higher frequencies of genes (p<0.05) are
associated with 58 biological processes and 30 pathways.
[0382] Biological processes classified by GO modulated by the 201
unique gene products:
TABLE-US-00010 # # genes % genes % GO Biological Processes (ref)
(ref) (expt) (expt) p-value 1. GO:42221: response to chemical
stimulus 130 0.53 23 11.56 2.50E-24 2. GO:42330: taxis 130 0.53 23
11.56 2.50E-24 3. GO:6935: chemotaxis 130 0.53 23 11.56 2.50E-24 4.
GO:9605: response to external stimulus 157 0.65 24 12.06 9.45E-24
5. GO:50896: response to stimulus 158 0.650 24 12.06 1.11E-23 6.
GO:9628: response to abiotic stimulus 150 0.62 23 11.56 7.83E-23 7.
GO:48513: organ development 116 0.48 11 5.53 3.06E-09 8. GO:7275:
development 827 3.40 23 11.56 3.27E-07 9. GO:16052: carbohydrate
catabolism 51 0.21 6 3.02 3.66E-06 10. GO:19320: hexose catabolism
51 0.21 6 3.02 3.66E-06 11. GO:44248: cellular catabolism 51 0.21 6
3.02 3.66E-06 12. GO:44265: cellular macromolecule catabolism 51
0.21 6 3.02 3.66E-06 13. GO:44275: cellular carbohydrate catabolism
51 0.21 6 3.02 3.66E-06 14. GO:46164: alcohol catabolism 51 0.21 6
3.02 3.66E-06 15. GO:46365: monosaccharide catabolism 51 0.21 6
3.02 3.66E-06 16. GO:6007: glucose catabolism 51 0.21 6 3.02
3.66E-06 17. GO:6096: glycolysis 51 0.21 6 3.02 3.66E-06 18.
GO:9057: macromolecule catabolism 51 0.21 6 3.02 3.66E-06 19.
GO:9056: catabolism 53 0.22 6 3.02 4.60E-06 20. GO:15980: energy
derivation by oxidation of organic 65 0.27 6 3.02 1.53E-05
compounds 21. GO:19318: hexose metabolism 65 0.27 6 3.02 1.53E-05
22. GO:44262: cellular carbohydrate metabolism 65 0.27 6 3.02
1.53E-05 23. GO:5975: carbohydrate metabolism 65 0.27 6 3.02
1.53E-05 24. GO:5996: monosaccharide metabolism 65 0.27 6 3.02
1.53E-05 25. GO:6006: glucose metabolism 65 0.27 6 3.02 1.53E-05
26. GO:6066: alcohol metabolism 65 0.27 6 3.02 1.53E-05 27.
GO:6091: generation of precursor metabolites and energ 65 0.27 6
3.02 1.53E-05 28. GO:6092: main pathways of carbohydrate metabolism
65 0.27 6 3.02 1.53E-05 29. GO:43170: macromolecule metabolism 571
2.35 16 8.04 2.03E-05 30. GO:1525: angiogenesis 44 0.18 5 2.51
2.90E-05 31. GO:1568: bloodvessel development 47 0.19 5 2.51
4.02E-05 32. GO:1944: vasculature development 47 0.19 5 2.51
4.02E-05 33. GO:48514: bloodvessel morphogenesis 47 0.19 5 2.51
4.02E-05 34. GO:6950: response to stress 10 0.04 3 1.51 6.18E-05
35. GO:9611: response to wounding 10 0.04 3 1.51 6.18E-05 36.
GO:1660: fever 2 0.01 2 1.01 6.64E-05 37. GO:31649: heat generation
2 0.01 2 1.01 6.64E-05 38. GO:43207: response to external biotic
stimulus 2 0.01 2 1.01 6.64E-05 39. GO:6952: defense response 2
0.01 2 1.01 6.64E-05 40. GO:6954: inflammatory response 2 0.01 2
1.01 6.64E-05 41. GO:6955: immune response 2 0.01 2 1.01 6.64E-05
42. GO:9607: response to biotic stimulus 2 0.01 2 1.01 6.64E-05 43.
GO:9613: response to pest, pathogen or parasite 2 0.01 2 1.01
6.64E-05 44. GO:9887: organ morphogenesis 53 0.22 5 2.51 7.23E-05
45. GO:1659: thermoregulation 3 0.01 2 1.01 1.98E-04 46. GO:30097:
hemopoiesis 26 0.11 3 1.51 1.22E-03 47. GO:48534: hemopoietic or
lymphoid organ development 26 0.11 3 1.51 1.22E-03 48. GO:1501:
skeletal development 38 0.16 3 1.51 3.67E-03 49. GO:1503:
ossification 38 0.16 3 1.51 3.67E-03 50. GO:31214: biomineral
formation 38 0.16 3 1.51 3.67E-03 51. GO:46849: bone remodeling 38
0.16 3 1.51 3.67E-03 52. GO:6793: phosphorus metabolism 13 0.05 2
1.01 4.88E-03 53. GO:6796: phosphate metabolism 13 0.05 2 1.01
4.88E-03 54. GO:9888: tissue development 54 0.22 3 1.51 9.82E-03
55. GO:45045: secretory pathway 34 0.14 2 1.01 3.14E-02 56.
GO:6887: exocytosis 34 0.14 2 1.01 3.14E-02 57. GO:46903: secretion
36 0.15 2 1.01 3.49E-02 58. GO:1570: vasculogenesis 5 0.02 1 0.50
4.02E-02 GO Pathways p-value 1. Cytokine-cytokine receptor
interaction {grave over ( )} 4.97E-47 2. ECM-receptor interaction
3.21E-17 3. Jak-STAT signaling pathway 1.19E-10 4. MAPK signaling
pathway 1.19E-08 5. Toll-like receptor signaling pathway 1.34E-05
6. TGF-beta signaling pathway 0.000292 7. mTOR signaling pathway
0.0143 8. Fc epsilon RI signaling pathway 0.00267 9. Epithelial
cell signaling in Helicobacter pylori infection 0.0183 10. Cell
Communication 1.17E-17 11. Gap junction 0.00077 12. Tight junction
0.0459 13. Focal adhesion 1.54E-21 14. Regulation of actin
cytoskeleton 2.65E-13 15. Leukocyte transendothelial migration
3.88E-05 16. Complement and coagulation cascades 0.0488 17. Antigen
processing and presentation 0.0102 18. Apoptosis 0.00648 19. T cell
receptor signaling pathway 0.000866 20. Hematopoietic cell lineage
3.32E-14 21. Type I diabetes mellitus 4.60E-06 22. Carbon fixation
0.000257 23. Glycolysis or Gluconeogenesis 0.00106 24. Stilbene,
coumarine and lignin biosynthesis 0.00348 25. Phenylalanine
metabolism 0.00488 26. Phenylalanine, tyrosine and tryptophan
biosynthesis 0.00567 27. Methane metabolism 0.00739 28. Reductive
carboxylate cycle (CO2 fixation) 0.00739 29. Inositol metabolism
0.0163 30. Citrate cycle (TCA cycle) 0.0404
[0383] The 58 biological processes could be approximated into three
major groups: metabolism, defense response and tissue
differentiation while the 30 pathways could be broadly categorized
into: receptor binding, signal transduction, cell-cell interaction,
cell migration, immune response and metabolism (FIG. 8, FIG. 9).
The postulated biological processes and pathways both suggest that
the secreted proteins have a major impact on the cellular
metabolism that will modulate energy production, breakdown,
biosynthesis and secretion of macromolecules, processes essential
for the removal of damaged tissues and regeneration of new tissues
(FIG. 8, FIG. 9).
[0384] Consistent with the predominant presence of cytokines and
chemokines in the MSC-conditioned media, the analysis also
predicted that the secreted factors could elicit many cellular
responses that are dependent on external stimuli .e.g. chemotaxis,
taxis and many immune responses (FIG. 8). Notably, the conditioned
media could also induce biological processes that are important in
tissue differentiation particularly processes that promote
vascularization, hematopoiesis and bone development (FIG. 8). In
those pathways predicted to be modulated by the secreted proteome,
receptor-mediated binding of cytokine and ECM pathways are
consistent with the predominance of cytokines and ECM components in
the secreted proteome (FIG. 9). The main signal transduction
pathways that could be activated by the secreted proteome include
Jak-STAT signaling pathway, MAPK signaling pathway, Toll-like
receptor signaling pathway, TGF-beta signaling pathway, mTOR
signaling pathway, Fc epsilon RI signaling pathway and Epithelial
cell signaling in Helicobacter pylori infection. The computational
analysis of the secreted proteome also suggested that MSC secretion
could enhance cell-cell interaction, migration and immune
responses.
Example 12
Analysis of Proteome of Human ESC-Derived MSCs (hESC-MSCs)
DISCUSSION
[0385] MSCs have been used in pre-clinical and clinical trials to
treat a myriad of diseases (3-5,24-27). However the underlying
mechanism has remained imprecisely understood. Although MSCs have
to potential to differentiate into numerous cell type e.g.
endothelial cells, cardiomyctes, chondrocytes that can potentially
repair or regenerate damaged tissues, the therapeutic effects of
MSCs cannot be solely mediated by generation of MSC-derived
reparative cell types as differentiation of MSCs is generally too
inefficient to mediate tissue repair or restore tissue function. It
has been increasingly proposed that some of the therapeutic effects
of MSCs may be mediated by paracrine factors secreted by MSCs (8).
Here we describe the composition of the secreted proteome of
hESC-MSCs through a combination of two techniques, LC-LC-MS and
antibody arrays.
[0386] Although shot-gun proteomic analysis by LC-LC-MS is a
sensitive technique and has high throughput capability, it is
difficult to detect small proteins/peptides that include most of
the cytokines, chemokines and growth factors. This is partially
mitigated by the use of antibody arrays. The qualitative proteomic
profile of the MSC secretion using the two techniques is highly
reproducible. Proteins identified by LC MS/MS are present in two
independently prepared batches of CM while those identified by
antibody array are present in at least three of four independently
prepared batches of CM. The resulting proteomic profile of
secretion by hESC-MSCs included almost all the factors that are
previously reportedly secreted by adult tissue-derived MSCs
(13,16,19-21) as well as many others that have not been described.
The robustness of the proteomic profiling is further substantiated
by the detection of transcripts for 86-88% of gene products in the
proteomic profile using a high throughput microarray-based gene
expression analysis.
[0387] To evaluate and assess the potential functions of the MSC
secretion on a global scale, we utilized the more readily available
computational tools for gene expression analysis, based on gene
products rather than on post-translationally modified proteins.
Consistent with the predominance of cytokines and chemokines in the
secretion, computational analysis predicted many processes and
pathways that are generally associated with the functions of
cytokines and chemokines such as chemotaxis, taxis, cellular
response to external stimuli, breakdown, biosynthesis and secretion
of macromolecules, cytokine-cytokine receptor interactions,
cell-cell communication, and basal metabolism e.g. glucose and
amino acid metabolism. The MSCs described here can be used to treat
diseases which these functions may have a role in.
[0388] Although these processes and pathways are not specific to
the process of injury, repair and regeneration in any particular
cell or tissue type, their facilitation of immune cell migration to
the site of injury, ECM remodelling and increase in the cellular
metabolism will have reparative effects on most injured or diseased
tissues.
[0389] Aside from these generic pathways associated with cytokines
and chemokines, computational analysis also predicted that the
secreted proteins regulate many processes involved in
vascularization, hematopoiesis and skeletal development.
Coincidentally, most reported MSC-mediated tissue repair or
regeneration are associated with cardiovascular, hematopoietic and
musculoskeletal tissues (3-5, 24-27). Pathway analysis further
uncovered candidate pathways that may be involved in mediating some
of the paracrine effects of MSCs. In fact, many of these candidate
pathways have already been implicated in many aspects of
cardiovascular, hematopoietic and musculoskeletal biology. For
example, Jak-STAT signaling is associated with cardioprotection
(28), hematopoiesis (29,30), and skeletal repair and remodelling
(31,32); MAPK signaling plays a crucial role in many aspects of
cardiovascular responses (33,34), skeletal repair and remodelling
(32,35), and hematopoiesis (36); Toll-like receptor signalling has
been implicated in the initiation and progression of cardiovascular
pathologies (37), and modulation of innate and adaptive immunity
(38); TGF-beta signalling is critical in correct heart development
cardiac remodeling, progression to heart failure and
vascularization (39-41) hematopoiesis (42), formation and
remodelling of bone and cartilage (27,43) as well as general wound
healing (44); and mTOR as an important regulator of cell growth and
proliferation plays a non-specific yet critical role in both normal
physiology and diseases (45-47).
[0390] In conclusion, our analysis of the secreted proteome in
hESC-derived MSCs which includes many of the cytokines reportedly
secreted by adult tissue-derived MSCs suggests that the secreted
proteome could potentially exert modulating effects on tissue
repair and regeneration particularly in the cardiovascular,
hematopoietic and musculoskeletal tissues, and therefore provide
molecular support for a MSC-mediated paracrine effect on tissue
repair and regeneration in MSC transplantation studies. This
secreted proteome also uncovered many highly testable hypotheses
for the molecular mechanisms in MSC-mediated tissue repair and also
potential "druggable" targets to modulate tissue repair and
regeneration. The significant similarity between hESC-derived MSCs
and adult tissue-derived MSCs suggest that conditioned media of
either MSC cultures are likely to have similar biological
activities.
[0391] Accordingly, this demonstrates that the MSCs derived by our
methods have significant biological similarities to their bone
marrow derived counterparts. These findings demonstrate the
resemblance of the hESC-MSCs to adult BM-derived MSCs in their
ability to secrete paracrine factors. Furthermore, any one or more
proteins secreted from the MSCs described here, including in the
form of conditioned media, may be used for the same purposes as the
MSCs described herein.
[0392] hESC-derived MSCs, however, have several advantages over
adult tissue-derived MSCs. The use of hESC cell lines as a tissue
source of MSC constitutes an infinitely renewable and expansible
tissue source, and enhances the reproducible and consistent batch
to batch preparation of MSCs and therefore CM in a clinically
compliant manner. It also boosts the scalability of preparing CM
and the potential of developing low cost off-the-shelf
therapeutics. In addition, the development of a serum-free
chemically defined medium for the preparation of hESC-derived MSC
CM reduces confounding and variable contaminants associated with
complex media supplements such as serum or serum replacement media.
Therefore, our elucidation of the CM is very relevant for the
translation of MSC-based biologics towards clinical
applications.
Example 13
Listing of 201 Genes in Each of the 58 Biological Processes
TABLE-US-00011 [0393] Alcohol Metabolism GI_16507966-S ENO2 enolase
2 (gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Angiogenesis GI_11321596-S KDR kinase insert domain receptor (a
type III receptor tyrosine kinase) GI_30172563-S VEGF vascular
endothelial growth factor GI_28610153-S IL8 interleukin 8
GI_42716312-S ANG angiogenin, ribonuclease, RNase A family, 5
GI_10337586-S FGF6 fibroblast growth factor 6 Biomineral Formation
GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast)
GI_5902810-A BMP1 bone morphogenetic protein 1 GI_4507170-S SPARC
secreted protein, acidic, cysteine-rich (osteonectin) Blood Vessel
Development GI_11321596-S KDR kinase insert domain receptor (a type
III receptor tyrosine kinase) GI_30172563-S VEGF vascular
endothelial growth factor GI_28610153-S IL8 interleukin 8
GI_42716312-S ANG angiogenin, ribonuclease, RNase A family, 5
GI_10337586-S FGF6 fibroblast growth factor 6 Blood Vessel
Morphogenesis GI_11321596-S KDR kinase insert domain receptor (a
type III receptor tyrosine kinase) GI_30172563-S VEGF vascular
endothelial growth factor GI_28610153-S IL8 interleukin 8
GI_42716312-S ANG angiogenin, ribonuclease, RNase A family, 5
GI_10337586-S FGF6 fibroblast growth factor 6 Bone Remodeling
GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast)
GI_5902810-A BMP1 bone morphogenetic protein 1 BMP1 GI_4507170-S
SPARC secreted protein, acidic, cysteine-rich (osteonectin)
Carbohydrate Metabolism GI_16507966-S ENO2 enolase 2 (gamma,
neuronal) GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Catabolism GI_16507966-S ENO2 enolase 2 (gamma, neuronal)
GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Cellular Carbohydrate Catabolism GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Cellular Carbohydrate Metabolism GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Cellular Catabolism GI_16507966-S ENO2 enolase 2 (gamma,
neuronal) GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Cellular Macromolecule Catabolism GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Chemotaxis GI_27262654-A IL16 interleukin 16 (lymphocyte
chemoattractant factor) GI_22538807-A CCL23 chemokine (C-C motif)
ligand 23 GI_34335180-A CCL15 chemokine (C-C motif) ligand 15
GI_40316922-I CXCL12 chemokine (C-X-C motif) ligand 12 (stromal
cell-derived factor 1) GI_41872613-S CXCL5 chemokine (C-X-C motif)
ligand 5 GI_22538812-S CCL2 chemokine (C-C motif) ligand 2
GI_4504098-S CXCR3 chemokine (C-X-C motif) receptor 3 GI_22538399-S
CCL11 chemokine (C-C motif) ligand 11 GI_5453576-S CXCL13 chemokine
(C-X-C motif) ligand 13 (B-cell chemoattractant) GI_22538815-S CCL8
chemokine (C-C motif) ligand 8 GI_34222286-S CYR61 cysteine-rich,
angiogenic inducer, 61 GI_4506832-S CCL1 chemokine (C-C motif)
ligand 1 GI_28610153-S IL8 interleukin 8 GI_22538800-S CCL16
chemokine (C-C motif) ligand 16 GI_14790145-S CXCL11 chemokine
(C-X-C motif) ligand 11 GI_22165426-S CCL24 chemokine (C-C motif)
ligand 24 GI_22538813-S CCL5 chemokine (C-C motif) ligand 5
GI_13435401-S CCL7 chemokine (C-C motif) ligand 7 GI_4505862-S PLAU
plasminogen activator, urokinase GI_4504152-S CXCL1 chemokine
(C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha) GI_27894329-S IL1A interleukin 1, alpha GI_22547151-S CCL26
chemokine (C-C motif) ligand 26 GI_4506852-S XCL1 chemokine (C
motif) ligand 1 Defense Response GI_27894305-S IL1B interleukin 1,
beta GI_27894329-S IL1A interleukin 1, alpha Development
GI_42716312-S ANG angiogenin, ribonuclease, RNase A family, 5
GI_5902810-A BMP1 bone morphogenetic protein 1 GI_27262662-A CDH11
cadherin 11, type 2, OB-cadherin (osteoblast) GI_27262662-A CSF1
colony stimulating factor 1 (macrophage) GI_27437029-S CSF2 colony
stimulating factor 2 (granulocyte-macrophage) GI_27437048-A CSF3
colony stimulating factor 3 (granulocyte) GI_34222286-S CYR61
cysteine-rich, angiogenic inducer, 61 GI_24430140-S FBN1 fibrillin
1 (Marfan syndrome) GI_4755135-S FBN2 fibrillin 2 (congenital
contractural arachnodactyly) GI_10337586-S FGF6 fibroblast growth
factor 6 GI_24430216-S IL10 interleukin 10 GI_28610153-S IL8
interleukin 8 GI_11321596-S KDR kinase insert domain receptor (a
type III receptor tyrosine kinase) GI_4580419-A KITLG KIT ligand
GI_6006018-S LIF leukemia inhibitory factor (cholinergic
differentiation factor) GI_7262388-S PCOLCE procollagen
C-endopeptidase enhancer GI_4507170-S SPARC secreted protein,
acidic, cysteine-rich (osteonectin) GI_10863872-S TGFB1
transforming growth factor, beta 1 (Camurati-Engelmann disease)
GI_4507470-S TGFBR3 transforming growth factor, beta receptor III
(betaglycan, 300 kDa) GI_40317625-S THBS1 thrombospondin 1
GI_40805871-S THPO thrombopoietin (myeloproliferative leukemia
virus oncogene ligand, megakaryocyte growth and development factor)
GI_4507508-S TIMP1 TIMP metallopeptidase inhibitor 1 GI_30172563-S
VEGF vascular endothelial growth factor Energy Derivation by
Oxidation of Organic Compounds GI_16507966-S ENO2 enolase 2 (gamma,
neuronal) GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Exocytosis GI_22538815-S CCL8 chemokine (C-C motif) ligand 8
GI_22538813-S CCL5 chemokine (C-C motif) ligand 5 Fever
GI_27894305-S IL1B interleukin 1, beta GI_27894329-S IL1A
interleukin 1, alpha Generation of Precursor Metabolites and Energy
GI_16507966-S ENO2 enolase 2 (gamma, neuronal) GI_16507965-S ENO1
enolase 1, (alpha) GI_22095338-S PGK1 phosphoglycerate kinase 1
GI_31543396-S PGK2 phosphoglycerate kinase 2 GI_21735620-S MDH2
malate dehydrogenase 2, NAD (mitochondrial) GI_26024330-S TPI1
triosephosphate isomerase 1 Glucose Catabolism GI_16507966-S ENO2
enolase 2 (gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Glucose Metabolism GI_16507966-S ENO2 enolase 2 (gamma, neuronal)
GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Glycolysis GI_16507966-S ENO2 enolase 2 (gamma, neuronal)
GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1 Heat
Generation GI_27894305-S IL1B interleukin 1, beta GI_27894329-S
IL1A interleukin 1, alpha Hemopoiesis GI_27262662-A CSF1 colony
stimulating factor 1 (macrophage) GI_4580419-A KITLG KIT ligand
GI_24430216-S IL10 interleukin 10 Hemopoietic or Lymphoid Organ
Development GI_27262662-A CSF1 colony stimulating factor 1
(macrophage) GI_4580419-A KITLG KIT ligand GI_24430216-S IL10
interleukin 10 Hexose Catabolism GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Hexose Metabolism GI_16507966-S ENO2 enolase 2 (gamma, neuronal)
GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Immune Response GI_27894305-S IL1B interleukin 1, beta
GI_27894329-S IL1A interleukin 1, alpha Inflammatory Response
GI_27894305-S IL1B interleukin 1, beta GI_27894329-S IL1A
interleukin 1, alpha Macromolecule Metabolism GI_5902810-A BMP1
bone morphogenetic protein 1 GI_31542249-S C1R complement component
1, r subcomponent GI_22538429-A CTSB cathepsin B GI_16507965-S ENO1
enolase 1, (alpha) GI_16507966-S ENO2 enolase 2 (gamma, neuronal)
GI_33859834-S HGF hepatocyte growth factor (hepapoietin A; scatter
factor) GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_13027798-S MMP1 matrix metallopeptidase 1
(interstitial collagenase) GI_4505204-S MMP10 matrix
metallopeptidase 10 (stromelysin 2) GI_13027796-S MMP13 matrix
metallopeptidase 13 (collagenase 3) GI_13027803-S MMP3 matrix
metallopeptidase 3 (stromelysin 1, progelatinase) GI_4826835-S MMP9
matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa
type IV collagenase) GI_22095338-S PGK1 phosphoglycerate kinase 1
GI_31543396-S PGK2 phosphoglycerate kinase 2 GI_4505862-S PLAU
plasminogen activator, urokinase GI_26024330-S TPI1 triosephosphate
isomerase 1 Macromolecule Catabolism GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Main Pathways of Carbohydrate Metabolism GI_16507966-S ENO2
enolase 2 (gamma, neuronal) GI_16507965-S ENO1 enolase 1, (alpha)
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 GI_21735620-S MDH2 malate dehydrogenase
2, NAD (mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase
1 Monosaccharide Catabolism GI_16507966-S ENO2 enolase 2 (gamma,
neuronal)
GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Monosaccharide Metabolism GI_16507966-S ENO2 enolase 2 (gamma,
neuronal) GI_16507965-S ENO1 enolase 1, (alpha) GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 GI_21735620-S MDH2 malate dehydrogenase 2, NAD
(mitochondrial) GI_26024330-S TPI1 triosephosphate isomerase 1
Organ Development GI_27262662-A CSF1 colony stimulating factor 1
(macrophage) GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin
(osteoblast) GI_4580419-A KITLG KIT ligand GI_5902810-A BMP1 bone
morphogenetic protein 1 GI_11321596-S KDR kinase insert domain
receptor (a type III receptor tyrosine kinase) GI_30172563-S VEGF
vascular endothelial growth factor GI_28610153-S IL8 interleukin 8
GI_4507170-S SPARC secreted protein, acidic, cysteine-rich
(osteonectin) GI_42716312-S ANG angiogenin, ribonuclease, RNase A
family, 5 GI_24430216-S IL10 interleukin 10 GI_10337586-S FGF6
fibroblast growth factor 6 Organ Morphogenesis GI_11321596-S KDR
kinase insert domain receptor (a type III receptor tyrosine kinase)
GI_30172563-S VEGF vascular endothelial growth factor GI_28610153-S
IL8 interleukin 8 GI_42716312-S ANG angiogenin, ribonuclease, RNase
A family, 5 GI_10337586-S FGF6 fibroblast growth factor 6
Ossification GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin
(osteoblast) GI_5902810-A BMP1 bone morphogenetic protein 1
GI_4507170-S SPARC secreted protein, acidic, cysteine-rich
(osteonectin) Phosphate Metabolism GI_22095338-S PGK1
phosphoglycerate kinase 1 GI_31543396-S PGK2 phosphoglycerate
kinase 2 Phosphorus Metabolism GI_22095338-S PGK1 phosphoglycerate
kinase 1 GI_31543396-S PGK2 phosphoglycerate kinase 2 Response to
Abiotic Stimulus GI_27262654-A IL16 interleukin 16 (lymphocyte
chemoattractant factor) GI_22538807-A CCL23 chemokine (C-C motif)
ligand 23 GI_34335180-A CCL15 chemokine (C-C motif) ligand 15
GI_40316922-I CXCL12 chemokine (C-X-C motif) ligand 12 (stromal
cell-derived factor 1) GI_41872613-S CXCL5 chemokine (C-X-C motif)
ligand 5 GI_22538812-S CCL2 chemokine (C-C motif) ligand 2
GI_4504098-S CXCR3 chemokine (C-X-C motif) receptor 3 GI_22538399-S
CCL11 chemokine (C-C motif) ligand 11 GI_5453576-S CXCL13 chemokine
(C-X-C motif) ligand 13 (B-cell chemoattractant) GI_22538815-S CCL8
chemokine (C-C motif) ligand 8 GI_34222286-S CYR61 cysteine-rich,
angiogenic inducer, 61 GI_4506832-S CCL1 chemokine (C-C motif)
ligand 1 GI_28610153-S IL8 interleukin 8 GI_22538800-S CCL16
chemokine (C-C motif) ligand 16 GI_14790145-S CXCL11 chemokine
(C-X-C motif) ligand 11 GI_22165426-S CCL24 chemokine (C-C motif)
ligand 24 GI_22538813-S CCL5 chemokine (C-C motif) ligand 5
GI_13435401-S CCL7 chemokine (C-C motif) ligand 7 GI_4505862-S PLAU
plasminogen activator, urokinase GI_4504152-S CXCL1 chemokine
(C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha) GI_27894329-S IL1A interleukin 1, alpha GI_22547151-S CCL26
chemokine (C-C motif) ligand 26 GI_4506852-S XCL1 chemokine (C
motif) ligand 1 Response to Biotic Stimulus GI_27894305-S IL1B
interleukin 1, beta GI_27894329-S IL1A interleukin 1, alpha
Response to Chemical Stimulus GI_27262654-A IL16 interleukin 16
(lymphocyte chemoattractant factor) GI_22538807-A CCL23 chemokine
(C-C motif) ligand 23 GI_34335180-A CCL15 chemokine (C-C motif)
ligand 15 GI_40316922-I CXCL12 chemokine (C-X-C motif) ligand 12
(stromal cell-derived factor 1) GI_41872613-S CXCL5 chemokine
(C-X-C motif) ligand 5 GI_22538812-S CCL2 chemokine (C-C motif)
ligand 2 GI_4504098-S CXCR3 chemokine (C-X-C motif) receptor 3
GI_22538399-S CCL11 chemokine (C-C motif) ligand 11 GI_5453576-S
CXCL13 chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
GI_22538815-S CCL8 chemokine (C-C motif) ligand 8 GI_34222286-S
CYR61 cysteine-rich, angiogenic inducer, 61 GI_4506832-S CCL1
chemokine (C-C motif) ligand 1 GI_28610153-S IL8 interleukin 8
GI_22538800-S CCL16 chemokine (C-C motif) ligand 16 GI_14790145-S
CXCL11 chemokine (C-X-C motif) ligand 11 GI_22165426-S CCL24
chemokine (C-C motif) ligand 24 GI_22538813-S CCL5 chemokine (C-C
motif) ligand 5 GI_13435401-S CCL7 chemokine (C-C motif) ligand 7
GI_4505862-S PLAU plasminogen activator, urokinase GI_4504152-S
CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating
activity, alpha) GI_27894329-S IL1A interleukin 1, alpha
GI_22547151-S CCL26 chemokine (C-C motif) ligand 26 GI_4506852-S
XCL1 chemokine (C motif) ligand 1 Response to External Biotic
Stimulus GI_27894305-S IL1B interleukin 1, beta GI_27894329-S IL1A
interleukin 1, alpha Response to External Stimulus GI_27262654-A
IL16 interleukin 16 (lymphocyte chemoattractant factor)
GI_22538807-A CCL23 chemokine (C-C motif) ligand 23 GI_34335180-A
CCL15 chemokine (C-C motif) ligand 15 GI_40316922-I CXCL12
chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)
GI_41872613-S CXCL5 chemokine (C-X-C motif) ligand 5 GI_22538812-S
CCL2 chemokine (C-C motif) ligand 2 GI_4504098-S CXCR3 chemokine
(C-X-C motif) receptor 3 GI_22538399-S CCL11 chemokine (C-C motif)
ligand 11 GI_5453576-S CXCL13 chemokine (C-X-C motif) ligand 13
(B-cell chemoattractant) GI_22538815-S CCL8 chemokine (C-C motif)
ligand 8 GI_34222286-S CYR61 cysteine-rich, angiogenic inducer, 61
GI_4506832-S CCL1 chemokine (C-C motif) ligand 1 GI_28610153-S IL8
interleukin 8 GI_22538800-S CCL16 chemokine (C-C motif) ligand 16
GI_14790145-S CXCL11 chemokine (C-X-C motif) ligand 11
GI_22165426-S CCL24 chemokine (C-C motif) ligand 24 GI_22538813-S
CCL5 chemokine (C-C motif) ligand 5 GI_27894305-S IL1B interleukin
1, beta GI_13435401-S CCL7 chemokine (C-C motif) ligand 7
GI_4505862-S PLAU plasminogen activator, urokinase GI_4504152-S
CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating
activity, alpha) GI_27894329-S IL1A interleukin 1, alpha
GI_22547151-S CCL26 chemokine (C-C motif) ligand 26 GI_4506852-S
XCL1 chemokine (C motif) ligand 1 Response to Pest, Pathogen or
Parasite GI_27894305-S IL1B interleukin 1, beta GI_27894329-S IL1A
interleukin 1, alpha Response to Stimulus GI_27262654-A IL16
interleukin 16 (lymphocyte chemoattractant factor) GI_22538807-A
CCL23 chemokine (C-C motif) ligand 23 GI_34335180-A CCL15 chemokine
(C-C motif) ligand 15 GI_40316922-I CXCL12 chemokine (C-X-C motif)
ligand 12 (stromal cell-derived factor 1) GI_41872613-S CXCL5
chemokine (C-X-C motif) ligand 5 GI_22538812-S CCL2 chemokine (C-C
motif) ligand 2 GI_4504098-S CXCR3 chemokine (C-X-C motif) receptor
3 GI_22538399-S CCL11 chemokine (C-C motif) ligand 11 GI_5453576-S
CXCL13 chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant)
GI_22538815-S CCL8 chemokine (C-C motif) ligand 8 GI_34222286-S
CYR61 cysteine-rich, angiogenic inducer, 61 GI_4506832-S CCL1
chemokine (C-C motif) ligand 1 GI_28610153-S IL8 interleukin 8
GI_22538800-S CCL16 chemokine (C-C motif) ligand 16 GI_14790145-S
CXCL11 chemokine (C-X-C motif) ligand 11 GI_22165426-S CCL24
chemokine (C-C motif) ligand 24 GI_22538813-S CCL5 chemokine (C-C
motif) ligand 5 GI_27894305-S IL1B interleukin 1, beta
GI_13435401-S CCL7 chemokine (C-C motif) ligand 7 GI_4505862-S PLAU
plasminogen activator, urokinase GI_4504152-S CXCL1 chemokine
(C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha) GI_27894329-S IL1A interleukin 1, alpha GI_22547151-S CCL26
chemokine (C-C motif) ligand 26 GI_4506852-S XCL1 chemokine (C
motif) ligand 1 Response to Stress GI_27894305-S IL1B interleukin
1, beta GI_4505862-S PLAU plasminogen activator, urokinase
GI_27894329-S IL1A interleukin 1, alpha Response to Wounding
GI_27894305-S IL1B interleukin 1, beta GI_4505862-S PLAU
plasminogen activator, urokinase GI_27894329-S IL1A interleukin 1,
alpha Secretion GI_22538815-S CCL8 chemokine (C-C motif) ligand 8
GI_22538813-S CCL5 chemokine (C-C motif) ligand 5 Secretory Pathway
GI_22538815-S CCL8 chemokine (C-C motif) ligand 8 GI_22538813-S
CCL5 chemokine (C-C motif) ligand 5 Skeletal Development
GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast)
GI_5902810-A BMP1 bone morphogenetic protein 1 BMP1 GI_4507170-S
SPARC secreted protein, acidic, cysteine-rich (osteonectin) Taxis
GI_27262654-A IL16 interleukin 16 (lymphocyte chemoattractant
factor) GI_22538807-A CCL23 chemokine (C-C motif) ligand 23
GI_34335180-A CCL15 chemokine (C-C motif) ligand 15 GI_40316922-I
CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived
factor 1) GI_41872613-S CXCL5 chemokine (C-X-C motif) ligand 5
GI_22538812-S CCL2 chemokine (C-C motif) ligand 2 GI_4504098-S
CXCR3 chemokine (C-X-C motif) receptor 3 GI_22538399-S CCL11
chemokine (C-C motif) ligand 11 GI_5453576-S CXCL13 chemokine
(C-X-C motif) ligand 13 (B-cell chemoattractant) GI_22538815-S CCL8
chemokine (C-C motif) ligand 8 GI_34222286-S CYR61 cysteine-rich,
angiogenic inducer, 61 GI_4506832-S CCL1 chemokine (C-C motif)
ligand 1 GI_28610153-S IL8 interleukin 8 GI_22538800-S CCL16
chemokine (C-C motif) ligand 16 GI_14790145-S CXCL11 chemokine
(C-X-C motif) ligand 11 GI_22165426-S CCL24 chemokine (C-C motif)
ligand 24 GI_22538813-S CCL5 chemokine (C-C motif) ligand 5
GI_13435401-S CCL7 chemokine (C-C motif) ligand 7 GI_4505862-S PLAU
plasminogen activator, urokinase GI_4504152-S CXCL1 chemokine
(C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha) GI_27894329-S IL1A interleukin 1, alpha GI_22547151-S CCL26
chemokine (C-C motif) ligand 26 GI_4506852-S XCL1 chemokine (C
motif) ligand 1 Thermoregulation GI_27894305-S IL1B interleukin 1,
beta GI_27894329-S IL1A interleukin 1, alpha Tissue Development
GI_27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast)
GI_5902810-A BMP1 bone morphogenetic protein 1 BMP1 GI_4507170-S
SPARC secreted protein, acidic, cysteine-rich (osteonectin)
Vasulogenesis GI_30172563-S VEGF vascular endothelial growth factor
Vasculature Development GI_11321596-S KDR kinase insert domain
receptor (a type III receptor tyrosine kinase) GI_30172563-S VEGF
vascular endothelial growth factor GI_28610153-S IL8 interleukin 8
GI_42716312-S ANG angiogenin, ribonuclease, RNase A family, 5
GI_10337586-S FGF6 fibroblast growth factor 6
Example 14
Listing of 201 Genes in Each of the 30 Pathways
TABLE-US-00012 [0394] Antigen processing and presentation - Homo
sapiens (human) GI_22538429-A CTSB cathepsin B GI_37704380-S B2M
beta-2-microglobulin GI_24234685-A HSPA8 heat shock 70 kDa protein
8 GI_30581139-A PSME1 proteasome (prosome, macropain) activator
subunit 1 (PA28 alpha) Apoptosis - Homo sapiens (human)
GI_41281560-S CLSTN1 calsyntenin 1 GI_28416914-S IL3 interleukin 3
(colony-stimulating factor, multiple) GI_27894329-S IL1A
interleukin 1, alpha GI_27894305-S IL1B interleukin 1, beta
GI_25952110-S TNF tumor necrosis factor (TNF superfamily, member 2)
Carbon fixation - Homo sapiens (human) GI_21735620-S MDH2 malate
dehydrogenase 2, NAD (mitochondrial) GI_26024330-S TPI1
triosephosphate isomerase 1 GI_22095338-S PGK1 phosphoglycerate
kinase 1 GI_31543396-S PGK2 phosphoglycerate kinase 2 Cell
Communication - Homo sapiens (human) GI_27436944-A LMNA lamin A/C
GI_5016088-S ACTB actin, beta GI_10938011-S ACTC actin, alpha,
cardiac muscle GI_11038618-S ACTG1 actin, gamma 1 GI_14719826-S
COL1A1 collagen, type I, alpha 1 GI_21536289-S COL1A2 collagen,
type I, alpha 2 GI_15149480-S COL3A1 collagen, type III, alpha 1
(Ehlers-Danlos syndrome type IV, autosomal dominant) GI_45580690-S
COL4A1 collagen, type IV, alpha 1 GI_17986276-S COL4A2 collagen,
type IV, alpha 2 GI_16554578-S COL5A1 collagen, type V, alpha 1
GI_16554580-S COL5A2 collagen, type V, alpha 2 GI_15011912-S COL6A1
collagen, type VI, alpha 1 GI_17402876-A COL6A2 collagen, type VI,
alpha 2 GI_17149810-A COL6A3 collagen, type VI, alpha 3
GI_18375521-A COL11A1 collagen, type XI, alpha 1 GI_16933543-A FN1
fibronectin 1 GI_9845497-S LAMC1 laminin, gamma 1 (formerly LAMB2)
GI_16554581-S COL5A3 collagen, type V, alpha 3 GI_40317625-S THBS1
thrombospondin 1 Citrate cycle (TCA cycle) - Homo sapiens (human)
GI_4504374-S CFH1/HF1 complement factor H GI_21735620-S MDH2 malate
dehydrogenase 2, NAD (mitochondrial) Complement and coagulation
cascades - Homo sapiens (human) GI_4505862-S PLAU plasminogen
activator, urokinase GI_4504374-S CFH1/HF1 complement factor H
GI_31542249-S C1R complement component 1, r subcomponent
Cytokine-cytokine receptor interaction - Homo sapiens (human)
GI_4506832-S CCL1 chemokine (C-C motif) ligand 1 GI_22538399-S
CCL11 chemokine (C-C motif) ligand 11 GI_34335180-A CCL15 chemokine
(C-C motif) ligand 15 GI_22538800-S CCL16 chemokine (C-C motif)
ligand 16 GI_22538812-S CCL2 chemokine (C-C motif) ligand 2
GI_22538807-A CCL23 chemokine (C-C motif) ligand 23 GI_22165426-S
CCL24 chemokine (C-C motif) ligand 24 GI_22547151-S CCL26 chemokine
(C-C motif) ligand 26 GI_22538813-S CCL5 chemokine (C-C motif)
ligand 5 GI_13435401-S CCL7 chemokine (C-C motif) ligand 7
GI_22538815-S CCL8 chemokine (C-C motif) ligand 8 GI_27262662-A
CSF1 colony stimulating factor 1 (macrophage) GI_27437029-S CSF2
colony stimulating factor 2 (granulocyte-macrophage) GI_27437048-A
CSF3 colony stimulating factor 3 (granulocyte) GI_4506856-S CX3CL1
chemokine (C-X3-C motif) ligand 1 GI_4504152-S CXCL1 chemokine
(C-X-C motif) ligand 1 (melanoma growth stimulating activity,
alpha) GI_14790145-S CXCL11 chemokine (C-X-C motif) ligand 11
GI_40316922-I CXCL12 chemokine (C-X-C motif) ligand 12 (stromal
cell-derived factor 1) GI_5453576-S CXCL13 chemokine (C-X-C motif)
ligand 13 (B-cell chemoattractant) GI_41872613-S CXCL5 chemokine
(C-X-C motif) ligand 5 GI_4504098-S CXCR3 chemokine (C-X-C motif)
receptor 3 GI_6031163-S EGF epidermal growth factor
(beta-urogastrone) GI_38455415-S FLT3LG fins-related tyrosine
kinase 3 ligand GI_33859834-S HGF hepatocyte growth factor
(hepapoietin A; scatter factor) GI_10835170-S IFNG interferon,
gamma GI_24430216-S IL10 interleukin 10 GI_24497437-S IL12B
interleukin 12B (natural killer cell stimulatory factor 2,
cytotoxic lymphocyte maturation factor 2, p40) GI_26787977-S IL13
interleukin 13 GI_27894329-S IL1A interleukin 1, alpha
GI_27894305-S IL1B interleukin 1, beta GI_28178860-S IL2
interleukin 2 GI_28416914-S IL3 interleukin 3 (colony-stimulating
factor, multiple) GI_10834983-S IL6 interleukin 6 (interferon, beta
2) GI_28610152-S IL7 interleukin 7 GI_28610153-S IL8 interleukin 8
GI_11321596-S KDR kinase insert domain receptor (a type III
receptor tyrosine kinase) GI_4580419-A KITLG KIT ligand
GI_4557714-S LEP leptin (obesity homolog, mouse) GI_6006018-S LIF
leukemia inhibitory factor (cholinergic differentiation factor)
GI_15451785-A PDGFB platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v- sis) oncogene homolog) GI_15451788-S
PDGFRB platelet-derived growth factor receptor, beta polypeptide
GI_10863872-S TGFB1 transforming growth factor, beta 1
(Camurati-Engelmann disease) GI_4507462-S TGFB2 transforming growth
factor, beta 2 GI_40805871-S THPO thrombopoietin
(myeloproliferative leukemia virus oncogene ligand, megakaryocyte
growth and development factor) GI_4507508-S TIMP1 TIMP
metallopeptidase inhibitor 1 GI_25952110-S TNF tumor necrosis
factor (TNF superfamily, member 2) GI_22547122-S TNFRSF11B tumor
necrosis factor receptor superfamily, member 11b (osteoprotegerin)
GI_30172563-S VEGF vascular endothelial growth factor GI_4506852-S
XCL1 chemokine (C motif) ligand 1 ECM-receptor interaction - Homo
sapiens (human) GI_9845497-S LAMC1 laminin, gamma 1 (formerly
LAMB2) GI_14719826-S COL1A1 collagen, type I, alpha 1 GI_21536289-S
COL1A2 collagen, type I, alpha 2 GI_15149480-S COL3A1 collagen,
type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal
dominant) GI_45580690-S COL4A1 collagen, type IV, alpha 1
GI_17986276-S COL4A2 collagen, type IV, alpha 2 GI_16554578-S
COL5A1 collagen, type V, alpha 1 GI_16554580-S COL5A2 collagen,
type V, alpha 2 GI_15011912-S COL6A1 collagen, type VI, alpha 1
GI_17402876-A COL6A2 collagen, type VI, alpha 2 GI_17149810-A
COL6A3 collagen, type VI, alpha 3 GI_18375521-A COL11A1 collagen,
type XI, alpha 1 GI_16554581-S COL5A3 collagen, type V, alpha 3
GI_16933543-A FN1 fibronectin 1 GI_40317625-S THBS1 thrombospondin
1 GI_7427516-S HSPG2 heparan sulfate proteoglycan 2 (perlecan)
GI_21361192-S CD44 CD44 antigen (homing function and Indian blood
group system) Epithelial cell signaling in Helicobacter pylori
infection - Homo sapiens (human) GI_22538813-S CCL5 chemokine (C-C
motif) ligand 5 GI_4504152-S CXCL1 chemokine (C-X-C motif) ligand 1
(melanoma growth stimulating activity, alpha) GI_28610153-S IL8
interleukin 8 Fc epsilon RI signaling pathway - Homo sapiens
(human) GI_25952110-S TNF tumor necrosis factor (TNF superfamily,
member 2) GI_26787977-S IL13 interleukin 13 GI_27437029-S CSF2
colony stimulating factor 2 (granulocyte-macrophage) GI_41281560-S
CLSTN1 calsyntenin 1 GI_28416914-S IL3 interleukin 3
(colony-stimulating factor, multiple) Focal adhesion - Homo sapiens
(human) GI_5016088-S ACTB actin, beta GI_10938011-S ACTC actin,
alpha, cardiac muscle GI_11038618-S ACTG1 actin, gamma 1
GI_41281560-S CLSTN1 calsyntenin 1 GI_18375521-A COL11A1 collagen,
type XI, alpha 1 GI_14719826-S COL1A1 collagen, type I, alpha 1
GI_21536289-S COL1A2 collagen, type I, alpha 2 GI_15149480-S COL3A1
collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV,
autosomal dominant) GI_45580690-S COL4A1 collagen, type IV, alpha 1
GI_17986276-S COL4A2 collagen, type IV, alpha 2 GI_16554578-S
COL5A1 collagen, type V, alpha 1 GI_16554580-S COL5A2 collagen,
type V, alpha 2 GI_16554581-S COL5A3 collagen, type V, alpha 3
GI_15011912-S COL6A1 collagen, type VI, alpha 1 GI_17402876-A
COL6A2 collagen, type VI, alpha 2 GI_17149810-A COL6A3 collagen,
type VI, alpha 3 GI_6031163-S EGF epidermal growth factor
(beta-urogastrone) GI_4503744-S FLNA filamin A, alpha (actin
binding protein 280) GI_16933543-A FN1 fibronectin 1 GI_33859834-S
HGF hepatocyte growth factor (hepapoietin A; scatter factor)
GI_19923111-S IGF1 insulin-like growth factor 1 (somatomedin C)
GI_10834983-S IL6 interleukin 6 (interferon, beta 2) GI_11321596-S
KDR kinase insert domain receptor (a type III receptor tyrosine
kinase) GI_9845497-S LAMC1 laminin, gamma 1 (formerly LAMB2)
GI_15451785-A PDGFB platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v- sis) oncogene homolog) GI_15451788-S
PDGFRB platelet-derived growth factor receptor, beta polypeptide
GI_40317625-S THBS1 thrombospondin 1 GI_30172563-S VEGF vascular
endothelial growth factor Gap junction - Homo sapiens (human)
GI_5174476-S K-ALPHA-1 tubulin, alpha, ubiquitous GI_17986282-S
TUBA3 tubulin, alpha 3 GI_31880337-S TUBA6 tubulin, alpha 6
GI_15451788-S PDGFRB platelet-derived growth factor receptor, beta
polypeptide GI_6031163-S EGF epidermal growth factor
(beta-urogastrone) GI_15451785-A PDGFB platelet-derived growth
factor beta polypeptide (simian sarcoma viral (v- sis) oncogene
homolog) Glycolysis or Gluconeogenesis - Homo sapiens (human)
GI_16507965-S ENO1 enolase 1, (alpha) GI_16507966-S ENO2 enolase 2
(gamma, neuronal) GI_26024330-S TPI1 triosephosphate isomerase 1
GI_22095338-S PGK1 phosphoglycerate kinase 1 GI_31543396-S PGK2
phosphoglycerate kinase 2 Hematopoietic cell lineage - Homo sapiens
(human) GI_21361192-S CD44 CD44 antigen (homing function and Indian
blood group system) GI_4503744-S FLNA filamin A, alpha (actin
binding protein 280) GI_40805871-S THPO thrombopoietin
(myeloproliferative leukemia virus oncogene ligand, megakaryocyte
growth and development factor) GI_4507508-S TIMP1 TIMP
metallopeptidase inhibitor 1 GI_25952110-S TNF tumor necrosis
factor (TNF superfamily, member 2) GI_27262662-A CSF1 colony
stimulating factor 1 (macrophage) GI_27437048-A CSF3 colony
stimulating factor 3 (granulocyte) GI_38455415-S FLT3LG fms-related
tyrosine kinase 3 ligand GI_10834983-S IL6 interleukin 6
(interferon, beta 2) GI_27437029-S CSF2 colony stimulating factor 2
(granulocyte-macrophage) GI_27894329-S IL1A interleukin 1, alpha
GI_27894305-S IL1B interleukin 1, beta GI_28416914-S IL3
interleukin 3 (colony-stimulating factor, multiple) GI_28610152-S
IL7 interleukin 7 GI_4580419-A KITLG KIT ligand Inositol metabolism
- Homo sapiens (human) GI_26024330-S TPI1 triosephosphate isomerase
1 Insulin signaling pathway - Homo sapiens (human) GI_31377794-S
CALM1 calmodulin 1 (phosphorylase kinase, delta) GI_41281560-S
CLSTN1 calsyntenin 1 Jak-STAT signaling pathway - Homo sapiens
(human) GI_41281560-S CLSTN1 calsyntenin 1 GI_27437029-S CSF2
colony stimulating factor 2 (granulocyte-macrophage) GI_27437048-A
CSF3 colony stimulating factor 3 (granulocyte) GI_10835170-S IFNG
interferon, gamma GI_24430216-S IL10 interleukin 10 GI_24497437-S
IL12B interleukin 12B (natural killer cell stimulatory factor 2,
cytotoxic lymphocyte maturation factor 2, p40) GI_26787977-S IL13
interleukin 13 GI_28178860-S IL2 interleukin 2 GI_28416914-S IL3
interleukin 3 (colony-stimulating factor, multiple) GI_10834983-S
IL6 interleukin 6 (interferon, beta 2) GI_28610152-S IL7
interleukin 7 GI_4557714-S LEP leptin (obesity homolog, mouse)
GI_6006018-S LIF leukemia inhibitory factor (cholinergic
differentiation factor) GI_40805871-S THPO thrombopoietin
(myeloproliferative leukemia virus oncogene ligand, megakaryocyte
growth and development factor) GI_4507508-S TIMP1 TIMP
metallopeptidase inhibitor 1 Leukocyte transendothelial migration -
Homo sapiens (human) GI_4826835-S MMP9 matrix metallopeptidase 9
(gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
GI_5453576-S CXCL13 chemokine (C-X-C motif) ligand 13 (B-cell
chemoattractant) GI_28610153-S IL8 interleukin 8 GI_40316922-I
CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived
factor 1) GI_41281560-S CLSTN1 calsyntenin 1 GI_5016088-S ACTB
actin, beta GI_10938011-S ACTC actin, alpha, cardiac muscle
GI_11038618-S ACTG1 actin, gamma 1 MAPK signaling pathway - Homo
sapiens (human) GI_34106709-A BDNF brain-derived neurotrophic
factor GI_6031163-S EGF epidermal growth factor (beta-urogastrone)
GI_4503692-S FGF17 fibroblast growth factor 17 GI_4503700-S FGF4
fibroblast growth factor 4 (heparin secretory transforming protein
1, Kaposi sarcoma oncogene) GI_10337586-S FGF6 fibroblast growth
factor 6 GI_15147344-S FGF7 fibroblast growth factor 7
(keratinocyte growth factor) GI_4503706-S FGF9 fibroblast growth
factor 9 (glia-activating factor) GI_13186266-A FGFR2 fibroblast
growth factor receptor 2 (bacteria-expressed kinase, keratinocyte
growth factor receptor, craniofacial dysostosis 1, Crouzon
syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) GI_4503744-S
FLNA filamin A, alpha (actin binding protein 280) GI_40549401-A
GDNF glial cell derived neurotrophic factor GI_24234685-A HSPA8
heat shock 70 kDa protein 8 GI_27894329-S IL1A interleukin 1, alpha
GI_27894305-S IL1B interleukin 1, beta GI_15451785-A PDGFB
platelet-derived growth factor beta polypeptide (simian sarcoma
viral (v- sis) oncogene homolog) GI_15451788-S PDGFRB
platelet-derived growth factor receptor, beta polypeptide
GI_10863872-S TGFB1 transforming growth factor, beta 1
(Camurati-Engelmann disease) GI_4507462-S TGFB2 transforming growth
factor, beta 2 GI_25952110-S TNF tumor necrosis factor (TNF
superfamily, member 2) Methane metabolism - Homo sapiens (human)
GI_40805871-S THPO thrombopoietin (myeloproliferative leukemia
virus oncogene ligand, megakaryocyte growth and development factor)
GI_32455261-A PRDX5 peroxiredoxin 5 mTOR signaling pathway - Homo
sapiens (human) GI_30172563-S VEGF vascular endothelial growth
factor GI_41281560-S CLSTN1 calsyntenin 1 GI_19923111-S IGF1
insulin-like growth factor 1 (somatomedin C) Phenylalanine,
tyrosine and tryptophan biosynthesis - Homo sapiens (human)
GI_16507965-S ENO1 enolase 1, (alpha) GI_16507966-S ENO2 enolase 2
(gamma, neuronal) Phenylalanine metabolism - Homo sapiens (human)
GI_4505184-S MIF macrophage migration inhibitory factor
(glycosylation-inhibiting factor) GI_40805871-S THPO thrombopoietin
(myeloproliferative leukemia virus oncogene ligand, megakaryocyte
growth and development factor) GI_32455261-A PRDX5 peroxiredoxin 5
Regulation of actin cytoskeleton - Homo sapiens (human)
GI_5016088-S ACTB actin, beta GI_10938011-S ACTC actin, alpha,
cardiac muscle GI_11038618-S ACTG1 actin, gamma 1 GI_5031634-S CFL1
cofilin 1 (non-muscle) GI_41281560-S CLSTN1 calsyntenin 1
GI_6031163-S EGF epidermal growth factor (beta-urogastrone)
GI_4503692-S FGF17 fibroblast growth factor 17 GI_4503700-S FGF4
fibroblast growth factor 4 (heparin secretory transforming protein
1, Kaposi sarcoma oncogene) GI_10337586-S FGF6 fibroblast growth
factor 6 GI_15147344-S FGF7 fibroblast growth factor 7
(keratinocyte growth factor) GI_4503706-S FGF9 fibroblast growth
factor 9 (glia-activating factor) GI_13186266-A FGFR2 fibroblast
growth factor receptor 2 (bacteria-expressed kinase, keratinocyte
growth factor receptor, craniofacial dysostosis 1, Crouzon
syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) GI_16933543-A
FN1 fibronectin 1 GI_38044287-A GSN gelsolin (amyloidosis, Finnish
type) GI_22507396-S MYH9 myosin, heavy polypeptide 9, non-muscle
GI_15451785-A PDGFB platelet-derived growth factor beta polypeptide
(simian sarcoma viral (v- sis) oncogene homolog) GI_15451788-S
PDGFRB platelet-derived growth factor receptor, beta polypeptide
GI_16753213-S PFN1 profilin 1 GI_34328943-S TMSB4X thymosin, beta
4, X-linked GI_34013529-S TMSL3 thymosin-like 3 Stilbene, coumarine
and lignin biosynthesis - Homo sapiens (human) GI_40805871-S THPO
thrombopoietin (myeloproliferative leukemia virus oncogene ligand,
megakaryocyte growth and development factor) GI_32455261-A PRDX5
peroxiredoxin 5 T cell receptor signaling pathway - Homo sapiens
(human) GI_25952110-S TNF tumor necrosis factor (TNF superfamily,
member 2) GI_27437029-S CSF2 colony stimulating factor 2
(granulocyte-macrophage) GI_10835170-S IFNG interferon, gamma
GI_24430216-S IL10 interleukin 10 GI_28178860-S IL2 interleukin 2
GI_41281560-S CLSTN1 calsyntenin 1 TGF-beta signaling pathway -
Homo sapiens (human) GI_4557730-S LTBP1 latent transforming growth
factor beta binding protein 1 GI_40317625-S THBS1 thrombospondin 1
GI_10863872-S TGFB1 transforming growth factor, beta 1
(Camurati-Engelmann disease) GI_4507462-S TGFB2 transforming growth
factor, beta 2 GI_25952110-S TNF tumor necrosis factor (TNF
superfamily, member 2) GI_10835170-S IFNG interferon, gamma Tight
junction - Homo sapiens (human) GI_5016088-S ACTB actin, beta
GI_10938011-S ACTC actin, alpha, cardiac muscle GI_11038618-S ACTG1
actin, gamma 1 GI_22507396-S MYH9 myosin, heavy polypeptide 9,
non-muscle Toll-like receptor signaling pathway - Homo sapiens
(human) GI_14790145-S CXCL11 chemokine (C-X-C motif) ligand 11
GI_28610153-S IL8 interleukin 8 GI_24497437-S IL12B interleukin 12B
(natural killer cell stimulatory factor 2, cytotoxic lymphocyte
maturation factor 2, p40) GI_27894305-S IL1B interleukin 1, beta
GI_41281560-S CLSTN1 calsyntenin 1 GI_10834983-S IL6 interleukin 6
(interferon, beta 2) GI_22538813-S CCL5 chemokine (C-C motif)
ligand 5 GI_25952110-S TNF tumor necrosis factor (TNF superfamily,
member 2) Type I diabetes mellitus - Homo sapiens (human)
GI_24497437-S IL12B interleukin 12B (natural killer cell
stimulatory factor 2, cytotoxic lymphocyte maturation factor 2,
p40) GI_28178860-S IL2 interleukin 2 GI_10835170-S IFNG interferon,
gamma GI_27894329-S IL1A interleukin 1, alpha GI_27894305-S IL1B
interleukin 1, beta GI_25952110-S TNF tumor necrosis factor (TNF
superfamily, member 2)
Example 15
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[0470] Each of the applications and patents mentioned in this
document, and each document cited or referenced in each of the
above applications and patents, including during the prosecution of
each of the applications and patents ("application cited
documents") and any manufacturer's instructions or catalogues for
any products cited or mentioned in each of the applications and
patents and in any of the application cited documents, are hereby
incorporated herein by reference. Furthermore, all documents cited
in this text, and all documents cited or referenced in documents
cited in this text, and any manufacturer's instructions or
catalogues for any products cited or mentioned in this text, are
hereby incorporated herein by reference.
[0471] Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments and that many modifications
and additions thereto may be made within the scope of the
invention. Indeed, various modifications of the described modes for
carrying out the invention which are obvious to those skilled in
molecular biology or related fields are intended to be within the
scope of the claims. Furthermore, various combinations of the
features of the following dependent claims can be made with the
features of the independent claims without departing from the scope
of the present invention.
* * * * *