U.S. patent application number 12/888724 was filed with the patent office on 2011-01-13 for method of deriving progenitor cell line.
This patent application is currently assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH. Invention is credited to Sai Kiang Lim, Elias Lye.
Application Number | 20110008298 12/888724 |
Document ID | / |
Family ID | 37075090 |
Filed Date | 2011-01-13 |
United States Patent
Application |
20110008298 |
Kind Code |
A1 |
Lim; Sai Kiang ; et
al. |
January 13, 2011 |
METHOD OF DERIVING PROGENITOR CELL LINE
Abstract
We disclose a method comprising: (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. Preferably, the method selects
against somatic cells based on their inability to self-renew.
Preferably, the progenitor cell line is derived or established in
the absence of co-culture, preferably in the absence of feeder
cells, which preferably selects against embryonic stem cells.
Optionally, the method comprises (d) deriving a differentiated cell
from the progenitor cell line.
Inventors: |
Lim; Sai Kiang; (Singapore,
SG) ; Lye; Elias; (Singapore, SG) |
Correspondence
Address: |
COHEN & GRIGSBY, P.C.
625 LIBERTY AVENUE
PITTSBURGH
PA
15222-3152
US
|
Assignee: |
AGENCY FOR SCIENCE, TECHNOLOGY AND
RESEARCH
Singapore
SG
|
Family ID: |
37075090 |
Appl. No.: |
12/888724 |
Filed: |
September 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12065549 |
Mar 3, 2008 |
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PCT/SG2006/000233 |
Aug 15, 2006 |
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12888724 |
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60713992 |
Sep 2, 2005 |
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Current U.S.
Class: |
424/93.7 ;
435/29; 435/325; 435/377 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 3/10 20180101; A61P 25/16 20180101; A61P 25/28 20180101; C12N
2502/1352 20130101; A61P 9/00 20180101; C12N 2506/02 20130101; C12Q
1/6809 20130101; C12N 2501/135 20130101; C12N 2501/115 20130101;
C12N 5/0662 20130101; A61P 35/00 20180101; G01N 33/5044 20130101;
A61P 35/02 20180101; C12N 5/0603 20130101; A61P 17/02 20180101;
C12N 2501/734 20130101 |
Class at
Publication: |
424/93.7 ;
435/377; 435/29; 435/325 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/00 20060101 C12N005/00; C12Q 1/02 20060101
C12Q001/02; A61P 25/28 20060101 A61P025/28; A61P 25/16 20060101
A61P025/16; A61P 35/00 20060101 A61P035/00; A61P 3/10 20060101
A61P003/10 |
Claims
1. A method of deriving a mesenchymal progenitor cell line from a
parental embryonic stem cell, wherein the mesenchymal progenitor
cell line comprises mesenchymal stem cells, the method comprising:
(a) providing the parental embryonic stem cell or descendents of
the parental embryonic stem cell obtained by dispersing an
embryonic stem cell colony with trypsin; (b) culturing the parental
embryonic stem cell in the absence of feeder cells in conditions
that promote growth of putative mesenchymal progenitor cells and at
least retard growth or propagation of embryonic stem cells; (c)
selecting mesenchymal progenitor cells which self-renew; and (d)
establishing in the absence of transformation a mesenchymal
progenitor cell line from the mesenchymal progenitor cells which
self-renew; wherein the mesenchymal progenitor cell line is lineage
restricted compared to the parental embryonic stem cell.
2. The method of claim 1, further comprising the step of deriving a
differentiated cell from the mesenchymal progenitor cell line.
3. The method of claim 1, further comprising (a) performing the
method in the presence of a candidate molecule; and (b) determining
an effect of the candidate molecule on the mesenchymal progenitor
cell line.
4. A method according to claim 1, further comprising the step of
using the progenitor cell line for the treatment of at least one of
a disease treatable by regenerative therapy, cardiac failure, bone
marrow disease, skin disease, burns, or a degenerative disease.
5. A differentiated cell produced by: (a) providing a parental
embryonic stem cell or descendents of the parental embryonic stem
cell obtained by dispersing an embryonic stem cell colony with
trypsin; (b) culturing the parental embryonic stem cell in the
absence of feeder cells in conditions that promote growth of
putative progenitor cells and at least retard growth or propagation
of embryonic stem cells; (c) selecting progenitor cells which
self-renew; (d) establishing in the absence of transformation a
progenitor cell line from the progenitor cells which self-renew,
and (e) deriving a differentiated cell from the progenitor cell
line, wherein the progenitor cell line is lineage restricted
compared to the parental embryonic stem cell.
6. The method according to claim 2, further comprising the step of
using the differentiated cell for the treatment of at least one of
a disease treatable by regenerative therapy, cardiac failure, bone
marrow disease, skin disease, burns, or a degenerative disease.
7. The method according to claim 1, further comprising the step of
using the mesenchymal progenitor cell line to prepare a
pharmaceutical composition for the treatment of at least one of a
disease treatable by regenerative therapy, cardiac failure, bone
marrow disease, skin disease, burns, or a degenerative disease.
8. The method according to claim 2, further comprising the step of
using the differentiated cell to prepare a pharmaceutical
composition for the treatment of at least one of a disease
treatable by regenerative therapy, cardiac failure, bone marrow
disease, skin disease, burns, or a degenerative disease.
9. The method according to claim 4, wherein the degenerative
disease is at least one of diabetes, Alzheimer's Disease,
Parkinson's Disease, or cancer.
10. The method according to claim 6, wherein the degenerative
disease is at least one of diabetes, Alzheimer's Disease,
Parkinson's Disease, or cancer.
11. The method according to claim 7, wherein the degenerative
disease is at least one of diabetes, Alzheimer's Disease,
Parkinson's Disease, or cancer.
12. The method according to claim 8, wherein the degenerative
disease is at least one of diabetes, Alzheimer's Disease,
Parkinson's Disease, or cancer.
Description
[0001] Reference is made to U.S. provisional application Ser. No.
60/713,992 filed 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 progenitor 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] This invention seeks to solve this and other problems with
methods in the art.
SUMMARY
[0007] According to a 1.sup.st aspect of the present invention, we
provide a method comprising: (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.
[0008] Preferably, the method selects against somatic cells based
on their inability to self-renew.
[0009] 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.
[0010] In preferred embodiments, the progenitor cell line is
established without transformation. Preferably, the progenitor cell
line is 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.
[0011] Preferably, the self-renewal-promoting conditions comprise
growth in rich media. More preferably, the self-renewal-promoting
conditions comprise growing cells in the absence of LIF.
[0012] Preferably, the self-renewal-promoting conditions comprise
serial passages. Preferably, the self-renewal promoting conditions
comprise at least 12 serial passages.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] Preferably, the embryonic stem cell or progenitor cell line
is a mammalian, preferably mouse or human, embryonic stem cell or
progenitor cell line.
[0021] 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.
[0022] In some embodiments, the method further comprises the step
of (d) deriving a differentiated cell from the progenitor cell
line.
[0023] Preferably, the progenitor cell line is propagated for at
least 5 generations prior to differentiation.
[0024] There is provided, according to a 2.sup.nd aspect of the
present invention, a method according to the 1.sup.st aspect of the
invention for generating a differentiated cell from an embryonic
stem (ES) cell.
[0025] Preferably, the differentiated cell is an endothelial cell
or a mesenchymal cell. More preferably, the differentiated cell is
an adipocyte or an osteocyte.
[0026] We provide, according to a 3.sup.rd aspect of the present
invention, a method according to the 1.sup.st or 2.sup.nd aspect of
the invention for up-regulating expression of mesenchymal or
endothelial markers of a cell.
[0027] As a 4.sup.th aspect of the present invention, there is
provided a method according to the 1.sup.st or 2.sup.nd aspect of
the invention for down-regulating expression of stem cell or
pluripotency markers of a cell.
[0028] We provide, according to a 5.sup.th aspect of the present
invention, a method of identifying an agent capable of promoting or
retarding self-renewal or differentiation of a stem cell, the
method comprising performing a method according to any preceding
aspect of the invention in the presence of a candidate molecule,
and determining an effect thereon.
[0029] The present invention, in a 6.sup.th aspect, provides 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.
[0030] In a 7.sup.th aspect of the present invention, there is
provided a progenitor cell line produced by a method according to
any preceding aspect of the invention.
[0031] According to an 8.sup.th aspect of the present invention, we
provide a differentiated cell produced by a method according to any
preceding aspect of the invention.
[0032] We provide, according to a 9.sup.th aspect of the invention,
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.
[0033] There is provided, in accordance with a 10.sup.th aspect of
the present invention, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1. Derivation of E-RoSH cell lines.
[0035] FIG. 1A. Embryonic stem cells are plated singly on
methycellulose based media to form embryoid bodies (EBs). At day
3-6, embryoid bodies are harvested, dissociated by collagenase and
cultured as a monolayer on gelatinized feeder plate. RoSH-like
colonies with adherent fibroblast-like cells and ring-like
structures are selected and propagated on gelatinized plates to
generate E-RoSH 1, 2, 3 . . . Each of the cultures are then plated
at a low density of 10-100 cells per 10 cm plate and single RoSH
like colonies are picked to established sublines, E-RoSH 2.1, 2.2,
2.3 . . . etc.
[0036] FIG. 1B. A putative RoSH-like colony consisting of adherent
short fibroblast-like cells with characteristic ring-like cells
(inset) expanding over time.
[0037] FIG. 1C. Morphological similarity between E-RoSH2.1 and
RoSH2 cells in sub-confluent cultures;
[0038] FIG. 1D. Alkaline phosphatase staining of E-RoSH2.1 and its
parental E14 embryonic stem cells;
[0039] FIG. 1E. Average chromosome number from 20 metaphase nuclei
in E-RoSH2.1 and 3.2 lines at passage 3 and 13;
[0040] FIG. 2. Relative gene expression analysis by quantitative
RT-PCR analysis. The expression level is normalized against that of
embryonic stem cells and expressed as a logarithmic function.
[0041] FIG. 2A. Gene expression profile of E-RoSH2.1 cells at three
different passages;
[0042] FIG. 2B. Comparative gene expression profiles in E-RoSH2.1,
E-RoSH3.2 and RoSH2 cells;
[0043] FIG. 2C and FIG. 2D Relative expression of genes associated
with pluripotency and endothelial potential in the parental E14
embryonic stem cells and E-RoSH2.1 cells as measured by
quantitative RT-PCR analysis.
[0044] FIG. 3. Characterization of E-RoSH cells.
[0045] FIG. 3A. In vitro differentiation of RoSH2.1 cells on
matrigel coated plate. In two weeks, RoSH2.1 cells differentiate to
form a network of tubular structures that covered the surface of
the entire tissue dish;
[0046] FIG. 3B. E-RoSH derived tubular structures have patent
lumens and endocytosed acetylated LDL. The structures are labeled
with CFDA, a cytoplasmic green fluorescent dye (Molecular Probe,
Eugene, Oreg.) and propidinm iodide, and viewed by confocal
microscopy (left panel). The tubular structures are incubated with
acetylated red fluoresecent diI-labelled LDL (Molecular Probe,
Eugene, Oreg.) for 24 hours and counterstained with SYTOX
Green.TM., a green fluorescent nuclear dye (Molecular Probe,
Eugene, Oreg.) before analysis by confocal microscopy;
[0047] FIG. 3C. Immunoreactivity for vWF on paraffin-embedded
sections of E-RoSH2.1 derived tubular structures are using
HRP-based detection system. Brown precipitates indicate positive
staining. The nuclei are stained with Mayer's hematoxylin.
[0048] FIG. 3D. Gene expression during endothelial differentiation
of E-RoSH2.1 cells as measured by quantitative RT-PCR analysis.
Relative gene expression is normalized against that at time 0 and
expressed as a logarithmic function.
[0049] FIG. 3E. Suspension cultures of embryonic stem cells and
E-RoSH at day 7
[0050] FIG. 3F. Quantitative RT-PCR profiling of gene expression by
embryonic stem cells and E-RoSH2.1 cells when cultured in
suspension cultures for 0, 2, 3 and 7 days. Relative gene
expression is normalized against that of embryonic stem cells at
time 0 and expressed as a logarithmic function.
[0051] FIG. 3G. In vivo differentiation. 1.times.10.sup.5 E-RoSH
cells labeled with Qdot.RTM. nanocrystals (655 nm emission) are
injected into a embryonic stem cell-derived teratoma that is
induced in SCID mice. Three days later, the mice are euthanized and
the tumors are removed. The tumors are fixed in 4% paraformaldehyde
and cryosectioned at 20 .mu.m thickness. The sections are assayed
for pecam-1 immunoreactivity using rat anit-pecam1 followed by
FITC-conjugated rabbit anti-rat antibody, and counterstained with
DAPI. The sections are viewed by light microscopy and then confocal
microscopy.
[0052] FIG. 4. Characterization of HuES9.E1 cells.
[0053] FIG. 4A. HuES9 colony grown on mitotically inactive MEFs
surrounded by proliferating fibrobastic stromal cells (arrow).
[0054] FIG. 4B. A representative confluent culture of HuES9.E1
MSC-like cells and BM-derived MSCs.
[0055] FIG. 4C. HuES9, a human embryonic stem cell line, E14, a
mouse embryonic stem cell line, mouse embryonic fibroblast (MEF)
and HuES9.E1 mesenchymal stem cell (MSC)-like cells are stained for
the presence of allcaline phosphatase activity.
[0056] FIG. 4D. HuES9 and HuES9.E1 MSC-like cells are tested for
the expression of Pou5f1 by quantitative RT-PCR analysis using
TaqMan.RTM. primers. Pou5f1 transcript level in HuES9 human
embryonic stem cell normalized to one.
[0057] FIG. 4E. Genomic PCR analysis for the presence of
human-specific Alu repeat sequence and mouse-specific c-mos repeat
sequences in HuES9.E1 MSC-like cells.
[0058] FIG. 4F. Karyotype analysis of HuES9.E1 at passage 4 and
passage 8.
[0059] FIG. 4G. Profile of surface antigens by FACS analysis.
HuES9.E1 cells are tested for immunoreactivity against CD29, CD44,
CD105, CD166, CD34 and CD45.
[0060] FIG. 4H. Differentiation of HuES9.E1 into adipocytes and
osteocytes. Confluent HuES9.E1 cells are cultured in standard
culture media for inducing adiogenesis or osteogenesis. After 12
days, cells that are induced to undergo adiogenesis are stained for
oil droplets by oil red and analyzed for the expression of
PPAR.gamma. by quantitative RT-PCR (top panel) while those that are
induced to undergo osteogenesis are stained for calcium deposits by
von Kossa staining and analyzed for the expression of bone-specific
alkaline phosphatase, ALP by quantitative RT-PCR (bottom
panel).
DETAILED DESCRIPTION
[0061] We demonstrate that it is possible to derive progenitor cell
lines from embryonic stem cells (ES), based on the ability of
progenitor cells to self-renew. Unlike terminally differentiating
cells, putative progenitor cells with self-renewing properties can
be selected and propagated without transformation.
[0062] Our methods therefore generally involve deriving progenitor
cell lines of limited potential from embryonic stem cells by
culturing the pluripotent cells in vitro. This enables the
expansion of a progenitor cell with a highly restricted
differentiation potential that, upon differentiation, will generate
a highly enriched population of a specific cell type with reduced
or abolished tumorigenic potential.
[0063] We therefore use this property of self-renewal of progenitor
cells as the underlying principle of self-selection for generating
lineage restricted progenitor cell lines from embryonic stem
cells.
[0064] Absence of Co-Culture
[0065] In preferred embodiments, however, the method further
includes culture of cells in conditions that promote growth of
progenitor cells, and optionally retard or prevent growth or
propagation of embryonic stem cells.
[0066] Thus, in highly preferred embodiments, our methods involve
culturing putative progenitor cells in the absence of co-culture,
as a monolayer or in the absence of feeder cells. 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.
According to preferred embodiments of the methods described here,
the embryonic stem cells are cultured in the absence of feeder
cells to establish a progenitor cell line.
[0067] Biasing Differentiation
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] Endodermal Progenitor Cells
[0073] 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.
[0074] Hematopoietic and Endothelial Progenitor Cells
[0075] 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.
[0076] Cardiac Mesoderm and Skeletal Myoblast Progenitor Cells
[0077] 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.
[0078] 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.
[0079] Formation of Embryoid Bodies
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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).
[0084] Rich Media
[0085] 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.
[0086] 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).
[0087] According to such embodiments, continued propagation will
selectively enrich for progenitor cells which can then be
cloned.
[0088] Long-Term Maintenance in Culture
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] Characteristics of Progenitor Cells
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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, BC052653), 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, BC039220), c-kit (GenBank Accession nos: NM.sub.--021099,
Y00864, AY536430, BC075716, AK047010, BC026713, BC052457,
AK046795), PDGFR.alpha. (NM.sub.--011058, M57683, M84607,
BC053036), Oct4 (GenBank Accession nos: NM.sub.--013633, X52437,
M34381, BC068268), Runx1 (GenBannk 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:
N.sub.--011562, M87321) and Tie-2 (GenBank Accession nos:
NM.sub.--013690, X67553, X71426, D13738, BC050824).
[0099] 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.
[0100] Regulators of Self-Renewal and Differentiation
[0101] 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.
[0102] 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.
[0103] 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.
[0104] The methods described here may be combined with decreasing
the activity of mTOR to promote differentiation, as described in
U.S. Ser. No. 60/609,216, herein incorporated by reference.
Progenitor Cells and Stem Cells
[0105] The methods described here are capable of producing
progenitor cells, and cell lines thereof.
[0106] When embryonic stem cells differentiate, they generally
recapitulate the complexity of early mammalian development where
embryonic stem cells transit through a series of lineage
restriction to generate progenitor cells of decreasing lineage
potential before finally generating terminally differentiated cells
representing all three germ layers (Wiles, Methods in Enzymology.
1993; 225:900-918). This is exemplified by the process of
hematopoiesis, where increasingly lineage-restricted hematopoietic
progenitors appearing in a sequential manner similar to that found
within the mouse embryo, can be identified within embryoid bodies
(Choi et al, Methods Mol Med. 2005; 105:359-368).
[0107] Typically, stem cells generate an intermediate cell type or
types before they achieve their fully differentiated state,
referred to as a precursor or progenitor cell. Progenitor or
precursor cells in foetal or adult tissues are partly
differentiated cells that divide and give rise to differentiated
cells. Such cells are usually regarded as "committed" to
differentiating along a particular cellular development pathway,
Progenitor cells are therefore sometimes referred to as "committed
stem cells".
[0108] Our methods are capable of producing of progenitor cells and
cell lines of various types.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
Differentiated Cells
[0117] Differentiated cells, such as terminally differentiated
cells, may be derived from the progenitor cells or cell lines made
according to the methods described. We therefore disclose methods
for generating differentiated cells, the methods comprising
generating progenitor cells or cell lines as described, and
deriving differentiated cells from these.
[0118] Differentiated cells which may be made according to the
methods described here may include any or all of the following:
[0119] 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
[0120] ii) cardiomyocytes: the functional muscle cell type of the
heart that allows it to beat continuously and rhythmically
[0121] 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
[0122] 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.
[0123] 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
[0124] 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.
[0125] vii) myocyte: the functional cell type of muscles
[0126] viii) neuron: the functional cell type of the brain that is
specialized in conducting impulses
[0127] ix) osteoblast: the functional cell type responsible for
making bone
[0128] 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 Progenitor Cells and Differentiated Cells
[0129] Progenitor cell lines 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.
[0130] For example, populations of undifferentiated 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.
[0131] These and other uses of progenitor cell lines and
differentiated cells are described in further detail below, and
elsewhere in this document. The progenitor cell lines 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.
[0132] Drug Screening
[0133] Progenitor cell lines 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 differentiated cells.
[0134] In some applications, progenitor cell lines 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 progenitor 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.
[0135] Furthermore, gene expression profiling of progenitor cell
lines 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 progenitor cell lines and differentiated cells.
[0136] 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.
[0137] 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.
[0138] Tissue Regeneration
[0139] Progenitor cell lines 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.
[0140] For example, the methods and compositions described here may
be used to modulate the differentiation of stem cells. Progenitor
cell lines 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.
[0141] In another example, neural progenitor cells are transplanted
directly into parenchymal or intrathecal sites of the central
nervous system, according to the disease being treated. Grafts are
done using single cell suspension or small aggregates at a density
of 25,000-500,000 cells per .mu.L (U.S. Pat. No. 5,968,829). The
efficacy of neural cell transplants can be assessed in a rat model
for acutely injured spinal cord as described by McDonald et al.
(Nat. Med. 5:1410, 1999. A successful transplant will show
transplant-derived cells present in the lesion 2-5 weeks later,
differentiated into astrocytes, oligodendrocytes, and/or neurons,
and migrating along the cord from the lesioned end, and an
improvement in gate, coordination, and weight-bearing.
[0142] Certain neural progenitor cells are designed for treatment
of acute or chronic damage to the nervous system. For example,
excitotoxicity has been implicated in a variety of conditions
including epilepsy, stroke, ischemia, Huntington's disease,
Parkinson's disease and Alzheimer's disease. Certain differentiated
cells as made according to the methods described here may also be
appropriate for treating dysmyelinating disorders, such as
Pelizaeus-Merzbacher disease, multiple sclerosis, leukodystrophies,
neuritis and neuropathies. Appropriate for these purposes are cell
cultures enriched in oligodendrocytes or oligodendrocyte precursors
to promote remyelination.
[0143] Hepatocytes and hepatocyte precursors prepared using our
methods can be assessed in animal models for ability to repair
liver damage. One such example is damage caused by intraperitoneal
injection of D-galactosamine (Dabeva et al., Am. J. Pathol.
143:1606, 1993). Efficacy of treatment can be determined by
immunohistochemical staining for liver cell markers, microscopic
determination of whether canalicular structures form in growing
tissue, and the ability of the treatment to restore synthesis of
liver-specific proteins. Liver cells can be used in therapy by
direct administration, or as part of a bioassist device that
provides temporary liver function while the subject's liver tissue
regenerates itself following fulminant hepatic failure.
[0144] The efficacy of cardiomyocytes prepared according to the
methods described here can be assessed in animal models for cardiac
cryoinjury, which causes 55% of the left ventricular wall tissue to
become scar tissue without treatment (Li et al., Ann. Thorac. Surg.
62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai
et al., J. Thorac. Cardiovasc. Surg. 118:715, 1999). Successful
treatment will reduce the area of the scar, limit scar expansion,
and improve heart function as determined by systolic, diastolic,
and developed pressure. Cardiac injury can also be modeled using an
embolization coil in the distal portion of the left anterior
descending artery (Watanabe et al., Cell Transplant. 7:239, 1998),
and efficacy of treatment can be evaluated by histology and cardiac
function. Cardiomyocyte preparations can be used in therapy to
regenerate cardiac muscle and treat insufficient cardiac function
(U.S. Pat. No. 5,919,449 and WO 99/03973).
[0145] Cancer
[0146] Progenitor cell lines and differentiated cells made by the
methods and compositions described here may be used for the
treatment of cancer.
[0147] 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.
[0148] 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.
[0149] In preferred embodiments, the progenitor cell lines and
differentiated cells made according to the methods and compositions
described here are used to treat T cell lymphoma, melanoma or lung
cancer.
[0150] The progenitor cell lines 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.
[0151] 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
[0152] 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.
[0153] Stem cells as referred to in this document may include
totipotent stem cells, pluripotent stem cells, and multipotent stem
cells.
[0154] Totipotent Stem Cells
[0155] 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.
[0156] Pluripotent Stem Cells
[0157] "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.
[0158] Embryonic Stem Cells
[0159] 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.
[0160] Embryonic Germ Cells
[0161] Embryonic Germ (EG) cells may be isolated from the precursor
to the gonads in aborted fetuses.
[0162] Embryonic Carcinoma Cells
[0163] 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.
[0164] Adult Stem Cells
[0165] 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.
[0166] Multipotent Stem Cells
[0167] 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.
[0168] 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.
[0169] 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
[0170] 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 progenitor cells, progenitor cell
lines and differentiated cells.
[0171] 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.
[0172] 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).
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] Media and Feeder Cells
[0178] Media for isolating and propagating pPS cells can have any
of several different formulas, as long as the cells obtained have
the desired characteristics, and can be propagated further.
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).
[0179] 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.
[0180] 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.
[0181] Embryonic Stem Cells
[0182] 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.
[0183] 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 Softer
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.
[0184] 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.
[0185] Embryonic Germ Cells
[0186] 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.
[0187] 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.
[0188] 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
[0189] Self-Renewal
[0190] Stem cells which are self-renewing may be identified by
various means known in the art, for example, morphology,
immunohistochemistry, molecular biology, etc.
[0191] 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.
[0192] 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 discernable 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.
[0193] 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.
[0194] 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 Lines from
Human Gem 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.).
[0195] 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.
[0196] Differentiation
[0197] Differentiating cells, including progenitor cell lines 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 lenghtened cell cycle compared
to stem cells which are not self-renewing.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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, glucolcinase, insulin growth factors 1 and
2, IGF-1 receptor, insulin receptor, and leptin. Markers dependent
on HNF-4 alpha expression include albumin, apoAI, apoAII, apoB,
apoCIII, apoCII, aldolase B, phenylalanine hydroxylase, L-type
fatty acid binding protein, transferrin, retinol binding protein,
and erythropoietin (EPO).
[0202] 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.
[0203] 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 (URL www.ncbi.nlm.nih.gov:80/entrez).
EXAMPLES
Example 1
Methods: Derivation of E-RoSH Cell Lines
[0204] Embryonic stem cells (ESCs) are induced to differentiate to
form embryoid bodies (Ebs) using the methycellulose-based approach
described in Lim et al, Blood. 1997; 90:1291-1299)
[0205] Day 3 to day 6 embryoid bodies are harvested, dissociated
into single cell suspensions by collagenase digestion (Robertson E
J. Embryo-derived stem cell lines. In: Robertson E J, ed.
Teratocarcinomas and embryonic stem cells: a practical approach.
Oxford: IRL Press Limited; 1987:71-112) and plated on at a density
of 1-5.times.10.sup.5 cells per 10 cm feeder plate. After about a
week, the cells proliferated and differentiated into a complex
mixture of cell types.
[0206] Colonies of rapidly dividing cells resembling embryo-derived
RoSH cells are picked and expanded sequentially to a 48-well plate,
24-well plate, 6-well plate and then a 10 cm plate. The culture
from each colony is named E-RoSH1, 2, 3 . . . in the sequence in
which each culture is established.
[0207] Each of these cell cultures are then replated at 10-100
cells per 10 cm plate. Colonies are then selected and expanded to
establish sublines that are named based on their parental lines
e.g. E-RoSH1.1, 1.2, 1.3, etc. For suspension cultures,
1.times.10.sup.6 cells are plated on 10 cm bacterial Petri dishes
that are placed on an orbital shaker.
[0208] Alkaline phosphatase assay, and MTT assays are performed
using assay kits from Chemicon (Temecula, Calif.) and Bioassay
Systems (Hayward, Calif.). Chromosomes counting is performed as
previously described (Robertson, supra).
Example 2
Methods: Derivation of HuES9.E Mesenchymal Stem Cell (MSC)-Like
Cell Lines
[0209] HuES9 cells are cultured as previously described in Cowan et
al, N Engl J Med. 2004; 350:1353-1356.
[0210] To derive HuES9.E MSC-like cells, HuES9 cells are split 1:4
onto gelatinized feeder-free plates in using HuES9 culture media.
Confluent cultures are typsinized and split 1:4. Differentiation
into adipocytes, and osteocytes is performed as previously
described (Barberi et al, PLoS Med. 2005; 2:e161).
[0211] BM MSCs are prepared as previously described in Pittenger et
al, Science. 1999; 284:143-147.
[0212] Genomic PCR for mouse- and human-specific repeat sequences
are performed as previously described in Que et al, In Vitro Cell
Dev Biol Anim. 2004; 40:143-149.
Example 3
Methods: RT-PCR Analysis
[0213] Total RNA is prepared using standard protocols and are
quantified using, respectively, the RiboGreen RNA Quantification
kit and the PicoGreen dsDNA Quantification kit (Molecular Probes,
Eugene, Oreg.).
[0214] Quantitative RT-PCR is performed using TaqMan.RTM. primers
(Applied Biosystems, Foster City, Calif.).
Example 4
Methods: In vitro Endothelial Differentiation
[0215] Endothelial differentiation of E-RoSH cells and acetylated
LDL uptake by differentiated E-RoSH cells are performed as
previously described (Yin et al, Arterioscler Thromb Vasc Biol.
2004; 24:691-696)
[0216] In vitro differentiated E-RoSH vascular structures are fixed
in formalin, embedded in paraffin, sectioned at 4 .mu.m and stained
for vWF using polyclonal, rabbit-generated antibody and Envision+
System-peroxidase (DakoCytomation, Gostrup, Denmark). The sections
are counterstained with Mayer's hematoxylin.
Example 5
Methods: In vivo Endothelial Differentiation
[0217] 1.times.10.sup.6 embryonic stem cells are transplanted
subcutaneously into SCID mice. At three weeks when embryonic stem
cell-derived tumors are about 1 cm in diameter, 1.times.10.sup.5
E-RoSH cells labeled with Qdot.RTM. nanocrystals (655 nm emission)
using a Qtracker.RTM. Cell Labeling Kit (Quantum Dot Corp, Hayward,
Calif.) are injected into the embryonic stem cell-derived
teratoma.
[0218] Three days later, the mice are euthanized with an overdose
of anesthesia and the tumors are removed. The tumors are fixed in
4% paraformaldehyde and cryosectioned at 20 .mu.m thickness. The
sections are assayed for pecam-1 immunoreactivity using rat
anit-pecam1 (Pharmingen, San Diego, Calif.) followed by
FITC-conjugated rabbit anti-rat antibody (Chemicon, Temecula,
Calif.), and counterstained with DAPI. The sections are analyzed by
confocal microscopy.
Example 6
Derivation of Lineage-Restricted Endothelial Progenitor Cell Lines
from Mouse Embryonic Stem Cells (mESCs)
[0219] To derive endothelial progenitor cell lines from mouse
embryonic stem cells (mESCs), we relied on our previous experience
of deriving RoSH endothelial progenitor cell lines from 5.5 dpc
delayed blastocysts and early post-implantation mouse embryos (Yin
et al, Arterioscler Thromb Vase Biol. 2004; 24:691-696).
[0220] We rationalized that since differentiation of embryonic stem
cells (ESCs) into embryoid bodies (EBs) recapitulates some of early
events in mammalian development, 3 to 6 days old embryoid bodies
that are developmentally analogous to 5.5 dpc delayed blastocysts
and early post-implantation embryos, will be enriched for cells
that gave rise to RoSH progenitor cells. Therefore,
[0221] 3 to 6 day old embryoid bodies are generated using a
semi-solid, methycellulose-based media (Lim et al, Blood. 1997;
90:1291-1299), dissociated into cell suspensions by collagenase
digestion to disrupt the differentiating microenvironment of the
embryoid bodies, and plated on gelatinized tissue culture plate at
a density of 1-5.times.10.sup.5 cells per 10 cm plate in embryonic
stem (ES) media without LIF supplementation to discourage
propagation of mouse embryonic stem cells (FIG. 1A).
[0222] Propagation of dissociated cells is enhanced if they are
plated on embryonic fibroblast feeder as previously noted for the
derivation of RoSH progenitor cells (Yin et al, Arterioscler Thromb
Vasc Biol. 2004; 24:691-696) but this tended to encourage growth of
embryonic stem cells. After about a week, most of the cells
differentiated into a heterogenous cell culture.
[0223] The cultures are then screened for RoSH-like colonies of
rapidly dividing cells with large nucleus to cytoplasm ratio and
ring-like cells that are immunoreactive for von Willebrand Factor
(or vWF) (Yin et al, Arterioscler Thromb Vasc Biol. 2004;
24:691-696) (FIG. 1B).
[0224] Only colonies that maintained a steady rate of proliferation
and a stable morphology are selected when they reached a size of
2-300 cells, and expanded on either embryonic fibroblast feeder or
gelatin-coated plates to generate lines, E-RoSH 1, 2, . . .
etc.
[0225] Each of these lines is then subcloned by plating the cells
at a low density of 10-100 cells per 10 cm plate, and colonies are
then picked to derive sublines E-RoSH1.1, 1.2 etc. Alternatively,
it is possible to enrich for self-renewing RoSH-like cells by
passaging at 1:4 about two or three times before cloning by plating
at low density. The most efficient yield of about one RoSH-like
colony per 1-5.times.10.sup.5 embryoid body cells is dependant on
the age of embryoid bodies and the parental embryonic stem lines.
For example, D3 to D5 embryoid bodies derived from the E14
embryonic stem cell line and D6 embryoid bodies derived from the
CSL3 embryonic stem cell line (Bourc'his et al, Science. 2001;
294:2536-2539) are most efficient for derivation of RoSH-like
lines.
[0226] On the other hand, derivation of RoSH-like lines from
differentiating embryonic stem cells grown in the absence of LIF or
other developmental stages of embryoid bodies is possible but much
less efficient. We have established nine independently derived
lines, five from CSL3 embryonic stem cell line and four from E14
embryonic stem cell line.
Example 7
Characterisation of E-RoSH Endothelial Progenitor Cells
[0227] E-RoSH cells, as typified by E-RoSH2.1, are morphologically
similar to embryo-derived RoSH cell lines (FIG. 1C), and unlike
their parental embryonic stem cell lines, do not have detectable
alkaline phosphatase activity (FIG. 1D).
[0228] Population doubling time is estimated be .about.15 hours by
MTT assay (data not shown). E-RoSH cells have been maintained in
continuous culture for >40 generations by passaging every two
days at 1:4 to 1:5 split (data not shown).
[0229] The karyotype of E-RoSH 2.1 and 3.2 as monitored by
chromosome number, is stable for at least 10 passages with a normal
mean chromosome number of 40 (FIG. 1E).
[0230] Gene expression in E-RoSH2.1 is monitored by quantitative
RT-PCR analysis of 15 genes and shown to be stable at different
passages (FIG. 2A). In addition, this gene expression profile is
similar to that in other independently derived E-RoSH lines as well
as the mouse embryo-derived RoSH lines (FIG. 2B).
[0231] Subcutaneous transplantation of E-RoSH cells into SCID or
Rag1 -/- immunodeficient mice did not induce teratoma formation
during a two to nine-months' observation period while similar
transplantation of the parental embryonic stem cells will
invariably generate a 2 cm teratoma within three weeks, suggesting
a loss of pluripotency in E-RoSH cells (data not shown).
[0232] This loss of pluripotency is further evidenced by reduced
expression of several genes associated with pluripotent cells such
as BMP4, FGF5, Oct4, Sox-2 and Utf1 (FIG. 2C) (Wei D, Xu G L, Lin C
S, Bollman B, Bestor T H. Dnmt3L and the establishment of maternal
genomic imprints. Stem Cells. 2005; 23:166485; Rao M. Dev Biol.
2004; 275:269-286).
[0233] In contrast, a set of genes consisting of runx-1, flk-1,
PDGFR.alpha., Tie-2, and c-kit whose expression is commonly
associated with endothelial progenitor cells (Jaffredo et al, Int J
Dev Biol. 2005; 49:269-277), is highly expressed (FIG. 2D).
[0234] Together these observations demonstrate that E-RoSH cells
are embryonic stem cell-derivatives that are no longer pluripotent
and have a restricted differentiation potential that is likely to
include endothelial potential.
Example 8
Differentiation of E-RoSH Cells into Endothelial Cells in vitro and
in vivo
[0235] To confirm their endothelial potential, E-RoSH cells are
plated on matrigel.
[0236] Within two weeks, the cells formed a network of
vascular-like tubules that covered the entire tissue culture dish
(FIG. 3A). These tubules are patent and cells lining the lumen
endocytosed acetylated LDL (FIG. 3B) and are immunoreactive for vWF
(FIG. 3C).
[0237] Expression of endothelial genes such as Tie-2 is also
increased (FIG. 3D).
[0238] When grown in suspension, E-RoSH cells, like embryonic stem
cells, formed spherical bodies. However, unlike the tightly packed
embryonic stem cell-derived embryoid bodies, E-RoSH-derived bodies
are morphologically distinct with a hollow center (FIG. 3E),
providing another distinguishing difference between embryonic stem
cells and E-RoSH cells.
[0239] Genes whose expressions are associated with early lineage
commitment during embryonic development, are significantly reduced
during formation of E-RoSH-derived bodies in comparison to that
during embryoid body formation. These genes include cerberus
(cer-1) which is expressed during early gastrulation (De Robertis
et al, Int J Dev Biol. 2001; 45:189-197), mix11 which is important
for axial mesendoderm morphogenesis and patterning (Hart et al,
Development. 2002; 129:3597-3608; Mohn et al, Dev Dyn. 2003;
226:446-459) and PCSK1 which is expressed in neuroendocrine tissues
(Seidah et al, Mol Endocrinol. 1991; 5:111-122; Benjannet et al,
Proc Natl Acad Sci USA. 1991; 88:3564-3568) (FIG. 3F).
[0240] The relatively low expression levels of these genes are
consistent with the reduced potency of E-RoSH, and suggest that
E-RoSH cells no longer have the capacity of embryonic stem cells to
differentiate into a wide repertoire of cell types from all three
germ layers.
[0241] In contrast, a ten-fold increase in the expression of
endothelial genes such as c-kit and Tie-2 (FIG. 3F), suggests that
E-RoSH cells preferentially differentiate into endothelial cells
with a ten-fold efficiency over its parental embryonic stem cells.
Although the gene expression of E-RoSH cells suggested that they
have the potential to differentiate into hematopoietic cells, we
have not been able to induce robust hematopoietic differentiation
of these cells using standard hematopoietic'differentiation
assays.
[0242] When E-RoSH cells are labeled with Q-tracker, a long term,
cell-permeable fluorescent cell label, and transplanted into a
parental embryonic stem cell-derived teratoma that will provide a
suitable microenvironment for differentiating E-RoSH cells, E-RoSH
cells are found to be incorporated into the capillary plexus in the
teratoma and are immunoreactive for pecam-1 (FIG. 3G). Many of the
transplanted cells that are not incorporated in the tumor
vasculature are not immunoreactive for pecam-1 (data not
shown).
Example 9
Derivation of Lineage-Restricted Progenitor Cell Lines from Human
Embryonic Stem (hES) Cell Lines
[0243] To illustrate the general applicability of our approach, we
derived MSC-like lines from huES9 a human embryonic stem cells line
(Cowan et al, N Engl J Med. 2004; 350:1353-1356).
[0244] We observed that in most human embryonic stem cell cultures,
human embryonic stem cell colonies grow in a state of equilibrium
with proliferating stromal fibroblastic cells (FIG. 4A) suggesting
that they are human embryonic stem cell-derived progenitor cells.
To encourage the propagation of these cells and discourage that of
human embryonic stem cells, huES9 cells are cultured and passaged
in the absence of feeder. A homogenous culture of fibroblast-like
cells that are morphologically similar to bone marrow derived MSC
(BM-MSC) cultures is generated (FIG. 4B).
[0245] Two polyclonal lines, named huES9.E1 and huES9.E3, are
independently generated. Unlike its parental huES9 cells, huES9.E1
did not have detectable alkaline phosphatase activity (FIG. 4C) or
express OCT4 (FIG. 4D).
[0246] As our previous experience suggests that fusion between
putative stem cell and feeder cell occurs to generate self-renewing
cells (Que et al, In Vitro Cell Dev Biol Anim. 2004; 40:143-149),
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. 4E).
[0247] The cells have 46, XX, chromosomes like its parental huES9
line (Cowan et al, N Engl I Med. 2004; 350:1353-1356) (FIG.
4F).
[0248] When grown in media supplemented with serum replacement
media, the population doubling time is 4-5 days, and in media
supplemented with 10% fetal calf serum, the population doubling is
about 36 hours.
[0249] Based on the close resemblance of huES9.E1 and huES9.E3 to
BM-MSC, we further approximated the lineage potential of huES9.E1
by comparing its surface antigen profile to that of BM-MSCs. These
cells exhibited typical MSC surface markers, CD29+, CD44+, CD105+
and CD166+ (Barry and Murphy, Int I Biochem Cell Biol. 2004;
36:568-584) and did not express CD34 and CD45 (FIG. 4F).
[0250] HuES9.E1 cells can be induced to differentiate into
adipocytes and osteocytes using standard differentiation conditions
(Barberi T et al, PLoS Med. 2005;2:e161). Adipocytic
differentiation is confirmed by the presence of oil droplets in the
differentiated cells and expression of PPAR.gamma. mRNA (FIG. 4G)
while osteogenesis is determined by von Kossa staining for calcium
deposits in the matrix and expression of bone-specific alkaline
phosphatase, Alp1 (FIG. 4H).
Example 9
Discusssion
[0251] In summary, our study provides proof that lineage-restricted
embryonic stem cell-derived progenitor cell lines can be
established using the principle that progenitor cells with their
unique ability to self-renew, can be propagated without
transformation. They can be distinguished from terminally
differentiating cells by their steady rate of proliferation without
senescence.
[0252] Based on this distinguishing feature, these progenitor cells
can be isolated by plating the differentiating cultures at low
density to select for steadily proliferating colonies or by
continual passaging of the culture to select for proliferating
cells while terminally differentiating cells will senesce and will
be lost from the cultures. One requirement is that the culture
media does not promote propagation of the parental embryonic stem
cells.
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[0274] 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.
[0275] 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.
* * * * *
References