U.S. patent application number 14/746599 was filed with the patent office on 2015-10-08 for differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells.
The applicant listed for this patent is Asterias Biotherapeutics, Inc.. Invention is credited to Joseph D. Gold, Mohammad Hassanipour.
Application Number | 20150284684 14/746599 |
Document ID | / |
Family ID | 37595761 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150284684 |
Kind Code |
A1 |
Gold; Joseph D. ; et
al. |
October 8, 2015 |
DIFFERENTIATION OF PRIMATE PLURIPOTENT STEM CELLS TO
CARDIOMYOCYTE-LINEAGE CELLS
Abstract
The present application describes the new methods for the
differentiation of primate pluripotent stem cells into
cardiomyocyte-lineage cells. The methods utilize sequential
culturing of the primate pluripotent stem cells in certain growth
factors to produce cardiomyocyte-lineage cells. In certain
embodiments of the invention, the population of cells produced by
the sequential culturing is further enriched for
cardiomyocyte-lineage cells so as to produce a higher percentage of
those cells.
Inventors: |
Gold; Joseph D.; (San
Francisco, CA) ; Hassanipour; Mohammad; (Danville,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Asterias Biotherapeutics, Inc. |
Menlo Park |
CA |
US |
|
|
Family ID: |
37595761 |
Appl. No.: |
14/746599 |
Filed: |
June 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11471916 |
Jun 20, 2006 |
9062289 |
|
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14746599 |
|
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60693141 |
Jun 22, 2005 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 5/0657 20130101;
C12N 2501/105 20130101; C12N 2501/16 20130101; C12N 2533/54
20130101; C12N 2506/02 20130101; C12N 2501/155 20130101; C12N
2500/90 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2006 |
US |
PCT/US2006/024060 |
Claims
1. A method for obtaining cardiomyocyte-lineage cells from human
embryonic stem (hES) cells, comprising a) culturing the hES cells
in the presence of Activin A in the absence of a BMP; and b)
subsequently culturing the cells in the presence of a BMP.
2. The method of claim 1, wherein the BMP is BMP-2, BMP-4, or
BMP-7.
3. The method of claim 2, wherein the BMP is BMP-2.
4. The method of claim 2, wherein the BMP is BMP-4.
5. The method of claim 2, wherein the BMP is BMP-7.
6. The method of claim 1, wherein the culturing in the presence of
a BMP is done in the absence of an Activin.
7. The method of claim 1, wherein the hES cells are cultured in the
presence of Activin A for about one day and then subsequently
cultured in the presence of the BMP for about four days.
8. The method of claim 1, further comprising subsequent to the BMP
culturing step a culturing step in which the medium does not
contain Activin A or a BMP.
9. The method of claim 8, wherein the further culturing step is
performed for at least one week.
10. The method of claim 8, wherein the further culturing step is
performed for at least two weeks.
11. The method of claim 8, wherein the further culturing step is
done in medium containing IGF-I or IGF-II.
12. The method of claim 11, wherein the further culturing step is
done in medium containing IGF-I.
13. The method of claim 1, wherein the method does not involve a
step in which embryoid bodies are formed.
14. The method of claim 1, wherein the method further comprises
harvesting cells from the culture subsequent to the BMP culturing
step and enriching the harvested cell population for
cardiomyocyte-lineage cells.
15. The method of claim 14, wherein the harvested cell population
is enriched by Percoll gradient.
16. The method of claim 14, wherein the enrichment involves the
formation of cardiac bodies.
17. A method of obtaining an enriched population of
cardiomyocyte-lineage cells from hES cells, comprising in the
following order: a) culturing the hES cells in a serum-free medium
in the presence of Activin A in the absence of a BMP for about one
day; b) subsequently culturing in a serum-free medium in the
presence of a BMP in the absence of an Activin for about four days;
c) harvesting cells from the culture; and d) enriching the
harvested cell population for cardiomyocyte-lineage cells.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
60/693,141, filed Jun. 22, 2005.
FIELD OF THE INVENTION
[0002] This invention relates to the field of in-vitro
differentiation of primate pluripotent stem cells into
cardiomyocyte-lineage cells.
BACKGROUND
[0003] A central challenge for research in regenerative medicine is
to develop cell compositions that can help reconstitute cardiac
function. It is estimated that nearly one in five men and women
have some form of cardiovascular disease (National Health and
Nutrition Examination Survey III, 1988-94, Center of Disease
Control and the American Heart Association). Widespread conditions
include coronary heart disease (5% of the population), congenital
cardiovascular defects (0.5%), and congestive heart failure (3%).
The pharmaceutical arts have produced small molecule drugs and
biological compounds that can help limit the damage that occurs as
a result of heart disease, but there is nothing commercially
available to help regenerate the damaged tissue.
[0004] With the objective of developing a cell population capable
of cardiac regeneration, research has been conducted on several
different fronts. Clinical trials are underway at several centers
to test the use of autologous bone marrow derived cells for therapy
after myocardial infarction (Perin et al., Circulation 107:2294,
2003; Strauer et al., Circulation 106:1913, 2002; Zeiher et al.,
Circulation 106:3009, 2002; Tse et al., Lancet 361:47, 2003; Stamm
et al., Lancet 3661:45, 2003). It has been hypothesized that the
cells may have a cleansing function to improve blood perfusion of
the heart tissue. Clinical trials are also underway to test the use
of autologous skeletal muscle myoblasts for heart therapy (Menasche
et al., J. Am. Coll. Cardiol. 41:1078, 2003; Pagani et al., J. Am.
Coll. Cardiol. 41:879, 2003; Hagege et al., Lancet 361:491, 2003).
However, it is unclear if the contraction of striatal muscle cells
can coordinate adequately with cardiac rhythm.
[0005] A more direct approach would be to use cells that are
already committed to be functional cardiomyocytes. Syngeneic
neonatal or postnatal cardiac cells have been used in animal models
to repair damage resulting from permanent coronary occlusion
(Reffelmann et al., J. Mol. Cell Cardiol. 35:607, 2003; Yao et al.,
J. Molec. Cell. Cardiol. 35:607, 2003). Accordingly, if such cells
were available for human therapy, they could be very effective for
the treatment of ischemic heart disease. In addition, cardiomyocyte
cells can be used for screening compounds such as
pharmaceuticals.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides methods of obtaining
cardiomyocyte-lineage cells from primate pluripotent stem cells.
Cardiomyocyte-lineage cells have many possible uses, including, but
not limited to, screening of potential pharmaceuticals, screening
for cytotoxic chemicals, and therapeutic applications such as in
vivo repair of damaged or diseased hearts.
[0007] In certain embodiments of the invention, the methods of
obtaining cardiomyocyte-lineage cells from primate pluripotent stem
cells comprise in the following order: culturing the primate
pluripotent stem cells in the presence of an Activin but in the
absence of a BMP; subsequently culturing the cells in the presence
of a BMP; and harvesting the resulting harvested cells from the
culture.
[0008] The present invention also provides methods of obtaining
enriched populations of cardiomyocyte-lineage cells. In certain
embodiments, those methods comprise in the following order:
culturing the primate pluripotent stem cells in the presence of an
Activin but in the absence of a BMP; subsequently culturing the
cells in the presence of a BMP; harvesting the cells from the
culture; and enriching the harvested cell population for
cardiomyocyte-lineage cells. In certain embodiments, those methods
comprise in the following order: culturing the primate pluripotent
stem cells in a serum-free medium in the presence of Activin A but
in the absence of a BMP for about one day; subsequently culturing
the cells in a serum-free medium in the presence of BMP-4 or BMP-2
in the absence of an Activin for about four days; harvesting the
cells from the culture; and enriching the harvested cell population
for cardiomyocyte-lineage cells.
[0009] In certain embodiments of the invention, the cells are
attached to a solid surface during the culturing steps. In certain
embodiments, the cells are allowed to form an embryoid body during
the culturing step with the BMP. In certain embodiments, the cells
are cultured in a single-cell suspension during the Activin and/or
BMP culture steps.
[0010] In certain embodiments, the cells are cultured for one day
or more in the presence of the Activin. In certain embodiments, the
cells are cultured for four days or more in the presence of the
BMP. In certain embodiments, the Activin is Activin A. In certain
embodiments, the BMP is BMP-4 or BMP-2.
[0011] In certain embodiments, the cells are cultured for an
additional time period after the BMP culture step without the
presence of an Activin or a BMP. In certain of those embodiments,
that additional culture step is two weeks or longer. In certain of
those embodiments, an IGF is included in the culture step. In
certain of those embodiments, the IGF is IGF-1.
[0012] In certain embodiments of the invention, the cell population
that results from the differentiation protocol is enriched for
cardiomyocyte-lineage cells. In certain of those embodiments, a
Percoll gradient is used to enrich the proportion of
cardiomyocyte-lineage cells.
[0013] In certain embodiments of the invention, the harvested cells
are at least 10% positive for .alpha.-myosin heavy chain
(.alpha.MHC). In certain embodiments of the invention, the
harvested cells are at least 10% cardiac troponin I (cTnI)
positive). In certain embodiments of the invention, the harvested
cells are at least 25% cardiac troponin I (cTnI) positive).
[0014] In certain embodiments of the invention, cardiac bodies are
formed to enrich and/or expand the population of
cardiomyocyte-lineage cells. In certain of those embodiments, the
methods further comprise separating cells in the enriched cell
population that are present as single cells from cells that are
present as clusters; resuspending the cells present as clusters in
nutrient medium; reculturing the resuspended cells in the nutrient
medium; and collecting and washing the recultured cells.
[0015] The invention also provides for population of
cardiomyocyte-lineage cells differentiated from primate pluripotent
stem cells according to the methods of the invention. The invention
also provides a plurality of cell populations cultured during
production of cardiomyocyte-lineage cells from human blastocyst
cells, comprising undifferentiated cells from a line of primate
pluripotent stem cells obtained from a human blastocyst; and a
population of cardiomyocyte-lineage cells differentiated from said
primate pluripotent stem cell line according to the methods of the
invention.
[0016] In certain embodiments of the invention, the differentiation
of primate pluripotent stem cells to cardiomyocyte-lineage cells
occurs in a serum-free medium. In certain embodiments of the
invention, the differentiation of primate pluripotent stem cells to
cardiomyocyte-lineage cells occurs in a medium that contains less
than 0.5% serum. In certain embodiments of the invention, the
differentiation of primate pluripotent stem cells to
cardiomyocyte-lineage cells occurs in a medium that contains less
than 1% serum. In certain embodiments of the invention, the
differentiation of primate pluripotent stem cells to
cardiomyocyte-lineage cells occurs in a medium that contains less
than 5% serum.
[0017] In certain embodiments of the invention, the cells are
adhered to a substrate that comprises s one or more of gelatin,
Matrigel, laminin, fibronectin, and/or vitronectin during the
differentiation of primate pluripotent stem cells to
cardiomyocyte-lineage cells.
[0018] In certain embodiments of the invention, the primate
pluripotent stem cells are cultured in MEM-CM plus bFGF for one to
seven days before the Activin culture step. In certain embodiments,
the primate pluripotent stem cells are cultured in MEM-CM plus bFGF
for about six days before the Activin culture step.
[0019] In certain embodiments, the medium RPMI plus 1.times.B27 is
used when culturing the cells in the presence of an Activin. In
certain embodiments, the medium RPMI plus 1.times.B27 is used when
culturing the cells in the presence of an a BMP. In certain
embodiments, the medium RPMI plus N2 is used when culturing the
cells in the presence of an Activin. In certain embodiments, the
medium RPMI plus N2 is used when culturing the cells in the
presence of a BMP.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1--H7 cells were plated onto wells coated with gelatin
and FBS and subsequently differentiated according to the method
described in Example 1. On day 24 after the original addition of
activin, cultures were dissociated with trypsin-EDTA, fixed,
permeablized, and stained with an antibody against cardic troponin
I. Prior to fixation, cells were incubated with EMA to distinguish
live cells (cells excluding EMA) from dead cells (cells
incorporating EMA). Samples were analyzed on a FACScalibur and dead
cells were excluded from the analysis. In this experiment,
approximately 54% of the cells survived the trypsin dissociation;
of these live cells, 24-27% were cardiomyocytes as determined by
labeling with the cardiac troponin I-specific antibody. Two
different gating methods were used in the 2 panels (FIG. 1A:
histogram- and FIG. 1B: scatter-plot based); the percentage of
cardiomyocytes was similar by either method.
[0021] FIG. 2--H7 cells were plated onto wells coated with Matrigel
and subsequently differentiated according to the method described
in Example 2. On day 21 after the original addition of activin,
cultures were dissociated with trypsin-EDTA, fixed, permeablized,
and stained with an antibody against cardic troponin I. Prior to
fixation, cells were incubated with EMA to distinguish live cells
(cells excluding EMA) from dead cells (cells incorporating EMA).
Samples were analyzed on a FACScalibur and dead cells were excluded
from the analysis. In this experiment, approximately 69% of the
cells survived the trypsin dissociation; of these live cells, 8.9%
were cardiomyocytes as determined by labeling with the cardiac
troponin I-specific antibody.
[0022] FIG. 3 shows the expression of cTnI measured in cardiac
bodies formed from each of the four Percoll fractions.
Undifferentiated hES cells are used as a negative control.
Culturing the Fraction IV cells as cardiac bodies enriched for
.alpha.MHC or cTnI expression by 100- to 500-fold.
[0023] FIG. 4 shows the expression of .alpha.MHC in cell
populations that result from the differentiation of hES cells using
different concentrations of BMP-2 and BMP-4.
DEFINITIONS
[0024] The term "cardiomyocyte-lineage cells" refers generally to
both cardiomyocyte precursor cells and mature cardiomyocytes.
Reference to cardiomyocyte-lineage cells, precursors, or
cardiomyocytes in this disclosure can be taken to apply equally to
cells at any stage of cardiomyocyte ontogeny without restriction,
as defined above, unless otherwise specified. As described below,
cardiomyocyte-lineage cells may have one or more markers (sometimes
at least 3 or 5 markers) from the following list: cardiac troponin
I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain
(MHC), GATA-4, Nkx2.5, N-cadherin, .beta.1-adrenoceptor
(.beta.1-AR), ANF, the MEF-2 family of transcription factors,
creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor
(ANF).
[0025] The term "embryoid bodies" refers to heterogeneous clusters
comprising differentiated and partly differentiated cells that
appear when primate pluripotent stem cells are allowed to
differentiate in a non-specific fashion in suspension cultures or
aggregates.
[0026] As used herein, "primate pluripotent stem cells" refers to
cells that are derived from any kind of embryonic tissue (fetal or
pre-fetal tissue) and that have the characteristic of being capable
under appropriate conditions of producing progeny of different cell
types that are derivatives of all of the 3 germinal layers
(endoderm, mesoderm, and ectoderm), according to a standard
art-accepted test such as the ability to form a teratoma in 8-12
week old SCID mice or the ability to form identifiable cells of all
three germ layers in tissue culture. Included in the definition of
primate pluripotent stem cells are embryonic cells of various
types, exemplified by human embryonic stem (hES) cells, (see, e.g.,
Thomson et al. (Science 282:1145, 1998)) and human embryonic germ
(hEG) cells (see, e.g., Shamblott et al., Proc. Natl. Acad. Sci.
USA 95:13726, 1998); embryonic stem cells from other primates, such
as Rhesus stem cells (see, e.g., Thomson et al., Proc. Natl. Acad.
Sci. USA 92:7844, 1995), marmoset stem cells (see, e.g., Thomson et
al., Biol. Reprod. 55:254, 1996).
[0027] As used herein, "undifferentiated primate pluripotent stem
cells" refers to a cell culture where a substantial proportion of
primate pluripotent stem cells and their derivatives in the
population display morphological characteristics of
undifferentiated cells. It is understood that colonies of
undifferentiated cells within the population will often be
surrounded by neighboring cells that are partly differentiated.
[0028] As used herein, "embryonic stem cell" refers to pluripotent
stem cells that are derived from a human embryo at the blastocyst
stage, or before substantial differentiation of the cells into the
three germ layers. Except where explicitly required otherwise, the
term includes primary tissue and established lines that bear
phenotypic characteristics of hES cells, and progeny of such lines
that still have the capacity of producing progeny of each of the
three germ layers. Prototype "human Embryonic Stem cells" (hES
cells) are described by Thomson et al. (Science 282:1145, 1998;
U.S. Pat. No. 6,200,806).
[0029] As used herein, "Activin" refers to a polypeptide growth
factor that is a member of the transforming growth factor-.beta.
(TGF-.beta.) superfamily. Currently there are four know
Activins--A, AB, B, and C.
[0030] As used herein, "Bone Morphogenetic Protein (BMP)" refers to
a polypeptide growth factor of the TGF-.beta. superfamily. There
are currently about 20 known members in the BMP family. For the
purposes of this application, the term "BMP" does not include
BMP-1. As used herein, "enrich" refers to increasing the level of a
component in a mixture. For example, in certain embodiments of the
present invention, a given cell population may be enriched by
increasing the proportion of cardiomyocyte-lineage cells in that
population.
[0031] As used herein, "cardiac body" refers to a cluster of
primate pluripotent stem cell-derived cells in suspension, bearing
two or more characteristics of human cardiomyocyte-lineage
cells.
[0032] As used herein, "direct differentiation" refers to a process
for differentiating primate pluripotent stem cells into progeny
that are enriched for cells of a particular tissue type without
forming embryoid bodies as an intermediate. To clarify, the term
direct differentiation encompasses processes in which a small
number of cell aggregates form inadvertently.
[0033] As used herein, "genetically altered", "transfected", or
"genetically transformed" refer to a process where a polynucleotide
has been transferred into a cell by any suitable means of
artificial manipulation, or where the cell is a progeny of the
originally altered cell and has inherited the polynucleotide. The
polynucleotide will often comprise a transcribable sequence
encoding a protein of interest, which enables the cell to express
the protein at an elevated level or may comprise a sequence
encoding a molecule such as siRNA or antisense RNA that affects the
expression of a protein (either expressed by the unmodified cell or
as the result of the introduction of another polynucleotide
sequence) without itself encoding a protein. The genetic alteration
is said to be "inheritable" if progeny of the altered cell have the
same alteration.
[0034] As used herein, "serum-free" refers to a condition where the
referenced composition contains no added serum.
[0035] As used herein, "feeder cells" refers to cells of a
different tissue type, and typically a different genome, that may
act to promote proliferation and/or control differentiation of
cells they are cocultured with. For example, undifferentiated
primate pluripotent stem cells can be cocultured with feeder cells
that help maintain the undifferentiated state, while primate
pluripotent stem cells in the process of being differentiated can
be cocultured with feeders that direct differentiation towards a
particular tissue type (e.g., cardiomyocyte-lineage cells).
[0036] As used herein, "feeder-free" refers to a condition where
the referenced composition contains no added feeder cells. To
clarify, the term feeder-free encompasses, inter alia, situations
where primate pluripotent stem cells are passaged from a culture
with feeders into a culture without added feeders even if some of
the feeders from the first culture are present in the second
culture.
[0037] As used herein, "culturing" refers to the process of
maintaining and/or expanding cells in vitro.
[0038] As used herein, "same genome" refers to the genomes of a
primate pluripotent stem cell and a differentiated cell derived
from that primate pluripotent stem cell and means that the
chromosomal DNA will be over 90% identical between the primate
pluripotent stem cell and the derived cell as determined by
Restriction Fragment Length Polymorphism ("RFLP") or SNP analysis.
Even if the primate pluripotent stem cell or the derived cell has
been genetically altered, those cells will be considered to have
the same genome as the cell from which it was derived or the cell
derived from it, since all non-manipulated genetic elements are
preserved.
[0039] As used herein, "Matrigel" refers to BD Matrigel.TM.
Basement Membrane Matrix, which is a commercial preparation of
basement membrane produced by Engelbreth-Holm-Swarm tumor cells and
containing extracellular matrix components such as laminin.
Matrigel is available commercially through Becton, Dickinson and
Company (Franklin Lakes, N.J.).
[0040] As used herein, "RPMI" refers to RPMI Medium 1640
(Invitrogen, Carlsbad, Calif.).
DETAILED DESCRIPTION OF THE INVENTION
[0041] General Techniques--
[0042] For further elaboration of general techniques useful in the
practice of this invention, the practitioner can refer to standard
textbooks and reviews in cell biology, tissue culture, embryology,
and cardiophysiology.
[0043] With respect to tissue and cell culture and embryonic stem
cells, the reader may wish to refer to Teratocarcinomas and
embryonic stem cells: A practical approach (E. J. Robertson, ed.,
IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P.
M. Wasserman et al. eds., Academic Press 1993); Embryonic Stem Cell
Differentiation in Vitro (M. V. Wiles, Meth. Enzymol. 225:900,
1993); Properties and uses of Embryonic Stem Cells: Prospects for
Application to Human Biology and Gene Therapy (P. D. Rathjen et
al., Reprod. Fertil. Dev. 10:31, 1998; and R. I. Freshney, Culture
of Animal Cells, Wiley-Liss, New York, 2000). With respect to the
culture of heart cells, standard references include The Heart Cell
in Culture (A. Pinson ed., CRC Press 1987), Isolated Adult
Cardiomyocytes (Vols. I & II, Piper & Isenberg eds., CRC
Press 1989), and Heart Development (Harvey & Rosenthal,
Academic Press 1998). General methods in molecular and cellular
biochemistry can be found in such standard textbooks as Short
Protocols in Molecular Biology, 4.sup.th Ed.; Immunology Methods
Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue
Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998).
[0044] Primate Pluripotent Stem Cells
[0045] The present invention provides methods for differentiating
primate pluripotent stem cells into cardiomyocyte-lineage cells.
Primate pluripotent stem cells that may be used in the methods of
the invention include, but are not limited to, embryonic stem
cells. Embryonic stem cells can be isolated from blastocysts of
primate species (U.S. Pat. No. 5,843,780; Thomson et al., Proc.
Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES)
cells can be prepared from human blastocyst cells using, for
example, the techniques described by Thomson et al. (U.S. Pat. No.
6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133
ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000. Other
primate pluripotent stem cell types include, but are not limited
to, primitive ectoderm-like (EPL) cells, outlined in WO 01/51610
(Bresagen) and human embryonic germ (hEG) cells (Shamblott et al.,
Proc. Natl. Acad. Sci. USA 95:13726, 1998).
[0046] Embryonic stem cells used in the invention may be chosen
from embryonic stem cell lines or may be obtained directly from
primary embryonic tissue. A large number of embryonic stem cell
lines have been established including, but not limited to, H1, H7,
H9, H13 or H14 (reference Thompson); hESBGN-01, hESBGN-02,
hESBGN-03 (BresaGen, Inc., Athens, Ga.); HES-1, HES-2, HES-3,
HES-4, HES-5, HES-6 (from ES Cell International, Inc., Singapore);
HSF-1, HSF-6 (from University of California at San Francisco); I 3,
I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J 3.2 (derived at the
Technion-Israel Institute of Technology, Haifa, Israel); UCSF-1 and
UCSF-2 (Genbacev et al., Fertil. Steril. 83(5):1517-29, 2005);
lines HUES 1-17 (Cowan et al., NEJM 350(13):1353-56, 2004); and
line ACT-14 (Klimanskaya et al., Lancet, 365(9471):1636-41,
2005).
[0047] In certain embodiments, primate pluripotent stem cells used
in the present invention may have been derived in a feeder-free
manner (see, e.g., Klimanskaya et al., Lancet, 365(9471):1636-41
(2005)).
[0048] Primate Pluripotent Stem Cell Culture
[0049] Primate pluripotent stem cells may be cultured using a
variety of substrates, media, and other supplements and factors
known in the art. Primate pluripotent stem cells can be propagated
continuously in culture, using culture conditions that promote
proliferation while inhibiting differentiation. Exemplary medium is
made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of either
defined fetal bovine serum (FBS, Hyclone) or serum replacement (US
2002/0076747 A1, Life Technologies Inc.), 1% non-essential amino
acids, 1 mM L-glutamine, and 0.1 mM .beta.-mercaptoethanol.
[0050] In certain embodiments, primate pluripotent stem cells are
cultured on a layer of feeder cells, typically fibroblasts derived
from embryonic or fetal tissue (Thomson et al., Science 282:1145,
1998). In certain embodiments, those feeder cells are from human or
mouse. Human feeder cells can be isolated from various human
tissues or derived by differentiation of human embryonic stem cells
into fibroblast cells (see, e.g., WO01/51616) In certain
embodiments, human feeder cells that may be used include, but are
not limited to, placental fibroblasts (see, e.g., Genbacev et al.,
Fertil. Steril. 83(5):1517-29, 2005), fallopian tube epithelial
cells (see, e.g., Richards et al., Nat. Biotechnol., 20:933-36,
2002), foreskin fibroblasts (see, e.g., Amit et al., Biol. Reprod.
68:2150-56, 2003), uterine endometrial cells (see, e.g., Lee et
al., Biol. Reprod. 72(1):42-49, 2005)
[0051] In certain embodiments, embryonic stem cells may be
maintained in an undifferentiated state without added feeder cells
(see, e.g., Rosler et al., Dev. Dynam. 229:259-274, 2004).
Feeder-free cultures are typically supported by a nutrient medium
containing factors that promote proliferation of the cells without
differentiation (see, e.g., U.S. Pat. No. 6,800,480). In certain
embodiments, such factors may be introduced into the medium by
culturing the medium with cells secreting such factors, such as
irradiated (.about.4,000 rad) primary mouse embryonic fibroblasts,
telomerized mouse fibroblasts, or fibroblast-like cells derived
from primate pluripotent stem cells (U.S. Pat. No. 6,642,048).
Medium can be conditioned by plating the feeders in a serum free
medium such as KO DMEM supplemented with 20% serum replacement and
4 ng/mL bFGF. Medium that has been conditioned for 1-2 days is
supplemented with further bFGF, and used to support primate
pluripotent stem cell culture for 1-2 days (see. e.g., WO 01/51616;
Xu et al., Nat. Biotechnol. 19:971, 2001).
[0052] Alternatively, fresh or non-conditioned medium can be used,
which has been supplemented with added factors (like a fibroblast
growth factor or forskolin) that promote proliferation of the cells
in an undifferentiated form. Exemplary is a base medium like
X-VIVO.TM. 10 (Biowhittaker) or QBSF.TM.-60 (Quality Biological
Inc.), supplemented with bFGF at 40-80 ng/mL, and optionally
containing stem cell factor (15 ng/mL), or Flt3 ligand (75 ng/mL)
(see, e.g., Xu et al., Stem Cells 23(3):315-23, 2005). These medium
formulations have the advantage of supporting cell growth at 2-3
times the rate in other systems (see, e.g., WO 03/020920).
[0053] For example, the primate pluripotent stem cells are plated
at >15,000 cells cm.sup.-2 (optimally 90,000 cm.sup.-2 to
170,000 cm.sup.-2). Typically, enzymatic digestion is halted before
cells become completely dispersed (say, .about.5 min with
collagenase IV). Clumps of .about.10 to 2,000 cells are then plated
directly onto the substrate without further dispersal.
Alternatively, the cells can be harvested without enzymes before
the plate reaches confluence by incubating .about.5 min in a
solution of 0.5 mM EDTA in PBS or by simply detaching the desired
cells from the plate mechanically, such as by scraping or isolation
with a fine pipet. After washing from the culture vessel, the cells
are plated into a new culture without further dispersal. In a
further illustration, confluent human embryonic stem cells cultured
in the absence of feeders are removed from the plates by incubating
with a solution of 0.05% (wt/vol) trypsin (Gibco) and 0.053 mM EDTA
for 5-15 min at 37.degree. C. The remaining cells in the plate are
removed and the cells are triturated into a suspension comprising
single cells and small clusters, and then plated at densities of
50,000-200,000 cells cm.sup.-2 to promote survival and limit
differentiation.
[0054] Under the microscope, primate pluripotent stem cells appear
with high nuclear/cytoplasmic ratios, prominent nucleoli, and
compact colony formation with poorly discernable cell junctions.
Primate primate pluripotent stem cells typically express the
stage-specific embryonic antigens (SSEA) 3 and 4, and markers
detectable using antibodies designated Tra-1-60 and Tra-1-81.
Undifferentiated human embryonic stem cells also typically express
the transcription factor Oct-3/4, Cripto, gastrin-releasing peptide
(GRP) receptor, podocalyxin-like protein (PODXL), and human
telomerase reverse transcriptase (hTERT) (US 2003/0224411 A1), as
detected by RT-PCR.
[0055] Differentiation of Primate Pluripotent Stem Cells to
Cardiomyocyte-Lineage Cells
[0056] The present invention provides, inter alia, methods for
differentiating primate pluripotent stem cells into
cardiomyocyte-lineage cells by the sequential culturing of the
primate pluripotent stem cells first in the presence of an Activin
with subsequent culturing in the presence of a BMP. Although the
BMP is excluded during the culturing step with Activin, the Activin
may optionally be included during the subsequent culturing step
with the BMP.
[0057] In certain embodiments, Activin is included in the culture
medium at a concentration between 10 ng/ml and 200 ng/ml, or
between 25 ng/ml and 100 ng/ml, or between 50 ng/ml and 100 ng/ml.
In certain embodiments, Activin is included in the culture medium
at a concentration below 10 ng/ml or above 200 ng/ml.
[0058] In certain embodiments, the BMP is included in the culture
medium at a concentration between 10 ng/ml and 200 ng/ml, or
between 25 ng/ml and 100 ng/ml, or between 50 ng/ml and 100 ng/ml.
In certain embodiments, the BMP is included in the culture medium
at a concentration below 10 ng/ml or above 200 ng/ml.
[0059] In certain embodiments, the Activin used in the
differentiation is Activin A, Activin B, Activin AB, or Activin C.
In certain embodiments, more than one Activin may be used. In
certain embodiments, other TGF.beta. superfamily members such as
TGF.beta., nodal, or lefty may be substituted instead of or in
addition to the Activin in the methods of the present
invention.
[0060] In certain embodiments, the BMP used in the differentiation
is BMP-2, BMP-4, or BMP-7. In certain embodiments, the BMP is a BMP
other than BMP-2, BMP-4 or BMP-7 (excluding BMP-1). In certain
embodiments, more than one BMP may be used.
[0061] In certain embodiments, the differentiating cells are
cultured in the absence of both Activin and BMP after the BMP step.
In certain of those embodiments, an IGF is included in that culture
step. In certain of those embodiments, the IGF is included at a
concentration between 10 ng/ml and 500 ng/ml; or between 25 ng/ml
and 100 ng/ml; or between 50 ng/ml and 100 ng/ml. In certain
embodiments, the IGF is included at concentrations less than 10
ng/ml or more than 500 ng/ml. The IGF may be IGF-1 or IGF-2. In
certain embodiments, insulin may be substituted for the IGF in the
methods of the present invention.
[0062] During the differentiation of the primate pluripotent stem
cells to cardiomyocyte-lineage cells, the cells are cultured in the
presence of the Activin, BMP, or IGF for various specified time
periods. In certain embodiments, the culture step with Activin is
between 12 hours and 36 hours in length, or between 12 hours and 2
days in length, or between 6 hours and 4 days in length, or between
4 hours and 5 days in length. In certain embodiments, the culture
step with Activin is longer than 5 days.
[0063] In certain embodiments, the culture step with the BMP is
between 3 days and 5 days in length, or between 2 days and 8 days
in length, or between 1 day and 14 days in length. In certain
embodiments, the culture step with the BMP is longer than 14
days.
[0064] In certain embodiments, the culture step with the IGF is
between 3 days and 5 days in length, or between 2 days and 8 days,
or between 1 day and 4 weeks in length. In certain embodiments, the
culture step with the IGF is longer than 4 weeks long.
[0065] For example, in certain embodiments, human embryonic stem
cells plated on Matrigel may be first cultured with 50 ng/ml
Activin A in the absence of a BMP for about one day, then cultured
with 50 ng/ml BMP-4 in the absence of an Activin for about four
days, and then cultured in the presence of 50 ng/ml IGF-1 in the
absence of both an Activin and a BMP for two weeks. In certain of
those embodiments, the resulting cardiomyocyte-lineage cells are
harvested and enriched by Percoll gradient as described in Example
3.
[0066] In certain embodiments, the primate pluripotent stem cells
may be differentiated into cardiomyocyte-lineage cells by direct
differentiation. Differentiation paradigms for primate pluripotent
stem cells traditionally involve the deliberate formation of
embryoid bodies, which allows cross-talk between different cell
types, thought to promote tissue formation in a manner reminiscent
of an embryo. However, it is often advantageous to eliminate the
need to form embryoid bodies, allowing the differentiation process
to be more controlled, and the resulting cell population tends to
be more uniform (see, e.g., WO 01/51616; US 2002/0151053 A1).
[0067] One of the advantages of the direct differentiation
technique is that a serum or serum substitute is not needed to
initiate or support the cardiomyocyte differentiation process, as
is typical of other methods. Instead, the medium can be formulated
so that it contains an artificial nutritional supplement that
supports differentiated cells like cardiomyocytes or neurons.
Exemplary are B27 supplement, N2 supplement, and G5 supplement
(Life Technologies/Gibco). In certain embodiments, supplements
comprise nutrients and cofactors like human insulin (500 .mu.g/L),
human transferrin (5-10 mg/mL), and selenium (0.5 .mu.g/mL), and
may also contain putrescine (1.5 mg/L), biotin (1 .mu.g/L),
hydrocortisone (0.4 .mu.g/L), or progesterone (0.6 .mu.g/L), and/or
low levels of mitogens like EGF or FGF (1 .mu.g/L). For purposes of
commercial scale production and human therapy, elimination of
components derived from non-human animals may be advantageous.
[0068] In certain embodiments, the culture medium used during the
differentiation steps is serum-free. In certain embodiments, the
culture medium used during the differentiation steps contains less
than 0.25% serum, or less than 0.5% serum, or less than 1.0% serum,
or less than 2.0% serum, or less than 5.0% serum, or less than 10%
serum.
[0069] Notwithstanding the advantages of the direct differentiation
method, in certain embodiments of the present invention, the
primate pluripotent stem cells may be differentiated by the methods
of the present invention into cardiomyocyte-lineage cells through
the formation of embryoid bodies at some point in the
differentiation protocol except for the Activin culture step.
Embryoid bodies can be formed in a variety of ways known in the
art.
[0070] In certain embodiments, the differentiating cells are
cultured on a substrate during the methods of the invention.
Substrates that can be used in this invention include, but are not
limited to collagen, laminin, fibronectin, vitronectin, hyaluronate
poly-L-lysine-coated tissue culture plastic, or Matrigel.
[0071] In the practice of the present invention, there are various
solid surfaces that may be used in the culturing of cells. Those
solid surfaces include, but are not limited to, standard cell
culturing plates such as 6-well, 24-well, 96-well, or 144-well
plates. Other solid surfaces include, but are not limited to,
microcarriers and disk. In certain embodiments, the microcarriers
are beads. Those beads come in various forms such as Cytodex
Dextran microcarrier beads with positive charge groups to augment
cell attachment, gelatin/collagen-coated beads for cell attachment,
and macroporous microcarrier beads with different porosities for
attachment of cells. The Cytodex dextran, gelatin-coated and the
macroporous microcarrier beads are commercially available
(Sigma-Aldrich, St. Loius, Mo. or Solohill Engineering Inc., Ann
Arbor, Mich.). In certain embodiments, the beads are 90-200 .mu.m
in size with an area of 350-500 cm.sup.2. Beads may be composed of
a variety of materials such as, but not limited to, glass or
plastic. In certain embodiments, disks may be used in stirred-tank
bioreactors for attachment of the cells. Disks are sold by
companies such as New Brunswick Scientific Co, Inc. (Edison, N.J.).
In certain embodiments, the disks are Fibra-cel Disks, which are
polyester/polypropylene disks. A gram of these disks provide a
surface area of 1200 cm.sup.2.
[0072] The solid surface may be made of a variety of substances
including, but not limited to, glass or plastic such as
polystyrene, polyvinylchloride, polycarobnate,
polytetrafluorethylene, melinex, or thermanox. In certain
embodiments of the invention, the solid surfaces may
three-dimensional in shape. Exemplary three-dimensional solid
surfaces are described, e.g., in US20050031598.
[0073] In certain embodiments, the cells are in a single-cell
suspension during the methods of the invention. The single-cell
suspension may be performed in various ways including, but not
limited to, culture in a spinner flask, in a shaker flask, or in a
fermentors. Fermentors that may be used include, but are not
limited to, Celligen Plus (New Brunswick Scientific Co, Inc.,
Edison, N.J.), and the STR or the Stirred-Tank Reactor (Applikon
Inc., Foster City, Calif.). In certain embodiments, the bioreactors
may be continuously perfused with media or used in a fed-batch
mode. Bioreactors come in different sizes like 2.2 L, 5 L, 7.5 L,
14 L or 20 L.
[0074] Enriching and Expanding Cardiomyocyte-Lineage Cells
[0075] The present invention provides methods for obtaining high
purity cardiomyocyte-lineage cell populations without an enrichment
step. However, the addition of one or more enrichment steps may
produce an even higher purity cardiomyocyte-lineage cell
population. Thus, methods of the invention may include steps for
enriching and/or expanding cardiomyocyte-lineage cells obtained by
the differentiation steps of the invention. Various methods for
enriching specific cell types are known in the art and include, but
are not limited to, mechanical separation, density separation, cell
sorting, magnetic sorting, and genetic selection techniques (for a
general discussion of cell separation, see Freshney, Culture of
Animal Cells, Wiley-Liss, New York, 2000--Chapter 14). Examples of
some of those methodologies are discussed below.
[0076] Density Gradients
[0077] In certain embodiments, cardiomyocyte-lineage cells are
enriched by density gradient separation using density gradient
mediums such as, but not limited to, Percoll (see, e.g., Example 3
herein and Xu et al., Circ. Res. 91(6):501-08, 2002), Ficoll
(Pharmacia), metrizamide (Nygaard), RediGrad (GE Healthcare) and
dextran.
[0078] Cell Sorting Techniques
[0079] Many cell sorting techniques are available for sorting
cardiomyocyte-lineage cells from non-cariomyocyte-lineage cells.
Those cell sorting techniques include, but are not limited to
negative immunoselection and positive immunoselection.
[0080] Immunoselection is a generic term that encompasses a variety
of techniques in which the specificity of a selection system is
conferred by an antibody or an antibody-like molecule such as a
lectin or hapten. An example of such specificity is the affinity of
an antibody for a specific cell surface antigen. Two general types
of immunoselection techniques are practiced. Negative
immunoselection involves the elimination of a specific
subpopulation of components from a heterogeneous population such as
the elimination on non-cardiomyocyte-lineage cells from the cell
population that results from the differentiation of primate
pluripotent stem cells according to the methods herein. In
contrast, positive immunoselection refers to the direct selection
and recovery of a specific component, such as the direct selection
and recovery of cardiomyocyte-lineage cells from the
differentiation of primate pluripotent stem cells according to the
methods herein. Various types of immunoselection may be used in the
practice of the present invention, including, but not limited to,
flow cytometry (FACS), immunomagnetic techniques, antibody columns,
immunoprecipitation, and immunopanning.
[0081] Cardiac Bodies--
[0082] In certain embodiments, cardiomyocyte-lineage cells may be
further expanded or enriched by allowing them to grow in clusters
that are referred to as cardiac bodies.
[0083] First, a cell population is generated that contains cells
with phenotype characteristics of cardiomyocyte-lineage cells, and
optionally enriched by density separation or other technique. The
cells are then allowed to form clusters, and single cells in the
suspension are removed. This can be accomplished by letting the
clusters settle, and pipetting out the supernatant containing
single cells. Before proceeding, it is sometimes beneficial to
break apart the clusters (for example, by brief trypsinization
and/or mechanical dispersion). The cells are then cultured in
suspension in low adhesion plates in fresh culture medium
(exemplified by medium containing fetal bovine serum, serum
substitute, or CCT), and allowed to reaggregate into "secondary"
cardiac bodies. Culturing then continues with periodic refeeding,
as necessary, with cardiomyocyte-lineage cells remaining as
clusters of 10 to 5000 cells (typically 50 to 1000 cells) in
size.
[0084] After a suitable period (typically 1 to 7 days), the
cultured cells can be harvested for characterization, or used in
drug screening or pharmaceutical manufacture. The purification
effect may improve if the cells are taken through further cycles of
removing single cells and reculturing the clusters, over a period
of 8 days or more. Each cycle can optionally incorporate a step in
which the clusters of cells are dispersed into single cells, or
smaller cell clusters, to allow for further expansion. Larger
clusters may form, either by aggregation of the suspended cells, or
by proliferation within the cluster, or both. It is a hypothesis of
this invention that cardiomyocyte-lineage cells have a tendency to
form such clusters under appropriate conditions, and that the
removal of single cells helps eliminate other cell types and
increase homogeneity.
[0085] The cardiac body technique can be used to expand and/or
enrich the cardiomyocytes in the cell population at any time in the
differentiation process. As exemplified below, the technique can be
used after a previous enrichment step by density separation.
Implementation of the technique has benefits that were not
anticipated before the making of this invention. In particular, the
expression of myosin heavy chain detected by real-time PCR
increases 10- to 100-fold when the cells are cultured for a 7 day
period. A large proportion of the clusters in the composition
exhibit spontaneous contractile activity: usually over 50%, and
potentially between about 80% and 100% when processed in the manner
described.
[0086] Characterization of ES-Differentiated Cardiomyocyte-Lineage
Cells
[0087] The cardiomyocyte-lineage cells obtained according to the
techniques of this invention can be characterized according to a
number of phenotypic criteria.
[0088] Cardiomyocytes and precursor cells derived from primate
pluripotent stem cell lines often have morphological
characteristics of cardiomyocytes from other sources. They can be
spindle, round, triangular or multi-angular shaped, and they may
show striations characteristic of sarcomeric structures detectable
by immunostaining (FIG. 1). They may form flattened sheets of
cells, or aggregates that stay attached to the substrate or float
in suspension, showing typical sarcomeres and atrial granules when
examined by electron microscopy.
[0089] Under appropriate circumstances, primate pluripotent stem
cell-derived cardiomyocytes often show spontaneous periodic
contractile activity. This means that when they are cultured in a
suitable tissue culture environment with an appropriate Ca.sup.++
concentration and electrolyte balance, the cells can be observed to
contract across one axis of the cell, and then release from
contraction, without having to add any additional components to the
culture medium. The contractions are periodic, which means that
they repeat on a regular or irregular basis, at a frequency between
.about.6 and 200 contractions per minute, and often between
.about.20 and .about.90 contractions per minute in normal buffer
(FIG. 2). Individual cells may show spontaneous periodic
contractile activity on their own, or they may show spontaneous
periodic contractile activity in concert with neighboring cells in
a tissue, cell aggregate, or cultured cell mass.
[0090] The contractile activity of the cells can be characterized
according to the influence of culture conditions on the nature and
frequency of contractions. Compounds that reduce available
Ca.sup.++ concentration or otherwise interfere with transmembrane
transport of Ca.sup.++ often affect contractile activity. For
example, the L-type calcium channel blocker diltiazem inhibits
contractile activity in a dose-dependent manner. On the other hand,
adrenoceptor agonists like isoprenaline and phenylephrine have a
positive chronotropic effect. Further characterization of
functional properties of the cell can involve characterizing
channels for Na.sup.+, K.sup.+, and Ca.sup.++. Electrophysiology
can be studied by patch clamp analysis for cardiomyocyte like
action potentials. See Igelmund et al., Pflugers Arch. 437:669,
1999; Wobus et al., Ann. N.Y. Acad. Sci. 27:752, 1995; and
Doevendans et al., J. Mol. Cell Cardiol. 32:839, 2000.
[0091] Cardiomyocyte-lineage cells typically have at least one of
the following cardiomyocyte specific markers: [0092] Cardiac
troponin I (cTnI), a subunit of troponin complex that provides a
calcium-sensitive molecular switch for the regulation of striated
muscle contraction [0093] Cardiac troponin T (cTnT) [0094] Nkx2.5,
a cardiac transcription factor expressed in cardiac mesoderm during
early mouse embryonic development, which persists in the developing
heart
[0095] The cells will also typically express at least one (and
often at least 3, 5, or more) of the following markers: [0096]
Atrial natriuretic factor (ANF), a hormone expressed in developing
heart and fetal cardiomyocytes but down-regulated in adults. It is
considered a good marker for cardiomyocytes because it is expressed
in a highly specific manner in cardiac cells but not skeletal
myocytes. [0097] myosin heavy chain (MHC), particularly the .beta.
chain which is cardiac specific [0098] Titin, tropomyosin,
.alpha.-sarcomeric actinin, and desmin [0099] GATA-4, a
transcription factor that is highly expressed in cardiac mesoderm
and persists in the developing heart. It regulates many cardiac
genes and plays a role in cardiogenesis [0100] MEF-2A, MEF-2B,
MEF-2C, MEF-2D; transcription factors that are expressed in cardiac
mesoderm and persist in developing heart [0101] N-cadherin, which
mediates adhesion among cardiac cells [0102] Connexin 43, which
forms the gap junction between cardiomyocytes. [0103]
.beta.1-adrenoceptor (.beta.1-AR) [0104] creatine kinase MB (CK-MB)
and myoglobin, which are elevated in serum following myocardial
infarction [0105] .alpha.-cardiac actin, early growth response-I,
and cyclin D2.
[0106] Tissue-specific markers may be detected using suitable
immunological techniques--such as flow immunocytometry or affinity
adsorption for cell-surface markers, immunocytochemistry (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. Antibodies that distinguish cardiac
markers like cTnI and cTnT from other isoforms are available
commercially from suppliers like Sigma and Spectral Diagnostics.
Expression of an antigen by a cell is said to be
antibody-detectable if a significantly detectable amount of
antibody will bind to the antigen in a standard immunocytochemistry
or flow cytometry assay, optionally after fixation of the cells,
and optionally using a labeled secondary antibody.
[0107] The expression of tissue-specific gene products may 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 using publicly available sequence
data (GenBank). Expression of tissue-specific markers as detected
at the protein or mRNA level is considered positive if the level is
at least 2-fold, and preferably more than 10- or 50-fold above that
of an undifferentiated primate pluripotent stem cell.
[0108] The expression of tissue-specific gene products may 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. See U.S. Pat. No. 5,843,780 for
further details. 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). Expression at the
mRNA level is said to be "detectable" according to one of the
assays described in this disclosure if the performance of the assay
on cell samples according to standard procedures in a typical
controlled experiment results in clearly discernable hybridization
or amplification product. Expression of tissue-specific markers as
detected at the protein or mRNA level is considered positive if the
level is at least 2-fold, and preferably more than 10- or 50-fold
above that of an undifferentiated primate pluripotent stem
cell.
[0109] Once markers have been identified on the surface of cells of
the desired phenotype, they can be used for immunoselection to
further enrich the population by techniques such as immunopanning
or antibody-mediated fluorescence-activated cell sorting.
[0110] Where derived from an established line of primate
pluripotent stem cells, the cell populations and isolated cells of
this invention can be characterized as having the same genome as
the line from which they are derived. This means that the
chromosomal DNA will be over 90% identical by RFLP or by SNP
analysis between the primate pluripotent stem cells and the cardiac
cells, which can be inferred if the cardiac cells are obtained from
the undifferentiated line through the course of normal mitotic
division. The characteristic that cardiomyocyte-lineage cells are
derived from the parent cell population is important in several
respects. In particular, the undifferentiated cell population can
be used for producing additional cells with a shared genome--either
a further batch of cardiac cells, or another cell type that may be
useful in therapy--such as a population that can pre-tolerize the
patient to the histocompatibility type of the cardiac allograft (US
2002/0086005 A1; WO 03/050251).
[0111] Genetic Alteration of Differentiated Cells
[0112] The cells of this invention can be made to contain one or
more genetic alterations by genetic engineering of the cells either
before or after differentiation (US 2002/0168766 A1). For example,
the cells can be processed to increase their replication potential
by genetically altering the cells to express telomerase reverse
transcriptase, either before or after they progress to restricted
developmental lineage cells or terminally differentiated cells (US
2003/0022367 A1).
[0113] The cells of this invention can also be genetically altered
in order to enhance their ability to be involved in tissue
regeneration, or to deliver a therapeutic gene to a site of
administration. A vector is designed using the known encoding
sequence for the desired gene, operatively linked to a promoter
that is either pan-specific or specifically active in the
differentiated cell type. Of particular interest are cells that are
genetically altered to express one or more growth factors of
various types such as FGF, cardiotropic factors such as atrial
natriuretic factor, cripto, and cardiac transcription regulation
factors, such as GATA-4, Nkx2.5, and MEF2-C. Production of these
factors at the site of administration may facilitate adoption of
the functional phenotype, enhance the beneficial effect of the
administered cell, or increase proliferation or activity of host
cells neighboring the treatment site.
[0114] In certain embodiments, it is desirable to genetically alter
non-human cardiomyocyte-lineage cells such that the expression of
one or more antigens is reduced or eliminated so that the
immunogenecity of those cells is reduced. This could be useful, for
example, in xenotransplantation of non-human cardiomyocyte-lineage
cells into a human.
[0115] Uses of ES-Differentiated Cardiomyocyte-Lineage Cells
[0116] This invention provides a method to produce large numbers of
cells of the cardiomyocyte-lineage. These cell populations can be
used for a number of important research, development, and
commercial purposes.
[0117] Screening
[0118] Cardiomyocytes of this invention can be used commercially to
screen for factors (such as solvents, small molecule drugs,
peptides, oligonucleotides) or environmental conditions (such as
culture conditions or manipulation) that affect the characteristics
of such cells and their various progeny.
[0119] In some applications, primate pluripotent stem cells
(undifferentiated or differentiated) are used to screen factors
that promote maturation into later-stage cardiomyocyte precursors,
or terminally differentiated cells, or to 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 cells in different wells, and then determining any
phenotypic change that results, according to desirable criteria for
further culture and use of the cells.
[0120] Other screening applications of this invention relate to the
testing of pharmaceutical compounds for their effect on cardiac
muscle tissue maintenance or repair. Screening may be done either
because the compound is designed to have a pharmacological effect
on the cells, or because a compound designed to have effects
elsewhere may have unintended side effects on cells of this tissue
type. The screening can be conducted using any of the precursor
cells or terminally differentiated cells of the invention.
[0121] 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. Assessment of the activity of candidate
pharmaceutical compounds generally involves combining the
differentiated cells of this invention with the candidate compound,
either alone or in combination with other drugs. The investigator
determines any change in the morphology, marker phenotype, or
functional activity of the cells that is attributable to the
compound (compared with untreated cells or cells treated with an
inert compound), and then correlates the effect of the compound
with the observed change.
[0122] Cytotoxicity can be determined in the first instance by the
effect on cell viability, survival, morphology, and the expression
of certain markers and receptors. 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.
[0123] Effect of cell function can be assessed using any standard
assay to observe phenotype or activity of cardiomyocytes, such as
marker expression, receptor binding, contractile activity, or
electrophysiology--either in cell culture or in vivo.
Pharmaceutical candidates can also be tested for their effect on
contractile activity--such as whether they increase or decrease the
extent or frequency of contraction. Where an effect is observed,
the concentration of the compound can be titrated to determine the
median effective dose (ED.sub.50).
[0124] Animal Testing
[0125] This invention also provides for the use of cardiomyocytes
and their precursors to enhance tissue maintenance or repair of
cardiac muscle for any perceived need, such as an inborn error in
metabolic function, the effect of a disease condition, or the
result of significant trauma.
[0126] To determine the suitability of cell compositions for
therapeutic administration, the cells can first be tested in a
suitable animal model. At one level, cells are assessed for their
ability to survive and maintain their phenotype in vivo. Cell
compositions are administered to immunodeficient animals (such as
nude mice, or animals rendered immunodeficient chemically or by
irradiation). Tissues are harvested after a period of regrowth, and
assessed as to whether pluripotent stem derived cells are still
present.
[0127] This can be performed by administering cells that express a
detectable label (such as green fluorescent protein, or
.beta.-galactosidase); that have been prelabeled (for example, with
BrdU or [.sup.3H]thymidine), or by subsequent detection of a
constitutive cell marker (for example, using human-specific
antibody). The presence and phenotype of the administered cells can
be assessed by immunohistochemistry or ELISA using human-specific
antibody, or by RT-PCR analysis using primers and hybridization
conditions that cause amplification to be specific for human
polynucleotides, according to published sequence data.
[0128] Suitability can also be determined by assessing the degree
of cardiac recuperation that ensues from treatment with a
population of cardiomyocyte-lineage cells. A number of animal
models are available for such testing. For example, hearts can be
cryoinjured by placing a precooled aluminum rod in contact with the
surface of the anterior left ventricle wall (Murry et al., J. Clin.
Invest. 98:2209, 1996; Reinecke et al., Circulation 100:193, 1999;
U.S. Pat. No. 6,099,832; Reinecke et al., Circ Res., Epub March
2004). In larger animals, cryoinjury can be effected by placing a
30-50 mm, copper disk probe cooled in liquid N.sub.2 on the
anterior wall of the left ventricle for .about.20 min (Chiu et al.,
Ann. Thorac. Surg. 60:12, 1995). Infarction can be induced by
ligating the left main coronary artery (Li et al., J. Clin. Invest.
100:1991, 1997) or by using an ameroid constriction device that
gradually swells to occlude an artery. Injured sites are treated
with cell preparations of this invention, and the heart tissue is
examined by histology for the presence of the cells in the damaged
area. Cardiac function can be monitored by determining such
parameters as left ventricular end-diastolic pressure, developed
pressure, rate of pressure rise, and rate of pressure decay.
[0129] Therapeutic Use in Humans
[0130] After adequate testing, differentiated cells of this
invention can be used for tissue reconstitution or regeneration in
a human patient or other subject in need of such treatment. The
cells are administered in a manner that permits them to graft or
migrate to the intended tissue site and reconstitute or regenerate
the functionally deficient area. Special devices are available that
are adapted for administering cells capable of reconstituting
cardiac function directly to the chambers of the heart, the
pericardium, or the interior of the cardiac muscle at the desired
location.
[0131] Where desirable, the patient receiving an allograft of
cardiomyocyte-lineage cells can be treated to reduce immune
rejection of the transplanted cells. Methods contemplated include
the administration of traditional immunosuppressive drugs like
cyclosporin A (Dunn et al., Drugs 61:1957, 2001), or inducing
immunotolerance using a matched population of pluripotent stem
derived cells (WO 02/44343; U.S. Pat. No. 6,280,718; WO 03/050251).
Another approach is to adapt the cardiomyocyte-lineage cell
population to decrease the amount of uric acid produced by the
cells upon transplantation into a subject, for example, by treating
them with allopurinol. Alternatively or in conjunction, the patient
is prepared by administering allopurinol, or an enzyme that
metabolizes uric acid, such as urate oxidase (PCT/US04/42917).
[0132] Patients suitable for receiving regenerative medicine
according to this invention include those having acute and chronic
heart conditions of various kinds, such as coronary heart disease,
cardiomyopathy, endocarditis, congenital cardiovascular defects,
and congestive heart failure. Efficacy of treatment can be
monitored by clinically accepted criteria, such as reduction in
area occupied by scar tissue or revascularization of scar tissue,
and in the frequency and severity of angina; or an improvement in
developed pressure, systolic pressure, end diastolic pressure,
.DELTA.pressure/.DELTA.time, patient mobility, and quality of
life.
[0133] The cardiomyocyte-lineage cells of this invention can be
supplied in the form of a pharmaceutical composition, comprising an
isotonic excipient prepared under sufficiently sterile conditions
for human administration. When the differentiation procedure has
involved culturing the cells as cardiac bodies, it may be desirable
to disperse the cells using a protease or by gentle mechanical
manipulation into a suspension of single cells or smaller clusters.
To reduce the risk of cell death upon engraftment, the cells may be
treated by heat shock or cultured with .about.0.5 U/mL
erythropoietin .about.24 hours before administration.
[0134] For general principles in medicinal formulation, the reader
is referred to Cell Therapy: Stem Cell Transplantation, Gene
Therapy, and Cellular Immunotherapy, by G. Morstyn & W.
Sheridan eds, Cambridge University Press, 1996; and Hematopoietic
Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill
Livingstone, 2000. Choice of the cellular excipient and any
accompanying elements of the composition will be adapted in
accordance with the route and device used for administration. The
composition may also comprise or be accompanied with one or more
other ingredients that facilitate the engraftment or functional
mobilization of the cardiomyocyte-lineage cells. Suitable
ingredients include matrix proteins that support or promote
adhesion of the cardiomyocyte-lineage cells, or complementary cell
types, especially endothelial cells.
[0135] This invention also includes a reagent system, comprising a
set or combination of cells that exist at any time during
manufacture, distribution, or use. The cell sets comprise any
combination of two or more cell populations described in this
disclosure, exemplified but not limited to a type of differentiated
pluripotent stem-derived cell (cardiomyocytes, cardiomyocyte
precursors, cardiac bodies, and so on), in combination with
undifferentiated primate pluripotent stem cells or other
differentiated cell types, often sharing the same genome. Each cell
type in the set may be packaged together, or in separate containers
in the same facility, or at different locations, at the same or
different times, under control of the same entity or different
entities sharing a business relationship.
[0136] Pharmaceutical compositions of this invention may optionally
be packaged in a suitable container with written instructions for a
desired purpose, such as the reconstitution of
cardiomyocyte-lineage cell function to improve a disease condition
or abnormality of the cardiac muscle.
[0137] The cells of this invention can be used to prepare a cDNA
library relatively uncontaminated with cDNA preferentially
expressed in cells from other lineages. For example,
cardiomyocyte-lineage cells are collected by centrifugation at 1000
rpm for 5 min, and then mRNA is prepared and reverse transcribed.
Expression patterns of the cardiomyocyte-lineage cells can be
compared with other cell types by microarray analysis, reviewed
generally by Fritz et al Science 288:316, 2000; "Microarray Biochip
Technology", L Shi, www.Gene-Chips.com.
[0138] The differentiated cells of this invention can also be used
to prepare antibodies that are specific for markers of
cardiomyocyte-lineage cells. Polyclonal antibodies can be prepared
by injecting a vertebrate animal with cells of this invention in an
immunogenic form. Production of monoclonal antibodies is described
in such standard references as Harrow & Lane (1988), U.S. Pat.
Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology
73B:3 (1981).
[0139] All publications and patents mentioned in the present
application are herein incorporated by reference for any
purpose.
Example 1
Three Factor Differentiation on Gelatin/FBS
[0140] Preparation of a Gelatin/FBS-Coated Surface:
[0141] 1 ml/well of 0.5% gelatin solution was added to the wells of
a 6-well plate and incubated at 37.degree. C. overnight. The
gelatin solution was removed and sufficient 20% FBS-containing
medium (e.g., 20% FBS (Sigma) in Knockout DMEM) was added to cover
the surface of the wells. The plate incubated at 37.degree. C. for
a further 5-6 hours. Prior to addition of the human embryonic stem
cells, the medium was removed from well.
[0142] Plating Undifferentiated Human Embryonic Stem Cells for
Subsequent Differentiation:
[0143] 1 well of a 6 well plate of undifferentiated human embryonic
stem cells was dissociated by a) removing medium; b) rinsing well
once with PBS; and c) adding 1 ml of 0.25% trypsin/500 mM EDTA
solution. The well was incubated at 37.degree. C. for 10 minutes
and then triturated ten times with 1 ml pipettor. The well was
examined under a microscope to see that the cells were dissociated
completely. Two ml of 20% FBS-containing medium (e.g., 20% FBS in
Knockout DMEM) was added to inactivate the trypsin. The cells were
counted and this number used to plate cells derived from the
remaining wells at the desired density.
[0144] The medium was removed from the remaining wells. A solution
of 20 unit/ml collagenase was added to the wells (1 ml/well). The
wells were incubated at 37 degrees for 10 minutes and the
collagenase solution removed. 1 ml of MEF-conditioned medium plus 8
ng/ml bFGF was added to the wells. The wells were scraped with a 5
ml pipet until the cells were detached (in small clusters); no
further trituration was performed. The cells were diluted to the
desired density and plated into 6 well plate prepared as described
above (in this case, 670,000 cells in a volume of 5 ml per well; 3
wells were plated). The ES cells were re-fed daily (for cells
plated on a Thursday, the feeding on Saturday is usually skipped)
by removing the spent medium and replacing it with new MEF-CM plus
8 ng/ml bFGF.
[0145] Growth Factor Treatment:
[0146] After 6 days of growth as described above, the cells' media
was removed and replaced with RPMI plus 1.times.B27 supplement
(Invitrogen) plus 50 ng/ml Activin A (R&D Systems). After 18-24
hours, the medium was removed and replaced with RPMI plus
1.times.B27 supplement plus 50 ng/ml BMP-4 (R&D Systems). After
a total of 4 days in BMP-4-containing medium, the medium was
removed and replaced with RPMI plus 1.times.B27 supplement plus 50
ng/ml IGF-1 (R&D Systems) without the Activin or BMP. The
cultures were re-fed every 2-3 days by removing spent medium and
replacing it with fresh RPMI plus 1.times.B27 supplement plus 50
ng/ml IGF-1 without the Activin or BMP.
[0147] Numerous beating clusters of cells became evident starting
approximately 12 days after the addition of Activin A. On day 24
after the addition of Activin A, cells were counted (9.1 million
cells from a total of 3 wells of a 6 well plate), and analyzed by
FACS for cardiac troponin I expression by the following
procedure:
[0148] FAGS Analysis--
[0149] Media was removed from cultures by aspiration. The wells
were rinsed once with 5 ml of Calcium/Magnesium-free PBS. One half
ml of a solution of 0.25% trypsin/500 mM EDTA was added per well,
and the cells were incubated at 37.degree. C. for 20 minutes. The
cells were triturated with a pipetor until a single cell suspension
was achieved. The trypsin digestion was stopped by the addition of
1 ml of 20% FBS-containing medium (20% FBS in Knockout DMEM). The
cell concentration was assessed by counting, and about 500,000
cells were allocated for each staining (EMA, isotype, cTnI, cTnI
plus EMA; each in a 15 ml conical tube).
[0150] Tubes containing cells were spun in a centrifuge at
400.times.g for 5 minutes. The medium was aspirated and the cell
pellets were resuspended in 1 ml of staining buffer (PBS plus 1%
heat inactivated goat serum and 0.1% sodium azide). For EMA
staining, cells received EMA to a final concentration of 5
micrograms/ml. These samples were incubated on ice in the dark for
15 minutes, then pelleted as described above. The EMA-treated
samples were resuspended in 500 microliters of PBS and exposed to
light for 10 minutes. The EMA-treated samples received 500
microliters of 4% paraformaldehyde and were incubated in the dark
at room temperature for 15 minutes. Samples that did not receive
EMA but that were subsequently stained with antibodies were
pelleted as described above, resuspended in 500 microliters of PBS
and then received 500 microliters of 4% paraformaldehyde and were
incubated in the dark at room temperature for 15 minutes.
[0151] All samples were pelleted as described above and resuspended
in 100 microliters of PBS. All samples next received 900
microliters of ice-cold 100% methanol and were incubated on ice for
30 minutes. All samples received 1 ml of staining buffer (PBS plus
1% heat inactivated goat serum and 0.1% sodium azide) and pelleted
as described above. The supernatant was aspirated and the cells
resuspended in blocking buffer (PBS plus 20% normal goat serum and
0.1% sodium azide) at a density of about 500,000 cells/50
microliters. Samples were incubated at 4 degrees for 10-15 minutes.
For each stained sample, a 50 microliter aliquot of cells was
dispensed into an individual 12.times.75 mm polystyrene tube. Each
sample to be stained received 50 microliters of either cardiac
troponin I antibody (Spectral Diagnostics) or isotype control
(final amount of antibody per tube was 1.2 micrograms). Samples
were incubated at 4 degrees for 30 minutes.
[0152] After the addition of 2 ml staining buffer, samples were
pelleted as described above. This wash step was repeated. After
removal of the 2.sup.nd wash supernatant, the samples were
resuspended in 50 microliters of 5% normal goat serum in PBS
containing 0.25 micrograms of the secondary antibody (Molecular
Probes goat antimouse IgG labeled with alexa 488). Samples were
incubated at 4 degrees for 30 minutes in the dark, and washed with
the addition of 2 ml staining buffer and pelleting as described
above. The supernatant was decanted and the samples were
resuspended in 300 microliters of staining buffer for flow
acquisition on a FACScalibur machine.
[0153] In this experiment, 54.49% of the total cells were viable
after trypsin treatment. Of these viable cells, 24.67-27.36% of the
cells were stained with an antibody directed against the
cardiomyocyte sarcomeric protein cardiac troponin I. These results
are shown in FIG. 1.
Example 2
Three Factor Direct Differentiation on Matrigel-Coated Surface
[0154] An aliquot of growth factor-reduced Matrigel (previously
diluted 1:2 with cold Knockout DMEM and stored at -20 degrees). The
Matrigel solution was diluted a further 1:15 with cold Knockout
DMEM. Empty wells of a 6-well plate were coated with the diluted
Matrigel solution at 1 ml/well, and the plate was incubated at room
temperature for 4-5 hours. The Matrigel solution was removed, and
human ES cells were plated as described below without a pre-rinsing
of the wells.
[0155] Plating Undifferentiated hES Cells for Subsequent
Differentiation:
[0156] 1) The cells in 1 well of the 6 well plate of
undifferentiated hES cells were dissociated by a) removing medium;
b) rinsing well once with PBS; c) adding 1 ml of 0.05% trypsin/500
mM EDTA solution. The well was incubated in a 37 degree incubator
for 10 minutes. The cells were triturated with a pipettor until
cells were dissociated completely. 2 ml of 20% FBS-containing
medium (e.g., 20% FBS in Knockout DMEM) were added to inactivate
trypsin. The cells were counted, and this number used to plate
cells derived from the remaining wells at the desired density.
[0157] The medium was removed from the remaining wells. A solution
of collagenase (200 units/ml) was added at 1 ml/well, and the well
incubated at 37.degree. C. for 10 minutes. The collagenase solution
was removed, and MEF-conditioned medium plus 8 ng/ml bFGF was
added. The well was scraped with a pipet until cells were detached
(in small clusters); no further trituration was performed. The
cells were diluted to a desired density and plated into a 6-well
plate prepared as described above (1.85 million cells in a volume
of 5 ml per well). The plated hES cells were fed daily (except not
on the second day) by removing the spent medium and replacing it
with new MEF-CM plus 8 ng/ml bFGF.
[0158] Growth Factor Treatment:
[0159] After 6 days of growth as described above, the cells' media
was removed and replaced with RPMI plus 1.times.B27 supplement plus
50 ng/ml Activin A. After 18-24 hours, the medium was removed and
replaced with RPMI plus 1.times.B27 supplement plus 50 ng/ml BMP-4
without the Activin A. After a total of 4 days in the
BMP-4-containing medium, the medium was removed and replaced with
RPMI plus 1.times.B27 supplement plus 50 ng/ml IGF-1 without the
Activin or BMP. The culture were re-fed every 2-3 days by removing
spent medium and replacing it with fresh RPMI plus 1.times.B27
supplement plus 50 ng/ml IGF-1 without the Activin or BMP.
[0160] Beating cells were evident 10-12 days after the addition of
the Activin A. On day 21, the cells were counted and analyzed by
FACS for cardiac troponin I expression as described below
[0161] FACS Analysis--
[0162] Media was removed from cultures by aspiration. The wells
were rinsed once with 5 ml of Calcium/Magnesium-free PBS. One ml of
a solution of 0.25% trypsin/500 mM EDTA was added per well, and the
cells were incubated at 37.degree. C. for 5 minutes. The detached
cells in trypsin were transferred to 15 ml conical tubes and
incubated at 37.degree. C. for a further 15-20 minutes. The cells
were triturated with a pipetor until a single cell suspension was
achieved. The trypsin digestion was stopped by the addition of 2 ml
of 20% FBS-containing medium (20% FBS in Knockout DMEM). The cell
concentration was assessed by counting, and about 500,000 cells
were allocated for each staining (EMA, isotype, cTnI, cTnI plus
EMA; each in a 15 ml conical tube). Tubes containing cells were
spun in a centrifuge at 400.times.g for 5 minutes. The medium was
aspirated and the cell pellets were resuspended in 1 ml of staining
buffer (PBS plus 2% heat inactivated fetal calf serum and 0.1%
sodium azide). For EMA staining, cells received EMA to a final
concentration of 5 micrograms/ml. These samples were incubated on
ice in the dark for 15 minutes, then pelleted as described above.
The EMA-treated samples were resuspended in 1 ml of PBS and exposed
to light for 10 minutes. The EMA-treated samples received 1 ml of
4% paraformaldehyde and were incubated in the dark at room
temperature for 15 minutes. Samples that did not receive EMA but
that were subsequently stained with antibodies were pelleted as
described above, resuspended in 500 microliters of PBS and then
received 500 microliters of 4% paraformaldehyde and were incubated
in the dark at room temperature for 15 minutes. All samples were
pelleted as described above and resuspended in 100 microliters of
PBS.
[0163] All samples next received 900 microliters of ice-cold 100%
methanol and were incubated on ice for 30 minutes. All samples
received 1 ml of staining buffer (PBS plus 2% heat inactivated
fetal calf serum and 0.2 microgram/0.5.times.10.sup.6 cells of rat
antimouse Fc block (BD) and pelleted as described above. The
supernatant was aspirated and the cells resuspended in blocking
buffer (PBS plus 20% normal goat serum and 0.1% sodium azide) at a
density of about 500,000 cells/100 microliters. Samples were
incubated at 4 degrees for 10-15 minutes. For each stained sample,
a 100 microliter aliquot of cells was dispensed into an individual
12.times.75 mm polystyrene tube. Each sample to be stained received
20 microliters of either cardiac troponin I antibody (Spectral
Diagnostics) or isotype control (final amount of antibody per tube
was 1.2 micrograms). Samples were incubated at 4 degrees for 30
minutes. After the addition of 4 ml staining buffer, samples were
pelleted as described above.
[0164] After removal of the 2.sup.nd wash supernatant, the samples
were resuspended in 50 microliters of 5% normal goat serum in PBS
containing 0.25 micrograms of the secondary antibody (Molecular
Probes goat antimouse IgG1 labeled with alexa 647). Samples were
incubated at 4 degrees for 30 minutes in the dark, and washed with
the addition of 4 ml staining buffer and pelleting as described
above. The supematant was decanted and the samples were resuspended
in 300 microliters of staining buffer plus 0.5% paraformaldehyde
for flow acquisition on a FACScalibur machine. The results were
analyzed using Flojo software. In this experiment, 69% of the total
cells were viable after the trypsin dissociation. Of these viable
cells, 8.9% were stained with an antibody against the cardiomyocyte
specific protein cardiac troponin I (see FIG. 2).
Example 3
Example of a Four-Phase Centrifugation Separation Method
Enrichment
[0165] Cardiomyocytes were generated from hES cells of the H7 line
by forming embryoid bodies for 4 days, and then proliferating on
gelatin-coated plates for 17 days (5-aza-deoxy-cytidine and growth
factors were not used). The cells were then dissociated using
collagenase B, resuspended in differentiation medium. The cell
suspension was then layered onto a discontinuous gradient of
Percoll, and centrifuged at 1500 g for 30 min. Four fractions were
collected: I. The upper interface; II. The 40.5% layer; III. The
lower interface; IV. The 58.5% layer. The cells were washed and
resuspended in differentiation medium. Cells for immunostaining
were seeded into chamber slides at 10.sup.4 cells per well,
cultured for 2 or 7, and then fixed and stained.
[0166] Results are shown in Table 3. Percentage of MHC positive
cells was determined by counting cells in 30 images from triplicate
wells for each fraction and presented as mean.+-.standard deviation
of cells from 3 wells.
TABLE-US-00001 TABLE 3 Percoll Separation % staining for MHC
Fraction Cell Count Beating Cells Day 2 Day 7 Before + 17 .+-. 4.4%
15 .+-. 4% separation I 9.0 .times. 10.sup.6 .+-. 2.6 .+-. 0.5% 2.5
.+-. 3.0% II 1.6 .times. 10.sup.6 + 4.5 .+-. 1.5% 2.4 .+-. 0.9% III
4.0 .times. 10.sup.6 ++ 35.7 .+-. 2.7% 28.3 .+-. 9.4% IV 1.3
.times. 10.sup.6 +++ 69. .+-. 5.0% 52.2 .+-. 14.5%
[0167] Beating cells were observed in all fractions, but Fractions
III and IV contained the highest percentage.
[0168] Phenotype of the cells as determined by indirect
immunocytochemistry is shown in Table 4.
TABLE-US-00002 TABLE 4 Characteristics of Separated Cell
Populations Epitope Cardiomyocyte-lineage Non-cardiac cells cTn1 ++
- cardiac-specific .alpha./.beta. MHC ++ - cardiac .beta. MHC ++ -
sarcomeric MHC ++ - N-cadherin ++ .+-. smooth muscle actin ++
subset myogenin - - .alpha.-fetoprotein - - .beta.-tubulin III - -
Ki67 subset subset BrdU subset subset SSEA-4 - - Tra-1-81 - -
[0169] Cardiomyocyte populations separated by density gradient
centrifugation could be distinguished by staining for cTnI and MHC.
Absence of staining for myogenin, .alpha.-fetoprotein, or
.beta.-tubulin III showed the absence of skeletal muscle, endoderm
cell types, and neurons. Lack of staining for SSEA-4 and Tra-1-81
confirms the absence of undifferentiated hES cells.
[0170] .alpha.-Smooth muscle actin (SMA) is reportedly present in
embryonic and fetal cardiomyocytes, but not adult cardiomyocytes
(Leor et al., Circulation 97:11332, 1996; Etzion et al., Mol. Cell
Cardiol. 33:1321, 2001). Virtually all cTnI-positive cells and a
subset of cTnI negative cells obtained in the cardiomyocyte
differentiation protocol were positive for SMA, suggesting that
they may be at an early stage and capable of proliferation.
[0171] Cells in fraction III and IV were replated, cultured for an
additional 2 days. 43.+-.4% of the MHC positive cells expressed
BrdU, indicating that they were in the S phase of the cell cycle.
In other experiments, a subset of cTnI-positive cells were found to
express Ki-67. These results show that about 20% or 40% of the
cardiomyocytes in the population were undergoing active
proliferation.
Example 4
Example of Enrichment of Contracting Cells by Making Cardiac
Bodies
[0172] This example illustrates the subsequent culturing of
cardiomyocyte clusters as cardiac bodies to enrich for cells having
characteristics desirable for therapeutic use and other
purposes.
[0173] Three 225 cm.sup.2 flasks of undifferentiated hES cells of
the H7 line were used to generate embryoid bodies as already
described. The EBs from each flask were resuspended in 75 mL of
medium and transferred to three low adhesion six well plates (4 mL
cell suspension per well), yielding nine plates of EBs in
suspension in total. The EBs were re-fed after one day in
suspension by transferring the newly formed EBs to 50 mL conical
tubes (one plate per tube), letting the EBs settle at room
temperature without agitation for 10 to 20 min, then removing the
medium and replacing with fresh medium (25 mL per tube).
[0174] The EBs were returned to their original low attachment
plates and maintained in suspension in 20% FBS containing medium
for 3 additional days, then transferred to a total of three
gelatin-coated 225 cm.sup.2 tissue culture flasks. Two days after
transfer to the gelatin coated flasks, the medium was removed and
each flask was re-fed with 75 mL 20% FBS containing medium. Similar
re-feedings occurred on day 8, 11, 13, 15, and 18. On day 20, the
differentiated cultures were dissociated with Blendzyme (Roche
Applied Sciences, Penzberg, Del.) and fractionated on discontinuous
Percoll gradients as before. Fraction IV (the highest density
fraction) was recovered and counted, yielding
.about.3.7.times.10.sup.6 single cells and small clusters.
[0175] The Fraction IV cells were resuspended in .about.6.5 mL of
20% FBS containing medium, transferred to a 15 mL conical tube, and
allowed to settle at room temperature without agitation for 10 min.
The medium (containing 2.8.times.10.sup.6 cells, which is most of
the single cells) was removed and replaced with fresh medium. The
cell suspension was transferred to a single low attachment six well
plate (.about.4 mL of cell suspension per well). The CBs were
re-fed in a similar manner (transfer to 50 mL tube, settling for 10
min, medium removal and replacement) every 48 h.
[0176] FIG. 3 shows the expression of the sarcomeric genes
.alpha.MHC and cardiac troponin I as measured by real-time PCR.
Relative to the expression after 20 days of culture on gelatin,
separating the cells by Percoll increased expression by 2-5 fold in
Fraction IV cells. Removing the single cells and collecting
clusters increased expression to 5-20 fold. After 8 days of
culturing the cells as cardiac bodies, the expression was 100- to
500-fold higher than the unseparated cells.
[0177] When CBs are replated onto gelatin or Matrigel, the clusters
adhere, flatten, and produce large patches of spontaneously
contracting cells. For use in animal testing, the cardiac bodies
may be implanted directly, or dispersed into suspensions of single
cells.
Example 5
[0178] A differentiation of 117 hES cells was performed as in
Example 1, except that the differentiation was performed in a
24-well plate instead of a 6-well plate and the volume for the
factors was 1 ml per well. In addition, BMP-2 and BMP-4 were used
at concentrations of 25 ng/ml, 50 ng/ml, and 100 ng/ml. Each
concentration was done in triplicate. FIG. 4 shows the results
expressed as a relative fold of the control, which involved
performing the protocol but without the addition of an Activin, a
BMP, or IGF-I. It can be seen that BMP-2 is also effective in the
differentiation protocol.
Example 6
[0179] A 6-well plate of confluent H7 hES cells were washed with 2
nil PBS. Then, 2 ml of 0.5 mM EDTA in PBS was added to each well,
and the plate was incubated for 10 minutes in 37.degree. C. The
EDTA solution was replaced with 1 ml mouse embryonic
fibroblast-conditioned medium (MEF-CM) plus 8 ng/ml bFGF ("Medium
A"). The undifferentiated ES cells were detached by pipetting 2-3
times and then seeded onto a 24-well plate at 400,000 cells/well in
Medium A. The cells were incubated for two days at 37.degree.
C.
[0180] To induce the hES cells to differentiate, Medium A was
replaced with 0.5 ml of B27:RPMI (1:50) (both reagents from
Invitrogen) with 100 ng/ml Activin A (R&D Systems) ("Medium B")
per well of 24-well plate. The cells were incubated for 24 hours.
Medium B was then replaced with 10 ng/ml BMP-4 (R&D Systems) in
1:50 B27:RPMI ("Medium C") at 1 ml per well of a 24-well plate. The
cells were incubated for 4 days.
[0181] Medium C was replaced with 1 ml of 1:50 B27:RPMI per well of
the 24-well plate, and the plate was incubated for 15 days. The
resulting cells were analyzed by FACS as in Example 2, except that
the cells were incubated in the 0.25% trypsin/500 mM EDTA solution
for 5 minutes instead of the 20 minutes used in Example 2. About
36% of the cells expressed cTnI.
* * * * *
References