U.S. patent application number 12/148059 was filed with the patent office on 2008-08-28 for embryonic stem cells capable of differentiating into desired cells lines.
This patent application is currently assigned to Diacrin, Inc.. Invention is credited to Jonathan H. Dinsmore, Judson Ratliff.
Application Number | 20080206863 12/148059 |
Document ID | / |
Family ID | 22511936 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080206863 |
Kind Code |
A1 |
Dinsmore; Jonathan H. ; et
al. |
August 28, 2008 |
Embryonic stem cells capable of differentiating into desired cells
lines
Abstract
An embryonic stem cell which may be induced to differentiate
homogeneously into a desired primary cell line. The embryonic stem
cell may be engineered with DNA, which encodes a protein or
polypeptide which promotes differentiation of the stem cell into a
specific cell line, such as, for example, a neuronal cell line, a
muscle cell line, or a hematopoietic cell line. The DNA may encode
a transcription factor found in the particular cell line. In
another alternative, a desired cell line is produced from embryonic
stem cells by culturing embryonic stem cells under conditions which
provide for a three-dimensional network of embryonic stem cells,
and then stimulating embryonic stem cells with an agent, such as
retinoic acid, or dimethylsulfoxide, which promotes differentiation
of the embryonic stem cells into the desired cell line, such as,
for example, a neuronal cell line, or a muscle cell line.
Inventors: |
Dinsmore; Jonathan H.;
(Brookline, MA) ; Ratliff; Judson; (Stoneham,
MA) |
Correspondence
Address: |
CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,;STEWART & OLSTEIN
5 BECKER FARM ROAD
ROSELAND
NJ
07068
US
|
Assignee: |
Diacrin, Inc.
Charlestown
MA
|
Family ID: |
22511936 |
Appl. No.: |
12/148059 |
Filed: |
April 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11249588 |
Oct 13, 2005 |
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12148059 |
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10214903 |
Aug 8, 2002 |
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11249588 |
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08333076 |
Nov 1, 1994 |
6432711 |
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10214903 |
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08145175 |
Nov 3, 1993 |
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08333076 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
G01N 33/5005 20130101;
C12N 2501/11 20130101; C12N 5/0619 20130101; C07K 14/475 20130101;
C12N 2500/84 20130101; C12N 2501/18 20130101; C12N 2500/36
20130101; C12N 5/0068 20130101; C07K 14/715 20130101; C12N 2501/395
20130101; C12N 2501/91 20130101; C12N 2506/02 20130101; C12N
2501/39 20130101; C12N 2501/36 20130101; C12N 2500/25 20130101;
C12N 2510/00 20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Claims
1-29. (canceled)
30. An embryonic stem cell, said embryonic stem cell having been
genetically engineered with DNA which encodes MyoD, which promotes
differentiation of said embryonic stem cell into a muscle cell.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 08/145,175, filed Nov. 3, 1993.
[0002] This invention relates to embryonic stem cells. More
particularly, this invention relates to embryonic stem cells which
are engineered with DNA and/or cultured in the presence of an
agent, whereby such cells become capable of differentiating
homogeneously into a desired primary cell line. Such homogeneous
differentiation has not and cannot be achieved unless the methods
described herein are applied.
[0003] Embryonic stem cells are pluripotent cells derived from the
inner cell mass of pre-implantation embryos. (Evans et al., Nature,
Vol. 292, pgs. 154-156 (1981)). Embryonic stem cells can
differentiate into any cell type in vivo (Bradley, et al., Nature,
Vol. 309, pgs. 255-256 (1984); Nagy, et al., Development, Vol. 110,
pgs. 815-821 (1990) and into a more limited variety of cells in
vitro (Doetschman, et al., J. Embryol. Exp. Morph., Vol. 87, pgs.
27-45 (1985); Wobus, et al., Biomed. Biochim. Acta, Vol. 47, pgs.
965-973 (1988); Robbins, et al., J. Biol. Chem., Vol. 265, pgs.
11905-11909 (1990); Schmitt, et al., Genes and Development, Vol. 5,
pgs. 728-740 (1991)). Embryonic stem cells, however, are more
difficult to maintain in the laboratory and require the addition of
a differentiation inhibitory factor (commonly referred to as
leukemia inhibitory factor (or LIF) in the culture medium to
prevent spontaneous differentiation (Williams, et al., Nature, Vol.
336, pgs. 684-687 (1988); Smith, et al., Nature, Vol. 336, pgs.
688-690 (1988); Gearing, et al., Biotechnology, Vol. 7, pgs.
1157-1161 (1989); Pease, et al., Dev. Biol., Vol. 141, pgs. 344-352
(1990). LIF is a secreted protein and can be provided by
maintaining embryonic stem cells on a feeder layer of cells that
produce LIF (Evans, et al., 1981; Robertson, Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, Washington, D.C.: IRL
Press (1987)) or by the addition of purified LIF (Williams, et al.,
1988; Smith, et al., 1988; Gearing, et al., 1989; Pease, et al.,
Exp. Cell Res., Vol. 190, pgs. 209-211 (1990) to the medium in the
absence of feeder layers. Differentiation of embryonic stem cells
into a heterogeneous mixture of cells occurs spontaneously if LIF
is removed, and can be induced further by manipulation of culture
conditions (Doetschmann, et al., 1985; Wobus, et al., 1988;
Robbins, et al., 1990; Schmitt, et al., 1991; Wiles, et al.,
Development, Vol. 111, pgs. 254-267 (1991); Gutierrez-Ramos, et
al., Proc. Nat. Acad. Sci., Vol. 89, pgs. 9111-9175 (1992)).
Differentiation of stem cells into a homogeneous population,
however, has not been achieved. Embryonic stem cell differentiation
can be variable between different established embryonic stem cell
lines and even between laboratories using the same embryonic stem
cell lines.
[0004] It is an object of the present invention to provide
embryonic stem cells which are capable of differentiating uniformly
into a specific and homogeneous cell line, not achievable by
previous methods.
[0005] In accordance with an aspect of the present invention, there
is provided a method of producing a desired cell line from
embryonic stem cells. The method comprises culturing embryonic stem
cells under conditions which promote growth of the embryonic stem
cells at an optimal growth rate. The embryonic stem cells then are
cultured under conditions which promote the growth of the cells at
a rate which is less than that of the optimal growth rate, and in
the presence of an agent which promotes differentiation of the
embryonic stem cells into the desired cell line.
[0006] In general, a growth rate which is less than the optimal
growth rate, is a growth rate from about 10% to about 80%,
preferably from about 20% to about 50%, of the maximum growth rate
for embryonic stem cells. The growth rates for embryonic stem cells
can be determined from the doubling times of the embryonic stem
cells. In general, the optimum doubling time for embryonic stem
cells is from about 13 hours to about 18 hours, and more
particularly, from about 15 hours to about 16 hours.
[0007] In one embodiment, when the embryonic cells are being
cultured under conditions which promote growth of the cells at an
optimal growth rate, the embryonic stem cells are cultured in the
presence of a medium including leukemia inhibitory factor (LIF),
and serum selected from the group consisting of: (i) horse serum at
a concentration of from about 5% by volume to about 30% by volume;
and (ii) fetal bovine serum at a concentration of from about 15% by
volume to about 30% by volume. In one embodiment, the serum is
horse serum at a concentration of about 10% by volume. In another
embodiment, the serum is fetal bovine serum at a concentration of
about 15% by volume.
[0008] In yet another embodiment, when the embryonic stem cells are
cultured at an optimal growth rate, the embryonic stem cells are
cultured in the absence of a feeder layer of cells.
[0009] In one embodiment, the agent which promotes differentiation
of the embryonic stem cells is selected from the group consisting
of retinoic acid and nerve growth factor, and the desired cell line
is a neuronal cell line.
[0010] In one embodiment, in addition to culturing the cells in the
presence of the stimulating agent selected from the group
consisting of retinoic acid and nerve growth factor, the embryonic
stem cells are grown in the presence of a cytokine. Cytokines which
may be employed include, but are not limited to, Interleukin-1,
Interleukin-3, Interleukin-4, Interleukin-6, colony stimulating
factors such as M-CSF, GM-CSF, and CSF-1, steel factor, and
erythropoietin.
[0011] In a further embodiment, the agent which promotes
differentiation of the embryonic stem cells is selected from the
group consisting of dimethylsulfoxide and hexamethylene
bis-acrylamide, and the desired cell line is a muscle cell line,
such as a smooth muscle cell line, or a skeletal muscle cell line,
or a cardiac muscle cell line. In one embodiment, the agent is
dimethylsulfoxide. In another embodiment, the agent is
hexamethylene bis-acrylamide.
[0012] In one embodiment, in addition to culturing the embryonic
stem cells in the presence of an agent which promotes
differentiation of the embryonic stem cells into a muscle cell
line, the embryonic stem cells also are grown in the presence of a
cytokine, examples of which are hereinabove described.
[0013] In yet another embodiment, when the embryonic stem cells are
cultured in the presence of the agent which promotes
differentiation of the embryonic stem cells into a desired cell
line, the embryonic stem cells also are cultured in the presence of
fetal bovine serum at a concentration of about 10% by volume.
[0014] In a further embodiment, when the embryonic stem cells are
cultured in the presence of the agent which promotes
differentiation of the embryonic cells into a desired cell line,
the embryonic stem cells also are cultured in a three-dimensional
format.
[0015] Thus, Applicants have found that one may produce a
homogenous desired cell line from embryonic stem cells by culturing
the embryonic stem cells initially under conditions which favor the
growth or proliferation of such embryonic stem cells at an optimal
growth rate, and then culturing the cells under conditions which
decrease the growth rate of the cells and promote differentiation
of the cells to a desired cell type.
[0016] In a preferred embodiment, the embryonic stem cells cultured
in a standard culture medium (such as, for example, Minimal
Essential Medium), which may include supplements such as, for
example, glutamine, and .beta.-mercaptoethanol. The medium may also
include leukemia inhibitory factor (LIF), or factors with LIF
activity, such as, for example, CNTF or IL-6, and horse serum. LIF,
and factors with LIF activity, prevents spontaneous differentiation
of the embryonic stem cells, and is removed prior to the addition
of the agent. Horse serum promotes differentiation of the embryonic
stem cells into the specific cell type after the addition of the
agent to the medium. After the cells have been cultured for a
period of time sufficient to permit the cells to proliferate to a
desired number, the cells are washed free of LIF, and then cultured
under conditions which provide for the growth of the cells at a
decreased growth rate but which also promote differentiation of the
cells. The cells are cultured in the presence of an agent which
promotes or stimulates differentiation of the embryonic stem cells
into a desired cell line, and in the presence of fetal bovine serum
at a concentration of from about 5% by volume to about 10% by
volume, preferably at about 10% by volume. The presence of the
fetal bovine serum at a concentration of from about 5% by volume to
about 10% by volume, and of the agent, provides for growth or
proliferation of the cells at a rate which is less than the optimal
rate, while favoring the differentiation of the cells into a
homogeneous desired cell type. The desired cell type is dependent
upon the agent which promotes or stimulates the differentiation of
the embryonic stem cells. The embryonic stem cells also are
cultured in a three-dimensional format. Examples of such
three-dimensional culturing formats are disclosed in Doetschman, et
al., (1985), and in Rudnicki, et al., (1987).
[0017] For example, the embryonic stem cells may be placed in a
culture vessel to which the cells do not adhere. Examples of
non-adherent substrates include, but are not limited to,
polystyrene and glass. The substrate may be untreated, or may be
treated such that a negative charge is imparted to the cell culture
surface. In addition, the cells may be plated in methylcellulose in
culture media, or in normal culture media in hanging drops
(Rudnicki, et al., 1987). Media which contains methylcellulose is
viscous, and the embryonic stem cells cannot adhere to the dish.
Instead, the cells remain isolated, and proliferate, and form
aggregates.
[0018] In order to form aggregates in hanging drops of media, cells
suspended in media are spotted onto the underside of a lid of a
culture dish, and the lid then is placed on the culture vessel. The
cells, due to gravity, collect on the undersurface of the drop and
form aggregates.
[0019] In accordance with another aspect of the present invention,
there is provided an embryonic stem cell. The embryonic stem cell
has been engineered with DNA which encodes a protein or polypeptide
which promotes differentiation of the cell into a specific cell
line.
[0020] The DNA which encodes a protein or polypeptide which
promotes differentiation of the embryonic stem cell into a specific
cell line is DNA encoding a protein or polypeptide which is
normally found in the specific differentiated cell line.
Preferably, the protein or polypeptide which is present in the
specific cell line is a protein or polypeptide which generally is
not present in other types of cells.
[0021] In one embodiment, the DNA which encodes a protein or
polypeptide which promotes differentiation of the embryonic stem
cell into a specific differentiated cell line is DNA encoding a
transcription factor present in the specific cell line to promote
differentiation of the cell into the specific cell line.
[0022] In one embodiment, the DNA encoding a transcription factor
is DNA encoding a transcription factor present in neuronal cells,
and the specific cell line is a neuronal cell line.
[0023] In another embodiment, the DNA encoding a transcription
factor is DNA encoding a transcription factor, such as the MyoD
gene, present in muscle cells, and the specific cell line is a
muscle cell line.
[0024] In yet another embodiment, the DNA encoding a transcription
factor is DNA encoding a transcription factor present in
hematopoietic cells, and the specific cell line is a hematopoietic
cell line.
[0025] The DNA which encodes a protein or polypeptide which
promotes differentiation of the embryonic cell into a specific cell
line may be isolated in accordance with standard genetic
engineering techniques (such as, for example, by isolating such DNA
from a cDNA library of the specific cell line) and placed into an
appropriate expression vector, which then is transfected into
embryonic stem cells.
[0026] Appropriate expression vectors are those which may be
employed for transfecting DNA into eukaryotic cells. Such vectors
include, but are not limited to, prokaryotic vectors such as, for
example, bacterial vectors; eukaryotic vectors, such as, for
example, yeast vectors and fungal vectors; and viral vectors, such
as, but not limited to, adenoviral vectors, adeno-associated viral
vectors, and retroviral vectors. Examples of retroviral vectors
which may be employed include, but are not limited to, those
derived from Moloney Murine Leukemia Virus, Moloney Murine Sarcoma
Virus, and Rous Sarcoma Virus.
[0027] In a preferred embodiment, cDNA is synthesized from RNA
isolated by the method of Chomczynski, et al., Anal. Biochem., Vol.
162, pgs. 156-159 (1987) from cells of interest. All RNA
preparations are screened for the presence of large RNAs with gene
probes that recognize high molecular weight mRNA (i.e., greater
than 6 kb) on Northern blots. For example, all RNA preparations
from neural cells may be screened for detection of MAP2 mRNA on
Northern blots. (MAP2 is a brain specific protein present in low
abundance and coded for by a messenger RNA of about 9 kb. The
ability to detect MAP2 messenger RNA on a Northern blot is a
stringent test for the presence of intact high quality RNA.)
[0028] For cDNA synthesis, a single tube method developed by
Gubler, Nucl. Acids Res., Vol. 16, pg. 2726 (1988) is employed, and
conditions are optimized to yield the greatest amount of full
length cDNA product (about 7.5 kb in length). The cDNA is inserted
into the pcDNA3 vector (Invitrogen), which allows for expression of
the cDNA insert in mammalian cells. The pcDNA3 vector contains the
cytomegalovirus (CMV) promoter, the SV40 origin of replication, the
neomycin resistance gene for selection in eukaryotic cells, and the
ampicillin resistance gene for selection in bacteria such as E.
coli.
[0029] cDNA libraries are constructed wherein all the clones are
oriented in the proper orientation for expression. Such is achieved
by synthesizing oligo (dT) primed libraries with an oligo (dT)
primer that includes a NotI site, and after cDNA synthesis, a BstXI
adapter is ligated to the cDNA. Finally, the cDNA is digested with
NotI (an enzyme that cuts infrequently in eukaryotic genes), thus
creating a cDNA with a NotI overhang at the 3' end and a BstXI
overhang at the 5' end. The cDNA then is ligated into pcDNA3
digested with BstXI and NotI. This places the 5' end of the cDNA
downstream from the CMV promoter.
[0030] To enrich for developmentally expressed genes, libraries
from uninduced embryonic stem cells are screened with labeled cDNA
from differentiated embryonic stem cells and all cross-hybridizing
clones are eliminated from further analysis. Such method allows the
removal of those elements common to differentiated and
undifferentiated cells. Also, subtractive cDNA libraries are
constructed according to the method of Sive, et als., Nucl. Acids
Res., Vol. 16, pg. 10937 (1988). Subtractive cDNA libraries are
cDNA libraries that are enriched for genes expressed in one cell
type but not in another. The method relies on removal of common DNA
sequences through hybridization of similar DNA sequences, and then
the removal of these hybridized double-stranded DNAs. A subtractive
cDNA library that contains sequences specific for a particular cell
type derived from induced embryonic stem cells is generated. Single
stranded cDNA is synthesized from uninduced cells. To select for
those genes that are specific for the desired cell line derived
from embryonic stem cells, genes that are expressed both in the
induced cells and the non-induced embryonic stem cells are removed.
Thus, RNA which is isolated and purified from embryonic stem cells
that have differentiated into a desired cell line is hybridized to
an excess of cDNA synthesized from uninduced embryonic stem cells
to insure that all common elements are removed. RNA and cDNA common
to both the induced and uninduced embryonic stem cells will
hybridize, and these hybrids are removed. To remove double-stranded
material, cDNA from uninduced embryonic stem cells is covalently
modified with photoactivatable biotin (Sive, et al., 1988), and the
hybrid can be removed by a simple phenol extraction because the
biotin on the cDNA will cause the hybrid to partition to the phenol
phase while the non-hybridized RNA will partition to the aqueous
phase. Following this selection, RNA species found specifically in
differentiated embryonic stem cells are used to construct cDNA
libraries as hereinabove described.
[0031] Plasmid DNA containing cDNA inserts then are electroporated
into embryonic stem cells. Cells are transfected with a plasmid
that contains sequences for neomycin resistance and stable
transfectants are isolated based on neomycin resistance. Stable
transfected clones are isolated and induced with an appropriate
agent, or with leukemia inhibitory factor (LIF) withdrawal alone,
and scored for an increased ability to differentiate in response to
these induction signals. Clones also are examined to determine if
they are differentiating spontaneously in the presence of LIF.
[0032] In accordance with another aspect of the present invention,
there is provided a method of producing a desired cell line from
embryonic stem cells. The method comprises engineering embryonic
stem cells with DNA which encodes a protein or polypeptide which
promotes differentiation of the embryonic stem cells into a
specific cell line. The embryonic stem cells then are stimulated
with an agent which promotes differentiation of the embryonic stem
cells into the desired cell line.
[0033] In one embodiment, the DNA which encodes a protein or
polypeptide which promotes differentiation of the embryonic stem
cells into a specific cell line is DNA encoding a transcription
factor present in neuronal cells and said agent is selected from
the group consisting of retinoic acid and nerve growth factor. In
one alternative, the cells also may be grown in the presence of a
cytokine such as those hereinabove described.
[0034] In another embodiment, the DNA which encodes a protein or
polypeptide which promotes differentiation of the embryonic stem
cells into a specific cell line is DNA encoding a transcription
factor, such as, for example, the MyoD gene, present in muscle
cells and said agent is a bipolar agent such as dimethylsulfoxide
or hexamethylene bis-acrylamide. In one alternative, the embryonic
stem cells also may be grown in the presence of a cytokine.
[0035] The embryonic stem cells may be engineered with the DNA and
cultured under conditions hereinabove described. For example, prior
to induction, the embryonic stem cells are engineered with DNA
which encodes a protein or polypeptide which promotes
differentiation of the embryonic stem cells into a specific cell
line. Then, the embryonic stem cells may be cultured under
conditions which provide for a three-dimensional network of such
cells.
[0036] Also, it is to be understood that, within the scope of the
present invention, that the embryonic stem cells may be used for
gene therapy purposes. The embryonic stem cells may be engineered
with DNA encoding a desired therapeutic agent. Such engineering may
be accomplished by using expression vectors such as those
hereinabove described. Once the cells are engineered with DNA
encoding a desired therapeutic agent, the cells then are engineered
with DNA which encodes a protein or polypeptide which promotes
differentiation of the embryonic stem cells into a specific desired
cell line, and/or stimulated with an agent which promotes
differentiation of the embryonic stem cells into a desired cell
line. The differentiated cells then may be administered to a host,
such as a human or non-human host, as part of a gene therapy
procedure.
[0037] In addition, there is also provided within the scope of the
present invention, a method of screening embryonic stem cells for
proteins which induce differentiation of embryonic stem cells into
desired cell lines. In such method, RNA is obtained from
specifically desired cells or tissues (such as for example, brain
cells), and cDNA libraries are then constructed and placed into
expression vectors. The libraries may be normal cDNA libraries or
they may be subtractive cDNA libraries, i.e., such DNA libraries
include DNA found in the desired cells or tissues but not in other
cells or tissues. The expression vectors are then transfected into
eukaryotic cells, such as COS cells. The cell culture supernatant
then may be applied to embryonic stem cell cultures to determine if
any secreted proteins from such cells induce differentiation of
embryonic stem cells to a specific cell type. The cDNA from cells
which induce differentiation of embryonic stem cells to a specific
cell type then is evaluated further in order to determine which
individual clones of such cDNA libraries induce differentiation of
embryonic stem cells to a specific cell type. Once a specific cDNA
which induces differentiation of embryonic stem cells to a desired
cell type is identified, such cDNA then may be isolated and cloned
into an appropriate expression vector, which may be transfected
into undifferentiated embryonic stem cells or the expressed,
purified protein may be added directly to cultured embryonic stem
cells.
[0038] In one embodiment, such screening may be carried out by
pooling bacterial clones, from the cDNA library prepared as
hereinabove described, into groups of 1,000, and isolating plasmid
DNA from the pooled clones. The plasmid DNA's then are
electroporated into COS cells, such as COS-7 cells, for expression.
After allowing from 48 to 72 hours for expression of transfected
genes, tissue culture supernatant from transfected COS cells is
harvested and applied to embryonic stem cells to determine if any
secreted proteins from the COS cells can induce differentiation of
embryonic stem cells. Supernatants from mock transfected cells
(cells transfected with the plasmid alone) are tested in parallel
to control for any non-specific effects of COS cell derived
proteins. Embryonic stem cell differentiation may be screened by
several means: (i) by microscopic observation of overt changes in
embryonic stem cell morphology; (ii) by measuring changes in neuron
specific gene expression on Northern blots with probes to neuron
specific markers such as neuron specific enolase, GAP-43, and MAP2;
and (iii) by loss of expression of a carbohydrate surface marker
present only on undifferentiated stem cells recognized by the
monoclonal antibody SSEA-1 (Ozawa, et al., Cell. Diff., Vol. 16,
pp. 169-173 (1985)).
[0039] When a pool has been identified that expresses inducing
capacity, that pool of cDNA clones is broken down further into
smaller pools of 100 clones, and these sub-pools are transfected
into COS cells. Supernatants are screened for inducing activity.
Once appropriate sub-pools are identified, the clones are plated in
96 well dishes, and rows and columns are combined. The pooled
columns and rows then are transfected into COS cells, and
supernatants again are screened for activity. By analyzing the
columns and rows that exhibit activity, the exact clone expressing
inducing activity can be identified. This clone then is tested for
ability to induce differentiation. After initial identification of
potential factors, full-length cDNA clones are isolated and
sequenced. Sequenced clones then are compared to other cloned genes
in the DNA data base for homology or identity with previously
cloned genes. Once a novel gene is identified, the gene is cloned
into a stable expression system, the protein is purified, and its
biological activity is tested. Sequencing of DNA is performed by
standard protocols. Biologically active protein is prepared by
standard chromatographic methods.
[0040] Alternatively, cDNA from differentiating embryonic stem
cells or from embryonic organs and brain regions can be introduced
directly into embryonic stem cells, and embryonic stem cell
supernatants are screened for inducing activity.
[0041] The differentiated stem cells may be employed by means known
to those skilled in the art to treat a variety of diseases or
injuries. For example, stem cells which have differentiated into
neuronal cells may be administered to a patient, such as, for
example, by transplanting such cells into a patient, to treat
diseases such as Huntington's disease, Parkinson's disease, and
Alzheimer's disease. Such neuronal cells also may be employed to
treat spinal cord injuries or chronic pain. Also, stem cells which
have differentiated into muscle cells may be employed in treating
muscular dystrophy, cardiomyopathy, congestive heart failure, and
myocardial infarction, for example.
[0042] The invention will now be described with respect to the
following examples; however, the scope of the present invention is
not intended to be limited thereby.
EXAMPLE 1
[0043] Undifferentiated embryonic stem cells (ES-E14TG2a, purchased
from the American Type Culture Collection, catalog no. ATCC CRL.
1821) are maintained in Dulbecco's modified Minimal Essential
Medium (DMEM) supplemented with glutamine, .beta.-mercaptoethanol,
10% (by volume) horse serum, and human recombinant Leukemia
Inhibitory Factor (LIF). The LIF replaces the need for maintaining
embryonic stem cells on feeder layers of cells, and is essential
for maintaining embryonic stem cells in an undifferentiated
state.
[0044] In order to promote the differentiation of the embryonic
stem cells into neuronal cells, the embryonic stem cells are
trypsinized and washed free of LIF, and placed in DMEM supplemented
with 10% (by volume) fetal bovine serum (FBS). After resuspension
in DMEM and 10% FBS, 1.times.10.sup.6 cells are plated in 5 ml DMEM
plus 10% FBS plus 0.5 .mu.M retinoic acid in a 60 mm Fisher brand
bacteriological grade Petri dish. In such Petri dishes, embryonic
stem cells cannot adhere to the dish, and instead adhere to each
other, thus forming small aggregates of cells. Aggregation of cells
aids in enabling proper cell differentiation. After two days,
aggregates of cells are collected and resuspended in fresh DMEM
plus 10% FBS plus 0.5 .mu.M retinoic acid, and replated in Petri
dishes for an additional two days. Aggregates, now induced four
days with retinoic acid, are trypsinized to form a single-cell
suspension, and plated in serum free medium on poly-D-lysine-coated
tissue culture grade dishes. The stem cell medium is formulated
with Kaighn's modified Ham's F12 as the basal medium with the
following supplements added:
[0045] 15 .mu.g/ml ascorbic acid
[0046] 0.25% (by volume) calf serum
[0047] 6.25 .mu.g/ml insulin
[0048] 6.25 .mu.g/ml transferrin
[0049] 6.25 ng/ml selenous acid
[0050] 5.35 .mu.g/ml linoleic acid
[0051] 30 pg/ml thyroxine (T3)
[0052] 3.7 ng/ml hydrocortisone
[0053] 1. ng/ml Heparin 10 ng/ml somatostatin
[0054] 10 ng/ml Gly-His-Lys (liver cell growth factor)
[0055] 0.1 .mu.g/ml epidermal growth factor (EGF)
[0056] 50 .mu.g/ml bovine pituitary extract (BPE)
[0057] Such medium provides for consistent differentiation of the
stem cells into neuronal cells, and provides for survival of the
neuronal cells for a period of time greater than 3 days, and
selectively removes dividing non-neuronal cells from the
population. The poly-D-lysine promotes the attachment of the
neuronal cells to the tissue culture plastic, and prevents
detachment of the cells from the dish and the forming of floating
aggregates of cells. The cells are cultured for 5 days. Upon
culturing of the cells in the above medium, a culture of cells in
which greater than 90% of the cells are neuronal cells is obtained.
Such neuronal cells, which express the neurotransmitter gamma amino
butyric acid (GABA), then may be employed for the treatment of the
neural degeneration disease Huntington's disease. Through genetic
engineering, these cells can be directed to express dopamine (for
the treatment of Parkinson's disease) or acetylcholine (for the
treatment of Alzheimer's disease).
EXAMPLE 2
[0058] Undifferentiated embryonic stem cells (ES-D3, purchased from
the American Type Culture Collection as ATCC catalog no. ATCC CRL
1934) are maintained in supplemented Dulbecco's modified Minimal
Essential Medium as described in Example 1. The embryonic stem
cells then are trypsinized and washed free of LIF and placed in 1%
(by volume) dimethylsulfoxide in DMEM plus 10% horse serum. Two
days after the addition of dimethylsulfoxide and plating of cells
in Petri dishes to form aggregates, the aggregates are collected
and resuspended in fresh medium plus 1% dimethylsulfoxide. The
aggregates are then plated onto multi-well untreated culture grade
dishes without trypsin treatment. One aggregate is plated per well.
The aggregates are cultured for 5 days. Upon culturing of the cells
in multi-well dishes, cell cultures in which greater than 80% of
the aggregates contain contracting muscle cells are obtained. Such
cells may be used to treat cardiomyopathies, myocardial infarction,
congestive heart failure, or muscular dystrophy.
EXAMPLE 3
Transfection of Embryonic Stem Cells with Mouse MyoD cDNA
[0059] For transfection of embryonic stem cells with mouse MyoD
cDNA, both the D3 (ATCC catalog no. CRL 1934) and E14 TG2a (ATCC
catalog no. CRL 1821) embryonic stem cell lines were used.
Embryonic stem cells were cultured as described in Robertson, 1987,
except that the cells were maintained in media containing 5 ng/ml
human recombinant leukemia inhibitory factor instead of on feeder
layers.
[0060] Embryonic stem cells were co-transfected with pKJ1-Neo
(Dinsmore, et al., Cell, Vol. 64, pgs. 817-826 (1991)), which
carries the neomycin resistance gene for selection of stable
transfectants, and with pEMCII (Davis, et al., Cell, Vol. 51, pgs.
987-1000 (1987)), which contains a portion of the mouse MyoD cDNA.
pKJ1-Neo was linearized at the unique NsiI site and pEMCII was
linearized at the unique ScaI site. In order to introduce the
linearized plasmids into the embryonic stem cells, the embryonic
stem cells were electroporated using a Gene Pulser (Bio Rad) in 0.4
cm gap distance electroporation cuvettes with the Gene Pulser set
at 240 volts, 500.mu. Farads. For electroporation, 8.times.10.sup.6
embryonic stem cells were suspended in 1 ml of HEPES-buffered
saline (25 mM HEPES, 134 mM NaCl, 5 mM KCl, 0.7 mM
Na.sub.2HPO.sub.4, pH 7.1) with 2 .mu.g of linearized pKJ1-Neo and
20 .mu.g pEMCII. After electroporation, the cells were plated at
5-7.times.10.sup.5 per 35 mm gelatin coated culture dish in growth
medium containing recombinant human leukemia inhibitory factor. The
cells were allowed to grow for 36 hours and then Geneticin
(Gibco-BRL), a commercial brand of neomycin, was added to the
medium at a concentration of 400 .mu.g/ml. The media containing the
Geneticin was changed daily until clones of neomycin resistant
cells could be identified (7 days after Geneticin addition).
Individual neomycin resistant clones were isolated using glass
cloning cylinders (Bellco).
[0061] Stable transfectants were isolated, expanded, frozen, and
then stored in liquid nitrogen. 35 independent stably transfected
embryonic stem cell lines were isolated. Ten of these cell lines
have been analyzed, and have been found to express different
amounts of MyoD as detected by Northern blots. Embryonic stem cell
lines that were found to express high levels of MyoD RNA were found
to have embryonic stem cells in the population that spontaneously
differentiated into muscle cells as assessed by the staining of
cells with a muscle specific myosin antibody. Those cell lines
which showed high levels of MyoD expression were characterized
further by inducing differentiation with dimethylsulfoxide. Cell
lines which expressed high amounts of MyoD differentiated almost
exclusively into skeletal muscle after dimethylsulfoxide induction.
The percentage of cells that differentiated into skeletal muscle
was greater than 90% as assessed by staining for muscle specific
myosin, and by the ability of these cells to fuse and form myotubes
that spontaneously twitch. In contrast, MyoD transformants that
expressed very low amounts of MyoD differentiated into a mix of
cardiac, smooth, and skeletal muscle indistinguishable from that
derived from non-transfected embryonic stem cells. Additionally,
there was no detectable difference between the D3 and E14 embryonic
stem cell lines for MyoD expression or differentiation.
[0062] It is to be understood, however, that the scope of the
present invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
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