U.S. patent application number 15/684617 was filed with the patent office on 2018-04-26 for high-throughput image-based chemical screening in zebrafish blastomere cell culture.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The applicant listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION, JOSLIN DIABETES CENTER INC., PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to C. Ronald KAHN, Amy J. WAGERS, Cong XU, Leonard I. ZON.
Application Number | 20180112182 15/684617 |
Document ID | / |
Family ID | 49161817 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180112182 |
Kind Code |
A1 |
ZON; Leonard I. ; et
al. |
April 26, 2018 |
HIGH-THROUGHPUT IMAGE-BASED CHEMICAL SCREENING IN ZEBRAFISH
BLASTOMERE CELL CULTURE
Abstract
Disclosed are methods of inducing differentiation of stem into
myogenic cells without gene manipulation and for inducing
proliferation of satellite cells. The cells can be used as a source
of cells for transplantation in a subject in need thereof. Also
disclosed is a screening assay for screening test compounds using
blastomere cultures.
Inventors: |
ZON; Leonard I.; (Wellesley,
MA) ; XU; Cong; (Boston, MA) ; WAGERS; Amy
J.; (Cambridge, MA) ; KAHN; C. Ronald;
(Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION
PRESIDENT AND FELLOWS OF HARVARD COLLEGE
JOSLIN DIABETES CENTER INC. |
Boston
Cambridge
Boston |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Cambridge
MA
JOSLIN DIABETES CENTER INC.
Boston
MA
|
Family ID: |
49161817 |
Appl. No.: |
15/684617 |
Filed: |
August 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14384514 |
Sep 11, 2014 |
9771560 |
|
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PCT/US2013/031504 |
Mar 14, 2013 |
|
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15684617 |
|
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|
|
61610668 |
Mar 14, 2012 |
|
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61720713 |
Oct 31, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/471 20180101;
C12N 2506/02 20130101; Y02A 50/30 20180101; A61P 21/00 20180101;
C12N 2501/16 20130101; G01N 33/5044 20130101; A61K 35/545 20130101;
A61K 38/1808 20130101; A61K 38/28 20130101; A61K 38/1825 20130101;
A61K 38/30 20130101; C12N 2501/01 20130101; C12N 2501/999 20130101;
C12N 5/0658 20130101; C12N 2506/45 20130101; A61K 38/1841 20130101;
A61K 38/1858 20130101; G01N 33/56983 20130101; C12N 2501/115
20130101; A61K 38/1825 20130101; A61K 2300/00 20130101; A61K
38/1808 20130101; A61K 2300/00 20130101; A61K 38/1841 20130101;
A61K 2300/00 20130101; A61K 38/1858 20130101; A61K 2300/00
20130101; A61K 38/28 20130101; A61K 2300/00 20130101; A61K 38/30
20130101; A61K 2300/00 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; A61K 38/18 20060101 A61K038/18; G01N 33/569 20060101
G01N033/569; G01N 33/50 20060101 G01N033/50; A61K 35/545 20060101
A61K035/545; A61K 38/30 20060101 A61K038/30; A61K 38/28 20060101
A61K038/28 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
nos. 5P30 DK49216-19, 5R01CA103846-10, DP2OD004345 and DK31036
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A composition comprising: at least two of: (i) a GSK3 pathway
inhibitor; (ii) a compound that increases intracellular levels of
3',5'-cyclic adenosine monophosphate (cAMP); and (iii) a FGF
pathway activator.
2. The composition of claim 1, wherein the composition further
comprises a stem cell.
3. The composition of claim 1, wherein the composition is a cell
culture media.
4. A reagent kit for inducing differentiation from a pluripotent
cell to a skeletal muscle progenitor cell, wherein the kit
comprises: at least two of: (i) a GSK3 pathway inhibitor; (ii) a
compound that increases intracellular levels of 3',5'-cyclic
adenosine monophosphate (cAMP); and (iii) a FGF pathway
activator.
5. The kit of claim 4, wherein the kit further comprises a stem
cell.
6. A method of increasing satellite cell proliferation, the method
comprising: culturing a satellite cell in the presence of a
compound that increases intracellular levels of 3',5'-cyclic
adenosine monophosphate.
7. The method of claim 6, wherein the compound is an activator of
adenylyl cyclase.
8. A method of treating a subject for damaged muscle tissue or for
increasing muscle mass, the method comprising co-administering to
the subject in need thereof at least two of: (i) a GSK3 pathway
inhibitor; (ii) a compound that increases intracellular levels of
3',5'-cyclic adenosine monophosphate (cAMP); and (iii) a FGF
pathway activator.
9. A screening assay comprising culturing a blastomere in presence
of a test compound, wherein the blastomere comprises a reporter
gene, wherein the reporter gene encodes a cell lineage specific
marker and produces a detectable signal when expressed; and
measuring/detecting the detectable signal, wherein a change in
level or amount of the detectable signal indicating that the test
compound modulates the development or function of cell lineage that
express the cell lineage specific marker.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 14/384,514 filed Sep. 11, 2014, which is a 35 U.S.C. .sctn. 371
National Stage Entry Application of International Application No.
PCT/US2013/031504 filed Mar. 14, 2013, which designates the United
States, and which claims benefit under 35 U.S.C. .sctn. 119(e) of
the U.S. Provisional Application No. 61/610,668, filed Mar. 14,
2012 and the U.S. Provisional Application No. 61/720,713, filed
Oct. 31, 2012, content of both of which is incorporated herein by
reference in their entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 23, 2017 is named 701039-073626-DIV_SL.txt and is 10, 204
bytes.
TECHNICAL FIELD
[0004] The disclosure relates generally to compositions and methods
for inducing differentiation of stem cells into myogenic cells and
uses thereof. More specifically, the disclosure is concerned with
compositions and methods of inducing differentiation of induced
pluripotent stem cells (iPSCs and embryonic stem cells (ESCs) into
myogenic cells. The present disclosure also provides methods for
inducing proliferation of satellite cells and uses thereof. The
present disclosure also provides screening assays for assaying
modulators of organ development.
BACKGROUND
[0005] Skeletal muscle is a highly specialized tissue composed of
non-dividing, multi-nucleated muscle fibers that contract to
generate force in a controlled and directed manner. Skeletal muscle
is formed during embryogenesis from a subset of muscle precursor
cells found in a region of the embryo known as the myotome. In
addition to generating differentiated muscle fibers, these cells
also give rise to specialized muscle-forming stem cells, known as
satellite cells, which remain associated with muscle fibers and are
responsible for muscle growth and repair throughout life (Gros et
al., 2006; Seale et al., 2000). Injury-induced satellite cell
proliferation both replenishes the satellite cell pool and produces
differentiated myoblasts which fuse with existing myofibers and
with one another to regenerate muscle tissue.
[0006] Satellite cells are defined anatomically by their
localization beneath the basal lamina of muscle fibers (Mauro,
1961) and molecularly by their expression of the paired-box
transcription factor Pax7 (Seale et al., 2000). In resting muscle,
satellite cells are maintained in a largely dormant state, but in
response to muscle damage, these cells become activated, an event
marked by their upregulation of MyoD, and enter the cell cycle
(Seale et al., 2000). Transplantation-based studies in animal
models have demonstrated the utility of engrafted satellite cells
for regenerating diseased muscle (Cerletti et al., 2008; Sherwood
et al., 2004) and analyses of mouse and human muscles indicate that
their loss during aging contributes to age-associated muscle
weakness (Cerletti et al., 2012). Thus, muscle satellite cells are
promising targets for cell therapies involving either cell
replacement or activation of endogenous repair mechanisms. However,
realization of this promise has been hindered by the paucity of
satellite cells that can be isolated from adult skeletal muscle and
a lack of methods to support their ex vivo expansion.
[0007] In contrast to satellite cells, embryonic stem cells (ESCs)
and, more recently, iPSCs process boundless expansion potential in
culture and are theoretically capable of generating an unlimited
supply of differentiated cell types, including myogenic cells.
Although some success has been achieved in directing the myogenic
differentiation of ESCs/IPSCs, largely through genetic manipulation
and cell sorting approaches (Barberi et al., 2007; Darabi et al.,
2008; Mizuno et al., 2010; Zheng et al., 2006), the generation of
well differentiated muscle cells from human or murine pluripotent
cells has proved very challenging. Thus, to realize the promise of
stem cell approaches or muscle biology and regeneration, it is
essential to uncover the molecular pathways that regulate the
myogenic specification of these cells, and to develop systems that
enable their robust and selective differentiation in ex vivo
systems.
SUMMARY
[0008] The present disclosure relates to the differentiation of
stem cells including embryonic stem cells and induced pluripotent
stem cells. More specifically, the present disclosure is concerned
with methods of inducing differentiation of stem cells into
myogenic cells.
[0009] In one aspect, there is provided a method of inducing
differentiation of stem cells in myogenic cells comprising,
culturing the cells in a medium comprising at least two of: (i) a
GSK3 pathway inhibitor; (ii) a compound that increases
intracellular levels of 3',5'-cyclic adenosine monophosphate
(cAMP); and (iii) a FGF pathway activator.
[0010] Also provided is a method of converting stem cells into
multipotent stem cells capable of giving rise to myogenic cells,
the method comprising culturing the stem cells in the presence of
at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that
increases intracellular levels cAMP; and (iii) a FGF pathway
activator.
[0011] In another aspect, the present invention provides a method
of conditioning stem cells for further differentiation into
myogenic cells comprising culturing the cells in in the presence of
at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that
increases intracellular levels of cAMP; and (iii) a FGF pathway
activator.
[0012] In one embodiment, the myogenic cells are cells of the
skeletal myogenic lineage.
[0013] In one embodiment, the myogenic cells are terminally
differentiated skeletal muscle cells.
[0014] In a further aspect, the disclosure provides a myogenic cell
prepared in accordance with the method disclosed herein. In an
embodiment, the myogenic cell is for implantation into a subject
for increasing muscle mass or preventing or treating a muscle
disease, the stem cell having been subjected to a differentiation
treatment according to the method disclosed herein prior to
implantation to convert (i.e., transform/differentiate) the stem
cell into a myogenic cell. The present invention also concerns a
method of transplanting myogenic cells in a subject comprising
implanting in the subject myogenic cells prepared in accordance
with the method disclosed herein.
[0015] In another aspect, the disclosure provides a method of
increasing muscle mass or of preventing or treating a muscle
disease in a subject comprising implanting into the subject
myogenic cells prepared in accordance with the method disclosed
herein.
[0016] In another aspect, the disclosure provides a use of the
myogenic cells prepared according to the method disclosed herein,
for transplantation into a subject.
[0017] In another aspect, the disclosure provides a use of the
myogenic cells prepared according to the method disclosed herein,
for increasing muscle mass or preventing or treating a muscle
disease in a subject.
[0018] In one aspect, there is provided a method of increasing
satellite cell proliferation, the method comprising: culturing a
satellite cell in the presence of a compound that increases
intracellular levels of 3',5'-cyclic adenosine monophosphate.
[0019] In a further aspect, the disclosure provides a satellite
cell prepared in accordance with the method disclosed herein. In an
embodiment, the satellite cell is for implantation into a subject
for increasing muscle mass or preventing or treating a muscle
disease, the satellite cell having been subjected to a
proliferation treatment according to the method disclosed herein
prior to implantation to convert (i.e., transform/differentiate).
The present invention also concerns a method of transplanting
satellite cells in a subject comprising implanting in the subject
satellite cells prepared in accordance with the method disclosed
herein.
[0020] In another aspect, the disclosure provides a method of
increasing muscle mass or of preventing or treating a muscle
disease in a subject comprising implanting into the subject
satellite cells prepared in accordance with the method disclosed
herein.
[0021] In another aspect, the disclosure provides a use of the
satellite cells prepared according to the method disclosed herein,
for transplantation into a subject.
[0022] In another aspect, the disclosure provides a use of the
satellite cells prepared according to the method disclosed herein,
for increasing muscle mass or preventing or treating a muscle
disease in a subject.
[0023] In another aspect, the disclosure provides a composition for
inducing differentiation of stem cells to myogenic cells, the
composition comprising at least two of: (i) a GSK3 pathway
inhibitor; (ii) a compound that increases intracellular levels of
cAMP; and (iii) a FGF pathway activator.
[0024] The disclosure also provides a method of treating subject
for damaged muscle tissue or increasing muscle mass, the method
comprising co-administering to the subject in need thereof at least
two of: (i) a GSK3 pathway inhibitor; (ii) a compound that
increases intracellular levels of cAMP; and (iii) a FGF pathway
activator.
[0025] In one aspect provided herein is a method for screening
chemical compounds or compositions in zebrafish blastomere cells
comprising the steps of culturing a population of zebrafish
blastomere cells in presence of a test compound, wherein the
blastomere cell comprises a cell lineage specific marker (CLSM)
fused with a fluorescent protein (i.e., a CSLM::FP construct); and
visualizing the FP. A change in level or amount of FP indicating
that the test compound modulates the development of organ that
expresses the cell lineage specific marker. The level or expression
of FP can be determined relative to a reference or control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1E show the chemical genetic screen to identify
modifiers of skeletal muscle development. FIG. 1A shows that
myf5-GFP; mylz2-mCherry double transgenic expression recapitulates
endogenous expression of the genes respectively. myf5-GFP is first
detected at the 11-somite stage in the newly formed somite. The
yolk of the double transgenic embryo is mCherry positive,
presumptively due to the maternal deposit of mCherry protein.
mylz2-mCherry expression is not observed until 32 hpf. Its
expression is first detected in anterior somites and then
progressively spreads to posterior somites. FIG. 1B shows myf5-GFP;
mylz2-mCherry embryos were disassociated at the oblong stage and
cultured in zESC medium with or without bFGF. Images were taken 48
hours after plating. Scale bars represent 250 .mu.M. FIG. 1C shows
the expression of myogenic genes measured by quantitative RT-PCR.
Blastomere cells from myf5-GFP; mylz2-mCherry embryos were cultured
with or without bFGF and harvested at 24 hours. Results of bFGF
treated cells are normalized to the expression of untreated cells.
Error bars represent standard deviation (SD) from the triplicate
quantitative RT-PCR reaction. All expressions are normalized to
.beta.-actin. FIG. 1D shows the expression of myogenic genes
measured by quantitative RT-PCR. Blastomere cells from myf5-GFP;
mylz2-mCherry embryos were cultured with bFGF and harvested at 24
hours. Different populations were isolated by FACS. Results are
normalized to the expression of cells of the double negative
population. Error bars represent standard deviation (SD) from the
triplicate quantitative RT-PCR reaction. All expressions are
normalized to .beta.-actin. FIG. 1E shows that ATRA blocks muscle
development in vitro. myf5-GFP; mylz2-mCherry embryos were
disassociated at the oblong stage and cultured in zESC medium with
bFGF. 0.1% DMSO or 100 nM ATRA was added immediately. Images were
taken 48 hours after plating. Scale bars represent 250 .mu.M.
[0027] FIGS. 2A-2C show the chemical genetic screens to identify
modifiers of skeletal muscle development. FIG. 2A is a schematic of
a high-throughput image-based chemical screening assay.
Approximately 800 myf5-GFP; mylz2-mCherry double transgenic embryos
were collected and disassociated at the oblong stage. The resulting
blastomere cells were aliquoted into four 384-well plates with
pre-added chemicals. We performed duplicates for each chemical to
mitigate false positives. After 2 days, the 384-well plates were
imaged using a Celigo cytometer. For each well, an image for each
signal channel was captured. GFP and mCherry signals were
determined by the Celigo cytometer's built-in software and
confirmed by eye. FIG. 2B shows sample images from the modifier
screening. Most of the hits could be grouped into three categories.
Category I has only myf5-GFP expression, examples being TCPOBOP and
CA-074-Me. The phenotype is presumptively due to inhibition of
differentiation of muscle progenitors into mature muscle. Category
II has a decreased amount of both fluorescent colors, examples
being Ro 41-1049 and tyrphostin AG 879. This phenotype is
presumptively due to the blocking of muscle progenitor commitment.
Category III has an increased expression of both markers presumably
due to accelerated muscle development. Scale bars represent 250
.mu.M. FIG. 2C shows hits from a screen without supplementing bFGF
to the culture medium. 6 chemicals were identified to increase the
GFP and mCherry signals. Scale bars represent 250 .mu.M.
[0028] FIGS. 3A-3F show that forskolin treatment elevates cAMP
level and increases proliferation of satellite cells from both
healthy and dystrophic mice. FIG. 3A, satellite cells from C57BL/6J
mice were cultured and treated with DMSO or forskolin in the
absence or presence of basic fibroblast growth factor (bFGF). Total
number of cells were counted after 5 days and are shown as fold
changes compared to the DMSO treated cells in the absence of bFGF
(mean+/-SEM, n=4). Forskolin expands satellite cells by .about.2 or
.about.2.3 folds compared to the DMSO treated cells in the absence
or presence of bFGF, respectively. FIG. 3B shows fold change of
number of satellite cells from mdx mice after 5 days in culture and
forskolin treatment as compared to the DMSO treated satellite
cells. Forskolin expands mdx satellite cells by .about.3.6 folds in
culture (mean+/-SEM, n=4). FIG. 3C shows that concentration of cAMP
in cultured satellite cells is increased by .about.4.5, .about.5.2
or .about.5.7 fold after treatment with 25 .mu.M, 50 .mu.M or 100
.mu.M of forskolin, respectively as compared to the DMSO treated
cells. (mean+/-SD, n=5). FIG. 3D, satellite cells were isolated
from C57BL/6J mice and a single cell was plated into each well of
96-well plates. Cells were cultured for 6 days and treated with
DMSO or forskolin. Number of wells containing a myogenic colony and
number of cells in each colony were counted after 6 days. FIG. 3E
shows that in vitro clonal plating efficiency of satellite cells is
not affected by forskolin treatment as compared to DMSO-treated
cells (mean+/-SD, n=4). FIG. 3F shows that forskolin treatment
increases the number of myogenic cells originated from a single
satellite cell in each well, showing an increase in cell
proliferation. .star-solid.: P<0.05, .star-solid..star-solid.:
P<0.01 (mean+/-SD, n=4).
[0029] FIGS. 4A-4F show that forskolin-treated satellite cells do
not exhibit defective differentiation in vitro, regardless of the
timing of compound exposure. FIG. 4A, satellite cells from C57BL/6J
mice were cultured in the presence of bFGF for 5 days. In order to
test the effect of forskolin on in vitro differentiation of
satellite cells, the cells were harvested on day 5 and equal
numbers of cells were induced to differentiation in the presence of
forskolin or DMSO. FIG. 4B shows pictures of satellite cells
differentiated in the presence of DMSO (left) or forskolin (right)
and stained for Myosin Heavy Chain (MHC, red) and nuclei (blue).
Scale bar: 200 .mu.m. FIG. 4C shows quantification of percentage of
nuclei in myotubes after differentiation of satellite cells in the
presence of forskolin or DMSO (mean+/-SEM, n=4). Differentiation
potential of satellite cells is not affected by presence of
forskolin in the medium as compared to DMSO. FIG. 4D, satellite
cells from C57BL/6J mice were cultured in the presence of bFGF and
forskolin/DMSO treatment for 5 days. In order to test
differentiation potential of forskolin treated satellite cells, the
cells were harvested on day 5 and equal numbers of cells were
induced to differentiation in the absence of the compound. FIG. 4E
shows pictures of DMSO (left) or forskolin (right) treated
satellite cells differentiated in the absence of compound and
stained for MHC (red) and nuclei (blue). Scale bar: 200 .mu.m. FIG.
4F shows the quantification of percentage of nuclei in myotubes
after differentiation of forskolin or DMSO treated cells
(mean+/-SEM, n=5). Forskolin-treated satellite cells don't show any
defect in in vitro myotube formation as compared to DMSO treated
cells.
[0030] FIGS. 5A-5E shows that forskolin-treated cultured satellite
cells retain immunophenotypic characteristics of freshly isolated
satellite cells and engraft into skeletal muscle in vivo. FIG. 5A
shows representative FACS plots depicting
CD45.sup.-SCA-1.sup.--MAC1.sup.- cells and show gating of
CXCR4.sup.+ and .beta.-1 Integrin.sup.+ cells for freshly isolated
(left panel) and cultured satellite cells treated with DMSO (middle
panel) or forskolin (right panel). FIG. 5B shows the average
frequency (mean+/-SEM, n=6) of CXCR4.sup.+ .beta.-1 Integrin.sup.+
cells among cultured satellite cells treated with DMSO or forskolin
was quantified by FACS. Results indicate that cultured satellite
cells treated with either DMSO or forskolin retain expression of
cell surface markers CXCR4 and .beta.-1 Integrin by .about.80%.
FIG. 5C, GFP.sup.+ satellite cells were harvested from .beta.-actin
GFP mice and were transplanted into TA muscle of recipient mdx
mice, injured 1 day previously by injection of cardiotoxin, either
right after isolation or after five days in culture with DMSO or
forskolin treatment. FIG. 5D shows the quantification of donor
derived myofibers in mdx muscle transplanted with freshly isolated,
cultured DMSO-treated or cultured forskolin-treated satellite cells
(n=6). FIG. 5E shows transverse frozen section of TA muscle from
mdx mice transplanted with 6000 freshly isolated satellite cells
(left panel), cultured DMSO-treated satellite cells expanded from
6000 freshly isolated cells (middle panel) or cultured
forskolin-treated satellite cells expanded from 6000 freshly
isolated cells (right panel). Scale bars represent 200 am.
[0031] FIGS. 6A-6E shows that treatment of human iPSCs with muscle
promoting cocktail induces skeletal muscle differentiation. FIGS.
6A and 6B show the gene expression analysis of EBs (FIG. 6A) and
monolayer cells (FIG. 6B). Cells were harvested at the times
indicated for RNA extraction. Ectodermal, endodermal and mesodermal
genes were analyzed by quantitative RT-PCR using GAPDH as a
housekeeping gene. Changes in gene expression levels are expressed
relative to undifferentiated iPSCs (Day 0). Bars represent the
standard deviation of three independent experiments (*p<0.05,
**<0.01, ***<0.001). FIG. 6C shows the immunostaining of
differentiated iPSCs. Under terminal differentiating procedures
(day 36) most of the cells express Desmin (red) and Myogenin
(green), forming multinucleated myofibers. Cells were also stained
with Hoechst (blue). The number on each panel represents the
percentage of cells expressing Desmin and Myogenin. Scale bars
represent 100 .mu.M. FIG. 6D shows representative electron
microscopy image of differentiated BJ cells at day 36,
magnification .times.52700. FIG. 6E shows immuno-electron
microscopy staining using skeletal muscle specific anti-myosin
heavy and light chain. Black dots indicate gold cross-linked
particles to secondary antibody, magnification .times.52700.
[0032] FIG. 7 shows engraftment of human iPSC-derived muscle
progenitors into immune-compromised mice. Representative images of
immunostaining and Hematoxylin/Eosin on tibialis anterior (TA)
sections of injured-NSG mice injected with 1.times.10.sup.5 iPSCs
at day 12 of differentiation. BJ, 00409, 05400 lines showed
positivity for human 6-Sarcoglycan (red) protein indicating
engraftment. No staining is observed in PBS injected mice and when
a fibroblast line was transplanted. Scale bars represent 100 .mu.M.
n=3 per sample.
[0033] FIGS. 8A-8C show that myogenesis suppressors identified in
vitro also affect muscle development in vivo (related to FIG. 1).
FIG. 8A shows the higher magnification of myf5-GFP; mylz2-mCherry
blastomere cells cultured in the zESC medium with bFGF. FIG. 8B
shows myf5-GFP; mylz2-mCherry embryos injected with 400 pg con-MO
or co-injected with 200 pg myf5-MO and 200 pg myoD-MO lack both
myf5-GFP and mylz2-mCherry expression. Images were taken at 30 hpf
FIG. 8C shows myf5-GFP; mylz2-mCherry embryos injected with 400 pg
con-MO or co-injected with 200 pg myf5-MO and 200 pg myoD-MO were
disassociated at the oblong stage and cultured in zESC medium with
bFGF. No myf5-GFP or mylz2-mCherry expression was detected in the
culture of double morphants. Images were taken 26 hours after
plating.
[0034] FIGS. 9A-9C show the chemical genetics screens to identify
modifiers of skeletal muscle development (related to FIG. 2) FIG.
9A, Embryos were treated with hits at the sphere stage. Once the
control embryos reached the 6-somite stage, they were collected for
whole-mount in situ hybridization with the myoD riboprobe. Examples
of hits verified to perturb muscle development in vivo. Flat
mounted embryos stained with the myoD riboprobe at the 6-somite
stage are shown with anterior to the left. Embryos were treated
with (.+-.)-6-Chloro-PB hydrobromide, LY-294002, 1-ACYL-PAF, or
ATRA. All chemicals were from the CHB library and were diluted
300-fold. The level of myoD reduction was determined by comparing
the intensity of myoD expression in these embryos with that in
control embryos treated with DMSO. FIG. 9B, myf5-GFP; mylz2-mCherry
embryos were collected at the oblong stage and disassociated. The
resulting blastomere cells were aliquoted into a 96-well plate with
pre-added chemicals. The culture medium contained 10 ng/ml bFGF to
promote muscle development. Cells were treated with 50 .mu.M
tyrphostin AG 879, 50 .mu.M tyrphostin AG 808, 50 .mu.M tyrphostin
AG 555, 50 .mu.M PD 98059, 10 .mu.M U0126, 10 .mu.M LY-294002, 10
.mu.M wortmannin, or 20 .mu.M rapamycin. Cells were imaged after 2
days by the Celigo cytometer for GFP, mCherry, and bright-field
signals. FIG. 9C, 1-cell-stage myf5-GFP; mylz2-m Cherry embryos
were injected with 250 pg fgf8-MO and 250 pg fgf24-MO and rescued
by adding 10 .mu.M forskolin at the dome stage.
[0035] FIGS. 10A and 10B show that forskolin treatment restores
proliferation of mdx satellite cells and transplant of
forskolin-treated satellite cells restores Dystrophin expression to
dystrophic mdx muscle (related to FIG. 3). FIG. 10A, satellite
cells from C57BL/6J (wild type) or mdx mice were cultured and
treated with DMSO or forskolin in the presence of bFGF. Cell
numbers were counted at day 5 and are shown as fold change of wild
type cells treated with DMSO. mdx satellite cells show defect in ex
vivo expansion and forskolin treatment restores the number of
cultured satellite cells (mean+/-SEM, n=4). FIG. 10B, shows
transverse frozen section of mdx muscle transplanted with cultured
forskolin-treated GFP.sup.+ satellite cells showing GFP (left,
green) and Dystrophin (right, red) in the engrafted fibers.
[0036] FIGS. 11A-11D show the pluripotency of 00409 and 05400 iPSCs
(related to FIG. 6). FIG. 11A shows the expression of nuclear
NANOG, OCT4 and cytoplasmic SSEA3, SSEA4, TRA1-60 in
undifferentiated 00409 and 05400 iPSCs. Images show merged colors
between blue (Hoechst) and red (indicated protein). FIG. 11B shows
bright-field images of BJ, 00409, 05400 iPSC colonies. FIG. 11C
shows the gene expression analysis of undifferentiated 00409 and
05400 iPSCs. Indicated pluripotency genes were analyzed by
quantitative RT-PCR using GAPDH as a housekeeping gene. Changes in
gene expression levels are expressed relative to undifferentiated
BJ iPSCs. Bars represent the standard deviation of three
independent experiments. FIG. 11D shows the bright-field images
showing EB formation of BJ, 00409 and 05400 iPSCs after 7 days of
differentiation. Scale bars represent 100 .mu.M.
[0037] FIGS. 12A-12E show that treatment of 00409 and 05400 iPSCs
with muscle promoting medium induces skeletal muscle
differentiation (related to FIG. 6). FIGS. 12A and 12B show the
gene expression analysis of EB (FIG. 12A) and monolayer cells (FIG.
12B). Cells were harvested at the times indicated for RNA
extraction. Ectodermal, endodermal and mesodermal genes were
analyzed by quantitative RT-PCR using GAPDH as housekeeping gene.
Changes in gene expression levels are expressed relative to
undifferentiated iPSCs (Day 0). Bars represent the standard
deviation of three independent experiments, (*p<0.05,
**<0.01, ***<0.001). FIG. 12C shows immunostaining of
differentiated 05400 iPSCs. Under terminal differentiating
procedures (day 36) most of the cells express Desmin (red) and
Myogenin (green), forming multinucleated myofibers. Cells were also
stained with Hoechst (blue). FIG. 12D shows representative electron
microscopy images of differentiated 00409 and 05400 cells at day
36, magnification .times.52700. FIG. 12E shows immuno-electron
microscopy staining using skeletal muscle specific anti-Myosin
heavy and light chain. Black dots indicate gold cross-linked
particles to secondary antibody, magnification .times.52700.
[0038] FIGS. 13A and 13B show the effect of combination of bFGF+BIO
and bFGF+forskolin on EB differentiation (related to FIG. 6). FIGS.
13A and 13B, BJ iPSC-derived EBs were stimulated with bFGF+BIO
(FIG. 13A) and bFGF+forskolin (FIG. 13B). Cells were harvested at
the times indicated for RNA extraction. Ectodermal, endodermal and
mesodermal genes were analyzed by quantitative RT-PCR using GAPDH
as a housekeeping gene. Changes in gene expression levels are
expressed relative to undifferentiated BJ iPSCs (Day 0).
DETAILED DESCRIPTION
[0039] The present disclosure is based, in part, on the inventors'
surprising and unexpected discovery that stem cells can be induced
to differentiate into skeletal muscle cells without gene
manipulation. Accordingly, in one aspect provided herein is a
method for inducing differentiation of a stem cell into a myogenic
cell. Generally, the method comprises contacting a stem cell with
at least two of: (i) a GSK3 pathway inhibitor; (ii) a compound that
increases intracellular levels of 3',5'-cyclic adenosine
monophosphate (cAMP); and (iii) a FGF pathway activator. Without
limitations, the stem cell to be contacted can be in vitro, ex vivo
or in vivo.
[0040] In some embodiments, the contacting is with the FGF pathway
activator and the GSK3 pathway inhibitor. In embodiments, the
contacting is with the FGF pathway activator and the compound that
increases intracellular levels of cAMP. In some embodiments,
contacting is with the GSK3 pathway inhibitor and the compound that
increases intracellular levels of cAMP.
[0041] In some embodiments, the method comprises contacting the
stem cell with all three, i.e., contacting with a GSK3 pathway
inhibitor, a compound that increases intracellular levels of
3',5'-cyclic adenosine monophosphate (cAMP), and a FGF pathway
activator.
[0042] The stem cell can be contacted with the combination of the
compounds that induce differentiation to a myogenic cell in a cell
culture e.g., in vitro or ex vivo, or the compounds can be
co-administrated to a subject, e.g., in vivo. In some embodiments,
the combination of compounds that induces differentiation of the
stem cell into a myogenic cell can be administrated to a subject
for repairing or regenerating a damaged muscle tissue.
[0043] As used herein, the term "stem cells" refers to cells
capable of differentiating into many cell types of an organism from
which it arises and includes totipotent, pluripotent and
multipotent cells (e.g., stem cells of embryonic origin (e.g.,
ESCs), induced stem cells (iPSCs) and multipotent progenitor
cells).
[0044] In some embodiments, the stem cell expresses at least one of
Rex-1, OCT4, SOX2, and Nanog. In some embodiments, the stem cell
expresses CD73. In some embodiments, the stem cell expresses CD73
and at least one of Rex-1, OCT4, SOX2, and Nanog.
[0045] As used herein, the term "myogenic cells" refers to cells
giving rise to or forming muscle tissue and includes cells
expressing one or more of the following markers Pax3, Pax7, MyoD,
Myf5, myogenin, GATA2, and MHC and low levels of embryogenic
markers such as Rex-1. In some embodiments, the myogenic cells
disclosed herein are capable of fusing and forming myotubes
comprising at least 10 (e.g., 10, 15, 20, 25, or more) nuclei. In
some embodiments, the myogenic cells disclosed herein are capable
of fusing and forming myotubes comprising from 5 to 20 nuclei.
[0046] In some embodiments, the myogenic cells are cells of the
skeletal myogenic lineage.
[0047] In some embodiments, the myogenic cell prepared in
accordance with the method disclosed herein express at least one,
at least two, at least three, at least 4, at least 5 and preferably
all of the following myogenic markers: Pax3, Pax7, MyoD, Myf5,
myogenin, GATA2, CD56, desmin and MHC. In some embodiments, the
myogenic cell prepared in accordance with the method disclosed
herein express at least Myogenin, MyoD and MHC. In some
embodiments, the myogenic cells prepared in accordance with the
method disclosed herein express at least MyoD1 and Myf5.
[0048] In some embodiments, the myogenic cells prepared in
accordance with the method disclosed herein express at least MyoD1,
Myf5, and Pax7.
[0049] In some embodiments, the myogenic cells prepared in
accordance with the method disclosed herein express at least MyoD1,
Myf5, Pax7, and GATA2.
[0050] In one embodiment, the myogenic cells disclosed herein
express lower levels of Rex-1, OCT4, SOX2 and/or Nanog than stem
cells which have not been treated in accordance with the method
disclosed herein.
[0051] In some embodiments, the stem cell is a pluripotent cell.
The term "pluripotent" as used herein refers to a cell with the
capacity, under different conditions, to differentiate to cell
types characteristic of all three germ cell layers (endoderm,
mesoderm and ectoderm). Pluripotent cells are characterized
primarily by their ability to differentiate to all three germ
layers, using, for example, a nude mouse teratoma formation assay.
Pluripotency is also evidenced by the expression of embryonic stem
(ES) cell markers, although the preferred test for pluripotency is
the demonstration of the capacity to differentiate into cells of
each of the three germ layers. In some embodiments, a pluripotent
cell is an undifferentiated cell.
[0052] Stem cells that can be used in accordance with the method
disclosed herein include embryonic stem cells, pluripotent stem
cells and multipotent progenitor cells. In some embodiments, the
stem cells are mammalian stem cells. In one embodiment, the stem
cells are human stem cells.
[0053] In some embodiments, the stem cell is an induced pluripotent
stem cell. As used herein, the term "induced pluripotent stem cell"
or "iPSC" or "iPS cell" refers to a cell derived from a complete
reversion or reprogramming of the differentiation state of a
differentiated cell (e.g. a somatic cell). As used herein, an iPSC
is fully reprogrammed and is a cell which has undergone complete
epigenetic reprogramming. As used herein, an iPSC is a cell which
cannot be further reprogrammed (e.g., an iPSC cell is terminally
reprogrammed). Human pluripotent cell lines exhibit a level of
developmental plasticity that is similar to the early embryo,
enabling in vitro differentiation into all three embryonic germ
layers. At the same time it is possible to maintain these
pluripotent cell lines for many passages in the undifferentiated.
These unique characteristics render human embryonic stem (ES) and
human induced pluripotent stem (iPS) cells a promising tool for
biomedical research. ES cell lines have already been established as
a model system for dissecting the cellular basis of monogenic human
diseases. The discovery of defined reprogramming methods (Takahashi
and Yamanaka, Cell, 2006, 126: 663-676) and their use in the
derivation of patient-specific iPS cell lines (Dimos et al.,
Science, 2008, 321: 1218-1221; and Park et al., Cell, 2008, 1'34:
877-886) has further expanded the utility of pluripotent cells for
monogenic disease modeling.
[0054] In one embodiment, the stem cells are iPSCs derived from a
subject in need of treatment for muscle repair or damage.
[0055] In some embodiments, the stem cell is an embryonic stem
cell. As used herein, the term "embryonic stem cell" refers to the
pluripotent stem cells of the inner cell mass of the embryonic
blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806, which are
incorporated herein by reference). Such cells can similarly be
obtained from the inner cell mass of blastocysts derived from
somatic cell nuclear transfer (see, for example, U.S. Pat. Nos.
5,945,577, 5,994,619, 6,235,970, which are incorporated herein by
reference). The distinguishing characteristics of an embryonic stem
cell define an embryonic stem cell phenotype. Accordingly, a cell
has the phenotype of an embryonic stem cell if it possesses one or
more of the unique characteristics of an embryonic stem cell such
that that cell can be distinguished from other cells. Exemplary
distinguishing embryonic stem cell characteristics include, without
limitation, gene expression profile, proliferative capacity,
differentiation capacity, karyotype, responsiveness to particular
culture conditions, and the like.
[0056] In one embodiment, the stem cells are ESs derived from a
subject in need of treatment for muscle repair or damage.
[0057] In some embodiments, the stem cell is a multipotent stem
cell. The term "multipotent" when used in reference to a
"multipotent cell" refers to a cell that is able to differentiate
into some but not all of the cells derived from all three germ
layers. Thus, a multipotent cell is a partially differentiated
cell. Multipotent cells are well known in the art, and examples of
multipotent cells include adult stem cells, such as for example,
hematopoietic stem cells and neural stem cells. Multipotent means a
stem cell may form many types of cells in a given lineage, but not
cells of other lineages. For example, a multipotent blood stem cell
can form the many different types of blood cells (red, white,
platelets, etc. . . . ), but it cannot form neurons.
[0058] In one embodiment, the stem cells are multipotent stem cells
derived from a subject in need of treatment for muscle repair or
damage.
[0059] In some embodiments, the stem cell is present in an embryoid
body or an embryoid body-analogous cellular aggregate. Accordingly,
in some embodiments, the method comprises forming an embryoid body
from the stem cell before contacting with the compounds and
activators.
[0060] The term "embryoid bodies" (EBs) is a term of art synonymous
with "aggregate bodies". The terms refer to aggregates of
differentiated and undifferentiated cells that appear when ES cells
overgrow in monolayer cultures, or are maintained in suspension
cultures. Embryoid bodies are a mixture of different cell types,
typically from several germ layers, distinguishable by
morphological criteria; see also infra. As used herein, "embryoid
body", "EB" or "EB cells" typically refers to a morphological
structure comprised of a population of cells, the majority of which
are derived from embryonic stem (ES) cells that have undergone
differentiation. Under culture conditions suitable for EB formation
(e.g., the removal of Leukemia inhibitory factor or other, similar
blocking factors), ES cells proliferate and form small mass of
cells that begin to differentiate. In the first phase of
differentiation, usually corresponding to about days 1-4 of
differentiation for humans, the small mass of cells forms a layer
of endodermal cells on the outer layer, and is considered a "simple
embryoid body". In the second phase, usually corresponding to about
days 3-20 post-differentiation for humans, "complex embryoid
bodies" are formed, which are characterized by extensive
differentiation of ectodermal and mesodermal cells and derivative
tissues. As used herein, the term "embryoid body" or "EB"
encompasses both simple and complex embryoid bodies unless
otherwise required by context. The determination of when embryoid
bodies have formed in a culture of pluripotent cells is routinely
made by persons of skill in the art by, for example, visual
inspection of the morphology. Floating masses of about 20 cells or
more are considered to be embryoid bodies; see. e.g., Schmitt et
al., Genes Dev. 5 (1991), 728-740; Doetschman et al., J. Embryol.
Exp. Morph. 87 (1985), 27-45. It is also understood that the term
"embryoid body", "EB", or "EB cells" as used herein encompasses a
population of cells, the majority of which being pluripotent cells
capable of developing into different cellular lineages when
cultured under appropriate conditions. As used herein, the term
also refers to equivalent structures derived from primordial germ
cells, which are primitive cells extracted from embryonic gonadal
regions; see, e.g., Shamblott, et al. (1998), supra. Primordial
germ cells, sometimes also referred to in the art as EG cells or
embryonic germ cells, when treated with appropriate factors form
pluripotent ES cells from which embryoid bodies can be derived;
see, e.g., U.S. Pat. No. 5,670,372.
[0061] Differentiation of the stem cells into myogenic cells can be
confirmed by, for example, analyzing the expression of one or more
myogenic cell markers. Markers characteristic of myogenic cells
include the expression of cell surface proteins or the encoding
genes, the expression of intracellular proteins or the encoding
genes, cell morphological characteristics, and the like. Those
skilled in the art will recognize that known immunofluorescent,
immunochemical, polymerase chain reaction, in situ hybridization,
Northern blot analysis, chemical or radiochemical or biological
methods can readily ascertain the presence or absence of satellite
cell specific characteristics.
[0062] If desired, the type(s) of cells in a population of cells
can be determined using techniques that are well known in the art.
For example, the use of cell-type specific stains. Alternatively,
one can perform immunofluorescence staining using antibodies
directed to various satellite cell specific proteins. In addition,
a cell type can be determined by its morphology using techniques
such as, for example, light microscopy, or electron microscopy.
Exemplary myogenic cell markers include, but are not limited to,
Pax3, Pax7, MyoD, Myf5, myogenin and MHC
[0063] The term "contacting" or "contact" as used herein in
connection with contacting a stem cell (or population of stem
cells) includes subjecting the stem cells to an appropriate culture
media which comprises the indicated compounds. Where the stem cell
is in vivo, "contacting" or "contact" includes co-administering the
compounds in the same or different pharmaceutical compositions to a
subject via an appropriate administration route such that the
compounds contacts the stem cell in vivo.
[0064] Cell culture media for differentiation induction include,
but are not limited to, serum-free minimal essential medium (MEM),
Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199
medium, F12 medium, mixtures thereof, and media prepared by
supplementing any one of the aforementioned media with appropriate
concentrations of publicly known medium additives in common use
[e.g., serum albumin, 2-mercaptoethanol, insulin, transferrin,
sodium selenite, ethanolamine, antibiotics (e.g., penicillin,
streptomycin) and the like] [e.g., S-Clone medium (e.g., SF-03,
Sanko Junyaku)] and the like.
[0065] The stem cell can be contacted with the combination of the
compounds for any desired period of time. For example, for inducing
differentiation, said contacting can be for hours, days or weeks.
In some embodiments, said contacting can be for at least one day,
e.g., one day, two days, three days, four days, five days, six
days, a week, two weeks, three weeks, four weeks or more.
[0066] Myogenic cells prepared in accordance with the method
disclosed herein can be used for transplantation. In some
embodiments, the myogenic cells prepared in accordance with the
method are autologous to the subject which will receive the
transplantation. Myogenic cells prepared in accordance with the
method disclosed herein can be used for transplantation in a
subject for repairing or regenerating a damaged muscle tissue or
increasing muscle mass of the subject.
[0067] The present disclosure also provides myogenic cells and
populations prepared according to the method described herein. The
cell population can be a purified population of myogenic cells. The
myogenic cells can be obtained by sorting out the cell culture
obtained by the method disclosed herein. In some embodiments, one
or more kind of cells other than the myogenic cells can be
co-present in the cell population. The present disclosure further
provides a composition comprising a myogenic cell prepared
according to the method described herein and a pharmaceutically
acceptable carrier.
[0068] The present disclosure also provides a method for repairing
or regenerating a damaged muscle tissue or increasing muscle mass
of a subject. The method comprising co-administering to the subject
therapeutically effective amount of at least two of: (i) a GSK3
pathway inhibitor; (ii) a compound that increases intracellular
levels of 3',5'-cyclic adenosine monophosphate (cAMP); and (iii) a
FGF pathway activator.
GSK3 Pathway
[0069] As used herein, an "inhibitor of the GSK3 pathway" refers to
compounds and compositions that can inhibit the activity of at
least one component of the GSK3 pathway. The definition and details
of the GSK3 pathway are disclosed in the art e.g., Biondi R. M. and
Nebreda A. R. Biochem J. 372, 1-13 (2003); Jope R. S. and Johnson
G. V. Trends Biochem Sci. 29, 95-102 (2004); and Polakis P. Curr.
Biol. 12, R499-R501 (2002), content of all of which is incorporated
herein by reference in its entirety.
[0070] In some embodiments, the GSK3 pathway inhibitor is a GSK
inhibitor, e.g., a GSK3.beta. inhibitor. GSK inhibitors are known
widely in the art and can be grouped into different chemical
classes such as pyrroloazepine, flavone, beruazepinone, bis-indole,
pyrrolopyrazine, pyridyloxadiazole, pyrazolopyridine,
pyrazolopyridazine, aminopyridine, pyrazoloquinoxaline, oxindole
(indolinone), thiazole, bisindolylmaleimide, azainodolylmaleimide,
arylindolemaleimide, aniliomaleimide, phenylaminopyridine,
triazole, pyrrolopyrimidine, pyrazolopyrimidine, and chloromethyl
thienyl ketone.
[0071] Exemplary GSK3 pathway inhibitors include, but are not
limited to, 6-Bromoindirubin-3'-oxime (BIO), CHIR98014; CHIR99021;
ARA014418; hymenialdisine; flavopiridol; aloisine A; aloisine B;
CT20026; SU9516; staurosporine; GF109203x; R0318220; SB216763;
SB415286; 15; CGP60474; kenpaullone (9-bromopaullone);
alsterpaullone; 2-cyanoethyl-al sterpullone; 1-aza-alsterpaullon;
1-aza-kenpaullone; 9-cyano-2,3-dimethoxypaullone; 2-iodopaullone;
2-bromo-9-nitropaullone; 2,3-dimethoxy-9-nitropaullone;
7-bromo-5-(4-nitrophenylhydrazono)-4,5-dihydro-1-H-[1]benzazepin2(3H)-one-
;
7,8-dimethoxy-5-(4-nitrophenylhydrazono)-4,5dihydro-1H-[1]benzazepin-2-(-
3H)-one; 9-cyanopaullone; 9-chloropaullone;
9-trifluoromethylpaullone; 2,3-dimethoxy-9-trifluoromethylpaullone;
9-bromo-12-methyloxycarbonylmethylpaullone; 9-fluoropaullone;
9-bromo-2,3-dimethoxypaullone; 9-bromo-2,3-dimethoxypaullone;
9-methylpaullone; 10-bromopaullone; 2-bromopaullone;
11-chloropaullone;
2-(3-hydroxy-1-propinyl)-9-trifluoromethylpaullone;
9-bromo-12-(2-hydroxyethyl)-paullone; kenpaullone; Alsterpaullone;
2-cyanoetyl-alsterpaullone; 1-aza-kenpaullone;
1-aza-alsterpaullone; 9-bromo-12-methylpaullone;
9-bromo-5-(methyloxycarbonylmethyl)paullone; 11-methylpaullone;
paullone; 11-ethylpaullone;
9-bromo-7,12-dihydro-6-(hydroxyamino)-indolo[2-3-d][1]benzazepine;
2,9-dibromopaullone; 11-bromopaullone; 2,3-dimethoxypaullone;
9-bromo-7,12dihydro-6-methylthio-indolo[2-3-d][1]benzazepine;
(E)-2(3-oxo-1-butenyl)-9-trifluoromethylpaullone;
9-bromo-12ethylpaullone;
9-bromo-7,12-dihydro-indolo[2-3-d][1]benzazepine-6(5H)-thione;
2-bromo-9-trifluoromethylpaullone;
2-[2-(1-hydroxycyclohexyl)ethinyl]-9-trifluoromethyl-paullone;
9-bromo-5methylpaullone; 9-methoxypaullone;
2-iodo-9-trifluoromethylpaullone;
9-bromo-12-(tert-butyloxycarbonyl)-paullone;
9-bromo-12-(2-propenyl)paullone; 9-bromo-4-hydroxypaullone;
8,10-dichloropaullone; 5-benzyl-9-bromopaullone;
9-bromo-4-methoxypaullone; 9-bromo-5-ethylpaullone;
9-bromo-5,7bis-(tert-butyloxycarbonyl)-paullone; 4-methoxypaullone;
9-bromo-5,6,7,12-tetrahydrobenzo[6-7]cyclohept[1,2.b]indole;
2-phenyl-4-(2-thienyl)-5H-pyrido[2-3-d][1]benzazepine-6(7H)-thione;
9-bromo-5,7,12-tri-(tert-butyloxycarbonyl)-paullone;
9-bromo-5,12-bis-(tert-butyloxycarbonyl)paullone;
4-(4-chlorophenyl)-2-(2-naphthyl)-5H-pyrido[23-d]
[1]benzazepine-6(7H)-thione;
5,6,7,12-tetrahydrobenzo[6-7]cyclohept[1,2-b]indole;
N-Butyl-N'-(5-nitro-1, 3-thiazol-2-yl) urea; N-(5-Nitro-1,
3-thiazol-2-yl) pentanamide;
1-{4-Amino-2-[(4-methoxyphenyl)amino]-1,3-thiazol-5-yl}ethanone;
N-Benzyl-N'-(5-nitro-1, 3-thiazol-2-yl) urea;
N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thaizol-2-yl) urea;
3-(4-Methoxyphenyl)-N-(5-nitro-1, 3-thiazol-2-yl) propanamide;
4-(4-Methoxyphenyl)-N-(5-nitro-1, 3-thiazol-2-yl) butanamide;
2-(3-Methoxyphenyl)-N-(5-nitro-1, 3-thiazol-2yl) acetamide;
2-(4-Fluorophenyl)-N-(5-nitro-1, 3-thiazol-2-yl) propanamide;
2-(3-Methylphenyl)-N-(5-nitro-1, 3-thiazol-2-yl) acetamide;
1-benzyl-3-naphthalen-1-yl-urea or
1-benzyl[1,3]dioxol-5-yl-3-benzyl-urea.
[0072] Additional GSK3 pathway inhibitors, e.g., GSK-3beta
inhibitors, amenable to the invention are described in U.S. Pat.
Nos. 7,056,939, 7,045,519, 7,037,918, 6,989,382, 6,949,547,
6,872,737, 6,800,632, 6,780,625, 6,608,063, 6,489,344, 6,479,490,
6,441,053, 6,417,185, 6,323,029, 6,316,259, 7,232,814, 7,393,953,
and 6,057,117; PCT Patent Application Publication Nos. WO07/017145,
WO03/089419, WO 99/65910, and WO09/010298; and Leost at al.,
Paullones Are Potent inhibitors of Glycogen Synthase Kinase-3B and
Cyclin-dependent Kinase 5/p25, Eur. J. Biochem. (2000), 267,
5983-5994, content of all of which is incorporated herein by
reference in their entirety of which each are incorporated herein
by reference in their entirety.
[0073] In one embodiment, the GSK3 pathway inhibitor is BIO.
[0074] In some embodiments, the GSK3 pathway inhibitor activates
Wnt signaling. The Wnt signaling pathway is known for its important
role in the inductive interactions that regulate growth and
differentiation, and likely also plays important roles in the
homeostatic maintenance of post-embryonic tissue integrity. Wnt
stabilizes cytoplasmic p-catenin, which stimulates the expression
of genes including c-myc, c jun, fra-1, and cyclin Dl. In addition,
misregulation of Wnt signaling can cause developmental defects and
is implicated in the genesis of several human cancers. More
recently, the Wnt pathway has been implicated in the maintenance of
stem or progenitor cells in a growing list of adult tissues that
now includes skin, blood, gut, prostate, muscle and the nervous
system. Wnt signaling affects fundamental development pathways by
regulating cell proliferation and differentiation. As such, the Wnt
signaling pathway can be instrumental in the regulation of cell
proliferation, differentiation and morphogenesis. Non-limiting
examples of Wnt activators include .beta.-catenin, APC, axinl,
axin2, GSK3, GSK-313 inhibitors, Disheveled, LRP5, LRP6, Frizzled,
Wnt proteins (e.g., Wnt-1, Wnt-3a, Wnt-5a, and Wnt-8a).
[0075] In some embodiments, concentration of the GSK3 pathway
inhibitor for contacting with the stem cell is from about 0.05
.mu.M to about 50 .mu.M, from about 0.1 .mu.M to about 25 .mu.M,
from about 0.2 .mu.M to about 15 .mu.M, from about 0.3 .mu.M to
about 5 .mu.M, from about 0.1 .mu.M to about 1 .mu.M, or from about
0.25 .mu.M to about 0.75 .mu.M. In one embodiment, concentration of
the GSK3 pathway inhibitor for contacting with the stem cell is
about 0.5 .mu.M.
cAMP
[0076] In some embodiments, the compound that increases
intracellular levels of cAMP is an activator of adenylyl cyclase.
Adenylyl cyclase catalyzes the conversion of ATP to 3',5'-cyclic
AMP (cAMP) and pyrophosphate. Divalent cations (usually Mg) are
generally required and appear to be closely involved in the
enzymatic mechanism. The cAMP produced by adenylyl cyclase then
serves as a regulatory signal via specific cAMP-binding proteins,
either transcription factors or other enzymes (e.g., cAMP-dependent
kinases).
[0077] An "activator" of an adenylyl cyclase is a compound or
composition which causes the adenylyl cyclase to become more
active, and thereby elevates the cAMP levels or signal of the cell.
The mode of action of the activator can be direct, e.g., through
binding the cyclase, or indirect, e.g., through binding another
molecule which otherwise interacts with the cyclase.
[0078] Exemplary activators of adenylate cyclase, include, but are
not limited to, forskolin; forskolin derivatives and analogues;
non-hydrolyzable analogues of cAMP including 8-bromo-cAMP,
8-chloro-cAMP, or dibutyryl cAMP (db-cAMP); isoprotenol; vasoactive
intestinal peptide; calcium ionophores; membrane depolarization;
macrophage-derived factors that stimulate cAMP; agents that
stimulate macrophage activation such as zymosan or IFN-.gamma.;
phosphodiesterase inhibitors such as pentoxifylline and
theophylline; specific phosphodiesterase IV (PDE IV) inhibitors;
pituitary adenylate cyclase activating peptide (PACAP); cholera
toxin; prostaglandin compounds such as prostaglandin E2 (PGE2); and
beta 2-adrenoreceptor agonists such as salbutamol.
[0079] In some embodiments, the compound that can increase
intracellular cAMP is a G-coupled receptor activator, e.g., a GPCR
ligand. The G protein-coupled receptors (GPCRs) form a vast
superfamily of cell surface receptors which are characterized by an
amino-terminal extracellular domain, a carboxyl-terminal
intracellular domain, and a serpentine structure that passes
through the cell membrane seven times. Hence, such receptors are
sometimes also referred to as seven transmembrane (7TM) receptors.
These seven transmembrane domains define three extracellular loops
and three intracellular loops, in addition to the amino- and
carboxy-terminal domains. The extracellular portions of the
receptor have a role in recognizing and binding one or more
extracellular binding partners (e.g., ligands), whereas the
intracellular portions have a role in recognizing and communicating
with downstream molecules in the signal transduction cascade.
[0080] As used herein, the term "GPCR ligand" refers to molecules
that bind GPCRs. The G protein-coupled receptors bind a variety of
ligands including calcium ions, hormones, chemokines,
neuropeptides, neurotransmitters, nucleotides, lipids, odorants,
and even photons, and are important in the normal (and sometimes
the aberrant) function of many cell types. [See generally
Strosberg, Eur. J Biochem. 196:1-10 (1991) and Bohm et al, Biochem
J. 322:1-18 (1997).] When a specific ligand binds to its
corresponding receptor, the ligand typically stimulates the
receptor to activate a specific heterotrimeric
guanine-nucleotide-binding regulatory protein (G-protein) that is
coupled to the intracellular portion of the receptor. The G protein
in turn transmits a signal to an effector molecule within the cell,
by either stimulating or inhibiting the activity of that effector
molecule. These effector molecules include adenylate cyclase,
phospholipases and ion channels. Adenylate cyclase and
phospholipases are enzymes that are involved in the production of
the second messenger molecules cAMP, inositol triphosphate and
diacyglycerol. It is through this sequence of events that an
extracellular ligand stimuli exerts intracellular changes through a
G protein-coupled receptor. Each such receptor has its own
characteristic primary structure, expression pattern,
ligand-binding profile, and intracellular effector system.
[0081] GPCRs include receptors for sensory signal mediators (e.g.,
light and olfactory stimulatory molecules); adenosine, bombesin,
bradykinin, endothelin, .gamma.-aminobutyric acid (GABA),
hepatocyte growth factor (HGF), melanocortins, neuropeptide Y,
opioid peptides, opsins, somatostatin, GH, tachykinins, members of
the vasoactive intestinal peptide family, and vasopressin; biogenic
amines (e.g., dopamine, epinephrine, norepinephrine, histamine,
glutamate (metabotropic effect), glucagon, acetylcholine
(muscarinic effect), and serotonin); chemokines; lipid mediators of
inflammation (e.g., prostaglandins, prostanoids,
platelet-activating factor, and leukotrienes); and peptide hormones
(e.g., calcitonin, C5a anaphylatoxin, follicle-stimulating hormone
(FSH), gonadotropin-releasing hormone (GnRH), neurokinin,
thyrotropin-releasing hormone (TRH), cannabinoids, and oxytocin).
GPCRs that act as receptors for stimuli that have not yet been
identified are known as orphan receptors. Whereas, in other types
of receptors that have been studied, wherein ligands bind
externally to the membrane, the ligands of GPCRs typically bind
within the transmembrane domain. However, protease-activated
receptors are activated by cleavage of part of their extracellular
domain.
[0082] Types of GPCR ligands include, but are not limited to:
agonists which shift the equilibrium in favor of active states;
inverse agonists which shift the equilibrium in favor of inactive
states; and neutral antagonists which do not affect the
equilibrium. When a GPCR in an active state encounters a G-protein,
it can activate the G-protein. GPCRs are the target of about 40% of
all prescription pharmaceuticals on the market. (Filmore, Modern
Drug Discovery, November 2004, pp. 11). Examples of commonly
prescribed GPCR-based drugs include Atenolol (TENORMIN.RTM.),
Albuterol (VENTOLIN.RTM.), Ranitidine (ZANTAC.RTM.), Loratadine
(CLARITIN.RTM.), Hydrocodone (VICODIN.RTM.) Theophylline
(THEODUR.RTM.), and Fluoxetine (PROZAC.RTM.).
[0083] Exemplary GPCR activators include, but are not limited to,
calcitonin, PGE2, corticotropin releasing factor (CRF), urocortin
1, urocortin 2, usorcortin 3, parathyroid hormone, PTH-related
hormone, TIP39, amylin, CGRP (CALCA and CALCB), adrenomedullin,
secretin, VIP, PACAP, glucagon, GHRH, GLP-1, GLP-2, Dynorphin A,
Dynorphin A amide, Dynorphin A (1-6), Dynorphin A (1-13), Dynorphin
A (2-13), Dynorphin A (2-17), MetEnk, Met-Enk-RF-amide,
Met-Enk-Arg-Phe, Met-Enk-Glyleu, [D-pGlul, D-Phe2, D-Trp3,6]-LH-RH,
gl-MSH amide, g2-MSH, [N-MePhel, D-Pro4]-Morphiceptin (PL017), ACTH
(Human), Leu-Enk, Adrenomedullin (22-52), Adrenomedullin (26-52)
(Human)(ADM antagonist), Agouti 1-40 Amide, Agouti Related Protein
(87-132)-Amide, Alpha-MSH, Alpha-Neo-Endorphin, Amylin Amide,
BAM(1-20), BAM(1-22), BAM(2-22), BAM(6-22), BAM(1-20), ANP (Atrial
Natriuretic Peptide), Anti-inflammatory Peptide 1,
Anti-inflammatory Peptide 2, (3-endorphin,
Benzylureido-Met-Leu-Phe, Beta-ANP, Beta-Endorphin, Beta-MSH, Big
Endothelin-1, Big Gastrin-1, BNP (Brain Natriuretic Peptide-32),
BNP-45 (Cardiac Natriuretic Peptide, Bombesin, BAM(8-25),
BAM(8-20), FLRF, Calcitonin Gene Related Peptide, NPFF, Calcitonin,
Calcitonin Gene Related Peptide (8-37), CART (55-1,02), CART
(55102)[Met(O)67, CART (61-102), CGRP (8-37), CGRP II,
Cholecystokinin Octapeptide [CCK(26-33)], Cholecystokinin-33,
CNP-22 (C-Type Natriuretic Peptide), Corticotropin Releasing
Factor, Cortistatin-14, NPAF, SST, NPY, FMRFamide (SEQ ID NO: 39),
OrpaninFQFMRF amide related peptide (SEQ ID NO: 40), YMRFamide (SEQ
ID NO: 41), YLPLRFamide (SEQ ID NO: 42), YFMRFamide (SEQ ID NO:
43), LPLRFamide (SEQ ID NO: 44), dFMRFamide, W-Nle-R-F-amide, and
ACEP. Polypeptide activstors of GPCRs include, but are not limited
to, vasopressin, oxytocin, somatostatin, neuropeptide Y, GnRH,
leutinizing hormone, follicle stimulating hormone, parathyroid
hormone, orexins, urotensin II, endorphins, enkephalins, and the
like. A list of GPCR modulators, including activators, is compiled
on the web at
pharminfo.pharm.kyoto-u.ac.jp/services/glida/ligand_classification.php.
[0084] In some embodiments, the G-coupled receptor activator is
calcitonin or PGE2.
[0085] In some embodiments, the compound that can increase
intracellular cAMP is forskolin.
[0086] In some embodiments, concentration of the compound that
increases intracellular cAMP is from about 0.1 .mu.M to about 500
.mu.M, from about 1 .mu.M to about 250 .mu.M, from about 2.5 .mu.M
to about 150 .mu.M, from about 5 .mu.M to about 100 .mu.M, from
about 7.5 .mu.M to about 75 .mu.M, from about 10 .mu.M to about 50
.mu.M, or from about 15 .mu.M to about 25 .mu.M. In one embodiment,
concentration of the compound is about 20 .mu.M.
FGF Pathway
[0087] As used herein, a "FGF pathway activator" refers to
compounds and compositions that can increase or enhance the
activity of at least one component of the FGF pathway. The
definition and details of the FGF pathway are disclosed in the art
e.g., Lee P. L. et al., Science. 245, 57-60 (1989); Mignatti P. et
al., J. Cell Physiol. 151, 81-93 (1992); Miki T. et al., Proc.
Natl. Acad. Sci. USA. 89, 246-250 (1992); Gringel S. et al., J.
Biol. Chem. 385, 1203-1208 (2004); Ornitz D. M. and Itoh, N. Genome
Biol. 2, 1-12 (2001); Sorensen V. et al., Bioessays. 28, 504-514
(2006); Coulson E. J. Prog. Brain Res. 146, 41-62 (2004); Huang E.
J. and Reichardt L. F. Annu. Rev. Biochem. 72, 609-642 (2003);
Miller F. D. and Kaplan D. R. Cell Mol. Life Sci. 58, 1045-1053
(2001); and Rabizadeh S. and Bredesen D. E. Cytokine Growth Factor
Rev. 14, 225-239 (2003), content of all of which is incorporated
herein by reference in their entirety.
[0088] In some embodiments, the FGF pathway activator is an
activator of PI-3K pathway. PI3Ks interact with the IRS (Insulin
receptor substrate) in order to regulate glucose uptake through a
series of phosphorylation events. The phosphoinositol-3-kinase
family is composed of Class I, II and Class III, with Class I the
only ones able to convert PI(4,5)P2 to PI(3,4,5)P3 on the inner
leaflet of the plasma membrane. Class I PI3K are heterodimeric
molecules composed of a regulatory and a catalytic subunit; they
are further divided between IA and IB subsets on sequence
similarity. The PI3K pathway also recruits many other proteins
downstream, including mTOR, GSK3.beta., and PSD-95. The PI3K-mTOR
pathway leads to the phosphorylation of p70S6K, a kinase which
facilitates translational activity.
[0089] In some embodiments, the activator of the PI-3K pathway
activates phosphatidylinositide 3-kinase (PI3K), phosphoinositide
dependent kinase (PDK) or protein kinase B (PKB, aka Akt).
Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) are a family of
related enzymes that are capable of phosphorylating the 3 position
hydroxyl group of the inositol ring of phosphatidylinositol
(PtdIns). They are also known as phosphatidylinositol-3-kinases. An
"activator" of a PI3K is a compound or composition which causes the
PI3K to become more active. The mode of action of the activator can
be direct, e.g., through binding the cyclase, or indirect, e.g.,
through binding another molecule which otherwise interacts with the
cyclase.
[0090] In some embodiments, the activator of the FGF pathway is a
growth factor, e.g., a fibroblast growth factor (FGF), epidermal
growth factor (EGF), nerve growth factor (NGF), or transforming
growth factor (tumor growth factor, TGF).
[0091] Exemplary activators of the FGF pathway include, but are not
limited to, basic fibroblast growth factor (bFGF, FGF2 or
FGF-.beta.); TGF.alpha.; TGF.beta.; EGF; NGF; Akt activators, such
as Ro-31-8220 (Wen, H. et al., Cellular signaling, 15:37-45
(2003)); Nicotine (West, K. et al., J. Clinical Investigation,
111:81-90 (2003)); carbachol (Cui Q L, Fogle E & Almazan G
Neurochem Int, 48:383-393 (2006));
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (West, K. et
al., J. Clinical Investigation, 111:81-90 (2003)); adrenomedullin
(AM) (Nikitenko, L L et al, British J. Cancer, 94:1-7 (2006));
lysophosphatidic acid; platelet activating factor, macrophage
simulating factor; sphingosine-1-phosphate; cAMP-elevating agents,
such as forskolin, chlorophenylthio-cAMP, prostaglandin-E1, and
8-bromo-cAMP (Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005)),
insulin and insulin growth factor-1 (Datta, S. R., et al., Cell,
91:231-241 (1997)), and platelet derived growth factor.
[0092] In one embodiment, activator of the FGF pathway is bFGF.
Other exemplary FGF pathway include FGF1, FGF2, FGF3, FGF4, FGF5,
FGF6, FGF7, FGF8, FGF9, FGF10, and the like.
[0093] In some embodiments, concentration of the FGF pathway
activator for contacting with the stem cell is about 0.25 ng/ml to
about 100 ng/ml, about 0.5 ng/ml to about 75 ng/ml, about 1 ng/ml
to about 50 ng/ml, about 2.5 ng/ml to about 25 ng/ml, from about 5
ng/ml to about 15 ng/ml, or about 7.5 ng/ml to about 12.5 ng/ml. In
one embodiment, concentration of the activator is about 10
ng/ml.
Proliferation of Satellite Cells
[0094] In one aspect, the present disclosure provides a method of
inducing, enhancing or increasing satellite cell proliferation. The
method comprising contacting a satellite cell with an compound that
increases intracellular levels of 3',5'-cyclic adenosine
monophosphate (cAMP). The satellite cell to be contacted can be in
vitro, ex vivo or in vivo. Compounds that increase intracellular
levels of cAMP are described elsewhere herein.
[0095] As used herein, the terms "proliferating" and
"proliferation" refer to an increase in the number of cells in a
population (growth) by means of cell division. Cell proliferation
is generally understood to result from the coordinated activation
of multiple signal transduction pathways in response to the
environment, including growth factors and other mitogens. Cell
proliferation can also be promoted by release from the actions of
intra- or extracellular signals and mechanisms that block or
negatively affect cell proliferation.
[0096] As used herein, "inducing,", "enhancing," or "increasing"
satellite cell proliferation means that satellite cells replicate
at a faster rate and/or more frequently. In some embodiments of
this and other aspects described herein, satellite cell
proliferation is increased by at least 5%, 10%, 20%, 30%, 40%, 50%,
50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more higher relative
to an untreated control. The % or fold increase in satellite cell
proliferation can be determined by measuring number of replicating
satellite cells while in contact with a compound described herein
relative to a control where the satellite cells are not in contact
with the compound. Increase in proliferation can also be based on
ratios of replicating cells to total number of cells in the
respective treated and untreated control. In some embodiments,
total number of cells in the treated and untreated controls is used
to determine the proliferation. Satellite cell proliferation can be
determined using the BrdU incorporation method described in U.S.
Patent Publication No. 2009/0136481, content of which is
incorporated herein by reference.
[0097] Myosatellite cells or satellite cells are small mononuclear
progenitor cells with virtually no cytoplasm found in mature
muscle. They are found sandwiched between the basement membrane and
sarcolemma (cell membrane) of individual muscle fibers, and can be
difficult to distinguish from the sub-sarcolemmal nuclei of the
fibers. Satellite cells are able to differentiate and fuse to
augment existing muscle fibers and to form new fibers. These cells
represent the oldest known adult stem cell niche, and are involved
in the normal growth of muscle, as well as regeneration following
injury or disease.
[0098] In undamaged muscle, the majority of satellite cells are
quiescent; they neither differentiate nor undergo cell division. In
response to mechanical strain, satellite cells become activated.
Activated satellite cells initially proliferate as skeletal
myoblasts before undergoing myogenic differentiation.
[0099] Markers characteristic of satellite cells include the
expression of cell surface proteins or the encoding genes, the
expression of intracellular proteins or the encoding genes, cell
morphological characteristics, and the like. Those skilled in the
art will recognize that known immunofluorescent, immunochemical,
polymerase chain reaction, in situ hybridization, Northern blot
analysis, chemical or radiochemical or biological methods can
readily ascertain the presence or absence of satellite cell
specific characteristics.
[0100] If desired, the type(s) of cells in a population of
satellite can be determined using techniques that are well known in
the art. For example, the use of cell-type specific stains.
Alternatively, one can perform immunofluorescence staining using
antibodies directed to various satellite cell specific proteins. In
addition, a cell type can be determined by its morphology using
techniques such as, for example, light microscopy, or electron
microscopy.
[0101] Satellite cells express a number of distinctive genetic
markers. For example, current thinking is that all satellite cells
express PAX7 and PAX3 (F. Rlaix et al. Nature, 2005, 435(7044):
898-899). Activated satellite cells express myogenic transcription
factors, such as Myf5 and MyoD. They also begin expressing
muscle-specific filament proteins such as desmin as they
differentiate. The data presented herein shows that CXCR4 and
.beta.-1 Integrins are also useful surrogate markers for satellite
cells, especially engraftable myogenic cells.
[0102] Little is known of the regulation of satellite cells. Whilst
together PAX3 and PAX7 currently form the definitive satellite
markers, Pax genes can be poor transcriptional activators. The
dynamics of activation and quiesence and the induction of the
myogenic program through the myogenic regulatory factors, Myf5,
MyoD, myogenin, and MRF4 remains to be determined. There is also
some research indicating that satellite cells are negatively
regulated by a protein called myostatin.
[0103] In some embodiments, the satellite cells are in a stabilized
state, e.g., the cells were taken from a subject and treated in
such a manner as to allow them to be stored for some period of
time. For example, the cells can be frozen, e.g., using methods
known in the art for freezing primary cells, such that the cells
are viable when thawed. For example, methods known in the art to
freeze and thaw embryos to generate live mammals can be adapted for
use in the present methods. Such methods can include the use of
liquid nitrogen, e.g., with one or more cryoprotectants, e.g.,
agents that prevent freeze-thaw damage to the cell.
[0104] The satellite cell population can be contacted with the
compound that increases intracellular level of cAMP in a cell
culture e.g., in vitro or ex vivo, or the compound can be
administrated to a subject, e.g., in vivo. In some embodiments of
the invention, a compound that increases intracellular level of
cAMP can be administrated to a subject for repairing or
regenerating a damaged muscle tissue.
[0105] The term "contacting" or "contact" as used herein in
connection with contacting a population of satellite cells includes
subjecting the satellite cells to an appropriate culture media
which comprises the indicated compound. Where the satellite cell
population is in vivo, "contacting" or "contact" includes
administering the compound in a pharmaceutical composition to a
subject via an appropriate administration route such that the
compound contacts the satellite cell population in vivo.
[0106] For in vivo methods, a therapeutically effective amount of
the indicated compound can be administered to a subject. Methods of
administering compounds to a subject are known in the art and
easily available to one of skill in the art. Promoting satellite
cell proliferation in a subject can lead to treatment, prevention
or amelioration of a number of diseases, disorders or conditions
which are caused by a damaged muscle tissue.
[0107] Satellite cells suitable for use in ex vivo methods can be
obtained from subject according to methods well known to those
skilled in the art. The term "ex vivo" refers to cells which are
removed from a living organism and cultured outside the organism
(e.g., in a test tube). For ex vivo methods, satellite cells can
include autologous satellite cells, i.e., a cell or cells taken
from a subject who is in need of treatment for muscle damage or
repair. Autologous satellite cells have the advantage of avoiding
any immunologically-based rejection of the cells. Alternatively,
the cells can be heterologous, e.g., taken from a donor. The second
subject can be of the same or different species. Typically, when
the cells come from a donor, they will be from a donor who is
sufficiently immunologically compatible with the recipient, i.e.,
will not be subject to transplant rejection, to lessen or remove
the need for immunosuppression. In some embodiments, the cells are
taken from a xenogeneic source, i.e., a non-human mammal that has
been genetically engineered to be sufficiently immunologically
compatible with the recipient, or the recipient's species. Methods
for determining immunological compatibility are known in the art,
and include tissue typing to assess donor-recipient compatibility
for HLA and ABO determinants. See, e.g., Transplantation
Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons,
Incorporated 1994).
[0108] Without wishing to be bound by theory any suitable cell
culture media can be used for the in vitro or ex vivo methods
described herein. After in vitro or ex vivo contact with a compound
that increases intracellular level of cAMP, when the satellite
cells have reached a desired population number or density, e.g.,
about 1.times.10.sup.6, 2.times.10.sup.6, 3.times.10.sup.6,
4.times.10.sup.6, 5.times.10.sup.6, 6.times.10.sup.6,
7.times.10.sup.6, 8.times.10.sup.6, 9.times.10.sup.6,
1.times.10.sup.7, 2.times.10.sup.7, or more cells, the cells can be
transplanted in a subject who is in need of treatment for muscle
repair or damage. The cells can be transplanted in a subject from
whom the cells were originally obtained or in different
subject.
[0109] Satellite cells prepared in accordance with the method
disclosed herein can be used for transplantation. In some
embodiments, the satellite cells prepared in accordance with the
method are autologous to the subject which will receive the
transplantation. Satellite cells prepared in accordance with the
method disclosed herein can be used for transplantation in a
subject for repairing or regenerating a damaged muscle tissue or
increasing muscle mass of the subject.
[0110] The present disclosure also provides a cell population
comprising satellite cells produced by the method disclosed herein.
The cell population can be a purified population of satellite
cells. The purified satellite cells can be obtained by sorting out
the cell culture obtained by the method disclosed herein. In some
embodiments, one or more kind of cells other than the satellite
cells can be co-present in the cell population.
[0111] In another aspect, provided herein is a method for repairing
or regenerating a damaged muscle tissue or increasing muscle mass
of a subject of a subject. The method comprising administering to
the subject a therapeutically effective amount of a compound that
increases intracellular levels of cAMP.
Compositions
[0112] In one aspect provided herein is a composition for inducing
differentiation of a stem cell into a myogenic cell, the
composition comprising: at least two of: (i) a GSK3 pathway
inhibitor; (ii) a compound that increases intracellular levels of
3',5'-cyclic adenosine monophosphate (cAMP); and (iii) a FGF
pathway activator.
[0113] Without wishing to be bound by a theory, the components of
the composition disclosed herein can have a synergistic effect on
differentiation of stem cell into myogenic cells. The term
"synergistic" as used herein is defined to mean a combination of
components wherein the activity of the combination is greater than
the additive of the individual activities of each component of the
combination. In some embodiments, the activity of the combination
is at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 1-fold, at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least
10-fold, at least 50-fold, at least 100-fold or greater than the
additive of the individual activities of each component of the
combination.
[0114] Ratio of the components of the composition can be chosen to
provide optimum activity. For example, when present, the GSK3
pathway inhibitor and the compound that increases intracellular
levels of cAMP can be in a ratio from about 10:1 to about 1:1,000;
from about 5:1 to about 1:500; from about 1:1 to about 1:250; from
about 1:5 to about 1:200; from about 1:10 to about 1:100; from
about 1:20 to about 1:75; or from about 1:25 to 1:75. In one
embodiment, the GSK3 pathway inhibitor and the compound that
increases intracellular levels of cAMP can be in a ratio of about
1:40. The ratio can be a molar ratio or mass ratio.
[0115] When present, FGF pathway activator and the GSK3 pathway
inhibitor can be in a ratio from about 1:50 to about 1:2,000; from
about 1:100 to about 1:1,750; from about 1:200 to about 1:1,500;
from about 1:500 to about 1:1,250; or from about 1:750 to about
1:1,000. In one embodiment, the FGF pathway activator to the GSK3
pathway inhibitor can be in ratio of about 1:875. The ratio can be
a molar ratio or mass ratio.
[0116] When present, FGF pathway activator and the compound that
increases intracellular levels of cAMP can be in a ratio from about
1:10,000 to about 1:100,000, from about 1:15,000 to about 1:75,000;
from about 1:20,000 to about 1:50,000; from about 1:25,000 to about
1:45,000, or from about 1:30,000 to about 1:40,000. In one
embodiment, the FGF pathway activator to the compound that
increases intracellular levels of cAMP can be in ratio of about
1:35,000. The ratio can be a molar ratio or mass ratio.
[0117] In some embodiments, the composition is formulated with a
pharmaceutically acceptable carrier (additive), i.e., a
pharmaceutically acceptable composition. In some embodiments, the
pharmaceutically acceptable composition comprises therapeutically
effective amount of at least two of: (i) a GSK3 pathway inhibitor;
(ii) a compound that increases intracellular levels of 3',5'-cyclic
adenosine monophosphate (cAMP); and (iii) a FGF pathway activator.
The composition can be specially formulated for administration in
solid or liquid form, including those adapted for the following:
(1) oral administration, for example, drenches (aqueous or
non-aqueous solutions or suspensions), lozenges, dragees, capsules,
pills, tablets (e.g., those targeted for buccal, sublingual, and
systemic absorption), boluses, powders, granules, pastes for
application to the tongue; (2) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (3) topical application, for
example, as a cream, ointment, or a controlled-release patch or
spray applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; (5) sublingually; (6)
ocularly; (7) transdermally; (8) transmucosally; or (9) nasally.
Additionally, compounds can be implanted into a patient or injected
using a drug delivery system. See, for example, Urquhart, et al.,
Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed.
"Controlled Release of Pesticides and Pharmaceuticals" (Plenum
Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No.
35 3,270,960.
[0118] As used here, the term "pharmaceutically acceptable" refers
to those compounds, materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0119] As used here, the term "pharmaceutically-acceptable carrier"
means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents,
such as magnesium stearate, sodium lauryl sulfate and talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl
oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; (22) bulking agents, such as
polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; (22) C.sub.2-C.sub.12 alcohols, such as
ethanol; and (23) other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, coloring agents,
release agents, coating agents, sweetening agents, flavoring
agents, perfuming agents, preservative and antioxidants can also be
present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable carrier" or the like are
used interchangeably herein.
Kits
[0120] In one aspect provided herein is a reagent kit for inducing
differentiation of a pluripotent stem cell into a skeletal muscle
cell, the kit comprising: (i) at least two of: (a) a GSK3 pathway
inhibitor; (b) a compound that increases intracellular levels of
3',5'-cyclic adenosine monophosphate (cAMP); and (c) a FGF pathway
activator; and (ii) instructions for use. In some embodiments, the
kit comprises a GSK3 pathway inhibitor; a compound that increases
intracellular levels of 3',5'-cyclic adenosine monophosphate
(cAMP); and a FGF pathway activator.
[0121] In some embodiments, the kit further comprises a stem
cell.
Screening Assay
[0122] In one aspect provided herein is a method for screening
chemical compounds or compositions in blastomere cells, e.g.,
zebrafish blastomeres. The embodiments of the disclosure provide a
system for screening organ development modulators. More
specifically, the present disclosure provides for screening,
including high throughput screening, for modulators that effect
organ development and function. In some embodiments, the screening
assay utilizes zebrafish blastomeres.
[0123] Using the present invention, one can distinguish at least
three different classes of compounds that increase, decrease or
otherwise modulate organ development and function. Specificity is
high in this well-defined novel assay, providing deeper and broader
information including obtaining compounds that can induce
differentiation of a pluripotent cell to a progenitor cell of a
specific lineage; compounds that can inhibit differentiation of
progenitor cells to fully differentiated cells; and compounds that
induce differentiation of progenitor cells to fully differentiated
cells. Thus, without wishing to be bound by a theory, the screening
assay can be used to probe the pathway of a pluripotent cell to a
fully differentiated cell and modulators of that pathway.
[0124] Generally, the method comprises the steps of culturing
blastomere in presence of a test compound, wherein at least one
cell in the cell culture comprises a reporter gene, wherein the
reporter gene encodes a cell lineage specific marker and produces a
detectable signal when expressed; and measuring/detecting the
detectable signal. A change in level or amount of the detectable
signal indicating that the test compound modulates the development
of cell lineage and organs and tissue comprising cells that express
the reporter gene. The level or amount of the detectable signal can
be determined relative to a reference or control. In some
embodiments, the reference or control can be a blastomere culture
without the test compound.
[0125] As used herein, the term "reporter gene" refers to a gene
that expresses a cell marker that is expressed in a cell lineage
specific manner and produces a detectable response or signal. As
used herein, the term "detectable" refers to a molecule or an
element or functional group in a molecule that allows for the
detection, imaging, and/or monitoring of the presence the molecule.
The reporter gene can be an endogenous gene, an exogenous gene, or
a transgene. A detectable response generally refers to a change in,
or occurrence of, a signal that is detectable either by observation
or instrumentally. Without limitations, the expressed molecule can
be detected directly or the molecule can produce a detectable
signal in the presence of a reagent. Further, any available method
for determining the amount of the reporter in a culture can be
employed. In some embodiments, detectable response is an optical
signal, i.e., the reporter is an optical reporter. Suitable optical
reporters include, but are not limited to, fluorescent reporters
and chemiluminescent groups.
[0126] In some embodiments, the detectable response is fluorescence
or a change in fluorescence, e.g., a change in fluorescence
intensity, fluorescence excitation or emission wavelength
distribution, fluorescence lifetime, and/or fluorescence
polarization. In some embodiments, the reporter gene encodes a
fusion protein comprising a cell lineage specific marker (CLSM)
fused with a fluorescent protein (i.e., a CLSM::FP construct or
fusion protein).
[0127] The cell lineage specific marker can be a marker that is
expressed only in the progenitor cells of the lineage; expressed
only in the terminally differentiated cells of the lineage; or
expressed in both the progenitor cells and the terminally
differentiated cells of the lineage.
[0128] Thus, in some embodiments, the blastomere cells can comprise
two or more different reporter genes, each reporter gene encoding a
different detectable cell lineage specific marker. One reporter
gene can be expressed in the terminally differentiated cells, a
second reporter gene can be expressed in the progenitor cells, and
at least one of the two reporter genes is not expressed in both the
progenitor cells and the terminally differentiated cells. In some
embodiments, the blastomere cells comprise two different reporter
genes, each encoding a fused protein comprising a cell lineage
specific marker fused to a different fluorescent protein. For
example, the cell can comprise a first reporter gene encoding a
first CLSM::FP construct/fusion protein and a second reporter gene
encoding a second CLSM::FP construct/fusion protein wherein the
CLSM and FP in the first construct/fusion protein are different
from the CLSM and FP in the second construct/fusion protein. In
some further embodiments of this, one of the CLSM::FP construct can
comprise a marker that is also expressed in a progenitor cell of
the cell lineage, while the second CLSM::FP construct comprises a
marker that is preferentially expressed in the terminally
differentiated cells of the cell lineage. For example, the first
reporter gene can encode a cell lineage progenitor cell specific
marker (CLPCSM) fused with a fluorescent protein (i.e., a
CLPCSM::FP construct) and the second reporter gene can encode a
terminally differentiated cell lineage specific marker with a
fluorescent protein (i.e., a DCSLM::FP construct). BY a "terminally
differentiated cell lineage marker" is meant a cell marker that is
present only in the terminally differentiated cells. Without
limitations, cells comprising the two different reporter genes can
be used to screen for compounds that modulate differentiation of
pluripotent cells into progenitor cells; differentiation of
progenitor cells into fully differentiated cells; or
differentiation of pluripotent cells into fully differentiation
cells.
[0129] In some embodiments, the cell lineage specific marker can be
a marker of a myogenic cell. In some embodiments, the cell lineage
specific marker is a marker of myogenic progenitor cells. In some
embodiments, the cell lineage specific marker is a maker of
mesodermal cells. Myogenic, myogenic progenitor, and mesodermal
lineage markers are described elsewhere in the disclosure.
[0130] Examples of fluorescent proteins suitable for use include,
but are not limited to, green fluorescent protein, red fluorescent
protein (e.g., DsRed), yellow fluorescent protein, cyan fluorescent
protein, blue fluorescent protein, and variants thereof (see, e.g.,
U.S. Pat. Nos. 6,403,374, 6,800,733, and 7,157,566). Specific
examples of GFP variants include, but are not limited to, enhanced
GFP (EGFP), destabilized EGFP, the GFP variants described in Doan
et al, Mol. Microbiol, 55:1767-1781 (2005), the GFP variant
described in Crameri et al, Nat. Biotechnol., 14:315319 (1996), the
cerulean fluorescent proteins described in Rizzo et al, Nat.
Biotechnol, 22:445 (2004) and Tsien, Annu. Rev. Biochem., 67:509
(1998), and the yellow fluorescent protein described in Nagal et
al, Nat. Biotechnol., 20:87-90 (2002). DsRed variants are described
in, e.g., Shaner et al, Nat. Biotechnol., 22:1567-1572 (2004), and
include mStrawberry, mCherry, morange, mBanana, mHoneydew, and
mTangerine. Additional DsRed variants are described in, e.g., Wang
et al, Proc. Natl. Acad. Sci. U.S.A., 101:16745-16749 (2004) and
include mRaspberry and mPlum. Further examples of DsRed variants
include mRFPmars described in Fischer et al, FEBS Lett.,
577:227-232 (2004) and mRFPruby described in Fischer et al, FEBS
Lett, 580:2495-2502 (2006).
[0131] Other non-limiting list of fluorescent proteins includes
AceGFP, AcGFP1, AmCyan1, AQ143, AsRed2, Azami-Green (mAG),
Cerulean, Cerulean, Citrine, cOFP, CopGFP, Cyan, CyPet, Dronpa,
DsRed/DsRed2/DsRed-Express, DsRed-Monomer, EBFP, ECFP, EGFP,
Emerald, eqFP611, EYFP, GFPs, HcRedl, HcRed-tandem, J-Red, Kaede,
KFP, KikGR, mBanana, mCFP, mCherry, mCitrine, mEosEP, mHoneydew,
MiCy, mKO, mOrange, mPlum, mRaspberry, mRFP1, mStrawberry,
mTangerine, mYFP, mYFP, mYFP, PA-GFP, PA-mRFP, PhiYFP, PS-CFP-2,
Renilla, tdEosFP, tdTomato, T-Sapphire, TurboGFP, UV-T-Sapphire,
Venus, YPet, ZsYellow1, and derivatives and analogs thereof. In one
embodiment, the fluorescent protein is Green Fluorescent Protein
(GFP).
[0132] One of skill in the art is well aware of methods for
constructing reporter genes that encode fusion proteins comprising
fluorescent proteins.
[0133] Specific devices or methods known in the art for the
detection of fluorescence, e.g., from fluorophores or fluorescent
proteins, include, but are not limited to, in vivo near-infrared
fluorescence (see, e.g., Frangioni, Curr. Opin. Chem. Biol,
7:626-634 (2003)), the Maestro.TM. in vivo fluorescence imaging
system (Cambridge Research & Instrumentation, Inc.; Woburn,
Mass.), in vivo fluorescence imaging using a flying-spot scanner
(see, e.g., Ramanujam et al, IEEE Transactions on Biomedical
Engineering, 48:1034-1041 (2001), and the like. Other methods or
devices for detecting an optical response include, without
limitation, visual inspection, CCD cameras, video cameras,
photographic film, laser-scanning devices, fluorometers,
photodiodes, quantum counters, epifluorescence microscopes,
scanning microscopes, flow cytometers, fluorescence microplate
readers, or signal amplification using photomultiplier tubes.
[0134] In some embodiments, the FP can be Green fluorescent protein
(GFP) or mCherry.
[0135] In some embodiments, the reporter gene encodes a fusion
protein comprising Myf5 and GFP. In some embodiments, the reporter
gene encodes a fusion protein comprising mylz2 and mCherry.
[0136] The term "blastomere" is used throughout to refer to at
least one blastomere (e.g., 1, 2, 3, 4, etc. . . . ) obtained from
an embryo. The term "cluster of two or more blastomeres" is used
interchangeably with "blastomere-derived outgrowths" to refer to
the cells generated during the in vitro culture of a blastomere.
For example, after a blastomere is obtained from an embryo and
initially cultured, it generally divides at least once to produce a
cluster of two or more blastomeres (also known as a
blastomere-derived outgrowth). The cluster can be further cultured
with embryonic or fetal cells. Ultimately, the blastomere-derived
outgrowths will continue to divide. From these structures, ES
cells, TS cells, and partially differentiated cell types can
develop over the course of the culture method.
[0137] The blastomere can be removed from an embryo at various
developmental stages prior to implantation including but not
limited to: before compaction of the morula, during compaction of
the morula, right after compaction of the morula, before formation
of the blastocoel or during the blastocyst stage. In some
embodiments, a blastomere (one blastomere, two blastomeres, or more
than two blastomeres) are from oblong-stage embryos. The
oblong-stage embryos can be dissociated to obtain the
blastomeres.
[0138] For the screening assay disclosed herein, the blastomere can
be obtained from any source available to the practitioner or one of
skill in the art. In some embodiments, the blastomere is from
zebrafish. As used herein, the term "zebrafish" refers to any fish
or strain of fish that is considered to be of the genus and species
Danio rerio. In some embodiments, the blastomere can be from a
transgenic species that expresses a cell lineage specific marker
fused with a fluorescent protein. One of skill in the ordinary
skill in the art is well aware of methods from producing transgenic
zebrafish and can easily use these methods for producing transgenic
zebrafush from which the blastomeres for the screening assay can be
obtained. See, for example, Gabriela, et al., BMC Developmental
Biology 2007, 7:62 (doi:10.1186/1471-213X-7-62), content of which
is incorporated herein by reference in its entirety.
[0139] Culture media for culturing the blastomere can be any
suitable media available to one of skill in the art for culturing
blastomeres. For example, one can use zESC medium, which is
composed of 70% LDF medium and 30% RTS34st-conditioned medium, as
discussed in the Examples section herein.
[0140] For the assay, blastomere can be optionally allowed to grow
for a period time before contacting with the test compound. In some
embodiments, a practitioner can obtain blastomeres that are already
planted in the appropriate vessel and allowed to grow for a period
of time. In other embodiments, the practitioner plates the
blastomeres in the appropriate vessel and allow them to grow for a
period time, e.g., at least one day, at least two days, at least
three days, at least four days, at least five days, at least six
days, at least seven days or more before contacting with the test
compound.
[0141] After the test compound has been in contact with the cell
for a sufficient period of time, amount of reporter (e.g.,
expression or activity) is measured and compared to a control or
reference. For example, contact time can be from seconds to days or
weeks. The practitioner can optimized the contact time for
obtaining an optimal signal-to-noise ratio, time constraints,
amount of test compound to be tested, number of cells, test volume,
availability of reagents for the assay, and the like.
[0142] As used herein, the term "test compound" refers to compounds
and/or compositions that are to be screened for their ability to
stimulate and/or increase and/or promote motor neuron survival. The
test compounds can include a wide variety of different compounds,
including chemical compounds and mixtures of chemical compounds,
e.g., small organic or inorganic molecules; saccharines;
oligosaccharides; polysaccharides; biological macromolecules, e.g.,
peptides, proteins, and peptide analogs and derivatives;
peptidomimetics; nucleic acids; nucleic acid analogs and
derivatives; an extract made from biological materials such as
bacteria, plants, fungi, or animal cells; animal tissues; naturally
occurring or synthetic compositions; and any combinations thereof.
In some embodiments, the test compound is a small molecule.
[0143] The number of possible test compounds runs into millions.
Methods for developing small molecule, polymeric and genome based
libraries are described, for example, in Ding, et al. J Am. Chem.
Soc. 124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123:
8155-8156 (2001). Commercially available compound libraries can be
obtained from, e.g., ArQule, Pharmacopia, graffinity, Panvera,
Vitas-M Lab, Biomol International and Oxford. These libraries can
be screened using the screening devices and methods described
herein. Chemical compound libraries such as those from NIH Roadmap,
Molecular Libraries Screening Centers Network (MLSCN) can also be
used. A comprehensive list of compound libraries can be found at
www.broad.harvard.edu/chembio/platform/screening/compound_libraries/index-
.htm. A chemical library or compound library is a collection of
stored chemicals usually used ultimately in high-throughput
screening or industrial manufacture. The chemical library can
consist in simple terms of a series of stored chemicals. Each
chemical has associated information stored in some kind of database
with information such as the chemical structure, purity, quantity,
and physiochemical characteristics of the compound.
[0144] As used herein, the term "small molecule" can refer to
compounds that are "natural product-like," however, the term "small
molecule" is not limited to "natural product-like" compounds.
Rather, a small molecule is typically characterized in that it
contains several carbon-carbon bonds, and has a molecular weight
more than about 50, but less than about 5000 Daltons (5 kD).
Preferably the small molecule has a molecular weight of less than 3
kD, still more preferably less than 2 kD, and most preferably less
than 1 kD. In some cases it is preferred that a small molecule have
a molecular mass equal to or less than 700 Daltons.
[0145] Depending upon the particular embodiment being practiced,
the test compounds can be provided free in solution, or may be
attached to a carrier, or a solid support, e.g., beads. A number of
suitable solid supports may be employed for immobilization of the
test compounds. Examples of suitable solid supports include
agarose, cellulose, dextran (commercially available as, i.e.,
Sephadex, Sepharose) carboxymethyl cellulose, polystyrene,
polyethylene glycol (PEG), filter paper, nitrocellulose, ion
exchange resins, plastic films, polyaminemethylvinylether maleic
acid copolymer, glass beads, amino acid copolymer, ethylene-maleic
acid copolymer, nylon, silk, etc. Additionally, for the methods
described herein, test compounds can be screened individually, or
in groups. Group screening is particularly useful where hit rates
for effective test compounds are expected to be low such that one
would not expect more than one positive result for a given group.
Group screening is also useful for determining hits that can act
synergistically.
[0146] The test compound can be tested at any desired
concentration. For example, the test compound can be tested at a
final concentration of from 0.01 nm to about 10 mM. Further, the
test can be tested at 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) different concentrations. This can be helpful if the test
compound is active only in a range of concentration. When the test
compound is tested at 2 or more different concentrations, the
concentration difference can range from 10-10,000 fold (e.g.,
10-5000 fold, 10-1000 fold, 10-500 fold, or 10-250 fold).
[0147] The screening assay can be performed in any suitable
container or apparatus available to one of skill in the art for
cell culturing. For example, the assay can be performed in 24-,
96-, or 384-well plates. In one embodiment, the assay is performed
in a 384-well plate.
[0148] In some embodiments, the screening method is a
high-throughput screening. High-throughput screening (HTS) is a
method for scientific experimentation that uses robotics, data
processing and control software, liquid handling devices, and
sensitive detectors. High-Throughput Screening or HTS allows a
researcher to quickly conduct millions of biochemical, genetic or
pharmacological tests. High-Throughput Screening are well known to
one skilled in the art, for example, those described in U.S. Pat.
Nos. 5,976,813; 6,472,144; 6,692,856; 6,824,982; and 7,091,048, and
contents of each of which is herein incorporated by reference in
its entirety.
[0149] HTS uses automation to run a screen of an assay against a
library of candidate compounds. An assay is a test for specific
activity: usually inhibition or stimulation of a biochemical or
biological mechanism. Typical HTS screening libraries or "decks"
can contain from 100,000 to more than 2,000,000 compounds.
[0150] The key labware or testing vessel of HTS is the microtiter
plate: a small container, usually disposable and made of plastic,
which features a grid of small, open divots called wells. Modern
microplates for HTS generally have either 384, 1536, or 3456 wells.
These are all multiples of 96, reflecting the original 96 well
microplate with 8.times.12 9 mm spaced wells.
[0151] To prepare for an assay, the researcher fills each well of
the plate with the appropriate reagents that he or she wishes to
conduct the experiment with, such as a blastomere cell or
population. After some incubation time has passed to allow the
reagent to absorb, bind to, or otherwise react (or fail to react)
with the compounds in the wells, measurements are taken across all
the plate's wells, either manually or by a machine. Manual
measurements are often necessary when the researcher is using
microscopy to (for example) seek changes that a computer could not
easily determine by itself. Otherwise, a specialized automated
analysis machine can run a number of experiments on the wells such
as colorimetric measurements, radioactivity counting, etc. In this
case, the machine outputs the result of each experiment as a grid
of numeric values, with each number mapping to the value obtained
from a single well. A high-capacity analysis machine can measure
dozens of plates in the space of a few minutes like this,
generating thousands of experimental data points very quickly.
[0152] Screening using zebrafish embryos is useful for the study of
development, disease, and stem cell biology. However, the required
manual manipulation of zebrafish embryos prevents capitalizing on
recent innovations in high-speed pipetting and imaging. Compared to
screening using zebrafish embryos, the screening assay described
herein is highly automatic, taking one sixth of the time, and
consumes only one tenth of the embryo. Such throughput enables
screening of larger chemical libraries and can be used for
screening of a genetic mutant suppressor. For example, several
zebrafish anemia mutants, including cia, edy, and weh, have defects
in iron metabolism and lack benzidine staining, a marker for mature
erythrocytes. The screening assay of the present disclosure can be
designed to screen for chemicals that restore benzidine staining.
Without wishing to be bound by a theory, the chemicals identified
can be used to treat these iron metabolism defects.
[0153] Transgenic zebrafish with a fluorescent reporter that labels
a cell or tissue of interest are widely used in the study of
development and stem cell biology. Despite the pioneering of
transgenic reporter lines for screening, readout by WISH is usually
preferred. Compared to WISH, transgenic reporters are usually
thought to be less sensitive to detect a difference. Moreover,
transgenic embryos need to be fixed and scored immediately.
However, the inventors surprising and unexpectedly found that
transgenic reporters were useful for the screening assay described
herein, mainly because transgenic reporters in a 2-dimensional
culture are more sensitive than those in a 3-D embryo. Moreover,
because images are automatically captured and stored by imaging
cytometers, the cells do not need to be fixed or scored
immediately. Further, the screening assay described herein enables
combining reporters of different colors, allowing the determination
of different developmental states or lineages simultaneously.
[0154] Since blastomere cells in culture lack spatial and temporal
information, the screening assay described herein uses cellular
markers, like transgenic fluorescent reporters. It can be
beneficial to combine reporters with different colors to better
define a lineage or state. Cell membrane markers, like F-actin and
Membrane-GFP, and nuclear markers, like DAPI and H2B-Tomato, can
also be used to acquire additional information regarding cellular
morphology and cell number. The possibility that a transgenic
reporter may behave differently in vitro requires researchers to
confirm that the reporter line in culture recapitulates its in vivo
expression pattern. Lastly, some lineages can be hard to derive
from zebrafish blastomere cells. The inventors circumvented this
problem by disassociating embryos at later stages. For example, to
study erythrocyte differentiation, the inventors disassociated
embryos at 24hpf, 27hpf, or 30hpf, and stained the cells with
benzidine. The inventors discovered that cells disassociated at
these various stages showed increasing staining, indicating that
they are in different stages of erythrocyte development (data not
shown). Thus, without wishing to be bound by a theory, a chemical
screen using the method disclosed in this disclosure to study
erythrocyte development can be designed using benzidine
staining.
[0155] The screening assay disclosed herein is also a substitute
for the ESC/iPSC in studies of lineage differentiation and
self-renewal of tissues. Because ESCs/iPSCs can theoretically be
induced into any desired tissue for organ repair, they hold promise
for treating diseases and injuries that are caused by cellular
deficiencies. One major challenge is being able to differentiate
ESCs/iPSCs into cells or tissues of interest efficiently. Knowledge
gained in developmental biology has guided the design of protocols
that differentiate ESCs/iPSCs in ways that recapitulate the
progression of embryonic development. However, to obtain
homogenous, functional, and transplantable cells or tissues of
interest, a more precise understanding of lineage differentiation
is needed. To this end, the screening assay described herein offers
several major advantages. Firstly, zebrafish blastomere cells
develop much faster than mouse and human ESCs/iPSCs, presumably
because embryogenesis programs are accelerated in zebrafish. For
example, zebrafish blastomere cells form skeletal muscle within 48
hours, while mouse ESCs/iPSCs need approximately 20 days. This
allows for faster screening with less variability. Secondly,
zebrafish blastomere cells are cultured at 28 degrees, a lower
temperature at which chemicals are more stable. Thirdly, the
screening assay does not require culture maintenance or sterile
manipulation. The fact that a large number of cells can be easily
derived from freshly spawned embryos makes the system very
cost-efficient. Lastly, the transparent adult zebrafish casper
mutant was developed for in vivo stem cell engraftment analysis.
Cells or tissues of interest can be transplanted into casper to
study engraftment of tissues in vivo.
[0156] The development of a robust chemical screening system using
blastomere cells is an important complement to studies using
embryos. Without wishing to be bound by a theory, other genetic
tools like morpholino/siRNA knockdown and mRNA overexpression can
be used to enable genome-wide loss-of-function and gain-of-function
genetic screening using blastomere cells. A casper based
transplantation system can be exploited for the study of
engraftment and self-renewal of cells derived from the system. In
summary, the data presented herein indicates that the system
described herein enables screening using well-established
fluorescent transgenic lines and capitalizes on advances in
high-speed pipetting and imaging systems. This system can be
modified for any cell lineage and can enhance our understanding of
developmental biology and to provide insights into cell-based
therapies for many diseases.
[0157] The disclosure also provides for a cell culture system
comprising a population of dissociated blastomere cells that
comprise two different fusion protein constructs, each construct
comprising a cell lineage specific marker fused with a fluorescent
protein.
[0158] The present disclosure also provides a compound selected by
the screening assay disclosed in this disclosure.
Some Selected Definitions
[0159] For convenience, certain terms employed herein, in the
specification, examples and appended claims are collected herein.
Unless stated otherwise, or implicit from context, the following
terms and phrases include the meanings provided below. Unless
explicitly stated otherwise, or apparent from context, the terms
and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments, and are not intended to limit the claimed invention,
because the scope of the invention is limited only by the claims.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the
singular.
[0160] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0161] As used herein the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0162] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise.
[0163] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.5% (e.g., .+-.4%, .+-.3%,
.+-.2%, or .+-.1%) of the value being referred to.
[0164] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
this disclosure, suitable methods and materials are described
below. The term "comprises" means "includes." The abbreviation,
"e.g." is derived from the Latin exempli gratia, and is used herein
to indicate a non-limiting example. Thus, the abbreviation "e.g."
is synonymous with the term "for example."
[0165] As used herein, the term "herein" is used to refer to the
whole disclosure and is not meant to be restricted to a specific
section or subsection of the disclosure.
[0166] The terms "decrease", "reduced", "reduction", "decrease" or
"inhibit" are all used herein generally to mean a decrease by a
statistically significant amount. However, for avoidance of doubt,
"reduced", "reduction" or "decrease" or "inhibit" means a decrease
by at least 10% as compared to a reference level, for example a
decrease by at least about 20%, or at least about 30%, or at least
about 40%, or at least about 50%, or at least about 60%, or at
least about 70%, or at least about 80%, or at least about 90% or up
to and including a 100% decrease (e.g. absent level as compared to
a reference sample), or any decrease between 10-100% as compared to
a reference level.
[0167] The terms "increased", "increase" or "enhance" or "activate"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of any doubt, the terms
"increased", "increase" or "enhance" or "activate" means an
increase of at least 10% as compared to a reference level, for
example an increase of at least about 20%, or at least about 30%,
or at least about 40%, or at least about 50%, or at least about
60%, or at least about 70%, or at least about 80%, or at least
about 90% or up to and including a 100% increase or any increase
between 10-100% as compared to a reference level, or at least about
a 2-fold, or at least about a 3-fold, or at least about a 4-fold,
or at least about a 5-fold or at least about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a
reference level.
[0168] The term "statistically significant" or "significantly"
refers to statistical significance and generally means at least two
standard deviation (2SD) away from a reference level. The term
refers to statistical evidence that there is a difference. It is
defined as the probability of making a decision to reject the null
hypothesis when the null hypothesis is actually true.
[0169] As used herein, the term "treating" and "treatment" refers
to administering to a subject an effective amount of a composition
so that the subject as a reduction in at least one symptom of the
disease or an improvement in the disease, for example, beneficial
or desired clinical results. For purposes of this invention,
beneficial or desired clinical results include, but are not limited
to, alleviation of one or more symptoms, diminishment of extent of
disease, stabilized (e.g., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. In some embodiments, treating
can refer to prolonging survival as compared to expected survival
if not receiving treatment. Thus, one of skill in the art realizes
that a treatment may improve the disease condition, but may not be
a complete cure for the disease. As used herein, the term
"treatment" includes prophylaxis. Alternatively, treatment is
"effective" if the progression of a disease is reduced or halted.
In some embodiments, the term "treatment" can also mean prolonging
survival as compared to expected survival if not receiving
treatment. Those in need of treatment include those already
diagnosed with a disease or condition, as well as those likely to
develop a disease or condition due to genetic susceptibility or
other factors which contribute to the disease or condition, such as
a non-limiting example, weight, diet and health of a subject are
factors which may contribute to a subject likely to develop
diabetes mellitus. Those in need of treatment also include subjects
in need of medical or surgical attention, care, or management. The
subject is usually ill or injured, or at an increased risk of
becoming ill relative to an average member of the population and in
need of such attention, care, or management.
[0170] As used herein, the terms "administering," "introducing" and
"transplanting" are used interchangeably in the context of the
placement of the cells, e.g., myogenic cells or satellite cells, as
disclosed herein, or their differentiated progeny into a subject,
by a method or route which results in at least partial localization
of the cells, or their differentiated progeny at a desired site.
The cells, or their differentiated progeny can be administered
directly to a tissue of interest, or alternatively be administered
by any appropriate route which results in delivery to a desired
location in the subject where at least a portion of the cells or
their progeny or components of the cells remain viable. The period
of viability of the cells after administration to a subject can be
as short as a few hours, e. g. twenty-four hours, to a few days, to
as long as several years.
[0171] The term "transplantation" as used herein refers to
introduction of new cells (e.g. myogenic cells or satellite cells),
tissues (such as differentiated cells produced from the cells), or
organs into a host (i.e. transplant recipient or transplant
subject).
[0172] The term "cell culture medium" (also referred to herein as a
"culture medium" or "medium") as referred to herein is a medium for
culturing cells containing nutrients that maintain cell viability
and support proliferation. The cell culture medium can contain any
of the following in an appropriate combination: salt(s), buffer(s),
amino acids, glucose or other sugar(s), antibiotics, serum or serum
replacement, and other components such as peptide growth factors,
etc. Cell culture media ordinarily used for particular cell types
are known to those skilled in the art.
[0173] The term "cell line" refers to a population of largely or
substantially identical cells that has typically been derived from
a single ancestor cell or from a defined and/or substantially
identical population of ancestor cells. The cell line may have been
or may be capable of being maintained in culture for an extended
period (e.g., months, years, for an unlimited period of time). It
may have undergone a spontaneous or induced process of
transformation conferring an unlimited culture lifespan on the
cells. Cell lines include all those cell lines recognized in the
art as such. It will be appreciated that cells acquire mutations
and possibly epigenetic changes over time such that at least some
properties of individual cells of a cell line may differ with
respect to each other.
[0174] The term "lineages" as used herein describes a cell with a
common ancestry or cells with a common developmental fate. By way
of an example only, a cell that is of endoderm origin or is
"endodermal lineage" means the cell was derived from an endodermal
cell and can differentiate along the endodermal lineage restricted
pathways, such as one or more developmental lineage pathways which
give rise to definitive endoderm cells, which in turn can
differentiate into liver cells, thymus, pancreas, lung and
intestine. A cell that is of mesoderm origin or is "mesodermal
lineage" means that the cell was derived from an myogenic cell and
can differentiate along the mesodermal lineage restricted pathways,
such as one or more myogenic lineage pathways, which give rise to
muscle cells, e.g., skeletal muscle cells.
[0175] The term "differentiation" as used herein refers to the
cellular development of a cell from a primitive stage towards a
more mature (i.e. less primitive) cell. The term "directed
differentiation" as used herein refers to forcing differentiation
of a cell from an undifferentiated (e.g. more primitive cell) to a
more mature cell type (i.e. less primitive cell) via non-genetic
manipulation. In some embodiments, a stem cell is subject to
directed differentiation into a myogenic cell.
[0176] The term "pluripotency" or a "pluripotent state" as used
herein refers to a cell with the ability to differentiate into all
three embryonic germ layers: endoderm (gut tissue), mesoderm
(including blood, muscle, and vessels), and ectoderm (such as skin
and nerve), and typically has the potential to divide in vitro for
a long period of time, e.g., greater than one year or more than 30
passages.
[0177] The term "multipotency" refers to a cell with the degree of
developmental versatility that is less than totipotent and
pluripotent.
[0178] The term "totipotency" refers to a cell with the degree of
differentiation describing a capacity to make all of the cells in
the adult body as well as the extra-embryonic tissues including the
placenta. The fertilized egg (zygote) is totipotent as are the
early cleaved cells (blastomeres)
[0179] The term "differentiated cell" is meant any primary cell
that is not, in its native form, pluripotent as that term is
defined herein. The term a "differentiated cell" also encompasses
cells that are partially differentiated, such as multipotent cells,
or cells that are stable non-pluripotent partially reprogrammed
cells. It should be noted that placing many primary cells in
culture can lead to some loss of fully differentiated
characteristics. Thus, simply culturing such cells are included in
the term differentiated cells and does not render these cells
non-differentiated cells (e.g. undifferentiated cells) or
pluripotent cells. The transition of a differentiated cell to
pluripotency requires a reprogramming stimulus beyond the stimuli
that lead to partial loss of differentiated character in culture.
Reprogrammed cells also have the characteristic of the capacity of
extended passaging without loss of growth potential, relative to
primary cell parents, which generally have capacity for only a
limited number of divisions in culture. In some embodiments, the
term "differentiated cell" also refers to a cell of a more
specialized cell type derived from a cell of a less specialized
cell type (e.g., from an undifferentiated cell or a reprogrammed
cell) where the cell has undergone a cellular differentiation
process.
[0180] As used herein, the term "somatic cell" refers to any cell
other than a germ cell, a cell present in or obtained from a
pre-implantation embryo, or a cell resulting from proliferation of
such a cell in vitro. Stated another way, a somatic cell refers to
any cells forming the body of an organism, as opposed to germline
cells. In mammals, germline cells (also known as "gametes") are the
spermatozoa and ova which fuse during fertilization to produce a
cell called a zygote, from which the entire mammalian embryo
develops. Every other cell type in the mammalian body--apart from
the sperm and ova, the cells from which they are made (gametocytes)
and undifferentiated stem cells--is a somatic cell: internal
organs, skin, bones, blood, and connective tissue are all made up
of somatic cells. In some embodiments the somatic cell is a
"non-embryonic somatic cell", by which is meant a somatic cell that
is not present in or obtained from an embryo and does not result
from proliferation of such a cell in vitro. In some embodiments the
somatic cell is an "adult somatic cell", by which is meant a cell
that is present in or obtained from an organism other than an
embryo or a fetus or results from proliferation of such a cell in
vitro.
[0181] As used herein, the term "adult cell" refers to a cell found
throughout the body after embryonic development.
[0182] In the context of cell ontogeny, the term "differentiate",
or "differentiating" is a relative term meaning a "differentiated
cell" is a cell that has progressed further down the developmental
pathway than its precursor cell. Thus in some embodiments, a
reprogrammed cell as this term is defined herein, can differentiate
to lineage-restricted precursor cells (such as a mesodermal stem
cell), which in turn can differentiate into other types of
precursor cells further down the pathway (such as an tissue
specific precursor, for example, a cardiomyocyte precursor), and
then to an end-stage differentiated cell, which plays a
characteristic role in a certain tissue type, and may or may not
retain the capacity to proliferate further.
[0183] The term "phenotype" refers to one or a number of total
biological characteristics that define the cell or organism under a
particular set of environmental conditions and factors, regardless
of the actual genotype.
[0184] The term "exogenous" refers to a substance present in a cell
other than its native source. The terms "exogenous" when used
herein refers to a nucleic acid (e.g. a nucleic acid encoding a
sox2 transcription factor) or a protein (e.g., a sox2 polypeptide)
that has been introduced by a process involving the hand of man
into a biological system such as a cell or organism in which it is
not normally found or in which it is found in lower amounts. A
substance (e.g. a nucleic acid encoding a sox2 transcription
factor, or a protein, e.g., a sox2 polypeptide) will be considered
exogenous if it is introduced into a cell or an ancestor of the
cell that inherits the substance. In contrast, the term
"endogenous" refers to a substance that is native to the biological
system or cell (e.g. differentiated cell).
[0185] The term "isolated" or "partially purified" as used herein
refers, in the case of a nucleic acid or polypeptide, to a nucleic
acid or polypeptide separated from at least one other component
(e.g., nucleic acid or polypeptide) that is present with the
nucleic acid or polypeptide as found in its natural source and/or
that would be present with the nucleic acid or polypeptide when
expressed by a cell, or secreted in the case of secreted
polypeptides. A chemically synthesized nucleic acid or polypeptide
or one synthesized using in vitro transcription/translation is
considered "isolated".
[0186] The term "isolated cell" as used herein refers to a cell
that has been removed from an organism in which it was originally
found or a descendant of such a cell. Optionally the cell has been
cultured in vitro, e.g., in the presence of other cells. Optionally
the cell is later introduced into a second organism or
re-introduced into the organism from which it (or the cell from
which it is descended) was isolated.
[0187] The term "isolated population" with respect to an isolated
population of cells as used herein refers to a population of cells
that has been removed and separated from a mixed or heterogeneous
population of cells. In some embodiments, an isolated population is
a substantially pure population of cells as compared to the
heterogeneous population from which the cells were isolated or
enriched from. In some embodiments, the isolated population is an
isolated population of reprogrammed cells which is a substantially
pure population of reprogrammed cells as compared to a
heterogeneous population of cells comprising reprogrammed cells and
cells from which the reprogrammed cells were derived.
[0188] The term "substantially pure", with respect to a particular
cell population, refers to a population of cells that is at least
about 75%, preferably at least about 85%, more preferably at least
about 90%, and most preferably at least about 95% pure, with
respect to the cells making up a total cell population. Recast, the
terms "substantially pure" or "essentially purified", with regard
to a population of reprogrammed cells, refers to a population of
cells that contain fewer than about 20%, more preferably fewer than
about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%,
3%, 2%, 1%, or less than 1%, of cells that are not reprogrammed
cells or their progeny as defined by the terms herein. In some
embodiments, the present invention encompasses methods to expand a
population of reprogrammed cells, wherein the expanded population
of reprogrammed cells is a substantially pure population of
reprogrammed cells.
[0189] As used herein, the term "marker" describes the
characteristics and/or phenotype of a cell. Markers can be used for
selection of cells comprising characteristics of interests. Markers
will vary with specific cells. Markers are characteristics, whether
morphological, functional or biochemical (enzymatic)
characteristics of the cell of a particular cell type, or molecules
expressed by the cell type. Preferably, such markers are proteins,
and more preferably, possess an epitope for antibodies or other
binding molecules available in the art. However, a marker may
consist of any molecule found in a cell including, but not limited
to, proteins (peptides and polypeptides), lipids, polysaccharides,
nucleic acids and steroids. Examples of morphological
characteristics or traits include, but are not limited to, shape,
size, and nuclear to cytoplasmic ratio. Examples of functional
characteristics or traits include, but are not limited to, the
ability to adhere to particular substrates, ability to incorporate
or exclude particular dyes, ability to migrate under particular
conditions, and the ability to differentiate along particular
lineages. Markers can be detected by any method available to one of
skill in the art. Markers can also be the absence of a
morphological characteristic or absence of proteins, lipids etc.
Markers can be a combination of a panel of unique characteristics
of the presence and absence of polypeptides and other morphological
characteristics.
[0190] The term "selectable marker" refers to a gene, RNA, or
protein that when expressed, confers upon cells a selectable
phenotype, such as resistance to a cytotoxic or cytostatic agent
(e.g., antibiotic resistance), nutritional prototrophy, or
expression of a particular protein that can be used as a basis to
distinguish cells that express the protein from cells that do not.
Proteins whose expression can be readily detected such as a
fluorescent or luminescent protein or an enzyme that acts on a
substrate to produce a colored, fluorescent, or luminescent
substance ("detectable markers") constitute a subset of selectable
markers. The presence of a selectable marker linked to expression
control elements native to a gene that is normally expressed
selectively or exclusively in pluripotent cells makes it possible
to identify and select somatic cells that have been reprogrammed to
a pluripotent state. A variety of selectable marker genes can be
used, such as neomycin resistance gene (neo), puromycin resistance
gene (puro), guanine phosphoribosyl transferase (gpt),
dihydrofolate reductase (DHFR), adenosine deaminase (ada),
puromycin-N-acetyltransferase (PAC), hygromycin resistance gene
(hyg), multidrug resistance gene (mdr), thymidine kinase (TK),
hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD
gene. Detectable markers include green fluorescent protein (GFP)
blue, sapphire, yellow, red, orange, and cyan fluorescent proteins
and variants of any of these. Luminescent proteins such as
luciferase (e.g., firefly or Renilla luciferase) are also of use.
As will be evident to one of skill in the art, the term "selectable
marker" as used herein can refer to a gene or to an expression
product of the gene, e.g., an encoded protein.
[0191] In some embodiments the selectable marker confers a
proliferation and/or survival advantage on cells that express it
relative to cells that do not express it or that express it at
significantly lower levels. Such proliferation and/or survival
advantage typically occurs when the cells are maintained under
certain conditions, e.g., "selective conditions". To ensure an
effective selection, a population of cells can be maintained for a
under conditions and for a sufficient period of time such that
cells that do not express the marker do not proliferate and/or do
not survive and are eliminated from the population or their number
is reduced to only a very small fraction of the population. The
process of selecting cells that express a marker that confers a
proliferation and/or survival advantage by maintaining a population
of cells under selective conditions so as to largely or completely
eliminate cells that do not express the marker is referred to
herein as "positive selection", and the marker is said to be
"useful for positive selection". Negative selection and markers
useful for negative selection are also of interest in certain of
the methods described herein. Expression of such markers confers a
proliferation and/or survival disadvantage on cells that express
the marker relative to cells that do not express the marker or
express it at significantly lower levels (or, considered another
way, cells that do not express the marker have a proliferation
and/or survival advantage relative to cells that express the
marker). Cells that express the marker can therefore be largely or
completely eliminated from a population of cells when maintained in
selective conditions for a sufficient period of time.
[0192] The term "drug screening" as used herein refers to the use
of cells and tissues in the laboratory to identify drugs with a
specific function. In some embodiments, the present invention
provides drug screening on myogenic cells or satellite cells
produced by the method disclosed herein to identify compounds or
drugs useful as therapies for diseases or illnesses (e.g. human
diseases or illnesses).
[0193] The term "co-administering," "co-administration," or
"co-administer" refers to the administration of two or more
compounds to the subject, wherein the two or more compounds can be
administered simultaneously, or at different times, as long as they
work additively or synergistically. The compounds can be
administered in the same formulation or in separate formulations.
When administered in separate formulations, the compounds can be
administered within any time of each other. For example, the
compounds can be administered within 24 hours, 12 hours, 6 hours, 5
hours, 4 hours, 3 hours, 2 hours, 1 hours, 45 minutes, 30 minute,
25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less
of each other. When administered in separate formulations, any
compound can be administered first. Additionally, co-administration
does not require the different compounds to be administered by the
same route, i.e., the components of the combination can be
administered to a subject by the same or different routes of
administration. As such, each can be administered independently or
as a common dosage form.
[0194] The disclosure is further illustrated by the following
examples which should not be construed as limiting. The examples
are illustrative only, and are not intended to limit, in any
manner, any of the aspects described herein. The following examples
do not in any way limit the invention.
EXAMPLES
Example 1: A Zebrafish Embryo Culture System Defines Factors that
Promote Vertebrate Myogenesis Across Species
[0195] Ex vivo expansion of satellite cells and directed
differentiation of pluripotent cells to mature skeletal muscle have
proved difficult challenges for regenerative biology. Using a
zebrafish embryo culture system with reporters of early and late
skeletal muscle differentiation, we examined the influence of 2,400
chemicals on myogenesis and identified six that expanded muscle
progenitors, including three GSK3.beta. inhibitors, two calpain
inhibitors and one adenylyl cyclase activator forskolin. Forskolin
also enhanced proliferation of mouse satellite cells in culture and
maintained their ability to engraft in vivo. A combination of bFGF,
forskolin and the GSK3.beta. inhibitor BIO induced skeletal muscle
differentiation in human induced pluripotent stem cells (iPSCs) and
produced engraftable myogenic progenitors that contributed to
muscle repair in vivo. Definition of these approaches to expand
satellite cells and differentiate iPSCs to muscle without genetic
manipulation provides new insights into skeletal muscle biology and
may lead to novel therapies for metabolic and neuromuscular
diseases.
[0196] In this study, we have taken an interdisciplinary,
cross-systems approach to identify evolutionarily conserved
molecular pathways that regulate muscle specification and satellite
cell expansion in three different vertebrate systems. Capitalizing
on the powerful chemical genetics approaches available in the
zebrafish, we first performed a high-throughput image-based
chemical screen using zebrafish blastomere cells and identified 29
compounds that perturbed muscle development and six that promoted
myogenesis. We then tested the muscle promoting compounds against
mouse muscle satellite cells and human iPSCs to identify conserved
activities. Forskolin, an adenylyl cyclase activator, significantly
increased satellite cell proliferation in culture, with the
majority of expanded cells retaining phenotypic and functional
characteristics of freshly isolated satellite cells, including the
capacity to regenerate dystrophic muscle upon transplantation. In
addition, a combination of bFGF, the GSK3.beta. inhibitor BIO and
forskolin drove skeletal muscle specification of human iPSCs
including spontaneous differentiation to mature myofibers and the
production of myogenic progenitors that could engraft and
contribute to mature muscle fibers when transplanted into
immune-compromised recipient mice. Our studies using a zebrafish
embryo culture system elucidated a combination of chemicals that
promotes muscle development in fish, mouse and human cell
populations. This work provides a novel and tractable system to
generate and expand mammalian muscle stem cells for functional
studies of normal muscle development and therapeutics development
for musculoskeletal diseases.
Materials and Methods
[0197] Zebrafish Care and Transgenic Lines:
[0198] The myf5-GFP; mylz2-mCherry double transgenic line was bred
and maintained using standard zebrafish husbandry (Westerfield,
1995). All zebrafish experiments and procedures were performed as
approved by the Children's Hospital Boston Institutional Animal
Care and Use Committee.
[0199] Culture of Disassociated Blastomere Cells:
[0200] Disassociated blastomere cells were grown in zESC medium,
which is composed of 70% LDF medium and 30% RTS34st-conditioned
medium. The LDF medium contains 50% Leibowitz's L-15 (Invitrogen),
35% DMEM (Invitrogen), and 15% Ham's F-12 (Invitrogen),
supplemented with 0.18 g/l sodium bicarbonate, 15 mM HEPES
(Invitrogen),1% L-Glutamine (Invitrogen), 10 .mu.g/ml ciprofloxacin
(Sigma-Aldrich), 100 .mu.g/ml piperacillin (Sigma-Aldrich), 10
.mu.g/ml amphotericin B (Sigma-Aldrich), 10 nM sodium selenite
(Sigma-Aldrich), 1% N2 (Invitrogen), and 2% B27 (Invitrogen). The
RTS34st conditioned medium was prepared by incubating fresh medium
(Leibowitz's L-15 plus 15% FBS) on a confluent culture of RTS34st
cells for 3 days.
[0201] High-Throughput Screen:
[0202] Four 384-well plates were coated with 0.1% gelatin.
[0203] Chemicals from the CHB library were diluted 150 times with
zESC medium containing 1 ng/ml bFGF and were aliquoted 30 .mu.l per
well. Males and females of the myf5-GFP; mylz2-mCherry transgenic
line were set up overnight and kept separated until morning when
they were mixed for mate. Approximately 800 oblong-stage embryos
were washed three times with E3 embryo water and were treated with
pronase to remove chorions. Dechorionated embryos were washed with
E3 embryo water three times and collected into a 50 ml falcon tube
with 25 ml zESC medium containing 1 ng/ml bFGF. Embryos were
disassociated by shaking approximately 20 times and were filtered
through a 70 .mu.m nylon mesh filter. The resulting single cells
were diluted to 60 ml with zESC medium containing 1 ng/ml bFGF and
aliquoted 30 .mu.l per well into the four 384-well plates with
pre-added chemicals. The cells were cultured in a 28-degree
incubator without CO.sub.2. Cells were imaged by a Celigo cytometer
(Cyntellect) under channels of GFP, mCherry, and transmitted light.
The images were analyzed by the built-in software and confirmed
eye.
[0204] Whole-Mount In Situ Hybridization:
[0205] Whole mount in situ hybridizations were performed as
previously described (Belting et al., 2001).
[0206] SMP Isolation and Culture:
[0207] SMP isolation was performed as previously described
(Cerletti et al., 2008; Sherwood et al., 2004). Briefly, MFA cells
were prepared from intact skeletal muscles (EDL, gastrocnemius,
quadriceps, soleus, TA, triceps brachii, abdominal) by digesting
the muscles with collagenase type II and then dispase enzymes. MFA
cells were stained for isolation of
CD45.sup.-Sca-1.sup.-Mac-1.sup.-CXCR4.sup.+b1-integrin.sup.+cell
population by FACS. For in vitro expansion experiments, SMPs were
seeded on collagen/laminin-coated plates in F10 (Gibco) containing
20% horse serum (Atlanta Biologics), 1% penicillin-streptomycin
(Invitrogen) and 1% glutamax (Invitrogen). In experiments that
cells were treated with bFGF, 5 ng/ml bFGF (Sigma) was added to the
medium daily. 50 .mu.M forskolin (Santa Cruz) or 0.1% DMSO was
added to the wells 24 hours after plating, medium was changed with
fresh medium containing the compound 48 hour after plating and the
treatment continued for 36 more hours, after which medium was
changed with fresh medium without the compound. Cells were counted
or used for transplantation after 5 days in culture. For Myogenic
colony formation assay cells were fixed and counted after 6 days in
culture. For the differentiation experiments cells were cultured
for 5 days with or without compound treatment, on day 5 cells were
harvested and equal numbers of cells (8,000 cells) were re-plated
in each well of a 96-well plate in growth medium. Medium was
changed to DMEM (gibco), containing 2% horse serum (Atlanta
Biologics), 1% penicillin-streptomycin (Invitrogen) and with or
without 50 .mu.M Forskolin or 0.1% DMSO after 4 hours. Cells were
fixed after 60 hours in differentiation medium.
[0208] Immunofluorescence and Imaging:
[0209] Cultured SMPs were fixed in 4% paraformaldehyde (PFA) and
stained with 10 .mu.g/ml Hoechst (Invitrogen). Pictures from the
whole well were taken using Celigo cytometer (Cyntellect) under the
UV channel. The images were analyzed and numbers of cells were
counted by the built-in software. Differentiated cells were stained
for myosin heavy chain (Primary antibody: anti-skeletal myosin type
II (fast-twitch) 1:200 and anti-skeletal myosin type I
(slow-twitch) 1:100, Sigma. Secondary: goat anti-mouse IgG
Alexa-555 conjugate (Molecular Probes) 1:250) and 10 .mu.g/ml
Hoechst (Invitrogen) and pictures from the whole well were taken
using Celigo cytometer (Cyntellect) under UV and red channels. The
images were analyzed and percentage of nuclei in myotubes was
calculated using a modified ImageJ macro developed in-house.
Sections of the transplanted muscles were stained for GFP (rabbit
anti-GFP Alexa 488 conjugate (Invitrogen) 1:250) and for dystrophin
(Primary: rabbit anti-dystrophin (Abcam) 1:50. Secondary: goat
anti-rabbit IgG Alexa-555 conjugate (Molecular probes) 1:250) and
imaged using an upright Zesis fluorescent microscope.
[0210] Camp Assay:
[0211] cAMP assay was performed using the cAMP-Glom Assay kit
(Promega) according to the manufacturer's protocol.
[0212] Flow Cytometry:
[0213] Flow cytometry analysis was performed using a BD LSR II,
provided through the Harvard Stem Cell Institute Flow Cytometry
Core Facility. Flow cytometry data were collected using DIVA
(Becton Dickinson (BD), Franklin Lakes, N.J.) software and analyzed
offline using Flowjo software (Tree Star, Inc., Macintosh version
8.1.1, Ashland, Oreg.). Antibodies used for flow cytometry
included: APC/Cy7 anti-mouse Terl 19, clone Terl 19 (1:200,
Biolegend 116223), APC/Cy7 anti-mouse CD45, clone 30-F11 (1:200,
Biolegend 103116), APC/Cy7 anti-mouse CD11b, clone M1/70 (1:200,
Biolegend 101226), APC Anti-mouse Ly-6A/E (Sca-1), clone D7 (1:200,
Biolegend 108112), Biotin anti-mouse CD184 (CXCR4, Fusin) (1:100,
BD Biosciences 551968), Streptavidin PE-Cy7 (1:100, eBioscience
25-4317-82), PE anti-mouse/rat CD29 Antibody (1:100, Biolegend
102208). Live cells were identified by positive staining with
calcein blue (1:1000) (Invitrogen, Carlsbad, Calif.) and negative
staining for propidium iodide (PI, 1 .mu.g/mL). Antibody
incubations were performed in staining medium (SM=Hank's Buffered
Saline Solution (HBSS, (Gibco))+2% donor horse serum), on ice for
15 min.
[0214] Tissue Injury and Mouse Satellite Cell Transplantation:
[0215] mdx mice were anesthetized and 25 .mu.l (0.03 mg/ml) of Naja
mossambica mossambica cardiotoxin (Sigma) was injected in their TA
muscle 1 day before cell transplantation. The next day, 6,000
freshly isolated double-sorted GFP.sup.+ SMPs or total number of
cells expanded from 6,000 SMPs after 5 days in culture and compound
treatment, were injected directly into these pre-injured muscles in
20 .mu.l PBS. The contralateral TA was injected with PBS only as
the negative control. 3-4 weeks after transplantation, transplanted
muscles were harvested and analyzed by cryosectioning and
microscopy.
[0216] Human iPSCs Lines Generation and Maintenance:
[0217] BJ iPSCs were obtained from Chad Cowan (Harvard Stem Cell
Institute, Cambridge, Mass.), 05400 and 00409 human iPSCs lines
were generated from adult skin fibroblasts by retroviral
transduction of KLF4, SOX2, OCT4 and c-MYC reprogramming factors as
previously described (Takahashi et al. 2007, Park et al. 2008).
Pluripotency was assessed by morphology, immunestaining for NANOG,
OCT4, SSEA3-4 and Tra1-60, gene expression of NANOG, OCT4, SOX2,
REX1, TERT, DNm3b pluripotency genes and EB formation. iPSCs were
grown on matrigel-coated dishes (BD) and maintained in mTeSR1
medium (STEMCELL technologies).
[0218] Skeletal Muscle Differentiation of iPSCs:
[0219] Cells were differentiated by generating EBs in suspension
culture 7 days and then transferred to matrigel-coated plates for
additional 29 days. During the suspension culture the cells
received the differentiation medium STEMDiff Apel medium (STEMCELL
technologies) supplied with 10 ng/ml bFGF (Invitrogen), 0.5 .mu.M
BIO (Santa Cruz Biotechnologies) and 20 .mu.M forskolin (Santa Cruz
Biotechnologies). Two-three days after plating EBs, the medium was
switched to DMEM complemented with 2% horse serum for the remaining
differentiation.
[0220] Gene Expression Analysis:
[0221] Total RNA was extracted from iPSCs using RNeasy Mini kits
(Qiagen, Mississagua, ON). RNA (1 g) was reverse-transcribed using
a high-capacity reverse transcription kit (Applied Biosystems)
according to the manufacturer's instructions. Real-time PCR was
performed using the SYBR green PCR master mix (Bio-Rad).
Fluorescence was detected by a multicolor real-time detection
system (Cycler IQ; Bio-Rad). All reactions were performed in
duplicate and GAPDH was used as a house-keeping control. Primers
used in the study are shown in Table 3:
TABLE-US-00001 TABLE 3 Primers used in this study. SEQ ID Gene
Location NO: Sequence (5'.fwdarw.3') NCAM Primer Forward 1
ATGGAAACTCTATTAAAGTGAACCTG Primer Reverse 2
TAGACCTCATACTCAGCATTCCAGT PAX6 Primer Forward 3
TCTAATCGAAGGGCCAAATG Primer Reverse 4 TGTGAGGGCTGTGTCTGTTC AFP
Primer Forward 5 AGCTTGGTGGTGGATGAAAC Primer Reverse 6
CCCTCTTCAGCAAAGCAGAC GATA4 Primer Forward 7 CTAGACCGTGGGTTTTGCAT
Primer Reverse 8 TGGGTTAAGTGCCCCTGTAG FLK1 Primer Forward 9
AGTGATCGGAAATGACACTGGA Primer Reverse 10 GCACAAAGTGACACGTTGAGAT
GATA2 Primer Forward 11 GCAACCCCTACTATGCCAACC Primer Reverse 12
CAGTGGCGTCTTGGAGAAG VECAD Primer Forward 13 CAGCCCAAAGTGTGTGAGAA
Primer Reverse 14 TGTGATGTTGGCCGTGTTAT GAPDH Primer Forward 15
TGGTATCGTGGAAGGACTCA Primer Reverse 16 TTCAGCTCAGGGATGACCTT PAX7
Primer Forward 17 CGTGCTCAGAATCAAGTTCG Primer Reverse 18
GTCAGGTTCCGACTCCACAT MYF5 Primer Forward 19 GCCTGAAGAAGGTCAACCAG
Primer Reverse 20 CCATCAGAGCAGTTGGAGGT MYOD1 Primer Forward 21
TGCCACAACGGACGACTT Primer Reverse 22 CGGGTCCAGGCTTCGAA Myo- Primer
Forward 23 AGATGTGTCTGTGGCCTTCC genin Primer Reverse 24
AGCTGGCTTCCTAGCATCAG MYHC Primer Forward 25 TTCATTGGGGTCTTGGACAT
Primer Reverse 26 AACGTCCACTCAATGCCTTC DNMT3B Primer Forward 27
ATAAGTCGAAGGTGCGTCGT Primer Reverse 28 GGCAACATCTGAAGCCATTT HTERT
Primer Forward 29 TGTGCACCAACATCTACAAG Primer Reverse 30
GCGTTCTTGGCTTTCAGGAT NANOG Primer Forward 31 TCCAACATCCTGAACCTCAG
Primer Reverse 32 GACTGGATGTTCTGGGTCTG OCT4 Primer Forward 33
GTGGAGGAAGCTGACAACAA Primer Reverse 34 CAGGTTTTCTTTCCCTAGCT REXI
Primer Forward 35 TGGACACGTCTGTGCTCTTC Primer Reverse 36
GTCTTGGCGTCTTCTCGAAC SOX2 Primer Forward 37 TTGTCGGAGACGGAGAAGCG
Primer Reverse 38 TTGTCGGAGACGGAGAAGCG
[0222] Immunofluorescence:
[0223] iPSCs were fixed in 4% paraformaldehyde and permeabilized
for 15 min with PBS containing 0.1% Triton. Cells were then blocked
for 1 hour with PBS containing 5% bovine serum albumin (BSA) and
incubated overnight at 4.degree. C. with anti-Desmin (Dako),
-Myogenin, -MYOD1 (Santa Cruz Biotechnologies), and -PAX7 (DSHB,
university of Iowa) primary antibodies. Alexa488- and
Alexa594-conjugated secondary antibodies (Invitrogen) were used for
detection as appropriate.
[0224] Transplantation Studies of iPSC-Derived Muscle
Progenitors:
[0225] For the in vivo study of transplantation, 5-6 week old NSG
mice from Jackson Laboratories (stock number 002378) were used. One
day before intramuscular cell transplantation, TA muscle were
pre-injured with 0.3 mg/ml cardiotoxin. After 24 hours, myogenic
progenitors (1.times.10.sup.5 cells/25 .mu.l PBS) were injected
into the left TA muscle, whereas the right leg of each mouse
received the same amount of PBS, as a negative control. One month
after transplantation, mice were sacrificed and muscles were
harvested and frozen in iso-pentane cooled in liquid nitrogen.
Serial cryosections were fixed with 4% PFA and stained with
anti-6-Sarcoglycan antibodies (Leica).
[0226] Electron Microscopy and Immune-Gold Staining:
[0227] For electron microscopy, samples were fixed in a solution of
2.5% glutaraldehyde in a 0.1 M phosphate buffer for 3 hours at room
temperature, washed several times with a 0.1 M phosphate buffer and
then post-fixed with a solution of 2% osmium tetroxide in a 0.1 M
phosphate buffer at room temperature for one hour. Subsequently,
samples were dehydrated and embedded in araldite resin blocks and
sectioned at 80 nanometers using an LKB Nova ultra-microtome with a
diatome diamond knife. The grids were stained with uranyl acetate
and reynolds lead citrate before being observed on the Philips
EM-301Transmission Electron Microscope.
[0228] For electron immune-gold staining, sample were fixed in 2%
paraformaldehyde, 1% glutaraldehyde, in a 0.1M phosphate buffer for
3 hours at room temperature, washed and directly embedded in
araldite resin blocks. Section of 80 nanometers were stained with
primary anti-skeletal myosin heavy and light chain antibodies
(AbCam) followed by a gold-conjugated secondary antibody. The
samples were then stained with uranyl acetate and lead citrate for
contrast and then observed in the Philips 301 Transmission Electron
Microscope.
Results
[0229] A Zebrafish Embryo Culture System to Examine Skeletal Muscle
Development:
[0230] To develop a platform for screening for factors involved in
myogenic specification, we sought to establish fluorescent
"reporter fish" in which different developmental states of skeletal
myogenesis could be distinguished. During zebrafish gastrulation,
mesoderm progenitors undergo involution and convergence extension
movements and begin myogenesis. Myogenic commitment is signified by
expression of myoD and myf5 (Weinberg et al., 1996), which are
functionally redundant and exhibit overlapping expression in the
earliest myogenic precursors (Hinits et al., 2009). Terminal
differentiation of these progenitors produces cells expressing
genes encoding muscle-specific structural proteins like myosin
light polypeptide 2 (mylz2), found in fast skeletal muscle (Ju et
al., 2003). To label different developmental states of skeletal
muscle cells in zebrafish embryos, we generated a myf5-GFP;
mylz2-mCherry double transgenic zebrafish line. At the 11-somite
stage, myf5-GFP expression was restricted to the newly formed
somite, while no mylz2-mCherry expression was detected (FIG. 1A).
Expression of mylz2-mCherry was first detected at 30 hours post
fertilization (hpf) in the anterior somites and later spread to the
posterior somites (FIG. 1A). These data indicate that expression of
myf5-GFP and mylz2-mCherry recapitulate the expression patterns of
their corresponding endogenous genes (Thisse et al., 2001), and
thus provide a useful surrogate to track myogenic specification and
progression during embryonic development. To test whether zebrafish
blastomere cells could form muscle in vitro, we disassociated
myf5-GFP; mylz2-mCherry embryos at the oblong stage and plated them
on gelatin-coated dishes. When cells were cultured in zebrafish ESC
(zESC) medium (Fan and Collodi, 2006), 1-10% became GFP-positive,
indicating upregulation of myf5 expression. Among the GFP-positive
cells, 1-5% were also mCherry (mylz2) positive, suggesting that
myogenic specification and differentiation had occurred in our in
vitro system (FIG. 1B).
[0231] FGF signaling appears to be required for muscle development
in early zebrafish embryos, as myogenesis is essentially blocked in
fgf24 and fgf8 double deficient embryos (Draper et al., 2003). FGF
signals directly activate myoD expression in Xenopus (Fisher et
al., 2002) and are important for fast skeletal muscle induction
(Groves et al., 2005). Based on these data, we added bFGF to the
embryo cultures and found that the majority of cells became GFP and
mCherry double positive, indicating a potent muscle-promoting
effect (FIGS. 1B and 8A). Other myogenic genes, including myoD,
mrf4, and myog, were also highly expressed by blastomere cells
cultured with bFGF (FIG. 1C). To confirm that the in vitro
generated cells labeled by GFP and mCherry corresponded to the
expected stages of muscle development, we isolated each population
by fluorescence-activated cell sorting (FACS) and analyzed its gene
expression. Compared to the GFP/mCherry double negative population,
both myf5-GFP.sup.+; mylz2-mCherry- and double positive populations
were enriched for myogenic gene expression and exhibited lower
expression of blood (embryonic globin) and liver (ifabp) genes.
Double positive cells also expressed higher levels of myoD and
mylz2 than the myf5-GFP.sup.+; mylz2-mCherry-population, suggesting
that, similar to the in vivo situation (FIG. 1A), myf5-GFP and
mylz2-mCherry faithfully label stage-specific myogenic populations
within these in vitro cultures (FIG. 1D).
[0232] To further validate this in vitro myogenesis system, we next
determined whether genes required for muscle development in vivo
were also required in culture. Zebrafish embryos injected with myf5
and myoD double morpholinos are immobile, presumably due to
defective muscle development (Schnapp et al., 2009). Expression of
key myogenic regulatory factors (mrfs), like myogenin and mrf4, and
genes encoding structural proteins, like myosin heavy chain (mhc)
and mylz2, are absent in the double morphant (Schnapp et al.,
2009). Consistent with these prior observations, embryos injected
with double morpholinos lack myf5-GFP and mylz2-mCherry expression
at 30 hpf (FIG. 8B). In addition, cultured blastomere cells from
the double morphant showed dramatically decreased expression of
myf5-GFP and mylz2-mCherry, indicating that Myf5 and MyoD are
likewise essential for muscle development in vitro as well (FIG.
8C). The embryonic myogensis program appears to be conserved during
in vitro differentiation of zebrafish blastomeres.
[0233] A Chemical Genetic Screen Identifies Modifiers of Skeletal
Muscle Development.
[0234] To evaluate the sensitivity of the embryo culture system to
a modifier screen, we treated cells in FGF with all-trans retinoic
acid (ATRA). ATRA was previously added to murine ESC culture to
promote neural lineage differentiation (Lu et al., 2009), and ATRA
treatment inhibits posterior mesoderm formation in zebrafish (de
Jong et al., 2010). Consistent with these prior studies, ATRA
treatment blocked skeletal muscle development in cultures of
zebrafish blastomere cells (FIG. 1E).
[0235] To enable a higher throughput analysis of candidate
modifiers of skeletal myogenesis, the embryo culture system was
adapted to a semi-automated chemical screening platform. myf5-GFP;
mylz2-mCherry embryos were collected from double transgenic parents
and disassociated at the oblong stage. The resulting individual
blastomere cells were aliquoted into four 384-well plates with
pre-added chemicals, and culture medium was supplemented with bFGF
to promote muscle development. After one day, the cells were
automatically imaged and analyzed using a Celigo cytometer for GFP,
mCherry, and bright-field signals, and image analysis was confirmed
by visual inspection (FIG. 2A).
[0236] We screened 2,400 chemicals and identified 29 that perturbed
the GFP or mCherry signals with undetectable toxicity. Based on the
changes of GFP (Myf5) and mCherry (Mylz2) signals, these 29 hits
could be divided into three categories. Category I compounds
produced cultures with only myf5-GFP-positive cells, a phenotype
likely caused by blocking differentiation of muscle progenitors
into mature muscle cells (FIG. 2B and Table 1). Eleven chemicals,
including inhibitors of the p38 pathway, which previously was shown
to be required for muscle formation (Cuenda and Cohen, 1999; Wu et
al., 2000; Zetser et al., 1999), fell into this category. Category
II included 17 chemicals, and resulted in fewer cells expressing
either fluorescent color, likely because these chemicals block
commitment of mesoderm progenitors to the muscle progenitor fate
(FIG. 2B and Table 2). Category III held only one compound
(SB415286, a GSK3.beta. inhibitor), which induced increased
expression of both markers, presumably by accelerating muscle
development (FIG. 2B).
TABLE-US-00002 TABLE 1 Exemplary chemicals and their targeting
pathway that block mature muscle differentiation. Chemical Pathway
CHIR 99021 GSK inhibitor Purmorphamine Hedgehog Activator TMB-8 ion
channel or pump Propafenone K+ channel or pump Ouabain K+ channel
or pump TCPOBOP Nuclear Receptor Ligand Ouabain other CA-o74-Me
Protease Inhibitor SB 202190 p38 inhibitor LY-3 67265 Serotonin
Table 1 lists chemicals that are identified specifically by their
reduction of mylz2-mCherry expression. The phenotype is
presumptively due to the blocking of muscle progenitor
differentiation. We used a chemical library with biologically known
chemicals. The pathway that a chemical targets is listed on the
right.
TABLE-US-00003 TABLE 2 Chemicals and their targeting pathway that
block skeletal muscle progenitor formation. Chemical Name Pathway
myoD expression (-)-Scopolamine, n-Butyl-, acetylcholine Level 1
bromide Miberfradil dihydrochloride Ca.sup.2+ channel or pump Level
2 Methotrexate Cell cycle No change Ro 41-1049 hydrochloride
COMT/MAO Level 4 (.+-.)-6-Chloro-PB dopamine Level 1 hydrochloride
Spermidine trihydrochloride glutamate Level 1 Tyrphostin AG 527
HER/EGF-R Inhibitor No Change Tyrphostin AG 527 HER/EGF-R Inhibitor
Level 1 Tyrphostin AG 879 HER2R/Trka inhibitor Toxic Geldanamycin
Hsp90 inhibitor No change ATRA Nuclear Receptor Ligand Level 4
1-Acyl-PAF PAF Level 3 LY-294,002 hydrochloride PI3K/mTOR inhibitor
Level 2 Rapamycin PI3K/mTOR inhibitor Level 2 SB-431542 TGFbR/ALKR
inhibitor No change Helenalin TNFa/IKK/NFkB Toxic Tyrphosfin AG 808
tyrosine kinase Level 1
Table 2 lists chemicals that are identified by their reduction of
both myf5-GFP and mylz2-mCherrz expression. This phenotype is
presumptively due to the blocking of skeletal muscle progenitor
formation. We used a chemical library with biologically known
chemicals. The pathway that a chemical targets is listed in the
middle. To test if the hits function in vivo, developing embryos
were treated with individual chemicals and assayed for myoD
expression. Two thirds of the hits showed decreased myoD staining
at different levels, which are indicated in FIG. 9A. The staining
results are listed in the third column.
[0237] To test whether these chemicals might also modulate
myogenesis in vivo, we treated zebrafish embryos with each of the
Category II chemicals, which blocked myogenesis in vitro, and
evaluated the effect on expression of myoD at the 6-somite stage.
Among the 17 compounds tested, 11 caused decreased myoD expression
in vivo. Two chemicals induced non-specific toxicity and four
produced no apparent effect (FIG. 9A and Table 2). Thus, 65% of the
hits identified in our initial in vitro screen also perturbed
muscle development in vivo, suggesting that the in vitro screening
system is valid and effective for revealing muscle biology in
vivo.
[0238] bFGF induces muscle development by activating the FGF
receptor tyrosine kinase. In support of this concept, we identified
three receptor tyrosine kinase (RTK) inhibitors that block the
muscle-promoting effect of bFGF (FIG. 9B). bFGF activates several
intracellular pathways, including those involving MEK/ERK and
phosphoinositide-3-kinase (PI3K)-AKT signaling. No MEK/ERK
inhibitor was found to inhibit muscle development (FIG. 9B). In
contrast, the muscle-promoting effect of bFGF could be blocked
completely by the PI3K inhibitor LY-294002 (FIG. 9B). Another PI3K
inhibitor, wortmannin, had a similar effect (FIG. 9B). Treatment
with rapamycin, an mTOR inhibitor, also blocked muscle development
induced by bFGF (FIG. 9B). These results suggest that bFGF promotes
myogenesis through the PI3K-mTOR signaling pathway, rather than the
MEK/ERK pathway.
[0239] A Sensitized Chemical Genetic Screen Identifies Enhancers of
Skeletal Muscle Development:
[0240] The strong muscle promoting effect of bFGF made it difficult
to identify enhancers of skeletal muscle development in our
original system. Only one out of 2,400 chemicals was identified to
promote muscle development. To sensitize the system for identifying
enhancers of skeletal muscle development, we repeated the screen in
the absence of bFGF. In this sensitized screen, six chemicals out
of 2,400 were identified to increase the GFP and mCherry signals
(FIG. 2C). Among the six hits were three GSK3.beta. inhibitor
(kenpaullone, SB415286, and SB216763), two calpain inhibitors
(E-64-D and MDL28170) and one cAMP activator forskolin.
[0241] To examine the ability of these chemicals to induce
myogenesis in vivo, embryos were incubated with each of the
chemicals and the impact on myoD expression was assessed. None of
the six chemicals increased myoD expression in wild-type embryos,
presumably due to a masking effect by the strong endogenous muscle
development program (data not shown). We next evaluated the
interaction of these chemicals with the FGF pathway. Embryos were
injected with morpholinos targeted to fgf8 and fgf24, which led to
a defect in myogenesis as previously reported (Draper et al.,
2003), and each chemical was added to the double fgf8fgf24 morphant
to evaluate its ability to rescue myogenesis. Of the six compounds
tested, only forskolin rescued the myogenesis defect, indicating a
unique activity of forskolin compared to the other compounds (FIG.
9C).
[0242] Forskolin Treatment Elevates cAMP Levels and Expands Mouse
Satellite Cells in Vitro:
[0243] We hypothesized that the chemical hits enhancing skeletal
muscle development in zebrafish blastomeres may likewise promote
muscle precursor cell formation and/or expansion in other species.
To test this hypothesis, we first tested the effects of these
compounds on satellite cells purified from adult mouse skeletal
muscle. Mouse satellite cells were isolated by FACS, as described
in (Cerletti et al., 2008; Sherwood et al., 2004) and seeded into
culture in the presence of different concentrations of each of the
(Cerletti et al., 2008; Sherwood et al., 2004) myogenesis promoting
chemicals. Cell number in the resulting colonies was determined
after 5 days in culture. Of the six chemicals tested, only
forskolin triggered dose-dependent expansion of mouse satellite
cell cultures (data not shown). The number of cells in these
cultures was significantly increased by forskolin treatment both in
the presence and absence of bFGF (FIG. 3A). Forskolin treatment
also increased cell number in satellite cell cultures seeded from
mdx mice, a mouse model of Duchenne muscular dystrophy (DMD)
(Sicinski et al., 1989). Satellite cells from mdx mice typically
exhibit defective ex vivo expansion under control conditions, and
forskolin treatment restored their proliferation to levels seen
normally in cultures of untreated wild-type satellite cells (FIGS.
3B and 10A).
[0244] To evaluate the mechanism by which forskolin drives
increased cell number in satellite cell cultures, we assayed cAMP
production and performed cell survival and proliferation assays in
forskolin-treated cultures. Consistent with forskolin's known role
as an activator of adenylate cyclase (Metzger and Lindner, 1981;
Seamon et al., 1981), forskolin treatment induced a dose dependent
increase in cAMP levels in mouse satellite cell cultures (FIG. 3C).
To test forskolin's effect on satellite cell survival and
proliferation, single cells were plated at one cell per well in
96-well plates and treated with forskolin or DMSO. After six days
in vitro culture, we quantified the number of wells containing any
number of myogenic cells (a measure of cell survival) and the
number of cells in those wells (a measure of cell proliferation)
(FIG. 3D). The frequency of myogenic colony formation did not
differ between forskolin and DMSO treated cells (FIG. 3E),
indicating that forskolin treatment does not affect cell survival.
Consistent with previous observations (FIG. 3A), myogenic colonies
formed in the presence of forskolin contained more cells as
compared to DMSO treated cells (FIG. 3F), most likely reflecting an
effect of the compound on myogenic cell proliferation.
[0245] To test if the increase in cell proliferation was caused by
an inhibitory effect of forskolin on satellite cell
differentiation, we expanded cultured satellite cells under control
conditions and then induced them to differentiate in the presence
or absence of forskolin. The percentage of nuclei in myotubes in
each culture was quantified as an indication of myogenic
differentiation (FIG. 4A). Under these conditions, myotube
formation from satellite cells was not different between the
forskolin and DMSO-treated groups (FIGS. 4B and 4C). Similarly,
satellite cells that were exposed to forskolin during growth and
then induced to differentiate after removal of the compound (FIG.
4D), formed myotubes with the same efficiency as control treated
cells (FIGS. 4E and 4F). Thus, forskolin-treated satellite cells
exhibit unperturbed differentiation in vitro, regardless of the
timing of compound exposure.
[0246] Forskolin-Treated Cells Retain Phenotypic Characteristics of
Satellite Cells and Engraft into Dystrophic Skeletal Muscle In
Vivo:
[0247] Muscle satellite cells are highly enriched within the subset
of myofiber associated cells that co-expresses Cxcr4 and .beta.-1
Integrin markers (Sherwood et al., 2004). Myogenic cells lacking
CXCR4 and .beta.-1 Integrin fail to engraft into mdx muscle
(Cerletti et al., 2008). High levels of Cxcr4 expression also
predict high levels of Pax7 expression, which identifies a subset
of serially transplantable satellite cells with
muscle-stem-cell-like properties (Rocheteau et al., 2012). These
data suggest that CXCR4 and .beta.-1 Integrin are useful surrogate
markers for engraftable myogenic cells. Flow cytometric analysis
revealed that 78.9%+/-1.52% of forskolin-expanded cells maintained
co-expression of both CXCR4 and .beta.-1 Integrin after 5 days in
culture (FIGS. 5A and 5B). Thus, a high percentage of satellite
cells cultured in forskolin retain phenotypic characteristics of
freshly isolated engraftable muscle stem cells.
[0248] To assess directly the engraftment potential of cultured
forskolin-treated satellite cells, we isolated cells from
.beta.-actin-GFP transgenic mice (Wright et al., 2001) for
transplantation experiments. 6,000 GFP-expressing satellite cells
were cultured for 5 days with forskolin, and the resulting cells
were transplanted into the pre-injured muscle of mdx mice (FIG.
5C). Recipient muscles were harvested 3-4 weeks after
transplantation and analyzed for the presence of donor-engrafted,
GFP-expressing myofibers. Consistent with the ability of forskolin
to expand a primitive and engraftable myogenic cell population, the
number of GFP.sup.+ fibers was significantly higher in animals
receiving expanded forskolin-treated cells, as compared to those
receiving the original number of freshly isolated satellite cells
or those receiving expanded DMSO-treated cells (FIGS. 5D and 5E).
Furthermore, myofibers engrafted by forskolin-treated cells stained
for Dystrophin, which normally is not present in mdx muscle (FIG.
10B). Thus, exposure to forskolin expands engraftable myogenic
cells from cultured satellite cells.
[0249] A Combination of bFGF, BIO and Forskolin Activates Skeletal
Muscle Program in iPSCs:
[0250] To examine further the conservation of muscle-inducing
potential of chemicals identified in our zebrafish screen, we next
evaluated their impact on the differentiation of human pluripotent
cells. It is currently difficult to differentiate human iPSCs or
ESCs to the skeletal muscle lineage, with the only major success
involving ectopic introduction and overexpression of
muscle-specific transcription factor genes (Darabi et al., 2012;
Tedesco et al., 2012) Strategies to reproducibly drive myogenic
differentiation in the absence of genome modification have thus far
been elusive.
[0251] We tested the effects of muscle-promoting compounds on the
differentiation of three different lines of iPSCs (00409, 05400,
and BJ) derived from healthy human donors (FIGS. 11 and 12).
Embryoid bodies (EBs) from BJ, 00409 or 05400 iPSCs were exposed
for 7 days to a serum-free differentiation medium containing bFGF
(10 ng/ml), BIO (0.5 .mu.M) and forskolin (20 .mu.M) (medium added
at day 1, 3 and 5). We used BIO, rather than the GSK3.beta.
inhibitors found in the screen, since we tried several GSK3.beta.
antagonists and BIO had lower toxicity (data not shown). After 7
days, we measured the expression of a panel of markers specific for
ectoderm, endoderm or mesoderm to quantify differentiation
outcomes. The mesodermal markers GATA2, PAX7, MYF5, MYOD1 genes
were significantly up-regulated after 2, 4 and 7 days of
differentiation compared to the corresponding undifferentiated
iPSCs lines (FIGS. 6A, 6B, and 13A), while ectodermal and
endodermal genes were not significantly altered. These data suggest
that this chemical mixture specifically favors the mesodermal
lineage and promotes myogenic specification (FIGS. 6A, 6B, and
13A). Two-way combinations, such as bFGF+BIO or bFGF+forskolin,
yielded significantly less potent myogenic induction as compared to
the bFGF/BIO/forskolin "triple cocktail" (FIGS. 13A and 13B).
[0252] To determine whether the iPSC-derived cells undergo terminal
skeletal muscle differentiation with bFGF/BIO/forskolin, EBs were
plated on matrigel-coated dishes for further differentiation. Gene
expression analysis of these cultures revealed that PAX7, MYOD1,
and MYF5 expression remained increased after 36 days of culture.
Expression of Myogenin and MyHC, two markers of late myogenic
differentiation, increased after 20 days, and this increase became
more significant after 36 days (FIG. 6B). At day 36, we observed
many multinucleated myotubes in our cultures, which were positive
for Desmin and Myogenin staining (FIG. 6C). Although iPSCs are
known to differentiate spontaneously to cardiac cells, gene
expression analysis revealed no increase in cardiac markers (NKX2.5
and GATA2) after 36 days of differentiation and no spontaneously
beating cells were found in the treated cultures. In contrast,
beating cells were observed in the untreated cells lines after
12-14 days (data not shown). Thus, the three compounds together
induced a skeletal myogenic program, rather than a cardiogenic
program.
[0253] We further confirmed the skeletal muscle fate of
iPSC-derived cells cultured in bFGF/BIO/forskolin by electron
microscopy (EM) and immunogold staining with anti-human skeletal
muscle Myosin, heavy and light chain antibodies
(sk-Myosin-HL-Chains). EM analysis of BJ iPSC-derived cells
harvested from culture after 36 days revealed the presence of
distinct sarcomere structures, which stained positively for
sk-Myosin-HL-Chains (FIGS. 6D and 6E). Similar results were
obtained in 00409 and 05400 iPSCs (FIGS. 12C, 12D, and 12E).
[0254] iPSCs-Derived Muscle Progenitors can En Graft into Skeletal
Muscle In Vivo:
[0255] To determine whether myogenic specification using the
bFGF/BIO/forskolin cocktail could generate or expand engraftable
muscle progenitors from human pluripotent cells, we assessed the
ability of myogenic cells derived from human iPSCs to support
muscle regeneration in vivo. BJ, 05400 or 00409 iPSCs were
differentiated by treatment with bFGF/BIO/forskolin and
transplanted into immunodeficient mice after 12 days of
differentiation. This time point was chosen because expression of
the satellite cell specific transcription factor PAX7 reaches its
peak at 12 days (FIG. 6B). iPSC-derived cells were transplanted
directly into the limb muscle of NOD/SCID/IL2R.gamma.-/- (NSG) mice
that had been pre-injured by cardiotoxin injection 24 hours before
transplantation. The contralateral muscles of these recipient mice
were injured but injected with only PBS. One month after
transplantation, muscles were harvested and analyzed for
engraftment by immunostaining with human muscle specific
.delta.-Sarcoglycan antibody. PBS-injected muscle showed no
detectable staining for human .delta.-Sarcoglycan (FIG. 7). Human
.delta.-Sarcoglycan.sup.+ fibers were also absent when the
00409-derived fibroblast cell line was transplanted (FIG. 7). By
contrast, transplantation of myogenic progenitors derived from any
of the three iPSCs lines (BJ, 05400 or 00409) yielded significant
engraftment of cells that showed clear reactivity for human
6-Sarcoglycan (FIG. 7). In addition, no tumor formation was
observed in the injected muscles (data not shown). These studies
confirm that the iPSC-derived progenitor cells generated by
treatment with bFGF/BIO/forskolin maintain myogenic commitment and
support regenerative muscle formation after in vivo
transplantation.
Discussion
[0256] In this study, we took advantage of chemical genetic
approaches available in zebrafish to reveal conserved mechanisms
controlling the specification and expansion of myogenic progenitor
cells. In the process, we defined new approaches to promote the
expansion of mouse satellite cells and specify myogenic
differentiation of human iPSCs. While many prior studies have
employed chemical genetics in whole zebrafish embryos to
interrogate development and disease, this approach requires manual
manipulation of embryos and often requires time-consuming readout
by whole mount in situ hybridization. Some laboratories have
screened whole embryos using high-speed pipetting and imaging, but
the three-dimensional structure of the embryo makes this approach
rather difficult. In comparison to screening using whole zebrafish
embryos, the culture-based screening system described here takes
one sixth the time and uses one tenth of the embryos. Such
throughput enables screening of significantly larger chemical
libraries and increasing the speed of conventional chemical
screening on mammalian cell lines. Transgenic zebrafish with a
fluorescent reporter known to label a particular cell type in vivo
can be used and images can be automatically captured and stored by
imaging cytometers such that the cells do not need to be fixed or
scored immediately. Recently, a zebrafish explant system with some
similarities to our embryo culture system identified compounds that
could expand angiogenic progenitors (Huang et al., 2012). Our
system enables the combination of reporters of different colors to
simultaneously interrogate multiple different developmental states
or lineages.
[0257] One remarkable aspect of this screening system is that it
appears to support critical developmental transitions in the
absence of the normal spatial and temporal information available in
the embryo. While some cell lineages may be difficult to derive
from zebrafish blastomere cells, disassociating embryos at later
stages could be used to study the desired tissue. Ultimately, we
predict that similar zebrafish cultures systems will prove highly
useful in screens for compounds and pathways affecting development
in many different organ systems.
[0258] Another noteworthy attribute of the zebrafish embryo culture
system is its ability to identify critical pathways that influence
tissue specification and progenitor cell expansion in analogous
systems across species. The chemicals found in the zebrafish system
expand postnatal muscle satellite cells from mice and specify
myogenic differentiation from human pluripotent cells. These
chemicals identified in our screen proved useful in addressing two
of the more vexing challenges in the production of mammalian muscle
precursors for experimental applications and, perhaps ultimately,
for cell therapy. Indeed, one of the major obstacles in using
purified skeletal muscle satellite cells for cell therapy is their
very low frequency in adult tissue. High numbers of engraftable
cells are required for functional recovery of skeletal muscles
throughout the body in genetic muscle disorders, and efforts to
expand purified satellite cells in culture while maintaining their
engraftment potential have been largely unsuccessful (Montarras et
al., 2005). Here, we show that mouse muscle progenitors treated
with the adenylyl cyclase activator forskolin have enhanced
proliferation in culture. These data are consistent with a previous
report showing that activation of cAMP signaling in transgenic mice
expressing an activated from of cAMP response element binding
protein (CREB) increases the in vitro proliferation of primary
myoblasts (Stewart et al., 2011). Forskolin does not inhibit
satellite cell differentiation in vitro, and forskolin-treated
muscle progenitor cells differentiate normally after removal of the
compound. Forskolin-treated progenitor cells also retain most
phenotypic characteristics of freshly isolated satellite cells. It
is possible that forskolin mimics activation of a natural G-protein
coupled receptor in the genesis or maintenance of muscle
progenitors. When transplanted into pre-injured mdx muscle,
forskolin-expanded satellite cells engrafted to generate
Dystrophin-expressing myofibers. Forskolin treatment dramatically
increased the proliferation of muscle progenitors isolated from
dystrophic mdx mice, which exhibited defective ex vivo expansion
under control conditions. Thus, forskolin treatment may be useful
to enhance the ex vivo expansion of human dystrophic satellite
cells for autologous therapy or to improve the efficiency of cell
replacement therapies for muscle disorders.
[0259] The capacity of forskolin to expand functional satellite
cells may also explain the enhanced myogenesis observed in
zebrafish blastomeres and human iPSCs upon forskolin exposure.
During culture, some pluripotent cells likely differentiate to form
skeletal muscle progenitors and forskolin may act to increase the
proliferation of these myogenic cells by activating cAMP signaling,
thereby enriching the muscle-forming capacity in the culture. Given
prior difficulties in obtaining robust skeletal muscle
differentiation from pluripotent cells, most previous studies have
relied on transgenic modification (Darabi et al., 2012; Tedesco et
al., 2012). By applying insights obtained from our zebrafish embryo
culture screen, we identified a novel chemical cocktail that
successfully enabled myogenic differentiation of iPSCs into mature
myofibers in vitro and in vivo in the absence of genomic
modification or transcription factor overexpression. A screen to
find such chemical inducers of muscle development using iPSCs would
have been highly reagent- and labor-intensive, particularly as
myogenic differentiation in the human system requires .about.36
days. In contrast, the zebrafish muscle differentiation protocol
takes only 1-2 days, enabling the completion of a small-scale
screen in about one week.
[0260] One remarkable aspect of the myogenic protocol defined here
is the selectivity with which the skeletal muscle fate is specified
during culture. Simultaneous inhibition of GSK3.beta. and
activation of cAMP and bFGF pathways during EB formation is
sufficient to specifically promote commitment of these cells to
skeletal muscle differentiation, and our marker analysis indicates
that alternative fates, including cardiac and neural lineages, are
relatively rare in cultures exposed to the triple cocktail.
Interestingly, prior studies indicate that cAMP signaling is
increased at discrete times during embryonic muscle differentiation
and promotes muscle regeneration and metabolic adaptation in
satellite cells (Carlsen, 1975; Chen et al., 2005; Le Peuch et al.,
1979; Zalin and Montague, 1974). Myogenesis of muscle stem cells
can also be induced by Wnt activation (Brack et al., 2008;
Polesskaya et al., 2003), and bFGF-mediated activation of PI3K-AKT
pathway has likewise been reported as a potent mediator of muscle
differentiation (Stitt et al., 2004). The data presented herein
unexpectedly shows that pathways can cooperate uniquely during
muscle differentiation, enabling ex vivo myogenic differentiation,
and generation of engraftable myogenic progenitors, which mix with
host muscle upon transplantation into damaged tissue and are
indistinguishable by hematoxylin and eosin staining from the
regenerated mouse muscle fibers. These results, together with the
absence of teratoma formation and the ability to achieve myogenic
specification of iPSCs by giving solely exogenous factors, shows
that iPSC-derived myogenic cells can be applied to regenerative
cell therapy for the treatment of musculoskeletal diseases. In any
event, the findings reported are highly beneficial to understanding
normal myogenesis and for in vitro studies of musculoskeletal and
metabolic disease in a variety of experimental settings.
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[0303] All patents and other publications identified in the
specification and examples are expressly incorporated herein by
reference for all purposes. These publications are provided solely
for their disclosure prior to the filing date of the present
application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention or for any other reason.
All statements as to the date or representation as to the contents
of these documents is based on the information available to the
applicants and does not constitute any admission as to the
correctness of the dates or contents of these documents.
[0304] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow. Further, to the extent not already indicated, it will be
understood by those of ordinary skill in the art that any one of
the various embodiments herein described and illustrated can be
further modified to incorporate features shown in any of the other
embodiments disclosed herein.
Sequence CWU 1
1
44126DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1atggaaactc tattaaagtg aacctg 26225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2tagacctcat actcagcatt ccagt 25320DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3tctaatcgaa gggccaaatg
20420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4tgtgagggct gtgtctgttc 20520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5agcttggtgg tggatgaaac 20620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6ccctcttcag caaagcagac
20720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7ctagaccgtg ggttttgcat 20820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8tgggttaagt gcccctgtag 20922DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9agtgatcgga aatgacactg ga
221022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10gcacaaagtg acacgttgag at 221121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11gcaaccccta ctatgccaac c 211219DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 12cagtggcgtc ttggagaag
191320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13cagcccaaag tgtgtgagaa 201420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14tgtgatgttg gccgtgttat 201520DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 15tggtatcgtg gaaggactca
201620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16ttcagctcag ggatgacctt 201720DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17cgtgctcaga atcaagttcg 201820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 18gtcaggttcc gactccacat
201920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19gcctgaagaa ggtcaaccag 202020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20ccatcagagc agttggaggt 202118DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 21tgccacaacg gacgactt
182217DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 22cgggtccagg cttcgaa 172320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23agatgtgtct gtggccttcc 202420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24agctggcttc ctagcatcag
202520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25ttcattgggg tcttggacat 202620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26aacgtccact caatgccttc 202720DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 27ataagtcgaa ggtgcgtcgt
202820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28ggcaacatct gaagccattt 202920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
29tgtgcaccaa catctacaag 203020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 30gcgttcttgg ctttcaggat
203120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31tccaacatcc tgaacctcag 203220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
32gactggatgt tctgggtctg 203320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 33gtggaggaag ctgacaacaa
203420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34caggttttct ttccctagct 203520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
35tggacacgtc tgtgctcttc 203620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 36gtcttggcgt cttctcgaac
203720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 37ttgtcggaga cggagaagcg 203820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38ttgtcggaga cggagaagcg 20394PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Phe Met Arg Phe 1
406PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Phe Gln Phe Met Arg Phe 1 5 414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Tyr
Met Arg Phe 1 426PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 42Tyr Leu Pro Leu Arg Phe 1 5
435PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Tyr Phe Met Arg Phe 1 5 445PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Leu
Pro Leu Arg Phe 1 5
* * * * *
References