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*Correspondence: khochedlinger@mgh.harvard.edu
https://doi.org/10.1016/j.stemcr.2018.04.009
SUMMARY
Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles
during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we
show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse
fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell
markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of
transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from
Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle
tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/pro-
genitor-like cells from multiple cell types.
Stem Cell Reports j Vol. 10 j 1505–1521 j May 8, 2018 j ª 2018 The Author(s). 1505
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. MyoD and Small Molecules Induce Skeletal Muscle Progenitor-like Program in Fibroblasts
(A) Experimental outline. MEFs, murine embryonic fibroblasts.
(B) Representative bright-field images (scale bar, 500 mm) and immunofluorescence images for MyHC (green; scale bar, 50 mm) of MEFs
induced with MyoD alone or MyoD in the presence of the indicated small molecules and medium containing fetal bovine serum (FBS), Serum
Replacement (SR), and basic FGF (bFGF). Three-dimensional, proliferative, and contractile colonies were obtained only in the presence of
the cyclic AMP agonist forskolin (F) and the TGF-b inhibitor RepSox (R), with or without GSK3b inhibitor (G). The experiment was validated
using at least three different MEF lines. Green arrowheads indicate three-dimensional colonies; red arrowheads indicate multinucleated
myofibers.
(legend continued on next page)
(C) Flow-cytometric analysis for EdU to measure proliferation in MEFs subjected to the indicated treatments.
(D) Expression of skeletal muscle- and cardiac-associated markers by microarray analysis in control MEFs, C2C12 myoblasts, and MEFs
undergoing conventional transdifferentiation (MEF + MyoD) or reprogramming (MEFs + MyoD + F/R) for 14 days.
(E) qRT-PCR analysis for the indicated skeletal muscle genes during tail-tip fibroblast reprogramming. **p < 0.005, ****p < 0.00005, n.s.,
not significant.
(F) Venn diagram showing the overlap of genes (>2-fold relative to MEFs) between quiescent satellite cells (QSCs), activated satellite cells
(ASCs), and either MEFs expressing MyoD for 14 days or MEFs expressing MyoD and exposed to F/R for 14 days. Expression data for QSCs and
ASCs were obtained from a previous publication (Liu et al., 2013).
(G) Graph showing the top differentially expressed genes by expression microarray analysis in MEFs expressing MyoD and exposed to F/R
in comparison with MEFs expressing MyoD alone. Arrows highlight examples of mature muscle markers detected exclusively under re-
programming conditions (MyoD + F/R).
(H) Functional annotation analysis for upregulated genes (>2-fold) in MEFs + MyoD + F/R relative to MEFs + MyoD alone. Benjamini-
Hochberg (BH) adjusted p values are presented. Top categories are shown together with the number of genes.
See also Figure S1.
(E) Experimental design to assess if iMPC formation requires passage through an Oct4+ pluripotent intermediate state using a DTA
(diphtheria toxin A) lineage ablation system.
(F) Quantification of contracting colonies generated with and without 4OHT administration from Oct4-CreER;Rosa26-LSL-DTA MEFs and
exposed to MyoD + F/R (n = 3 independent replicates; error bars denote SD).
(G) Representative immunofluorescence images show staining for Pax7 in Oct4-CreER;Rosa26-LSL-DTA MEFs exposed to MyoD + F/R with and
without 4OHT. Scale bar, 100 mm.
(H) Quantification of Pax7+ nuclei in three random fields taken from Oct4-CreER;Rosa26-LSL-DTA MEFs exposed to MyoD + F/R with and
without 4OHT (n = 3 independent replicates; error bars denote SD).
See also Figure S1.
Figure 3. iMPCs Self-Renew and Express Markers of Muscle Stem, Progenitor, and Mature Cells
(A) Representative images of dox-independent iMPC cultures containing spheroid structures, mononucleated cells, and multinucleated
myotubes. Scale bar, 500 mm.
(B) qRT-PCR analysis for skeletal muscle-specific genes in low-passage (P < 5) and high-passage (P > 15) iMPC lines expanded from in-
dividual colonies. MEFs were used as negative control and C2C12 myoblasts as positive control (n = 3–4 biological replicates; error bars
denote SD; *p < 0.05, **p < 0.005).
(C) Representative immunofluorescence images for the indicated muscle-specific proteins in a representative iMPC line. Scale bar, 100 mm.
(D) Representative images of single cell-derived iMPCs subcloned from CAG-RFP+ iMPCs. Scale bar, 250 mm.
(E) qRT-PCR analysis for skeletal muscle-specific genes in single cell-derived iMPCs. MEFs were used as a negative control (n = 3 biological
replicates; error bars denote SD; *p < 0.05, **p < 0.005).
(F) Representative immunofluorescence images for the indicated muscle-specific proteins in a single cell-derived iMPC clone. Scale bar,
100 mm.
(G) Representative immunofluorescence images for Pax7 (red) and Myf5 (green) or Pax7 (red) and MyoD (green) expression in iMPCs#1
cultured in F/R or F/R/G conditions for at least five passages before analysis. Scale bar, 100 mm.
(H) Quantification of (G).
(I) Top: flow-cytometric analysis of GFP expression using low-passage iMPCs derived from Pax7-nGFP MEFs subjected to MyoD + F/R/G
condition. Bottom: forward/side scatter flow-cytometric plot using Pax7-nGFP+ cells (green) compared with all mononucleated cells
(gray).
(J) Representative images of sorted Pax7-nGFP+ cells using bright-field and GFP channels. Zoomed images show sorted Pax7-nGFP+
doublets. Arrowheads indicate Pax7-nGFP+ cell doublets. Scale bar, 100 mm.
(K) Venn diagram based on RNA sequencing data showing the overlap of upregulated genes (>2-fold, FDR < 0.05 relative to MEFs) between
quiescent satellite cells (QSCs), activated satellite cells (ASCs), and sorted Pax7-nGFP+ cells purified from iMPCs. See Supplemental
Experimental Procedures and main text for definition of QSCs and ASCs.
(L) Heatmap depicting expression of skeletal muscle stem and progenitor associated genes based on RNA sequencing data obtained from
Pax-nGFP+ iMPCs, QSCs, ASCs, and MEFs.
(M) Integrative genomic viewer tracks for the indicated genes based on RNA sequencing data.
See also Figure S3.
muscle cross-section
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H2B-RFP-iMPCs (CTX injury model) H2B-RFP iMPCs (CTX injury model)
Dystrophin
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400 Dystrophin
DAPI
Dystrophin
DAPI
H2B-RFP
DAPI
Pax7-/RFP- Pax7+/RFP+
Pax7+/RFP- Pax7-/RFP+
muscle cross-section
*** 3.14%
#Dys+ myofibers per
300
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Dystrophin Dystrophin Dystrophin Pax7
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H I
Serial injury model 1 month post injury 3 days post reinjury 1 month post reinjury
Day0 BaCl2 injury CAG-RFP CAG-RFP CAG-RFP
Control
Day28 Image+reinjury
Satellite
Day31
cells
Image muscle
Recovery
Day61 Image+harvest
iMPCs
Analysis
J K
Control Satellite cells iMPCs 800 n.s
CAG-RFP
muscle cross-section
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Laminin
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Figure 4. iMPCs Contribute to Skeletal Muscle Regeneration and the Satellite Cell Niche In Vivo
(A) Experimental design to assess the engraftment and differentiation potential of iMPCs in comparison with satellite cells.
(B) Immunofluorescence images for dystrophin (red) and DAPI (blue) for the indicated samples. One million cells were transplanted into
the tibialis anterior or gastrocnemius of 12-week-old homozygous mdx dystrophic mice, and the muscles were isolated 1 month after
transplantation for analysis. Non-transplanted or PBS-injected mdx muscle sections were used as negative controls. Rare dystrophin-
positive myofibers, present in non-transplanted or PBS-injected control sections, are due to spontaneous reversion of the mdx mutation.
Scale bars, 1,000 mm (top) and 100 mm (bottom).
(C) Quantification of (B) (n = 3 biological replicates; error bars denote SD; *p < 0.05).
(D) Immunofluorescence images of cardiotoxin (CTX)-injured SCID-mdx muscles transplanted with 1 3 106 iMPCs carrying a nuclear
H2B-RFP reporter and analyzed 1 month after injury/transplantation. CTX injury was carried out 24 hr prior to transplantation. Successful
engraftment was assessed by measuring dystrophin expression (purple) and H2B-RFP expression (red). Scale bar, 100 mm.
(E) Quantification of (D) (n = 3 biological replicates; error bars denote SD; ***p < 0.0005).
(F) Immunofluorescence images for dystrophin (purple) and Pax7 (green) expression within an engrafted area. Insets indicate Pax7
nuclear staining co-localizing with the H2B-RFP reporter. Scale bar, 100 mm.
(G) Quantification of (F) (n = 3 biological replicates; error bars denote SD).
(H) Experimental outline for serial injury experiment.
(I) Representative images of tibialis anterior muscles transplanted with 1 3 106 iMPCs or 7.5 3 104 purified satellite cells carrying a CAG-
RFP fluorescent reporter following BaCl2-induced muscle injury (48 hr prior to transplantation). Muscles transplanted with satellite cells or
iMPCs were subjected to the same procedures as illustrated in (H). Note reduction of CAG-RFP signal 3 days after reinjury and restoration of
CAG-RFP signal 1 month after reinjury, indicating successful regeneration by donor cells.
(J) Immunofluorescence images of a tibialis anterior muscle section transplanted with CAG-RFP iMPCs and analyzed 1 month after reinjury
compared with a non-transplanted control. Scale bar, 100 mm.
(K) Quantification of serial injury procedure (n = 4 independent transplantation experiments per cell type). n.s., not significant.
See also Figure S4.
(D) Representative images of EYFP+ myotubes emerging from Pax7-CreER;Rosa26-LSL-EYFP MEFs after exposure to MyoD + F/R in the
presence of 4OHT. Scale bar, 100 mm.
(E) Cell ablation system to determine if Pax7+ cells are required for the generation of iMPCs.
(F) qRT-PCR analysis for skeletal muscle-specific genes in Pax7-CreER;Rosa26-LSL-DTA MEFs undergoing reprogramming into iMPCs using
the indicated conditions (n = 3 biological replicates; error bars denote SD; **p < 0.005, ****p < 0.00005; n.s., not significant).
(G) Representative images of Pax7-CreER;Rosa26-LSL-DTA MEFs undergoing reprogramming into iMPCs using MyoD + F/R/G in the presence
or absence of 4OHT. Scale bar, 250 mm.
(H) Representative immunofluorescence images for Pax7 expression (green) for the experiment depicted in (G). Scale bar, 50 mm.
(I) Schematic of Pax7 knockout (KO) alleles used to test whether iMPC generation requires Pax7 gene function.
(J) Bright-field images show myotubes derived from Pax7+/ and Pax7/ MEFs upon MyoD overexpression (left panels), and an iMPC clone
derived from Pax7+/ MEFs (top right) but not from Pax7/ MEFs (bottom right). Scale bar, 500 mm.
(K) qRT-PCR analysis for indicated samples at day 21 of reprogramming. **p < 0.005, ***p < 0.0005; ****p < 0.00005.
(L) Representative immunofluorescence images show staining for MyHC (green) and Pax7 expression (red) in Pax7+/ and Pax7/ MEFs
exposed to MyoD or MyoD + F/R/G conditions followed by several passages without exogenous MyoD expression. Scale bar, 50 mm.
See also Figure S5.
and Myf5. Consistent with this notion, TGF-b inhibition stem cells directly from fibroblasts and muscle tissue. Our
reportedly induces a mesenchymal-to-epithelial transition results also raise the intriguing possibility that any other
(Polo and Hochedlinger, 2010), whereas ascorbic acid, a key stem/progenitor populations of interest could be derived
component of Serum Replacement (Stadtfeld et al., 2012), using similar principles, i.e., overexpression of differentia-
triggers epigenetic remodeling of targets via its role as a tion-associated transcription factors and pharmacological
cofactor for TET enzymes and Jumonji-containing histone suppression of pathways that resist reprogramming.
demethylases during the generation of iPSCs from fibro-
blasts (Chen et al., 2013). Once the endogenous MyoD,
EXPERIMENTAL PROCEDURES
Myf5, and Pax7 loci have been activated in iMPCs, these
small molecules may then be required to stabilize and Details on reprogramming conditions, cell culture and antibody
maintain a self-renewing stem/progenitor cell state. Of reagents, vectors, expression analysis, animals, transplantation,
note, these compounds also appear to promote terminal and injury assays can be found in Supplemental Information. All
differentiation and maturation of myogenic stem/progeni- procedures, including maintenance of animals, were performed
tor-like cells based on our finding that myofibers spontane- in compliance with active IACUC protocols and according to insti-
ously contract and express markers associated with adult tutional guidelines.
muscle (e.g., Myh1, Myh4, Myh6, Car3, Casq1, Mstn), which
we rarely observed during transdifferentiation induced ACCESSION NUMBERS
with MyoD alone. The accession numbers for the RNA sequencing and microarrays
In addition to providing mechanistic insights and a use- reported in this paper are GEO: GSE108543 and GSE92336.
ful tool to study the role of transcription factors and
external stimuli in cell fate control, our data may have ther- SUPPLEMENTAL INFORMATION
apeutic implications once translated to human cells. For
example, patient-specific iMPCs might be useful to study Supplemental Information includes Supplemental Experimental
Procedures, six figures, and seven videos and can be found
myogenic disorders ex vivo as well as to screen for small
with this article online at https://doi.org/10.1016/j.stemcr.2018.
molecules that could reverse disease phenotypes. Similarly,
04.009.
iMPCs derived from patients with Duchenne muscular dys-
trophy could in principle be used for cell therapy following AUTHOR CONTRIBUTIONS
restoration of dystrophin expression using CRISPR/Cas9
technology, as was recently shown in mouse satellite cells O.B.-N., M.F.M.G., and K.H. conceived the experiments; O.B.-N.
(Long et al., 2016; Nelson et al., 2016; Tabebordbar et al., and M.F.M.G. conducted the majority of experiments with assis-
tance from B.D.S., A.G., A.C., A.H., C.V., P.C., and D.P.-D.; A.E.A
2016). Lastly, our observation that iMPC-derived myotubes
and A.J.W. helped with transplantation experiments; P.F. and
express adult muscle markers and display vigorous contrac-
M.A.R. provided Pax7+/ and Pax7/ MEFs; S.P. and S.T. provided
tions may provide a valuable source of material for tissue Pax7-nGFP MEFs; O.B.-N., B.D.S., A.A., and R.I.S. performed RNA
engineering purposes (Madden et al., 2015). sequencing and microarray analyses; H.C.O., A.J.W., M.A.R.,
In conclusion, our study reports on a simple and and S.T. provided critical advice and contributed to discussion
robust approach to generate expandable, non-transformed of results; O.B.-N., M.F.M.G., and K.H. wrote and revised the
myogenic cell populations with characteristics of muscle manuscript.
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Supplemental Information
Figure S1: Derivation and molecular analysis of MEF-derived iMPCs using MyoD
expression and small molecules
(A) Doxycycline-dependent lentiviral system to induce MyoD expression in fibroblasts.
MEFs were infected with lentiviral vectors expressing the reverse tetracycline
transactivator (rtTA) and the tetOP-MyoD gene, respectively. Representative
immunofluorescence images show staining for MyoD in indicated samples following 24
hours of doxycycline (dox) administration (scale bar, 100µm). (B) Representative
immunofluorescence images of MyHC (green) and Pax7 (red) expression in MEFs
expressing MyoD in the presence of the indicated small molecules (scale bar, 50µm). (C)
Venn-diagram showing the overlap of upregulated genes (>2-fold or more) between
quiescent satellite cells (QSCs), activated satellite cells (ASCs), and MEF line #2 vs. MEF
line #1. Expression data for QSCs and ASCs were previously published (Liu et al., 2013).
(D) Experimental design to assess the temporal requirement for MyoD expression or
small molecule treatment to generate stable Pax7 + iMPCs. (E) Representative
immunofluorescence images of MyHC (green) and Pax7 (red) expression in MEFs
expressing MyoD for the indicated days and cultured continuously in the presence of F/R.
Analysis was performed 7 days after the last time point (day 12) (scale bar, 50µm). (F)
Graph showing the temporal requirement for MyoD expression or small molecule
treatment to generate skeletal muscle progenitor-like cells. Dox and F/R treatment were
applied to infected MEFs for the indicated lengths of time. Following dox withdrawal, cells
were propagated in the presence or absence of F/R and scored for Pax7 expression by
immunofluorescence 7 days after the last time point (day 12). This experiment was
validated using 3 different MEF lines; for each replicate, 1*10 5 cells were used per time
point.
Figure S5: Surface marker analysis of iMPCs and inspection of iMPC generation
using a Pax7-CreER lineage reporter
(A) Flow cytometric analysis of VCAM-1 expression in iMPCs. The BV421 channel was
used to control for autofluorescence. (B) Live antibody staining of iMPC cultures for the
satellite cell and myoblast marker VCAM-1(Fukada et al., 2007; Liu et al., 2015), which is
present on mononucleated cells but absent on myotubes (scale bar, 100µm). (C)
Quantitative RT-qPCR analysis of MyHC expression in purified VCAM-1+Sca1-CD31-
CD45- cells isolated from iMPC cultures and compared to sorted bulk iMPCs immediately
after sorting as well as 9 days after sorting and explantation (n=3 biological replicates;
error bars s.d. *P<0.05). (D) Representative images of the indicated sorted VCAM-
1+Sca1-CD31-CD45- or VCAM-1- cells at indicated reprogramming time points. Note that
iMPCs originate only from VCAM-1+ cells. Equal numbers of VCAM-1+ and VCAM-1- cells
were plated for this experiment (scale bar, 500µm). (E) Flow cytometric analysis of Pax7-
CreER; Rosa26-LSL-EYFP MEFs treated with 4OHT for 24 hours confirms absence of
contaminating Pax7+ cells. The PE-Cy7 channel was used to control for autofluorescence.
(F) Flow cytometric analysis of Pax7-CreER; Rosa26-LSL-EYFP MEFs treated with
4OHT and exposed to the indicated conditions for 14 days. Note significant EYFP
expression only when MyoD expression is combined with F/R or F/R/G treatment but not
with individual small molecules. A graph summarizing the flow cytometric analysis is
shown on the bottom right. This experiment was validated using 3 different Pax7-CreER;
Rosa26-LSL-EYFP MEF lines; for each replicate 1*105 cells were used per treatment.
The PE-Cy7 channel was used to control for autofluorescence. (G) Representative
images of emerging EYFP+ iMPCs derived from sorted Thy1+ cells during the
reprogramming process (scale bar, 50µm). (H) Flow cytometric analysis of sorted Pax7-
CreER; Rosa26-LSL-EYFP Thy1+ cells exposed to 4OHT with and without MyoD
overexpression and F/R treatment. The PE-Cy7 channel was used to control for
autofluorescence. (I) Representative images of emerging EYFP+ iMPCs derived from
adult Pax7-CreER; Rosa26-LSL-EYFP TTFs (scale bars, 100µm). (J) Flow cytometric
analysis of Pax7-CreER; Rosa26-LSL-EYFP TTFs exposed to the indicated conditions
and analyzed at day 18. Note activation of the Pax7 reporter only in the MyoD+F/R+4OHT
condition. The PE-Cy7 channel was used to control for autofluorescence.
Figure S6: iMPC cultures contain satellite-like cells that recapitulate myogenesis in
vitro
(A) Flow cytometric analysis of iMPC line #1 showing 4.71% Pax7+ cells after 3 days of
4OHT treatment. The PE-Cy7 channel was used to control for autofluorescence. (B) Flow
cytometric analysis of 3 single cell-derived Pax7-CreER; Rosa26-LSL-EYFP iMPC
subclones derived from iMPC line #1; 4OHT was administered for 3 days before analysis.
The PE-Cy7 channel was used to control for autofluorescence. (C) Flow cytometric
analysis of Pax7-CreER; Rosa26-LSL-EYFP iMPC line #1 cultured without 4OHT and
analyzed at indicated passages. Note lack of lineage label even after 9 passages. The
PE-Cy7 channel was used to control for autofluorescence. (D) Flow cytometric analysis
of Pax7-CreER; Rosa26-LSL-EYFP iMPC line #1 labeled with 4OHT at passage 5 and
cultured in the presence of 4OHT for 9 passages. Note increased labeling of iMPCs with
passaging, reaching 87% by passage 14. The PE-Cy7 channel was used to control for
autofluorescence. (E) Flow cytometric analysis of VCAM-1 expression in Pax7-CreER;
Rosa26-LSL-EYFP iMPCs treated with 4OHT for 3 days. Note that 4.6% of the total cells
are EYFP+ of which 4.31% are VCAM-1+ (93% EYFP+/VCAM-1+). Satellite cells derived
from tamoxifen-treated Pax7-CreER; Rosa26-LSL-EYFP mice (“QSCs”) were sorted and
explanted, giving rise to proliferative EYFP+ progeny (“ASCs”), which served as positive
control.
Animals
The following mouse strains were obtained from Jackson Laboratories and used in this
study: (i) B10ScSn.Cg-Prkdcscid Dmdmdx/J, stock number 018018; (ii) C57BL/10ScSn-
Dmdmdx/J, stock number 001801; (iii) B6.Cg-Pax7tm1(cre/ERT2)Gaka/J, stock number
017763; (iv) B6 (SJL)-Pou5f1tm1.1(cre/Esr1*)Yseg/J, stock number 016829; (v)
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J, stock number 00618; (vi) B6.Cg-Tg(CAG-
mRFP1)1F1Hadj/J, stock number 005884; (vii) B6.129P2-
Gt(ROSA)26Sortm1(DTA)Lky/J, stock number 009669. Pax7-nGFP MEFs (from
Tg:Pax7-nGFP/C57BL6;DBA2 mice) were derived from previously reported animals
(Sambasivan et al., 2009). MEFs deficient for Pax7 were derived from previously reported
knockout animals (Mansouri et al., 1996).
Cell culture
Mouse embryonic fibroblasts (MEFs), tail-tip fibroblasts (TTFs) and the commercial
myoblast cell line C2C12 (ATCC®CRL-1772TM) were cultured in “MEF medium”
containing DMEM (ThermoFisher Scientific, catalog number 10313-021), supplemented
with 10% Fetal Bovine Serum (FBS) (HyClone catalog number SH30396.03), 1%
GlutaMAX (ThermoFisher Scientific, catalog number 35050061), 1% non-essential amino
acids (ThermoFisher Scientific, catalog number 11140050), 1% penicillin-streptomycin
(ThermoFisher Scientific, catalog number 15140122), 0.05% β-mercaptoethanol
(ThermoFisher Scientific, catalog number 21985-023). Freshly isolated satellite cells and
derivative myoblasts were cultured using a 1:1 ratio of DMEM and F-10 (1X) Nutrient mix
(ThermoFisher Scientific, catalog number 11550-043) supplemented with 10% horse
serum (ThermoFisher Scientific, catalog number 16050-122), 20% FBS (HyClone catalog
number SH30396.03) and 10ng/ml basic-FGF (R&D Systems 233-FB). Satellite cells and
myoblasts were cultured on plates coated with Matrigel Basement Membrane Matrix
(Catalog number 356237, Corning) or laminin (Catalog number 23017015, Thermo Fisher
Scientific).
Immunocytochemistry
For immunocytochemistry, cultured cells were first washed with 1xPBS, cross-linked with
4% paraformaldehyde (PFA) (EMS, 15710) for 5 minutes, washed with 1xPBS and
blocked for 0.5hr at room temperature (RT). The blocking solution consisted of 2% BSA
dissolved in 1xPBS and 0.1% Triton-X-100. Primary antibodies were diluted in blocking
solution and incubated for 1hr at RT. The primary antibodies used in this study were:
Rabbit anti-MyoD1 (sc-760, Santa Cruz Biotechnology, 1:200), Mouse IgG anti-Myogenin
(sc-17320, Santa Cruz Biotechnology, 1:200), Mouse IgG anti-Myogenin (sc-13137,
Santa Cruz Biotechnology, 1:200), Mouse IgG1 anti-Pax7 (MAB1675, clone Pax7, R&D
Systems, 5μg/ml), Mouse IgG2B anti-Myosin HC (MAB4479, clone MF20, R&D Systems,
1:500), Rabbit IgG anti-Myf5 (sc-302, clone C-20, Santa Cruz Biotechnology, 1:200). The
secondary antibodies used in this study were: A11008 Alexa Fluor 488 goat anti-rabbit
IgG, A21202 Alexa Fluor 488 donkey anti-mouse IgG, A21141 Alexa Fluor 488 goat anti-
mouse IgG2B, A11056 Alexa Fluor 546 donkey anti-goat IgG, A21123 Alexa Fluor 546
goat anti-mouse IgG1, A21121 Alexa Fluor 488 goat anti-mouse IgG1, A10040 Alexa
Fluor 546 donkey anti-rabbit IgG, A11055 Alexa Fluor 488 donkey anti-goat IgG and
A21151 Alexa Fluor 488 anti-mouse IgG3, all at a 1:400 dilution. DAPI was used for nuclear
counterstaining. ProLong® Gold Antifade reagent was used to prevent photobleaching
(Life Technologies, P36934).
For immunocytochemistry of muscle tissue, slides containing muscle sections
were cross-linked with 4% PFA, washed with 1xPBS, incubated for 0.5hr with 2% BSA,
dissolved in 1xPBS and 0.1% Triton-X-100, followed by 0.25hr incubation with 10%
donkey serum (Sigma-Aldrich, D9663) and 10% goat serum (Sigma-Aldrich, G9023-10ML)
diluted in 1xPBS. Cells were then incubated for 1hr in primary antibody at RT followed by
1xPBS rinse (twice) and incubation in secondary antibodies (1hr at RT). Primary
antibodies used were Mouse IgG1 anti-Pax7 (MAB1675, R&D Systems, 5μg/ml), Rabbit
anti-Dystrophin (ab15277, Abcam, 1:200), Rabbit anti-Laminin (ab11575, Abcam,
1:1000) and chicken anti-EYFP/GFP (GFP-1020, AVES, 1:300). Secondary antibodies
used were: A21121 Alexa Fluor 488 goat anti-mouse IgG1, A10040 Alexa Fluor 546
donkey anti-rabbit IgG, A31573 Alexa Fluor 647 Donkey anti-rabbit IgG, and A11039
Alexa Fluor 488 goat anti-chicken IgG, all at 1:400 dilution.
RNA sequencing
DNase I-treated total RNA was extracted using RNeasy Micro Kit (74004) according to
the manufacturer’s instructions (Qiagen). RNA-Seq libraries were constructed from
polyA-selected RNA using NEBNext Ultra Directional RNA Library Prep Kit for Illumina
(New England Biolabs) and sequenced on Illumina HiSeq2500 instrument, resulting in
approximately 30 million reads per sample on average. Transcriptome mapping was
performed with STAR version 2.3.0 (Anders et al., 2015) using the Ensembl 67
release exon/splice-junction annotations. Read counts for individual genes were
produced using the unstranded count feature in HTSeq v.0.6.0 (Anders et al., 2013).
Differential expression analysis was performed using the EdgeR package(Dobin et al.,
2013) after normalizing read counts and including only those genes with cpm > 1 for one
or more samples (Robinson et al., 2010). Differentially expressed genes were defined
based on the criteria of >2-fold change in expression value and false discovery rate (FDR)
< 0.05. The RNA-Seq data has been deposited in NCBI’s Gene Expression Omnibus
(GEO, accession number: GSE108543).
Prospective isolation of satellite cells and myoblasts
Muscles were harvested from all four limbs of the animals and subjected to mechanical
dissociation using fine surgical scissors. Muscle slurry was then subjected to enzymatic
dissociation using Collagenase Type II (17101015, Thermo Fisher Scientific) for 1.5hrs in
a 37C shaking hot bath. The mix was then centrifuged and subjected to another round of
enzymatic digestion for 30minutes in Collagenase Type II / Dispase Neutral Protease
solution (LS02100, Worthington Biochemical Corporation). Digested tissues were filtered
through 70μM and 50μM strainers before sorting satellite cells using either a genetic
reporter or surface markers as previously described(Maesner et al., 2016). To generate
myoblast cultures, satellite cells were sorted onto plates coated with Matrigel Basement
Membrane Matrix (Catalog number 356237, Corning) or Laminin (Catalog number
23017015, Thermo Fisher Scientific) and cultured for several passages in myoblast
medium as illustrated in the “Cell Culture” section.
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