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Pluripotent stem (PS) cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous therefore clinical translation requires development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1 and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and cGMP-compatible magnetic-based sorting technologies, and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step towards enabling translation of PS cell-based therapies for muscle diseases.
Pluripotent stem cells are an attractive option for regenerative medicine as they can repeatedly produce large amounts of differentiated tissue, thus representing an unlimited source of cells for therapeutic application. Nevertheless, a major requirement remains the development of protocols, compatible with Current Good Manufacturing Practice (cGMP) standards, for the isolation of specific cell populations with proven in vivo regenerative potential whilst preventing the presence of contaminating teratogenic undifferentiated pluripotent stem cells.
Cell-based therapies are particularly attractive for muscular dystrophies (Tedesco and Cossu, 2012). These are a heterogeneous group of genetic diseases characterized by relentless and catastrophic progression of muscle wasting leading to premature death in high risk disorders, such as Duchenne Muscular Dystrophy (DMD) (Emery, 2002). Following the expansion of the PS cell field, several pioneering studies (Darabi et al., 2012; Darabi et al., 2008; Darabi et al., 2011a; Darabi et al., 2011b; Filareto et al., 2012; Filareto et al., 2013), have demonstrated functionally beneficial engraftment and many others (Borchin et al., 2013; Chal et al., 2015; Choi et al., 2016; Shelton et al., 2014; Xi et al., 2017; Xu et al., 2013), have demonstrated skeletal myogenic differentiation in vitro of both embryonic (ES) and induced-pluripotent (iPS) stem cells. We have shown that upon controlled expression of PAX7 followed by GFP-mediated sorting, human ES/iPS derived myogenic progenitors proliferate robustly in vitro and contribute to muscle regeneration in vivo, as transplanted cells generate functional myofibers while also seeding the muscle stem compartment (Darabi et al., 2012).
To successfully translate this approach toward the clinic, we envisioned the need to identify specific surface markers for the isolation of this progenitor cell population, which would avoid potentially immunogenic transgenes (Ansari et al., 2016) (e.g. GFP or mCherry) as well as establishing quality control for the cGMP process. For this, we performed whole transcriptome sequencing analysis, in which we systematically evaluated different time points along the PAX7-dependent myogenic commitment from human pluripotent stem cells. Our results revealed a subset of genes differentially expressed at various stages of this differentiation process, including a discrete number of surface markers. After Fluorescence Activated Cell Sorting (FACS)-mediated screening, we identified integrin α9β1, CD54 and SDC2, as potential surface markers to be used for the prospective isolation of human PS cell-derived myogenic progenitors. We demonstrate that these surface molecules reproducibly allow the isolation of myogenic progenitors from multiple human ES/iPS cell lines and that α9β1+CD54+SDC2+ (triple+) cells represent a homogenous population of PAX7+ cells endowed with in vivo muscle regeneration potential. These findings provide a clinically relevant method for the purification of PS cell-derived muscle progenitors for therapeutic applications.
Controlled expression of PAX7 in differentiating human ES/iPS cells results in the specification of a highly proliferative population of myogenic progenitors with considerable regenerative potential in vivo. However in order for this approach to be suitable for clinical translation, strategies for the purification of PAX7-induced (referred as PAX7+ hereafter) myogenic progenitors that do not rely on fluorescent reporters (e.g. GFP), but rather specific native surface markers that are compatible with clinical-grade purification methods, are required. To address this important issue, we performed a systematic analysis to identify the gene expression changes induced by PAX7 during the commitment of the skeletal myogenic lineage from human H9 ES cells in vitro. As schematized in Fig. 1A, we focused on the following time points: PAX7+ mesodermal cells (day 14), PAX7+ proliferating myogenic progenitors (approximately day 23), and differentiated myocytes (differentiation stage – around day 30; 7 days in the absence of PAX7 induction). Since PAX7 expression is doxycycline inducible, we also collected uninduced control samples at the same time points (termed mesodermal cells and proliferating cells; Fig. 1A, left). RNA-sequencing (RNA-seq) identified about 2000 genes whose expression levels displayed a fold change >4 and TPM (Transcripts Per Million reads) >64 in at least one of the replicates (Fig. 1B). Unsupervised analysis of these differentially expressed genes identified 4 distinct clusters representing cells undergoing PAX7-dependent myogenic commitment (PAX7+ mesodermal cells, PAX7+ proliferating myogenic progenitors, and differentiation stage) as well as uninduced cells (Fig. 1B). Importantly, upon principal component analysis (PCA), these clusters were easily separated according to PAX7 expression (dox-treatment – on PC2 axis), and progression toward the differentiated skeletal myogenic state (on PC1 axis) (Fig. 1C). As shown by the heat map in Fig. 1B and Spearman correlations map in Fig. S1A, gene expression changes follow a pattern suggesting the gradual activation of the skeletal myogenic program induced by PAX7, which is also discernible at the single gene level (Fig. 1D and Fig. S1B). In agreement with our previous studies using mouse ES cells (Magli et al., 2012; Magli et al., 2014), these results show that genes associated with paraxial mesoderm development, such as DLL1, MEOX1 and LFNG, are also up-regulated by PAX7 expression, indicating the ability of this transcription factor to induce a population of paraxial mesodermal progenitors (Fig. 1D). Moreover, taking advantage of recently published RNAseq data from developing human embryos (Xi et al., 2017), we demonstrate that both PAX7+ mesodermal cells and PAX7+ proliferating myogenic progenitors are more closely related to cells isolated from somites (SM and SM_DEV) than presomitic mesoderm (PSM - Fig S1C), thus supporting the capability of our system to recapitulate cellular processes occurring during human development.
After validating selected candidates by RT-qPCR (Fig. S1D), we chose sets of genes from each cluster that are up-regulated with respect to the uninduced samples (boxed areas, Fig. 1b) and applied Gene Ontology (GO)-based functional annotation to each set. This revealed that each cluster is characterized by up-regulated genes with distinct biological function based on GO classification (Fig. 1E and Fig. S1E). As expected, while both PAX7+ mesodermal cells and PAX7+ proliferating myogenic progenitors are enriched for genes involved in cell motility/adhesion and communication (Fig. 1E), differentiated myocytes (differentiation stage) are characterized by the expression of genes involved in terminal differentiation (Fig. S1E).
To gain further insight on the PAX7-mediated activation of the skeletal myogenic program, we also performed Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) for PAX7 in PAX7+ proliferating myogenic progenitors. As expected, we identified putative PAX7 binding sites in several differentially regulated genes (approximately 50%), including known PAX3/7 targets MET, MYF5 and MYOD (Bajard et al., 2006; Epstein et al., 1996; Soleimani et al., 2012) (Fig. 1F and Fig. S1F). Following annotation, we observed that PAX7 peaks are located within introns and in intergenic regions (Fig. 1G), while the gene type distribution highlighted a preference for protein-coding genes and long non-coding RNAs (Fig. S1G). Analysis of the top 3000 peaks showed enrichment of both the PAX Paired- and homeo-domain dependent motifs (Fig. 1H), demonstrating the specificity of our approach in identifying bona fide PAX7 binding sites. These data are in agreement with previous work in murine primary myoblasts (Soleimani et al., 2012) and, collectively, represent a detailed analysis of PAX7 function during human skeletal myogenesis, allowing us to interrogate specific questions such as the molecular identity of the myogenic progenitor population endowed with muscle regenerative potential.
Next, we focused on the identification of candidate surface markers specific for cells undergoing PAX7-dependent myogenic commitment. GO analysis and manual inspection of the RNAseq data (available in Table S1) identified a group of 50 genes associated with cell adhesion, which were found up-regulated following PAX7 induction (Table S2) in PAX7+ mesodermal cells and PAX7+ proliferating myogenic progenitors. RNA molecules are subjected to post-transcriptional regulation and protein expression might not reflect such variations. Therefore, using available commercial antibodies, we screened several candidate surface markers to identify changes in protein expression levels between PAX7+ mesodermal cells (identified based on GFP expression, which is encoded by the bicistronic transcript PAX7-ires-GFP) and control uninduced cells (PAX7-negative). As shown in Fig. 2A and Fig. S2A, FACS analysis confirmed distinct up-regulation of 3 surface markers following PAX7 induction (compare GFP+ and GFP-cells). These surface markers are the Intercellular Adhesion Molecule 1 (ICAM1 or CD54), Syndecan 2 (SDC2 or CD362), and Alpha9 Integrin (ITGA9) subunit. Since Integrin B1 is the main and almost exclusive beta integrin subunit expressed in these cells (Fig. S2B), we studied the expression of ITGA9 using the antibody for the integrin α9β1 dimer (referred as α9β1 hereafter).
Because RNA-seq data showed time dependent changes in gene expression during the PAX7-mediated myogenic commitment, we next evaluated the expression of these markers over time. Following PAX7 induction, we detected a population of CD54+ (bright) cells in PAX7+ mesodermal fraction (day 14; Fig. 2B, left). This population was also found positive for α9β1 integrin, and it was clearly distinct from control populations (no dox) in the PAX7+ mesodermal and PAX7+ proliferating myogenic progenitor stages (day 23; Fig. 2B, right). Quantification of the frequency of these populations confirmed the up-regulation of these surface markers upon PAX7 induction (Fig. 2C). Similar results were obtained when cells were analyzed based on PAX7 expression using the GFP reporter (GFP+ vs. GFP−) (Fig. S2C–D). Not surprisingly, upon inspection of our ChIP-seq data, we were able to identify PAX7 binding at the 5’ region of CD54 (ICAM1) and in the intronic region of SDC2 (Fig. S1F), strongly supporting the notion that PAX7 directly regulates these genes. Because the PAX7-expressing population positive for these markers is clearly separated from the negative fraction at the proliferating myogenic progenitor stage (Fig. 2B, right panel), we focused our purification efforts at this specific time point. Of note, CD54+ α9β1+ proliferating myogenic progenitors are ~95% SDC2+ (Triple+; Fig. 2D–E), and retrospective analysis of GFP expression shows that Triple+ cells are nearly 100% GFP+ (Fig. 2D–E), thus suggesting that these markers can replace the GFP transgene for the purification of human PS cell-derived PAX7+ myogenic progenitors.
To confirm this hypothesis, we FACS-purified different cell populations from dox-treated and untreated cultures using different combinations of these three antibodies, and assessed their ability to generate Myosin Heavy Chain positive (MYHC+) skeletal myocytes in vitro. Remarkably, MYHC+ terminal differentiation was robust and indistinguishable between the dox-treated CD54+α9β1+SDC2+ (Triple+) cell fraction and the control GFP+ (PAX7+) cell population (Fig. 2F and Fig S2F). In contrast, dox-treated CD54-α9β1- or CD54-α9β1-SDC2-(Triple-) cells and CD54-α9β1-, CD54+α9β1+, SDC2+ or Triple+ cell fractions from uninduced cultures showed virtually no myogenic potential, as demonstrated by the extremely low number of MYHC+ cells (Fig. 2F and Fig S2E–F). These results highlight the transcriptional importance of PAX7 in the specification of the skeletal myogenic lineage since cells without PAX7 expression have no myogenic potential. Importantly, both GFP+ and Triple+ purified cells displayed equivalent proliferative potential (Fig. S2G). Also of extreme relevance, these surface markers were detected upon PAX7 induction in multiple different differentiating pluripotent stem cell lines (Fig. S3A–B), and were consistently found to purify myogenic progenitors with robust skeletal myogenic differentiation potential (Fig. S3C). These data clearly demonstrate that the isolation of human PS cell-derived PAX7+ myogenic progenitors can be achieved using CD54, α9β1 and SDC2.
A distinct population of CD54+α9β1+SDC2+ cells was also identified upon PAX7 induction in pluripotent stem cells differentiated using a chemically-defined serum-free monolayer protocol, (Fig. 3A–B), which was confirmed to be endowed with high myogenic potential (Fig. 3C and Fig S3D), and indistinguishable from cells sorted based on GFP expression. These data indicate that PAX7 is capable of inducing a population of CD54+α9β1+SDC2+ myogenic progenitors independently of the culture conditions used.
Since the use of lentiviral vectors is required for the consistent in vitro generation of PAX7+ proliferating myogenic progenitors, we have tested the pCCL lentiviral backbone vector system, which is a third generation self-inactivating vector (Dull et al., 1998) demonstrated to be safer than standard lentiviral vectors (Aiuti et al., 2013; Biffi et al., 2013; Romero et al., 2013a) in recent gene therapy trials. Validation studies for doxycycline-dependent PAX7 expression in the absence of a GFP reporter in pluripotent stem cells transduced with this construct (pCCL-PAX7) show indistinguishable expression of CD54, α9β1 and SDC2 compared to inducible PAX7-ires-GFP cells and importantly, allow FACS-mediated isolation of myogenic progenitors, which robustly differentiate into MYHC+ cells (Fig S3E–F).
As shown above, myogenic progenitors are homogeneously defined by the expression of CD54, α9β1 and SDC2. Nevertheless, because methods compatible with clinical application involve the use of single marker purification by magnetic beads, we predicted that sequential purification of 3 surface markers would be inefficient for this purpose. Although CD54 is expressed in uninduced cells, this receptor is clearly upregulated following PAX7 induction (Fig. 2A), such that cells prepared for FACS can be visually identified based on doxycycline treatment (Fig S3G). Considering that CD54bright cells are also positive for both α9β1 and SDC2 (Fig. 2D), we investigated whether CD54 could be used as a single marker for the isolation of PS cell-derived myogenic progenitors. For this, we isolated CD54-labeled human PS cell-derived myogenic progenitors using either FACS or magnetic cell sorting (MACS). Retrospective analysis of GFP expression revealed that both CD54 FACS- and MACS-purified cells are highly enriched for GFP (Fig. 3D–E) and, as expected, both methods yielded a population of myogenic progenitors endowed with robust myogenic differentiation potential, comparable to the GFP+ (PAX7+) control cell population (Fig. 3F). Altogether these data demonstrate that a single step purification strategy can provide cultures with optimal enrichment of human PS cell-derived PAX7+ proliferating myogenic progenitors and support the translational potential of these identified markers for future therapeutic applications.
One of the main concerns associated with potential cell therapies involving PS cell-derived cultures is the presence of contaminating undifferentiated pluripotent cells which may give rise to teratomas. We investigated whether the cell fraction negative for the aforementioned identified markers displayed pluripotency features, which would associate the negative fraction with teratoma risk. FACS-purified CD54-α9β1-SDC2- (Triple-) cells maintained in culture conditions specific to PS cells were not able to form human pluripotent stem cell-like colonies (Fig. S4A), and consistently, both CD54-α9β1-SDC2- and CD54+α9β1+SDC2+ cells lacked expression of the pluripotency markers OCT3/4, SOX2 and NANOG (Fig. S4B). To further demonstrate the safety of human PS cell-derived myogenic progenitors, we injected CD54+ MACS-sorted (representing the potential cGMP product) and CD54- FACS-sorted (to specifically evaluate their teratoma-forming ability) cell preparations into immunodeficient (NSG) mice to determine their teratoma-forming potential. To date, 12 months post-injection, no signs of potential teratoma-forming activity have been observed in mice injected with CD54+ or CD54- cells. This is in contrast to undifferentiated human PS cells, which gave rise to evident teratomas 2 months after cell injection (Fig. S4C).
A fundamental requirement for a prospective cell population to be used for therapy is the demonstration of in vivo regenerative potential using animal models. We previously demonstrated that human ES/iPS-derived myogenic progenitors efficiently participate in muscle regeneration by producing fibers positive for both human DYSTROPHIN (hDYS) and human LAMIN A/C. To assess whether human myogenic progenitors isolated using these identified surface markers have the same regenerative potential as their GFP+ control counterparts, we injected CD54+α9β1+SDC2+ sorted myogenic progenitors into cardiotoxin-injured Tibialis Anterior (TA) muscles of immunodeficient (NSG) mice. Whereas no detection of human DYS and LAMIN A/C was observed in PBS-injected controls (Fig. 4A, left), TA muscles that had been transplanted with myogenic progenitors purified based on the expression of CD54, α9β1 and SDC2 displayed large numbers of hDYS+/hLAMIN A/C+ fibers (Fig. 4a, right panel) both 8 weeks and 10 months post-intramuscular cell delivery. Similar results were obtained upon transplantation of these cells in the DMD mouse model NSG-mdx4cv (Fig. S4D). Importantly, the regenerative potential of CD54+α9β1+SDC2+ sorted myogenic progenitors was found equivalent to that observed with the GFP+ (PAX7+) control cell population (Fig. 4A–B).
One of the most attractive features of stem cell-based therapies is the contribution to the tissue-resident stem cell pool responsible for the long-term homeostasis. We show CD54+α9β1+SDC2+ sorted myogenic progenitors provide long-term regenerative potential, as demonstrated by the detection of hDYS+/hLAMIN A/C+ fibers as late as 10 months post-transplantation (Fig 4A, lower panels). Consistently, we observed the presence of human donor-derived (hLAMIN A/C+) cells occupying the satellite cell position and co-expressing the muscle stem cell markers M-cadherin (Cooper et al., 1999; Donalies et al., 1991; Irintchev et al., 1994) (Fig. 4C) or Pax7 (Seale et al., 2000) (Fig. S4E). All together, these data support the application of this purification strategy for clinically-compatible production of human PS cell-derived myogenic progenitors endowed with long term regenerative potential.
The genetic nature of myopathies such as Duchenne Muscular Dystrophy requires therapeutic approaches able to both repair the existing myofibers and repopulate the stem cell pool. After initial attempts involving myoblast transplantation (Huard et al., 1991; Partridge et al., 1989) (Mendell et al., 1995; Partridge et al., 1998; Tremblay et al., 1993; Vilquin, 2005), which produced no clinical benefit, the recognition that stemness is lost upon ex vivo culture has led many to focus on satellite cells, particularly their self-renewal and efforts to enable their ex vitro expansion (Arpke et al., 2013; Bernet et al., 2014; Cosgrove et al., 2014; Dumont et al., 2015; Gilbert et al., 2010; Quarta et al., 2016; Sousa-Victor et al., 2014). In addition, several other adult stem cell populations have been reported to be endowed with myogenic potential (Mitchell et al., 2010; Sampaolesi et al., 2003; Torrente et al., 2004). Although this is encouraging, definitive demonstration of their clinical efficacy remains to be proven.
Since the isolation of human ES cells in 1998 (Thomson et al., 1998), regenerative medicine based on PS cells has attracted remarkable interest from both academia and industry, which promoted the rapid expansion of this field and, among various achievements, resulted in the development of iPS cell technology (Takahashi and Yamanaka, 2006). This has the potential to revolutionize the field of regenerative medicine as it potentially enables for the use of autologous genome-edited or allogeneic HLA-matched iPS cells. Importantly, the future success of regenerative medicine relies on the safe and reproducible production of tissue-specific progenitors from PS cells (Carpenter and Rao, 2015). Methods for the isolation of the target cell population represent one of the crucial requirements for clinical application of PS cell-derived stem cell products, and their development requires a detailed characterization of the PS cell differentiation process. Based on our earlier findings, in this study we took advantage of the conditional PAX7 expression system to investigate the process leading to the specification of the skeletal myogenic lineage by performing RNA- and ChIP-seq experiments. Of note, this work represents the first detailed investigation of PAX7 function in human myogenesis and identifies several potential regulators of the muscle lineage. Specifically, these analyses revealed a group of differentially expressed genes accounting for the unbiased separation of the samples into 4 independent clusters and whose localization on the PCA graph is reminiscent of the time-dependent commitment toward the skeletal myogenic lineage (Fig. 1B–C). Furthermore, many up-regulated genes contains putative PAX7 binding sites (Fig. 1F and Fig. S1F) and upon functional classification of these data, we observed that genes involved in cell adhesion and motility represent the most significant category up-regulated by PAX7 (Fig. 1E). Using this knowledge, we first performed a screen to identify markers enriched on the surface of PS cell-derived myogenic progenitors (Fig. 2A–B and Fig. S2A), and then we demonstrated that CD54, α9β1 and SDC2 mark and allow for the isolation of these progenitors from cultures of differentiating pluripotent stem cells (Fig. 2C–E and Fig. 3A–C). Importantly, myogenic progenitors isolated based on the expression of these identified markers are indistinguishable from the ones sorted using intracellular fluorescent reporters, and accordingly are endowed with robust in vitro proliferative potential and in vivo regenerative capacity, including contribution to the satellite cell compartment (Fig. 4 and Fig. S4D–E). Issues related to safety represent significant hurdles when translating initial studies at the bench into viable clinical protocols. Defining the global PAX7 DNA-binding and transcriptional profiles allowed us to discover and use surface markers to significantly purify the cell product. In addition, these markers eliminate the need for a transduction reporter (GFP- Fig. S3E–F) and guarantee the selection of myogenic cells in which we know PAX7 is functionally active.
Although transgene-free monolayer cultures for the differentiation of hPS cells toward the skeletal myogenic lineage have been reported, findings produced so far have not been sufficient to justifying their application to regenerative medicine. Overall, these protocols allow for the derivation of myocytes, however their translational applicability is uncertain since engraftment capability, in most cases, was not reported (Borchin et al., 2013; Chal et al., 2015; Loh et al., 2016; Shelton et al., 2014; Xi et al., 2017) or was very limited (Xu et al., 2013). Notably, in absence of cell purification, these protocols generate heterogeneous populations including neural cells (Chal et al., 2016; Kim J, 2017). Moreover, based on MYOGENIN expression (Chal et al., 2016; Choi et al., 2016; Shelton et al., 2014), these PS cell-derived myogenic cells more resemble myoblasts, a population already tested in several clinical trials without beneficial improvements to date (reviewed by (Tedesco and Cossu, 2012). In contrast, a differentiation strategy incorporating inducible PAX7 expression produces homogeneous cultures of proliferating PAX7+ myogenic progenitors, with minimum spontaneous terminal differentiation, as previously demonstrated by negligible expression levels of MYOGENIN and MHC in the expansion phase (Darabi et al, 2012). Consistently, these PAX7+ myogenic progenitors are capable of in vivo skeletal muscle regeneration and contribution to the muscle stem cell pool, essential aspects for safe and effective cell therapy for muscle diseases.
Based on these findings, we postulate these surface markers will be instrumental for the large scale cGMP-compatible production of PS cell-derived myogenic progenitors. This purification strategy combined with PAX7 expression obtained using third generation self-inactivating lentiviral vectors (Fig. S3E–F), currently in gene therapy clinical trials for hematological disorders with encouraging results in terms of safety (Aiuti et al., 2013; Biffi et al., 2013; Romero et al., 2013b), or expression cassettes integrated in a safe harbor locus (DeKelver et al., 2010; Hockemeyer et al., 2009) (e.g. AAVS1), has the potential to enable clinical translation of this myogenic progenitor cell population in patients with muscular dystrophy.
Inducible PAX7 Human H9 ES cells and PLZ wt iPS cells were generated previously (Darabi et al., 2012) and they were maintained on matrigel-coated flasks using mTeSR (Stem Cell Technology). Inducible PAX7 DMD1705 and DMD0907 and LGMD2a-1509 human iPS cells were generated by lentiviral transduction of the pSAM2-PAX7-ires-GFP and pFUGW-rtTA constructs (Darabi et al., 2012). The third generation lentiviral construct pCCL-c-MCS (Zufferey et al., 1998) was kindly provided by Donald Kohn. The tetracycline-responsive-element (TRE) followed by PAX7 or PAX7-ires-GFP sequences and Ubiquitin-C (UBC) promoter followed by the rtTA sequences was subcloned into the pCCL vector to generate respectively pCCL-PAX7, pCCL-PAX7-ires-GFP and pCCL-rtTA. Details on the cloning strategy are available upon request. These constructs were used to transduce H9 human ES cells as described above. Cells were passaged at 90% of plate density using Accumax (3 min incubation) followed by gentle resuspension. After centrifuge for 5 min at 300g, cell pellets were resuspended in mTeSR supplemented with 10µM of the ROCK inhibitor Y-27632 and replated in matrigel-coated flasks or used for differentiation. For skeletal myogenic differentiation (protocol described in (Darabi and Perlingeiro, 2016)), cells were plated in 6cm non-adherent Petri dishes and incubated at 37°C, 5% CO2 on a shaker at 60 RPM. After 2 days, mTeSR was replaced with EB Myogenic (EBM) Media supplemented with 10µM Y-27632 and 5µM GSK3 inhibitor (CHIR990217). 2 days later, media was replaced to remove GSK3 inhibitor and the formed EBs were cultured in suspension on the shaker up to day 7. After plating EBs on gelatin-coated flasks using EBM + 10ng/ml human basic FGF (bFGF), EBs were let to adhere on the flasks for 3 days and then supplemented with 1µg/ml doxycycline (dox) for PAX7 induction. At day 14, cells were harvested using Trypsin+EDTA solution and FACS sorted based on GFP expression. Alternatively, unsorted single cells were re-seeded on gelatin-coated flasks using EBM supplemented with 5ng/ml bFGF, 1µg/ml dox and expanded as monolayer until day 23 for FACS sorting using specific antibodies. GFP+ or Marker+ cells were then plated at 1.5*10^6 cells per T25 on gelatin-coated flasks using EBM supplemented with 5ng/ml bFGF, 1µg/ml dox and 5µM ROCK inhibitor (ROCK inhibitor was removed the day after) and cultured as monolayer. At ~90% cell density, cells were passaged using Trypsin+EDTA and replated on new gelatin-coated flasks. Terminal differentiation was induced by switching 100% confluent cultures to KOSR differentiation media (KnockOut™ DMEM + 20% KnockOut™ Serum Replacement + 1% Penicillin/Streptomycin + 2mM GlutaMAX™ - all products from Gibco). Serum-free differentiation using inducible-PAX7 cell lines is based on the protocol published by Chal and colleagues (Chal et al., 2015). At day 9 of the protocol, cultures were supplemented with 1µg/ml doxycycline (dox) for PAX7 induction. Cells were passaged 2 times and then FACS sorted using specific markers or GFP. Sorted cells were then plated at 1.5*10^6 cells per T25 on matrigel-coated flasks using DMEM-F12 + KOSR + Insulin-Transferrin-Selenium (ITS) supplemented with 10ng/ml HGF, 2ng/ml IGF, 1µg/ml dox and 5µM ROCK inhibitor (ROCK inhibitor was removed the day after). Terminal differentiation was induced by withdrawing doxycycline from the medium in 100% confluent cultures. Data presented in the manuscript are originated from biological replicates (at least 3 independent experiments for at least 3 independent cell lines).
A detailed description is provided in Supplemental Experimental Procedures.
A detailed description is provided in Supplemental Experimental Procedures.
Genes were filtered to select only those with median-normalized TPM (Transcripts Per Million reads) greater than 64 in at least one sample, and a fold change greater than 4 between the median-normalized Expected Counts (EC) of any two samples. Heatmaps of relative expression display for each gene G and sample S the value log2 ( (1+median-normalized EC(G,S))/(1+mean over all Si median-normalized EC(G, Si)). Functional annotation of genes (From Supplemental file 1) was performed using the online tool DAVID (Huang da et al., 2009). Graphs represent the output of PANTHER biological process category. Additional information is available in Supplemental Experimental Procedures.
A detailed description is provided in Supplemental Experimental Procedures.
Animal experiments were carried out according to protocols approved by the University of Minnesota Institutional Animal Care and Use Committee. Both tibialis anterior (TA) muscles of 6–8 weeks old male NOD-scid IL2Rgnull (NSG - Jackson lab) or NOD-scid IL2Rgnull mdx4cv (Arpke et al., 2013) (NSG-mdx4cv) mice were pre-injured with 15 µl of cardiotoxin 10 µM (Latoxan). 24 hrs after cardiotoxin, cells were collected at approximately 60–80% cell density using Enzyme-free cell dissociation buffer (Gibco), then washed with PBS and resuspended in PBS supplemented with 10ng/ml bFGF at the concentration of 5*10^5 cells/10µl.
Cells were injected in one TA per mouse while the contralateral TA received the same volume of PBS as internal control. 8 weeks and 10 months after transplantation, mice were euthanized and TAs collected for immunostaining analysis as previously described (Darabi et al., 2008). Briefly, muscles were frozen in isopentane cooled in liquid nitrogen, and serial 10 µm-thick cryosections were collected. Teratoma studies were performed by injecting 1.5×10^6 H9 ES cells (positive control) or 5×10^6 MACS-purified CD54+ (representing the potential cGMP product) or FACS-purified CD54- (to specifically evaluate their teratoma-forming ability) cells in the quadriceps of NSG mice. Before injection, cells were resuspended in 1:1 solution DMEM-F12 and matrigel (final volume including cells: 65µl).
A detailed description is provided in Supplemental Experimental Procedures.
Differences between samples were assessed by using the unpaired two-tailed Student’s t-test. p values < 0.05 were considered significant.
Raw and processed RNA-seq and ChIP-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession code GSE98976.
List of genes analyzed by gene ontology described in Figure 1E and Figure S1E. Each column represents the list of genes analyzed using DAVID.
List of genes associated with cell adhesion, which were found up-regulated following PAX7 induction in PAX7+ proliferating myogenic progenitors.
This project was supported by NIH grants R01 AR055299 (R.C.R.P.), U01 HL100407R01 (R.C.R.P. and J.A.T.), the Greg Marzolf Jr Foundation (R.C.R.P.), ADVault, Inc and MyDirectives.com (R.C.R.P.) and by Regenerative Medicine Minnesota (A.M.). The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH Grant #P30 CA077598-09.
The authors thank Dr. Jaemin Kim for his assistance with the serum-free differentiation protocol and Dr. Nelio Oliveira for his assistance with the teratoma experiments.
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CONTRIBUTIONSA.M. and T.I. designed and performed the research, analyzed the data and wrote the manuscript; J.K. performed the research and analyzed the data; S.A.S and C.Y. performed the bioinformatic analyses; R.S. and J.A.T. supervised the bioinformatic analyses; R.D., F.R., S.S. and A.Y. performed research; J.T. supervised iPSC facility; R.C.R.P. contributed with experimental design, interpretation of the data and wrote the manuscript.