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• W2401496253 abstract "•Isolation of functional myoblasts from multiple hiPSC lines using a defined system•Concordant but heterogeneous phenotypes among myoblasts from DMD patients•Genetic and pharmacological rescue of DMD-related phenotypes•Myotube formation in DMD-myoblasts and genetically corrected isogenic myoblasts Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling. Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling. Embryonic development has been successfully modeled in vitro by differentiating human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This occurs by modulating relevant signaling pathways via chemical compounds, as demonstrated in the neuroectodermal, cardiac, and endodermal lineages (Borowiak et al., 2009Borowiak M. Maehr R. Chen S. Chen A.E. Tang W. Fox J.L. Schreiber S.L. Melton D.A. Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.Cell Stem Cell. 2009; 4: 348-358Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, Burridge et al., 2014Burridge P.W. Matsa E. Shukla P. Lin Z.C. Churko J.M. Ebert A.D. Lan F. Diecke S. Huber B. Mordwinkin N.M. et al.Chemically defined generation of human cardiomyocytes.Nat. Methods. 2014; 11: 855-860Crossref PubMed Google Scholar, Chambers et al., 2009Chambers S.M. Fasano C.A. Papapetrou E.P. Tomishima M. Sadelain M. Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat. Biotechnol. 2009; 27: 275-280Crossref PubMed Scopus (2421) Google Scholar, Di Giorgio et al., 2008Di Giorgio F.P. Boulting G.L. Bobrowicz S. Eggan K.C. Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation.Cell Stem Cell. 2008; 3: 637-648Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Combined with a strategy to isolate homogenous populations, disease-specific hiPSC-derived cells have facilitated our understanding of the pathogenesis of human genetic disorders by providing symptom-relevant cell types in a patient-specific manner (Chen et al., 2014Chen H. Qian K. Du Z. Cao J. Petersen A. Liu H. Blackbourn 4th, L.W. Huang C.L. Errigo A. Yin Y. et al.Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons.Cell Stem Cell. 2014; 14: 796-809Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, Kiskinis et al., 2014Kiskinis E. Sandoe J. Williams L.A. Boulting G.L. Moccia R. Wainger B.J. Han S. Peng T. Thams S. Mikkilineni S. et al.Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1.Cell Stem Cell. 2014; 14: 781-795Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, Wainger et al., 2014Wainger B.J. Kiskinis E. Mellin C. Wiskow O. Han S.S. Sandoe J. Perez N.P. Williams L.A. Lee S. Boulting G. et al.Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons.Cell Rep. 2014; 7: 1-11Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). This has lead researchers to validate potential therapeutic small molecules that can rescue in vitro phenotypes (Brennand et al., 2011Brennand K.J. Simone A. Jou J. Gelboin-Burkhart C. Tran N. Sangar S. Li Y. Mu Y. Chen G. Yu D. et al.Modelling schizophrenia using human induced pluripotent stem cells.Nature. 2011; 473: 221-225Crossref PubMed Scopus (1035) Google Scholar, de Boer et al., 2014de Boer A.S. Koszka K. Kiskinis E. Suzuki N. Davis-Dusenbery B.N. Eggan K. Genetic validation of a therapeutic target in a mouse model of ALS.Sci. Transl. Med. 2014; 6: 248ra104Crossref PubMed Scopus (23) Google Scholar). However, efforts such as those mentioned above have not yet been fully applied to the hiPSCs of muscular dystrophy, mainly due to the absence of a successful strategy for isolating expandable functional myoblasts. Previous efforts to derive myogenic cells from hESCs and hiPSCs were based on the ectopic expression of myogenic transcription factors, such as PAX3, PAX7, and MYOD1, by viral gene delivery (Darabi et al., 2012Darabi R. Arpke R.W. Irion S. Dimos J.T. Grskovic M. Kyba M. Perlingeiro R.C. Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice.Cell Stem Cell. 2012; 10: 610-619Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Tedesco et al., 2012Tedesco F.S. Gerli M.F. Perani L. Benedetti S. Ungaro F. Cassano M. Antonini S. Tagliafico E. Artusi V. Longa E. et al.Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy.Sci. Transl. Med. 2012; 4: 140ra89Crossref PubMed Scopus (212) Google Scholar). Although this approach can produce certain myogenic cells, the random integration of viral DNA can jeopardize disease modeling and mask any unknown potential disease phenotype. In addition, other previous methods rely on animal-derived factors and require arduous long-term culture (4 months or more) (Barberi et al., 2007Barberi T. Bradbury M. Dincer Z. Panagiotakos G. Socci N.D. Studer L. Derivation of engraftable skeletal myoblasts from human embryonic stem cells.Nat. Med. 2007; 13: 642-648Crossref PubMed Scopus (256) Google Scholar). More importantly, high-purity, disease-specific myoblasts must be isolated and expanded to study their transcriptional profiles or functional deficits. Considering the high disease prevalence and severity, as well as a lack of meaningful therapies for most skeletal muscle disorders, it is critical to develop a human cellular model system. One of the most common muscular dystrophies is Duchenne muscular dystrophy (DMD), which affects approximately 1 in 5,000 live male births. DMD is caused by mutations in DYSTROPHIN (Hoffman et al., 1987Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus.Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3658) Google Scholar), and more than 1,000 different sequence variations in the culprit gene (http://www.dmd.nl) have been discovered. Patients with DMD have heterogeneous disease severity due to specific DYSTROPHIN mutations as well as modifier genes, as demonstrated by a wide range of loss of ambulation and end of life (Vo and McNally, 2015Vo A.H. McNally E.M. Modifier genes and their effect on Duchenne muscular dystrophy.Curr. Opin. Neurol. 2015; 28: 528-534Crossref PubMed Scopus (38) Google Scholar). Although zebrafish, mouse, and dog models have provided DMD-related data on pathogenesis, it is generally recognized that each of these models have limitations (Kornegay et al., 2012Kornegay J.N. Childers M.K. Bogan D.J. Bogan J.R. Nghiem P. Wang J. Fan Z. Howard Jr., J.F. Schatzberg S.J. Dow J.L. et al.The paradox of muscle hypertrophy in muscular dystrophy.Phys. Med. Rehabil. Clin. N. Am. 2012; 23: 149-172, xiiAbstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, Partridge, 2013Partridge T.A. The mdx mouse model as a surrogate for Duchenne muscular dystrophy.FEBS J. 2013; 280: 4177-4186Crossref PubMed Scopus (128) Google Scholar). A DMD study using mouse embryonic stem cells (mESCs) from a DMD mouse model was published recently (Chal et al., 2015Chal J. Oginuma M. Al Tanoury Z. Gobert B. Sumara O. Hick A. Bousson F. Zidouni Y. Mursch C. Moncuquet P. et al.Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.Nat. Biotechnol. 2015; 33: 962-969Crossref PubMed Scopus (244) Google Scholar). However, it is still questionable whether any of the mESC-based studies can sufficiently model such varied severity in a mutation-dependent or patient-specific manner. Humanized DMD models carrying patient-specific DYSTROPHIN mutations will be complementary to current animal models of DMD. One such potential humanized DMD cell model is DMD-specific hiPSCs. Here, we planned to establish a defined, robust, and efficient system to direct hPSCs into myogenic specification, demonstrating the feasibility of myoblast derivation from DMD-specific hiPSCs. Recapitulation of the heterogeneous severity of disease among patients could be a step toward comprehensive mechanistic studies on DMD pathogenesis. In addition, harnessing the potential of hiPSC technology may lead to a more personalized approach for DMD treatment. To harness the potential of hPSCs, we developed a protocol to direct hPSCs into the skeletal muscle lineage. As the somite is an intermediate stage between hPSCs and myogenic progenitor cells (Bentzinger et al., 2012Bentzinger C.F. Wang Y.X. Rudnicki M.A. Building muscle: molecular regulation of myogenesis.Cold Spring Harb. Perspect. Biol. 2012; 4: a008342Crossref Scopus (268) Google Scholar, Dequéant and Pourquié, 2008Dequéant M.L. Pourquié O. Segmental patterning of the vertebrate embryonic axis.Nat. Rev. Genet. 2008; 9: 370-382Crossref PubMed Scopus (295) Google Scholar) (Figure S1A), we generated a MESOGENIN1::eGFP reporter hESC line with the CRISPR/Cas9 system (Mali et al., 2013Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6424) Google Scholar) (Figures S1B–S1D). MESOGENIN1 is a genetic marker for the pre-somite mesoderm fate (Fior et al., 2012Fior R. Maxwell A.A. Ma T.P. Vezzaro A. Moens C.B. Amacher S.L. Lewis J. Saúde L. The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1.Development. 2012; 139: 4656-4665Crossref PubMed Scopus (45) Google Scholar). Brief treatment (4 days after day 0 of differentiation) with CHIR99021, a GSK-3β inhibitor (Bennett et al., 2002Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. Regulation of Wnt signaling during adipogenesis.J. Biol. Chem. 2002; 277: 30998-31004Crossref PubMed Scopus (582) Google Scholar), significantly increased expression of MESOGENIN1::eGFP (80.8% ± 11.3% cells out of total cells in a dish), TBX6 (67.4% ± 10.4%), and PAX3 in a dose-dependent manner (Figures S1E–S1G) at day 4 and gave rise to myogenic cells expressing MyHC (MF20), MYOG, and MYOD at day 40 (30.4% ± 13.7%, 37.7% ± 5.78%, and 30.4% ± 13.70%, respectively) (Figure S1H). CHIR99021 appeared to activate the canonical WNT signaling pathway, confirmed by β-catenin translocation into the nucleus (Figure S1I). Further data analysis suggests that WNT activation and inhibition of the PI3K pathway (Figures S1J–S1L) are sufficient for induction of MESOGENIN1::eGFP from hPSCs. These data are partially explained by fostering myogenic specification from somite cells as well as enhanced WNT activation (Figures S1M–S1P), and they are in agreement with results of studies from other groups (Borchin et al., 2013Borchin B. Chen J. Barberi T. Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells.Stem Cell Reports. 2013; 1: 620-631Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Chal et al., 2015Chal J. Oginuma M. Al Tanoury Z. Gobert B. Sumara O. Hick A. Bousson F. Zidouni Y. Mursch C. Moncuquet P. et al.Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.Nat. Biotechnol. 2015; 33: 962-969Crossref PubMed Scopus (244) Google Scholar, Wang et al., 2007Wang J. Li S. Chen Y. Ding X. Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis.Dev. Biol. 2007; 304: 836-847Crossref PubMed Scopus (15) Google Scholar, Xu et al., 2013Xu C. Tabebordbar M. Iovino S. Ciarlo C. Liu J. Castiglioni A. Price E. Liu M. Barton E.R. Kahn C.R. et al.A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species.Cell. 2013; 155: 909-921Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To increase the speed and efficacy of myogenic specification, we found that treatment from day 4 to day 12 with DAPT, a γ-secretase inhibitor that blocks Notch signaling (Dovey et al., 2001Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. et al.Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (796) Google Scholar) (Figure 1A), promoted a robust and fast myogenic differentiation. At day 30, 63.6% ± 9.68% of cells were MF20+, and 61.5% ± 11.0% were MYOGENIN+ (Figures 1B and S1Q–S1U), which is consistent with data from recent rodent studies (Mayeuf-Louchart et al., 2014Mayeuf-Louchart A. Lagha M. Danckaert A. Rocancourt D. Relaix F. Vincent S.D. Buckingham M. Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb.Proc. Natl. Acad. Sci. USA. 2014; 111: 8844-8849Crossref PubMed Scopus (46) Google Scholar, Mourikis et al., 2012Mourikis P. Gopalakrishnan S. Sambasivan R. Tajbakhsh S. Cell-autonomous Notch activity maintains the temporal specification potential of skeletal muscle stem cells.Development. 2012; 139: 4536-4548Crossref PubMed Scopus (85) Google Scholar). The resulting “CHIR99021-DAPT culture” in defined N2 media (Figure 1A) was tested on multiple hiPSC lines (>10 different clones) and consistently resulted in differentiation of myoblasts into multinucleated and spontaneously contractile myotubes (Movies S1, S2, and S3; skeletal muscle cells derived from hESC [H9] and normal hiPSCs [GM01582, GM02036]). The hESC- and hiPSC-derived myotubes in CHIR99021-DAPT culture were further characterized by transmission electron microscopy. The spontaneously contracting myotubes showed a highly organized structure, including intact sarcomeres with distinct Z-lines, M-lines, and I-bands (Figures 1C and S1V). To determine the in vivo engraftment potential, we transplanted the dissociated CHIR99021-DAPT culture cells into the injured tibialis anterior (TA) muscle of NOD-Rag1nullIL2rγnull (NRG) mice. 6 weeks after transplantation, immunohistochemistry performed with two human specific antibodies (human-specific Lamin A/C and human-specific Laminin) confirmed that the transplanted human myoblasts formed extensive myofibers without tumor formation (n = 33 mice) (Figure 1D, left). Importantly, a small proportion of human nuclei (human Lamin A/C +) were also observed to express PAX7 underneath a human Laminin basal lamina, indicating that some of the transplanted cells occupied the niche of adult muscle stem cells, known as satellite cells (Figure 1D, right). In contrast, no expression of human antigens was detected in sham-transplanted control mice. To determine the presence of fusion-competent myoblasts, we re-plated the dissociated cells from the CHIR99021-DAPT culture (days 25–30). Most of the attached and surviving cells were mono-nucleated at day 2 after re-plating, and they could form multi-nucleated myotubes at day 10 after re-plating with typical striations and expression of myotube marker proteins, including DYSTROPHIN (35.55 ± 6.4% cells were positive), TITIN (37.5% ± 5.25%), and α-ACTININ (40.8% ± 9.7%, sarcomeric organization) (Figures 1E, 1F, and S1W). Isolation of fusion-competent myoblasts could pave the way for disease modeling. As shown in Figures 1E, 1F, and S1T, our CHIR99021-DAPT culture contains fusion-competent myoblasts as well as differentiated myotubes with well-organized sarcomeres. To isolate myoblasts, we tested multiple cell surface markers to facilitate fluorescence-activated cell sorting (FACS) purification of myoblasts in CHIR99021-DAPT culture. Positive selection with the NCAM (5.1H11) antibody (Webster et al., 1988Webster C. Pavlath G.K. Parks D.R. Walsh F.S. Blau H.M. Isolation of human myoblasts with the fluorescence-activated cell sorter.Exp. Cell Res. 1988; 174: 252-265Crossref PubMed Scopus (110) Google Scholar) combined with negative selection using the HNK1 antibody (Figures 2A and S2A) enriched skeletal muscle progenitor cells. This determination was based upon significantly increased expression levels of MYOD1, MYOG, and MyHC (Figures 2B, 2C, S2B, and S2C) in the NCAM+/HNK1− fraction over the NCAM− or NCAM+/HNK1+ fractions. Furthermore, single-cell qRT-PCR showed that 98% (95 out of 96) of single NCAM+/HNK1− cells had higher expression of MYOG and PAX7 than a sample of human fetal skeletal muscle and undifferentiated hESCs (Figures S2D and S2E). To identify the global mRNA profiles, we performed unbiased gene expression analysis, which showed a hierarchical clustering between the hPSC-derived NCAM+/HNK1− population and fetal skeletal muscle (18 to 19 weeks of gestation) over undifferentiated hESCs (Figure 2D). Transcripts highly enriched in the NCAM+/HNK1− fraction included key markers of skeletal muscle structure development (Millay et al., 2013Millay D.P. O’Rourke J.R. Sutherland L.B. Bezprozvannaya S. Shelton J.M. Bassel-Duby R. Olson E.N. Myomaker is a membrane activator of myoblast fusion and muscle formation.Nature. 2013; 499: 301-305Crossref PubMed Scopus (317) Google Scholar, Wang et al., 1979Wang K. McClure J. Tu A. Titin: major myofibrillar components of striated muscle.Proc. Natl. Acad. Sci. USA. 1979; 76: 3698-3702Crossref PubMed Scopus (443) Google Scholar, Wohlgemuth et al., 2007Wohlgemuth S.L. Crawford B.D. Pilgrim D.B. The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish.Dev. Biol. 2007; 303: 483-492Crossref PubMed Scopus (89) Google Scholar) and key transcription factors (L’Honoré et al., 2007L’Honoré A. Coulon V. Marcil A. Lebel M. Lafrance-Vanasse J. Gage P. Camper S. Drouin J. Sequential expression and redundancy of Pitx2 and Pitx3 genes during muscle development.Dev. Biol. 2007; 307: 421-433Crossref PubMed Scopus (69) Google Scholar, Martin et al., 1993Martin J.F. Schwarz J.J. Olson E.N. Myocyte enhancer factor (MEF) 2C: a tissue-restricted member of the MEF-2 family of transcription factors.Proc. Natl. Acad. Sci. USA. 1993; 90: 5282-5286Crossref PubMed Scopus (220) Google Scholar) (Figure 2E). Gene ontology (GO) analysis revealed statistically significant over-representation of GO terms among the upregulated genes in the NCAM+/HNK1− fraction, including those of “embryonic skeletal muscle development” (p = 4.52 × 10−22), “muscle structure system” (p = 2.10 × 10−29), and “muscle contraction” (p = 2.61 × 10−24) (Figure 2F). The gene expression array results were confirmed by qRT-PCR with primer sets for selected genes (Figure S2F). The isolated NCAM+/HNK1− population was markedly proliferative during exposure to expansion-permissive conditions for 6 weeks and could be easily expanded up to hundreds of millions of cells (a population doubling time for hESC and hiPSCs of 50.8 ± 18.5 hr and 47.9 ± 17.0 hr, respectively) (Figure S2G). The hiPSC-derived myoblasts could be successfully cryopreserved and maintained their myotube-forming potential upon thawing (Figure S2H). To test whether our myoblast specification and isolation protocol could be applied to hiPSCs derived from DMD patients, we generated patient-specific hiPSCs from five different genotypes (Table 1 and Figures S3A–S3E). In order to find transcriptional changes between NCAM+/HNK1− cells (Figure S3F) of DMD-hiPSC (GM05169, exon 4–43 deletion) and the control (healthy)-hiPSC population, we applied unbiased global transcription analysis and subsequent GO analysis (Figure 3A). The differentially upregulated genes in NCAM+/HNK1− cells of the DMD-hiPSC population (hereafter referred to as DMD-myoblasts) were largely classified into wound healing, inflammation, and signaling pathways. To validate these findings in different genotypes and mutations in DMD patient hiPSCs (Table 1), we chose a set of significantly upregulated genes in DMD-myoblasts (fold change ≥ 2-fold; corrected p value of 0.05), which included 17 over-represented genes in the five categories mentioned above, to perform qRT-PCR analysis. Using additional DMD-hiPSC lines with different mutations (two exon 3–17 deletion, one exon 5–7 duplication, and one nonsense mutation; at least three iPSC clones per genotypes), we generated myoblasts from each clone of DMD-hiPSC lines using previously mentioned protocols (Figure S1) of myogenic specification and FACS purification. No DYSTROPHIN protein was detectable in the DMD-hiPSC-derived myoblasts using western blot and immunohistochemistry (Figures S3G and S3H), whereas control hPSC-derived myoblasts showed detectable levels of DYSTROPHIN expression (Figure S3I). Furthermore, DMD-hiPSC-derived myoblasts showed distinctively different myogenic marker gene expression patterns than fibroblasts (Figure S3J). In vivo transplantation experiments with two different mouse strains, NOD-Rag1nullIL2rγnull mice (immuno-deficient healthy recipients) and NOD-SCID-IL2rγnull-mdx4Cv mice (immuno-deficient mdx mice lacking dystrophin) (Arpke et al., 2013Arpke R.W. Darabi R. Mader T.L. Zhang Y. Toyama A. Lonetree C.L. Nash N. Lowe D.A. Perlingeiro R.C. Kyba M. A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation.Stem Cells. 2013; 31: 1611-1620Crossref PubMed Scopus (72) Google Scholar), demonstrated that myogenic culture of DMD-hiPSCs could participate in muscle regeneration processes after cardiotoxin injury (Figure S3K).Table 1Details of Fibroblasts Used for Generation of DMD-hiPSC LinesID in Coriell CatalogDescriptionMutationsAge (yr)GM05169DMD (deletion)EX4-43DEL9GM05127DMD (non-sense mutation)c.5533G > T, p.E1845X18GM04327DMD (duplication)EX5-7DUP23GM03781DMD (deletion)EX3-17DEL11GM03783DMD (deletion)EX3-17DEL10 Open table in a new tab The different DMD-myoblasts each had varying levels of gene expression in the DMD qRT-PCR analysis, although all assayed genes were aberrantly expressed in DMD-myoblasts compared to control hiPSCs. In particular, all lines showed upregulated expression of BMP4 and TGFβ genes (Figure 3B). Increased levels of BMP4 and TGFβ signaling were confirmed by a significantly increased nuclear localization of phosphorylated SMAD (pSMAD 1/5 and 2/3) in DMD-myoblasts compared to control myoblasts (Figures 3C, 3D, and S3L). We also found increased protein expression levels of interleukin 6 and 8 and collagen 3 in DMD-myoblasts, whereas the expression level of collagen 1 remained unchanged (Figures 3E–3G and S3M). Interestingly, myoblasts from individual DMD-hiPSC lines showed different patterns. For example, they all had higher levels of nuclear localized phospho-SMAD 1/5 and 2/3, but expression levels of interleukin 6 and 8 and collagen 3 were varied among different DMD-hiPSC lines. Furthermore, analysis of myotube formation as measured by the fusion index (the ratio of number of nuclei inside DESMIN+ myotubes to the number of total nuclei × 100) showed that all the DMD-myoblasts from different DMD-hiPSC lines had decreased myotube formation compared to control myoblasts (p < 0.0001) (Figures 3H and 3I). To test whether this observation was specific to the DMD-myoblasts, we compared the hiPSC lines we developed from facioscapulohumeral muscular dystrophy (FSHD) and amyotrophic lateral sclerosis (ALS, C9ORF72 mutation) (Figures S3A–S3D). Myoblast cultures derived from hiPSC lines of FSHD and ALS could form myotubes with expression of disease biomarkers (Figures S3N–S3P). As shown in Movie S4, we often found spontaneously twitching cells during myogenic specification of ALS-hiPSCs, but we have not found any spontaneously contracting cells during DMD-hiPSC differentiation (n = 71 repeats). To confirm our findings, we used a previously published protocol (Shelton et al., 2014Shelton M. Metz J. Liu J. Carpenedo R.L. Demers S.P. Stanford W.L. Skerjanc I.S. Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells.Stem Cell Reports. 2014; 3: 516-529Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and showed robust myogenic specification (comparable levels to our protocol) in healthy control hPSCs (Figures S3Q and S3R). However, there were differential transcriptional profiles of DMD-hiPSCs (Figure S3S). In addition, the DMD-hiPSC-derived myoblasts showed aberrant myotube formation with many branched arms, reminiscent of another group’s study using mdx mESCs (Chal et al., 2015Chal J. Oginuma M. Al Tanoury Z. Gobert B. Sumara O. Hick A. Bousson F. Zidouni Y. Mursch C. Moncuquet P. et al.Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.Nat. Biotechnol. 2015; 33: 962-969Crossref PubMed Scopus (244) Google Scholar) (Figure S3T). Our data suggest that myoblasts of DMD-hiPSCs display transcriptional, translational, and functional in vitro phenotypes. After confirming the transcriptional and translational phenotypes of DMD-myoblasts, we hypothesized that there might be a significant correlation among the list of aberrantly expressed genes (Figure 3B). A correlation heatmap was generated using the gene-to-gene Pearson correlations for the 16 genes initially chosen from global transcriptional analysis (Figure 3J). Among the positively co-correlated genes, SPP1, BMP4, CASPASE1, BMP2, and TGFβ3 formed the clearest cluster. Furthermore, additional correlation analysis (Table 2) confirmed a statistically significant correlation (p value, 0.0211 ∼ <0.0001) between SPP1 and the other 12 genes. Interestingly, SPP1 (Osteopontin) has been shown to be a potent genetic modifier of disease severity in DMD (Pegoraro et al., 2011Pegoraro E. Hoffman E.P. Piva L. Gavassini B.F. Cagnin S. Ermani M. Bello L. Soraru G. Pacchioni B. Bonifati M.D. et al.Cooperative International Neuromuscular Research GroupSPP1 genotype is a determinant of disease severity in Duchenne muscular dystrophy.Neurology. 2011; 76: 219-226Crossref PubMed Scopus (167) Google Scholar, Zatz et al., 2014Zatz M. Pavanello R.C. Lazar M. Yamamoto G.L. Lourenço N.C. Cerqueira A. Nogueira L. Vainzof M. Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin.Neuromuscul. Disord. 2014; 24: 986-989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).Table 2Correlation between SPP Expression and Other “Potentially Culprit Genes”r95% Confidence IntervalR squaredp (Two-Tailed)p Value SummarySignificant? (Alpha = 0.05)SPP1 versus BMP20.9740.9071 to 0.99290.9486<0.0001∗∗∗∗YesSPP1 versus BMP40.98510.9461 to 0.99590.9704<0.0001∗∗∗∗YesSPP1 versus TGFB1−0.6537−0.8927 to −0.12780.42740.0211∗YesSPP1 versus TGFB2−0.8336−0.9520 to −0.49780.69480.0008∗∗∗YesSPP1 versus TGFB30.97030.8945 to 0.99190.9415<0.0001∗∗∗∗YesSPP1 versus TNFRSF210.70230.2151 to 0.90960.49320.0109∗YesSPP1 versus EGF0.4151−0.2085 to 0.79870.17230.1797nsNoSPP1 versus CASPASE10.98390.9416 to 0.99560.968<0.0001∗∗∗∗YesSPP1 versus CASPASE40.93970.7942 to 0.98330.8831<0.0001∗∗∗∗YesSPP1 versus COL1A1−0.2073−0.6981 to 0.41610.042970.518nsNoSPP1 versus COL3A10.8490.5365 to 0.95670.72080.0005∗∗∗YesSPP1 versus COL4A50.72750.2637 to 0.91810.52930.0073∗∗YesSPP1 versus COL4A60.88460.6312 to 0.96740.78260.0001∗∗∗YesSPP1 versus IL60.89930.6725 to 0.97170.8088<0.0001∗∗∗∗YesSPP1 versus IL80.2953−0.3354 to 0.74330.087220.3514nsNo" @default.
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• W2401496253 date "2016-06-01" @default.
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• W2401496253 title "Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model" @default.
• W2401496253 cites W1153484958 @default.
• W2401496253 cites W124023198 @default.
• W2401496253 cites W1564806441 @default.
• W2401496253 cites W1754645121 @default.
• W2401496253 cites W1953958400 @default.
• W2401496253 cites W1970561503 @default.
• W2401496253 cites W1971436489 @default.
• W2401496253 cites W1980845430 @default.
• W2401496253 cites W1990897270 @default.
• W2401496253 cites W1992121070 @default.
• W2401496253 cites W1992879938 @default.
• W2401496253 cites W1994971898 @default.
• W2401496253 cites W1997223407 @default.
• W2401496253 cites W1997394079 @default.
• W2401496253 cites W2003171404 @default.
• W2401496253 cites W2005703297 @default.
• W2401496253 cites W2007786482 @default.
• W2401496253 cites W2009012143 @default.
• W2401496253 cites W2009100350 @default.
• W2401496253 cites W2011354459 @default.
• W2401496253 cites W2012799716 @default.
• W2401496253 cites W2014178753 @default.
• W2401496253 cites W2015703333 @default.
• W2401496253 cites W2021440286 @default.
• W2401496253 cites W2025174672 @default.
• W2401496253 cites W2026975258 @default.
• W2401496253 cites W2037216224 @default.
• W2401496253 cites W2037438288 @default.
• W2401496253 cites W2043126651 @default.
• W2401496253 cites W2043317882 @default.
• W2401496253 cites W2055710769 @default.
• W2401496253 cites W2056431144 @default.
• W2401496253 cites W2060801754 @default.
• W2401496253 cites W2066180106 @default.
• W2401496253 cites W2066390373 @default.
• W2401496253 cites W2073998126 @default.
• W2401496253 cites W2074283287 @default.
• W2401496253 cites W2076462595 @default.
• W2401496253 cites W2087152939 @default.
• W2401496253 cites W2087786585 @default.
• W2401496253 cites W2093441343 @default.
• W2401496253 cites W2101687573 @default.
• W2401496253 cites W2106594964 @default.
• W2401496253 cites W2129171086 @default.
• W2401496253 cites W2129442835 @default.
• W2401496253 cites W2134665278 @default.
• W2401496253 cites W2137007484 @default.
• W2401496253 cites W2144073625 @default.
• W2401496253 cites W2144993965 @default.
• W2401496253 cites W2145931730 @default.
• W2401496253 cites W2156131965 @default.
• W2401496253 cites W2166059867 @default.
• W2401496253 cites W2194012442 @default.
• W2401496253 cites W4241560578 @default.
• W2401496253 cites W4380290265 @default.
• W2401496253 doi "https://doi.org/10.1016/j.celrep.2016.05.016" @default.
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