Myogenic differentiation of muscular dystrophy-specific induced pluripotent stem cells for use in drug discovery

Abstract

Human induced pluripotent stem cells (iPSCs) represent a scalable source of potentially any cell type for disease modeling and therapeutic screening. We have a particular interest in modeling skeletal muscle from various genetic backgrounds; however, efficient and reproducible methods for the myogenic differentiation of iPSCs have not previously been demonstrated. Ectopic myogenic differentiation 1 (MyoD) expression has been shown to induce myogenesis in primary cell types, but the same effect has been unexpectedly challenging to reproduce in human iPSCs. In this study, we report that optimization of culture conditions enabled direct MyoD-mediated differentiation of iPSCs into myoblasts without the need for an intermediate step or cell sorting. MyoD induction mediated efficient cell fusion of mature myocytes yielding multinucleated myosin heavy chain-positive myotubes. We applied the same approach to dystrophic iPSCs, generating 16 iPSC lines from fibroblasts of four patients with Duchenne and Becker muscular dystrophies. As seen with iPSCs from healthy donors, within 36 hours from MyoD induction there was a clear commitment toward the myogenic identity by the majority of iPSCs in culture (50%-70%). The patient iPSC-derived myotubes successfully adopted the skeletal muscle program, as determined by global gene expression profiling, and were functionally responsive to treatment with hypertrophic proteins insulin-like growth factor 1 (IGF-1) and wingless-type MMTV integration site family, member 7A (Wnt7a), which are being investigated as potential treatments for muscular dystrophy in clinical and preclinical studies, respectively. Our results demonstrate that iPSCs have no intrinsic barriers preventing MyoD from inducing efficient and rapid myogenesis and thus providing a scalable source of normal and dystrophic myoblasts for use in disease modeling and drug discovery.

Keywords: Differentiation; Induced pluripotent stem cells; Muscular dystrophy; Skeletal muscle.

Figure 1.

Expression of MyoD induces efficient myogenic differentiation of iPSCs. (A): Schematic sketch of iPSC generation and myogenic differentiation protocols. (B): Phase images of MyoD-infected FTc01-C2 iPSC line (derived from healthy donor) on indicated days following Dox treatment. (C): MyoD-infected FTc01-C2 iPSC line stained for MYHC (green) and MyoD (red) untreated (lower panels) or on day 6 post-Dox treatment (upper panels). Nuclei were stained with DAPI (blue). Scale bar = 100 µm. (D): Top panel shows highlighted area in (C) at higher resolution. Lower panel shows highlighted area above. Arrows point to examples of striated pattern of staining for MYHC. Scale bar = 50 µm. (E): iPSCs stained for MYHC (green) and Myogenin (red) untreated (lower panels) or on day 6 post-Dox treatment (upper panels). Nuclei were stained with DAPI (blue). Scale bar = 200 µm. (F): Efficiency of myogenic differentiation without induction of MyoD (−Dox) or 6 days post-MyoD induction (+Dox) determined as percentage of Myogenin⁺ nuclei of total number of nuclei. Average of triplicate samples (three wells; n ≥ 100 each from at least 3 fields) and standard deviations are shown from a representative experiment. (G): Fusion index determined as the percentage of nuclei occurring in myotubes (>2 nuclei) stained with MYHC. Average of triplicate samples (three wells; n ≥ 100 each from at least three fields) and standard deviations are shown from a representative experiment. Abbreviations: bFGF, basic fibroblast factor; DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; FBS, fetal bovine serum; HS, horse serum; iPSCs, induced pluripotent stem cells; MYHC, myosin-heavy chain; MyoD, myogenic differentiation 1; RF, reprogramming factor; ROCKi, ρ-associated protein kinase 1 inhibitor; rtTA, reverse tetracycline-controlled transactivator; SMC4, small molecule cocktail 4.

Figure 2.

Generation of DMD- and BMD-specific iPSC lines. (A): Real-time reverse transcription polymerase chain reaction expression analyses of pluripotency genes OCT4 and SOX2 and reprogramming transgene (WPRE viral element) in indicated induced pluripotent stem cell (iPSC) lines, H1 human embryonic stem cells, and uninfected and/or infected (with reprogramming virus, day 4) fibroblasts. (B): Flow cytometry analysis of indicated undifferentiated iPSC lines for expression of SSEA4 and TRA-1-81. (C): Karyotype analysis of GM05112-M5.1 iPSCs at passage 16 showing normal genome. (D): Immunofluorescence staining of GM05169-M20.3 iPSCs for pluripotency markers Nanog, Oct4, TRA-1-60, and TRA-1-81. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. (E): In vitro trilineage differentiation potential of GM05112-M5.1 iPSCs. Immunofluorescence staining for markers of neural stem cells (Nestin), α-SMA, and definitive endoderm (Sox17) is shown in red. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. (F): In vivo trilineage differentiation potential of GM05169-M20.3 iPSCs. Histological sections of teratoma derived from GM05169-M20.3 iPSCs. Arrows point to areas of interest: neuroectoderm (left panel), endoderm (glands; middle), mesoderm (muscle; right). Scale bar = 200 µm. Abbreviations: BMD, Becker muscular dystrophy; DAPI, 4′,6-diamidino-2-phenylindole; DMD, Duchenne muscular dystrophy; Nanog, Nanog homeobox; Oct4, POU class 5 homeobox 1 (POU5F1/Oct4); SMA, α-smooth muscle Actin; Sox2, SRY (sex determining region Y)-box 2; Sox17, SRY (sex determining region Y)-box 17; SSEA4, stage-specific embryonic antigen-4; TRA, tumor-related antigen.

Figure 3.

MyoD-induced myogenic differentiation of Duchenne muscular dystrophy (DMD)-specific iPSCs (GM05112-M5.1). (A): Phase images of DMD-specific GM05112-M5.1 iPSCs on indicated days following Dox treatment. Day 0 refers to iPSCs 1 day after seeding in seeding media and before adding Dox. Numbers on top represent seeding cell densities (cells per cm²). Scale bar = 1,000 µm. (B): Immunofluorescence staining for NCAM (red) and MyoD (green) 8 days following incubation of iPSCs with or without Dox. Nuclei were stained with DAPI (blue). Scale bar = 200 µm. (C): Flow cytometry analysis of NCAM (CD56), CD44, and CD29 expression in MyoD-infected GM05112-M5.1 iPSCs cultured without Dox (purple) or with Dox for 36 hours (blue) and human primary myoblasts (red). Control (black line) is unstained pooled cells. Numbers on the right reflect percentage of indicated cells relative to total cell population. (D): Immunofluorescence staining for MYHC (green) and nuclei (blue). Cells were seeded at indicated densities and cultured for 5 days in media containing 15% FBS and Dox or 5% SR and Dox before switching to differentiation media (5% HS) for 3 more days. Scale bar = 200 µm. (E): Immunofluorescence staining for MYHC (green), Myogenin (red), and nuclei (blue). Cells were cultured with (left) or without Dox (right). Scale bar = 200 µm. (F): Left panel shows iPSC-derived multinucleated myotube stained for MYHC (green), MyoD (red), and nuclei (blue). Right panel shows enclosed region of interest displaying striated pattern of staining. Scale bar = 50 µm. (G): Efficiency of myogenic differentiation with or without Dox treatment. Average of triplicate samples (three wells; n ≥ 100 each from at least three fields) and standard deviations are shown from a representative experiment. (H): Fusion index. Average of triplicate samples (3 wells; n ≥ 100 each from at least 3 fields) and standard deviations are shown from a representative experiment. Abbreviations: FBS, fetal bovine serum; DAPI, 4′,6-diamidino-2-phenylindole; Dox, doxycycline; iPSCs, induced pluripotent stem cells; MYHC, myosin-heavy chain; MyoD, myogenic differentiation 1; NCAM, neural cell adhesion molecule; SR, serum replacement.

Figure 4.

MyoD mediates direct and rapid induction of the myogenic program in dystrophic iPSCs. (A–F): Gene expression analysis by real-time reverse transcription polymerase chain reaction of untreated (day 0) and Dox-treated MyoD-infected GM05112-M5.1 iPSCs (days 3, 4, 5, 6, and 8). (A): Expression of pluripotency marker NANOG, total MYOD1, and endogenous MYOD1. (B): Expression of mesoderm marker T (Brachyury) and skeletal muscle specification genes PAX3 and PAX7. (C): Expression of skeletal muscle markers MYH1, MYOG, DES, TNNC, and RYR1. (D): Expression of DMD as detected using TaqMan probes specific to exons 63 and 64 or exons 44 and 45. Note that parental fibroblasts of GM05112-M5.1 iPSCs have a deletion of exon 45 of the DMD gene. (E, F): Expression of components of the Wnt signaling pathways: indicated Wnt genes (E) and Wnt receptors Frizzled (FZD) genes (F). (G–I): Genome-wide gene expression analysis (Affymetrix Human Genome U133 Plus 2.0) of MyoD-infected GM05112-M5.1 iPSCs on days 0 (iPSCs-3), 3, and 8 post-Dox treatment, human primary myoblasts (undifferentiated and as differentiated myotubes), and undifferentiated iPSCs from healthy donors (iPSCs-1 and iPSCs-2). (G): The Pearson correlation coefficients for all possible sample pairs were visualized in a correlation matrix as a heat map. (H): Data for the top 100 genes (unselected; see supplemental online Table 2) with highest expression levels in primary myotubes were extracted for all the samples and visualized as a heat map. (I): Genes upregulated (>fourfold) in the Dox-treated iPSCs (day 8) relative to the undifferentiated GM05112-M5.1 iPSCs were subjected to Ingenuity Pathway Analysis to reveal the most significant associated functions. Abbreviations: DES, Desmin; DMD, dystrophin; Dox, doxycycline; Endo-MYOD1, endogenous MYOD1; iPSCs, induced pluripotent stem cell; MYH1, myosin, heavy chain 1, skeletal muscle, adult; MYOD1, myogenic differentiation 1; MYOG, Myogenin; Nanog, Nanog homeobox; Pax3, paired box 7; Pax7, paired box 7; RYR1, Ryanodine receptor 1, skeletal; TNNC1, Troponin C type 1; WNT, wingless-type MMTV integration site family, member.

Figure 5.

IGF-1 and Wnt7a induce hypertrophy in DMD- and Becker muscular dystrophy (BMD)-specific myotubes. Human primary myotubes and dystrophic iPSC-derived myotubes (day 8 post-doxycycline addition) were treated with either formulation control, 100 ng/ml Wnt7a, or 100 ng/ml IGF-1 for 2 days. Cells were stained with myosin-heavy chain and 4′,6-diamidino-2-phenylindole, and images were analyzed by Axiovision software for measurement of fiber diameter (n = 100 fibers per treatment). (A): Representative images of primary myotubes (lower panels) and DMD-specific GM05112-M5.1 iPSC-derived myotubes treated as indicated. Scale bar = 200 µm. (B–E): Fiber diameter measurements for human primary myotubes (B) and DMD-specific GM05112-M5.1 (C), DMD-specific GM05169-M20.3 (D), and BMD-specific GM05081-C3 (E) iPSC-derived myotubes. Bars show median values with interquartile ranges. ∗∗∗∗, p < .0001. Abbreviations: DMD, Duchenne muscular dystrophy; IGF-1, insulin-like growth factor 1; iPSC, induced pluripotent stem cell; Wnt7a, wingless-type MMTV integration site family, member 7A.