Abstract
Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.
Highlights
The technologies for reprogramming human somatic cells into induced pluripotent stem cells (Takahashi et al, 2007) and for inducing specific differentiated cell types are providing extraordinary opportunities for investigating mechanisms of human tissue differentiation, the molecular pathology of diseases, and therapeutic development
Gene expression was assayed in the FSHD1 and Ctrl Embryonic Stem Cell (ESC) lines originally used to develop and optimize this induction protocol to assure that induced pluripotent stem cells (iPSCs) and ESCs respond to this transgene-free myogenesis induction protocol (Figure 1—figure supplement 1B)
These studies established that Ctrl and disease iPSC and ESC lines robustly upregulated expression of PAX3, MYOD1, and MYH8 on the order of 1000-fold during S1, S2, and S3 stages of myogenic induction, validating the consistency and efficiency of the transgene-free induction protocol
Summary
The technologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) (Takahashi et al, 2007) and for inducing specific differentiated cell types are providing extraordinary opportunities for investigating mechanisms of human tissue differentiation, the molecular pathology of diseases, and therapeutic development. Less attention has been given to generation of lineage-specific human stem cells and progenitors to enable studies of tissue and organ development, genetic and epigenetic disease mechanisms, and stem cell therapeutics. The goal of our study has been to isolate and propagate myogenic stem cells from human iPSC cultures in response to gene-free myogenic induction and cell growth selection and to establish the utility of these myoblast stem cells for molecular investigations of human myogenesis and muscular dystrophies. We show that iMyoblasts generated from Facioscapulohumeral Muscular Dystrophy (FSHD) Type 1 (FSHD1), Limb-Girdle Muscular Dystrophy (LGMD) R7 and R9 (formerly LGMD2G and 2I), and Walker Warburg Syndrome (WWS) patient iPSCs model the molecular pathologies of these diseases
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