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A self-renewing biomimetic skeletal muscle construct engineered using induced myogenic progenitor cells

ABSTRACT: Skeletal muscle is a highly organized and regenerative tissue that maintains its homeostasis primarily by activation and differentiation of muscle stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self-renewing adult muscle stem cells and aligned myotubes has been challenging. Here, we set out to engineer a biomimetic skeletal muscle construct that can self-regenerate and produce aligned myotubes using induced myogenic progenitor cells (iMPCs), a heterogeneous culture consisting of skeletal muscle stem, progenitor and differentiated cells. Utilizing electrospinning, we fabricated polycaprolactone (PCL) substrates that enabled iMPC-differentiation into aligned myotubes by controlling PCL fiber orientation. Newly-conceived constructs contained highly organized multinucleated myotubes in conjunction with self-renewing muscle stem cells, whose differentiation capacity was augmented by Matrigel supplementation. Additionally, we demonstrate using single cell RNA sequencing (scRNA-seq) that iMPC-derived constructs faithfully recapitulate a step-wise myogenic differentiation program. Notably, when the constructs were subjected to a damaging myonecrotic agent, self-renewing muscle stem cells rapidly differentiated into aligned myotubes, akin to skeletal muscle repair in vivo. Taken together, we report on a novel in vitro system that mirrors myogenic regeneration and muscle fiber alignment, and can serve as a platform to study myogenesis, model muscular dystrophies or perform drug screens.

ORGANISM(S): Mus musculus

PROVIDER: GSE222162 | GEO | 2023/09/27

REPOSITORIES: GEO

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Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.

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A self-renewing biomimetic skeletal muscle construct engineered using induced myogenic progenitor cells

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