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

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. Overall design: Single cell transcriptional analysis of induced myogenic progenitors (iMPCs) grown on electrospun PCL substrates with or without Matrigel

骨骼肌（Skeletal muscle）是一类高度有序且具备再生能力的组织，其稳态维持主要依赖于肌肉干细胞（muscle stem cells）的激活与分化。构建包含自我更新型成体肌肉干细胞与对齐肌管的体外骨骼肌分化程序一直以来颇具挑战。本研究旨在利用诱导性肌源性祖细胞（induced myogenic progenitor cells, iMPCs）——一种由骨骼肌干细胞、祖细胞与分化细胞构成的异质性培养体系——工程化制备可自我再生并产生对齐肌管的仿生骨骼肌构建体。研究人员借助静电纺丝（electrospinning）技术制备了聚己内酯（polycaprolactone, PCL）基底，通过调控PCL纤维取向实现了iMPCs向对齐肌管的分化。所构建的体系包含高度有序的多核肌管与自我更新的肌肉干细胞，且基质胶（Matrigel）的添加可增强该体系中细胞的分化能力。此外，本研究通过单细胞RNA测序（single cell RNA sequencing, scRNA-seq）证实，iMPCs来源的构建体能够忠实再现阶梯式肌源性分化程序。值得注意的是，当构建体经肌坏死性损伤试剂处理后，自我更新的肌肉干细胞可快速分化为对齐肌管，这一过程与体内骨骼肌修复过程高度相似。综上，本研究报道了一种新型体外系统，该系统可模拟肌源性再生与肌纤维对齐过程，可作为研究肌发生、构建肌营养不良疾病模型或开展药物筛选的平台。
总体实验设计：对在添加或不添加基质胶的静电纺丝PCL基底上培养的诱导性肌源性祖细胞（iMPCs）进行单细胞转录组分析。