Kim Inseon, Lee Seunghun S, Ghosh Adhideb, Ferguson Stephen J, Bar-Nur Ori
Laboratory of Regenerative and Movement Biology, Department of Health Sciences and Technology, ETH Zurich, Schwerzenbach 8603, Switzerland.
Institute for Biomechanics, Department of Health Sciences and Technology, ETH Zurich, Zurich 8092, Switzerland.
Adv Funct Mater. 2023 Sep 27;34(1). doi: 10.1002/adfm.202300571. eCollection 2024 Jan.
Skeletal muscle represents a highly organized tissue that primarily regenerates by myogenic stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self-renewing muscle stem cells and aligned myotubes is considered challenging. This study presents the engineering of a biomimetic 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, polycaprolactone (PCL) substrates are fabricated to facilitate iMPC-differentiation into aligned myotubes by controlling PCL fiber orientation. Newly-conceived constructs contain organized multinucleated myotubes alongside self-renewing stem cells, whose differentiation capacity is augmented by Matrigel supplementation. Furthermore, this work utilizes single-cell RNA-sequencing (scRNA-seq) to demonstrate that iMPC-derived constructs faithfully recapitulate a step-wise myogenic differentiation program. Notably, when subjected to a damaging myonecrotic agent, self-renewing stem cells rapidly differentiate into aligned myotubes within the constructs, akin to skeletal muscle repair in vivo. Finally, this study demonstrates that the iMPC derivation protocol can be adapted to engineer human myoblast-derived muscle constructs containing aligned myotubes, showcasing potential for translational applicability. Taken together, this work reports a novel in vitro system that mirrors myogenic regeneration and skeletal muscle alignment for basic research and regenerative medicine.
骨骼肌是一种高度组织化的组织，主要通过肌源性干细胞进行再生。模拟包含自我更新的肌肉干细胞和排列整齐的肌管的体外骨骼肌分化程序被认为具有挑战性。本研究展示了一种仿生肌肉构建体的工程化，该构建体能够利用诱导性肌源性祖细胞（iMPC）进行自我再生并产生排列整齐的肌管，iMPC是一种由骨骼肌干细胞、祖细胞和分化细胞组成的异质培养物。利用静电纺丝技术，制备聚己内酯（PCL）底物，通过控制PCL纤维取向促进iMPC分化为排列整齐的肌管。新构建的结构包含有组织的多核肌管以及自我更新的干细胞，补充基质胶可增强其分化能力。此外，这项工作利用单细胞RNA测序（scRNA-seq）来证明iMPC衍生的构建体忠实地重现了逐步的肌源性分化程序。值得注意的是，当受到损伤性肌坏死剂作用时，自我更新的干细胞会在构建体内迅速分化为排列整齐的肌管，类似于体内骨骼肌的修复。最后，本研究表明iMPC衍生方案可用于构建含排列整齐肌管的人成肌细胞衍生的肌肉构建体，展示了其转化应用的潜力。综上所述，这项工作报道了一种新的体外系统，可反映肌源性再生和骨骼肌排列，用于基础研究和再生医学。
