Hydrogel biomaterials that stiffen and soften on demand reveal that skeletal muscle stem cells harbor a mechanical memory

Muscle stem cells (MuSCs) are specialized cells that reside in adult skeletal muscle poised to repair muscle tissue. The ability of MuSCs to regenerate damaged tissues declines markedly with aging and in diseases such as Duchenne muscular dystrophy, but the underlying causes of MuSC dysfunction remain poorly understood. Both aging and disease result in dramatic increases in the stiffness of the muscle tissue microenvironment from fibrosis. MuSCs are known to lose their regenerative potential if cultured on stiff plastic substrates. We sought to determine if muscle stem cells harbor a memory of their past microenvironment and if it can be overcome. We tested MuSCs in situ using dynamic hydrogel biomaterials that soften or stiffen on demand in response to light and found that freshly isolated MuSCs develop a persistent memory of substrate stiffness characterized by loss of proliferative progenitors within the first three days of culture on stiff substrates. MuSCs cultured on soft hydrogels had altered cytoskeletal organization and activity of Rho and Rac GTPase and YAP mechanotransduction pathways compared to those on stiff hydrogels. Pharmacologic inhibition identified RhoA activation as responsible for the mechanical memory phenotype, and single cell RNA sequencing revealed a molecular signature of the mechanical memory. These studies highlight that microenvironmental stiffness regulates MuSC fate and leads to MuSC dysfunction that is not readily reversed by changing stiffness. Our results suggest that stiffness can be circumvented by targeting downstream signaling pathways to overcome stem cell dysfunction in aged and disease states with aberrant fibrotic tissue mechanics. Primary skeletal muscle stem cells were isolated from the hindlimbs of young (2 mo) or aged (>24 mo) C57BL/6 mice and were subsequently cultured on poly(ethylene glycol) hydrogels with either 12 kPa or 40 kPa stiffness for 1, 3, or 7 days. A subset of cells were treated with rhosin, a RhoA inhibitor, for three days prior to collection. Cells were enzymatically detached from the hydrogels and processed for single cell sequencing according to the Parse Biosciences Evercode WT version 1 protocol.

肌肉干细胞（Muscle stem cells, MuSCs）是驻留于成年骨骼肌中、待命修复肌肉组织的特化细胞。其修复受损组织的能力会随衰老以及杜兴氏肌营养不良症等疾病显著下降，但目前学界对肌肉干细胞功能异常的核心诱因仍知之甚少。衰老与疾病均会因纤维化导致肌肉组织微环境的刚度大幅升高。已有研究表明，若在硬质塑料基质上培养肌肉干细胞，其再生潜能会丧失。本研究旨在探究肌肉干细胞是否留存有其过往微环境的记忆，以及该记忆是否可被逆转。我们利用可响应光照、按需实现软化或硬化的动态水凝胶生物材料在原位对肌肉干细胞进行测试，发现新鲜分离的肌肉干细胞会形成对基质刚度的持久性记忆，具体表现为在硬质基质上培养的前3天内增殖性祖细胞丢失。与硬质水凝胶培养的细胞相比，在软性水凝胶上培养的肌肉干细胞，其细胞骨架结构、Rho与Rac GTP酶的活性以及YAP机械转导通路均发生显著改变。通过药物抑制实验确认，RhoA的激活是导致机械记忆表型的关键因素；单细胞RNA测序（single cell RNA sequencing）则揭示了该机械记忆的分子特征。本研究证实，微环境刚度可调控肌肉干细胞的命运，并导致其功能异常，且这种异常难以通过改变基质刚度得到逆转。研究结果提示，针对下游信号通路进行干预，可规避基质刚度带来的不良影响，从而改善纤维化组织力学异常的衰老及疾病状态下的干细胞功能障碍。本研究从年轻（2月龄）或老年（>24月龄）C57BL/6小鼠的后肢中分离原代骨骼肌干细胞，随后将其分别在刚度为12 kPa或40 kPa的聚乙二醇（poly(ethylene glycol), PEG）水凝胶上培养1、3或7天。另有部分细胞在收集前，使用RhoA抑制剂rhosin处理3天。随后通过酶解法将细胞从水凝胶上解离，并按照Parse Biosciences公司的Evercode WT V1实验流程完成单细胞测序样本制备与测序。