Engineered Asymmetric Hydrogel for Muscle Stem Cell Polarity and Fate Specification

摘要 骨骼肌干细胞移植(MUSC)是一种很有前途的治疗肌肉移植的策略。 相关的创伤和疾病。然而，MSC会自发地失去它们的“茎”和植入性。 传统2D培养的潜力，严重限制了它们在基于细胞的治疗中的适用性。这种无能为力 培养间充质干细胞进一步限制了细胞工程策略的应用，如体外基因编辑患者 派生的细胞。因此，迫切需要一种策略来维持和扩大具有治疗效力的MUSC。 体外长期的。到目前为止，MUSC培养系统概括了不对称的MUSC生态位，包括 肌纤维和基板，需要建立细胞极性，支持生理细胞分裂，并引导 命运的规范，并不存在。主动机械力在调制MUSC极性中的贡献， 分工，命运的规范也依然被忽视。我们认为，建立MUSC极性是一个 通过生化和生物物理手段支持体外长期对称扩张的要求。 为此，建议的研究将设计一个扩展自我更新的非对称设计器平台 MUSCs体外长期通过指导细胞的极性和命运规范。在目标1中，我们将最小限度地确定 通过设计不对称水凝胶来确定MUSC极性的基本不对称线索。在目标2中，我们 将确定生化促进对称细胞分裂对MUSCs长期扩增的影响 使用不对称水凝胶。抑制不对称分裂和促进对称分裂的生化策略 事业部实现MUSC的体外扩增将得到检验。在目标3中，我们将确定动态 生物力学拉伸对非对称水凝胶调节对称骨髓间充质干细胞扩张的影响。 外力对齐有丝分裂纺锤体方向和调节细胞分裂的机制 将对极化的MSC进行测试。这项研究的成功成果不仅将为 通过指导干细胞的极性和命运规范来扩大MUSCs用于细胞治疗，但也提供了 在再生细胞生物材料中建立利用干细胞极性的新范例 医药。

ABSTRACT Skeletal muscle stem cell (MuSC) transplantation is emerging as a promising strategy for treating muscle- associated trauma and diseases. However, MuSCs spontaneously lose their “stemness” and engraftment potential in conventional 2D culture, critically limiting their applicability for cell-based therapy. This inability to culture MuSCs further limits the employment of cell engineering strategies, such as ex vivo gene editing patient derived cells. Thus, there is a critical need for a strategy to maintain and expand therapeutically potent MuSC ex vivo long-term. To date, MuSC culture systems that recapitulate the asymmetric MuSC niche, consisting of myofiber and basal lamina, required to establish cellular polarity, support physiologic cell division, and guide fate specification, do not exist. The contribution of active mechanical forces in modulating MuSC polarity, division, and fate specification also remains overlooked. We believe that establishing MuSC polarity is a requirement for supporting long-term symmetric expansion ex vivo through biochemical and biophysical means. To this end, the proposed research will engineer an asymmetric designer platform for expanding self-renewing MuSCs ex vivo long-term by directing cellular polarity and fate specification. In Aim 1, we will identify minimally essential asymmetric cues for establishing MuSC polarity by engineering an asymmetric hydrogel. In Aim 2, we will determine the effects of biochemically promoting symmetric cell division on long-term expansion of MuSCs using the asymmetric hydrogel. Biochemical strategies to inhibit asymmetric division and to promote symmetric division to achieve ex vivo MuSC expansion will be tested. In Aim 3, we will determine the effects of dynamic biomechanical stretch on regulation of symmetric MuSC expansion using the asymmetric hydrogel. Mechanisms by which externally applied forces align mitotic spindle orientation and regulate cell division of polarized MuSCs will be tested. Successful outcomes of this research will not only develop a platform for expanding MuSCs for cell therapies by directing stem cell polarity and fate specification but also provide establish a new paradigm for harnessing stem cell polarity in cell-instructive biomaterials for regenerative medicine.