Regenerating Vascularized and Innervated Skeletal Muscle to Treat VML Defects

骨骼肌几乎占人类瘦体重的一半，约占所有创伤的40% 损伤涉及骨骼肌损伤。这导致了大约60亿美元的全球经济负担。而当 在体积肌的临床方案中，骨骼肌具有内在的自我再生能力 肌肉丧失(VML)当肌肉的自然修复机制被压垮时，再生就会失败。组织 人骨骼肌干/祖细胞与新型生物材料结合的工程策略 具有前所未有的潜力来提供有效的治疗方法。在这项研究中，我们建议利用肌源性 分离的骨骼肌干/祖报告细胞(PAX7：：GFP+)的潜能和再生能力 来源于人类多能干细胞(HPSCs)。具体地说，我们假设PAX7：：GFP+肌源性 生长在电纺纤维微纤维束上的祖细胞将增殖，上调其表达 肌源性基因和形成排列的多核肌管组装成3D肌肉移植物。这些 工程移植物将用于再生骨骼肌组织，并在VML后恢复正常功能。 我们进一步假设，集聚蛋白与胰岛素样生长因子-1(IGF-1)的联合使用将 促进工程肌肉中PAX7：：GFP+衍生肌管的形成 移植并使再生骨骼肌中形成成熟的神经肌肉连接(NMJ)。 我们将在三个具体目标上检验这些假设。在Sp.目标1，我们将设计制服，密集播种 (I)电纺PAX7：：GFP+细胞聚集体进入纤维蛋白微纤维束的骨骼肌移植 (Ii)用PAX7：：GFP+细胞接种的散体纤维蛋白包覆微纤维束。我们将促进他们的成熟 利用生物物理刺激转化为可收缩的3D骨骼肌组织。我们将对细胞进行定量评估 形态、增殖、多核和肌源性分化，并利用单细胞RNA测序 比较天然肌细胞的细胞异质性和肌源性基因表达谱。 在Sp.目的2，我们将单独和联合评价可溶性和拴系聚集素/IGF-1的潜能。 促进PAX7：：GFP+细胞的增殖和成肌作用。我们还将描述以下方面的影响 将这些分子捆绑在改性支架的物理化学和促肌原特性上。在……里面 SP.目的3，我们将植入PAX7：：GFP+来源的肌肉移植物，含有和不含可溶性或栓系 集聚蛋白/胰岛素样生长因子-1进入免疫缺陷小鼠胫前肌的细胞评估 生存、整合和再生潜力。我们将使用这些数据来优化工程骨骼 我们将植入VML缺损处的肌肉移植物，以定量评估肌肉再生、血管和 神经浸润、成熟神经肌肉接头的形成和1个月和3个月的功能恢复 移植后。为了成功地实现这些目标，我们结合了组织方面的互补专业知识 工程学、干细胞生物学、生物材料、VML小鼠模型和骨骼肌生理学。

Skeletal muscle makes up almost half of the human lean body mass and approximately 40% of all traumatic injuries involve skeletal muscle damage. This results in a global economic burden of roughly $6 billion. While skeletal muscle possesses an intrinsic self-regeneration capacity, in clinical scenarios of volumetric muscle loss (VML) where the muscle's natural repair mechanisms are overwhelmed, regeneration fails. Tissue engineering strategies using human skeletal muscle stem or progenitor cells combined with novel biomaterials have unprecedented potential to provide effective therapies. In this study, we propose to harness the myogenic potential and regenerative capacity of sorted skeletal muscle stem/progenitor reporter cells (PAX7::GFP+) derived from human pluripotent stem cells (hPSCs). Specifically, we hypothesize that PAX7::GFP+ myogenic progenitors grown on electrospun fibrin microfiber bundles will proliferate, upregulate their expression of myogenic genes and form aligned, multi-nucleated myotubes assembled into 3D muscle grafts. These engineered grafts will be used to regenerate skeletal muscle tissue and restore normal function following VML. We further hypothesize that the use of agrin in combination with insulin-like growth factor-1 (IGF-1) will promote the formation of more densely packed PAX7::GFP+ derived myotubes in the engineered muscle grafts and enable the formation of mature neuromuscular junctions (NMJs) in the regenerating skeletal muscle. We will test these hypotheses in three Specific Aims. In Sp. Aim 1, we will engineer uniform, densely seeded skeletal muscle grafts by (i) electrospinning PAX7::GFP+ cell aggregates into the fibrin microfiber bundles and (ii) coating the microfiber bundles with PAX7::GFP+ cell-seeded bulk fibrin. We will stimulate their maturation into contractile 3D skeletal muscle tissues using biophysical stimulation. We will quantitatively evaluate cell morphology, proliferation, multi-nucleation, and myogenic differentiation and utilize single-cell RNA-sequencing to compare the cellular heterogeneity and myogenic gene expression profiles with that of native muscle cells. In Sp. Aim 2, we will evaluate the potential of soluble and tethered agrin/IGF-1 individually and in combination to enhance the proliferation and myogenesis of PAX7::GFP+ cells. We will also characterize the effects of tethering these molecules on the physicochemical and pro-myogenic properties of the modified scaffolds. In Sp. Aim 3, we will implant PAX7::GFP+ derived muscle grafts engineered with and without soluble or tethered agrin/IGF-1 into small incisions into the tibialis anterior (TA) muscle of immunodeficient mice to assess cell survival, integration, and regenerative potential. We will use these data to optimize the engineered skeletal muscle grafts that we will implant into VML defects to quantitatively assess muscle regeneration, vascular and neural infiltration, the formation of mature neuromuscular junctions, and functional recovery at 1 and 3 months post-transplantation. To successfully accomplish these aims, we combine complementary expertise in tissue engineering, stem cell biology, biomaterials, murine models of VML, and skeletal muscle physiology.