Regenerating Vascularized and Innervated Skeletal Muscle to Treat VML Defects

再生血管化和神经支配的骨骼肌来治疗 VML 缺陷

• 批准号: 10748834
• 负责人: Warren L Grayson
• 金额: $ 0.64万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748834
• 关键词: 3-Dimensional; Agrin; Biocompatible Materials; Biophysics; Blood Vessels; Cell Aggregation; Cell Survival; Cells; Cellular Morphology; Clinical; Data; Defect; Economic Burden; Electrospinning; Engineering; Fiber; Fibrin; Gene Expression; Genes; Growth; Heterogeneity; Human; Immunodeficient Mouse; Implant; Individual; Infiltration; Insulin-Like Growth Factor I; Muscle; Muscle Cells; Muscle Fibers; Muscle satellite cell; Muscular Atrophy; Natural regeneration; Neuromuscular Junction; PAX7 gene; Proliferating; Property; Recovery of Function; Regenerative capacity; Reporter; Skeletal Muscle; Sorting; Surgical incisions; Testing; Tissue Engineering; Traumatic injury; Vascularization; effective therapy; human pluripotent stem cell; lean body mass; mouse model; muscle engineering; muscle grafts; muscle physiology; muscle regeneration; myogenesis; neural; neuromuscular; novel; post-transplant; progenitor; regeneration potential; repaired; scaffold; single-cell RNA sequencing; stem; stem cell biology; stem cells; tibialis anterior muscle; volumetric muscle loss

PROJECT SUMMARY 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.

项目概要 骨骼肌几乎占人体去脂体重的一半，约占所有创伤的 40% 伤害涉及骨骼肌损伤。这导致全球经济负担约 60 亿美元。尽管 在体积肌的临床场景中，骨骼肌具有内在的自我再生能力 肌肉的自然修复机制被淹没，再生失败。组织 使用人类骨骼肌干细胞或祖细胞与新型生物材料相结合的工程策略 具有提供有效治疗的前所未有的潜力。在这项研究中，我们建议利用生肌 分选的骨骼肌干/祖报告细胞的潜力和再生能力（PAX7::GFP+） 源自人类多能干细胞 (hPSC)。具体来说，我们假设 PAX7::GFP+ 肌原性 在电纺纤维蛋白微纤维束上生长的祖细胞会增殖，上调其表达 肌源基因并形成排列整齐的多核肌管，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恢复 VML 后的正常功能。 我们进一步假设集聚蛋白与胰岛素样生长因子-1 (IGF-1) 联合使用将 促进工程肌肉中更密集的 PAX7::GFP+ 衍生肌管的形成 移植物并能够在再生骨骼肌中形成成熟的神经肌肉接头（NMJ）。 我们将在三个具体目标中测试这些假设。在 Sp。目标1，我们将设计均匀、密实的种子 通过 (i) 静电纺丝 PAX7::GFP+ 细胞聚集成纤维蛋白微纤维束和 (ii) 用 PAX7::GFP+ 细胞接种的散装纤维蛋白涂覆微纤维束。我们将促进他们的成熟 使用生物物理刺激进入可收缩的 3D 骨骼肌组织。我们将定量评估细胞 形态、增殖、多核和肌原性分化，并利用单细胞 RNA 测序 将细胞异质性和肌源性基因表达谱与天然肌肉细胞进行比较。 在 Sp。目标 2，我们将单独和组合评估可溶性和束缚的 agrin/IGF-1 的潜力 增强 PAX7::GFP+ 细胞的增殖和肌生成。我们还将描述其影响 将这些分子束缚在修饰支架的物理化学和促肌原性特性上。在 Sp。目标 3，我们将植入 PAX7::GFP+ 衍生的肌肉移植物，无论是否可溶或系留 将 agrin/IGF-1 放入免疫缺陷小鼠胫骨前肌 (TA) 的小切口中以评估细胞 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 我们将把肌肉移植物植入 VML 缺陷中，以定量评估肌肉再生、血管和 1个月和3个月时神经浸润、成熟神经肌肉接头的形成以及功能恢复 移植后。为了成功实现这些目标，我们结合了组织领域的互补专业知识 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。