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.

项目摘要 骨骼肌几乎占人类瘦体重的一半，大约40％ 伤害涉及骨骼肌损伤。这导致全球经济负担约为60亿美元。尽管 在体积肌肉的临床情况下，骨骼肌具有内在的自我再生能力 损失（VML），其中肌肉的自然修复机制不堪重负，再生失败。组织 使用人类骨骼肌茎或祖细胞结合新生物材料的工程策略 具有提供有效疗法的空前潜力。在这项研究中，我们建议利用肌原性 排序的骨骼肌茎/祖细胞的潜在和再生能力（PAX7 :: GFP+） 源自人多能干细胞（HPSC）。具体来说，我们假设PAX7 :: GFP+肌原性 在电纺纤维蛋白超细纤维束上生长的祖细胞会增殖，上调其表达 肌原基因并形成组装成3D肌肉移植物的多核肌管。这些 工程的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，使用Agrin与胰岛素样生长因子1（IGF-1）结合使用将 在工程肌肉中促进更密集的Pax7 :: GFP+衍生的肌管的形成 移植物并能够形成再生骨骼肌中成熟的神经肌肉连接（NMJS）。 我们将以三个特定目标来检验这些假设。在sp。 AIM 1，我们将设计制服，密集的种子 （i）静电纺丝PAX7 :: GFP+细胞聚集到纤维蛋白微生物束和 （ii）将超细纤维束与PAX7 :: GFP+细胞种子纤维蛋白涂层。我们将刺激他们的成熟 使用生物物理刺激进入收缩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序列 比较细胞异质性和肌源性基因表达谱与天然肌肉细胞的表达。 在sp。 AIM 2，我们将分别和组合评估可溶性和束缚的Agrin/IGF-1的潜力 为了增强PAX7 :: GFP+细胞的增殖和肌发生。我们还将表征 将这些分子绑在修饰支架的物理化学和促肌原性上。在 sp。 AIM 3，我们将植入以有或不可溶解的或束缚的肌肉移植物+衍生的肌肉移植 Agrin/igf-1进入胫骨前（TA）肌肉的小切口，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的骨骼 我们将植入VML缺陷的肌肉移植物，以定量评估肌肉再生，血管和 神经浸润，成熟神经肌肉连接的形成和1和3个月的功能恢复 移植后。为了成功实现这些目标，我们结合了组织中的互补专业知识 工程，干细胞生物学，生物材料，VML的鼠模型和骨骼肌生理。