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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

基本信息

批准号：
10229561
负责人：
Warren L Grayson
金额：
$ 42.85万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-05 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10229561
关键词：
3-Dimensional Acetylcholine Action Potentials Agrin Biocompatible Materials Biophysics Blood Vessels Cell Survival Cell Therapy Cells Cellular Morphology Cholinergic Receptors Clinical Data Defect Development Economic Burden Electrospinning Engineering Fibrin Fibrosis Gene Expression Genes Genetic Growth Harvest Heparan Sulfate Proteoglycan Heterogeneity Human Immunodeficient Mouse Implant In Vitro Individual Infiltration Injury Insulin-Like Growth Factor I Intercept Kinetics Modulus Morphology Motor Neurons Mus Muscle Muscle Cells Muscle Contraction Muscle Fibers Muscle satellite cell Muscular Atrophy Myoblasts Natural regeneration Nerve Neuromuscular Junction PAX7 gene Phenotype Play Population Preparation Prevention Proliferating Property Recovery of Function Regenerative capacity Reporter Role Site Skeletal Muscle Surgical incisions Testing Tissue Engineering Transplantation Traumatic injury Treatment Protocols cell growth effective therapy functional restoration human pluripotent stem cell in vivo lean body mass monolayer mouse model muscle engineering muscle grafts muscle physiology muscle regeneration myogenesis nerve supply neuromuscular neurotransmission novel organizational structure post-transplant postsynaptic progenitor regeneration potential relating to nervous system repaired scaffold scale up single-cell RNA sequencing stem stem cell biology stem cells tibialis anterior muscle volumetric muscle loss

项目摘要

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.

骨骼肌几乎占人类瘦体重的一半，约40%的所有创伤性损伤包括骨骼肌损伤。这造成了大约60亿美元的全球经济负担。而骨骼肌具有内在的自我再生能力，在临床情况下，体积肌肉肌肉的自然修复机制不堪重负，再生失败。组织使用人骨骼肌干细胞或祖细胞结合新型生物材料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，我们建议利用肌源性分选的骨骼肌干/祖细胞报告细胞（PAX 7：：GFP+）的潜力和再生能力来源于人多能干细胞（hPSC）。具体来说，我们假设PAX 7：：GFP+肌源性在电纺纤维蛋白微纤维束上生长的祖细胞会增殖，上调其表达成肌基因，并形成对齐的多核肌管，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，聚集蛋白与胰岛素样生长因子-1（IGF-1）联合使用，促进在工程化肌肉中形成更密集的PAX 7：：GFP+衍生的肌管移植物，并使成熟的神经肌肉接头（NMJ）的再生骨骼肌的形成。我们将在三个具体目标中检验这些假设。在特殊目标1中，我们将设计均匀，密集的种子（i）将PAX 7：：GFP+细胞聚集体电纺成纤维蛋白微纤维束， (ii)用PAX 7：：GFP+细胞接种的大量纤维蛋白包被微纤维束。我们会刺激他们的成熟植入可收缩的3D骨骼肌组织。我们将定量评估细胞形态、增殖、多核化和肌源性分化，并利用单细胞RNA测序与天然肌细胞的细胞异质性和肌源性基因表达谱进行比较。在Sp. Aim 2中，我们将评估可溶性和拴系的聚集蛋白/IGF-1单独和组合的潜力。以增强PAX 7：：GFP+细胞的增殖和肌生成。我们还将描述将这些分子束缚在改性支架的物理化学和促肌生成性质上。在 Sp.目的3，我们将植入PAX 7：：GFP+衍生的肌肉移植物，其工程化有和没有可溶性或栓系的将聚集蛋白/IGF-1植入免疫缺陷小鼠的胫骨前肌（TA）的小切口中，以评估细胞增殖。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缺损的肌肉移植物，以定量评估肌肉再生、血管和 1个月和3个月时神经浸润、成熟神经肌肉接头形成和功能恢复移植后。为了成功实现这些目标，我们将联合收割机在组织切割方面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学。