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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：
10653183
负责人：
Warren L Grayson
金额：
$ 42.42万
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/10653183
关键词：
3-Dimensional Acetylcholine Action Potentials Agrin Binding Biocompatible Materials Biophysics Blood Vessels Cell Aggregation Cell Survival Cell Therapy Cells Cellular Morphology Cholinergic Receptors Clinical Data Defect Development Economic Burden Electrospinning Engineering Fiber Fibrin Fibrosis Functional Regeneration Gene Expression Genes Genetic Growth Harvest Heparan Sulfate Proteoglycan Heterogeneity Human Immunodeficient Mouse Immunologic Deficiency Syndromes 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 Phenotype Play Population Porosity Preparation Prevention Proliferating Property Recovery of Function Regenerative capacity Reporter Role Site Skeletal Muscle Sorting Surgical incisions Testing Tissue Engineering Transplantation Traumatic injury Treatment Protocols Vascularization 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 neural neuromuscular neurotransmission novel post-transplant postsynaptic progenitor regeneration potential reinnervation 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，约占所有创伤性肌肉的40%