Interrogating functional and molecular properties of PAX7+ putative skeletal muscle stem/progenitor cells derived from human iPSCs of healthy donors and Duchenne muscular dystrophy patients

探究源自健康捐献者和杜氏肌营养不良症患者 iPSC 的 PAX7 推定骨骼肌干/祖细胞的功能和分子特性

• 批准号: 9215162
• 负责人: Gabsang Lee
• 金额: $ 37.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9215162
• 关键词: Address; Adopted; Adult; Aging; Allogenic; Autologous; Behavior; CRISPR/Cas technology; Cachexia; Cell Line; Cell Maintenance; Cell Transplants; Cells; Child; Clinical; Clinical Trials; Conceptions; DNA Sequence Alteration; Development; Disease; Disease model; Duchenne muscular dystrophy; Dystrophin; Ectopic Expression; Embryo; Environment; Event; Extracellular Matrix; Genes; Genetic; Genetic Transcription; Healthcare Systems; Hereditary Disease; Human; In Vitro; Injury; Knowledge; Length; Life; Membrane; Mesoderm; Methodology; Molecular; Mus; Muscle; Muscle Fibers; Muscle function; Muscle satellite cell; Muscular Atrophy; Muscular Dystrophies; Mutation; Myoblasts; Myopathy; Neuromuscular Diseases; Non-Insulin-Dependent Diabetes Mellitus; PAX7 gene; Patients; Phenotype; Phospholipids; Pluripotent Stem Cells; Population; Process; Proliferating; Property; Proteins; Regenerative Medicine; Regulation; Replacement Therapy; Reporter; Reporting; Research; Role; Site; Skeletal Muscle; Stem cells; System; Techniques; Teenagers; Telomere Shortening; Time; Transplantation; Traumatic injury; Untranslated RNA; Variant; base; blastomere structure; cancer cachexia; cell type; experimental study; functional disability; human embryonic stem cell; in vivo; in vivo regeneration; induced pluripotent stem cell; innovation; loss of function; mdx mouse; motor function improvement; mouse model; muscle regeneration; myogenesis; novel; novel strategies; postnatal; programs; regenerative; repaired; satellite cell; skeletal muscle wasting; socioeconomics; stem; stemness; transcription factor

Muscle wasting, caused by aging, genetic mutations, cancan-associated cachexia, or traumatic injury, can result in significant functional impairment, and is a challenging clinical problem with a significant socioeconomic burden on our healthcare system. We have shown that functional myoblasts are readily derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), allowing us to begin to study Duchenne muscular dystrophy (DMD), the most common genetic disorder of muscle. However, we know little about i) how early myogenic events are genetically controlled during development, ii) whether embryonic PAX7 expressing myogenic stem/progenitor cells adopt postnatal `satellite-like' fate, and iii) how DMD is occurred in skeletal muscle stem/progenitor cell stage as well as their relevance for cell replacement therapy. First, using multiple genetic reporter lines to recapitulate human myogenic events, we will depict a time- course analysis of transcriptional landscape followed by `loss of function' analysis to address essential questions regarding which critical cell intrinsic/extrinsic component(s) govern the skeletal muscle specification process and stem cell maintenance. Secondly, by performing serial transplantation of human PAX7::GFP+ putative skeletal muscle stem/progenitor cells in mouse model, we will interrogate how the embryonic cells become to postnatal satellite-like cell fate. Thirdly, based on our observation on DYSTROPHIN expression in human skeletal muscle stem/progenitor cells, we will investigate stage-specific role(s) of DYSTROPHIN and its long intergenic non- coding RNAs (LincRNAs), in healthy and DMD condition. In addition, we will interrogate in vivo regeneration ability of patient-specific PAX7::GFP+ cells of genetically corrected DMD-hiPSC lines. Our proposed experiments are expected to expand and strengthen our current conception of myogenic specification events, and to accelerate a wide range of research on skeletal muscle disorders, e.g. traumatic muscle damages, genetic muscular dystrophies, neuromuscular diseases, type II diabetes and cancer-induced cachexia.

由衰老、基因突变、癌症相关恶病质或创伤性损伤引起的肌肉萎缩， 可导致显著的功能损害，并且是具有显著的 对我们的医疗系统造成了巨大的社会经济负担。 我们已经证明，功能性成肌细胞很容易从人胚胎干细胞中获得 人胚胎干细胞（hESC）和人诱导多能干细胞（hiPSC），使我们能够开始研究杜氏肌营养不良症。 营养不良（DMD），最常见的肌肉遗传性疾病。然而，我们对i）多早 ii）胚胎PAX 7表达是否受发育过程中的遗传控制， 肌源性干/祖细胞采用出生后的“卫星样”命运，和iii）DMD如何在骨骼肌中发生， 肌肉干/祖细胞阶段以及它们与细胞替代疗法的相关性。 首先，使用多个遗传报告细胞系来概括人类肌生成事件，我们将描绘一个时间- 转录景观的课程分析，然后是“功能丧失”分析，以解决基本的 关于哪些关键细胞内在/外在成分控制骨骼肌规格的问题 过程和干细胞维护。 其次，通过进行人PAX 7：：GFP+推定骨骼肌的连续移植， 干/祖细胞在小鼠模型中，我们将询问胚胎细胞如何成为出生后 卫星般的细胞命运 第三，基于我们对肌营养不良蛋白在人骨骼肌中表达的观察， 干/祖细胞，我们将研究阶段特异性作用的肌营养不良蛋白和其长的基因间非- 编码RNA（LincRNA），在健康和DMD条件下。此外，我们将询问体内再生 图2显示了遗传校正的DMD-hiPSC系的患者特异性PAX 7：：GFP+细胞的能力。 我们提出的实验有望扩大和加强我们目前的肌源性的概念， 规范事件，并加速对骨骼肌疾病的广泛研究，例如创伤性 肌肉损伤、遗传性肌营养不良症、神经肌肉疾病、II型糖尿病和癌症引起的 恶病质