Reprogramming and Directed Differentiation of Skeletal Muscle Cells from hPSCs

摘要 在最普遍的肌营养不良症，杜氏肌营养不良症（DMD），反复肌肉 退化和再生导致肌肉卫星细胞（SC）功能障碍和/或衰竭， 无法治愈在上一个融资期，我们确定了新的差异化和丰富化战略， 从人类诱导多能干细胞（hiPSC）中产生迄今为止最可移植的细胞，以及 CRISPR矫正策略恢复肌营养不良蛋白适用于60%的患者。这项工作的下一阶段是 现在专注于提高我们对骨骼肌祖细胞（SMPC）功能状态的理解 来源于wt、DMD和CRISPR校正的品系。这项工作将提高我们对分子生物学的理解。 以及人PAX 7+干细胞和祖细胞在人胎儿发育中的差异 通过成年期，并告知我们有能力从hiPSC中产生最多的再生细胞， 期在目标1中，我们将定义在发育中和从hiPSC产生的人SMPC和SC，并鉴定 在人类发育和体外衍生细胞中祖细胞和SC状态之间的功能差异 使用单细胞测序和评估SMPC和SC状态之间不同的候选途径。在 目的2：探讨宿主微环境（包括内源性PAX 7细胞）对干细胞增殖的影响。 移植和能力过渡到SC和居住在SC利基。在目标3中，我们将利用DMD， 等基因CRISPR/Cas9 SMPC/SC，以评估DMD的规格、细胞生物学和功能潜力， CRISPR纠正了患病mdx-NSG和mdx-D2-NSG微环境中的细胞。这将改善我们的 了解人类肌肉祖细胞和干细胞状态之间的差异和转变， 在长期研究中提高我们产生能够重新填充小生境的细胞的能力。

ABSTRACT In the most prevalent muscular dystrophy, Duchenne Muscular Dystrophy (DMD), repeated muscle degeneration and regeneration leads to muscle satellite cell (SC) dysfunction and/or exhaustion and there is no cure. In the previous funding period, we have identified novel differentiation and enrichment strategies to generate the most engraftable cells to date from human induced pluripotent stem cells (hiPSCs), as well as a CRISPR correction strategy to restore dystrophin applicable to 60% of patients. The next phase of this work is now focused on improving our understanding of the functional status of skeletal muscle progenitors (SMPCs) derived from wt, DMD and CRISPR corrected lines. This work will improve our understanding of the molecular and differences between human PAX7+ stem cells and progenitor cells across human fetal development through adulthood, and inform our ability to generate the most regenerative cells from hiPSCs in this funding period. In Aim 1, we will define human SMPCs and SCs arising in development and from hiPSCs and identify functional differences between progenitor and SC states across human development and in vitro derived cells using single cell sequencing and evaluation of candidate pathways different between SMPC and SC states. In Aim 2, we will evaluate the role of the host microenvironment including endogenous PAX7 cells on stem cell engraftment and ability to transition to SCs and reside in the SC niche. In Aim 3, we will utilize DMD and isogenic CRISPR/Cas9 SMPCs/SCs to evaluate specification, cell biology and functional potential of DMD and CRISPR corrected cells in diseased mdx-NSG and mdx-D2-NSG microenvironments. This will improve our understanding of differences and transitions between human muscle progenitor and stem cell states and will improve our ability to generate cells capable of repopulating the niche in long term studies.