Reprogramming and Directed Differentiation of Skeletal Muscle Cells from hPSCs

摘要 在最常见的肌营养不良症中，Duchenne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 没有解药。在上一个资助期，我们确定了新的差异化和丰富战略，以 从人类诱导的多能干细胞(HiPSCs)中产生迄今最可植入的细胞，以及 CRISPR矫正策略恢复dystrophin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功能状态的理解 源自WT、DMD和CRISPR校正后的线条。这项工作将提高我们对分子的理解。 人类PAX7+干细胞和祖细胞在人类胚胎发育过程中的差异 在成年期，并告知我们有能力在这笔资金中从HiPSCs中产生最具再生能力的细胞 句号。在目标1中，我们将定义在发育过程中和从HiPSC产生的人类SMPC和SCs，并确定 祖细胞和干细胞状态在人类发育和体外衍生细胞中的功能差异 使用单细胞测序和评估SMPC和SC状态之间不同的候选通路。在……里面 目的2，我们将评估宿主微环境包括内源性PAX7细胞对干细胞的作用 嫁接能力和过渡到SC的能力，并居住在SC利基。在目标3中，我们将使用DMD和 等位基因CRISPR/Cas9 SMPC/SCs评价DMD的生物学特性、细胞生物学和功能潜能 CRISPR纠正了病态MDX-NSG和MDX-D2-NSG微环境中的细胞。这将改善我们的 对人类肌肉祖细胞和干细胞状态和意志的差异和转变的理解 在长期研究中提高我们产生能够重新填充利基的细胞的能力。

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.