Ex vivo rejuvenation and expansion of muscle stem cells from aged mice

描述(申请人提供)：肌肉干细胞(MuSCs)，也被称为卫星细胞，对肌肉终生再生是必不可少的[1]。随着年龄的增长，骨骼肌质量和损伤后的再生能力逐渐下降，导致生活质量下降。 在老年人中[2]。解释衰老骨骼肌组织功能障碍的努力主要集中在与衰老相关的组织微环境的变化，限制MUSC功能的因素[3]。在我的博士后研究中，我使用组织再生的非侵入性成像分析方法证明，与年轻的MUSCs相比，预期从老年小鼠分离的MUSCs的再生能力显著降低，揭示了老年MUSCs存在以前未发现的固有干细胞缺陷。此外，我已经确定了一种新的体外策略来克服旧的MuSCs的再生功能障碍；在软的仿生水凝胶平台上用p38丝裂原相关蛋白激酶的小分子抑制剂[4]治疗旧的MuSCs，可以产生功能干细胞绝对数的扩大，并恢复它们的再生功能，恢复到年轻的MuSCs的功能。这种方法为恢复和增加MuSCs的数量提供了希望，并可以使局部自体干细胞治疗老年人的肌肉萎缩成为可能，目前临床上还没有药物治疗。我建议将以前的生物工程和干细胞生物学方面的培训与肌肉生理学和系统生物学方面的新培训相结合，以进一步研究MSC在衰老过程中的再生功能障碍及其体外救援。在目标1(K99阶段)，我将评估体外治疗的老年MuSCs能否挽救老年小鼠有缺陷的肌肉再生，增加肌肉力量，并能够响应连续的再生需求而长期恢复功能。在目标2(桥接K99/R00期)中，我将结合多参数质量细胞术(CyTOF)[5]和SPADE算法[6]结合敏感的移植试验来鉴定和比较从幼年和老年小鼠分离的MUSC亚群的再生功能，以阐明老年MUSCs中有缺陷的再生功能是同质性还是异质性的表型。在目标3(R00阶段)，我将阐明旧的MUSCs干细胞功能障碍背后的失调信号网络机制，以利用信号网络水平的系统生物学方法改进治疗[7]。这项向独立过渡的提案描述了研究和职业发展活动，包括出席会议和课程培训，这些活动将使我成为独立教职的竞争性候选人，并帮助我在生物学和干细胞老化治疗方面发展一个创新的、成功的研究计划。这些活动将由斯坦福大学的海伦·布劳博士(初级导师)、斯科特·德尔普博士(共同导师)和加里·诺兰博士(共同导师)指导，斯坦福大学是世界级的干细胞生物学研究机构。

DESCRIPTION (provided by applicant): Muscle stem cells (MuSCs), also known as satellite cells, are essential to muscle regeneration throughout life [1]. In aging, skeletal muscle mass and regenerative capacity after injury progressively decline, leading to diminished quality of life in aged individuals [2]. Efforts to explain the dysfunction of aged skeletal muscle tissue have focused on aging-related changes in tissue microenvironment factors restricting MuSC function [3]. In my postdoctoral research, I have demonstrated, using non-invasive imaging assays of tissue regeneration, that MuSCs prospectively isolated from old mice have a marked reduction in regenerative capacity relative to young MuSCs, revealing a previously undetected intrinsic stem cell defect in old MuSCs. Further, I have identified a novel ex vivo strategy to overcome the regenerative dysfunction of old MuSCs; treatment of old MuSCs maintained on a soft biomimetic hydrogel platform [4] with a small molecule inhibitor of p38 mitogen- associated protein kinase yields an expansion in absolute numbers of functional stem cells and restores their function in regeneration to that of young MuSCs. This approach offers promise for rejuvenating and increasing the numbers of MuSCs and could enable localized autologous stem cell therapy for muscle wasting in aged individuals, for which there are no pharmacologic treatments in clinical use. I propose to merge prior training in bioengineering and stem cell biology with new training in muscle physiology and systems biology to further investigate the regenerative dysfunction of MuSCs in aging and its rescue ex vivo. In Aim 1 (K99 phase), I will evaluate whether ex vivo-treated old MuSCs can rescue defective muscle regeneration and increase muscle strength in old mice and are capable of long-term rejuvenated function in response to successive regenerative demands. In Aim 2 (bridging K99/R00 phases), I will elucidate whether defective regenerative function is a homogeneous or heterogeneous phenotype in old MuSCs by combining multi-parameter mass cytometry (CyTOF) [5] and SPADE algorithm [6] analysis with sensitive transplantation assays to identify and compare the regenerative functions of MuSC sub-populations isolated from young and old mice. In Aim 3 (R00 phase), I will elucidate dysregulated signaling network mechanisms underlying the stem cell dysfunction of old MuSCs for improved therapeutic treatment using signaling network- level systems biology approaches [7]. This Transition to Independence proposal describes research and career development activities, including conference attendance and course training that will establish me as a competitive candidate for an independent faculty position and aid my development of an innovative, successful research program in the biology and treatment of stem cell aging. These activities will be mentored by Drs. Helen Blau (primary), Scott Delp (co-mentor), and Garry Nolan (co-mentor) at Stanford University, which is a world-class stem cell biology research institution.