Improving human pluripotent stem cell derived beta cell transplantation using genetic lineage tracing.

项目概要： 1型糖尿病（T1D）的特征在于产生胰岛素的β细胞被自身免疫性疾病破坏。 攻击目前还没有治愈方法，T1D患者依赖于通过注射提供内源性胰岛素， 但长期并发症和由于胰岛素控制不良而导致的低血糖风险仍然存在。移植 将分离的尸体胰岛移植到长期T1D患者中导致约35个月的胰岛素非依赖性， 极大地提高了生活质量。虽然这种β细胞替代疗法已经成为一种潜在的治疗方法， 对于T1D，其进展由于缺乏丰富的β细胞来源而受到阻碍。我们最近 证实了从人中大规模生产葡萄糖响应性胰岛素产生β样细胞 多能干细胞这些细胞可以纠正动物模型中的糖尿病，从而直接针对供体 缺乏这种可行的方法。然而，目前细胞治疗研究中一个很大程度上被忽视的方面 最大的努力是在移植时干细胞衍生的β细胞（sBC）的急剧和立即损失。 这是一个关键问题，如果成功解决，将大大提高虚拟化的效率。 所有目前提出递送sBC用于细胞替代疗法目的的方法，无论是裸的还是 封装的。我们的总体假设是，sBC的移植导致大比例的 这些细胞，由于几种机制，包括：（1）缺氧和营养剥夺介导的细胞死亡，（2） 转分化成其他激素表达细胞类型和（3）剩余的sBC的一部分采用较少的 功能性β细胞表型。本次资助申请的重点有两点：（1）准确全面 采用遗传谱系追踪阐明sBC在移植后的命运和（2）防止表型 通过使用mRNA组合的抗凋亡基因BCL 2的时间表达的变化和sBC死亡 我们显示生理氧适应显著提高移植物存活。特别是我们的血统 追踪分析将全面了解移植后sBC的细胞变化。我们进一步 预计将准确定义驱动它们的相关分子变化，从而可能提供 未确认的目标，以改善SBC生存。此外，我们希望提供一个有效的， 这是一种简单的方法，可以防止目前移植时发生的功能性sBC质量损失。采取 结合我们独特的专业知识，本提案中所概述的及时方法已经准备就绪 为了成功解决T1D患者细胞替代疗法的关键问题， 移植后sBC的存活和功能。

Project summary: Type one diabetes (T1D) is characterized by the destruction of insulin-producing beta cells by an autoimmune attack. Currently there is no cure available and T1D patients rely on supply of endogenous insulin via injections, but long-term complications and the risk of hypoglycemia due to poor insulin control persist. Transplantation of isolated cadaveric islets into long-standing T1D patients results in ~35 months of insulin independence, tremendously improving quality of life. While this beta cell replacement therapy has emerged as a potential cure for T1D, its advancement has been hampered by the lack of an abundant beta cell source. We have recently demonstrated the large-scale production of glucose responsive insulin producing beta-like cells from human pluripotent stem cells. These cells can correct diabetes in animal models, thus directly addressing donor shortage with this viable approach. However, a largely neglected aspect of current cell therapy research efforts is the dramatic and immediate loss of stem cell derived beta cells (sBCs) upon transplantation. This is a critical problem that, if successfully resolved, will tremendously increase the efficacy of virtually all current approaches proposing to deliver sBCs for cell replacement therapy purposes, both naked and encapsulated. Our overall hypothesis is that transplantation of sBCs results in the loss of a large proportion of these cells, due to several mechanisms, including: (1) hypoxia and nutrition deprivation mediated cell death, (2) transdifferentiate into other hormone expressing cell types and (3) that part of the remaining sBCs adopt a less functional beta cell phenotype. The focus of this grant application is two-fold: (1) accurately and comprehensively elucidate the fate of sBCs upon transplantation employing genetic lineage tracing and (2) to prevent phenotypical changes and sBC death by temporal expression of the anti-apoptotic gene BCL2 using mRNAs in combination with physiological oxygen adaption shown by us to significantly improve graft survival. Specifically, our lineage tracing analysis will comprehensively define the cellular changes of sBC upon transplantation. We further anticipate to accurately define the associated molecular changes driving them, thus likely providing unacknowledged targets to improve sBC survival thereafter. Furthermore, we except to provide an effective and simple way to prevent the loss of functional sBC mass that is currently occurring upon transplantation. Taken together, the timely approach outlined within this proposal combined with our unique expertise is poised to successfully address a critical problem of cell replacement therapy for T1D patients, the preservation of sBC survival and function immediately upon transplantation.