Understanding Pancreatic Progenitors for Diabetes Cell-Replacement Therapy

描述(申请人提供)：糖尿病是一个日益严重的全球健康问题。1型和2型糖尿病都会导致产生胰岛素的β细胞逐渐丧失。我们的长期目标是开发β细胞替代策略，以克服糖尿病患者的胰岛素缺乏。为了实现这一目标，我们开发了新的、更安全的方法，利用化学物质和遗传因素将患者成纤维细胞转化为诱导多能干细胞(IPSCs)。使用为人类胚胎干细胞(HESCs)开发的方案，这些IPSCs可以分化为多潜能的胰腺前体细胞，即β细胞的前体细胞。然而，我们对胰腺前体细胞的基本生物学的了解仍然很初级，这为开发有效的策略来扩增和进一步分化这些治疗用的前体细胞提供了障碍。从我们自己和其他实验室的研究中发现的证据表明，胰腺前体细胞是异质性的，可能由具有不同生理功能的亚群组成。然而，这些亚群之间的分子和功能差异却知之甚少。目前还不清楚发育中的胰腺中的哪个亚群(S)能够产生功能性的β细胞，以及特定的亚群在多大程度上能够自我更新。从人类胚胎干细胞分化而来的胰腺前体细胞的生理功能和自我更新特性更是知之甚少。我们的目标是在这项提案中解决这些关键问题。我们假设体内的胰脏祖细胞包含不同的亚群：(I)共同的多能胰脏祖细胞，负责生成所有三种胰腺谱系；(Ii)不同的谱系特异性祖细胞，负责生成一到两个特定的胰腺谱系。基于这一假设，这一提议的总体目标是定义胰腺祖细胞 在分子特征和生理功能方面，以及确定自我更新的机制。我们预计，我们的研究将产生以下结果。目的1揭示在胚胎发育和hESC分化培养过程中存在不同的胰腺祖细胞亚群。这将确定能够产生功能细胞的祖细胞亚群(S)。目的为进一步�分化为�细胞寻找合适的祖细胞亚群(S)的细胞表面标志物，为祖细胞扩增的研究奠定基础。目的3将阐明不同祖细胞亚群自我更新的机制，这对于开发新的、有效的策略以扩大胰岛前体细胞用于β细胞替代治疗至关重要。此外，大量产生人胰腺前体细胞的能力将为研究这些细胞的生物学提供一种新的方法，以补充小鼠的遗传学方法。总的来说，拟议的研究将带来新的发现，以填补关键 我们目前对人类胰腺发育的了解存在差距。

DESCRIPTION (provided by applicant): Diabetes mellitus is an escalating global health problem. Both Type 1 and Type 2 diabetes lead to the gradual loss of insulin-producing beta cells. Our long-term goal is to develop beta cell replacement strategies to overcome the insulin deficiency in diabetic patients. To achieve this goal, we have developed novel, safer approaches to convert patient fibroblasts to induced pluripotent stem cells (iPSCs) using chemicals together with genetic factors. These iPSCs can be differentiated into multipotent pancreatic progenitors, the precursors to beta cells, using protocols developed for human embryonic stem cells (hESCs). However, our understanding of the basic biology of pancreatic progenitor cells is still rudimentary, which presents hurdles in the development of effective strategies to expand and further differentiate these progenitor cells for therapy. Evidence emerged from our own studies and from those of other laboratories suggests that pancreatic progenitor cells are heterogeneous, and likely consist of subpopulations with different physiological functions. Yet, molecular and functional distinctions between these subpopulations are poorly understood. It is unclear which subpopulation(s) in the developing pancreas is(are) capable of generating functional beta cells, and to which extent a specific subpopulation is capable of self-renewal. Even less is known about the physiological functions and self-renewal properties of pancreatic progenitor cells differentiated from hESCs. We aim to address these critical questions in this proposal. We hypothesize that the pancreatic progenitor cell compartment in vivo contains heterogeneous subpopulations: (i) a common multipotent pancreatic progenitor responsible for generating all three pancreatic lineages; and (ii) distinct lineage-specific progenitors responsible for generating one or two specific pancreatic lineages. Based on this hypothesis, the overall objective of this proposal is to define pancreatic progenitor subpopulations in terms of their molecular characteristics and physiological functions, as well as to identify mechanisms for self-renewal. We anticipate that our study will yield the following outcomes. Aim 1 will reveal the presence of distinct pancreatic progenitor subpopulations both during embryo development and in hESC differentiation culture. This will identify the progenitor subpopulation(s) capable of generating functional cells. Aim 2 will identify cell surface markers for enrichment of the appropriate progenitor subpopulation(s) for further � differentiation into � cells, and set the stage for studies on expansion of progenitor cells. Aim 3 will elucidate the mechanism of self-renewal of distinct progenitor subpopulations, which are critical for the development of novel, effective strategies to expand pancreatic progenitors for beta cell replacement therapy. Additionally, the ability to generate a large quantity of human pancreatic progenitor cells will provide a new way to study the biology of these cells to complement mouse genetics approaches. Broadly, the proposed research will lead to novel findings to fill in critical gaps in our current knowledge of human pancreatic development.