A functional genomics and candidate gene validation approach to identify genes regulating pancreatic development and/or stem cell fitness

功能基因组学和候选基因验证方法，用于识别调节胰腺发育和/或干细胞适应性的基因

• 批准号: 214644051
• 负责人: Professor Dr. Alexander Kleger
• 金额: --
• 依托单位: Klinik für Innere Medizin I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/214644051?language=en
• 关键词: functional; genomics; candidate; gene; validation

Pluripotent stem cells are characterized by continuous self-renewal while maintaining the potential to differentiate into cells of all three germ layers. Despite extensive knowledge in this regard, a gap remains in the understanding of the precise signaling clues governing commitment to the respective cellular lineages particularly endodermal derivate. Differentiation strategies based on the use of cytokines and small molecules already allow the generation of virtually every cell type in the brain but also the heart while e.g. endocrine and exocrine pancreatic differentiation strategies remain rather inefficient. Forward genetic screening using RNA interference techniques and large-scale compound screens may provide novel and unorthodox avenues to reach this goal also during endodermal differentiation of human pluripotent stem cells. The first part of the current proposal aims to validate the results of an already performed shRNA screen while differentiating human embryonic stem cells towards definitive endoderm. Disease- and age-associated decreases in regenerative capacity and organ maintenance could represent major factors limiting the quality of life and life expectancy. In the second part of the proposal, we aim to identify such factors limiting stem cell fitness and therefore regenerative capacity in shRNA-based reprogramming screens. To do so, we have identified Dkk3 as a gene, which limits cellular reprogramming, but also liver and hematopoietic stem cell fitness. Herein, we aim to extend these findings towards pancreatic regeneration and try to define the molecular events governing this process. Taken together, the current proposal will provide novel clues to improve pancreatic differentiation of human pluripotent stem cells. This is the perquisite of applying these techniques to cell replacement therapies in e.g. diabetic patients. On the other, we will shed light on Dkk3 a stem cell fitness limiting gene to develop potential strategies for improved organ maintenance and regeneration.

多能干细胞的特征在于持续自我更新，同时保持分化为所有三个胚层细胞的潜力。尽管在这方面有广泛的知识，但在理解控制对相应细胞谱系特别是内胚层衍生物的承诺的精确信号线索方面仍然存在差距。基于使用细胞因子和小分子的分化策略已经允许在脑中以及心脏中产生几乎每种细胞类型，而例如内分泌和外分泌胰腺分化策略仍然相当低效。使用RNA干扰技术和大规模化合物筛选的正向遗传筛选可能提供新的和非正统的途径，以达到这一目标，也在人多能干细胞的内胚层分化。当前提案的第一部分旨在验证已经进行的shRNA筛选的结果，同时将人类胚胎干细胞分化为定形内胚层。与疾病和年龄相关的再生能力和器官维持能力下降可能是限制生活质量和预期寿命的主要因素。在提案的第二部分，我们的目标是确定限制干细胞适应性的因素，从而在基于shRNA的重编程筛选中确定再生能力。为此，我们已经确定Dkk3是一种基因，它限制了细胞重编程，但也限制了肝脏和造血干细胞的适应性。在此，我们的目标是将这些发现扩展到胰腺再生，并试图定义控制这一过程的分子事件。综上所述，目前的建议将提供新的线索，以改善胰腺分化的人多能干细胞。这是将这些技术应用于例如糖尿病患者的细胞替代疗法的先决条件。另一方面，我们将阐明Dkk3干细胞健身限制基因，以开发改善器官维护和再生的潜在策略。