Developmental Paradigms to Direct Human Endoderm into Foregut Lineage

描述(申请人提供)：这项建议的目标是了解如何有效地引导人类胚胎干细胞(HESCs)分化为不同的内胚层谱系，如肺、肝、胰腺和肠道，用于治疗用来替代患病或受损的组织。我推测，目前的ES细胞分化方案可以通过加入一个关键的前后(A-P)模式事件来极大地改进，这种模式通常发生在内胚层器官发育的早期。此外，我还假设操纵信号 负责A-P图案化将使我们能够系统地生成包括肺、甲状腺和肠道在内的各种不同的内胚层谱系。我们和其他实验室发表的研究表明，在肠管形成之前控制成纤维细胞生长因子和Wnt信号的水平对于沿着前后(A-P)轴形成内胚层图案和建立器官结构域至关重要。在hESCs中，成纤维细胞生长因子和Wnt信号通路的内胚层构型活性是保守的。根据我们的初步数据，我假设成纤维细胞生长因子和Wnt通路以两种方式协同促进人内胚层细胞的命运，第一是通过调节关键A-P决定因素的表达，第二是通过引导细胞沿A-P轴迁移。此外，我假设，通过控制成纤维细胞生长因子和Wnt信号的水平，我可以有效地将最终内胚层(DE)定向到特定的前肠谱系，包括胰腺和肝脏。拟议的实验将首次允许询问成纤维细胞生长因子和Wnt通路如何引导人类前肠指定的早期阶段，并更有效地将hESCs引导到具有治疗意义的重要组织中。此外，细胞培养系统的优势将使我能够测试成纤维细胞生长因子和Wnt信号之间的机械相互作用，这在动物模型中是很难解决的。具体目标1：确定成纤维细胞生长因子和Wnt信号如何协同作用来塑造人类内胚层和直接前肠谱系规范。特定目的2：研究HESC来源的前肠内胚层在体内的分化潜能。具体目标3：确定FGF4如何协调人体内胚层迁移。公共卫生相关性：这项建议的目标是了解如何有效地将人类胚胎干细胞(HESCs)定向分化为不同的内胚层谱系，如肺、肝、胰腺和肠道，用于治疗，以取代患病或受损的组织。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how to efficiently direct differentiation of human embryonic stem cells (HESCs) into different endoderm lineages, such as lung, liver, pancreas and intestine for therapeutic use to replace diseased or damaged tissue. I hypothesize that current ES cell differentiation protocols can be greatly improved by incorporating a critical anterior-posterior (A-P) patterning event that normally occurs during early endoderm organ development. In addition, I also hypothesize that manipulating signals responsible for A-P patterning will allow us to systematically generate the full range of different endoderm lineages including lung, thyroid and intestine. Published studies from our and other labs have demonstrated that controlling the levels of FGF and Wnt signaling prior to formation of a gut tube is critical for patterning the endoderm along the anterior-posterior (A-P) axis and for establishing the organ domains. The endoderm patterning activity of FGF and Wnt signaling pathways is conserved in HESCs. From our preliminary data I hypothesize that the FGF and Wnt pathways synergize to promote human endoderm cell fate in two ways, first by regulating expression of key A-P determinants and second by directing cell migration along the A-P axis. Furthermore I hypothesize that by manipulating the levels of FGF and Wnt signaling I can efficiently direct definitive endoderm (DE) into specific foregut lineages, including pancreas and liver. The proposed experiments will allow, for the first time, interrogation of how FGF and Wnt pathways direct the early stages of foregut specification in humans and to more efficiently direct HESCs into therapeutically important tissues. Moreover, the advantages of a cell culture system will allow me to test mechanistic interactions between FGF and Wnt signaling which are difficult to address in animal models. Specific Aim 1: Determine how FGF and Wnt signaling synergize to pattern human endoderm and direct foregut lineage specification. Specific Aim 2: Investigate the differentiation potential of HESC-derived foregut endoderm in vivo. Specific Aim 3: Determine how FGF4 coordinates human endoderm migration. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand how to efficiently direct differentiation of human embryonic stem cells (HESCs) into different endoderm lineages, such as lung, liver, pancreas and intestine for therapeutic use to replace diseased or damaged tissue.