Mesodermal Commitment of the Definitive Hematopoietic Program

项目概要/摘要 我们研究的总体目标是了解胚胎干细胞的起源、发育和调控， 造血干细胞（HSC）的祖细胞，生血内皮细胞（HE）。本提案的重点是 使用人类多能干细胞（hPSC）和小鼠胚胎发育作为互补模型 系统，以确定必要的信号和基因的HE规范。目前，信号要求 在胚胎发育过程中，新生中胚层的HE特化仍然是一个知之甚少的过程。 我们提出的研究建立在我们最近在定向分化确定性造血干细胞方面的进展之上。 来自hPSC的祖细胞，已经确定了一个WNT依赖性中胚层群体， 通过CDX 4表达确定的造血潜能。我们现在已经确定了一个新的早期 中胚层完全依赖于视黄酸（RA）信号传导，用于产生HOXA+ HE， 谱系决定性造血潜能。由于RA是胚胎发育过程中HE功能所必需的，我们 假设hPSC衍生RA依赖性HE衍生自非常独特的中胚层亚群， 个体发育上与具有HSC独立HE潜能的中胚层不同。我们将检验这一假设 三个具体目标。在目标1中，我们将评估ALDH 1A 2+衍生的HE的生理相关性， 体外和体内。这些研究的目的是了解ALDH 1A 2+中胚层是否引起 HSC胜任HE。在目标2中，我们将定义RA依赖HE的规范的机制。客观 这些研究的目的是确定RA信号如何影响HE的发展。在目标3中，我们将确定 选择性剪接在RA依赖的确定性造血特化。这些研究的目的是 以确定早期中胚层中HE发生所必需的转录调控因子。的 这些研究的成功完成将使我们更全面地了解 造血发育这对基础生物学至关重要， 这些研究将具有临床意义，例如在体外产生广泛的HSC， 再生医学的应用我们独特的细胞和分子工具，结合我们的专业知识， 造血发育，干细胞生物学和生物信息学使我们处于一个理想的位置， 在这个领域的影响。

PROJECT SUMMARY / ABSTRACT The overall goal of our research is to understand the origin, development and regulation of the embryonic progenitor to the hematopoietic stem cell (HSC), hemogenic endothelium (HE). The focus of this proposal is to use human pluripotent stem cells (hPSCs) and mouse embryonic development as complementary model systems to identify the essential signals and genes for HE specification. Currently, the signal requirement(s) for HE specification from nascent mesoderm during embryonic development remains a poorly understood process. Our proposed studies build on our recent advances in the directed differentiation of definitive hematopoietic progenitors from hPSCs, having identified a WNT-dependent mesodermal population that harbors exclusively definitive hematopoietic potential, via CDX4 expression. We have now identified a novel subset of early mesoderm that is entirely dependent on retinoic acid (RA) signaling for the generation of HOXA+ HE with multi- lineage definitive hematopoietic potential. As RA is required for HE function during embryogenesis, we hypothesize that hPSC-derived RA-dependent HE is derived from a very unique mesodermal subset, which is ontogenically distinct from mesoderm harboring HSC-independent HE potential. We will test this hypothesis across 3 Specific Aims. In Aim 1, we will assess the physiological relevance of ALDH1A2+ derived HE, both in vitro and in vivo. The objective of these studies is to understand whether ALDH1A2+ mesoderm gives rise to HSC-competent HE. In Aim 2, we will define the mechanism the specification of RA-dependent HE. The objective of these studies is to determine how RA signaling affects HE development. In Aim 3, we will identify the role of alternative splicing during RA-dependent definitive hematopoietic specification. The objective of these studies is to define the essential transcriptional regulators within early mesoderm for the development of HE. The successful completion of these studies will provide us with a more comprehensive understanding of hematopoietic development. This is of fundamental importance to basic biology, and the insights generated from these studies will have clinical implications, such as the in vitro generation of HSCs for a wide array of regenerative medicine applications. Our unique cellular and molecular tools, combined with our expertise in hematopoietic development, stem cell biology and bioinformatics puts us in an ideal position to make a significant impact in this field.