PDGF-B signaling in fetal hematopoietic stem cell microenvironments

描述(由申请人提供)：我们的目标是确定胎儿造血器官中微环境线索决定造血干细胞命运选择的机制。我们假设PDGF-B信号是调节胎儿HSC巢中HSC/祖细胞平衡的主要信号通路。缺乏PDGF-B基因或其受体PDGF-R的胚胎在其造血系统中表现出异常，并表现出胎盘迷路结构完整性的丧失，胎盘迷路是发育过程中的主要造血器官。我们现在将确定缺乏PDGF-B信号是否会影响胎儿造血过程中HSC池的建立，并使HSC过早分化(目标1)。这将通过量化PDGF-B-胚胎中胎儿造血器官中的HSC和承诺的祖细胞，并通过确定与造血相关的PDGF-B的主要细胞来源来实现。我们随后将评估PDGF-B信号是直接在胎儿造血干/祖细胞中所需，还是间接在它们的壁龛中所需(目标2)。这将通过确定PDGF受体在小鼠和人类整个个体发育过程中的造血干/祖细胞亚群中的表达模式，并通过诱导组织特异性地去除造血细胞或其壁龛中的PDGF受体来实现。最后，我们将从机制上阐述PDGF-B信号如何调控造血(目标3)。这将通过研究体外PDGF-B刺激对HSC/祖细胞命运的功能后果以及通过定义PDGF-B在造血环境中激活的关键信号分子和转录程序来实现。我们已经建立了一套独特的小鼠模型和实验工具，可以在小鼠和人类的胎儿造血龛中剖析PDGF-B信号的关键成分。除了使用已建立的CRE小鼠品系进行组织特异性基因消融外，我们还开发了一种新的技术，通过在胚泡阶段利用慢病毒感染来靶向胎盘滋养层细胞特异性基因。我们希望这些研究将揭示这一众所周知的途径在调节造血干细胞命运中的一个新的、重要的作用，并开始确定在发育过程中用于造血干细胞与其生态位之间沟通的关键生态位细胞和分子线索。了解HSC的行为如何受其利基环境的影响，对于建立成功的HSC体外扩增方法或从多能细胞中产生具有治疗目的的HSC至关重要。 公共卫生相关性：该项目的目标是研究PDGF-B信号如何调节胎儿造血龛中的造血干/祖细胞平衡。我们将使用组织特异性基因去除的小鼠模型以及人胎儿造血组织来评估PDGF-B信号通路作为造血细胞及其生态位中关键调节和/或标记的重要性。这些研究将开始揭开用于胎儿造血干细胞定位的独特分子线索，最终可能使我们能够改进用于治疗目的的造血干细胞/祖细胞培养方法。

DESCRIPTION (provided by applicant): Our goal is to define the mechanisms by which microenvironmental cues in fetal hematopoietic organs dictate hematopoietic stem cell fate choices. We hypothesize that PDGF-B signaling is a major signaling pathway that regulates HSC/progenitor balance in fetal HSC niches. Embryos that lack the PDGF-B gene or its receptor PDGF-R-¿ display abnormalities in their hematopoietic system and exhibit loss of structural integrity of the placental labyrinth, a major hematopoietic organ during development. We will now define whether the lack of PDGF-B signaling compromises the establishment of the HSC pool during fetal hematopoiesis and subjects HSCs to premature differentiation (Aim 1). This will be accomplished by quantifying HSCs and committed progenitors in fetal hematopoietic organs in PDGF-B-/- embryos, and by defining the main cellular source of PDGF-B relevant for hematopoiesis. We will subsequently assess whether PDGF-B signaling is required directly in fetal hematopoietic stem/progenitor cells or indirectly in their niches (Aim 2.) This will be achieved by defining the expression pattern of PDGF-receptors in hematopoietic stem/progenitor subsets in mice and humans throughout ontogeny, and by inducing tissue specific ablation of PDGF-receptors in hematopoietic cells or in their niches. Finally, we will address mechanistically how PDGF-B signaling regulates hematopoiesis (Aim 3). This will be accomplished by investigating the functional consequences of in vitro PDGF-B stimulation on HSC/progenitor fate and by defining the key signaling molecules and transcriptional programs that PDGF-B activates in a hematopoietic context. We have established a unique collection of mouse models and experimental tools that allow dissection of the key components in PDGF-B signaling in fetal hematopoietic niches both in mice and humans. In addition to using established Cre mouse strains for tissue specific gene ablation, we have developed a novel technology for placental trophoblast specific gene targeting by using lentiviral infection at the blastocyst stage. We hope that these studies will uncover a new, important role for this well known pathway in regulating hematopoietic stem cell fates, and begin to define the key niche cells and molecular cues that are utilized for communication between HSCs and their niche during development. Understanding how the behavior of the HSC is influenced by its niche will be essential for the establishment of successful in vitro methods for HSC expansion or generation of functional HSCs from pluripotent cells for therapeutic purposes. PUBLIC HEALTH RELEVANCE: The goal of this project is to investigate how PDGF-B signaling regulates hematopoietic stem/progenitor cell balance in fetal hematopoietic niches. We will use mouse models with tissue specific gene ablation as well as human fetal hematopoietic tissues to assess the significance of PDGF-B signaling pathway as a key regulator and/or a marker in hematopoietic cells and their niche. These studies will begin to unravel unique molecular cues utilized in fetal hematopoietic stem cell niches that may ultimately allow us to improve the methods for culturing hematopoietic stem/progenitor cells for therapeutic purposes.