Skeletal Stem Cell PDGFR-beta Signaling as a New Regulator of the HSC Niche

项目摘要/摘要 血小板衍生生长因子及其相应受体(α和β)调节 器官发生和组织内稳态。异常的受体信号与发育有关 综合征和成人疾病，如纤维化、心血管疾病和癌症。因此，PDGF受体 激活及其下游信号已被认为是潜在的治疗靶点。最近的报道 涉及PDGFRβ信号在调节骨髓微环境和造血干细胞中的作用 小区(HSC)维护。尽管已知骨骼干细胞(SSCs)表达PDGFRβ，但它具有调节作用 血小板衍生生长因子受体β在肝星状细胞壁龛中的表达尚不清楚。我们的目标是确定PDGFRβ在干细胞利基中的作用 维护和SSC功能。我们的中心假设是SSCs中的PDGFRβ信号调节 骨髓的HSC生态位和造血功能。凭借我们在PDGF信号和新基因方面的专业知识 方法，本项目将解决以下两个目标的中心假说：1)表征 SSc PDGFRβ信号在造血干细胞池维持和骨髓增生性肿瘤中的作用 发展和2)确定改变的PDGFRβ对干细胞/基质谱系的功能影响 发信号。在目标1中，他莫昔芬可诱导Gli1 Cre重组酶小鼠系(Gli1-Creer)将用于 有条件地诱导一个功能增强的PDGFRβ敲入等位基因或灭活内源性PDGFRb基因 Gli1+SSCs。使用WT和突变体的实验将集中于评估BM和 SSCs中PDGFRβ改变的血液。我们将把PDGFRβ突变体的骨髓移植到照射后的野生型(WT) 小鼠(反之亦然)，以确定造血细胞获得永久功能的程度 来自PDGFRβ突变环境的改变。最后，我们将确定修改后的PDGFRβ是否- JAK2-V617F突变型BM骨髓移植调节基质环境改变MPN进展 细胞。在目标2中，为了描述支持干细胞生态位的干细胞的细胞变化和功能，以及 在PDGFRβ突变体中的基质谱系，我们将使用体外干细胞试验来评估干细胞功能的变化 茎、分化和细胞因子的产生。最后，从PDGFRβ突变体的骨髓中分选出SSCs 将对照移植到WT肾胶囊内，以确定SSc PDGFRβ信号转导对肾功能的影响 骨髓形成和HSC体内定植的调控。这些项目的成果将产生新的 PDGFRβ信号介导的基质到造血干细胞小生境串扰的知识和促进 骨髓和血液系统疾病的潜在治疗靶点。

PROJECT SUMMARY/ABSTRACT Platelet-derived growth factor (PDGF) and its corresponding receptors (PDGFRα and PDGFRβ) regulate organogenesis and tissue homeostasis. Abnormal receptor signaling has been implicated in developmental syndromes and adult diseases such as fibrosis, cardiovascular disease, and cancer. Therefore, PDGF receptor activation and its downstream signaling have been proposed as potential therapeutic targets. Recent reports have implicated PDGFRβ signaling in regulating bone marrow (BM) microenvironment and hematopoietic stem cell (HSC) maintenance. Although skeletal stem cells (SSCs) are known to express PDGFRβ, a regulatory role of PDGFRβ in HSC niche is unknown. Our goal is to determine the roles of PDGFRβ in HSC niche maintenance and SSC function. Our central hypothesis is that PDGFRβ signaling in SSCs regulates the size of the HSC niche and blood-forming functions of the BM. With our expertise in PDGF signaling and novel genetic approaches, this project will address the central hypothesis with the two following aims: 1) Characterize the impact of SSC PDGFRβ signaling on the HSC pool maintenance and myeloproliferative neoplasia (MPN) development and 2) Define the functional consequences on SSCs/stromal lineages with altered PDGFRβ signaling. In Aim 1, tamoxifen-inducible Gli1 Cre recombinase mouse line (Gli1-CreER) will be used to conditionally induce a gain-of-function PDGFRβ knock-in allele or to inactivate the endogenous Pdgfrb gene in Gli1+ SSCs. Experiments using WT and mutants will focus on the evaluation of functional changes in BM and blood with altered PDGFRβ in SSCs. We will transplant BM from PDGFRβ mutants to irradiated wild type (WT) mice (or vice versa) to determine the extent at which blood forming cells acquire permanent functional alterations from the PDGFRβ mutant environment. Finally, we will determine whether altered PDGFRβ- regulated stromal environment alters MPN progression using BM transplantation with JAK2-V617F mutant BM cells. In Aim 2, in order to characterize cellular changes and functionality in HSC niche-supportive SSCs and stromal lineages in PDGFRβ mutants, we will use in vitro SSC assays to evaluate functional changes in SSC stemness, differentiation, and cytokine production. Finally, SSCs sorted from BM of PDGFRβ mutants or controls will be transplanted into WT kidney capsules to determine the impact of SSC PDGFRβ signaling on the modulation of BM formation and HSC colonization in vivo. The results of these projects will generate new knowledge of PDGFRβ signaling-mediated stroma-to-HSC niche crosstalk and facilitate the development of potential therapeutic targets on BM and hematopoietic diseases.