Characterizing transient hematopoietic multipotent and megakaryocyte progenitor cells during postnatal development

摘要 胎儿造血具有不同于成人造血的特性，解释了 婴儿骨髓增生性疾病和白血病。成人型造血干细胞(HSCs)是在 小鼠胚胎9.5-E11.5天的腹主动脉-性腺-中肾区域的主要动脉定植于 胎肝(FL)和胎儿骨髓(BM)，并在一个生物体的一生中保留在成年BM中。以前的研究 假设胎儿向成人的造血转变是成人型造血干细胞逐渐成熟的过程。 然而，有两种胎儿型造血祖细胞(HPC)起源于卵黄囊造血(e7.5和 也定植于FL和胎儿BM，与成熟的HSCs重叠，是主要的贡献者 胎儿发育过程中的造血作用。越来越多的证据表明，这种转变是一个综合的过程 胎儿型HPC的减少和成人型HSC的成熟。然而，细胞和分子机制 在很大程度上，对造血转化的调控仍是未知的。 绿色荧光蛋白(GFP)在Ctnnal1-GFP报告小鼠中的表达被证明是特异性标记 年轻成人的造血干细胞，但不包括下游多能祖细胞(MPPs)或巨核细胞祖细胞 (MKPS)。除了新生小鼠的HSC外，我还发现GFP标记了MPP1、MPP2和MkPs的一个子集。这些 GFP-MPP1、MPP2和MkPs在出生后4周逐渐下降，直至消失。AS 成体HSC成熟发生在这一时期，GFP亚群代表新的发育受限的HPC 具有未知的来源、功能和调节机制。体外研究表明，GFP MPP1、MPP2 MKP与GFP-HPC不同，表现出胎儿型HPC的许多特性。例如, 尽管GFP和GFP-MPP2s均可分化为单核细胞、粒细胞和巨核细胞，但GFP 与胎儿HPC一样，MPP2s偏向于单核细胞和Mk分化，而GFP-MPP2s偏向于 粒细胞分化。与GFP-MKP产生的MKs相比，GFP MKP产生的MKP更小、更低 倍体MKS类似于胎儿BM中发现的MKS。我假设Ctnnal1-GFP MPP1、MPP2和MkP是 在表型和功能上与其Ctnnal1-GFP-对应基因不同，它们来自于胎儿型HPC 独立于成年的造血干细胞，并代表能够启动婴儿的新的暂住人口 血液病。我打算通过(1)充分描述细胞的生物学和功能来解释这一假说 GFP MPP1、MPP2和MKPS；以及(2)确定它们的来源以及它们是否来自胎儿类型 HPC独立于成体HSCs。为此，我将使用三种小鼠模型：CtnNA1-GFP小鼠，用于体外和 流式细胞术实验，CtnNA1-GFP/rosa26-td番茄小鼠移植，CDH5Cre-ERT/Ctnal1- 用GFP/rosa26-td番茄小鼠确定GFP细胞的来源。这项研究的成功不仅有助于 了解胎儿到成人的造血转换机制，也可以作为假说的模型 与婴儿/儿童白血病发展有关的一代人。

Abstract Fetal hematopoiesis has properties distinct from adult hematopoiesis, explaining the unique features of infant myeloproliferative disorders and leukemia. Adult-type hematopoietic stem cells (HSCs) are generated in the major arteries in the aorta-gonad-mesonephros region at embryonic day (E)9.5-E11.5 in mice, colonize the fetal liver (FL) and fetal bone marrow (BM), and remain in adult BM for an organism’s lifetime. Previous studies hypothesized that the fetal-to-adult hematopoietic transition is a gradual maturation process of adult type HSCs. However, two fetal-type hematopoietic progenitors (HPCs) originate from the yolk sac hematopoiesis (E7.5 and E8.5) that also colonize the FL and fetal BM, overlap with maturing HSCs, and are the primary contributors to hematopoiesis during fetal development. Increased evidence suggests this transition is a combined process of declining fetal-type HPCs and maturation of adult-type HSCs. However, the cellular and molecular mechanisms regulating the hematopoietic transition are still largely unknown. Green fluorescent protein (GFP) expression in Ctnnal1-GFP reporter mice was shown to specifically label HSCs in young adults, but not downstream multipotent progenitors (MPPs) or megakaryocyte progenitors (MkPs). In addition to HSCs in neonatal mice, I found GFP labels a subset of MPP1, MPP2 and MkPs. These GFP+ HPCs gradually decline until absent by week 4 post-birth, while GFP- MPP1, MPP2, and MkPs remain. As adult HSC maturation occurs during this period, GFP+ subsets represent novel developmentally restricted HPCs with unknown origin, function, and regulatory mechanisms. In vitro study demonstrates that GFP+ MPP1, MPP2 and MkPs are distinct from their GFP- counterparts and display many properties of fetal type HPCs. For example, although both GFP+ and GFP- MPP2s differentiate to monocytes, granulocytes and megakaryocytes (Mk), GFP+ MPP2s are biased to monocyte and Mk differentiation like fetal HPCs, while GFP- MPP2s are biased to granulocytic differentiation. Compared to Mks produced by GFP- MkPs, GFP+ MkPs produce smaller, lower ploidy Mks similar to Mks found in fetal BM. I hypothesize that Ctnnal1-GFP+ MPP1, MPP2, and MkPs are phenotypically and functionally distinct from their Ctnnal1-GFP- counterparts, are derived from fetal type HPCs independent of adult HSCs, and represent novel transient populations that are capable of initiating infant hematological diseases. I intend to address this hypothesis by (1) fully characterizing the biology and function of GFP+ MPP1, MPP2, and MkPs; and (2) determining where they arise and if they are derived from fetal type HPCs independent of adult HSCs. To do so, I will use three mouse models: Ctnna1-GFP mice for in vitro and flow cytometry experiments, Ctnna1-GFP/Rosa26-tdTomato mice for transplantations, and Cdh5Cre-Ert/Ctnnal1- GFP/Rosa26-tdTomato mice to determine the origin of GFP+ cells. Success of this study will not only help to understand the mechanism of fetal to adult hematopoietic transition, but also serve as a model for hypothesis generation related to the development of infant/pediatric leukemia.