Regulation and function of hematopoietic stem cell niches

项目概要： 造血干细胞（HSC）龛是一个复杂的结构，被认为在造血干细胞的分化过程中起着重要作用。 调节HSC的功能和数量。在生态位成分中，MSC表达 主要生态位因素的最高水平。然而，它们离体维持HSC的能力已经被证明是不稳定的。 有限到目前为止。MSC在培养中确实快速地失去主要生态位因子的表达。我们有 测试了MSC的转录机制在培养条件下改变的假设， 恢复关键转录调节因子的表达将使生态位活动恢复活力。我们 已经使用28种转录调节因子进行了遗传筛选，这些转录调节因子在高水平表达， 在体内Nestin-GFP+ niche细胞中表达水平，但在培养中下调，并鉴定了 Ostf 1、Xbp 1、Irf 3、Irf 7和Klf 7 5个基因对骨髓间充质干细胞的再生有重要作用。再生MSC（rMSC） 表现出更高的小生境因子表达和扩增鼠或人HSC的能力 与对照MSC相比。使用RNA-seq和ATAC-seq分析，我们已经鉴定了Mef 2c 作为再生过程的下游效应物。这一延续建议将评估 这一假说认为，造血干细胞是由一个小生境精心调控，以维持其数量。在 具体目标1，我们将评估5个因子的人类直系同源物在再生中的功能 人MSC。我们将在细胞培养中使用氨基酸的稳定同位素标记（SILAC）， 通过叠氮基高丙氨酸（AHA）标记以通过质谱鉴定新的可溶性因子 来源于rMSCs。我们将检测rMSCs在胚胎干细胞成熟过程中的作用 细胞来源的HSC，以促进它们在免疫缺陷小鼠中的植入。在具体目标2中， 将评估关键转录调节因子在HSC生态位活性中的作用，将我们的研究集中在 Snai 2、Ostf 1和Mef 2c，使用floxed小鼠系进行条件性缺失。在具体目标3中， 我们将评估HSC数量是否受当地生态位可用性或系统感知的调节 使用移植、局部照射和数学建模。拟议的研究将 阐明调控HSC数量的机制，并提供新的方法 进行体外扩增。

PROJECT SUMMARY: The hematopoietic stem cell (HSC) niche is a complex structure thought to play an important role in regulating HSC function and numbers. Among the niche constituents, MSCs express the highest levels of the major niche factors. Yet, their ability to maintain HSCs ex vivo has been limited thus far. MSCs indeed rapidly loose expression of major niche factors in culture. We have tested the hypothesis that MSCs’ transcriptional machinery was altered in culture conditions and that reinstating the expression of key transcription regulators would revitalize niche activity. We have carried out a genetic screen using 28 transcription regulators that were expressed at high levels in Nestin-GFP+ niche cells in vivo but downregulated in culture, and identified a combination of 5 genes (Ostf1, Xbp1, Irf3, Irf7 and Klf7) that can revitalize MSCs. Revitalized MSCs (rMSCs) exhibit higher niche factor expression and capacity to expand either murine or human HSCs compared to control MSCs. Using RNA-seq and ATAC-seq analyses, we have identified Mef2c as downstream effector of the revitalization process. This continuation proposal will evaluate the hypothesis that HSCs are finely regulated by a niche programmed to maintain their numbers. In Specific Aim 1, we will assess the function of human orthologues of the 5 factors in revitalization of human MSCs. We will use stable isotope labeling with amino acids in cell culture (SILAC) and labeling by azidohomoalanine (AHA) to identify by mass spectrometry novel soluble factors derived from rMSCs. We will test the function of rMSCs in the maturation of embryonic stem (ES) cell-derived HSCs to promote their engraftment in immunodeficient mice. In Specific Aim 2, we will evaluate the role of key transcription regulators in HSC niche activity, focusing our studies on Snai2, Ostf1 and Mef2c using conditional deletions using floxed mouse lines. In Specific Aim 3, we will assess whether HSC numbers are regulated by local niche availability or systemic sensing using transplantation, local irradiation, and mathematical modeling. The proposed studies will shed new light on mechanisms by which HSC numbers are regulated and provide new methods toward their ex vivo expansion.