Regulation and function of hematopoietic stem cell niches

项目总结： 造血干细胞(HSC)生态位是一种复杂的结构，被认为起着重要的作用 在调节HSC的功能和数量方面。在利基成分中，MSCs表达 最高水平的主要生态位因素。然而，他们在体外维持造血干细胞的能力一直是 到目前为止还很有限。MSCs确实在培养中迅速松散了主要利基因子的表达。我们有 测试了MSCs的转录机制在培养条件下发生改变的假设 恢复关键转录调节因子的表达将重振利基活动。我们 已经使用28个高表达的转录调控因子进行了基因筛查 体内Nestin-GFP+NICE细胞的水平，但在培养中下调，并确定了一种组合 有5个基因(Ostf1、XBP1、IRF3、IRF7和Klf7)可以激活MSCs。重振旗鼓的MSCs(RMSCs) 表现出更高的利基因子表达和扩增鼠或人HSCs的能力 与对照组间充质干细胞相比。利用RNA-SEQ和ATAC-SEQ分析，我们已经确定了MEF2C 作为振兴进程的下游效应者。这份延续提案将评估 假设肝星状细胞被编程为维持其数量的利基所精细调控。在……里面 具体目标1，我们将评估人类同源基因在振兴中的作用 人类间充质干细胞。我们将在细胞培养中使用稳定的氨基酸同位素标记(SILAC)和 叠氮高丙氨酸(AHA)标记质谱法鉴定新的可溶性因子 来源于rMSCs。我们将测试rMSCs在胚胎干细胞成熟过程中的作用。 细胞来源的造血干细胞促进其在免疫缺陷小鼠中的植入。在具体目标2中，我们 将评估关键转录调节因子在HSC生态位活动中的作用，重点是我们的研究 SNAI2、Ostf1和MEF2C使用带花的小鼠品系进行条件删除。在具体目标3中， 我们将评估HSC数量是否受本地生态位可用性或系统感知的影响 使用移植、局部照射和数学建模。拟议的研究将 揭示了调节HSC数量的机制，并提供了新的方法 它们的体外扩增。

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.