Functional study of novel transcriptional regulators of hematopoietic stem cells

描述（由申请人提供）：造血干细胞（HSC）是被广泛研究的成体干细胞。这些细胞补充造血功能的能力被常规利用于通过骨髓移植治疗血液病。然而，由于缺乏合适的供体，许多患者无法获得BMT。因此，临床可行的造血干细胞的替代来源将扩大BMT治疗疾病的途径。多能干细胞和胚胎干细胞（P/ESC）可能被诱导产生临床可行的造血干细胞，因此代表了一种可能的替代供体来源。然而，真正的HSC不会在P/ESC分化过程中自发出现，也从未在实验中被诱导过。ESC- hsc可以在小鼠ESC分化过程中通过操纵同源盒基因表达（如HoxB4和Cdx4）产生。然而，这些细胞具有有限的再生潜力，不能忠实地重建淋巴样细胞，并且显示出与妊娠中期造血祖细胞相似的细胞表面表型。这些数据表明，以这种方式衍生的ESC- HSC存在发育障碍，并且可以在适当的分子提示下诱导ESC产生成骨样HSC。不幸的是，目前对HSC规范和发育成熟的关键分子调节因子知之甚少。因此，为了鉴定新的HSC发育的分子调控因子，我们使用Affymetrix基因芯片技术获得了小鼠胚胎第9天至成年期HSC及其前体的基因表达谱。结果数据的计算分析暗示了无数的新基因作为假定的HSC个体发生的调节因子。我们的分析预测了RNA结合蛋白Zfp36l1在主动脉-性腺-中肾（AGM）中调节HSC规格。这个未被充分研究的基因在AGM造血内皮中表达，已知直接靶向几种已建立的HSC功能调节因子的RNA转录本（即VEGF， Stat5, Notch1）。我们建议根据以下目的对Zfp36l1和其他选择的候选基因在HSC发育中的作用进行功能评估：测试假设Zfp36l1是确定HSC规范所必需的。为此，我们将评估异位Zfp36l1在分化P/ESC中诱导造血承诺的能力，并通过体外和体内HSC功能测定，研究该基因从造血谱系中有条件删除的小鼠。具体目标2。通过慢病毒传递功能评估优先的候选基因

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSC) are heavily studied adult stem cells. The ability of these cells to replenish hematopoiesis is exploited routinely to treat hematologic disease via bone marrow transplantation (BMT). However, many patients lack access to BMT because they lack suitable donors. Thus, an alternative source of clinically viable HSC would expand access to BMT for the treatment of disease. Pluripotent and embryonic stem cells (P/ESC) could potentially be coaxed to generate clinically viable HSC and thus represent one possible alternative source for donors. However, bona fide HSC do not spontaneously emerge during P/ESC differentiation and have never been robustly induced experimentally. ESC-HSC can emerge from murine ESC by manipulating homeobox gene expression (e.g. HoxB4 and Cdx4) during their differentiation. However, these cells have limited repopulating potential, do not faithfully reconstitute lymphoid cells, and display a cell surface phenotype reminiscent of mid-gestation hematopoietic progenitors. These data suggest that ESC- HSC derived in this manner suffer from a developmental block and that ESC could be coaxed to yield bone fide adult-like HSC with the appropriate molecular cues. Unfortunately, little is currently known about the key molecular regulators of HSC specification and developmental maturation. Thus, to identify novel molecular regulators of HSC development, we acquired the gene expression profiles of HSC and their precursors from mouse embryonic day 9 through adulthood using Affymetrix gene chip technology. Computational analyses of the resulting data implicates a myriad of novel genes as putative regulators of HSC ontogeny. The RNA- binding protein, Zfp36l1, was predicted by our analyses to regulate HSC specification in the aorta-gonads- mesonephros (AGM). This understudied gene is expressed in the AGM hemogenic endothelium and known to directly target the RNA transcripts of several established regulators of HSC function (i.e. VEGF, Stat5, Notch1). We propose to functionally evaluate a role for Zfp36l1 and other select gene candidates in HSC development according to the following aims: Specific Aim 1. Test the hypothesis that Zfp36l1 is required for definitive HSC specification. Towards this aim, we will assess the ability of ectopic Zfp36l1 to induce hematopoietic commitment in differentiating P/ESC and study mice in which this gene is conditionally deleted from the hematopoietic lineage using assays for in vitro and in vivo HSC function. Specific Aim 2. Functionally evaluate prioritized gene candidates via lentiviral delivery to HSC. Here, we will use lentivirus to delivery genes to enriched WBM HSC prior to their transplantation into lethally irradiated mice and assess the ability of these genes to impact HSC repopulating activity. For these studies, we will focus on 20 genes computationally predicted to function as transcriptional regulators of HSC specification. Via these aims, we will establish Zfp36l1 as a novel regulator of HSC specification and identify additional critical regulators of ths process. Each of these newly identified genes may ultimately serve as directors of P/ESC fate. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells, the source of all blood, often behave aberrantly in hematologic disease. By uncovering novel molecular regulators of their function and development, we can achieve a better understanding of their aberrant function and develop novel therapies to treat hematologic disease.