Functional study of novel transcriptional regulators of hematopoietic stem cells

描述(申请人提供)：造血干细胞(HSC)是对成体干细胞进行了大量研究。这些细胞补充造血的能力是通过骨髓移植(BMT)治疗血液病的常规方法。然而，许多患者无法获得骨髓移植，因为他们缺乏合适的捐赠者。因此，临床上可行的造血干细胞的替代来源将扩大骨髓移植治疗疾病的机会。多能胚胎干细胞(P/ESC)有可能被诱使产生临床上可行的HSC，从而成为捐赠者的一种可能的替代来源。然而，在P/ESC分化过程中，真正的HSC并不会自发出现，也从未在实验上被有力地诱导出来。ESC-HSC可以通过调控同源框基因(如HOXB4和CDX4)在分化过程中的表达而从小鼠ESC分化而来。然而，这些细胞的再生潜力有限，不能忠实地重建淋巴细胞，并且显示出一种让人想起中期造血祖细胞的细胞表面表型。这些数据表明，以这种方式获得的ESC-HSC受到发育障碍的影响，可以通过适当的分子提示诱使ESC产生真正的成体样HSC。遗憾的是，目前对HSC规范和发育成熟的关键分子调控因子知之甚少。因此，我们利用Affymetrix基因芯片技术获得了小鼠胚胎第9天至成年期的HSC及其前体细胞的基因表达谱，以寻找HSC发育的新的分子调控因子。对所得数据的计算分析表明，无数新基因可能是HSC个体发育的调节因子。通过我们的分析，我们预测了RNA结合蛋白Zfp36l1，以调节主动脉-性腺-中肾(AGM)的HSC规格。这个未被研究的基因在AGM血源性内皮细胞中表达，并已知直接靶向几个已建立的HSC功能调节因子(即VEGF、Stat5、Notch1)的RNA转录产物。我们建议根据以下目的从功能上评估Zfp36l1和其他选择的候选基因在HSC发育中的作用：特定目标1.验证Zfp36l1是确定HSC规范所必需的假设。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，并研究该基因有条件地从造血谱系中缺失的小鼠，使用体外和体内HSC功能的检测。特定目标2.通过慢病毒传递对优先基因候选进行功能评估 致HSC。在这里，我们将使用慢病毒传递基因，在WBM HSC移植到致死照射的小鼠之前，将其富含，并评估这些基因影响HSC重新繁殖活动的能力。对于这些研究，我们将集中在20个基因，通过计算预测作为HSC规格的转录调节功能。通过这些目标，我们将Zfp36l1确立为HSC规范的新型调节器，并确定该工艺的其他关键调节器。这些新发现的基因中的每一个都可能最终成为P/ESC命运的导向者。 公共卫生相关性：造血干细胞是所有血液的来源，在血液病中经常表现异常。通过揭示它们的功能和发育的新的分子调控机制，我们可以更好地了解它们的异常功能，并开发治疗血液病的新疗法。

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.