Functional study of novel transcriptional regulators of hematopoietic stem cells

新型造血干细胞转录调控因子的功能研究

• 批准号: 8386114
• 负责人: SHANNON L MCKINNEY-FREEMAN
• 金额: $ 8.75万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386114
• 关键词: 3' Untranslated Regions; Adult; Alleles; Aorta; Binding; Blood; Bone Marrow; Bone Marrow Transplantation; Candidate Disease Gene; Cell Ontogeny; Cell physiology; Cell surface; Cells; Coculture Techniques; Cues; Data; Data Set; Derivation procedure; Development; Disease; Donor person; Dorsal; Ectopic Expression; Embryo; Endothelium; Fetal Liver; Gene Chips; Gene Delivery; Gene Expression; Gene Expression Profile; Genes; Germ Layers; Goals; Gonadal structure; Growth; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Histocytochemistry; Homeobox Genes; In Situ; Indium; Laboratories; Lentivirus Vector; Lymphoid Cell; Mesonephric structure; Messenger RNA; Molecular; Molecular Profiling; Mus; Open Reading Frames; Patients; Phenotype; Play; Pluripotent Stem Cells; Pregnancy; Process; RNA; RNA-Binding Proteins; Role; Site; Source; Specific qualifier value; Stem Cell Development; Subfamily lentivirinae; Sum; Surface; Technology; Testing; Therapeutic; Time; Transcript; Transgenes; Translations; Transplantation; Vascular Endothelial Growth Factors; Work; adult stem cell; bone; cell type; embryonic stem cell; gene correction; in vitro Assay; in vivo; irradiation; novel; peripheral blood; progenitor; reconstitution; stem cell biology; stem cell differentiation; stem cell fate; stem cell fate specification; stem cell therapy

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.

描述（由申请人提供）：造血干细胞（HSC）是大量研究的成体干细胞。这些细胞补充造血的能力被常规地用于通过骨髓移植（BMT）治疗血液病。然而，许多患者无法获得BMT，因为他们缺乏合适的供体。因此，临床上可行的HSC的替代来源将扩大BMT用于治疗疾病的途径。多能干细胞和胚胎干细胞（P/ESC）可能被诱导产生临床上可行的HSC，因此代表了一种可能的供体替代来源。然而，真正的HSC在P/ESC分化过程中不会自发出现，并且从未在实验中被稳健地诱导。ESC-HSC可以通过在其分化期间操纵同源框基因表达（例如HoxB 4和Cdx 4）从鼠ESC中出现。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，并显示出妊娠中期造血祖细胞的细胞表面表型。这些数据表明，以这种方式衍生的ESC-HSC遭受发育阻滞，并且ESC可以被诱导产生具有适当分子线索的真正成人样HSC。不幸的是，目前对HSC特化和发育成熟的关键分子调节因子知之甚少。因此，为了确定新的HSC发育的分子调控因子，我们使用Affyssin基因芯片技术获得了从小鼠胚胎第9天到成年的HSC及其前体的基因表达谱。计算分析所得数据暗示了无数的新基因作为HSC个体发育的假定调节因子。通过我们的分析预测，RNA结合蛋白Zfp 36 l1在性腺-中肾（AGM）中调节HSC特化。该未充分研究的基因在AGM造血内皮中表达，并且已知其直接靶向HSC功能的几种已建立的调节剂（即VEGF、Stat 5、Notch 1）的RNA转录物。我们建议根据以下目标对Zfp 36 l1和其他选择的候选基因在HSC发育中的作用进行功能评估：具体目标1。检验Zfp 36 l1是确定HSC规格所需的假设。为此，我们将评估异位Zfp 36 l1诱导分化P/ESC中造血定型的能力，并使用体外和体内HSC功能测定法研究该基因从造血谱系中有条件地缺失的小鼠。具体目标2。通过慢病毒递送对优先考虑的候选基因进行功能评估 到HSC。在此，我们将使用慢病毒将基因递送至富集的WBM HSC，然后将其移植至致死性照射的小鼠中，并评估这些基因影响HSC重建活性的能力。对于这些研究，我们将集中在20个基因的计算预测功能作为转录调控HSC规格。通过这些目标，我们将建立Zfp 36 l1作为HSC规范的一种新型调节剂，并确定该过程的其他关键调节剂。这些新发现的基因中的每一个都可能最终成为P/ESC命运的导演。 公共卫生相关性：造血干细胞，所有血液的来源，经常在血液病中表现异常。通过揭示其功能和发育的新分子调节因子，我们可以更好地了解其异常功能，并开发治疗血液病的新疗法。