HEMATOPOIETIC STEM CELL BIOLOGY

造血干细胞生物学

• 批准号: 6681484
• 负责人: DAVID M. BODINE
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6681484
• 关键词: bone marrow; bone marrow transplantation; cell differentiation; cell growth regulation; colony stimulating factor; cytokine receptors; gene expression; gene therapy; genetic mapping; green fluorescent proteins; growth factor; hematopoietic stem cells; laboratory mouse; microarray technology; nucleic acid sequence; polymerase chain reaction; receptor expression; transfection /expression vector

The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self- renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. In addition, cells highly enriched for HSC can generate cardiac myocytes when injected into the healthy tissue surrounding a myocardial infarct or when mobilized into the peripheral blood with cytokines (e.g. G-CSF and SCF). Others have shown that enriched populations of HSC can give rise to vascular, skeletal muscle, hepatic and neuronal cells. Project 1 will examine stem cell biology. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We hypothesized that the same hematopoietic stem cells that repopulated the bone marrow were responsible for the regeneration of cardiac myocytes after experimental myocardial infraction (MI). To test this hypothesis, we transplanted mice with bone marrow cells marked with a retrovirus vector containing the GFP gene. These mice then underwent a coronary artery ligation and stem and progenitor cells were mobilized into the peripheral blood with G-CSF and SCF. The infarct region was repopulated with new cells that contain the identical retrovirus markers found in peripheral blood cells. These studies are ongoing, but we conclude that the new cardiac myocytes are the progeny of cells that also give rise to peripheral blood cells. Specific Aim 1.2: We hypothesized that the repopulation of the injured regions of the heart was due to engraftment and expansion of new cells as opposed to fusion of a primitive cell with surrounding cardiac cells. To test this hypothesis we have isolated cells from the regenerating infarct and used in situ hybridization to demonstrate that the cells express proteins in primitive cardiac cells and are diploid, arguing against the fusion hypothesis. Future studies will involve mice genetically deficient in HSC to see whether G-CSF/SCF treatment can repopulate an infarct region. Specific Aim 1.3: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. To test this hypothesis, we have isolated the ?side population? stem cells from mouse skeletal muscle cultures and used cDNA subtraction to generate a library of sequences expressed in these cells. The muscle stem cell transcripts will be compared to those we have identified previously in a cDNA subtraction library of HSC. We will select transcripts common to both libraries for analysis in transgenic mice. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Project 2 will examine these problems separately. Project 2: Gene therapy for the hemoglobinopathies Specific aim 2.1: We have evidence that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells. We hypothesize that these high levels of receptor will result in a higher frequency of human HSC transduction. We begun to test this hypothesis by simultaneously transducing human HSC with a control GALV pseudotyped vector and either RD114 or FeLV-C pseudotyped vectors for transplantation into fetal sheep. Peripheral blood and bone marrow cells from the the first 5 sheep chimeras showed greater than 10 fold higher levels of transduction with RD114 or FeLV-C pseudotyped vectors compared to GALV pseudotyped vectors. This work is on going. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have generated stable retroviruses with this construct which will be evaluated in the mouse b-thalassemia model.

造血部分的研究集中在干细胞的基本生物学以及使用干细胞作为细胞和基因治疗的车辆上。造血干细胞（HSC）是罕见的自我更新细胞群，会导致外周血中的所有细胞，使其成为遗传性造血疾病的基因替代疗法的理想车辆。另外，当注射心肌梗死的健康组织或动员到周围血液中时，高度富集HSC的细胞会产生心肌细胞，例如G-CSF和SCF。其他人则表明，富集的HSC种群会引起血管，骨骼肌，肝和神经元细胞。项目1将检查干细胞生物学。项目1：造血干细胞的生物学特异性目标1.1：我们假设在实验性心肌违规后（MI）后，同一造血的造血干细胞负责心肌细胞的再生。为了检验这一假设，我们用带有含GFP基因的逆转录病毒载体的骨髓细胞移植小鼠。然后，这些小鼠接受了冠状动脉连接，茎和祖细胞用G-CSF和SCF动员到外周血中。梗塞区域用新细胞重新植入，其中包含外周血细胞中发现的相同逆转录病毒标记。这些研究正在进行中，但我们得出的结论是，新的心肌细胞是细胞的后代，也会引起周围血细胞。具体目标1.2：我们假设心脏受伤区域的重生是由于新细胞的植入和扩展，而不是与周围心脏细胞的原始细胞融合在一起。为了检验该假设，我们从再生梗塞中有分离的细胞，并原位杂交用于证明细胞在原始心脏细胞中表达蛋白质，并且是二倍体，反对融合假设。未来的研究将涉及HSC遗传缺陷的小鼠，以查看G-CSF/SCF治疗是否可以重新填充梗塞区域。特定目标1.3：我们假设在HSC和从骨骼肌分离的干细胞中表达的特定基因负责维持未分化的状态。为了检验这一假设，我们已经隔离了？小鼠骨骼肌培养物的干细胞并利用cDNA减法产生在这些细胞中表达的序列库。将将肌肉干细胞转录本与我们先前在HSC的cDNA减法库中鉴定的肌肉转录本进行比较。我们将选择两个库共有的成绩单以在转基因小鼠中进行分析。最近已显示出基因转移到HSC可以治愈严重的合并免疫缺陷，这表明HSC基因治疗可以应用于更常见的疾病。我们想开发用于镰状细胞疾病的基因疗法。但是，当前基因转移到HSC的水平太低，无法治疗该疾病。我们发现，基因转移如此之低的一个重要原因是，HSC上的常规逆转录病毒受体几乎无法检测到。第二个问题是含有球蛋白基因的逆转录病毒载体的不稳定性。项目2将分别检查这些问题。项目2：针对血红蛋白病的基因治疗特定目的2.1：我们有证据表明，RD114和FELV-C逆转录病毒的受体在造血干细胞上以高水平表达。我们假设这些高水平的受体将导致人类HSC转导的频率更高。我们开始通过用对照Galv伪型载体和RD114或FELV-C PSEUDOTYPECTER媒介转导人HSC来检验这一假设，以移植到胎儿绵羊中。与galv pseudotyped载体相比，前5只绵羊嵌合体的外周血和骨髓细胞显示出使用RD114或FELV-C-C-PSEUDOTYPECTORS的转导水平更高10倍。这项工作正在进行中。具体目的2.2：我们假设可以产生与红细胞细胞中表达的基因启动子有关的稳定逆转录病毒载体，可以产生能够以足以治疗镰状细胞病和B-心理亚无原的水平表达球蛋白mRNA。我们使用转基因小鼠测定法对红细胞基因启动子表达的相对表达水平的评估表明，与鸡肉绝缘子元件相关的AE-1启动子可以独立，均匀，高级和拷贝数依赖性表达来指导位置。我们已经与该构建体产生了稳定的逆转录病毒，该构建病毒将在小鼠B-心助乳模型中进行评估。