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.

造血科的研究重点是干细胞的基础生物学，以及干细胞作为细胞和基因治疗载体的使用。造血干细胞(HSC)是一种罕见的自我更新细胞群，可在外周血中产生所有细胞，使其成为遗传性血液病基因替代治疗的理想载体。此外，高度富含HSC的细胞在注射到心肌梗死周围的健康组织中或在细胞因子(如G-CSF和SCF)动员到外周血中时，可以生成心肌细胞。其他研究表明，丰富的HSC群体可以产生血管、骨骼肌、肝脏和神经细胞。项目1将研究干细胞生物学。项目1：造血干细胞生物学特定目标1.1：我们假设，在实验性心肌梗死(MI)后，重新填充骨髓的相同的造血干细胞负责心肌细胞的再生。为了验证这一假设，我们将带有GFP基因的逆转录病毒载体标记的骨髓细胞移植到小鼠体内。然后，这些小鼠接受冠状动脉结扎，并用G-CSF和SCF动员干细胞和祖细胞进入外周血。梗死区重新填充了新的细胞，这些细胞含有在外周血细胞中发现的相同的逆转录病毒标志物。这些研究正在进行中，但我们得出结论，新的心肌细胞是同时产生外周血细胞的细胞的后代。特定目的1.2：我们假设心脏受损区域的再生是由于新细胞的植入和扩张，而不是原始细胞与周围心肌细胞的融合。为了验证这一假设，我们从再生的心肌梗死中分离出细胞，并使用原位杂交来证明这些细胞在原始心肌细胞中表达蛋白质，并且是二倍体，这与融合假说背道而驰。未来的研究将涉及HSC基因缺陷的小鼠，看看G-CSF/SCF治疗是否能重新填充梗死区。具体目标1.3：我们假设在从骨骼肌分离的HSC和干细胞中表达的特定基因负责维持未分化状态。为了验证这一假设，我们分离了？侧种群？从小鼠骨骼肌培养的干细胞，并使用cDNA差减来生成在这些细胞中表达的序列的文库。肌肉干细胞转录本将与我们之前在HSC的cDNA消减文库中鉴定的转录本进行比较。我们将选择两个文库共有的转录本用于转基因小鼠的分析。将基因转移到HSC可以治愈严重的联合免疫缺陷，表明HSC基因治疗可以应用于更常见的疾病。我们想开发一种治疗镰刀细胞病的基因疗法。然而，目前向HSC转移的基因水平太低，无法治疗这种疾病。我们发现，基因转移率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检测不到。第二个问题是含有珠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：血红蛋白疾病的基因治疗特定目标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高水平表达。我们假设这些高水平的受体将导致更高频率的人类HSC转导。我们开始通过同时转导人HSC和对照Galv伪型载体以及RD114或FeLV-C伪型载体将其移植到胎羊体内来验证这一假设。来自前5个绵羊嵌合体的外周血和骨髓细胞显示，RD114或FeLV-C伪型载体的转导水平是Galv伪型载体的10倍以上。这项工作正在进行中。具体目的2.2：我们假设可以产生稳定的逆转录病毒载体，该载体含有与红系细胞中表达的基因启动子相连的珠蛋白基因，从而使珠蛋白mRNA的表达水平足以治疗镰刀细胞疾病和b地中海贫血。我们使用转基因小鼠实验对红细胞基因启动子的相对表达水平进行了评估，结果表明，连接到鸡绝缘元件的AE-1启动子引导位置无关、均匀、高水平和拷贝数相关的表达。我们已经用这个结构产生了稳定的逆转录病毒，将在小鼠b地中海贫血模型中进行评估。