Basic Research on Hematopoietic Human Stem Cells

人类造血干细胞基础研究

• 批准号: 7964580
• 负责人: Harry L Malech
• 金额: $ 52.56万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/7964580
• 关键词: Adhesions; Affect; Allogenic; Animals; Apoptosis; Autologous; Basic Science; Biochemical; Biology; Blood; Blood Cells; Bone Marrow; Bone Marrow Stem Cell; Busulfan; CD34 gene; CXCR4 gene; Carmustine; Cell Therapy; Cells; Chronic Granulomatous Disease; Cleaved cell; Clinical; Defect; Dipeptides; Dipeptidyl-Peptidase IV; Disease; Elements; Engraftment; Failure; Gene Transfer; Genes; Goals; Growth; Harvest; Hematopoietic; Hematopoietic stem cells; Homologous Transplantation; Human; Hydrogen Peroxide; Immune; Immune system; Immunity; Immunologic Deficiency Syndromes; Infection; Inherited; Leukocytes; Ligands; Link; Lymphocyte; Marrow; Mesenchymal; Methods; Modeling; NOD/SCID mouse; Outcome; Pathway interactions; Patients; Peptide Hydrolases; Phenotype; Process; Production; Property; Regulation; Regulatory Element; Research; Role; Signal Transduction; Staging; Stem cells; Stream; Stromal Cell-Derived Factor 1; System; Therapeutic; Transferase; Transplantation; Treatment Protocols; Umbilical Cord Blood; Universities; Xenograft Model; Xenograft procedure; base; chemokine receptor; conditioning; design; diprotin A; embryonic stem cell; gene correction; gene repair; gene therapy; gene transfer vector; human adult stem cell; human stem cells; improved; in vivo; inhibitor/antagonist; microbicide; migration; monocyte; mutant; neutrophil; nonhuman primate; overexpression; peripheral blood; progenitor; receptor; release of sequestered calcium ion into cytoplasm; research study; response; stem cell population; success; therapeutic gene; tool; trafficking

This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy or for use in allogeneic transplantation in the treatment of inherited diseases affecting cells of the immune system. This project also studies CD34 positive hematopoietic adult human stem cells from patients with inherited immune deficiencies with the ultimate goal of developing hematopoietic stem cell based therapies for these disorders. A new component of this research which we have incorporated into this project during the past year is an initiative to establish from patients with primary immune deficiencies induced pleuripotent stem cells (iPS cells) with the primitive properties of embryonic stem cells capable of differentiating into hematopoietic stem cells, and into mature neutrophils and other cells of the immune system. We have developed new methods and materials which improve our ability to get new genes into human blood stem cells. We are also exploring the use of those gene transfer systems to correct the genes and gene defects causing primary immune deficiencies in iPS cells derived from patients with PIDs. We are also exploring the potential of busulfan as a more stem cell specific and immune system sparing conditioning regimen for transplant so we need to understand better the effect of such agents on human CD34 positive hematopoietic adult human stem cells and upon more primitive stem cells capable of differentiating into CD34 positive hematopoietic stem cells. Evidence from human and animal studies of gene therapy suggest that providing an in vivo growth or survival advantage to genetically corrected blood cells can improve the outcome of gene therapy by increasing the percent of corrected cells in the body. One approach to this is to co-express the therapeutic gene (such as the corrective gene for X linked CGD) with a gene that allows for selective enrichment. In studies with collaborators we have explored the use of the methyguanine methyl transferase (MGMT) which protects against alykating agents such as BCNU in a non-human primate model achieving marking rates of up to 20%. Thus, we need to understand better how to culture and subject to selection human stem cells. As noted, a major goal of this project is to examine in CD34 positive hematopoietic adult human stem cells the role of the CXCR4 chemokine receptor (ligand is SDF-1) on engraftment in marrow. We showed that overexpression of CXCR4 in human CD34 hematopoietic stem cells enhanced engraftment of these cells in the NOD/SCID mouse xenotransplant model. The immunodeficiency, WHIM (warts, hypogammaglobulinemia, infections, myelokathexis apoptosis of neutrophils), is caused by truncations in the C-terminus of CXCR4. We created gene transfer vectors to over express the WHIM type mutant CXCR4 in CD34 stem cells and showed that this resulted in increased migration, adhesion and intracellular calcium flux in response to SDF-1. We showed that this was caused by a failure to downregulate or to internalize the mutant receptor providing a biochemical basis for the dominant hyperfunction abnormality of CXCR4 activity associated with WHIM. We also find that the mutant CXCR4 enhances engraftment of cells expressing this mutant receptor and it may be a useful tool to enhance engraftment. CD26 is a protease expressed on bone marrow stroma and also on some CD34 positive hematopoietic adult human stem cells. CD26 is a type IV dipeptide proteinase that can cleave and inactive SDF-1. We show that treatment of NOD/SCID mice with Diprotin A, an inhibitor of CD26 on marrow stroma, markedly enhances engraftment of CD34 positive hematopoietic adult human stem cells in this xenograft model. We believe that this could be an important therapeutic method to enhance engraftment. In the same mouse NOD/SCID xenograft system, we have during the past year demonstrated that forcing excess expression of the growth regulatory element HOXB4 in human hematopoietic stem cells will enhance engraftment of those human stem cells in the NOD/SCID mouse xenograft model. We did this by transferring the gene for an element upstream of HOXB4 regulation rather than by just overexpressing HOXB4 directly. These important experiments are ongoing, but we hope that similar to the observations of others, the overexpression of HOXB4 could be a means of increasing the number of primitive human hematopoietic stem cells that engraft. As noted above, we have together with our collaborator, Dr. Linzhao Cheng at Johns Hopkins University initiated a project to develop iPS cells from patients with primary immune deficiencies, focussing initially on patients with X-linked chronic granulomatous disease (X-CGD) (a PID caused by a defect of microbicidal hydrogen peroxide production by blood neutrophils). We have successfully completed the first stage of this project by establishing X-CGD iPS cells from bone marrow mesenchymal cells. Our next goal is to determine the conditions to cause differentiation of X-CGD iPS cells into mature neutrophils. This will establish a model that will allow exploration of the potential of applying methods of gene repair to the X-CGD iPS cells to correct the CGD phenotype in neutrophils that arise from the gene corrected iPS cells.

该项目将外周血造血祖细胞（PBHP）研究为基因治疗的靶标或用于同种异体移植时，用于治疗影响免疫系统细胞的遗传疾病。该项目还研究了来自遗传性免疫缺陷患者的CD34阳性造血成人人类干细胞，其最终目标是为这些疾病开发造血干细胞疗法。 在过去的一年中，我们已将本研究纳入该项目的一项新组成部分是一项主动性，是从具有原发性免疫缺乏症的患者中建立的，诱导的胸膜干细胞（IPS细胞）具有能够分化为血肿细胞的胚胎干细胞的原始特性，并将其分化为血肿的中性粒细胞和其他免疫系统。我们开发了新的方法和材料，这些方法和材料提高了我们将新基因进入人血干细胞的能力。 我们还正在探索这些基因转移系统的使用来纠正从PID患者衍​​生的IPS细胞中产生原发性免疫缺陷的基因和基因缺陷。 我们还正在探索Busulfan作为移植的更为干细胞特异性和免疫系统的较高的特异性和免疫系统，因此可以更好地理解此类药物对人CD34阳性造血成人人类干细胞的影响，以及对能够区分为CD34阳性阳性造血干细胞的更原始的干细胞。人类和动物研究的基因疗法研究的证据表明，为遗传校正的血细胞提供体内生长或生存优势可以通过增加体内校正细胞的百分比来改善基因治疗的结果。一种方法是与允许选择性富集的基因共表达治疗基因（例如X链接CGD的矫正基因）。在与合作者的研究中，我们探讨了甲基甲基转移酶（MGMT）的使用，该酶在非人类灵长类动物模型中可预防诸如BCNU之类的Alykating剂，可实现高达20％的标记率。 因此，我们需要更好地了解如何培养并受到选择人类干细胞的选择。 如前所述，该项目的主要目标是在CD34阳性造血成年人类干细胞中检查CXCR4趋化因子受体（配体是SDF-1）在骨髓植入中的作用。我们表明，在人类CD34造血干细胞中，CXCR4的过表达增强了这些细胞在点点/SCID小鼠异种移植模型中的植入。免疫缺陷（Warts，低磁性血症，感染，骨髓触及中性粒细胞的骨凋亡）是由CXCR4的C末端截断引起的。我们创建了基因转移载体，以过度表达CD34干细胞中的异想天开的突变体CXCR4，并表明这会导致迁移，粘附和细胞内钙通量增加，以响应SDF-1。我们表明，这是由于未能下调或内部化突变受体提供的，从而为与异想天开有关的CXCR4活性的显性高功能异常提供了生化基础。我们还发现，突变的CXCR4增强了表达这种突变受体的细胞的植入，它可能是增强植入的有用工具。 CD26是一种在骨髓基质和一些CD34阳性造血成人人类干细胞上表达的蛋白酶。 CD26是可以裂解和非活性SDF-1的IV型二肽蛋白酶。 我们表明，在这种异种移植模型中，用二吡啶A（一种CD26的抑制剂）Anod/SCID小鼠（一种CD26的抑制剂）显着增强了CD34阳性造血成人人类干细胞的植入。 我们认为，这可能是增强植入的重要治疗方法。 在同一小鼠点头/SCID异种移植系统中，我们在过去的一年中表明，在人造血干细胞中迫使生长调节元件HOXB4过量表达将增强在NOD/SCID小鼠异种移植模型中植入那些人类干细胞的植入。 我们通过转移HOXB4调节上游的元素而不是直接过表达HOXB4的基因来做到这一点。 这些重要的实验正在进行中，但我们希望与他人的观察相似，Hoxb4的过表达可能是增加植入原始人类造血干细胞数量的一种手段。 如上所述，我们与约翰·霍普金斯大学（Johns Hopkins University）的合作者Linzhao Cheng博士一起发起了一个项目，该项目旨在开发来自原发性免疫缺陷患者的IPS细胞，最初集中在X-连接的慢性肉芽肿性疾病（X-CGD）的患者（X-CGD）（X-CGD）（X-CGD）（一种由微生物氢化氧化氧化氧化物的缺陷引起的，是由微生物氢过氧化氢过氧化物的缺陷引起的。 我们通过从骨髓间充质细胞中建立X-CGD IPS细胞成功完成了该项目的第一阶段。 我们的下一个目标是确定导致X-CGD IPS细胞分化为成熟中性粒细胞的条件。 这将建立一个模型，该模型将允许探索将基因修复方法应用于X-CGD IPS细胞的潜力，以纠正由基因校正的IPS细胞引起的中性粒细胞中的CGD表型。