HEMOPOIETIC STEM CELL SELF RENEWAL AND RETINOID ANTAGONISTS

造血干细胞自我更新和类维生素A拮抗剂

• 批准号: 6110531
• 负责人: STEVEN Collins COLLINS
• 金额: $ 20.18万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110531
• 关键词: Retroviridae; SCID mouse; bone marrow; cell differentiation; cell transplantation; flow cytometry; growth factor; growth factor receptors; growth media; hematopoietic stem cells; human tissue; laboratory mouse; receptor expression; retinoate; tissue /cell culture; transfection /expression vector; vitamin receptor

Progress in utilizing retroviral vectors to correct hereditary disorders involving the hematopoietic stem cell (HSC) has been exceedingly slow. One of the major stumbling blocks relates to the observation that retroviral vectors preferentially infect mitotically active cells, but the normally quiescent HSC, when stimulated to divide in vitro, frequently commits itself to differentiate into a more mature, lineage committed stem cell exhibiting a limited clonal lifespan. It is these lineage committed proliferating progenitors exhibiting both a limited lifespan and poor long-term marrow repopulating ability that are preferentially infected by the retroviral vectors. Thus the successful use of retroviral vectors to correct HSC genetic disorders requires that self renewing HSC be preferentially expanded and transduced by these vectors. We wish to determine the specific culture conditions that will promote the self- renewal of HSC in vitro and will lead to more efficient retroviral vector mediated transduction of HSC. Our experimental approach will be based on recent observations in our laboratory indicating that blocking the activity of retinoic acid (RA) receptors in hematopoietic stem cells inhibits their lineage commitment and enhances their self renewal. Our specific goals are as follows: SPECIFIC AIM I) Determine the in vivo marrow repopulating capability of the cultured murine lymphohematopoietic EML cells. We wish to determine the capacity of primitive SCF-dependent mouse cell lines (designated EML) derived by transducing normal mouse bone marrow with a dominant negative RA receptor construct, to function in vivo as stem cells in irradiated syngeneic or in SCID mice. SPECIFIC AIM II) Determine the optimal in vitro conditions that enhance the self renewal of murine and human hematopoietic stem cells. Highly enriched fractions of murine and human HSCs will be cultured in vitro under various conditions with hematopoietic stem cell growth factors together with synthetic retinoids exhibiting RA receptor antagonism. The self renewal of HSCs in these cultures will be evaluated by various techniques. SPECIFIC AIM III) Determine in vitro conditions for optimizing retroviral mediated gene transduction into human hematopoietic stem cells. Utilizing culture conditions determined in Specific Aim Il we will infect self renewing hematopoietic stem cells with retroviral vectors harboring specific markers and assess the efficiency of successfully transducing these HSCs with these vectors. These studies directly address the problem related to inefficient retroviral vector mediated gene transduction into hematopoietic stem cells. Our approach, if successful, will have broad applicability to gene therapy of patients with a variety of different genetic disorders of hematopoietic stem cells.

利用逆转录病毒载体纠正遗传疾病的进展 涉及造血干细胞（HSC）的速度非常慢。一 主要的绊脚石与逆转录病毒有关 载体优先感染有丝分裂活性细胞，但通常是 静止的HSC，当刺激体外分裂时，经常提交 本身可以分化为更成熟，谱系犯的干细胞 表现出有限的克隆寿命。这些宗族犯了 增殖的祖细胞表现出有限的寿命和差 长期骨髓再现能力优先感染 逆转录病毒载体。因此，成功使用逆转录病毒向量 正确的HSC遗传疾病要求自我更新HSC是 这些向量优先扩展和转导。我们希望 确定将促进自我的特定文化条件 在体外更新HSC，将导致更有效的逆转录病毒载体 介导的HSC转导。我们的实验方法将基于 我们实验室的最新观察表明，阻止 视黄酸（RA）受体在造血干细胞中的活性 抑制他们的血统承诺并增强他们的自我更新。我们的 具体目标如下： 具体目的i）确定体内骨髓重现的能力 培养的鼠淋巴肿瘤EML细胞。我们希望确定 原始依赖SCF的小鼠细胞系的容量（指定的EML） 通过转导正常小鼠骨髓来得出 RA受体构建体，在辐射中作为干细胞在体内发挥作用 合成生或SCID小鼠。 特定目标ii）确定增强的最佳体外条件 鼠和人造血干细胞的自我更新。高度 富集的鼠和人类HSC的富集将在体外培养 在造血干细胞生长因子的各种条件下 以及表现出RA受体拮抗作用的合成类视网膜类似。这 这些文化中HSC的自我更新将由各种评估 技术。 特定目标iii）确定优化逆转录病毒的体外条件 介导的基因转导向人造血干细胞。利用 在特定目的中确定的文化条件，我们将感染自我 带有逆转录病毒载体的更新造血干细胞 特定标记并评估成功传递的效率 这些HSC与这些向量。 这些研究直接解决了与效率低下有关的问题 逆转录病毒载体介导的基因转导向造血茎 细胞。如果成功的话，我们的方法将对基因具有广泛的适用性 患有多种不同遗传疾病的患者的治疗 造血干细胞。