ENGINEERING STEM CELLS TO CONFER PROLIFERATIVE ADVANTAGE

工程干细胞赋予增殖优势

• 批准号: 6650013
• 负责人: MARCUS O MUENCH
• 金额: $ 13.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6650013
• 关键词: SCID mouse; biological models; biotechnology; cell growth regulation; cell proliferation; cord blood; erythropoietin; growth factor receptors; hematopoiesis; hematopoietic stem cells; human fetus tissue; human tissue; in utero transplantation; sickle cell anemia; stem cell transplantation

In utero hematopoietic stem cell transplantation (IUT) offers the hope of curing a number of hematological diseases by generating a state of hematopoietic stem cells (HSCs), the levels of donor cell engraftment that can be achieved by IUT are low, limiting the use of this therapy. Our aim is to extend the use of IUT to the treatment of diseases such as sickle cell anemia.. To achieve the high levels of donor cell engraftment needed to treat this disease it is the goal of this proposal to engineer HSCs to have a proliferative advantage over normal HSCs. This proposal will test the hypothesis that introduction of the erythropoietin (EpoR) into HSCs will render these altered cells responsive to erythropoietin (EPO). This will result in the altered HSCs and their progeny having a proliferative advantage over normal progenitors. Truncated forms of EpoR (tEpoR) will also be tested. These tEpoR, having deletions in the negative regulatory region of their cytoplasmic domains, deliver stronger proliferative signals, than EpoR. Fetal HSCs will be used as targets since they offer proliferative advantages over adult cells and are, therefore, susceptible to transduction by retroviral vectors. Lentiviral vectors will also be tested for their capacity to modify fetal as well as postnatal sources of HSCs. The effects of introducing the EpoR genes on the proliferation and differentiation of HSCs will be determined using various in vitro culture systems. It is hypothesized that ectopic expression of either EpoR or tEpoR expression on HSCs can make these cells more competitive than their normal counterparts, modified HSCs will be tested against control HSCs in a mouse model of human fetal hematopoiesis. The in vivo model will also be used to test the effects of EPO administration on the expansion of the modified HSCs. The ability of experiments will further determine if making HSCs responsive to HPO will have detrimental effect on the long-term reconstituting- and multi- lineage potential of HSCs.

子宫内造血干细胞移植（IUT）通过产生造血干细胞（HSC）的状态提供了治愈许多血液病的希望，通过IUT可以实现的供体细胞植入水平低，限制了这种疗法的使用。我们的目标是将IUT的使用扩展到镰状细胞贫血等疾病的治疗。为了实现治疗这种疾病所需的高水平的供体细胞植入，该提议的目标是工程化HSC以具有超过正常HSC的增殖优势。该提议将检验将促红细胞生成素（EpoR）引入HSC将使这些改变的细胞对促红细胞生成素（EPO）应答的假设。这将导致改变的HSC及其后代具有超过正常祖细胞的增殖优势。还将检测EpoR的截短形式（tEpoR）。这些tEpoR在其胞质结构域的负调控区中具有缺失，比EpoR递送更强的增殖信号。胎儿HSC将被用作靶，因为它们提供了比成人细胞更好的增殖优势，因此，对逆转录病毒载体的转导敏感。还将测试慢病毒载体修饰胎儿以及出生后来源的HSC的能力。引入EpoR基因对HSC增殖和分化的影响将使用各种体外培养系统来确定。假设HSC上EpoR或tEpoR表达的异位表达可以使这些细胞比它们的正常对应物更具竞争力，将在人胎儿造血的小鼠模型中针对对照HSC测试修饰的HSC。体内模型还将用于测试EPO施用对经修饰的HSC的扩增的影响。实验的能力将进一步确定使HSC响应于HPO是否会对HSC的长期重建和多谱系潜力具有不利影响。