DEVELOPMENT OF HEMATOPOIETIC STEM CELLS

造血干细胞的发育

• 批准号: 6540658
• 负责人: Roland Jurecic
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540658
• 关键词: bioassay; early embryonic stage; genetically modified animals; hematopoietic stem cells; laboratory mouse; method development; microinjections; stem cell transplantation; vertebrate embryology

Despite recent advances in understanding of mammalian embryonic hematopoiesis, site of origin and developmental potential of embryonic hematopoietic stem cells (HSC) remain unclear. Furthermore, recent observation that neuronal stem cells could have a much broader developmental capacity than anticipated, calls for reassessment of the full developmental potential of other tissue-specific stem cell types (e.g. HSC, mesenchymal, hepatic, epidermal stem cells etc.). New in utero transplantation assay termed blastocyst engraftment assay (BEA) will be used to further study embryonic hematopoiesis. BEA is based on microinjection of purified HSC into mouse preimplantation embryos (blastocysts), similar to ES cell technology. This new assay does not interfere with embryo development and results in engraftment of donor HSC in embryonic and adult hematopoietic tissues of recipients. The advantage of BEA is that the trafficking of donor HSC takes place during normal embryonic development, and secondly, the engraftment of donor HSC occurs through competitive repopulation of newly forming cell niches. The overall goal of the proposed project is to (a) study homing and engraftment of microinjected transgenic fetal and adult HSC during mouse embryo development in vivo and in vitro, and (b) comparatively analyze the potential of mouse fetal and adult HSC for development into non-hematopoietic cells/tissues. Towards this goal, the first part of the proposal focuses on the extensive in vivo analysis of the fate of transgenic fetal and adult HSC (isolated from ROSAbeta-geo26 and GFP transgenic mice) after microinjection into embryos of wild type and GATA-2 and SCL knockout mice. Engraftment of donor HSC in embryonic hematopoietic tissues will be assessed by analysis of embryos, tissues and cells using histochemical X-gal staining, fluorescence microscopy, histological and molecular techniques and in vitro hematopoietic colony forming assays (CFU-C). To complement in vivo studies, the second part deals with in vitro analysis of the fate of microinjected transgenic HSC. Culture of whole mouse embryos (reaching developmental stage of day 10 embryos in vivo) will be used as an experimental system for real time tracking of GFP-expressing transgenic HSC within the same embryos over the course of culture. The third part encompasses in vivo analysis of the potential of fetal and adult mouse HSC to differentiate into non-hematopoietic cells during mouse embryo development. The outcome of proposed studies could have important impact on the fields of embryonic hematopoiesis and clinical in utero and adult transplantation of HSC, .and perhaps on the development of new therapeutic approaches using autologous HSC to treat disorders or injuries affecting non-hematopoietic, tissues where the specific stem cells are not readily available for isolation.

尽管最近对哺乳动物胚胎造血作用的了解取得了进展，但胚胎造血干细胞（HSC）的起源位点和发育潜力仍不清楚。此外，最近观察到神经元干细胞可能具有比预期更广泛的发育能力，因此需要重新评估其他组织特异性干细胞类型（例如 HSC、间充质、肝脏、表皮干细胞等）的全部发育潜力。新的子宫内移植试验称为囊胚植入试验（BEA）将用于进一步研究胚胎造血作用。 BEA 基于将纯化的 HSC 显微注射到小鼠植入前胚胎（囊胚）中，类似于 ES 细胞技术。这种新的检测方法不会干扰胚胎发育，并导致供体 HSC 植入受体的胚胎和成人造血组织中。 BEA 的优点是，供体 HSC 的运输发生在正常胚胎发育过程中，其次，供体 HSC 的植入是通过新形成的细胞生态位的竞争性重新增殖而发生的。该项目的总体目标是（a）研究显微注射的转基因胎儿和成人HSC在体内和体外小鼠胚胎发育过程中的归巢和植入，以及（b）比较分析小鼠胎儿和成人HSC发育成非造血细胞/组织的潜力。为了实现这一目标，该提案的第一部分重点是对转基因胎儿和成年 HSC（从 ROSAbeta-geo26 和 GFP 转基因小鼠分离）显微注射到野生型和 GATA-2 和 SCL 敲除小鼠胚胎后的命运进行广泛的体内分析。将使用组织化学 X-gal 染色、荧光显微镜、组织学和分子技术以及体外造血集落形成测定 (CFU-C) 对胚胎、组织和细胞进行分析，评估供体 HSC 在胚胎造血组织中的植入情况。为了补充体内研究，第二部分涉及显微注射转基因 HSC 命运的体外分析。整个小鼠胚胎的培养（在体内达到第 10 天胚胎的发育阶段）将用作实验系统，用于在培养过程中实时跟踪同一胚胎内表达 GFP 的转基因 HSC。第三部分包括对胎儿和成年小鼠 HSC 在小鼠胚胎发育过程中分化为非造血细胞的潜力进行体内分析。拟议研究的结果可能对胚胎造血和临床宫内和成人 HSC 移植领域产生重要影响，并且可能对使用自体 HSC 治疗影响非造血组织的疾病或损伤（其中特定干细胞不易分离）的新治疗方法的开发产生重要影响。