DEVELOPMENT OF HEMATOPOIETIC STEM CELLS

造血干细胞的发育

• 批准号: 6639881
• 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/6639881
• 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.

尽管近年来对哺乳动物胚胎造血的研究取得了很大进展，但胚胎造血干细胞（HSC）的起源和发育潜能仍不清楚。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，需要重新评估其他组织特异性干细胞类型（例如HSC，间充质干细胞，肝干细胞，表皮干细胞等）的全部发育潜力。新的子宫内移植试验称为胚泡植入试验（blastocyst engraftment assay，简称EMT），将用于进一步研究胚胎造血。胚胎干细胞移植是基于将纯化的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干扰胚胎发育，并导致供体HSC在受体的胚胎和成体造血组织中植入。移植的优点是供体HSC的运输发生在正常的胚胎发育过程中，其次，供体HSC的植入通过新形成的细胞小生境的竞争性再增殖发生。本项目的总体目标是：（a）研究小鼠胚胎发育过程中显微注射转基因胚胎和成体HSC的体内和体外归巢和植入，（B）比较分析小鼠胚胎和成体HSC发育为非造血细胞/组织的潜力。为了实现这一目标，该提案的第一部分集中在转基因胎儿和成年HSC（分离自ROSA β-geo 26和GFP转基因小鼠）显微注射到野生型和加塔-2和SCL敲除小鼠的胚胎后的命运的广泛体内分析。将通过使用组织化学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试验（CFU-C）分析胚胎、组织和细胞来评估供体HSC在胚胎造血组织中的植入。为了补充体内研究，第二部分涉及显微注射转基因HSC的命运的体外分析。将整个小鼠胚胎（体内达到第10天胚胎的发育阶段）的培养物用作实验系统，用于在培养过程中对相同胚胎内表达GFP的转基因HSC进行真实的时间追踪。第三部分包括在小鼠胚胎发育过程中胎儿和成年小鼠HSC分化为非造血细胞的潜力的体内分析。所提出的研究结果可能对胚胎造血和HSC的子宫内和成人临床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的新治疗方法的开发产生重要影响，其中特定干细胞不易于分离。