DEVELOPMENT OF HEMATOPOIETIC STEM CELLS

尽管最近对哺乳动物胚胎造血的了解有所进展，但胚胎造血干细胞（HSC）的起源位置和发育潜力仍不清楚。此外，最近观察到神经干细胞可能具有比预期更广泛的发育能力，这要求重新评估其他组织特异性干细胞类型（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囊胚植入试验（BEA）将用于进一步研究胚胎造血。BEA是基于将纯化的造血干细胞显微注射到小鼠着床前胚胎（囊胚）中，类似于胚胎干细胞技术。这种新的试验不干扰胚胎发育，并导致供体HSC在受者的胚胎和成人造血组织中植入。BEA的优势在于供体造血干细胞的移植是在正常胚胎发育过程中进行的，其次，供体造血干细胞的移植是通过新形成的细胞龛的竞争性再生来实现的。本课题的总体目标是：(a)研究微注射转基因胚胎和成人造血干细胞在小鼠胚胎体内和体外发育过程中的归巢和植入，(b)比较分析小鼠胚胎和成人造血干细胞向非造血细胞/组织发育的潜力。为了实现这一目标，该提案的第一部分侧重于广泛的体内分析转基因胚胎和成年HSC（从ROSAbeta-geo26和GFP转基因小鼠中分离）在显微注射到野生型和GATA-2和SCL敲除小鼠胚胎后的命运。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）对胚胎、组织和细胞进行分析来评估供体HSC在胚胎造血组织中的植入情况。为了补充体内研究，第二部分涉及微注射转基因HSC的体外命运分析。整个小鼠胚胎（在体内达到发育阶段的第10天胚胎）的培养将作为一个实验系统，在培养过程中实时跟踪在同一胚胎中表达gfp的转基因HSC。第三部分包括在体内分析胚胎和成年小鼠HSC在小鼠胚胎发育过程中分化为非造血细胞的潜力。本研究的结果可能对胚胎造血、临床子宫内和成人造血干细胞移植等领域产生重要影响。也可能是开发新的治疗方法，使用自体造血干细胞来治疗影响非造血组织的疾病或损伤，这些组织的特定干细胞不易分离。

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.