HEMATOPOIETIC STEM CELL--BIOLOGY, PRECURSORS, AND PROGEN

造血干细胞被定义为至少在相当长的一段时间内，可以产生所有血细胞谱系并自我更新的克隆细胞。干细胞（HSC）和多能祖细胞有3个亚群，只有长期的（LT-HSC）在宿主的生命周期内自我更新，而ST-HSC的更新时间长达8周，MPP的更新时间更短。HSC/MPP的下游是2类低效祖细胞-共同淋巴祖细胞（CLP）和共同髓祖细胞（CMP）。这些祖细胞已被纯化，可以通过表型和分离进行前瞻性鉴定。在赠款的第一部分，将在整体水平和单细胞水平上对分裂的LT-HSC、ST-HSC和MPP进行自我更新或分化到一种或另一种下游命运的测试，以确定这些多能细胞做出的细胞命运决定在很大程度上是对称的还是不对称的。HSC不能通过任何已知的方案在体外扩增，但在各种体内环境中证明其扩增几个数量级的能力相对简单。为了确定哪些内在或外在程序可能调节LT-HSC自我更新，将在HSC和其他多能祖细胞中测试细胞生存基因（如bcl-2）、端粒延伸基因（如TERT和TPC3）以及特定信号转导途径下游基因（如β - catenin）的强制表达，并在体内环境中分析它们的后代。在过去的一年中，大量的干细胞，从全能干细胞到器官特异性干细胞（如中枢神经系统）已经被发现或声称，它们与造血系统干细胞之间具有非常惊人的转分化可能性。在这项拨款中，将努力建立明确的多能干细胞克隆测定方法，并研究各种组织特异性干细胞、多能干细胞和造血干细胞之间的关系。利用造血干细胞相关基因的表达对造血干细胞进行原位观察，可以加强对造血干细胞生物学的研究。作为该提案一部分的一个长期项目是开始在大量最近发现的蛋白质（如绿色荧光蛋白）中识别有用的荧光蛋白，并从那些似乎有用的蛋白质中“敲入”不同颜色的基因作为这些已知干细胞相关基因（ckit, Sca1, Thy- 1.1, Flk2/Flt3， SCL）的融合蛋白，以及其他最近确定的干细胞特异性表达(例如，一个新的蛇形蛋白基因克隆在这个实验室)。

Hematopoietic stem cells are defined as clonogenic cells that can give rise to all blood cell lineages, as well as self-renew, at least for a significant period of time. There are 3 subsets of stem cells (HSC) and multipotent progenitors, and only the long-term (LT-HSC) self-renew for the life of the host, while ST-HSC renew for up to 8 weeks, and MPP for much less. Downstream of HSC/MPP are 2 classes of oligopotent progenitors - the common lymphoid progenitor (CLP) and the common myeloid progenitor (CMP). Each of these progenitors have been purified and can be prospectively identified by phenotype and isolated. In the first section of the grant, dividing LT-HSC, ST-HSC, and MPP will be tested for self-renewal or differentiation to one or the other downstream fates at the bulk level and at the single cell level to determine whether the cell fate decisions these multipotent cells make are largely symmetrical or asymmetrical. HSC cannot be expanded in vitro by any known protocol, but it is relatively simple to demonstrate their ability to expand by several orders of magnitude in various in vivo settings. To determine what intrinsic or extrinsic programs might regulate LT-HSC self-renewal, enforced expression of cell survival genes such as bcl-2, telomere extending genes such as TERT and TPC3, and genes downstream of particular signal transduction pathways such as beta catenin will be tested in HSC and other multipotent progenitors, and their progeny analyzed in an in vivo setting. Within the past year a large number of stem cells ranging from totipotent stem cells to organ specific stem cells (such as for the central nervous system) have been identified or claimed, with very surprising putative transdifferentiation possibilities between them and stem cells of the hematopoietic system. In this grant an effort will be made to establish unequivocal clonogenic assays for totipotent stem cells, and the relationship between various tissue specific stem cells, totipotent stem cells, and HSC will be examined. The study of HSC biology could be enhanced if HSC could be observed in situ by means of their expression of stem cell related genes. A long-term project that is part of this proposal is to begin to identify proteins that are useful fluorochromes among a plethora of recent discovered proteins such as the green fluorescent protein, and from amongst those that appear to be useful, to "knockin" different color genes as fusion proteins for these stem cell related genes already known (ckit, Sca1, Thy- 1.1, Flk2/Flt3, SCL) as well as others recently identified as being stem cell specific in their expression (e.g., a new serpin gene cloned in this laboratory).