HEMATOPOIETIC STEM CELL--BIOLOGY, PRECURSORS, AND PROGEN

造血干细胞被定义为能够产生所有血细胞谱系以及自我更新（至少在相当长的一段时间内）的克隆原细胞。 造血干细胞（HSC）和多能祖细胞（MPP）有3个亚群，其中LT-HSC能在宿主生命周期内自我更新，而ST-HSC能更新8周，MPP更新时间更短，其下游是两类寡能祖细胞--共同淋巴样祖细胞（CLP）和共同髓样祖细胞（CMP）。 这些祖细胞中的每一个都已经被纯化，并且可以通过表型进行前瞻性鉴定并分离。在资助的第一部分中，将在批量水平和单细胞水平测试分裂的LT-HSC、ST-HSC和MPP的自我更新或分化为一种或另一种下游命运，以确定这些多能细胞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，但证明它们在各种体内环境中扩增几个数量级的能力相对简单。 为了确定什么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存活基因如bcl-2、端粒延伸基因如TERT和TPC 3以及特定信号转导途径下游基因如β连环蛋白的强制表达，并在体内环境中分析其后代。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）已被确定或声称，非常令人惊讶的推定转分化的可能性之间的造血系统的干细胞。 在这项资助中，将努力建立全能干细胞的明确克隆形成测定，并检查各种组织特异性干细胞，全能干细胞和HSC之间的关系。如果能通过干细胞相关基因的表达来原位观察HSC，将有助于对HSC生物学的研究。 作为该提议的一部分的长期项目是开始在最近发现的大量蛋白质如绿色荧光蛋白中鉴定作为有用荧光染料的蛋白质，并且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的干细胞相关基因的融合蛋白（ckit，Sca 1，Thy- 1.1，Flk 2/Flt 3，SCL）以及最近鉴定为在其表达中具有干细胞特异性的其它（例如，该实验室克隆了一个新的serpin基因）。

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).