HEMATOPOIETIC DIFFERENTIATION IN EMBRYONAL STEM CELLS

这项提案的中心目标是调查蜂窝和 原始干细胞植入早期的分子事件 到了造血系。我们研究了小鼠的全能胚胎 干细胞(ES细胞)在甲基纤维素培养中并观察到 克隆扩张的ES集落中的红系细胞和巨噬细胞。我们有 还从一只小鼠的文库中分离到两个小鼠的cdna克隆。 多潜能造血细胞系(FDCP-Mix)的杂交 带有两个人类造血特异性基因Vav和HS-1，它们的功能 都是未知的。我们的具体目标是确定VaV和HS-1何时 在造血干细胞分化的早期阶段表达 以及它们表达的紊乱是如何影响造血的。因此， 我们建议：1)确定ES细胞的最佳生长条件 使用各种细胞因子的造血细胞，包括最近的 鉴定的干细胞因子，SCF-1；2)鉴定和测序 我们分离的小鼠cDNA克隆，确定其与人的同源性 克隆及RNA分析鉴定其在大肠杆菌中表达的特异性 小鼠造血细胞；3)制备抗VaV多克隆抗体和 HS-1使用融合蛋白并使用抗体进一步表征 用SDS-聚丙烯酰胺凝胶分析其蛋白对造血细胞和 应用免疫细胞化学技术定位和建立动力学 在ES细胞来源的造血细胞中的蛋白表达和4)使用 、反义RNA载体和反义寡核苷酸阻断 这些蛋白在ES细胞中的表达，以了解其 在造血分化中的作用。 这些实验应该1)确定这样一种方法在 用于未来造血研究的体外模型系统，2)揭示 造血特异性基因的功能和意义3) 有助于研究造血系统的病理变化 细胞和4)与我们对基本过程的理解有关 对细胞分化的影响。

The central goal of this proposal is to investigate the cellular and molecular events during the early stages of primitive stem cell commitment to the hematopoietic lineages. We have studied murine totipotent embryonal stem (ES) cells in methyl-cellulose culture and observed the emergence of erythroid cells and macrophages in clonally expanding ES colonies. We have also isolated two murine cDNA clones from the library of a murine multipotential hematopoietic cell line (FDCP-Mix) which cross hybridize with two human hematopoietic specific genes, vav and HS-1, whose functions are unknown. Our specific aims are to determine when vav and HS-1 are expressed in the early stages of hematopoietic stem cell differentiation and how perturbation of their expression affect hematopoiesis. Therefore, we propose: 1) to define the optimal growth conditions for ES cell derived hematopoietic cells using various cytokines including the recently identified stem cell factor, SCF-1; 2) to characterize and sequence the murine cDNA clones we have isolated, determine their homology to human clones and evaluate by RNA analysis the specificity-of their expression in murine hematopoietic cells; 3) to generate polyclonal antibodies to vav and HS-1 using fusion proteins and use the antibodies to further characterize their protein by SDS polyacrylamide gel analysis of hematopoietic cells and employ immunocytochemical techniques to localize and establish the kinetics of protein expression in ES cell derived hematopoietic cells and 4) to use ,anti-sense RNA vectors and anti-sense oligonucleotides to block the expression of these proteins in ES cells in order to understand their function in hematopoietic differentiation. These experiments should 1) ascertain the usefulness of such an in vitro model system for future studies in hematopoiesis, 2) shed light on the function and significance of hematopoietic specific genes, 3) contribute to the investigations of pathological changes in hematopoietic cells and 4) be relevant to our understanding of the ,fundamental process of cellular differentiation.