Analysis of the maintenance of hematopoietic stem cells during embryogenesis

1.KS-13卫星信号分析。通过KS-13的数据库，我们鉴定了在红系分化过程中表达下调的基因Hmgn2。Hmgn2基因在白血病细胞系中的过表达导致体外抑制红系分化(Kulkeaw等人，2012年)。识别调节造血干细胞分化的新分子。胎肝由造血细胞、肝母细胞和肝窦内皮细胞组成。通过流式细胞术，我们筛选出DLK-1(+)肝母细胞，主要表达细胞外基质，如纤维连接蛋白和玻璃体连接蛋白，以及造血细胞因子、SCF和EPO(Sugiyama等，2011)。我们已经提交了关于细胞外基质的论文(Sugiyama等人，正在修订中)。为了进一步研究肝母细胞的作用，我们使用了既缺乏肝母细胞又缺乏肝细胞的突变小鼠。我们从E19中分离出CD45(+)、c-Kit(+)、SCA-1(+)造血干细胞。5将突变株和野生型对照的胎肝移植到受照小鼠体内。移植后3~6个月，用流式细胞仪分析移植后的造血重建情况。野生型对照的嵌合率达到5%以上，而突变体的嵌合率小于0。5%。利用这些样本，我们进行了微阵列分析，并确定了几个可能调节造血干细胞分化的候选基因。

1. Analysis of KS-13 signal. By database of KS-13, we identified Hmgn2, which gene expression is down-regulated during erythroid differentiation. Over-expression of Hmgn2 gene into leukemic cell lines resulted in suppression of erythroid differentiation in vitro(Kulkeaw et al., 2012).2. Identification of novel molecules that regulate differentiation of hematopoietic stem cells. Fetal liver comprises hematopoietic cells, hepatoblasts and sinusoid endothelial cells. By Flow cytometry, we sorted out Dlk-1(+) hepatoblasts, which primarily expressed extra-cellular matrix such as fibronectin and vitronectin, in addition to hematopoietic cytokines, SCF and EPO(Sugiyama et al., 2011). We have submitted the paper about extra-cellular matrix(Sugiyama et al., in revision).To further investigate the roles of hepatoblasts, we used the mutant mice lacking both hepatoblasts and hepatocytes. We sorted out CD45(+) c-Kit(+) Sca-1(+) hematopoietic stem cells from E19. 5 fetal liver of both mutants and wild type control and transplanted into irradiated recipient mice. Three to six months after transplantation, hematopoietic reconstitution was analyzed by flowcytometry. The chimerism of wild type control was achieved to over 5%, whereas that of mutants was less than 0. 5%. Using these samples, we performed micro-array analysis and identified several candidate genes that likely regulate differentiation of hematopoietic stem cells.