Development of efficient hematopoietic stem cell proliferation systems using our own established ES cells of small monkey, common marmoset

为了从我们已建立的普通绒毛(CM)胚胎干细胞(ES)中建立高效的造血干细胞和造血分化系统，我们首先建立了TALL/SCL基因转导的CME(tal1/scl)细胞系。我们前期的研究发现，TALL/SCL基因转导可以在体外有效地将CME细胞分化为造血细胞。我们首先发现TAL1/SCL细胞在体外具有向淋巴细胞和巨核细胞分化的能力，但分化效率较低。另一方面，我们使用免疫缺陷的NOG小鼠进行的体内研究表明，由于早期肺癌的产生和死亡，骨髓内注射TALL/SCL细胞的造血重建非常差。然后，我们试图寻找第二个基因，该基因可能有助于体内TALL/SCL细胞的造血细胞分化。为此，我们构建了表达人胎肝VSV-G伪型慢病毒载体文库。该文库由8×10~(-7)个个体克隆组成，平均长度为2.1kb。对几个组分的DNA测序分析表明，其中60%含有全长cDNA。当我们用293T细胞文库生产病毒时，100%的细胞进行了基因转导。转导基因的cDNA平均长度约为1kb。用抗血糖素抗体进行流式细胞仪分析表明，转导基因的细胞正确表达了血糖素，这是已知在人胎肝中大量表达的基因之一。利用这个新的人胎肝cDNA文库，我们已经克隆了几个促进TALL/SCL细胞造血或诱导细胞因子依赖性白血病细胞系细胞因子非依赖性的候选基因。通过在体外和体内有效地将ES细胞分化为造血细胞的基因的鉴定，将为将来实现更安全、更有效的细胞补充治疗奠定基础。

Toward the goal of establishing efficient hematopoietic stem cells and hematopoietic differentiation system from our own established common marmoset(CM) embryonic stem(ES) cells, we first established tall/scl gene transduced CMES (tal1/scl) cell lines. Tall/scl gene transduction was found to differentiate CMES cells efficiently to hematopoietic cells in vitro in our previous studies. We first of all found in vitro differentiating capacity of the tal1/scl cells to lymphocytes and megakaryocytes, although the efficiency was low. On the other hand, our in vivo studies using immune deficient NOG mice demonstrated very poor hematopoietic reconstitution with intramarrow injected tall/scl cells because of early lung tumor production and death. Then we tried to find out second gene which may assist the hematopoietic differentiation of tall/scl cells in vivo. For this purpose, we constructed human fetal liver cDNA expression VSV-G pseudotyped lentiviral vector library. This library consisted of more than 8x10^7 individual clones with average cDNA length of 2.1kb. DNA sequencing analysis of several fractions showed 60% of them contained full length cDNAs. When we produced virus using the library with 293T cells, 100% of cells demonstrated gene transduction. Average length of the gene transduced cDNA was about 1 kb. Flow cytometric analysis with antiglycophorin antibody showed the gene transduced cells expressed glycophorin, which is known to be one of the highly abundantly expressed genes in human fetal liver, properly. Using this novel human fetal liver cDNA library, we have already cloned several candidate genes which promotes hematopoiesis of tall/scl cells or induces cytokine independency of cytokine dependent leukemia cell lines. By the identification of genes which differentiate ES cells efficiently to hematopoietic cells in vitro and in vivo, safer and more effective cell supplementation therapy would be realized in future.