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

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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.