472. Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector

Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete their defective function as realistic medicine. However, the safety and efficacies of using differentiated human ES cells in vivo cannot be confirmed because of ethical regulation. So the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro differentiation of CMES cells to hematopoietic cells and developed the exogenous gene transfer methods suitable for CMES cells in order to study the feasibility of future gene modified ES cell therapy. First, in our in vitro culture condition, we could mainly obtain CFU-M colonies, occasionally CFU-GM and BFU-E colonies, although the frequency of inducing multipotent hematopoietic stem cells from CMES cells was low. To estimate this result as molecular level, we examined the expression patterns for various hematopoietic gene markers by RT-PCR during in vitro differentiation. The results showed that CD34, one of stem cell markers, was firstly detected at culture day14 and other markers, c-kit, and AC133, were also expressed in early phase. However, the expression of CD45 and gata1 could not be detected at least in this condition. These data were quite correspond to the results of CFU assay, suggesting that our culture condition is incomplete for hematopoietic induction although ES cells differentiated into mesodermal cells to some extent. Next we investigated the possibility of hematopoietic stem cell induction by gene transduction. To introduce exogenous DNA into CMES, VSV-G pseudotyped human immunodeficiency virus (lentiviral) vectors containing several kinds of promoters and GFP gene were constructed. When the HIV vector containing GFP gene under EF1a promoter was used, higher efficient transgenes of GFP could be confirmed in both undifferentiated and differentiated CMES cells. Using this vector system, we are now transducing several genes related to hematopoiesis such as tal1, gata1, gata2, bmi1, and hoxB4 genes into CMES cells to induce hematopoietic stem cell differentiation efficiently in vitro and in vivo.