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 its defective function. For the realistic medicine, 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 and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.