Hematopoietic Development of Multipotent Germline Stem Cells Derived from Neonatal Mouse Testis In Vitro and In Vivo.

Recently multipotent stem cells have been established from neonatal mouse testis (Shinohara et al, Cell 2004). This multipotent germline stem cell (mGS) is very similar to embryonic stem cells. It differentiates into various types of somatic cells in vitro and produces teratomas after inoculation into mice. Here we show the characteristics of hematopoietic development from mGS cells.

mGS cells were maintained on mitomycin C treated mouse embryonic fibroblasts. In undifferentiated state, surface markers were almost the same among mGS, ES and EG cell; E-cadherin⁺, β1 integrin⁺, CD31⁺ and c-kit⁺. For induction, undifferentiated mGS cells were cultured on OP9 stromal cell line. Four days after induction, we detected and sorted FLK1⁺ cells as much as ES cells. Flk1⁺ cells were further cultured on OP9 with various cytokines. Erythroid, myeloid, lymphoid, megakaryocyte and mast cells as well as endothelial cells and beating cells were obtained in the same manner as the induction of ES cell and EG cells. mGS cells had colony forming abilities including mix and magakaryocyte colonies. The number of mixed colony was dependent on the combination of cytokines. In erythroid differentiation, mGS showed the two waves of embryonic and definitive erythroid production, which was proved by RT-PCR and immunochemistry. When hematopoietic cells derived from GFP⁺ mGS cells that were cultured on OP9 for 6 days were transplanted into BM of NOD^γ mice directly, GFP⁺ cells were detected in the BM and Spleen by FACS analysis and PCR 4 months after transplantation. FACS analysis showed that GFP⁺ cells were detected in the side population when BM cells of transplanted mice were stained with Hoechst33324. Immunostaining of the slice of BM showed some GFP⁺ cells were attached to the endosteal region that is thought to be the niche for hematopoietic stem cells.

Thus, mGS cells have as much capabilities of blood cell production as ES cells and were showed having hematopoietic reconstitution ability. Considering mGS cells can be obtained from postnatal mice, they have strong advantages in research for clinical application.