Mouse Spermatogonial Stem Cells Isolated Culture And Induced Differentiation

In order to isolate and culture the spermatogonial stem cell of domestic animal successfully, apply this method for sex control and transgene breeding in domestic animal, lay the foundation for the application of illustrate differentiation and regulation mechanism of spermatogonial stem cell in domestic animal, the following experiment used the mouse as animal model and combined with traditional isolate technology, improved isolate and culture method by based on the hinge that affect the separation efficiency and culture conditions, strived to restart spermatogenesis process and differentiation into the sperm-like cells in vitro culture.Used the one step enzyme digestion method, we use the enzyme of dispaseⅡto digest the 7-day-old mouse testicular tube after bathing on 37℃water , and separates the spermatogonial stem cells (mSSCs) successfully. In vitro cultured on feeder cell after 12h, treated with the diapaseⅡ(2～3 s) again , the mSSCs colony which on adherence or half- adherence were falled off and floated in culture media. Then through the slight shake culture dish to make sure that mSSCs colony were all fell off, but the other cells and feeder cells were not fell off by enzyme, so it improved the purification of mSSCs. Isolated mouse spermatogonial stem cells (mSSCs) by the method of one-step enzymatic digestion, the mSSCs colonies were identified by morphology observation, AKP staining, immunohistochemistry. Compared the effect ofα-MEM or DMEM and the mouse embryonic fibroblasts(MEF) or the mouse seminiferous tubule cells feeder (MSF) as the feeder cells on the mSSCs colonies growth condition. Cultured the mSSCs colonies on the mouse seminiferous tubule cells feeder (MSF), treated with the media contained RA (2μmol/L) for 24 h and observed the shape change of mSSCs colonies when continuing cultured by the media ofα-MEM containing 10%FBS.The results showed that through the one-step enzymatic digestion, mSSCs were separated succinctly and successfully; the AKP staining showed the positive colour of mulberry; under the red fluorescent, the Oct-4 neucleoprotein of mSSCs colonies cells expressed positive and the membrane protein of C-Kit,β1-Integrin and Gfrα-1 expressed positive. Used mouse embryonic fibroblasts (MEF) as feeder cell, mSSCs colonies shaped the grape bunch-like or rosary-like; cultured byα-MEM and used the mouse seminiferous tubule cells feeder (MSF) , the mSSCs colonies growth state and anchoring ability were better than MEF as the feeder cells; cultured on MSF after 96 h mSSCs were obviously shaped on the board game piece distribution. After Treated with RA (2μmol/L) for 24 h, replaced new culture media withα-MEM contained 10%FBS and continue cultured for 4 days, observed that the part of mSSCs cells changed the circular cells into the cone ones, small tail-like protrusion appeared in cells. After continues cultured for 4 days, the cone cells increased and the conical head of cell were inflated, the tail changed into spiked-like protrusions and the cells appeared in sperm-like cells. It was concluded that used the method of one-step enzymatic digestion could isolate mSSCs successfully andα-MEM was more suitable for mSSCs in vitro culture.In conclusion, the above mentioned research demonstrated that the feasibility of isolating mSSCs by one step enzymatic method, explored the different mediums and feeder layers affected on the growth state of mSSCs, discovered the new culture system that MSF andα-MEM were more suitable for mSSCs in vitro culture, mSSCs could be induced to differentiate into sperm like cells by RA in this culture system.