The Experimental Studies Of The Committed Differentiation Of HESC And HiPSC In Vitro

Objective:To study an experimental protocol that human Embryonic stem cell (hEsc) and human inducible pluripotent stem cells (hiPSC) can generate the progenitors of the inner hair cells and nerve cells in vitro.The differentiation stages and the expression of the markers were discussed.Methods:Human Embryonic stem cell and human inducible pluripotent stem cells were were separately suspended in the Suspension Culture Flask with the suspension culture medium composed of the inhibitors (DKK1, DMH1, A8301), the cytokines (insulin-like Growth Factor1,Basic Fibroblast Growth Factor) and the basal medium.And then the cells were cultured with the adherent culture methods. The experession of marker gene was analyzed by RT-PCR and immunocytochemistry.Results:The cells could aggregate to produce the embryoid body in the Suspension Culture Flask. And with the embryoid bodies grow up, they became mellow and full. When the embryoid bodies were culture by the adherent culture methods, they could form neural rosettes.As the cells grew up, the expression of the pluripotency genes nanog and otc4decreased; the expression of the specific genes otx2, nestin, pax6, pax2and pax8increased and then decreased.The expression level of the markers in the experiment group was more than the control. There was statistical significance between the experiment group and the control (p<0.05).The progenitors of the experiment group expressed pax6, pax2and nestin. Pax6-positive cells coexpressed nestin.Conclusion:1The experimental protocol that can promote the human embryonic stem cell and human inducible pluripotent stem cells to differentiate into the progenitors of the inner hair cells and nerve cells was finally determined.2The inhibitors (DKK1, DMHI, A8301) and the cytokines (IFG) can effectively promote the human embryonic stem cell and human inducible pluripotent stem cells to differentiate into the progenitors of the inner hair cells and nerve cells.3The progenitor markers temporal expression periods do not completely overlap.