An In Vitro Fundamental Study:hADSCs Differentiate Towards RPEs And MiR-29a Regulates RPC Proliferation And Differentiation

objectiveTo investigate whether retinal pigment epithelial cell-conditioned medium(RPECM)can induce adipose derived stem cells(ADSCs)to differentiate towards RPE with enhanced proliferation and migration potentials and the effects of miR-29a on retinal progenitor cell(RPC)proliferation and differentiation for future application in retinal degenerative diseasesMethods1.qPCR,western blotting and immunocytochemistry were used to detect the proliferation of induced cells and the expression of related markers of retinal pigment epithelial cells under retinal pigment epithelial cell-conditioned medium.Cell migration assay was performed to detect cell migration potential2.Under proliferation medium,the proliferation potential of RPC treated with miR-29a mimics or inhibitors was evaluated by immunocytochemistry,Western blotting and CCK8 assay;under differentiation medium,the effects of miR-29a on RPC differentiation were detected by qPCR,immunocytochemistry and Western blottingResults1.Retinal pigment epithelial cell-conditioned medium could induce adipose derived stem cells to express RPE markers such as RPE65,CK8 and bestrophin.The percentage of Ki-67-and BrdU-positive cells among hADSCs incubated with RPECM was higher than that of the hADSC control or the RPE control.The cell migration rate(migration cell number/total cell number)in the hADSCs incubated with RPECM was higher(34.43%±0.57%)than in the hADSCs control(24.47%±0.80%)and the RPE control(14.57%±0.63%).2.RPCs treated with miR-29a mimics or inhibitors were cultured under proliferation or differentiation conditions,the overexpression of miR-29a showed a notable decrease in the nestin,Pax6 and Ki67 expression levels.Conversely,miR-29a knockdown induced the opposite effect.miR-29a mimic-treated RPC cultures exhibited higher levels of retinal neuronal cell markers(Rhodopsin,β3-tubulin,PKC-α,Brn-3a(a ganglion cell marker)and Calbindin(horizontal and ganglion cell marker)),and GFAP than the control cells.However,when the cells were treated with the miR-29a inhibitor,the levels of these markers were downregulated.conclusion1.RPECM could induce hADSCs to differentiate towards RPE with higher proliferation and migration potentials,which may aid in applications for hADSCs in RPE regenerative therapy2.miR-29a can promote the differentiation of RPCs but inhibit their proliferation.