Induction Of MiPSCs And Construction Of Mammary-like Organoids With MiPSCs

Objective:1.To establish mouse induced pluripotent stem cells(mi PSCs)derived from mouse embryonic fibroblasts(MEF)and confirm the effect of ROCK inhibitor on the reprogramming rate and efficiency.2.To investigate the roles of ROCK inhibitor in maintenance culture of mi PSCs.3.To generate mouse mammary-like organoids from mi PSCs in vitro and establish a model for exploring the mechanism of breast cancer initiation.Methods:Firstly,we used mouse embryonic fibroblasts(MEF)from OG2-OSKM transgenic mice as the somatic cells and then activated expression of Oct4,Sox2,Klf4 and c-Myc by Doxycycline(Dox)to induce MEF reprogramming.During the process of induction,we supplemented with Y27632,a competitive inhibitor of Rho-associated coiled-coil containing kinase(ROCK).Then,we observed and recorded the date of colony formation and GFP fluorescence,and we detected pluripotency by alkaline phosphatase(AP)staining.The comparison of GFP positive and AP positive clones reveals the effect of ROCK inhibitor on reprogramming.We treated MEF derived from C57BL/6 mice with Mitomycin C(MMC)as the feeder layer cells.Next,we picked and cultured monoclone in mouse embryonic stem cells medium(ESM)to establish mi PSCs.The effect of ROCK inhibitor on survival and passage rate of mi PSCs was reflected by colony number,size,GFP fluorescence and AP staining.Moreover,we detected the expression of pluripotent marker genes such as Oct4 and Nanog by Western Blot to detect the change of stemness by the addition of Y27632.Then we generated mouse mammary-like organoids by mi PSCs differentiation.In the first step,mi PSCs were cultured in the Mammo Cult medium to induce cells differentiation into non-neural ectoderm cells,referred to as m EB(Mammo Cult medium-cultured EB),and enrich mammary stem cells.In the second step,we embedded m EB in Matrigel floating gel and cultured in the Epi Cult B medium for 5days,and then we supplemented the medium with hormones and growth factors such as hydrocortisone,insulin and FGF10 to stimulate the generation of mammary cells.Finally,the luminal marker CK8/18 and basal marker P63 were detected by IF staining to validate existence of mammary cells in the organoids.Results:1.mi PSCs had been successfully constructed,and we have built a more efficient induction method by the addition of ROCK inhibitor.The rate and efficiency of MEF reprogramming and the survival of mi PSCs passage were promoted by ROCK inhibitor(Y27632).The stemness maintenance of mi PSCs was enhanced as well.2.Mouse mammary-like organoids had been generated.mi PSCs were differentiated into m EB with emerging cavities after 10-day suspension Mammo Cult culture.Then the branched alveolar and acinar mammary-like structures formed in the cell mass after 13-day 3D Epi Cult-B culture.Furthermore,hydrocortisone,insulin,FGF10 and FGF2 play important roles in the process of mammary-like organoids generation.Conclusion:1.ROCK inhibitor can improve the technique of mi PSCs establishment.2.Two-step differentiation procedure composed of suspension culture and 3D culture can be used to generate mouse mammary-like organoids.