Establishment Of 3i Culture Systemfor Porcine Pluripotent Stem Cells

The development of pluripotent stem cells provides ideal materials for regenerative medicine,disease models and drug screening studies.Pigs are ideal animal models since they are of similar physiological structure and organ size compared to human,with relative easy availability compared to other animal models.The progress of porcine pluripotent stem cells is very slow compared with that of humans and mice,and the difficulty is mainly to find suitable conditions for pig pluripotent stem cells.In this study,the drug-inducible pig iPS cells were used as a cell model to screen the proper culture condition which suitable for maintaining self-renewal of porcine iPS cells.1.Establishment and verification of pig DOX-iPSCs.Porcine embryonic fibroblasts(PEFs)were transduced with Doxycycline-inducible lentiviral expression vector which carrying murine four factors,Oct4,Sox2,Klf4 and c-Myc.Cells were induced in LF2 i medium supplemented with Dox for 5-7 days.When Mouse ES cell-like clones formed,the clones were picked and cultured in the induction medium.The results of PCR and RT-PCR results showed that FUW-TetO-OSKM and FUW-M2 rt TA constructs were inserted into the genome of three DOX-iPS cells,and the pluripotency gene was expressed.The immunofluorescence results showed that OCT4 and SOX2 were highly expressed,and the proteins were located in the nucleus.SSEA-1 was weakly expressed and was located in the cell membrane.NANOG expression was relatively low in these cells.These DOX-iPS cells are of normal karyotype(36+XY).After suspension culture,embryoid bodies(EB)could be formed,and could differentiated into cells which express three germ-layer markers,AFP,TUJ1 and DESMIN.The above results showed that these established pig DOX-i PS cells are of proper pluripotency.2.Establishment of reprogramming system induced by Dox.Since Dox induces the expression of exogenous pluripotent genes.To detect the effect of Dox in DOX-iPS cells,we observed the changes of pluripotency relative genes with/without Dox supplement.When Dox was removed from the LF2 i medium,the colony morphology disappeared gradually,and the DOX-iPS cells completely differentiated into fibroblast-likecells without AP activity when the cytokines were removed from the medium.The results of genomic PCR showed that the genomic insertions were still retained in the differentiated cells.RT-PCR results showed increased fibroblast marker.When the differentiated DOX-iPS cells were re-placed in Dox-containing LF2 i medium for 4 days,AP positive secondary iPS colonies were formed again.Quantitative RT-PCR showed that the second generation iPS(2ed DOX-iPS cells)cells were of similar gene expression pattern compared with the original ones.In this study,Dox-induced cell re-reprogramming system was established and provides experimental model for screening pig iPS culture conditions.3.Culture condition screening and slection 2i medium for DOX-iPSCs.To facilitate the selection of proper culture condition for porcine pluripotency,we tried to establish a screening system using DOX-iPS cells.Firstly,once LIF and b-FGF were removed from the LF2 i medium collectively,the morphology of iPS colonies seemingly much stereoscopic.The cells presented with stronger AP activity and expresses higher endogenous OCT4,SOX2,and NANOG.Secondly,when the re-reprogramming experiments were carried out using differentiated DOX-iPS cells in the above mentioned conditions,only LF2 i condition could reprogram these cells into iPSCs colonies with sufficient quantity and quality.While the newly confirmed 2i condition which was better for maintain of DOX-iPS cells presented with lower quantity and quality in the colony formation during the re-reprogramming procedure.The above results indicated that LF2 i medium is much suitable for reprogramming of porcine cells,while 2i medium is more suitable for maintaining the status of iPS cells.While all these tested conditions were insufficient for long term maintain of pluripotency once the exogenous genes were shut down by Dox withdrew.4.Establishment of 3i culture system.Although 2i media can increase the expression of endogenous pluripotent genes in DOX-iPS cells,Dox is still dependent on the self-renewal of iPS cells.We added different cytokines and small molecules on the basis of 2i,and tried to establish a culture condition without Dox and capable of maintaining DOX-iPS cell self-renewal.The expression of endogenous OCT4 and NANOG in DOX-i PS cells was significantly increased after adding BMP4,SCF,IL-6 and platelet lysate(PL)instead of serum.After removal of Dox,some iPS clones could still maintain AP positive state,But still can not continue to pass on.DOX-iPS cells maintained good clonal morphology and high expression of endogenous pluripotent genes under no Dox conditions after PD0325901 was added to the culture system.Thus,3i culture conditions were established.The pluripotent stem cells of different origins werecultured in 3i condition,and their self-renewal ability was maintained,and the expression of endogenous pluripotent gene was improved in different degree.The above results show that we have established a 3i culture condition without Dox without LIF / bFGF,and this condition can maintain the self-renewal ability of pig pluripotent stem cells.