Generation And Characterization Of Porcine Induced Pluripotent Stem Cells Reprogrammed From Adipose-tlerived Stem Cells

Induced pluripotent stem cells (iPSCs) technology not only sheds new light onembryonic stem cells (ESCs) researches in farm species but also provides a valuablealternative for derivation of bona fide ESCs in ungulates. However and unfortunately, onlyvery few porcine iPSCs (piPSCs) has been shown to be able to support full termdevelopment of chimerae or cloned embryos, and high rates of abortion, mortality andabnormalities of such embryos usually were encountered. All these are drive force thatattract scientists to seek ways of getting high-quality i.e. germ-line transmissable piPSCs,which is becoming the focus and tough job in the piPSCs area. Our objective, therefore, isto use porcine adipose-derived stem cells (pASCs) as source cell to generate piPSCs byreprogramming, and we will try to optimize somatic cell reprogramming protocols of usingpASCs as source cell type in order to harvest more safe and reliable iPSC lines, layingsolid basis for future production of cloned pigs from piPSCs, enriching methods to createtransgenic pigs used as pre-clinical models in evaluation of safety and efficacy of humaniPSCs. In this study, the following experiments were carried out.Experiment one: Isolation, culture and charaterization of pASCs. Collagenase wasused to digest fat tissue from28-day-old Landrace pig, and the primary pASCs wereisolated and purified, and followed by subculture. We performed comprehensivecharacterization of primary and subcultured pASCs of different passages. The results were:The proliferative activity of pASCs is significantly higher than porcine ear fibroblasts(pEFs) from the same pig; pASCs can be subcultured in vitro for long-term, and often havehigh aneuploidy rate which will be more severe as the passages increase; pASCs expressmesenchymal stem cell-specific markers CD44, CD90, CD29at a rate of100%,96.5%,and41.7%respectively, and0.148%for lymphocyte marker CD45,0.404%forendothelial cell marker CD31,0.08%for fibroblast marker HLA-DR; under appropriateinduction conditions, vertical differentiation shows that pASCs can produce oil red Ostaining positive mature adipocytes, and produce alizarin red staining positive osteoblastscalcified nodules after horizontal differentiation; pASCs express endogenous OCT4, SOX2,C-MYC, KLF4, NANOG and LIN28genes, but in addition to KLF4gene whose expressionlevel is a little higher than pEFs at the same passage, the remaining number ofreprogramming transcription factors express at a rare low level in pASCs; genomic DNAdemethylation intermediate5-hydroxymethylcytosine(5-hmC) in pASCs were significantlyhigher than in the same passaged pEFs (P<0.01). These results suggest that the pASCs we obtained hold high purity and possess the characteristic of differentiation plastics,indicating we successfully obtained pASCs; certain transcription factors associated directlywith the reprogramming express at low levels in the pASCs; a lower genomic DNAmethylation status of pASCs may helps itselves to regain pluripotency; high aneuploidyrate of pASCs warrants the need of screening normal karyotype cell lines before itsapplication in every use.Experiment two: Generation and characterisation of piPSCs reprogrammed by usingdrug inducible defined factors and pASCs as starting cells. Based on thefeeder-independent and serum-free iPSCs induction protocols used in mouse and human,we set pASCs as the source cell to obtain piPSCs through exogenously transducing fourhuman reprogramming factors OCT4, SOX2, KLF4, C-MYC gene under a drug inducible(RevTet-On) lentiviral expression system without contamination of heterogenous cells.The results were: In the presence of feeder cells, the formation rate of ESCs-like alkalinephosphatase (AP) staining positive colony generated by pASCs is6.28times better thanEFs, meanwhile in the absence of feeder layer, AP+clonies from pASCs are approximately6.33times more than those from EFs; under iPSCs culture medium supplemented with twosmall molecule compounds (2i, PD0325901and CHIR99021), the piPSCs resemblemorphological and passaging characteristics of mouse ESCs; after lentiviral infection,pASCs aneuploidy rate tends to arise; pASCs-derived piPSCs colonies are of AP stainingpositive, meanwhile these colonies also express stem cell-specific protein markers OCT4,SOX2, NANOG, SSEA-3and SSEA-4; key transcription factors OCT4, NANOG,DNMT3B, TERT gene are endogenously activated, while the other pluripotency relatedgenes LIN28, ESRRB, UTF1and DPPA5are also up-regulated; piPSCs can differentiateinto embryoid bodies in vitro, expressing marker genes of all the three germ layers, and toproduce teratomas in vivo; during cultivation, direct removal of doxycycline leaded thecells to rapidly differentiation. These results indicate that the reprogramming efficiency forpASCs is significantly higher than the pEFs (P<0.01), and could be successfully induced toiPSCs under feeder-independent and serum-free condition; exogenous lentivirus-mediatedgene transfer may result in iPSCs aneuploidy rate in a rise; at the late reprogramming stage,2i treatment can promote ASCs fully reprogrammed to achieve a stable pluripotent state;during the reprogramming process, sustained expression of the transcription factors hadresulted in the cells dependent on the exogenous transcription, and2i medium systemcould not sustain piPSCs self-renewal independently.In summary, we firstly, to our knowledge, established a repertoire for derivation of piPSC lines from pASCs under feeder-independent and serum-free condition, withexogenous transcription factors regulated by a drug-inducible system. These particularmesenchymal origined piPSC lines would provide excellent material in the follow-up studyof iPSCs trait, epigentic modification status and animal production, and on the other handfacilitate optimization of porcine ESCs culture system.