| Within the past decades, stem cell research has progressed remarkably,it hasplayed a huge role in promoting human medicine development. Mesenchyme stemcells were found as a new cell type, although its self-renewal and differentiationpotential were weaker than embryonic stem cells, but researchers payed moreattention on them for harvesting and culturing easily. Studies reported that in somespecies, the same regulating mechanism was existed in mesenchyme stem cells andembryonic stem cells. This found provided a new insight to study the mechanism ofembryonic stem cells. The establishment of porcine ESCs has proven elusive in spiteof decades of intense trials and a lack of success. In2006, the acquisition of inducedpluripotent stem cells injected fresh blood into the field of stem cell research. Becausethe function of induced pluripotent stem cells was similar to embryonic stem cells,which makes it be the best model for embryonic stem cell research. In light of theircompatibility with human physiology, pigs are the most attractive animal model forpreclinical studies. Up to now, there were only few researches on the pig mesenchymestem cells and induced pluripotent stem cells.Different passages of porcine MSCs (AMSCs and BMSCs) were used toinvestigate:(1) the effects on cells proliferation and differentiation ability when cellswere cultured for long time;(2) whether the different passage pMSCs had thedifferent reprogramming transcription factors expressions or not, such as Nanog, Oct4,Klf4, Sox2and c-Myc, and determined the expression locations of these factors in thepMSCs;(3) compared the efficiency and quality of the induced pluripotent stem cellswhen different types and passages pMSCs were induced. We performed the presentstudy to research on the proliferative and differentiation ability of porcinemesenchyme stem cells in vitro, and investigate advantages and disadvantages whenthe mesenchyme stem cells were induced to the pluripotent stem cells. Our resultsprovided important experimental basis for studying cellular senescence mechanism of porcine mesenchyme stem cells in vitro, gene expression regulation, as well as thequality of iPSCs which generated from different passage cells. The results arefollowing:Firstly, we determined the proliferation and differentiation ability of AMSCs andBMSCs with apoptosis detection, Semi-quantitative PCR and induced differentiationmethods. We cultured AMSCs and BMSCs to P20in vitro. The P0, P5, P10, P15andP20cells were collected for testing. The results showed that: as passage numberincreased, both types of pASCs growth capacity displayed a downward trend. Under10passages, the survival rate of pMSCs did not have obvious change; however, after10passages, there was significant decline in the survival of AMSCs (P <0.01). Inboth pMSCs, as passage number increased, the results of the CFU-F assay declinedsignificantly for all samples (P <0.01). Furthermore, different passage pMSCsexpressed apoptosis-related genes Bax and Bcl2at different level. After long-termculturing, the expression of Bcl2declined significantly for pMSCs; however theexpression of Bax increased significantly (P <0.01). The percentages of apoptosiscells in BMSCs tended to be lower than AMSCs. We successly induced the P0, P10and P20pMSCs to adipogenic and osteogenic cells, it illustrated that the pMSCs,which were cultured for long time, had the differentiation potential.Secondly, we investigated the expression of reprogramming transcription factorson different passage pMSCs and determined the localization of these factors on cellswith fluorescence quantitative-PCR and immunofluorescence staining techniques. Theresults showed that: except Klf4, the Oct4, Sox2, c-Myc and Nanog expressions hadlittle difference as the AMSCs passage number increasing, illustrated that these fourgenes expressed stabily; the mRNA of five reprogramming transcription factors wassimilar on the different passage BMSCs; BMSCs sample expressed c-Myc and Nanogat the similar levels with AMSCs; Except P20cells, other AMSCs samples expressedKlf4at a higher level than BMSCs(P>0.05). Each BMSCs sample expressed Oct4and Sox2at a higher level than AMSCs (P <0.05). The immunofluorescence stainingresults showed that the morphological appearance of pMSCs attaching to the culturedish was similar to that of fibroblasts. These reprogramming transcriptional factorsOct4, Nanog, Sox2, Klf4and c-Myc were expressed in both the nuclei and cytoplasmof the individual cellsFinally, we studied the speed and efficiency when inducing the pMSCs into iPSCs with iPS induction technology, Semi-quantitative PCR and AP stainingtechniques. We used the P0, P5and P10pMSCs as the target cells. The results showedthat: different passage cells had different reprogramming speed and efficiency. Thespeed and efficiency of P0pMSCs was significantly higher than fibroblasts (P <0.01);the speed and efficiency of P5pMSCs were lower than P0cells, but not obvious (P>0.05). P10AMSCs was not reprogrammed into iPSCs successfully. Although P10BMSCs could be induced to iPSCs, the reprogramming speed, efficiency and qualitydecreased significantly (P <0.01). Using Semi-quantitative PCR, we found that allobtained clones still expressed exogenous genes, it illustrated that cells have not beenfully reprogrammed.In conclusion, this study has confirmed that porcine AMSCs and BMSCs werepassaged to P20in vitro; proved that as the culturing time increased, cells growthability was decreasing; the expressions of reprogramming transcription factors (Oct4,Sox2, c-Myc and Nanog) on the AMSCs and BMSCs were stable, and these factorsexpressed in both the nuclei and cytoplasm of pMSCs. Inducing the cells whichexpressed endogenous transcription factors to iPSCs would be more quickly andefficiently; target cells growth capacity had significant influence on cellreprogramming. Results of this study will provide new evidence to research thesenescence and differentiation mechanisms of pMSCs; study the efficiency, speed andquality of iPSCs from endogenous gene expression and cell proliferative capacityaspects. Finally, our results provide theoretical basis for exploring pMSCs senescencemechanism and optimizing pig iPSCs induced system. |
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