The Role Of Linc-iNSC In The Safety Of Mouse Embryonic Stem Cell Transplantation

Background and Objective: Our previous study found that the expression level of long intergenic noncoding RNA(linc-iNSC)on chromosome 16 in neural stem cells(NSCs)/induced neural stem cells(i NSCs)was 1100 times higher than that of embryonic stem cells(ESCs)/induced pluripotent stem cells(i PSCs).Given that both NSCs/i NSCs and ESCs/i PSCs have pluripotency,but the latter has strong tumorigenicity,it is speculated that linc-iNSC may be related to the nerve differentiation,proliferation and tumorigenesis.In this study,the effects of linc-iNSC on the proliferation,pluripotency and nerve cell differentiation of mouse embryonic stem cells(mESCs)were investigated in vitro,and then mESCs overexpressed by linc-iNSC were transplanted into the cerebral cortex of mice.The effects of linc-iNSC on mESCs proliferation,tumorigenicity and nerve cell differentiation were investigated in vivo.Methods: 1)mESCs cell lines were constructed with lentivirus overexpressing linci NSC(OE group)and no-load control lentivirus(CTRL group),and the gene overexpression was verified by quantitative fluorescence PCR(QPCR).2)Cell proliferation was detected by CCK-8 and verified by immunofluorescence staining with Ki-67 antibody.3)Cell apoptosis and cell cycle were detected by flow cytometry.4)Cell pluripotency was detected by Nanog,Oct4 and Sox2 antibodies,and the expression level of RNA was detected by QPCR.5)Nerve cell markers Neu N,Olig2 and GFAP antibodies were used to detect the differentiation ability of nerve cells,and RNA expression levels were detected by QPCR.6)The two groups of cell lines obtained in vitro were injected into the cerebral cortex of C57BL/6 mice,and the grams of tumors generated by the transplanted cells were compared 14 days after cell transplantation and the nature of the tumor was determined by HE staining.The differentiation of nerve cells was detected with Neu N,Olig2 and GFAP antibodies 28 days after cell transplantation.The protein expression level was detected by WB.Results: 1)mESCs cell lines with stable overexpression of linc-iNSC were successfully constructed.2)Cell proliferation in OE group was slower than that in CTRL group;Ki-67 antibody staining showed that the fluorescence expression intensity of OE group was also weaker.3)The apoptosis rate of OE group was higher than CTRL group,especially the early apoptotic cells were significantly more than CTRL group;The G1 phase was prolonged and the G2/M phase was shortened in the OE group.4)The result of staining with Nanog,Oct4 and Sox2 antibodies showed that the fluorescence intensity of OE group was weaker than that of CTRL group,which was consistent with the results of QPCR.5)The result of staining with Neu N,Olig2 and GFAP antibodies showed that the fluorescence intensity of OE group was stronger than that of CTRL group,which was consistent with the results of QPCR.6)At 14 days after transplantation,HE staining showed that malignant tumor could be formed in both groups,but the tumor in OE group was smaller than that in CTRL group.7)At 28 days after cell transplantation,the result of staining with Neu N,Olig2 and GFAP antibodies showed that OE group had stronger fluorescence intensity and higher protein expression than CTRL group.Conclusions: 1)linc-iNSC can reduce the pluripotency of mESCs;2)linc-iNSC can inhibit the proliferation of mESCs;3)linc-iNSC can promote apoptosis of mESCs;4)linc-iNSCs can promote the differentiation of mESCs to nerve cells.