The Identification Of Porcine Naive-like Embryonic Stem Cells And The Study Of Directed Differentiation In Vitro

Background: Naive embryonic stem cells(ESCs)have long been considered to be true embryonic stem cells.However,to date,naive embryonic stem cells have not been successfully established in large hoofed animals.Recent studies have shown that pig blastocyst inner cell mass can be transfected with lentiviral vector carrying transcription factor and inoculated into 2i-containing LIF culture medium to obtain porcine naive embryonic stem cells.However,these ESCs probably do not have uniformity due to their originating from different blastomeres probably with different copies of foreign genes.In our previous study,we had transferred the classical mouse derived pluripotent transcriptional factors Oct4?Sox2?Klf4 and c-Myc(m OSKM)expression vectors into porcine embryonic fibroblasts(PEF)cells by nuclear translocation technology,and then obtained embryos by somatic cell nuclear transfer with the OSKM transgenic cells as donor cell.The inner cell mass were isolated and seeded into the the 3i LIF culture medium,the pig naive-like embryonic stem cell line was successfully established.However,the expression of exogenous pluripotent genes m OSKM and the initiation of expression of the endogenous porcine OSKM in the naive-like ESCs had not been investigated.To my knowledge,there is little report on the differentiation of pig pluripotent stem cells into neural cells.The differentiation of porcine pluripotent stem cells into neural cells in vitro lacks the relevant literature.Therefore,there is no available method for the differentiation of porcine embryonic stem cells into neural cells.In addition,there is no definite standard for the differentiation of porcine embryonic stem cells into mesoderm in vitro.So far,the differentiation of human and mouse embryonic stem cells into mesodermal lineage-like renal cells are mainly induced by specific induction factors,co-culture with nephrocyte and the embryoid body differentiation method.Based on the porcine naive-like embryonic stem cells established in our laboratory,this study was designed to explore the stem cell characteristics of the porcine naive-like ESCs and study the effects of the expression of the exogenous transcription factors on the start of the expression of endogenous Oct4,Sox2,Klf4,c-Myc and the differentiation potential of porcine naive-like embryonic stem cells in vitro.This study will provide some references for the establishment of authentic porcine naive embryonic stem cells and the method of in vitro differentiation of these stem cells.Objective: To identify the characteristics of porcine naive-like embryonic stem cells and the expression of endogenous and exogenous transcriptional factors OSKM.To study the differentiation potential of porcine naive-like embryonic stem cells into ectodermal lineage neural precursor cells and mesoderm lineage in vitro.Methods: The stem cell characteristics of porcine naive-like ESCs were identified by Q-PCR and immunofluorescence staining.The cell lines 1 and 4 were cultured with or without DOX.The expression of the endogenous p OSKM and exogenous m OSKM in the cell lines were evaluated via RT-PCR and sequencing.The ratio of the expression of the endogenous and exogenous OSKM was compared;the neural induction medium composed of the small molecule compound ?-mercaptoethanol,B-27 and heparin,which was used to differentiate the porcine naive-like ESCs into neural precursor cells.RT-PCR and Q-PCR were used to identify nerve-specific gene expression,and to detect whether the differentiated cells had lost pluripotency.The expression of neuronal markers Nf-l and ?III-Tubulin was detected by Western Blot and cellular immunofluorescence.The small molecule BMP4,Activin A,CHIR99021,FGF9,Herparin and RA were used to differentiate the porcine naive-like ESCs into the renal precursor cells by three-step method.The expression of the kidney-specific genes in the derived renal precursor cells was investigated by RT-PCR and Q-PCR.The expression of the renal cell marker Wt-1 in the renal precursor cells was identified by Western Blot and immunofluorescence staining.Results: The porcine naive-like embryonic stem cells were subjected to pluripotency immunofluorescence staining,the high level expression of Oct4 and Sox2 in the porcine naive-like ESCs was confirmed.The naive-like ESCs was also confirmed to be closer to the naive state since the Xist expression in them was lower than that in the primed state ESCs and the H3k27me3 ‘spots' did not existin the naive-like ESCs but existed in the primed ESCs;No.1 porcine naive-like embryonic stem cell line express endogenous transcriptional factors Oct4,Sox2,Klf4 and c-Myc at 3.2% ?35.0%?9.4% and 9.5% percentages when DOX was contained in the medium.After removing DOX,the expression of endogenous Sox2 and c-Myc reached 78.3% and21.7%.However,the expression of endogenous Oct4 and c-Myc had not increased.For No.4 naive-like ESCs cell line,only endogenous Sox2 showed expression when DOX was contained in the medium and the percentage was 10.0%.After removing DOX,the expression of endogenous Sox2 reached 100%.However,the endogenous Oct4?Klf4 and c-Myc had not initiated their expression.The neural progenitor cells obtained by small molecule induced differentiation have obvious neuronal cell structure.RT-PCR showed that the differentiated neural progenitor cells expressed a variety of neural cell-specific genes,and the Q-PCR results showed the expression of multipotent factors after differentiation significantly reduced,nerve-specific gene expression was significantly elevated.Immunofluorescence and Western Blot results showed the presence of neuronal markers ?III-Tubulin and Nf-l in the the differentiated neural progenitor cells.The naive-like embryonic stem cells were firstly induced into the middle mesoderm followed by induction into renal progenitor cells by small molecules or different small molecule combinations,which were identified by RT-PCR and Q-PCR.The mesoderm lineage and renal cell-associated gene expression in the renal progenitor cells were significantly increased.Western Blot and immunofluorescence showed that the expression of the kidney marker William's tumor suppressor gene Wt-1 was present in the differentiated renal precursor cells.Conclusion: The characteristics and endogenous transcriptional factors expression of the porcine naive-like embryonic stem cell line were verified.The expression characteristics of the endogenous and the exogenous transcriptional factors were different in different porcine naive-like ESCs lines.However,all the naive-like ESCs lines partially relayed on the expression of exogenous transcriptional factors and the initiate of endogenous Sox2 was vitally important to maintain the ESCs growth and selfrenew after DOX was removed from the medium.The differentiation of porcine naive-like embryonic stem cells into neural precursor cells and renal precursor cells was realized via direct induction in vitro.These results will be helpful for the establishment of an authentic porcine naive ESCs and the method for porcine naive ESCs differentiation in vitro.