Over-expression Of H1foo In Reprogramming Of Porcine Fibroblast Into Porcine IPSCs

H1foo（oocyte-specific linker histone）,the member in histone families, is locatedspecificly in mammalian oocytes and primary embryos.There exists fast replacement betweenH1foo and its counterpart, which do help to open the chromosome in donor nuclear andcomplete reprogramming.iPSC technology is another reprogramming strategy different from somatic nucleartransfer. Many reports show that an open chromosomal structure facilitates somaticreprogramming and increases the induction efficiency of iPSCs. Therefore, in order toinvestigate whether H1foo has an effect on the induction efficiency of iPSCs, a transientexpression of H1foo was conducted in the induction of porcine iPSCs.Two consecutive infections were conducted to the porcine fetal fibroblasts (PFF) byadding DOX-inducible lentiviral vectors expressing OCT4, SOX2, KLF4and c-MYC.Another vector pVenus-H1foo was electro-transferred into the OSKM-PFF and the cellstransferred pVenus were acted as negative control. After several days, the obtained colonieswere identified by IF and AP staining as iPSCs. By counting the number of iPSCs colonies,the results are as follows:1. The lentiviral vectors of OSKM-PFF expressing the four factors were controlled byDOX and porcine iPSCs can be induced by the system.2. The transfection efficiency of pVenus-H1foo and pVenus is about60%. H1foo islocated in the nuclear of PFF while pVenus is in the cytoplasma.3. The cells transferred pVenus-H1foo and pVenus can be induced into iPSCs identifiedby AP and IF.4. The number of AP-positive colonies obtained in PFF with pVenus-H1foo or pVenusis almost the same.Besides, when the PFF were first induced into iPSCs by using Yamanaka four factorsOCT4/SOX2/KLF4/MYC in our lab, there is another kinds of colonies occurred before thegeneration of iPSCs. They are named as induced Trophoblast (iTR) showing an epithelialphenotype. Interestingly, some factors related to trophoblast lineage specification wereup-regulated in iTR, such as GATA2, DLX3, CDX2and ELF5. Moreover, iTR colonies can self-renew and showed high telomerase activity. So, we want to know whether the hypothesisthat iTR colonies contain TR stem cells is true.Two kinds of iTR were divided by their phenotypes when culturing, named iTR-Type2and iTR-Type3. The results of PCR and microarray indicated that iTR-Type2may be the TRstem cells and iTR-Type3may be the differentiated TR cells.Finally, we compared porcine iPSCs, iTR-Type2and iTR-Type3by using WesternBlotting and immunofluorescence. The results show that the pluripotent marker POU5F1canbe detected in iTR-Type2together with some TR-associated transcription factors, forexample, GATA2, GATA3and CDX2while those differentiation-related markers such asGATA4, SOX17and ELF5can be detected in iTR-Type3.