Generation Of Induced Pluripotent Stem Cells From Porcine Fetal Fibroblasts By Defined Factors

Generation of Induced Pluripotent Stem Cells from Porcine Fetal Fibroblasts by Defined FactorsPluripotent cells, which derived from any differentiated cell type through ectopic expression of transcription factors, were designated as induced pluripotent stem (iPS) cells, exhibiting morphology and growth properties indistinguishible to ES cells and expressing ES cell marker genes. With the ability to differentiate into all types of cells, iPS cells technology has enormous potential values in generation of human disease models, drug screening, toxicology and regenerative medicine. The current research was attempted to generate porcine iPS by defined factors EGFP fusion protein, which would be of great help to better our understanding of nuclear reprogramming mechanisms, and enrich our means of examine safety and effectiveness of iPS cells in human, as well as to extend the applications of swine iPS cells.Experiment 1. To construct lentiviral expression vector with defined factors geneOpen reading frame (ORF) of porcine Sox2, Klf4 and c-Myc gene was successfully amplified respectively by RT-PCR from the primordial genital ridges and blastula. Human OCT4 gene was amplified directly by PCR from plasmid pMX-hOCT4. After confirmation by DNA sequencing, such correct sequences of defined factors were inserted individually into retroviral vector pLEGFP-N1. Recombinant retroviral vector pLEGFP-hOCT4/pSox2/pKlf4/pMyc was obtained respectively. Next, DNA sequences containing CMV promoter, defined factors and EGFP were amplified respectively from pLEGFP-hOCT4/pSox2/pMyc/pKlf4 by PCR. The lentiviral vector pLL3.7 was modified by removing U6 promoter, CMV promoter and EGFP sequences. Lastly, modified pLL3.7 was ligated to DNA sequence containing CMV promoter, defined factors and EGFP by T4 DNA ligase. Thus, recombinant lentiviral vector pLL-hOCT4/pSox2/pKlf4/pMyc-EGFP was constructed well respectively. Then 293T cells were transfected with recombinant lentiviral plasmids respectively. The results showed that the cloned sequences of pig Sox2, Klf4 and c-Myc gene were highly homologous to corresponding genes ORF published in genebank respectively. All the four fusion proteins hOCT4/pSox2/pKlf4/pMyc-EGFP was located in the nucleus of 293T cells as their natural localization. This confirmed that recombinant lentiviral vector pLL-hOCT4/pSox2/pKlf4/pMyc-EGFP was constructed correctly.Experiment 2. To generate pig iPS cells by lentiviral vector with defined factorsSeven porcine fetal fibroblasts (PFFs) cell lines were established from 7 porcine fetuses of Duroc-Landrace-Yorkshire crossbred at 57d of gestation by using explants-seeding method. Lentiviral plasmids harboring four defined factors and packaging helper plasmids were co-transfected into 293T cells by calcium phosphate transfection method. Four defined factors lentivirus generated by 293T were concentrated to reprogram PFFs. PPFs, expressed exogenous defined factors genes, were then sub-cultured in stem cell medium supplemented with 1000U/mL LIF and 4ng/mL bFGF, and as a consequence the clear-cut cell colonies were gradually derived. The cells in such colonies had large translucent nuclei and a high nucleocytoplasmic ratio. All of the cells grew at similar rates, requiring sub-culture at a roughly 1:4 ratio every 3⁴ days. They exhibited normal karyotype, and expressed alkaline phosphatase (AP), Oct4 and Nanog. Unlike human ES cells, such porcine cell colonies were positive for SSEA-1, but negative for SSEA-3/4 and Tra-1-61/81. Teratomas containing tissues of ectoderm, mesoderm, and endoderm formed gradually after subcutaneous injection of these cells into BALB/cA nude mice dorsal skin. Taken together, our results demonstrated that these derived cell colonies can exhibit very similar morphology and characteristics to ES cells, could be termed as pig iPS cells.Experiment 3. To generate pig iPS cells by defined factors recombinant proteinsAs exogenous genes were randomly inserted into the target cells genome, it possibly brought the potential hazard of insertional mutagenesis. Therefore it is necessary to seek for some new methods to induce somatic cell reprogramming without virus. In view of above study results, we attempted to use defined factors recombinant proteins carring cell-penetrating peptide (CPP) for generation of porcine iPS cells, which would be of tremendous benefits for the safe applications of iPS cells. Defined factors genes were amplified by PCR with specific primers of 9 arginines (R₉) from recombinant plasmid pLL-hOCT4/pSox2/pMyc/pKlf4-EGFP and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the four recombinant plasmids were then transformed into BL21 (Escherichia coli) strain respectively. After IPTG induction, hOCT4/pSox2/pMyc/pKlf4-R₉-EGFP fusion proteins were purified using Novagen His-Bind kit and confirmed by SDS-PAGE respectively. Then defiend factors recombinant proteins were added into PFFs medium every 48h to generate pig iPS cells. Our results showed that such purified hOCT4/pSox2/pMyc/pKlf4-R₉-EGFP fusion proteins could enter into PFFs efficiently, and most of them located in nuclei. PPFs were sub-cultured in stem cell medium condition and treated with defined factors recombinant proteins for 6 cycles simultaneously; the clear-cut cell colonies were gradually derived. These cells had large translucent nuclei and a high nucleocytoplasmic ratio. They were positive for AP, Oct4 and Nanog. It confirmed that recombinant prokaryotic expression vector for the hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP was constructed correctly. And the transduction activity of hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP fusion protein to PFFs was proved respectively. Recombinants proteins hOCT4/pSox2/pKlf4/pMyc-R₉-EGFP can reprogram PFFs, and reprogrammed cells exhibited the morphology of ES cells and express ES cell-specific genes, such as AP, Oct4 and Nanog.Form above results, we concluded that:1. The open reading frame (ORF) of pig Sox2, Klf4 and c-Myc genes were obtained from the primordial genital ridges and blastula by RT-PCR. Lentiviral vectors of defined factors (including pig Sox2, Klf4 and c-Myc genes, human OCT4) EGFP fusion protein were constructed successfully by genetic engineering technology. The mechanism and characteristics of defined factors in cells reprogramming could be tracked effectively by constructed defined factors EGFP fusion protein.2. Pig iPS cells were generated successfully from PFFs by defined factors EGFP fusion proteins lentivirus. Some cells in pig iPS colonies expressed defined factors EGFP fusion proteins, the others showed there was no or weak expression of defined factors EGFP fusion proteins under fluorescence microscope. It suggested that different PFFs may require different time to complete reprogramming process, or different cells may start reprogramming process at different time points.3. Reprogrammed cells exhibited the morphology of ES cells and expressed ES cell marker genes, such as AP, Oct4 and Nanog, were generated from PFFs by defined factors recombinant proteins with cell penetrating peptide------R₉. This would provide new ideas for the induction of porcine somatic cells reprogramming.The innovation of the present study was for the first time, to our knowledge, to establish pig iPS cells by defined factors (including pig Sox2, Klf4 and c-Myc genes, human OCT4) EGFP fusion proteins lentivirus. Moreover, first attempt to reprogram directly PFFs by defined factors recombinant proteins.