Generation Of Induced Pluripotent Stem Cells From Adult Bovine Dermal Fibroblasts

In the study, we firstly reported the generation of bovine induced pluripotent stem cells (iPSCs) from adult Holstein cow dermal fibroblasts through retrovirus-mediated transduction of bovine transcription factors Oct4, Sox2, Nanog and Lin28. In the present study, these important results are as follows:1. Cloning of the ORF sequences of bovine Nanog and Lin28 gene.Total RNA was extracted using Trizol reagent from bovine primordial germ ridges (PGR) of fetal cattle at 50-60 days gestation. cDNA sequences were synthesized by reverse transcription (RT). The ORF full sequences of Nanog and Lin28 gene were amplified by PCR. Two gene fragments were respectively subcloned into pMD18-T vectors, then two recombinant plasmids were verified by sequencing. The sequence homology of cloned Nanog and Lin28 gene were 100% respectively to the reference sequence in GenBank sequence.2. Construction and packaging of the recombinant retroviral plasmids pMSCV-Nanog and pMSCV-Lin28.(1) The cloned Nanog and Lin28 gene were inserted into retroviral vectors pMSCVneo to construct the recombinant retroviral expression vectors pMSCV-Nanog (MN) and pMSCV-Lin28 (ML).(2) Two recombinant retroviral plasmid pMSCV-Nanog and pMSCV-Lin28 were transfected into PT67 packaging cells with lipofectamine 2000. After G418 screening, the PT67 packaging cell lines containing recombinant retroviral plasmid MN and ML were established. The titer of recombinant retroviruses were determined using NIH3T3 cells. The titers of retroviruses MN and ML reached respectively 9.19×107 cfu/mL and 7.5×107 cfu/mL. The retroviral particles shown spherical and ellipsoidal shape under transmission electron microscopy. The retroviral particle diameter was approximately 50-100nm. To assess the safety of recombinant retroviruses, the NIH3T3 cells were infected by viral supernatants. But the wild type retrovirus could not detected from infected NIH3T3 cells. The result revealed that the plasmids MN and ML could not replicate in other cells. 3. Generation of bovine iPS cells from bovine dermal fibroblasts using defined four transcription factors.(1) Bovine dermal fibroblasts (BDFs) were isolated from the ear skin of a 1.5-year-old adult female Holstein cow. The biological characteristics of BDFs, including growth curve, population doubling time and cell cycle were detected. Immunocytochemistry shown that BDFs expressed fibroblast cell-specific protein vimentin and fibronectin.(2) Equal amounts of supernatants containing each of the retrovirus pMSCV-Oct4, pMSCV-Sox2, pMSCV-Nanog and pMSCV-Lin28 (denoted:OSNL) were supplemented with 8μg/mL polybrene, mixed, and transferred into the BDFs dishes. Approximately 20 days later, the distinct colonies were appeared on cell-free human amniotic membrane (HAM). The reprogramming efficiency was approximately 0.034%. In the study, we successfully replaced murine embryonic fibroblasts (MEFs) with cell-free human amniotic membranes (HAM) as the feeder layer for cultivating bovine iPSCs (biPSCs).(3) Immunocytochemistry shown that the resulting biPSCs were strongly positive for alkaline phosphatase (AP). They strongly expressed pluripotent stem cell-specific surface antigens, including stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81, and Oct4, Sox2, Nanog and telomerase reverse transcriptase (TERT) proteins. The cells did not expressed SSEA-1 and CD117 proteins. The karyotypes analysis of the biPSCs shown a normal karyotype and number of 60 XX. The optimal frozen media for biPSCs was 70%biPSCs media+20%FBS+10%DMSO. The survival rate of biPSCs was 90.9% after thawing.4. Identification of the pluripotency of biPSCs.(1) RT-PCR revealed that the biPSCs expressed many pluripotent stem cell-specific genes, including Oct4, Sox2, Nanog and Lin28. But exogenous retroviral genes were silent using transgene-specific primers. The quantitative PCR (qPCR) revealed that the expression levels of Oct4, Sox2, Nanog, Lin28 and TERT in biPSCs were markedly increased by 103-104-fold releative to those in BDFs, the levels were comparable to those of bovine PGR. However, the expression levels of c-Myc and Klf4 were approximately 10-fold greater than those in BDFs. The expression levels shown slight changes, but the differences were not significant in the different passages of biPSCs. Western blotting analysis revealed that Oct4, Sox2, Nanog, Lin28, and Klf4 proteins were expressed in the biPSCs as in bovine PGR.(2) biPSCs effectively formed ball-shaped and cavity-like EBs in suspension culture. The attached EBs differentiated randomly into cells with diverse morphologies that resembled neuron-like cells, fibroblast-like cells and epithelial cells. Immunocytochemistry shown that these differentiated cells derived from EBs were positive for a-fetoprotein (AFP, endoderm), cell keratin-7 (CK7, endoderm) and pancreatic duodenal homeobox-1 (PDX-1, endoderm); a-Actin (mesoderm), vimentin (mesoderm and parietal endoderm);βⅢ-tubulin (ectoderm), nestin (ectoderm), glial fibrillary acidic protein (GFAP, ectoderm). The biPSCs were directed differentiate into neural cells under induced condition by embryoid body-mediated. The induced neural cells were positive for Nestin,GFAP and NSE protein. These data demonstrated that biPSCs could differentiate into the three germ layers in vitro.(3) To investigate the developmental pluripotency of biPSCs in vivo, approximately 1.3×106 of biPSCs were injected subcutaneously into the dorsal flanks of nude mice. Teratomas were observed after injection of 6-9 weeks. Histological examination demonstrated the tumors were well-defined structures, including vascular structures (mesoderm), striated muscle (mesoderm), cartilage (mesoderm), glandular structures (endoderm) and neural rosettes structures (ectoderm). These results shown that the biPSCs had multi-lineage potential in vivo.(4) To generate cattle-goat chimeras with biPSCs, approximately 1.1×106 biPSCs were injected into the peritoneal cavity of each goat fetus at 55 to 60 days of gestation via laparotomy. After full-term pregnancy, one died kid exhibited extensive coat-color chimerism. PCR was used to amplify bovine mitochondrial D-loop sequences from different tissues of chimeric kid. The result revealed that many tissues contained bovine mitochondrial D-loop sequences besides pancreas, skeleton and cartilage. These findings demonstrated that established biPSCs had the ability to form chimera in vivo.