Generation Of Induced Puluripotent Stem Cells From New-Born Bovine Fibroblasts By Defined Factors

Embryonic stem cells (ESCs) commonly were derived from the inner cell mass (ICM) of mammalian blastocysts, and they remain the unique capacity to proliferate unlimitedly while maintain pluripotency. The study about human ESCs was restricted by law and ethics because of the use of human early embryos. Recently, The direct reprogramming of mouse somatic cells to induced pluripotent stem cells (iPSCs) was first succeeded through retroviral transduction of some transcription factors. The mouse iPSCs showed marked similarities to mouse ES cells in morphology, proliferation property, gene expression profiles and in vivo/ in vitro differentiation potential. Subsequently human iPSC lines were successfully established from human somatic cells, enabling the generation of patient-specific cells of any lineage without the use of human embryos. The generation of human iPSCs provides an invaluable resource for cell therapy and regenerative medicine. The iPSCs of other species, including rhesus monkey, rat and pig, were established from somatic cells in succession, which can be used in medicinal field such as establishing animal disease models and filtering medicine in vitro. Up to now the study about bovine iPSCs has not been reported. So in this study we attempt to generate the bovine iPSCs from bovine somatic cells through retroviral transduction of defined factors which used in induced process of mouse and human iPSCs. The bovine iPSCs has been established successfully and its properties similar to ES cells have been demonstrated with scientific test. The important results were listed below.(1) The ORF(open reading frame) sequence of bovine sox2 gene (963 bp) was cloned from the primodial genital ridges of fetal cattle by RT-PCR. Compare to the published sox2 gene sequence (NM-001105463), the nucleotide homology is 99.6% and the related amino acids homology is 99.7%. The ORF sequence of bovine klf4 gene (1434 bp) was cloned from MDBK cells in contact inhibition growth. Compare to the published klf4 gene sequence (NM-001105385), both the nucleotide and the related amino acids homology is 99.9%.(2) The two genes ORF sequence were respectively inserted into the restriction sites of retroviral vector pMSCVneo to construt recombinant retroviral expression vector pMSCV-sox2 (pMS) and pMSCV-klf4 (pMK). The pMS and pMK was transfected into packaging cell line(PT67). Then we successfully got stable virus producing cell strains which viral titer respectively was up to 8.16×107 CFU/mL(MSCV-sox2) and 7.16×107 CFU/mL(MSCV-klf4). The typical infectious retroviral particles in the supernatant can be found under electron microscope.(3) In this study the four transcription factors (Oct4, Sox2, c-Myc and Klf4) were transducted into new-born bovine fibroblasts(NBF) through the retroviral pMSCVnoe vector. The infected NBF cells were cultured on the cell-free human amniotic membrane (HAM) support. Subsequently ESCs-like cell colonies were gained successfully and were named as "iPS-NBF" cells.(4) The bovine iPS-NBF colonies were strongly positive for alkaline phosphatase (AKP). The results of immunocytochemistry stain showed that the bovine iPS-NBF colonies were negative for stage-specific embryonic antigen-1 (SSEA-1), while they were positive for SSEA-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81, Nanog, Oct4, Sox2, and telomerase reverse transcriptase (TERT) proteins. The real-time quantitative polymerase chain reaction (qPCR) analysis showed that the related pluripotency genes, nanog, oct4 and sox2, have been effectively activated and their expression levels were comparable to the bovine PGCs. In addition, the bovine iPS-NBF cells about passage 15 retained normal diploid karyotypes by karyotype analysis.(5) The bovine iPS-NBF cells could differentiate randomly into cells with various morphologies through embryoid body (EB)-mediated differentiation. Immunocytochemistry stain and RT-PCR analysis showed that the bovine iPS-NBF cells could differentiate into all cell types of three germ layers in vitro.(6) The bovine iPS-NBF cells were injected subcutaneously into NOD/SCID mice and formed teratoma. Hematoxylin and eosin staining showed that the teratoma contained mesoderm tissues (muscle,blood vessel and cartilage) and endoderm tissue(viscus epithelium),but no ectoderm tissues.(7) 10～15 bovine iPS-NBF cells were microinjected into mouse 8-cell embryos or morula. Then six neonatal bovine-mouse chimeric mice were obtained. Detection of bovine specific D-loop region of mitochondrial DNA sequences demonstrated that the bovine iPS-NBDF cells contributed to the development of chimeric mice various organs, including heart, blood, lung, gastrointestinal tract, liver, kidney, neural tissues, skeletal muscle and gonad.(8) The six transcription factors(Nanog, Oct4, Sox2, c-Myc, Klf4 and Lin28) were splited into different combinations:2-factors group(SO),3-factors group(NOS),4-factors group(OSCK and NOSL),5-factors group(NOSKL) and 6-factors group(NOSCKL). The NBF cells were infected respectively with different fators combinations. The results showed all the six combinations could induced the NBF cells to ESCs-like colonies successfully. Comparing to the 4-factors group, the induced efficiency of 2-factors and 3-factors groups was lower, while the induced efficiency of 5-factors and 6-factors groups were increased significantly.