Study Of Bovine Induced Pluripotent Stem Cells

Ectopically expressing of transcription factors could reprogram somatic cells into induced pluripotent stem cells(iPSCs),which possessed all characteristics of pluripotent stem cells,such as morphology,gene expression,epigenetic status,proliferation,self-renewal,EBs,teratomas and the ability to differentiate into different functional cells lineages.iPSCs was superior to other stem cell in many aspects,such as providing a simple stabilization method for establishment of pluripotent stem cell lines,bypassing the ethical concerns of using embryonic stem cells(ESCs)or fetus.The iPSCs technology provided abundance of cell resource for the study of reprogramming mechanism,disease pathogenesis,drug screening and the application of regenerative medicine in clinical.The appearance of iPSCs also initiated a new strategy to establish livestock stem cells and genetic modification livestock.In this study,bovine iPSCs was generated with lentiviral vector(containing bovine OSKMNL)transfection.The characterizations of biPSCs were similar to ESC confirmed by gene-protein level assay.The difference of Proteomic and phosphoproteomic was compared between BEFs and biPS cells.All these data will provide more information for the study and application of biPSCs in the future.Part Ⅰ:Cloning of bovine iPSCs-related transcription factors and constructions of lentiviral vectorsAccording to the published sequence of bovine iPSCs-related transcription factors in GenBank,primers were designed to amplify the coding sequences(CDS)of these transcription factors,including Oct4,Sox2,Klf4,c-Myc,Nanog and Lin28 gene(OSKMNLT).Total RNA were extracted from fetus tissues and were used as the template in RT-PCR.The sequence results showed that the CDS length of OSKMNLT were 1083bp,963bp,1434bp,1320bp,903bp and 618bp respectively.Amino acid sequence homology of OSKMNL between bovine and other species,including buffalo,pig,goat,human and mouse,was Oct4(98%、99%、100%、99%and 98%),Sox2(98%、99%、100%、99%and 98%),Klf4(97%、98%、97%、86%and 90%),c-Myc(98%、96%、98%、92%、and 91%),Nanog(98%、81%、76%、69%and 54%)and Lin28(98%、99%、97%、93%and 95%).These sequences exhibited a high conversation in evolution,raveling the important role in maintaining cellular functions.The CDS of OSKMNL were successfully inserted into a lentiviral vector to construct recombinant lentiviral vectors,including Lent-EF1α-Oct4-IRES-EGFP、Lent-EF1α-Sox2-IRES-EGFP、Lent-EF1α-Klf4-IRES-EGFP、Lent-EF 1α-cMyc-IRES-EGFP、Lent-EF1α-Lin28-IRES-EGFP、and Lent-EF 1α-Nanog-IRES-EGFP、respectively。Part Ⅱ:viral particles packaging and generation of bovine iPSCs The lentiviral system(Lent-EF1α-IRES-EGFP vector,containing bovine OSKMNL CDS)and the retrovirus systems(pMX-GFP vector,containing mouse OSKM CDS)were employed to investigate the biPSCs generation.These recombinant vectors were used to pack viral particles in 293T cells along with their assistant vectors.48h and 72h after transfection,the viral particles were collected and tittered respectively.the viral showed a titration of 5×106～5×107IU/mL and could be use to transfect 293T cells directly.Meanwhile,primary BEFs were derived from bovine fetus by a combination of mechanical and enzymatic means.BEFs were transfected with lentivirus mixture and retroviral mixture respectively at a multiplicity of infection(MOI)5,10,and 20.After transfection,the BEFs experienced.a series of change in morphology.biPSC-like cells colonies were picked and expanded at about 3 weeks.More of biPSC-like cells colonies were obtained in lentiviral system than retroviral system.Compared with MOI 5 and 10,these two systems obtained maximum colonies At MOI 20.The colony formation efficiency was 0.062%and 0.017%in lentiviral system and retroviral system respectively.Seven cell lines were obtained from lentiviral vectors system(contained bovine OSKMNL)could be cultured over 20 passages and still maintained the characters of the stem cell.While the other 2 cell lines obtained from retroviral system(contained mouse OSKM)could only be expanded for less than 10 passages only.Part Ⅲ:Characterization of biPSC-like cellsThe biology characterizations of biPSC-like cells were assayed at molecular level,cellular level and animal level,respectively.At molecular level,biPSC-like cells expressed discriminatory gene of pluripotency,including Oct4、Sox2、Nanog、Klf4、c-Myc、Lin28、Stat3、GP130、FOXD3、CDH1、bFGF2、DPPA3 and SALL4;Compared with BFFs,the promoters of Oct4,Nanog and Sox2 were hypomethylated in biPSCs.Meanwhile,the telomerase activity of biPSCs was higher than BEFs and 293T cells.At cellular level,these cells showed dome-shaped,tightly packed colonies with clear borders,which identify as the same morphology of bovine ES like cells and mouse ESCs/iPS.biPSC-like cells exhibited strong AP activity by AP staining,maintained a normal karyotype(2n=60)at passages 20,and expressed ES cell-related markers,including Oct4,Sox2,Nanog,SSEA-1,SSEA-3,SSEA-4,TRA-1-81 and TRA-1-60 by immunofluorescence assayed.At animal tissue level,the differentiation ability of biPSCs was tested in vitro and in vivo analysis.biPSCs can form embryoid bodies(EBs)after suspension culture for 7-10days in vitro.These EBs can differentiate into epithelioid cell,nerve cell and adipose cell.RT-PCR confirmed that these EBs expressed three germ layers marker genes,including AFP and Soxl7(endoderm),GATA4 and ACTA2(mesoderm),GFAP and β-3Tubulin(ectoderm).biPSCs can also efficiently form teratomas in immunodeficiency nude mice after subcutaneous for about 2 months.Histological examination results showed that the teratomas contained three germ layers,including pigmentary epithelium,neural tissues(ectoderm),osseous tissue,cartilage tissue(mesoderm),respiratory epithelium(endoderm).The teratomas were from bovine but not mice Confirmed by PCR with species-specific gene primers.Part Ⅳ:Phosphoproteomic comparison of bEF cells and biPS cellsCombining high-mass-accuracy mass spectrometry and phosphopeptide enriched with TiO2 IMAC,phosphoproteomic of BEFs and biPSCs were identified.This two samples comparison resulted in a very large set of identified phosphorylated protein and phosphorylation sites.A total of 363(containing 502 phosphorylation sites)and 806(containing 1131 phosphorylation sites)proteins were identified in BEFs and biPSCs respectively.Among them,272 proteins were identified in these 2 samples,while 86 proteins and 529 proteins were exclusively identified in BEFs and biPSCs respectively.These phosphoproteins participtated in many cellular process,and represented the difference cell type between BEFs and biPSCs.Phosphoproteomic comparisong of BEFs and biPSCs will provide guidance for investigating protein function in biPSCs,and for broadening our understanding of somatic cell reprogramming to biPSCs.Conclusion:With the transfection of lentiviral system,biPSCs successfully generated from BEFs.The biPSCs exhibited all characteristic of pluripotent stem cell,and could be cultured and passaged stably in vivo.BEFs and biPSCs exhibited difference between Proteomic and phosphoproteomic.Understanding of key intracellular signaling pathways and protein targets that control development of biPSCs from somatic cells is essential for designing new approaches to improve reprogramming efficiency.