The Establishment Of Transgene-free Mouse IPS Cell Lines And High-throughput Screening Method For Reprogramming Genes

Induced pluripotent stem(iPS)cells exhibit similar differentiation potential to embryonic stem(ES)cells and avoid the limitation of ethics.Therefore,iPS cells hold great promise for regenerative medicine,and for the study of human biology and pathology.However,the very high risks associated with the use of such iPS cells for human therapy have limited the applications of iPS cells.Viral vectors that are routinely used to generate iPS cells have been demonstrated to result in the induction of cancer.Also,random integration of the exogenous reprogramming genes in mouse iPS cells increases tumor formation,morbidity,and mortality in mice generated from these cells.Therefore,the safety and quality of iPS cells are of critical importance to their anticipated use for human cell-based therapies.To circumvent some of these safety issues,non-viral methods for the introduction of the reprogramming factors into somatic cells are being developed.But none of the iPS cells generated by these non-viral methods have been rigorously tested for their quality and in particular for their ability to generate germline chimeras.Here,we constructed three episomal vectors pMasterl,pMaster3 and pMasterl2.These episomal vectors carried positive selection gene neo,negative selection gene HSVtk and different combinations of reprogramming genes.We transfected Oct4-GFP mouse embryonic fibroblast cells(MEFs)with pMasterl plasmid to generate iPS cells.PCR analysis showed persistence of the OKSM genes in these iPS cells.Next pMaster3 and pMasterl2 were evaluated.PCR analysis of pMaster3-derived iPS cells showed persistence of the NR5A2 gene in most of cells.PCR analysis of pMasterl2-derived iPS cells validated the complete removal of all of the episomal sequences in 8 cell lines(iPSZX11-3-1,iPSZX11-3-2,iPSZX11-18-2 and so on).Among of these cell lines,iPSZX11-18-2 exhibited mouse ES cell-like morphology,normal karyotype,activation of endogenous pluripotency markers.In particular,iPSZX11-18-2 can generate germline chimeras.To further explore underlying reprogramming mechanism,we compared the transcriptome of pMaster iPS cell clones derived from each vector with that of the G4 ES cells.Microarray expression analysis revealed pMasterl 2-derived iPS cells were closest to G4 ES cells.The 2i medium significantly improved the attainment of germline competency for pMaster-derived iPS cells.To explore the mechanism of how 2i medium improves iPS cell quality,we performed genome-wide microarray analysis of seven iPS cell lines and two ES cell lines,cultured in defined 2i medium or serum containing medium.Microarray expression analysis revealed 2i medium greatly inhibited expression of genes related to cell differentiation and tissue and organ development.Incomplete reprogramming probably resulted in persistence of exogenous genes in pMaster-derived iPS cells.To optimize generation of iPS cells,we aimed to establish a gain-of-function screening strategy to systematically identify genes that participate in reprogramming.To determine an efficient screening scheme,we first found a practical combination of reprogramming factors OKS as sensitive background and constructed piggyBac transposon(PB)vector pFindl as screening vector.We next started OKS+pFindl screening method at a small-scale through manually picking-300 OKS/pFind1 iPS cell clones.In these-300 iPS cell clones we identified 213 genes including known pluripotency genes,such as Nr5al,Klf5 and miR205.Quantitative RT-PCR assay demonstrated that pFind1 insertions activated neighboring genes at the transcription level.Further,we found that the effect range of pFind1 could reach 120 kb.Because the identification of pFind1 insertion sites in single colonies is tedious and rate limiting,we designed a high-throughput method to detect pFind1 insertion sites with next generation sequencing(NGS).We collected 50 thousand OKS/pFind1 iPS cell clones and created pFind1 distribution histograms of insertion frequencies for 19 mouse autosomes and 2 sex chromosomes with NGS and bioinformatical analysis.Through statistical analysis of genes located around pFind1 integration sites,a ranking list of 12,634 genes was obtained by counting pFindl hit times around each gene.Bioinformatical analysis showed genes that were disrupted by pFindl exhibited an enrichment of terms relating to differentiation that was considered as a barrier to self-renewal;genes that were up-regulated by pFind1 showed high enrichment in innate immunity response.To further validate our gene ranking list,we selected 10 genes covering different segments to examine their reprogramming capability.All these genes can promote generation of iPS cells to various extents.A trend was that high ranking genes markedly enhanced iPS cell generation whereas low ranking genes only had slight effect,although there was no strict correlation.These validation results suggest that our gene ranking list from the screen can indeed provide a meaningful reference to evaluate each gene's reprogramming ability in the future.The transgene-free iPS cells that were established in our study should bring us closer to the use of such iPS cell lines for human cell-based therapy.The high-throughput screening method may have general use to discover genes involved in other biological processes.