Derivation And Evaluation Of Transgene-free Rat Induced Pluripotent Stem Cells

Forced expression of transcription factors can convert somatic cells into induced pluripotent stem cells?iPS cells?.Besides the pluripotency similarity to embryonic stem cells?ES cells?,the iPS cells can avoid the ethical issue resulted from the dependence on embryos.As a consequence,they are considered holding tremendous application value in the future.To reveal their full advantages,scientists around the world have made great efforts on the generation of high quality transgene-free iPS cells.Although a variety of reprogramming strategies have been reported to create transgene-free iPS cells from different cell types,the mouse is still the only species which has derived transgene-free iPS cells with germline transmission competency.Rat is the only species other than mouse that has commonly recognized ground state ES cells.Current rat iPS cell generation mainly relies on techniques that lead to permanent integration of transcription factors in the iPS cell genome.Thus,direct comparison with ES cells could provide an excellent opportunity to better understand technical limitations in generating germline-competent iPS cells for rat and pave the way for the generation of high quality iPS cells in other animals.We used the non-integrative episomal system that had been successfully used in mice to generate transgene-free iPS cells with competency for efficient germline transmission.Three such vectors were used in the current study.Vector pMaster3 contained 7 human transcription factor genes including OCT4/POU5f,SOX2,KLF4,C-MYC,NANOG,LIN28 and NR5A2.The pMasterl2 vector contained additional human miR-302/367 gene cluster.In this study we constructed a new vector,pMaster22,by replacing human miR-302/367 gene cluster with rat miR-302/367 gene cluster.The three reprogramming vectors were separately used to reprogram rat embryonic fibroblasts into iPS cells.We found that in normal culture condition?²0%O₂?reprogramming by pMaster3 failed,but succeeded by pMasterl2 or pMaster22 with very low efficiency,however the obtained iPS cells grew slowly.On the contrary,hypoxic culture conditions?5%O₂?played a key role in rat iPS cell generation,and greatly improved reprogramming efficiency.All three vectors could successfully generate iPS cells under hypoxic conditions.for pMaster12 and pMaster22,reprogramming efficiency increased by 10-fold and the generated iPS cells grew vigorously with lower doubling time.Next,FIAU negative selection was applied to promote the generation of transgene-free iPS cells.The obtained transgene-free iPS cells were confirmed by vigorous PCR.These transgene-free rat iPS cells exhibit typical morphology of rat ES cells.Colonies are dense with clear boundary and prone to detach from feeder layers.To evaluate the pluripotency of transgene-free iPS cells,we found they could be maintained in 3i medium for up to 40 passages without differentiation.In addition to the expression of typical ES cell surface marker alkaline phosphatase,transgene-free iPS cell lines also express multiple ES cell-specific pluripotency markers as validated by immunofluorescence staining.Importantly,quantitative real-time PCR?q-PCR?results show that endogenous pluripotency factors are expressed at levels comparable to rat ES cells.To further characterize the differentiation potential of transgene-free iPS cells,the formation of embryoid bodies?EBs?in vitro and teratoma in vivo were conducted.The formed EBs and teratoma could differentiate into cell types of three germ layers as validated by immunofluorescence staining and tissue staining with hematoxylin-eosin?H&E?,respectively.These results indicate that our transgene-free iPS cells display differentiation potential approximating that of ES cells.Next,we obtained Leptin gene knockout iPS cell lines by homologous recombination with a targeting vector,indicating that transgene-free iPS cells are amenable to genetic modification.Finally,after the transgene-free rat iPS cells were microinjected into blastocysts,we obtained good chimeric animals with typical coat color demonstrating their capability of contribution to the chimera formation.In summary,we achieved the derivation of transgene-free rat iPS cells that approximate the quality of ES cells.Although no germline transmission has been achieved,they can contribute significantly to chimeric formation.While this partial success in achieving competency is encouraging,it suggests more efforts are still needed to derive ground state rat iPS cells.Lessons learned from rat iPS cells are critical for the advancement of the entire iPS and ES cell fields.