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Single Transcription Factor Reprogramming of Hair Follicle Dermal Papilla Cells to Induced Pluripotent Stem Cells

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From the June 2011 Issue of Stem Cells
Paper Commentary by Stuart P. Atkinson

A safe and accessible source of somatic cells which are amenable to reprogramming and the generation of induced pluripotent stem cells (iPSCs) is currently very much sought after. While some adult stem cell sources are readily reprogrammable with one or two factors, neural progenitor cells are not a readily accessible population (Kim et al and Kim et al). So, can we find another source? Skin dermal papilla (DP) cells are a specialised mesenchymal stem cell type involved in hair morphogenesis and regeneration. Previous studies into the reprogramming of DP cells have shown that Sox2, Klf4 and Myc were already expressed and so DP cells could be reprogrammed using forced expression of Oct4 and Klf4 alone (Rendl et al and Tsai et al). The next step was to try the reprogramming process with only one factor, and a study (Tsai et al) which is presented in the June edition of Stem cells from the group of Michael Rendl at the Mount Sinai School of Medicine, New York, USA does just that – with Oct4.

First, hair follicle cell suspensions from the skin of Lef1-RFP/Oct4-GFP7Rosa26-LacZ triple-transgenic mice were stained with antibodies against Itga9 and then FACS sorted (using RFP and Itga9) to give a cell population of 97% purity. The Oct4-GFP construct is used to monitor endogenous Oct4 expression (to indicate the initiation of pluripotency); the Lef1-RFP construct and Itga9 were used to identify dermal papillae cells, while Rosa26-LacZ was used as a ubiquitous marker to trace cells during in vivo experiments. Reprogramming of DP cells with Oct4 alone remarkably led to the detection of GFP-positive colonies after 18 days and with well-defined embryonic stem cell (ESC)-like colonies arising between 3-5 weeks. Reprogramming efficiency was estimated at 0.088%, comparable to other 4 factor reprogramming experiments, so suggesting that overall, reprogramming of DP cells to iPSC is fast and efficient.

In total 8 lines were analysed which all demonstrated Nanog, Oct4 and SSEA1 expression, while the DP-specific gene expression signature was strongly downregulated. As only one transgene was used, the risk of mutational insertion is reduced compared to other methods, and analysis demonstrated the integration of only 2-4 copies in each iPSC line, each in a distinct pattern, suggesting that the insertion sites were not specific. Importantly, these transgenes were completely silenced after three passages. Global gene expression analysis showed a strong correlation between the DP-iPSC and mouse ESCs, which included the faithful reprogramming of imprinted genes. DP-iPSC could also form embryoid bodies and differentiate down all three germ layers as demonstrated by the detection of Foxa2 (endoderm), Vimentin (mesoderm) and Tubb3 (ectoderm) amongst other markers. Oct4 and Nanog transcripts were also faithfully downregulated upon differentiation. Importantly, chimeric pups with some evidence of germline contribution were also generated with no tumorigenesis reported. Further mating of the chimeric mice with wild type mice led to the birth of several pups bearing Lef1-RFP and Oct4-GDP.

Together these data suggest that the DP-iPSCs are bona fide pluripotent stem cells, with a similar developmental potential as ESCs and, further, show no signs of tumorigenesis. The advantages that these data entail for iPSC reprogramming are two-fold. 1) The realisation of an accessible cell source which is very amenable to reprogramming and 2) the reduction in the number of integrative and possibly tumorigenic elements utilised. The next step is to look into the possibilities of reprogramming human DP cells, which have been noted to express endogenous SOX2 (Laga et al), suggesting that they may be feasible for a similar reprogramming strategy.

 

References

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