ONSL and OSKM cocktails act synergistically in reprogramming human somatic cells into induced pluripotent stem cells

Abstract

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of >2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

“…Clonal iPSC lines were manually isolated at day 30 post-transduction, and further cultured individually on Laminin-521 and Nutristem. The iPSC lines derived in the laboratory of S. Viville, named STBG-iPSCs, were established using different reprogramming methods, as listed in the Table S1 and as described in Jung et al. (2014).…”

A Compendium of Preparation and Application of Stem Cells in Parkinson's Disease: Current Status and Future Prospects

“…No matter Yamanaka's OSKM reprogramming formula, Yu's ONSL (Oct4, Nanog, Sox2, and Lin28), recombination recipe or other versatile combinations, iPSCs have been proving to exhibit pluripotency at levels similar to ESCs. And a recent study indicates that OSKM and ONSL cocktails can act synergistically to reprogramme human somatic cells into iPSCs (Jung et al, 2014 ). Although highly efficient lineage trans-differentiation and long-term expression can be achieved with viral vectors, however, iPSCs technology is complicated by the potential risks posed by genome-integrating viruses, which are randomly but permanently integrated into the host genome at multiple sites together with viral vector backbone.…”

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells

“…To improve the efficiency of reprogramming, various methods and techniques have been reported. The use of cocktails consisting of OSKM and ONSL (i.e., OCT4, NANOG, SOX2, and Lin28) and the combination of OCT4, SOX2, and NANOG have been shown to be somewhat effective in improving the reprogramming efficiency of human somatic cells [10, 11]. …”