Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells.

Somatic cells can be reprogrammed toward pluripotency by overexpression of a set of transcription factors, yielding induced pluripotent stem cells (iPSCs) with features similar to embryonic stem cells. Little is known to date about stoichiometric requirements of the individual reprogramming factors (RFs) for efficient reprogramming and especially about whether stoichiometry also influences the quality of derived iPSCs. To address this important issue, we chose bicistronic lentiviral vectors coexpressing fluorescent reporters (eGFP, dTomato, Cerulean, or Venus) along with the canonical RFs to transduce a bulk of murine embryonic fibroblasts (MEFs). Using a flow cytometric approach, we were able to independently and proportionally quantify all fluorophores in multiple-infected MEFs and more importantly could sort these cells into all 16 stoichiometric combinations of high or moderate expression of the four factors. On average, we obtained about 600 alkaline phosphatase-expressing colonies from 20,000 seeded cells. Interestingly, only seven different stoichiometric ratios gave rise to any colonies at all. The by far most colonies were obtained from those fractions, where Oct4 was in excess over the other three factors (2,386 colonies/20,000 cells), or where both Oct4 and c-Myc were in excess over Sox2 and Klf4 (1,593 colonies/20,000 cells). Our findings suggest that increased Oct4 levels opposite to modest ones for Sox2 and Klf4 are required for satisfying reprogramming efficiencies and that these stoichiometries are also highly beneficial for achieving a stable pluripotent state independent of ectopic RF expression. Finally, the eligible Oct4(high) , Sox2(low) , and Klf4(low) subpopulation only resembles a small fraction of cells targeted by equal vector amounts, suggesting the necessity to address stoichiometry also in alternative approaches for iPSC generation or between different experimental systems.

Tiemann U ，Sgodda M ，Warlich E ，Ballmaier M ，Schöler HR ，Schambach A ，Cantz T ... -  《-》

Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc.

The first successful reprogramming of differentiated cells to a pluripotent state was done by retroviral introduction of four transcription factors (Oct4, Sox2, Klf4, cMyc) by the group of Yamanaka in 2006. Since then, scientists all over the world have attempted various methods to avoid insertional mutagenesis, a major limitation of the retrovirus-based method, however no technique was found to completely avoid DNA integration. Recently, a non-viral mRNA-based approach, inherent to avoid genomic integration, was implemented to generate stem cell-like cells, yet, seventeen daily transfections were required, inducing substantial stress on the cells. In this work, we demonstrate successful activation of pluripotency-associated genes in mouse embryonic fibroblasts by means of cationic lipid-mediated introduction of mRNAs encoding the four factors. Moreover, our transfection protocol required maximally three transfections. Up-regulation of the transfected factors as well as Nanog and SSEA-1, typical mouse pluripotency markers, was detected already after the first transfection. Nuclear localization of the introduced factors was confirmed. Positive alkaline phosphatase staining of cell clusters further confirmed the onset of the reprogramming process. In conclusion, the transfection method presented here holds great promise for safe generation of induced pluripotent stem cells of mouse origin.

Tavernier G ，Wolfrum K ，Demeester J ，De Smedt SC ，Adjaye J ，Rejman J ... -  《-》

Klf4 interacts directly with Oct4 and Sox2 to promote reprogramming.

Wei Z ，Yang Y ，Zhang P ，Andrianakos R ，Hasegawa K ，Lyu J ，Chen X ，Bai G ，Liu C ，Pera M ，Lu W ... -  《-》

Oct4-enhanced green fluorescent protein transgenic pigs: a new large animal model for reprogramming studies.

Nowak-Imialek M ，Kues WA ，Petersen B ，Lucas-Hahn A ，Herrmann D ，Haridoss S ，Oropeza M ，Lemme E ，Schöler HR ，Carnwath JW ，Niemann H ... -  《-》

Endogenous KLF4 expression in human fetal endothelial cells allows for reprogramming to pluripotency with just OCT3/4 and SOX2--brief report.

Ho PJ ，Yen ML ，Lin JD ，Chen LS ，Hu HI ，Yeh CK ，Peng CY ，Lin CY ，Yet SF ，Yen BL ... -  《-》