Reprogramming of mouse and human somatic cells by high‐performance engineered factors

Abstract

Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with enhanced efficiency and accelerated kinetics. Remarkably, Oct4-VP16 alone could efficiently reprogramme mouse embryonic fibroblasts (MEFs) into germline-competent iPSCs. Furthermore, episomally delivered synthetic factors could reproducibly generate integration-free iPSCs from MEFs with enhanced efficiency. Our results not only demonstrate the feasibility of engineering more potent reprogramming factors, but also suggest that transcriptional reactivation of OCT4 target genes might be a rate-limiting step in the conversion of somatic cells to pluripotent cells. Synthetic factor-based reprogramming might lead to a paradigm shift in reprogramming research.

Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers

“…For example, the transactivation domains of MyoD (residues 3–56) and VP16 have each been attached to Oct4 to make it a more robust activator of the pluripotency circuitry, thereby increasing the number of iPS cell colonies produced [140,141]. VP64 and KRAB (Krüppel-associated box) are the most commonly used activation and repression domains respectively; however, the degree to which these domains can induce or silence transcription depends on the target, and in some instances, another activation or repression domain could be more effective due to the local chromatin structure and co-operative binding of other protein complexes at that site [142,143].…”

Generation of Induced Pluripotent Stem Cells from Human Renal Proximal Tubular Cells with Only Two Transcription Factors, Oct4 and Sox2

“…Accordingly, we further engineered a tricistronic vector encoding for Oct4 and Sox2 and separated by the VP16 transactivation domain, which has been described as an enhancer of reprogramming (supplemental Fig. S4) (9). Although this combination was not sufficient to reprogram human fibroblasts, reprogrammed proximal tubular epithelial cells, hereafter referred to as pt-iPSCs, were observed as early as 13 days after viral transduction.…”

“…To alleviate these concerns, a range of different methodologies is being developed to generate "safer and higher quality" iPSCs suitable for transplantation and disease modeling. Such approaches include a reduction in the number of factors used for reprogramming (6 -8), the engineering of more potent transcription factors (9), the combination of chemical compounds alongside different reprogramming factors (10), as well as non-integrative approaches (11)(12)(13)(14)(15)(16)(17)(18). However, and despite the progress achieved, current methodologies still suffer from low reproducibility as well as a large variability in the efficiency of the reprogramming process.…”

Programming and reprogramming the brain: a meeting of minds in neural fate

“…Several talks highlighted the importance of somatic transcription factor depletion to improve reprogramming efficiency. Kathrin Plath (University of California, Los Angeles, USA) presented her work investigating how pluripotent reprogramming factors, such as Sox2 and Oct4 (POU5F1), which have been shown to act as transcriptional activators (Wang et al, 2011;Hammachi et al, 2012), mediate the erasure of differentiated cell identity. The transition during pluripotent reprogramming from murine embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs) was analysed at four distinct time points, using genome-wide approaches to map the binding of the Oct4, Sox2, Klf4 and Myc (OSKM) transcription factors, as well as the histone modification landscape, chromatin accessibility and the transcriptomic profile of the cells.…”