A Study Of Cellular Reprogramming At The Protein Level With The TMP Regulation System

Somatic cells of many species can be reprogrammed to embryonic stem cell (ESC)-like cells, known as induced pluripotent stem cells (iPSCs), classically by expression of transcription factors, Oct4, Klf4, Sox2, and c-Myc, or only the first three factors. By regulating the stoichiometry and temporal of reprogramming factors, we can reveal more mechanisms of reprogramming and improve the efficiency and quality of reprogramming in multiple species. So far, Doxycycline (Dox) inducible system is the sole regulation system for reprogramming, which has many limitations. We sought to find a more flexible system with fine-tuning capabilities to further dissect the molecular mechanisms of reprogramming.TMP regulation system is a new flexible inducible system to regulate protein stability by a commercially available small molecule, trimethoprim (TMP). The addition of TMP stabilizes the fusion protein in a rapid, reversible, and dose-dependent manner.We generated a destabilizing domain (dd) inducible system based on piggyBac transposon which included three factors of dd-human OCT4(dd-hOCT4), KLF4(hKLF4) and SOX2(hSOX2). Using this vector we successfully reprogrammed mouse Oct4-GFP embryonic fibroblasts into mouse iPSCs. They were homogenously AP positive, and expressed the stem cell markers, OCT4, NANOG, and SSEA1. Transplantation of these cells into nude mice resulted in teratomas that consisted of tissues derived from all three germ layers, indicating that these cells are pluripotent. Further experiment revealed that, stability of the fusion protein was precisely regulated by the addition of the small molecule TMP, in a dose-dependent manner, and the reprogramming function is also under the same dosage constraints. With the change of doses of TMP addition, the efficiency of mouse iPSC generation varied from0to0.4%. We could not acquired GFP+colonies without TMP treatment; and in addition of1μM TMP, the highest reprogramming efficiency was achieved by0.4%.Using TMP system to regulate the expression of ectopic hOCT4、hKLF4or hSOX2, we found that no matter where the dd fusion protein located in2A-peptide linked single polycistronic vector chain, the transfected cells could be reprogrammed efficiently (-0.4%). Interestingly, during the early and middle stages of reprogramming, exogenous OCT4or KLF4could be omitted, while the two stages of exogenous SOX2expression was required for successful reprogramming.We reprogrammed pig embryonic fibroblasts with TMP inducible vector, which included four factors of dd-hOCT4, hKLF4, hSOX2and hc-MYC, and hOCT4can be regulated by TMP. Upon TMP removal, pig iPSCs established lost their domed colony morphology and differentiated in contrast to the same iPS cell lines cultured with TMP. Pig iPS cell lines cultured with TMP were AP+and OCT4+, but in the absence of TMP, they lost most of the AP staining and were OCT4", revealing that pig iPS cell lines established total relied on the continued expression of ectopic OCT4.With these new tools, we found that mRNA and protein levels of ectopic OCT4appear to be regulated independently, which reminds us to emphasize the role of protein levels of core transcription factors. There is the first time to introduce the protein regulation system into the research of induce pluripotent stem cells. dd inducible system can be used to investigate the stoichiometry and temporal of reprogramming factors interested, and provide a platform to research reprogramming mechanism at protein level.