chir-99021 and pifithrin

Generation of induced pluripotent stem cells (iPSCs) from human urine-derived cells (hUCs) provides a convenient and non-invasive way to obtain patient-specific iPSCs. However, many isolated hUCs exhibit very poor proliferation and are difficult to reprogram. In this study, we optimized reprogramming approaches for hUCs with very poor proliferation. We report here that a compound cocktail containing cyclic pifithrin-a (a P53 inhibitor), A-83-01, CHIR99021, thiazovivin, NaB, and PD0325901 significantly improves the reprogramming efficiency (170-fold more) for hUCs. In addition, we showed that replacement of Matrigel with autologous hUC feeders can overcome the reprogramming failure due to the massive cell death that occurs during delivery of reprogramming factors. In summary, we describe improved approaches to enable iPSC generation from hUCs that were otherwise difficult to reprogram, a valuable asset for banking patient-specific iPSCs.

Topics: Benzamides; Benzothiazoles; Cell Differentiation; Cell Proliferation; Cellular Reprogramming; Diphenylamine; Humans; Induced Pluripotent Stem Cells; Pyrazoles; Pyridines; Pyrimidines; Thiazoles; Thiosemicarbazones; Toluene; Urine

High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.

Topics: Animals; Benzothiazoles; Cell Count; Chromones; Culture Media; Embryo Culture Techniques; Embryo, Mammalian; Embryonic Development; Embryonic Stem Cells; Enzyme Activation; Female; Germ Layers; Glycogen Synthase Kinase 3; Homeodomain Proteins; Immunohistochemistry; Insulin; Isoquinolines; Male; Mice; Mice, Inbred C57BL; Morpholines; Nanog Homeobox Protein; Octamer Transcription Factor-3; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Pyrimidines; Receptor, Insulin; Signal Transduction; Sulfonamides; Toluene; Transcription, Genetic; Tumor Suppressor Protein p53