In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogramme mouse embryonic fibroblasts towards neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even after switching off the reprogramming transgenes. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP positive cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cell output was achieved by inhibition of the BMP and TGFβ pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype.
Keywords: (allylamino)-17-demethoxygeldanamycin; 17AAD; Bmp; Bone Morphogenetic Protein; Cerebral cortex; FL; FPEL; FPL; FPd; Fibroblasts reprogramming; Foxg1; Foxg1-Lhx2; Foxg1-Pax6- Lhx2; Foxg1-Pax6-Emx2-Lhx2; Foxg1-Pax6-drugs; HDAC; HMT; MEF; NPC; NSC; Neural stem cells; Neuronal differentiation; PL; Pax6; Pax6-Lhx2; RMP; TF; Tgfb; Transforming Growth Factor b; VPA; histone deacetylase; histone methyltransferase; iPSC; induced pluripotent stem cell; moi; mouse embryonic fibroblast; multiplicity of infection; neural precursor cell; neural stem cell; resting membrane potential; t2PCPA; trans-2-Phenyl-cyclopropylamine hydrochloride; transcription factor; valproic acid.
© 2013.