Reprogramming fibroblasts to neural-stem-like cells by structured overexpression of pallial patterning genes

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 reprogramm mouse embryonic fibroblasts toward 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 upon transgenes switching off. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cells output was achieved by tonic inhibition of BMP and TGFb pathways. These Tau-EGFP cells showed a negative resting potential and displayed active electric responses, following injection of depolarizing currents.