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

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• 作者：Marilena Raciti ; Marilena Granzotto ; Minh Do Duc ; Cristina Fimiani ; Giada Cellot ; Enrico Cherubini ; Antonello Mallamaci
• 关键词：17AAD ; (allylamino)-17-demethoxygeldanamycin ; Bmp ; Bone Morphogenetic Protein ; FL ; Foxg1-Lhx2 ; FPd ; Foxg1-Pax6-drugs ; FPEL ; Foxg1-Pax6-Emx2-Lhx2 ; FPL ; Foxg1-Pax6- Lhx2 ; HDAC ; histone deacetylase ; HMT ; histone methyltransferase ; iPSC ; induced pluripotent stem cell ; MEF ; mouse embryonic fibroblast ; moi ; multiplicity of infection ; NPC ; neural precursor cell ; NSC ; neural stem cell ; PL ; Pax6-Lhx2 ; RMP ; resting membrane potential ; t2PCPA ; trans-2-Phenyl-cyclopropylamine hydrochloride ; TF ; transcriptio
• 刊名：Molecular and Cellular Neuroscience
• 出版年：November, 2013
• 年：2013
• 卷：57
• 期：Complete
• 页码：42-53
• 全文大小：1760 K

文摘

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 TGF尾 pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype.