title: Reprogramming fibroblasts to neural-precursor-like cells by structured overexpression of pallial patterning genes.
abstract: 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.
authors.affiliation: SISSA, Neuroscience Area, via Bonomea 265, 34136 Trieste, Italy. Electronic address: racitim@sissa.it.
authors.forename: Marilena
authors.initials: M
authors.lastname: Raciti
authors.forename: Marilena
authors.initials: M
authors.lastname: Granzotto
authors.forename: Minh Do
authors.initials: MD
authors.lastname: Duc
authors.forename: Cristina
authors.initials: C
authors.lastname: Fimiani
authors.forename: Giada
authors.initials: G
authors.lastname: Cellot
authors.forename: Enrico
authors.initials: E
authors.lastname: Cherubini
authors.forename: Antonello
authors.initials: A
authors.lastname: Mallamaci
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D065150:Cellular Reprogramming; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D060850:LIM-Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D055495:Neurogenesis; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism; D000099260:Empty Spiracles Homeobox Proteins
chemical_list: D019485:Bone Morphogenetic Proteins; D005136:Eye Proteins; D051858:Forkhead Transcription Factors; D056572:Histone Deacetylase Inhibitors; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53; D016875:tau Proteins; D000099260:Empty Spiracles Homeobox Proteins; C489565:Foxg1 protein, mouse; C489784:Lhx2 protein, mouse; C558298:Mapt protein, mouse; C000605732:Pax6 protein, mouse; C098693:Sox1 protein, mouse
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