Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming

Abstract

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

Direct neuronal reprogramming: learning from and for development

“…Co-expression of Sox2, FoxG1, Ascl1, Dlx5 and Lhx6 (Figs 1, 3) converts murine and human fibroblasts into telencephalic, mainly parvalbumin-positive GABAergic interneurons (Colasante et al, 2015). Interestingly, Dlx2, a key regulator of GABAergic neuronal fate (Petryniak et al, 2007), was induced upon Sox2 and FoxG1 co-expression (Colasante et al, 2015). Sox2 was shown to interact with Ascl1, while FoxG1 was essential for Dlx2 upregulation both in vitro and in vivo.…”

“…How does the presence or absence of other transcription factors in the starting cell type affect the outcome of Ascl1 overexpression? Co-expression of Sox2, FoxG1, Ascl1, Dlx5 and Lhx6 (Figs 1, 3) converts murine and human fibroblasts into telencephalic, mainly parvalbumin-positive GABAergic interneurons (Colasante et al, 2015). Interestingly, Dlx2, a key regulator of GABAergic neuronal fate (Petryniak et al, 2007), was induced upon Sox2 and FoxG1 co-expression (Colasante et al, 2015).…”

“…Interestingly, co-transduction of adult human brain-derived pericytes with Ascl1 and Sox2 was sufficient to induce functional neurons (Karow et al, 2012). A possible scenario for the functional interaction between Ascl1 and Sox2 could be that their biochemical interaction is required for the activation of the key neuronal genes in cells otherwise refractory to neuronal reprogramming (see also Colasante et al, 2015). Chromatin remodelling is also required for Pax6-mediated reprogramming, as Brg1 (Smarca4)-deficient glia cannot be reprogrammed into neurons (Ninkovic et al, 2013), and the above-mentioned miR-9/9* and miR-124 regulate the composition of the Brg1-associated factors (BAF) complex during neural development (Yoo et al, 2009).…”

Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience

“…c | To facilitate neurotransmitter-specific iN conversion, cocktails of specific transcription factors can be added to shape a specific neuronal identity. These lineage-specifying transcription factors are typically well known for their essential roles during the development of the targeted neuronal subtype in vivo 88,89,99,119,122–124,196 . d | Manipulation of signal transduction pathways through growth factors and small molecules that inhibit the transforming growth factor-β (TGFβ)–ALK–SMAD pathway and glycogen synthase kinase 3β (GSK3β), as well as the promotion of cyclic AMP signalling, increases iN conversion efficiencies.…”

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming

“…Importantly, our method can be applied to hPSCs or hNPCs, and, with small adjustments, it can be also useful in other differentiation paradigms such as the direct conversion of somatic cells into post-mitotic neurons. Our group and others have developed protocols to obtain specific subtypes of neurons from fibroblast direct reprogramming without passing through an induced pluripotent stem cell20212223242526. Direct reprogramming can represent an interesting alternative strategy for neuronal modeling2728 in particular for late-onset neurological diseases since hPSCs generate immature neurons that may need long time in culture to recapitulate the disease phenotype2930.…”