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Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming

ABSTRACT: Transplantation of GABAergic interneurons (INs) can sustain long-standing benefits in animal models of epilepsy and other neurological disorders. In a therapeutic perspective, a renewable source of functional GABAergic INs is needed. Here, we identified five factors (Foxg1, Sox2, Ascl1, Dlx5 and Lhx6) able to convert fibroblasts directly into induced GABAergic INs (iGABA-INs), displaying the molecular signature of telencephalic INs. The selected factors recapitulate in fibroblasts the activation of transcriptional networks required for the specification of GABAergic fate during telencephalon development. iGABA-INs exhibited progressively maturing firing patterns comparable to those of cortical INs, had synaptic currents and released GABA. Importantly, upon grafting in the hippocampus, iGABA-INs survived, matured and their optogenetic stimulation triggered GABAergic transmission and inhibited the activity of connected granule cells. The five factors also converted human cells into functional GABAergic neurons. These properties define iGABA-INs as a promising tool for disease modeling and cell-based therapeutic approaches.

ORGANISM(S): Mus musculus

PROVIDER: GSE74065 | GEO | 2015/12/15

SECONDARY ACCESSION(S): PRJNA298895

REPOSITORIES: GEO

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Project description:GABAergic interneurons are lost in conditions including epilepsy and CNS injury, but there are few culture models available to study their function. Towards the goal of obtaining renewable sources of GABAergic neurons, we used the molecular profile of a functionally-incomplete GABAergic precursor clone to screen 17 new clones isolated from GFP+ rat E14.5 cortex and ganglionic eminence (GE) that were generated by viral introduction of v-myc. The clones grow as neurospheres in medium with FGF2, and after withdrawal of FGF2 they exhibit varying patterns of differentiation. Transcriptional profiling and qPCR indicated that one clone (GE6) expresses high levels of mRNAs encoding Dlx1, 2, 5 and 6, glutamate decarboxylases, and presynaptic proteins including neuropeptide Y and somatostatin. Protein expression confirmed that GE6 is a progenitor with restricted differentiation giving rise mostly to neurons with GABAergic markers. In co-cultures with hippocampal neurons, GE6 neurons became electrically excitable and received both inhibitory and excitatory synapses. After withdrawal of FGF2 in cultures of GE6 alone, neurons matured to express BetaIII-tubulin, and staining for synaptophysin and vesicular GABA transporter (VGAT) were robust after 1-2 weeks of differentiation. GE6 neurons also became electrically excitable and displayed synaptic activity, but synaptic currents were carried by chloride and were blocked by bicuculline. The results suggest that the GE6 clone, which is ventrally derived from the GE, resembles GABAergic interneuron progenitors that migrate into the developing forebrain. This is the first report of a relatively stable fetal clone that can be differentiated into GABAergic interneurons with functional synapses. The purpose was to compare differentiation patterns of several different immortalized rat neural progenitor clones to identify early stages in differentiation. The cell clones studies were: GE6 (GABAergic neuronal precursor), GE2 (non-neuronal precursor, CTX8 (multipotential precursor), L2.2 (interneuronal precursor), and L2.3 (multipotential precursor). Five rat neural precursor cell clones were compared at three different time points following FGF2 withdrawal, which triggers differentiation. Three sister culture replicates were performed for each cell clone and time point, yielding 45 samples. One microarray failed so we have 44 microarray results in the dataset.

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Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming

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