MicroRNA-mediated conversion of human fibroblasts to neurons

Abstract

Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.

Figure 1. miRNA-induced transformation of human fibroblasts

a, MAP2 expression in miR-9/9*-124 only (left) and miR-9/9*-124-NEUROD2 (right) converted cells. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 20 µm. b, Quantification of MAP2-positive cells with processes at least three times the length of the cell body from ten random fields. The graph represents the percentage of MAP2-positive cells over DAPI-positive cells. miR-9/9*-124 only: n = 558; miR-9/9*-124-NEUROD2: n = 658 cells. The error bars are s.e.m. MAP2 signal was undetectable in fibroblasts infected with miR-NS, with or without NEUROD2. Scale bar, 20 µm. c, Expression of SCN1a, synapsin 1 and NMDAR1 in miR-9/9*-124-NEUROD2-converted cells. Scale bar, 20 µm. d, Representative traces of action potentials recorded in current clamp in miR-9/9*-124-NEUROD2-converted cells. Twelve out of 22 cells showed single action potentials and 2 cells showed repetitive firing. e, A representative example of a series of voltage steps applied to an miR-9/9*-124-NEUROD2-induced neuron held at−70 mV. An inward current was observed, blocked by 1 µM TTX, and reversed after TTX washout (n = 6). f, I–V curve for the peak inward (left) and outward (right) currents. g, An example of Ca²⁺ influx in induced neurons as measured by Fluo2-AM imaging. Images show the peak Fluo2-AM signal on stimulation before and during TTX application, and after TTX washout, respectively. The graph represents the changes in Fluo2 signal over time (circles, no TTX; triangles, with TTX). AP, action potential. Field stimulation is indicated by the black bar. Scale bar, 2 µm. h, An example of vesicle recycling measured by FM1-43 imaging in induced neurons. Top diagram illustrates the protocol of FM uptake or release experiments. The images represent the typical FM1-43 dye uptake signal (middle) in a converted cell marked by tRFP (left). The left graph shows the measurement of FM1-43 loading in 2 mM Ca²⁺, which was significantly reduced in low Ca²⁺ concentration (0.1 mM). The right graph quantifies FM1-43 release (destaining) during stimulations. FM1-43 signal was measured from n > 600 boutons (arrows indicate examples of boutons) from 4 cultures. All error bars are s.e.m. In some cases, the s.e.m. is too small to be resolved. Scale bar, 4 µm.

Figure 2. Additional neural factors enhance the conversion to neurons

a, MAP2 (left) and β-III tubulin (right) immunostaining of miR-9/9*-124-DAM-converted cells. Scale bar, 40 µm. The graph represents the percentage of MAP2-positive cells over DAPI-positive cells. The error bars are s.e.m.; n = 150 cells. b, A representative current clamp recording from a cell with typical neuronal morphology (see inset; scale bar, 50 µm). Voltage deflections were elicited by somatic current injections of various amplitudes (Δ = 5 pA). c, A representative voltage clamp recording of the net current at various membrane potentials (−40 to +20 mV, ΔV = 10 mV, V_hold = −90 mV). d, A representative trace of spontaneously active cells recorded in cell-attached mode. e, A representative trace demonstrating spontaneous EPSCs. f, Representative traces of evoked postsynaptic currents (left, EPSC; right, IPSC) obtained in response to local field stimulation with single current pulses (1 ms) of various amplitudes (left, 0.25 and 0.3 mA; right, 0.3 and 0.4 mA) at different membrane holding potentials (left, −70 mV; right, 0 mV). The arrows indicate the time when stimulation was applied. Stimulation artefacts were eliminated for clarity.

Figure 3. Characterization of induced neurons and nBAF subunit expression

a, Multiplex quantitative polymerase chain reaction (qPCR) of 45 induced neurons for genes of specific brain regions and cell types. Cb, cerebellum; Dpa, dopaminergic; Exc, excitatory; Inh, inhibitory; Mb, midbrain; P, peripheral nervous system; S, serotonergic; Str, striatum; Ubq, ubiquitous; Vg, voltage gated. b, Fibroblasts stained negative for neuron-specific subunits of BAF complexes (top) whereas induced neurons expressed BAF45b, BAF45c and BAF53b (bottom). Scale bar, 20 µm.

Figure 4. Conversion of adult fibroblasts by miR-9/9*-124-DAM

a, Immunostaining of β-III tubulin (left), MAP2 (middle) and neurofilament (right) in dermal fibroblasts of a 30-year-old individual converted by miR-9/9*-124-DAM. Scale bar, 20 µm. b, A representative current clamp recording. Voltage deflections were elicited by somatic current injections of various amplitudes (Δ = 2 pA). c, A representative voltage clamp recording of the net current at various membrane potentials (−40 to +20 mV, ΔV = 10 mV, V_hold = −90 mV). d, A representative trace of spontaneous EPSCs. e, Representative traces of evoked postsynaptic currents obtained from converted adult cells (see inset; scale bar, 50 µm) in response to local field stimulation with single current pulses (1 ms) of various amplitudes (left, 0.2 and 0.25 mA; right, 0.4 and 0.5 mA). Evoked EPSPs and IPSCs were recorded at −70 mV and +30 mV in the presence of picrotoxin and NBQX/APV, respectively. S, stimulator.