Nonviral direct conversion of primary mouse embryonic fibroblasts to neuronal cells

Abstract

Transdifferentiation, where differentiated cells are reprogrammed into another lineage without going through an intermediate proliferative stem cell-like stage, is the next frontier of regenerative medicine. Wernig et al. first described the direct conversion of fibroblasts into functional induced neuronal cells (iNs). Subsequent reports of transdifferentiation into clinically relevant neuronal subtypes have further endorsed the prospect of autologous cell therapy for neurodegenerative disorders. So far, all published neuronal transdifferentiation protocols rely on lentiviruses, which likely precludes their clinical translation. Instead, we delivered plasmids encoding neuronal transcription factors (Brn2, Ascl1, Myt1l) to primary mouse embryonic fibroblasts with a bioreducible linear poly(amido amine). The low toxicity and high transfection efficiency of this gene carrier allowed repeated dosing to sustain high transgene expression levels. Serial 0.5 µg cm(-2) doses of reprogramming factors delivered at 48-hour intervals produced up to 7.6% Tuj1(+) (neuron-specific class III β-tubulin) cells, a subset of which expressed MAP2 (microtubule-associated protein 2), tau, and synaptophysin. A synapsin-red fluorescent protein (RFP) reporter helped to identify more mature, electrophysiologically active cells, with 24/26 patch-clamped RFP(+) cells firing action potentials. Some non-virally induced neuronal cells (NiNs) were observed firing multiple and spontaneous action potentials. This study demonstrates the feasibility of nonviral neuronal transdifferentiation, and may be amenable to other transdifferentiation processes.

Figure 1

Optimization of nonviral GFP reporter plasmid transfection in PMEFs. (a) Fluorescence micrographs of PMEFs transfected with increasing doses of DNA in p(CBA-ABOL)/pmax-GFP polyplexes (Bar = 500 µm), with 1.0 µg of pmax-GFP delivered with Lipofectamine 2000 (LF2k) included for comparison. Images were taken 24 hours after transfection. (b) AlamarBlue toxicity assay multiplexed with (c) flow cytometric measurement of GFP transfection efficiency and (d) % median fluorescence intensity increase of GFP⁺ cells compared to nonfluorescent control transfections for one and two doses of p(CBA-ABOL)/pmax-GFP polyplexes, assayed 24 hours after transfection; 1.0 µg of pmax-GFP delivered with LF2k is again included for comparison. Error bars represent mean ± SEM of three separate experiments performed in triplicate. Two-way ANOVA were performed with P < 0.05 considered significant. The main effect of DNA mass was significant in b–d, and the main effect of DNA dose number was significant in b. Letters (x,y,z) not shared between columns denote significant comparisons between DNA masses by Tukey post-hoc tests (P < 0.05) of one-way ANOVA across DNA masses, with data from one and two doses pooled in c and d, due to absence of a main effect of dose number by two-way ANOVA. All comparisons of viability (as shown in b) across DNA mass within a given dose number were significant, other than those between 0.5 and 1.0 µg pmax-GFP in p(CBA-ABOL) polyplexes (marked ns–not significant). ANOVA, analysis of variance; GFP, green fluorescent protein; NC, negative control; PMEF, primary mouse embryonic fibroblast.

Figure 2

NiN generation schedule and resulting course of reprogramming factor gene expression. (a) NiN generation scheme. On day 0, PMEFs were seeded on TCPS wells or poly-D-lysine/laminin-coated glass coverslips in complete medium containing serum. Twenty-hour hours later, the first transfection was performed. Two to four additional doses were then administered every 48 hours. Beginning 48 hours after administration of the final dose, the cells were cultured for a minimum of 10 additional days before assay in serum-free N3 neural induction medium containing FGF2, which was replaced every 48 hours, or every 24 hours beyond day 10 in N3 for longer-term experiments. (b) Real-time comparative C_T RT-PCR of ectopic neuronal reprogramming factors (Ascl1, Brn2, Myt1l) and endogenous Ascl1 (Ascl1 Endo) mRNA expression. PMEFs were transfected with one or three doses of 1.0 µg pmax-BAM factors complexed with p(CBA-ABOL), and total mRNA was collected 24 hours after transfection (day 2) or after 10 days of culture in N3. Error bars represent the range of transcript produced by the SD about a mean C_T value (n = 3). FGF, fibroblast growth factor; mRNA, messenger RNA; NiN, non-virally induced neuronal cells; PMEF, primary mouse embryonic fibroblast; RT-PCR, reverse transcription-PCR; TCPS, tissue culture polystyrene.

Figure 3

Tuj1 expression induced in cells transfected with multiple doses of reprogramming factor plasmids. (a) Tuj1 stain of cells transfected with one to five doses of 1.0 µg pmax-BAM factors in p(CBA-ABOL) polyplexes after a minimum of 14 days in N3 medium following transfection. Bar = 200 µm. (b) Large mosaic images of the entire culture area (Bar = 2.5 mm) acquired for image analysis-based quantification of the number of Tuj1⁺ cells normalized to the number of PMEFs seeded (c), after a minimum of 10 days in N3 following transfection. Letters (x,y) not shared between columns denote significant comparisons by Tukey post-hoc tests of a one-way ANOVA (P < 0.05). Error bars represent the mean ± SEM of three separate experiments performed in triplicate. ANOVA, analysis of variance; PMEF, primary mouse embryonic fibroblast.

Figure 4

Immunofluorochemistry and synapsin reporter activity in NiNs generated with p(CBA-ABOL)/DNA polyplexes. All bars are 50 µm. Tau stain (first row): three doses of 1.0 µg pmax-BAM factors, ≥10 days in N3 medium, on TCPS. MAP2 stain (second row): three doses of 1.0 µg pmax-BAM factors (left panel), or 2.0 µg pUNO-AM/pmax-B factors (center and right panels), 16 days in N3, on poly-D-lysine/laminin-coated coverslips. Synaptophysin stain (third row): five (left and center panels) or three (right panel) doses of 1.0 µg pmax-BAM factors, 17 days in N3, on PDL/laminin-coated coverslips. Expression of RFP under control of the synapsin promoter (fourth row): three doses of 1.0 µg pmax-BAM factors (left and center panels), or 2.0 µg pUNO-AM/pmax-B factors (right panel), ≥10 days in N3, on PDL/laminin-coated coverslips. Synapsin-RFP images have not been false-colored red, in order to maximize visual contrast for thin cellular processes. Arrows indicate synapsin-RFP⁺ cell bodies. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein; TCPS, tissue culture polystyrene.

Figure 5

Current clamp in the whole-cell configuration of synapsin-RFP⁺ NiNs generated with p(CBA-ABOL)/DNA polyplexes. (a,b) Traces of cells that received three doses of 1.0 µg pmax-BAM factors, after 16–17 days of culture in N3 medium, on poly-D-lysine/laminin-coated coverslips. (c,d) Traces of cells that received three doses of 2.0 µg pUNO-AM/pmax-B factors, on day 12 of culture in N3, on PDL/laminin-coated coverslips. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein.