Human multipotent mesenchymal stem cells (hMSC) were isolated from umbilical cords donated with informed consent of the pregnant mothers at maternity Januário Cicco, Federal University of Rio Grande do Norte, Natal, Brazil. The study was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (Project Number 508.459), and in strict agreement with Brazilian law (Resolution 196/96). All subjects gave written informed consent in accordance with the Declaration of Helsinki.

In this study, Wharton's jelly mesenchymal stem cells were isolated from umbilical cord. Following isolation from the subendothelium vein, according to the method previously published (Duarte et al., 2012), the remaining umbilical cord tissue was cut in small pieces and washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.47 mM KH₂PO₄; Merck), supplemented with 3% antibiotic–antimycotic solution (prepared with 10,000 units/ml penicillin G sodium, 10,000 μg/ml streptomycin sulfate and 25 μg/ml amphotericin B; HyClone). Then, the tissue was centrifuged at 200 g for 10 min, and the pellet resuspended in 10 mL of 0.1% collagenase type IV (Worthington) diluted in PBS. After that, the explants were incubated for 16 h at 37°C in a water bath. The tissue was centrifuged again at 200 g for 10 min, the pellet washed twice with PBS and then gently dissociated in a digestion solution containing 0.25% trypsin and 0.02% EDTA (Invitrogen) for 15 min at room temperature. To interrupt trypsin activity, we added fetal bovine serum (FBS; HyClone). Once again, the cell suspension was centrifuged, and the cell pellet resuspended in minimum essential medium a (α MEM; Gibco Invitrogen) supplemented with 10% FBS and 1% antibiotic solution. Cells were plated onto T25 tissue culture flasks (TPP) and these cultures maintained at 37°C in a humidified atmosphere containing 5% CO₂. After 2 or 4 days, the medium was changed and non-adherent cells were removed. Cultures consisting of small, adherent and spindle shaped fibroblastoid cells reaching 60–70% of confluence were detached and subcultured at 4,000 cells/cm².

The cells isolated from Wharton's jelly human umbilical cord were characterized as MSCs, according to the criteria proposed by the International Society for Cellular Therapy (Horwitz et al., 2005; Dominici et al., 2006). The hMSCs were labeled with a panel of monoclonal antibodies against several cell markers, including CD105-FITC, CD90PE-Cy5 (Bioscience), CD73PE, CD34PE, HLA-DR-FITC, CD45-FITC, and CD14PE (Becton Dickinson's). Briefly, the cells were detached of the tissue culture plates using 0.25% trypsin/EDTA, washed, and homogenized with PBS. They were then incubated with monoclonal antibody for 30 min in darkness at room temperature. At the end of this period, the cell suspension was centrifuged, washed in PBS, and re-suspended in cold fixing solution, 0.5% formaldehyde in PBS. For each test, isotype-matched monoclonal antibodies were used as negative controls (IgG1-FITC, PE, and PE-Cy5; Becton Dickinson's). The fluorescence intensity of labeled cells was determined with a fluorescence-activated cell analyzer (FACScan) using cell quest software (Cell Quest™ Software, Becton Dickinson Immunocytometry Systems), a total of 20,000 events for each sample were recorded. The following parameters were considered: forward scatter in linear scale (which evaluates cell size), side scatter in linear scale (assessing cell complexity), and cell marker expression in fluorescence analysis by FL1, FL2, and FL3 in logarithmical scale, representing the antigen–antibody reaction conjugated to FITC, PE, and PE-Cy5, respectively. Results were expressed as a percentage of cells labeled with monoclonal antibodies. Osteogenic, adipogenic, and chondrogenic differentiation assays were carried out according to methodology previously published (Duarte et al., 2012).

The pro-neural genes Ascl1, Neurog2, or Sox2 were expressed under control of an internal chicken β-actin promoter with cytomegalovirus enhancer (pCAG) together with DsRed or GFP behind an internal ribosomal entry site (pCAG-Ascl1-IRES-DsRed, pCAG-Neurog2-IRES-DsRed, and pCAG-Sox2-IRES-GFP). For control experiments, cultures were transfected with plasmids encoding only DsRed or GFP (pCAG-IRES-DsRed or pCAG-IRES-GFP) (Heinrich et al., 2010; Karow et al., 2012). Plasmid stocks were prepared in Escherichia coli and purified using the endotoxin-free Maxiprep plasmid kit (Invitrogen). DNA concentration was adjusted to 1 μg/μL in TE buffer endotoxin free, and plasmids were stored at −20°C.

For transfections, hMSC were seeded in 24-well plates onto poly-D-lysine (Sigma-Aldrich) and laminin (L-2020; Sigma Aldrich) coated glass coverslips at a density of 3 × 10⁴ cells per well in 0.5 mL α MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin/streptomycin). The cells were grown in these conditions for 1–3 days until 70–80% confluent.

Both DNA plasmids (1 μg/μL) and a lipophilic cationic reagent (Lipofectamine 2000, Invitrogen) were diluted in 50 μL Opti-MEM (Reduced Serum Medium, Invitrogen). Mixtures were incubated for 5 min and then combined for a further 20 min according to the manufacturer's instructions. Complexes were added to the cells in a total volume of 0.5 mL Opti-MEM (Gibco) and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 10–12 h. Antibiotics and serum were not used during transfection procedures.

For co-culture experiments, mouse hippocampus at postnatal day 0 to 4 (P0-4) were dissected in ice-cold PBS and dissociated in a the digestion solution for 10 min at 37°C. Trypsin action was interrupted with fetal bovine serum and the tissue dissociated mechanically with a fire-polished glass Pasteur pipette. The suspension was then centrifuged at 200 g for 5 min and washed twice in DMEM/F12 10% FBS in DMEM/F12 medium (Gibco). Mouse hippocampal cells were added to the human cultures 1–2 days after transfection at a density of 50,000 cells per well. The local University Animal Care and Use Committee (CEUA/UFRN) approved experiments involving mice. All experiments were carried out in accordance with international guidelines and regulations for animal use.

Cell cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Primary antibodies were diluted in PBS, 0.5% Triton X-100 and 5% normal goat serum. Specimens were incubated overnight at 4°C. After three washes with PBS, cells were incubated with species-specific secondary antibodies conjugated to fluorophores for 2 h at room temperature. Once again, samples were washed with PBS three times. For nuclei staining, cells were incubated for 5 min with 0.1 μg/mL DAPI (4′6′-diamino-2-phenylindone) in PBS 0.1 M. Coverslips were finally mounted onto a glass slide with a mounting medium (Aqua Poly/Mount; Polysciences). The following primary antibodies and dilutions were used: chicken anti-Green Fluorescent Protein (GFP, Aves Labs, 1:1,000), rabbit anti-Red Fluorescent Protein (RFP, Rockland, 1:1,000), mouse anti-major microtubule associated protein (MAP2; Sigma, 1:500), guinea pig polyclonal anti-vesicular GABA transporter (vGAT, Synaptic Systems, 1:200), and polyclonal anti-vesicular glutamate transporter 1 (vGLUT11, Synaptic Systems, 1:1,000).

Cell cultures with induced neurons were transferred to a recording chamber mounted on the stage of a microscope equipped with a water immersion 40X objective (Zeiss Examiner. A1, 1 NA) and perfused with oxygenated external solution (1–1.25 ml/min) at 37°C. Data were acquired using a patch-clamp amplifier Axopatch 200B (Molecular Devices) in current or voltage clamp mode, a 16-bit data acquisition card (National Instruments), and WinWCP or WinEDR software implemented by Dr. John Dempster (University of Strathclyde). Patch-pipettes of borosilicate glass capillaries (GC150F-10 Harvard Apparatus) were pulled on a vertical puller (Narishige) with resistances from 5–7 MΩ. Pipettes were filled with internal solution (~290 Osm) containing (in mM) 130 K⁺-gluconate, 7 NaCl, 0.1 EGTA, 0.3 MgCl₂, 0.8 CaCl₂, 2 Mg-ATP, 0.5 NaGTP, 10 HEPES, and 2 EGTA (pH 7.2 adjusted with KOH 1M). The external solution (~300 Osm) contained (in mM) 120 NaCl, 3 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 23 NaHCO₃, 5 HEPES, and 11 Glucose (pH 7.4 adjusted with NaOH 1M).

Patch-clamped cells were measured for input resistance, resting membrane potential, and capacitance. Recordings were analyzed with custom routines in MATLAB. Action potentials were triggered by 400-ms depolarizing current injections from 100 pA, 400 ms, with 10 pA increments. The first fired action potential in response to minimal current injection was analyzed for amplitude (peak to afterhyperpolarization voltage), half-width (halfway between threshold voltage and peak), and afterhyperpolarization amplitude (threshold to minimum of voltage trough between the first and the second action potential in a spike train). Instantaneous and steady-state voltage were analyzed in response to hyperpolarizing current injections (−100 pA, 400 ms). Excitatory postsynaptic currents were analyzed for amplitude and rise time in free-run traces of 150 s. Active and passive electrophysiological membrane properties, including action potential parameters were analyzed using a Student's unpaired, two-tailed t-test.

Calcium imaging was performed on human MSC 3 weeks post-transfection using Oregon green 488 BAPTA-1 (Invitrogen, 10 μM). Imaging was performed in physiological saline solution containing (in mM) 140 NaCl, 5 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES, 10 glucose, and 6 sucrose (pH 7.35). Images were acquired approximately every 10 ms using a scientific CMOS camera (Andor). The microscope was controlled by Micro-Manager software together with the image processor ImageJ. Changes in fluorescence were measured for individual cells and average of the first 10 time-lapse images for each region of interest (ROI) was defined as initial fluorescence (F0).

After electrophysiological recordings, the cell was sucked into the recording pipette. Pipettes were quickly removed and broken into 1.5 mL tubes containing 20 U of RNase inhibitor and 8.3 mM DTT. Samples were frozen immediately on dry ice and stored at −80°C. Immediately after thaw, the samples were treated to eliminate contaminating DNA molecules. Complementary DNA (cDNA) synthesis and pre-amplified reactions were performed with the RT² PreAMP cDNA Synthesis Kit following the manufacturer's procedure (QIAGEN). Amplification was performed on the Applied Biosystems ViiA 7 Real-Time PCR (Applied Biosystems). RT² Profiler PCR Array were customized in 96-well plates, designed for analyzing the expression of the following genes: Choline O-acetyltransferase (CHAT), Tyrosine hydroxylase (TH), Tryptophan hydroxylase 2 (TPH2), Vesicular glutamate transporter 1 (VGLUT1 or SLC17A7), GABA Vesicular transporter (VGAT or SLC32A1), FEZ family zinc finger 2 (FEZF2), T-box brain 1 (TBR1), SATB homeobox 2 (SATB2), COUP-TF-interacting protein 2 (CTIP2 or BCL11B), Platelet-derived growth factor receptor, beta polypeptide (PDGFRB), Thy-1 cell surface antigen (THY-1), Atonal homolog 8 (ATOH8), Neurogenic differentiation 1 (NEUROD1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Hypoxanthine phosphoribosyltransferase 1 (HPRT1). The RT-qPCR was performed using the RT² profiler PCR customized array (QIAGEN). Each array included genomic DNA control primer set, reverse transcription control, positive PCR control to report the efficiency of the polymerase chain reaction itself, and the endogenous reference genes GAPDH and HPRT1.

A single-cell RT-qPCR pre-processing was performed based on method described by Ståhlberg et al. (2013). Melting curve analysis performed elimination of false positives. Next, relative quantities were calculated using a cycle of quantification cutoff (Cq-cutoff) and relative-quantities of cDNA molecule equation. Missing data were imputed with absolute value 0.5, followed by conversion to log₂-scale. Mean center and auto scale, for each gene mean center and auto scale were calculated separately using log₂-values. Heat map and Principal Component Analysis (PCA) were used to visualize expression differences between groups. Statistical analysis and plotting were performed using the software R version 3.3.3.

All statistical data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistically significant differences were assessed by Student's unpaired t-test or one-way Analysis of variance (ANOVA), comparing two or more groups, respectively. P < 0.05 was considered a significant difference (^*).

Mesenchymal stem cells (MSCs) can be isolated from different sources in adult humans, including the bone marrow and umbilical cord (Ding et al., 2011). These cells are highly plastic, retaining the potential to generate chondroblasts, adipocytes, and osteoblasts (Caplan, 1991; Dominici et al., 2006; Afanasyev et al., 2010; Keating, 2012). In order to characterize the cells isolated from Wharton's jelly umbilical cord, we first evaluated the expression of MSC-specific antigens using flow cytometry. Virtually all cells exhibited expression of CD105, CD73, and CD90 markers, and lacked the expression of hematopoietic lineage markers, such as CD14, CD34, and CD45 (Supplementary Figures 1A–I). The MSCs also demonstrated capacity for osteogenic, adipogenic, and chondrogenic differentiation (Supplementary Figures 1J–L). Given this versatility, we hypothesized that expression of neurogenic transcription factors in MSCs could directly reprogram these cells into neurons. To test this possibility, we transfected plasmids carrying the genes encoding for Sox2, Neurog2, or Ascl1 into human umbilical cord mesenchymal stem cells (hUCMSCs) using lipophilic cationic reagent. To monitor transduced cells, all vectors carried a fluorescent protein (GFP or DsRed) under control of an internal chicken β-actin promoter with cytomegalovirus enhancer (pCAG). Vectors expressing GFP or DsRed alone were used as control (Figures 1A,B). One day after transfection, cultured medium of hUCMSCs was replaced with neuronal differentiation medium containing B27. In this medium, most transfected hUCMSCs underwent cell death precluding analysis of lineage reprogramming (Supplementary Figure 2). To overcome this limitation, we co-cultured neonatal mouse hippocampal cells with hUCMSCs. We found an average of about 50% GFP+/DsRed+ hUCMSC per field 15 days after transfection and in the presence of co-cultured neonatal mouse hippocampal cells. In contrast, the average number of GFP+/DsRed+ hUCMSCs in the absence of co-cultured cells was <4% (Supplementary Figure 2). Low number of cells was also observed in Hucmsc cultures transfected with control plasmids, indicating that the cell death under these culture conditions was independent of lineage-reprogramming. It is likely that the withdrawal of serum performed after transfection (aiming at the differentiation of induced neurons) affects the survival of hUCMSCs, whereas addition of co-cultured cells, somehow, counteracts this cell-death effect. We, therefore, concluded that co-cultures are necessary to support hUCMSC in the culture conditions used.

A few days after transfection, we observed that some hUCMSCs transfected with proneural genes acquired neuronal-like morphology. To confirm this possible lineage conversion of hUCMSCs into iNs, we further analyzed the expression of the neuronal-specific microtubule-associate protein 2 (MAP2) 15 days after transfection with proneural genes (Figure 1). A fraction of cells transfected with proneural genes expressed MAP2 and acquired small-round cell bodies and thin and long processes, resembling immature neurons. Whereas, hUCMSCs transfected with control plasmids displayed classical mesenchymal cell morphologies and did not express MAP2 (Figures 1A–U). Expression of Sox2, Ascl1, or Neurog2 alone was sufficient to reprogram hUCMSCs into iNs, albeit at low rates (Figure 1V). However, the combination of Sox2 and Ascl1 increased the efficiency of reprogramming up to 49%, whereas the combination of Sox2 and Neurog2 increased the efficiency up to 35% (Figure 1V). We obtained these results using Wharton's jelly mesenchymal stem cells isolated from tree different donors and did not observed any heterogeneity in the potential of reprogramming (data not shown). These data indicate that single proneural TFs have potential to elicit lineage reprogramming of hUCMSCs into iNs, but that the synergistic action of the TFs Sox2/Ascl1 or Sox2/Neurog2 is sufficient to induce neuronal phenotype in a high number of hUCMSCs.

It has been shown that cells from the mesenchymal lineage can fuse with other cell types in culture (Terada et al., 2002; Alvarez-Dolado et al., 2003). To rule out the possibility that hUCMSCs could be fusing with mouse hippocampal neurons present in our co-cultures, we performed similar experiments co-culturing reprogrammed hUCMSCs with purified postnatal mouse cortical astrocytes (Heinrich et al., 2011). Similar to cultures containing neonatal hippocampal cells, we observed that hUCMSCs transfected with Sox2/Ascl1 or Sox2/Neurog2 survived in astrocyte monolayers and acquired neuronal-like morphologies (Figures 2A–I). Thus, lineage reprogramming of hUCMSCs into iN after Sox2/Ascl1 or Sox2/Neurog2 expression is unlikely to be attributed to cell fusion with primary co-cultured neurons. However, we observed a lower lineage conversion efficiency when plating hUCMSCs on astrocytes (Figure 2J) compared to hippocampal cells suggesting that additional factors released by co-cultured neurons may affect either the reprogramming efficiency or survival of iNs.

To investigate if iNs could establish synaptic connections with neighboring neurons, we studied the dynamics of calcium transients using calcium sensitive dye imaging (Rosenberg and Spitzer, 2011). We measured the spontaneous changes in fluorescence intensity (ΔF/F0) during the total period of imaging (17 s) and compared their responses with primary murine hippocampal neurons. We found that both human iNs and mouse hippocampal neurons present in the co-culture displayed fast calcium-transients as indicated by rapid variations in the fluorescence (Figures 3A–I, Supplementary Movies 1, 2). The iNs reprogrammed with Sox2/Ascl1, Sox2/Neurog2, and the mouse hippocampal neurons showed a mean variation in fluorescence intensity of 39.66% (Figure 3J, gray bar), 33.05% (Figure 3J, blue bar), of 60.83% (Figure 3J, white bar), respectively. In contrast, the change in fluorescence intensity observed in hUCMSCs transfected with control plasmids presented a mean value of 1.1% (Figure 3J, black bar), significantly lower than the responses observed in iNs and hippocampal neurons (ANOVA followed by Tukey's post-test, p < 0.0001). These observations indicate that hUCMSCs-derived iNs present fast calcium transients qualitatively similar of those observed in primary neurons.

Notably, this sudden increase in fluorescence intensity in hUCMSC-derived iNs was temporally synchronized mouse hippocampal neurons in the same field of observation (Figures 4A–I). To quantify this phenomenon, we measured the percentage of hippocampal neurons showing elevation in the fluorescence intensity within a time-range (ms) of the fluorescence fluctuation observed in a single iN within the same field of observation. The time of the iN calcium transient was considered as t = 0. We found that the majority of mouse hippocampal neurons showed changes in fluorescence intensity within 15 ms of fluctuations observed in iNs (Figure 4J), indicating a strong synchronization of calcium transients among primary neurons and iNs. Such a strong synchronization within the frame of milliseconds may suggest that cells are synaptically connected (Dawitz et al., 2011). To further confirm that hUCMSCs-derived iNs could receive synaptic inputs, we performed patch clamp recordings on these cells.

We performed patch-clamp recordings on iNs reprogrammed with one transcription factor (1 TF; n = 5) and compared their active and passive properties to cells reprogrammed with two transcription factors (2 TF; n = 10). Cells with 1 TF had a mean input resistance of 726 ± 119 MΩ, resting membrane potential of −61 ± 2 mV and capacitance of 22 ± 2 pF. Out of the 5 cells, 2 responded with regular spiking pattern (Figure 5A, top left), 1 responded with startle onset (Figure 5A, top right), and 2 with a spikelet in response to depolarizing current injections (0–100 pA, 400 ms, with 10 pA increments). Spikes were analyzed for action potential amplitude, action potential half-width and afterhyperpolarization amplitude. Spikes of cells with 1 TF had a mean action potential amplitude of 44 ± 8 mV, action potential half-width of 13 ± 0.5 ms and afterhyperpolarization amplitude of −6 ± 2 mV (Figure 5C). In comparison, cells reprogrammed with 2 TF had a mean input resistance of 605 ± 110 MΩ (p = 0.51), resting membrane potential of −59 ± 2 mV (p = 0.54) and capacitance of 26 ± 1 pF (p = 0.13). Of the 10 cells with 2 TF, 6 responded with regular spiking pattern and 4 responded with startle onset (0–100 pA, 400 ms, with 10 pA increments). Spikes of cells with 2 TF had a mean action potential amplitude of 76 ± 3 mV (p = 0.0004), action potential half-width of 4 ± 1 ms (p = 0.0006) and afterhyperpolarization amplitude of −11 ± 2 mV (p = 0.22; Figure 5B). While hyperpolarizing current injections (0 to −100 pA, 400 ms, with 10 pA decrements) caused some cells to rebound (n = 3), prominent membrane sags could only be detected in cells transfected with Sox2/Neurog2 (n = 7; Figure 5B, right) suggesting that these cells have a sizeable hyperpolarization-activated current. Comparing instantaneous and steady state voltage in response to negative current injections (−100 pA, 400 ms) showed a significant difference between instantaneous (−71 ± 2 mV) and steady state (−66 ± 1 mV) values (p = 0.0427).

We also observed spontaneous excitatory postsynaptic currents (EPSCs) in iN during voltage clamp (Figure 5D), suggesting that lineage-reprogrammed iNs could receive synaptic contacts from other neurons. We compared the first 100 events of 1 TF cells and 2 TF cells against each other. Postsynaptic currents of cells with 1 TF had an amplitude of 82 ± 9.7 pA and rise time of 11 ± 0.4 ms. The postsynaptic currents of cells with 2 TF had an amplitude of 86 ± 15.1 pA (p = 0.8312) and a mean rise time of 9 ± 0.3 ms (p = 0.0211).

Next, we set out to evaluate the expression of messenger RNAs (mRNA) of genes commonly expressed in either hMSCs or neurons. To that, we collected single cells using a glass-micropipette, isolated the total mRNA, reverse transcribed, and pre-amplified cDNAs that were used in RT-qPCR reactions. We observed that the average expression level of common MSCs genes THY1 and PDGFB was decreased in the iNs (Supplementary Figure 3A), whereas the expression of the neuronal genes ATHO8 or NEUROD1 increased after expression of Sox2/Ascl1 or Sox2/Neurog2 in hMSCs, respectively (Supplementary Figure 3B). Combined with our previous observations, these data indicate that hMSCs were effectively converted into iNs by forced expression of proneural TFs.

To evaluate the possible phenotypes adopted by hMSC-derived iNs, we analysed the expression of known genes expressed by cholinergic (CHAT), dopaminergic (TH), serotoninergic (TPH2), glutamatergic (SLC17A7), and GABAergic (SLC32A1) neurons, as well as genes encoding for transcription factors associated with specific classes of glutamatergic neurons within the cerebral cortex (FEZF2 and BCL11B—corticofugal neurons; TBR1—cortico-thalamic neurons; SATB2—callosal neurons). Relative expression of these transcripts was calculated using a cycle of quantification cutoff (Cq-cutoff) and relative-quantities of cDNA molecule equation (Ståhlberg et al., 2013). Next, we used unsupervised PCA analysis to classify iNs obtained from hUCMSCs expressing either Sox2/Neurog2 or Sox2/Ascl1. We observed that the expression levels of the transcripts for CHAT, TH, TPH2, SLC17A7, SLC32A1, FEZF2, BCL11B, TBR1, and SATB2 could not clearly distinguish the two populations of cells (Figure 6A), indicating that similar genes were regulated by both combinations of TFs in hUCMSCs-derived iNs. Indeed, we observed that both Sox2/Ascl1 and Sox2/Neurog2 could induce the expression of genes associated with distinct neurochemical phenotypes in hUCMSCs-derived iNs, although some phenotypes were more commonly observed for a given TF combination. For instance, Sox2/Ascl1 generated more iNs expressing high levels of TPH2, whereas Sox2/Neurog2 generated more CHAT expressing iNs. Nevertheless, the expression of all transcripts analyzed was regulated by both combinations of TFs, suggesting that Ascl1 and Neurog2 do not have a unique role in the phenotypic specification of lineage reprogrammed hUCMSC-derived iNs (Figure 6B). These data suggest that the expression of Sox2/Ascl1 or Sox2/Neurog2 in hUCMSC activates a transcriptional program associated with loss of mesenchymal phenotype and acquisition of multiple neuronal phenotypes.

To further evaluate the neurochemical phenotypes of hUCMSC-derived iNs, we investigated the expression of SLC17A7 (Vesicular Glutamate Transporter 1 or VGLUT1) and SLC32A1 (GABA Vesicular Transporter or VGAT) using immunocytochemistry (Figure 7). We observed that only a few Sox2/Ascl1-derived iNs showed expression of VGAT fifteen days after reprogramming (Figures 7I–L), whereas most of the hUCMSC-derived iNs did not express any of these markers days after (Figures 7M–Y). Although iNs expressed MAP2 15 days after transfection with proneural genes (Figure 1), expression of vesicular neurotransmitter transporters is likely to occur at later stages of neuronal differentiation. Further analyses and immunostaining for other vesicular transporters isoforms are necessary to confirm the phenotypes of hUCMSCs-derived iNs.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.