Induction of neurons via single defined transcription factors

We initially identified that of the four transcription factors (TFs), that is, Brn2, Ascl1, Myt1L and NeuroD1, used by Pang et al.12 to reprogram human fibroblasts to neurons, only Ascl1 and NeuroD1 could individually induce neuronal conversion of iPS cells, as also reported elsewhere10. Early neurons induced by NeuroD1 expression exhibited complex neuronal morphologies and express mature neuronal markers, so all subsequent studies were done using NeuroD1 as the single TF for generating iN cells. We transduced human iPS cells with lentiviruses encoding tetracycline-inducible, tetOn-NeuroD1, rtTA, and, in selected cases, tetOn-EGFP, and maintained them as undifferentiated iPS cells in mTeSR medium for multiple passages in the absence of doxycycline (dox; Supplementary Fig. 1a). We refer to these cells as ‘RN-iPS’ cells, since these cells were transduced with rtTA and NeuroD1. RN-iPS cells maintain expression of pluripotency markers SSEA-4 and Oct-4 over multiple passages in the absence of dox, indicating that they remain undifferentiated after transduction (Supplementary Fig. 1a).

To assess neuronal induction from RN-iPS cells, dense cultures of RN-iPS cells were seeded and dox was added 24 h after plating (Supplementary Fig. 1b). Early stage iN cells expressed neuronal marker βIII-tubulin and residual undifferentiated iPS cells expressed Oct-4 (Supplementary Fig. 1c). Dox addition rapidly induces the loss of undifferentiated morphology and the acquisition of bipolar, early neuronal morphologies in a subset of cells within 48 h (Supplementary Fig. 1c–e). Dox also rapidly induces EGFP expression in control cells transduced with tetOn-EGFP. By contrast, cells transduced with only tetOn-EGFP and rtTA (lacking tetOn-NeuroD1) show strong GFP fluorescence but maintain undifferentiated hPSC morphology upon dox addition (Supplementary Fig. 1e).

Despite the rapid, dox-induced neuronal conversion of a subset of RN-iPS cells, the remaining undifferentiated cells proliferated and rapidly overtook iN cultures. To address this, cells were replated 4 days post dox treatment, which established more uniform human neuronal cultures and eliminated many undifferentiated cells (Fig. 1a). The replating process may disrupt the cell–cell contacts necessary for undifferentiated iPS cell survival. After replating, enriched iN cells could be maintained for 4 weeks or longer before characterization.

Figure 1: Characterization of neuronal conversion and maturation in human induced-neuronal cells. (a) Schematic of long-term iN reprogramming to functional neurons. (b) After 12 days of conversion, iN cells express βIII-tubulin and MAP2. (c) Further differentiation on glial cells for 28 days allows specification of several neuronal subtypes, including vesicular GABA transporter-expressing GABAergic neurons and tyrosine hydroxylase-expressing dopaminergic neurons, in addition to the predominantly glutamate vescular transporter-expressing glutamatergic neurons. (d) Day 28 iN cells also extensively express mature neuronal makers such as the synaptic vesicle protein synaptophysin. (e) Whole-cell current recordings demonstrate that iN cells after 14 days culture in the absence of glia are predominantly electrically active with functional voltage-dependent Na+ channels, as well as voltage-dependent K+ channels, and (f) fire repetitive induced-action potentials (n=27/27). Scale bar, 50 μm. Inset: Scale bar, 10 μm. Full size image

As expected, human iN cells robustly express βIII-tubulin and microtubule-associated protein 2 (MAP2) by day 12–14 after dox addition (Fig. 1b). To identify the neuronal subtypes generated by this procedure, iN cells were replated 4 days after dox addition onto glial cell monolayers as reported elsewhere23. Immunocytochemistry on 28 day iNs post replating revealed that most cells expressed glutamate vesicular transporter VGLUT1, indicating that these cells are predominantly excitatory glutamatergic neurons (Fig. 1c). Occasional cells expressing markers of other neuronal subtypes were also observed, including inhibitory GABAergic neurons (expressing vesicular GABA transporter) and dopaminergic neurons (expressing tyrosine hydroxylase; Fig. 1c). Most cells also robustly expressed the pre-synaptic protein synaptophysin (Fig. 1d). Finally, patch-clamp electrophysiology demonstrated that iN cells are electrically active and express functional voltage-dependent Na+ channels as well as voltage-dependent K+ channels as revealed by whole-cell current recordings (Fig. 1e). Strikingly, these human iN cells were capable of firing repetitive action potentials (Fig. 1f). Taken together, these data indicate the robust generation of functional neuronal cells via NeuroD1 overexpression.

Generation of functional neurons on 3D electrospun fibres

Next, we investigated whether NeuroD1 expression would similarly induce neuronal conversion and maturation within model 3D electrospun substrates. We constructed fibrous substrates by electrospinning poly(desaminotyrosyl tyrosine ethyl ester carbonate) (pDTEc), into two architectures, which will be referred to as ‘thin’ and ‘thick’ fibre substrates with average fibre diameters of 1.25±0.05 μm and 3.23±0.06 μm, respectively24 (Fig. 2a–d). pDTEc is the lead candidate polymer from a combinatorial library of tyrosine-derived polycarbonates25, as it effectively supports pluripotent stem cell culture when fabricated into microscale fibrous substrates24, and is biocompatible26. Similarly, our results with 3D polymeric substrates compared with 2D polymeric substrates suggest that the fibrous architecture governs the longer term cellular behaviours observed in contrast to the polymer composition that plays a role on early interfacial phenomena. The thick fibre scaffolds are volumetrically permeable to cellular infiltration, whereas the thin fibre scaffolds are relatively impermeable, due to decreased void space between fibres. Without additional material modifications, it is difficult to produce equal fibre sizes with variable porosity, as both properties are simultaneously modulated when altering electrospinning parameters. We hypothesize that cell permeable, thick fibre substrates will support improved iN maturation and functionality by promoting enhanced 3D organization and cell–cell contacts relative to less permeable, thin fibre substrates and 2D controls.

Figure 2: Characterization of electrospun polymer fibres and validation for support of iN differentiation. (a,b) Scanning electron microscopy and (c,d) reflectance images of 2D fibrous and 3D electrospun pDTEc fibres, with substantially variable fibre architectures and porosities that respectively do not allow and allow cellular infiltration. (e) Schematic of RN-iPS cell reprogramming on 3D electrospun fibres. (f,g) RN-iPS reprogramming was carried out on 3D electrospun fibres, demonstrating that generation of βIII-tubulin and MAP2 positive iN cells on 3D electrospun fibres after 12 days proceeds similarly to 2D controls shown in Fig. 1. (h) Whole-cell current recordings demonstrate that iN cells cultured for 10 days on 3D electrospun fibres, 14 days total were predominantly electrically active with functional voltage-dependent Na+ channels, as well as voltage-dependent K+ channels, and (i) fire repetitive induced action potentials (n=18/20). Scale bar, 50 μm (c–d,f). Scale bar, 10 μm (g). Full size image

Human iN cells were generated within 3D constructs by treating RN-iPS cells, cultured on 2D tissue-culture plates, with dox for 4 days, followed by replating onto 3D electrospun substrates or 2D controls, according to the time course schematic shown in Fig. 2e. Human iN cells on 3D electrospun fibres showed complex morphology with extensive neurite outgrowth and expressed βIII-tubulin, MAP2 and synaptophysin after 12 days of reprogramming, similarly to 2D cultures (Fig. 2f,g). In addition, electrophysiological recordings revealed iN cells in electrospun substrates fired action potentials, demonstrating the derivation of functional iN cells (Fig. 2h,i).

Effect of 3D scaffold architecture on human iN conversion

Next, we examined whether the fibre architecture could be tuned to enhance human iN maturation, as we and others have shown that geometric cues can influence both human iPS cell and neural cell behaviours24,27. RN-iPS cells were treated with dox for 4 days, then replated onto 3D fibrous substrates or 2D controls, including 2D polymer-coated controls, for an additional 8 days of culture. Immunocytochemistry for proliferation marker Ki67 and Oct-4 revealed that significantly more proliferative and pluripotent cells were retained when iN cells were replated onto 2D substrates compared with 3D fibrous substrates (P<0.05, one-way ANOVA; Fig. 3a,c, Supplementary Figs 2,3). This suggests that the fibrous architectures may be able to selectively reduce the presence of residual proliferative and pluripotent iPS cells.

Figure 3: Comparison of neuronal selection and maturation in 2D and 3D substrates. (a) Human iN populations robustly express MAP2 in 2D and 3D conditions, while populations of unconverted, proliferative Ki67-expressing iPS cells persist in iN populations plated in 2D conditions. Scale bar, 100 μm. (b) Quantification reveals an enhancement of maturation as assessed by MAP2 expression in 3D electrospun thick fibres relative to thin fibres, as well as in both 3D fibrous conditions relative to 2D conditions. n=3, *P<0.05, ***P<0.001 by one-way ANOVA. (c) Quantification reveals an enhancement of neuronal selection, as assessed by Ki67 expression, fewer residual proliferative cells remained in iNs replated into 3D fibrous conditions relative to 2D conditions. Scale bar, 100 μm. n=3; *P<0.05, by one-way ANOVA. (d) Heat map images from calcium recordings of human iN cells before (left), during (middle) and after field electrical stimulation). Scale bar, 75 μm. (e) Quantification of the fraction of cells that respond to electrical stimulation with a substantial increase in fluorescence intensity reveals highly active cell populations in 2D and 3D thick fibre substrates relative to cells in the thin fibre substrates. n=3; *P<0.05 by one-way ANOVA, all error bars presented as mean±1 s.d. Full size image

Human iN cells in all conditions expressed extensive βIII-tubulin-positive processes, along with robust MAP2 expression (Fig. 3a, Supplementary Fig. 2). Significantly greater numbers of human iN cells expressed MAP2 in thick fibre substrates relative to 2D controls (P<0.0001, one-way ANOVA) and thin fibre substrates (P<0.05, one-way ANOVA), indicating accelerated maturation (Fig. 3b). qRT–PCR also revealed increasing trends in expression of several neuronal genes in 3D substrates relative to 2D controls, including βIII-tubulin, MAP2, synapsin 1 and VGLUT1, though not statistically significant (Supplementary Fig. 4). Most importantly, calcium imaging to identify the fraction of cells that respond to a field electrical stimulation indicated that thick fibre substrates yielded a high degree of activity, namely, >70% electrically active cells by day 12 of culture (Fig. 3d–e), which was significantly greater than those on the thin fibre substrates (P<0.05, one-way ANOVA). Inhibition of E-cadherin-dependent cell–cell contacts markedly reduced neuronal outgrowth in 2D, and decreased activity measured by calcium imaging for iNs on 2D and 3D thick fibre substrates but not thin fibre substrates (Supplementary Fig. 5). This indicates that 3D microfibrous architectures establish neuronal networks with enhanced cell–cell contacts and influence both functional and phenotypic maturity of iN cellular networks.

Microscale scaffolds support outgrowth and survival in brain

Next, transplantable constructs were designed to deliver human iN cells into the brain for regenerative therapies. The large mats of electrospun fibres (0.3–2 cm diameter discs) conventionally fabricated for in vitro studies cannot be easily transplanted into the CNS. To allow for injection in vivo, 100 μm square ‘microscale scaffolds’ that could be injected through a 21-gauge needle were created by downscaling thick fibre electrospun substrates with a Vibratome. Human iN cells were seeded in suspension onto microscale scaffolds analogous to our previous studies, which resulted in efficient population of microscale scaffolds with iN cells. Cells in microscale scaffolds matured into βIII-tubulin and MAP2-expressing neuronal cells, similarly to cells on macroscale fibrous substrates (Supplementary Fig. 6). The average number of live human iN cells in each scaffold was 83±13 (n=19; Supplementary Fig. 7).

The ability of microscale electrospun scaffolds to promote human iN cell survival and engraftment was first assessed using an ex vivo model consisting of organotypic hippocampal slice cultures from NOD-SCID IL2Rγc null mice (Fig. 4a). Human iN cells on 4–5 microscale scaffolds were injected into hippocampal slices, alongside equivalent numbers of dissociated cells on paired slices, and engraftment and functionality was assessed. Immunocytochemistry revealed that 3 days after transplantation, injected scaffold-supported iN cells had average neurite lengths of 831±169 μm, which was significantly greater (P<0.0001, one-way ANOVA) than those of injected dissociated iNs, which had neurite lengths of 241±42 μm (Fig. 4b–e). Electrophysiological recordings from human iN cells after 3 weeks indicated that both dissociated and scaffold-supported iN cells fire action potentials in response to current injection (Fig. 4f,g), with scaffold-seeded iN cells displaying enhanced excitability in response to injected currents (Fig. 4h). In addition, both modes of transplanted cells had mature Na+ channel expression (Fig. 4i). This suggests that microscale scaffolds enhance engraftment and functionality of transplanted iN cells.

Figure 4: iNs supported by scaffolds support outgrowth and survival ex vivo. (a) Hundred-micrometre edge scaffolds were cut from electrospun fibres before seeding with iNs, followed by injection onto ex vivo cultured mouse pup brain slices. (b–e) Neurite length was found to be significantly enhanced in transplanted scaffold-supported iNs when comparing dissociated GFP-labelled iNs (b) with iNs seeded on scaffolds (c) injected onto mouse ex vivo brain slices (n=8 brain slices for each transplantation mode). (f–h) Both scaffold-supported iNs (n=6) and dissociated iNs (n=5) fire action potentials in response to current injection 14 days after transplantation onto mouse ex vivo brain slices, however, scaffold-supported iNs displayed enhanced excitability relative to dissociated iNs. Both modes of transplanted iNs were observed to have mature sodium channel expression (i). ***P<0.0001 by one-way ANOVA. Scale bar, 20 μm. All error bars presented as mean±1 s.d. Full size image

Next, human iN cell survival was assessed after transplantation of scaffold-supported or dissociated cells into the mouse striatum in vivo. Three weeks after transplantation (Fig. 5a), immunocytochemistry of a 1.8 mm × 1.8 mm field including the injection site (Fig. 5b–e) revealed an average survival rate of 5.74±3.16% based on injected scaffold-seeded iN cells, a 38-fold improvement (P<0.05, one-way ANOVA) compared with an average survival rate of 0.15±0.15% of 100,000 injected dissociated iN cells (Fig. 5g). The survival rate of dissociated iN cell controls was comparable to that reported by Zhang et al.10 When magnitude of injected cells were matched at ∼1,000 cells, quantification of surviving cells yielded an average of 7.58±4.60% of injected scaffold-seeded iN cells compared with 0±0% out of 1,000 injected dissociated iN cells. The scaffold-seeded iNs maintained neurite length (35±8 μm) comparable to those of viable, dissociated iNs (39±15 μm) in the experiment when number of injected cells was unmatched. No overt difference in inflammatory response was detected between injection modes, and some ingrowth of host tissue into scaffolds was observed (Supplementary Fig. 8). Surviving transplanted iNs expressed neuronal cell-adhesion molecule CD56, βIII-tubulin and synaptophysin (Fig. 5f, Supplementary Fig. 9). Post-synaptic density protein 95 (PSD95, depicted in blue, with downward-pointing arrows) was detected adjacent or co-localized to transplanted GFP-labelled and synaptophysin-expressing iN neurite terminals, suggestive of synaptic integration with host tissue (Fig. 5f; Supplementary Movie 1). Notably, when microscale scaffolds were used to transplant multiple subtypes of neurons, we observed retention of neurons of distinct specificity in close apposition in vivo (Fig. 5i,j). Similar differences in survival were also seen when glutamatergic and dopamine iNs (Supplementary Fig. 10) were co-transplanted either dissociated or on microscaffolds. In all in vivo experiments, some human iNs were observed to migrate off scaffolds, and minimal scaffold degradation was observed in the 1–3 weeks post-transplantation.