Robust generation of retinal neural progenitors from human pluripotent stem cells under defined serum- and feeder-free condition

We developed a simple and efficient method to generate highly enriched retinal cells from human PSCs using a multi-step strategy (Supplementary Fig. S1). First, human PSCs were directly induced to differentiation in retinal induction medium (RIM) followed by exposure to neural differentiation medium (NDM) containing Noggin. After short exposure (usually 4 days) to RIM, cells at the edge of colonies became elongated and exhibited a columnar phenotype (Supplementary Fig. S2a), and small cells dominated the center of these colonies after further differentiation (Supplementary Fig. S2b). After 10 days of in vitro cell differentiation, more than 90% of cells expressed paired box 6 (PAX6), nestin and sex determining region Y-box 2 (SOX2) (Supplementary Fig. S2c,d), suggesting they were committed to neural stem cells. Approximately two weeks after differentiation and expansion in NDM, small cells dominated the whole culture with some large cells located at the edge of neural stem cell colonies (Supplementary Fig. S3a). Rapid upregulation of PAX6 and retinal homeobox gene 1 (RX1) transcription factors was observed, and the majority of them (>90%) were double positive for PAX6 and RX1 (Fig. 1a,b) at day 18. RT-PCR analyses showed expression of PAX6, RX1, LIM Homeobox 2 (LHX2), SIX homeobox 3 (SIX3), SIX homeobox 6 (SIX6) and T-Box 3 (TBX3) in these cells (Supplementary Fig. S3b). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) also demonstrated a dramatic decrease in the expression of octamer-binding transcription factor 4 (OCT4) and nanog, two critical pluripotent genes (Supplementary Fig. S3c). These results demonstrated a robust differentiation of hESCs towards retinal neural cells with gene profiles corresponding to eye field progenitors (EFP).

Figure 1: In vitro Differentiation of Human Embryonic Stem Cells towards Retinal Neural Progenitors. (a) Immunofluorescence staining shows co-expression of PAX6 and RX1 on day 13 eye field progenitors. (b) Quantification of PAX6 and RX1double positive eye field progenitors by flow cytometry analysis which shows >90% of them expressing both PAX6 and RX1 proteins. (c) Phase contrast image shows neural rosette structures of retinal neuronal progenitor cells (RNPC, far left) and immunofluorescence staining shows expression of PAX6 and CHX10 on RNPC at about day 30 after initial differentiation in vitro. Scale bar, 50 μm. Full size image

At day 19, EFP cells were collected and plated to form neural spheres in a suspension culture. After 3 days, neural spheres were re-plated on matrigel coated surface. Neural spheres were rapidly attached and cell bodies were spread out on the matrigel surface (Supplementary Fig. S4a) probably due to cell migration or active cell proliferation. Cells continually expanded and formed neural rosettes within a week (Fig. 1c (far left), Supplementary Fig. S4b). More than 95% of cells at this stage, including migrated-out neurons or neurons within aggregates, co-expressed PAX6 and Ceh-10 homeodomain containing homolog (CHX10) (Fig. 1c), suggesting they were retinal neural progenitor cells (RNPC). Recently published 3 dimensional (3D) retinal differentiation methods have demonstrated the formation of eyecup-like structures from human pluripotent stem cells. While this 3D method generates both retinal and forebrain neurons, the CHX10 positive RNPs are restricted to spheres with eyecup morphology. To check if our ESC-derived RNPs could form eyecup like structure in a suspension culture, we mechanically lifted RNPs from matrigel coated surface and plated them on an ultra-low attachment culture dish to form RNP spheres. Within 7 days, all spheres formed eyecup like structure (Figure S4c).

To validate the methodology, 6 hESC and 6 iPSC lines (Table S1), generated with different strategies, were subjected to retinal neuron differentiation and analyzed for the expression of PAX6 and RX1 by flow cytometry. Results demonstrated that all tested cell lines generated a near-homogenous population expressing these markers (Table S1), confirming the robustness of the retinal differentiation platform.

Generation of photoreceptor-like progenitors and photoreceptor-like cells

We next tested whether PSC-RNPCs were able to further differentiate into more mature retinal neurons such as photoreceptor progenitors (PhRP) and photoreceptors. After culturing H9-RNPCs in NDM without noggin supplementation, cells further differentiated into photoreceptor-like progenitors, as characterized by the expression of transcription factors involved in photoreceptor development. After four passage (approximately 3 months in NDM without noggin), dramatic morphological changes were observed (Supplementary Fig. S5a). Although neurons formed short neurites at this stage, neurite branching from the cell body was reduced or absent. Cells at this stage lost expression of CHX10, but were stained positive for Cone-Rod homeobox (CRX), neural retina leucine zipper (NRL), and nuclear receptor subfamily 2, group E, member 3 (NR2E3), key transcription factors that are essential for photoreceptor fate determination and development (Fig. 2). Less than 10% of them are positive for Ki67, a cellular marker that is strictly associated with cell proliferation (Supplementary Fig. S5b). Both neuN antibody and secondary antibody alone showed negative stains in these cells (Fig. 2a, bottom). These results indicate that cells were differentiating towards retinal photoreceptors, probably at the stage of PhRPs. Near-homogenous PhRPs were similarly generated from a human iPSC line, which expressed rod specific transcription factor, NRL (Supplementary Fig. S5c). Approximately 100 fold increase of cell number was observed from PSCs to PhRPs in three experiments using both ESC and iPSC lines.

Figure 2: In vitro Differentiation of Retinal Neural Progenitors towards Photoreceptor-like Progenitors. (a) Immunofluorescence staining shows the expression of transcription factors NRL, NR2E3 and CRX in PhRPs at 90 –100 days after in vitro differentiation. CHX10 and neuN genes are negative in PhRPs at this stage; the upper right corner of the CHX10 image shows positive expression of CHX10 in RNPCs at day 30 (positive control); the upper right corner of the neuN image shows positive expression of neuN in mouse central nerve cells (positive control). 2nd antibody only also shows negative staining. (b) Quatification of intracellular staining of NRL, NR2E3 and CRX as determined by Flow Cytometry analyses. Scale bar, 50 μm. Full size image

To generate photoreceptor-like cells, PhRPs were further cultured in medium containing retinoic acid, DAPT, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). After two weeks, most cells (≈95%) became rod-like photoreceptor cells expressing rhodopsin, recoverin and phosphodiesterase 6 alpha (PDE6α) (Fig. 3a). Congruently, cells expressing photoreceptor-specific markers were also generated from iPS-PhRPs (Fig. 3b).

Figure 3: In vitro Generation of Mature Photoreceptor-like Cells from Human ESC/iPS-Derived PhRPs. Expression of rod photoreceptor markers, rhodopsin, recoverin and PDE6α in hESC (a) and iPSC-derived (b) photoreceptor-like cells two week after in vitro maturation. Scale bar, 50 μm. Full size image

Human PSC-derived PhRPs survived in the subretinal space of wild type mice

H9-ESC and HA-iPSC derived PhRPs were selected for further in vivo investigation due to their relevance for clinical application; the H9 ESC line is registered with NIH for use in academic collaboration and the HA-iPS line was generated using mRNA reprogramming technology38 which eliminates the risk of genomic integration and harmful alteration of the host genome. In order to discern transplanted human cells following transplantation, PhRP spheres were transduced by a recombinant serotype2 capsid-mutant adeno-associated virus (rAAV2/2 Y444F), expressing GFP under the photoreceptor-specific human rhodopsin kinase promoter. GFP expression was achieved in vitro 7 days post transduction in 70–90% of cells (Supplementary Fig. S6). To reduce culture time prior to transplantation, and validate the contained transduction of donor cells by AAV, PhRP derived from H9-ESC (ESC-PhRP) were first subretinally transplanted to WT mice (n = 4) 48 hours post transfection (before peak GFP expression). Transplanted human cells, positive for both GFP and human nuclear antigen (HNA), were observed in the subretinal space of WT mice 7 days post transplantation, without transduction of the host retina by GFP (Supplementary Fig. S7), confirming the contained expression of GFP in donor cells. Hence for transplantation in the severely degenerate retina, dissociated single PhRPs were transplanted 48 hours following AAV transduction.

Human PSC-derived PhRPs survived in the severely degenerated subretinal space of rd1 mice

Rd1 mice were used as a host model of rapid and progressive RP with end-stage retinal degeneration. In these mice, retinal degeneration is caused by a null mutation in the rod photoreceptor cyclic GMP (cGMP) phosphodiesterase β subunit (Pde6b) gene39,40. Most rod photoreceptor cells are lost in the rd1 mouse by 3 weeks of age41, followed by progressive rod and cone photoreceptor degeneration over the first 8 weeks of life42 with absence of a functional ONL by 6–10 weeks of age4,43,44. At this late age, rd1 mice represent a relevant model for studying transplantation in patients with end stage RP, as in rd1 mice there is sever structural degeneration of the ONL, as appose to other retinal disease models such as the CRX knockout mouse, in which the retina is severely compromised functionally, but there is no structural degeneration.

Human PhRPs were transplanted into the subretinal space of rd1 mice aged 10–12 weeks, at the end-stage of ONL degeneration (supplementary Fig. S8 shows the vast loss of ONL in the adult rd1 by comparing WT and rd1 retinal structures at 10 weeks). To prevent immune-rejection of human cells, immunosuppression with cyclosporine was administered to host mice45, starting 2 days prior to transplantation and continuing throughout the experiment.

A first group of rd1 mice received transplantation of ESC-PhRPs, a second group received transplantation of iPSC-PhRPs (n = 8 per group, unilateral injections). A third group received transplantation of H9-ESC derived retinal neural progenitor cells (RNPCs)(n = 5) to control for PhRP cell survival. And a final group received a sham transplantation of PBS to control for the surgical manipulation (n = 8). All cells were transduced by rAAV2 Y444F. GFP prior to transplantation and animals in the RNPC transplantation group were investigated alongside PhRP groups to control for the undesired transfection of the host ONL by free AAV particles that could potentially be delivered with cells.

Three weeks post transplantation a distinct layer of GFP+ cells was observed in vivo in animals injected with ESC-PhRPs (Fig. 4a) and iPSC-PhRPs (Fig. 4b). In the same mice, histology revealed GFP+ human cells residing in the subretinal space of animals in both ESC-PhRP (Fig. 4c,c’) and iPSC-PhRP (Fig. 4d,d’) treated groups. On average 6.45 ± 1.1% (mean ± 1 S.E.M) of transplanted ESC-PhRPs and 5.7 ± 1% of iPSC-PhRPs survived in the subretinal space of rd1 mice 3 weeks post transplantation, (n = 8 specimens each). Less than 0.001% of 2 × 105 GFP-RNPCs, which were transplanted as a control for cell survival, were observed in the subretinal space of rd1 mice, and cells were only evident in eyes of 2 of 5 transplanted animals (see Supplementary Fig. S9). This validates the significance of appropriate developmental stage of PSC-derived retinal cells for survival following transplantation. The absence of GFP+ cells in the control group also further confirms that GFP+ cells observed in the PhRP-transplanted treatment groups were indeed donor-derived cells and not a result of host retina transduction.

Figure 4: Transplanted Human ESC-PhRPs and iPSC-PhRPs Survive in the Subretinal Space of rd1 Mice. Scanning laser ophthalmoscopy (SLO) was performed in vivo three weeks post transplantation to assess the extent of surviving donor cells; GFP positive cells are observed in autofluorescence (AF) mode as white dots or clusters (black areas represent areas of retina which were not seeded with transplanted cells, due to incomplete detachment of the retina around the optic nerve head). Representative near-infrared (NIR) and AF fundus images of rd1 mice show a homogeneous presence of GFP+ cells in the two treatment groups: ESC-PhRPs (a) and iPSC-PhRPs (b). Histological assessment 3 weeks post transplantation revealed ESC-PhRP (c-c’) and iPSC-PhRP (d-d’) derived cell layers (green) between the retinal pigment epithelium (RPE) and inner nuclear layer (INL) of the rd1 retina, replacing the absent outer nuclear layer (ONL) in the adult rd1 mice; (e-e’) GFP+ cells were stained with human nuclear antigen (HNA) which co-localized with GFP; indicating that the GFP signal observed in vivo in treated animals was indeed an indicator of transplanted human PhRPs. Scale bar, 25 μm. Full size image

Grafted GFP-positive cells expressed human nuclear antigen, and developed processes to interact with host circuitry. However, similar to a previous a report of cell transplantation in the severely degenerate adult rd1 mouse4, transplanted PSC-PhRPs in this study did not adopt normal photoreceptor morphology and orientation (Fig. 4e,e’) as described when retinae with a residual ONL were treated with mouse rod progenitors5,46,47,48 or human PhRPs6.

Transplanted human PSC-derived PhRPs mature in vivo in the severely degenerated retina

Three weeks post transplantation in the adult rd1 eye, PSC-PhRP located in the subretinal niche, examined by immunohistochemistry, expressed mature photoreceptor proteins: The pan-photoreceptor marker recoverin was expressed by both ESC-PhRPs and iPSC-PhRPs (Fig. 5a–c, respectively). The rod-specific cGMP phosphodiesterase β6 (PDE6β) was observed in the outer processes of transplanted cells (Fig. 5d-d’); a deficit in this phototransduction-enzyme is the underlying cause of retinal degeneration in the rd1 mouse, certifying its expression by donor rods. The rod specific protein rhodopsin was also expressed in transplanted cells of both treatment groups (Fig. 5f,g). Localization of rhodopsin and PDE6β proteins was correctly confined to the developing outer processes of cells, demonstrating maturation of human rod cells in the degenerate rd1 subretinal niche. Cone arrestin protein was observed in GFP+ cells (Fig. 5h,i), indicating maturation of human cone photoreceptors within the transplanted photoreceptor cohort. Normal photoreceptors signaling can be identified by the synaptic vesicle glycoprotein, synaptophysin which is normally expressed in photoreceptor synaptic terminals in contact with bipolar cells. Three weeks post transplantation synaptophysin protein was present between the human graft and the host retina, indicated an interaction between donor cells and host circuitry (Fig. 6a,a”’). Gliosis is a limiting factor in CNS regeneration and has been proposed to represent a cellular attempt to protect remaining tissue from further damage49. Gliosis in the retina predominantly involves Müller glia cells, which upregulate the glial fibrillary acidic protein (GFAP) of the Müller cell processes at the edge of the neural retina50. The presence of a glial scar in the retina may stand as a physical barrier to cell integration51. Host Müller glia (GFAP positive) were found to form a glial barrier at some areas of the retina, however glial processes were also found to extend into the engrafted human PhRPs (Fig. 6b,b”’), and grafted cell extended processes into the host INL (see Supplementary Fig. S10), supporting a degree of integration between the host and graft layers without a significant glial barrier forming between them. Confocal stacks from a whole-retina flatmount demonstrated the morphology and formation of cell processes in transplanted PhRPs 3 weeks after transplantation into the rd1 subretinal space (see Supplementary Fig. S10).

Figure 5: Transplanted Human ESC-PhRPs and iPSC-PhRPs Express Mature Photoreceptor Markers in vivo. Immunofluorescence staining 3 weeks post transplantation shows expression of mature photoreceptor markers in transplanted human PhRPs (green). In all images cells are located in the subretinal space and oriented so that the host INL is located at the top of the image and the RPE at the bottom. The pan-photoreceptor marker recoverin was observed within the reconstructed layer of cells in animals treated with both ESC-PhRPs (a) and iPSC-PhRPs (b,c). The rod specific enzyme phosphodiesterase β6 (PDE6b), which is necessary in phototransduction and is absent in rd1 mice due to mutation was reinstated in the retina and located in the outer processes of transplanted ESC-PhRPs (d-d’) and iPSC-PhRPs (e-e’). The rod specific protein rhodopsin, which is normally located in outer segment membrane disk was also observed in outer segments of ESC-PhRPs (f) and iPSC-PhRPs (g). Cone arrestin was observed in GFP+ cells, indicating that a subset of human cells matured to produce cone photoreceptors. Scale bar, 20 μm. Full size image

Figure 6: Graft-Host Connectivity. (a-a”’) Expression of synaptophysin, a synaptic marker, was present between the host INL and the GFP-positive graft. Synaptophysin is localized between the host and graft and is expressed in transplanted cells (white arrows) indicating synaptic transmission between human iPSC-derived grafted cells and the host rd1 retina; (a) GFP (human cells); (a’) synaptophysin; (a”) merged image of GFP and synaptophysin; and (a”’) merged image of GFP, synaptophysin and DAPI. The dashed line delineates the boundary between the host INL and the graft. (b-b”’) Glial fibrillary acidic protein (GFAP), a protein expressed by inner retinal astrocytes and activated Müller glia, is expressed by the glial cells of the host retina (red). Gliosis in the degenerate retina may occur to protect the retina from further damage, and a horizontal glial scar at the edge of the host ONL was observed in some areas of the retina (black arrow). However, glial processes were also observed to extend into the graft (green), without formation of a complete glial barrier between host and graft (white arrows). (b) GFP (human cells); (b’) GFAP+ host glial cells; (b”) merged image of ((b) GFP) and ((b’) GFAP); (b”’) merged image of GFP, GFAP and DAPI. Scale bar, 20 μm. Full size image

Recovery of basic visual function in animals with end-stage retinal degeneration

In order to further assess transplanted cell maturation and integration into host circuitry, we tested for changes in basic behavioral function in treated rd1 mice. Behavioral testing was conducted after dark adaptation and using dim (~10 lux) 510 nm illumination which is close to the wavelength of light preferentially detected by rod cells. This light stimulus was selected in order to target transplanted rod cells while eliminating the contribution of potentially functional host cone cells or intrinsically-photosensitive retinal ganglion cells (ipRGCs).

We assessed the presence of an optomotor response (OMR) to a rotating grating, by adapting a previous protocol52. The test animal is placed in the center of a rotating striped drum. An OMR is recorded when the animal turns its head to track the rotating grating. Tracking in each direction is independently driven by one eye53: an OMR elicited by rotation in a clockwise direction is driven by the left eye, and a response to anti-clockwise rotation is driven by the right eye (the treated eye in this study) (Fig. 7a). A significant increase in the number of direction-dependent head tracks was found when comparing head tracks driven by treated eyes compared to untreated eyes of animals transplanted with ESC-PhRPs (Paired t test, t = 2.86, p < 0.05) and iPSC-PhRPs (Paired t test, t = 5.02, p < 0.01), but not in the shame treatment group (paired t test, t = 0.31, ns) (Fig. 7b). A difference was also found between the three treatment groups in the number of treated eye-derived head tracks (ANOVA, F = 7.8, p < 0.01), with post hoc analysis revealing improvements in ESC-PhRP (p < 0.05) and iPSC-PhRP (p < 0.01) groups compared to sham treatment, but no difference between the two PhRP treatments (ns) (Fig. 7b).

Figure 7: Recovery of Basic Visual Responses in rd1 Mice Following Transplantation of Human PhRPs Correlates to Number of Engrafted Cells. (a) Schematic of the optomotor response (OMR) test arena and expected response to the direction of drum rotation. (b) Mean OMR 3 weeks post-transplantation indicating an improvement in OMR driven by treated eyes (dark grey) compared to paired untreated eyes (light grey) after transplantation of ESC-PhRPs (paired sample t-test, t = 2.86, p = 0.024) and iPSC-PhRPs (paired sample t-test, t = 5.02, p = 0.002); In the sham treated group there were no differences in OMR driven by treated and untreated eyes (paired sample t test, t = 0.31, ns). Furthermore, OMR was improved in PhRP treatment groups compared to sham treatment (one way-ANOVA, F = 7.8, p = 0.003), with an increase in the response in both ESC-PhRP (p < 0.05) and iPSC-PhRP (p < 0.005) treated animals (Bonferroni test for multiple comparisons). (c) A positive correlation was observed between number of head tracks and number of GFP+ cells in animals treated with ESC-PhRPs (n = 8, R2 = 0.729, F = 16.13, p < 0.01) and (d) iPSC-PhRPs (n = 8, R2 = 0.612, F = 9.46, p < 0.05). (*p < 0.5, **p < 0.01). (e) Schematic of the light avoidance apparatus. (f) There were no differences between the three groups in mean light avoidance responses (F = 1.43, p = 0.261 [ns]). However, a positive correlation was observed between number of GFP+ cells and light avoidance behavior in individual animals of (g) ESC-PhRP (R2 = 0.729, F = 16.13, p < 0.01) and (h) iPSC-PhRP treated group (R2 = 0.612, F = 9.46, p < 0.05). (i) Comparing only animals with above-median numbers of GFP+ cells (encircled in (g,h)) a significant difference was observed between the three groups (X2 = 6 (df2), p < 0.05) showing improvement in ESC-PhRP treated (n = 4, p < 0.05) and iPSC-PhRP treated (n = 4, p < 0.05) subgroups. The dashed line in (b,f) represents the mean response of age-matched wild-type mice. Error bars represent ± S.E.M. Full size image

We correlated behavioral performance to the number of positively identified human cells in each animal (Table S2), and found a positive correlation between the number of GFP+ human cells and performance in OMR for both ESC-PhRP (R2 = 0.626, F = 10.0, p < 0.05; Fig. 7c) and iPSC-PhRP (R2 = 0.518, F = 6.45, p < 0.05; Fig. 7d) treated animals.

To qualify further the observed behavior, we conducted a light avoidance assay, as previously described4. Mice were free to transition between light and dark compartments of a test arena, and the percent of time spent avoiding light was quantified (for schematic representation see Fig. 7e); As mice are nocturnal animals, they tend to avoid bright environments54, however, this is dependent on the perception of light, thus the tendency to avoid light is measured to infer visual function. Here, rd1 mice in the three treatment groups did not differ in their tendency to avoid light (ANOVA, F = 1.4, p = 0.261 (ns); Fig. 7f). Measures of anxiety-related behavior were examined in order to assess whether a difference in anxiety between the groups may contribute to the result, but no differences in anxiety-related behavior was observed (see Supplementary Fig. S11). Nevertheless, a trend was observed, in which some mice in the PSC-PhRP treated groups responded more than sham transplanted mice, hence in order to assess the reason for variability in the results, light avoidance behavior was qualified to the number of engrafted cells in each animal. As with the OMR, we found that number of engrafted cells in individual animals strongly correlated with light avoidance behavior in both ESC-PhRP (R2 = 0.729, F = 16.1, p < 0.01; Fig. 7g) and iPSC-PhRP (R2 = 0.612, F = 9.46, p < 0.05; Fig. 7h) treated animals (n = 8 per group) (individual results are displayed in Table S2). Since light avoidance behavior is driven by light-intensity, which might have a threshold effect to the number of light-sensitive cells, we performed a sub-analysis of the three groups, including only animals with above-median number of surviving cells (top 50%) in the treated groups. In this case, though it should be qualified that numbers were small in this sub-analysis, a difference was observed between treatment groups (χ2 = 6.0, p = 0.041) with an increase in light avoidance in both ESC-PhRP (n = 4, p < 0.05) and iPSC-PhRP (n = 4, p < 0.05) treated animals compared to sham treatment (Fig. 7i).

In order to exclude the possibility that the functional improvements presented here were due to neuroprotection of residual host cone-photoreceptors, remaining cones in treated rd1 mice were examined histologically using staining against cone-arrestin. As expected, some residual host cones were present in PSC-treated and sham rd1 mice55; however remaining cones were morphologically abnormal, with absence of inner and outer segments (see Supplementary Fig. S12). Furthermore, no difference was observed in the number of residual host-cones between ESC-PhRP, iPSC-PhRP or sham treatment groups (One way ANOVA, n = 5 per group, F = 0.56, p = 0.58, ns) (Supplementary Fig. S12).