Differentiation of Human Embryonic Stem Cell–Derived Retinal Progenitors into Retinal Cells by Sonic Hedgehog and/or Retinal Pigmented Epithelium and Transplantation into the Subretinal Space of Sodium Iodate

Abstract

Transplantation of retinal cells has recently provided a promising therapeutic approach for retinal degeneration. Here, we differentiated initially retinal progenitors (RPs) from adherent feeder-free human embryonic stem cells (hESCs) with the use of defined media supplemented with a specific combination of growth factors. The differentiated RPs highly (>80%) expressed related molecular features that included Six3 at an early stage in addition to Crx, Rx, Pax6, Otx2, and Chx10 at later stage. Next, we examined the induction of photoreceptors by Shh and/or the coculture of rabbit retinal pigmented epithelium with hESCs-derived RPs. The differentiation of retinal cells was demonstrated by protein and gene expression in all groups. However, S-Opsin, a cone photoreceptor marker, had higher expression in the presence of Shh, whereas expressions of Gli and Hes1 decreased in the same group. Finally, hESC-derived RPs were treated with Shh transplanted into the subretinal space of sodium iodate–injected albino-type adult rabbits and analyzed 4 weeks later. Transplanted retinal cells survived, migrated into retinal layers, and restored a small but significant B-wave. The grafted cells expressed photoreceptor markers, S-Opsin and Rhodopsin. Our results indicate that putative hESC-derived retinal cells express related genes and proteins. Further, our results show that retinal-like cells can be useful replacements for photoreceptors in retinal diseases.

Introduction

R etinal degeneration diseases that result from photoreceptor degeneration or genetic defects account for blindness and visual disorders in millions of people worldwide [1]. As with the central nervous system (CNS), there is no effective self-repair in the human retina. Therefore, cell therapy has been considered to be a therapeutic approach for retinal disease [2,3]. Several cell sources have been investigated as potential replacements for photoreceptors, including fetal retinal progenitors (RPs), which have the capacity to generate photoreceptor cells [4]. Additionally, it has been shown that postmitotic rod photoreceptors can integrate into the adult or degenerating retina [5]. These transplanted cells form synaptic connections and improve visual function [5]. However, obtaining a sufficient number of suitable photoreceptors remains a major obstacle. The discovery of human embryonic stem cells (hESCs) [6] and, recently, induced pluripotent stem cells (hiPSCs) [7 –9] have raised the hope for curing diseases with poor prognoses. Several reports have documented the generation of retinal cells, including retinal pigmented epithelium (RPE) and photoreceptors, from hESCs and hiPSCs [10 –20].

RPE is a densely pigmented epithelial monolayer present between the choroid and neural retina. This layer is critical for photoreceptor survival, phagocytosis of shed rods and cones in the outer segments, and secretion of several neurotrophic growth factors, which have important roles in the differentiation of photoreceptors and proper ocular development [21 –23]. On the other hand, during eye development, sonic hedgehog (Shh) is a regulatory factor involved in differentiation of RPs into photoreceptors [24 –29].

Therefore, in the present study, we initially differentiated RPs from adherent feeder-free hESCs. Next, Shh and/or a monolayer of in vivo derived RPE, prepared as an inducing cell layer that replicated the retinal microenvironment, were used to induce photoreceptor differentiation. Finally, the differentiated retinal cells from the Shh-treated group were transplanted into the subretinal space of sodium iodate–injected rabbits to evaluate both their ability to incorporate into retinas and improve functionality.

Materials and Methods

Culture of hESCs

The hESC line, Royan H5 [30], was used in these experiments. The cells were expanded and passaged on matrigel under feeder-free culture conditions in hESC medium that contained 100 ng/mL basic fibroblast growth factor (bFGF) as previously described [31]. The medium was changed every other day, until day 7.

Differentiation to RPs

The differentiation procedure is outlined in Fig. 1A. For neural differentiation induction, feeder-free hESCs were allowed to proliferate in hESC medium for 7 days. hESCs were subsequently induced to neural ectoderm in NE medium that contained noggin (1 ng/mL; R&D, 1976-NG), human recombinant Dkk-1 (1 ng/mL; R&D, 5439-DK/CF), and human recombinant insulin-like growth factor-1 (IGF-1, 5 ng/mL; R&D, 291-GI) in DMEM-F12 medium supplemented with 10% knockout serum replacement, 0.1 mM nonessential amino acids, 2 mM l-glutamine, and 1% B27 (Gibco, 17504-044), for 2 days. Subsequently, the medium was changed to retinal determination (RD) medium that consisted of DMEM:F12 supplemented with 1% B27, 2% N2 (Gibco, 17502-048), 10 ng/mL noggin, 10 ng/mL Dkk-1, 10 ng/mL IGF-1, and 5 ng/mL bFGF as previously described [32]. The medium was renewed every other day up to 2 weeks to form neural tube (NT)-like structures. NTs were manually separated from the surrounding flat cells with a sterile pulled-glass pipette while viewing under a phase-contrast microscope (10×, CKX41; Olympus) at day 16. Mechanical selection reduces contamination with nonneural cells. The NTs were dissociated and replated on 1 mg/mL laminin and 15 μg/mL poly-l-ornithine (both from Sigma-Aldrich)–coated 6-well tissue culture plates (TPP, 92406) in the same medium (20–30 NTs per well).

FIG. 1.

Generation of retinal progenitors (RPs) from human embryonic stem cells (hESCs). (A) A schematic illustration outlining the differentiation protocol to generate RPs and maturating retinal cells and their transplantation into an animal model. (B) Phase-contrast photomicrographs of a colony of hESCs grown under feeder-free conditions on matrigel. Cells demonstrate a typical undifferentiated morphology with a clear border. hESCs were subsequently induced to neural ectoderm in NE medium supplemented with noggin, DKK, insulin-like growth factor, and basic fibroblast growth factor. The periphery of the colony was thickened and NTs appeared at day 16. NTs (arrowheads) were isolated mechanically and replated on laminin/poly-l-ornithine–coated plates. (C) Reverse transcription (RT)–polymerase chain reaction (PCR) analysis showing the loss of pluripotency markers and expression of RP genes during retinal induction. HE, human embryo eye. (D) A schematic illustration for temporal gene expression during retinal cell differentiation from hESCs. (E) Immunocytofluorescence staining of RPs demonstrates the expression of RP markers. (F) Quantitative immunofluorescence staining showed high expression of RP markers at day 9 for Six3 and day 17 for others. More than 500 cells were counted in each group. Scale bars: 200 μm. Color images available online at www.liebertonline.com/scd

Animals

Subjects of this study were albino and pigmented rabbits (Department of Physiology, Isfahan University, Iran) that weighed between 1.5 and 2.0 kg. All animal care, surgical processes, and postoperative euthanasia were undertaken in strict accordance with the approval of the Ethical Committee of Royan Institute. All surgical procedures were performed under anesthesia produced by a combination of ketamine (35 mg/kg) and xylazine (5 mg/kg).

Isolation and culturing of rabbit RPE

Pigmented rabbits were sacrificed by an overdose of ketamine and xylazine. Then, RPE was isolated according to established protocols [33]. In brief, the globes were enucleated and washed in Ca²⁺- and Mg²⁺-free phosphate-buffered saline supplemented with penicillin/streptomycin. The intact globe was incubated in 2% dispase (Gibco, 17105-041) for 20 min at 37°C. The incision of the globe was made 3 mm posterior of the limbus circumferentially. The anterior segment was discarded, and the posterior segment was extended by 4 incisions radially and incubated in DMEM/F12 supplemented with 10% FBS for 2 h at 37°C. The majority of the RPE sheet was lifted carefully from the choroid and neural retina as visualized under a stereo microscope (Olympus, SZX16). The isolated sheet was cultured on filter insert (0.4 mm; Millicell-CM-PICMO3050) in the presence of RD medium. Filter inserts were maintained in 6-well plates. The medium was changed every other day for 14 days.

MTS assay

To investigate whether RD medium affected survival of RPE cells, a cell proliferation assay was carried out by MTS assay. Briefly, 2×10⁵ RPE cells were cultured in 96-well tissue culture plates under induction medium. Two weeks after plating, cell proliferation assay was carried out by MTS/PMS (Promega, G5421) according to the manufacturer's protocol. Absorbance was measured at 492 nm by an ELISA reader (Awareness).

Experimental design

One day after replating of dissociated NTs on laminin/poly-l-ornithine at day 17, the cells were cultured in the presence or absence of Shh (6 nM; Sigma, S0191) and/or cocultured by RPE in RD medium. To coculture, the filter inserts consisting of cultured RPE cells were transferred into 6-well plates that include dissociated NTs. The medium was changed every other day for 14 days. On day 31, the cells from all groups were cultured for an additional 10 days in neurobasal medium (NBM; Invitrogen, 21-103) supplemented with 2% N2 in the absence of RPE and/or Shh. Half of medium was renewed every other day.

Preparation of cells for transplantation

Immediately before transplantation, the hESC-derived retinal cells were harvested by trypsinization, washed 3 times in PBS, and resuspended the cells to the desired concentration. For identification after transplantation, we utilized human nuclear antigen (HNu), a selective nuclear marker of cells that originate from humans or PKH-26 (PKH26-GL; Sigma-Aldrich), according to manufacturer's protocol. PKH-26 staining provides the identification of grafted cells in vivo. To ensure labeling procedure, we examined the cells using fluorescence microscopy. A sample of the resulting single-cell suspensions was stained with trypan blue dye exclusion (Sigma) and counted with a Neubauer hemocytometer.

Surgical procedure, experimental grouping, and transplantation

Sodium iodate–induced model of RPE degeneration

A single injection of sterile 50 mg/kg solution of sodium iodate (NaIO₃; Sigma) in saline was administered into the ear marginal vein of each rabbit [34,35]. After 7 days, cell transplantation was performed.

Experimental grouping and transplantation

Eighteen adult albino rabbits with normal eye function were selected and randomly assigned into normal and NaIO₃-injured groups. The normal group (n=6) had normal, intact eye function and received no treatment. Seven days after NaIO₃ injury, the animals were assigned into 3 groups. The sham group (n=6) received DMEM/F12 in the subretinal space. hESC-derived retinal cells were transplanted (transplanted group) into the left eyes of 6 other NaIO₃-injured animals and the right eyes of the same animals were used as controls that underwent no additional manipulations.

For sham or cell transplantation, NaIO₃-treated albino rabbits were anesthetized with ketamine and xylazine. To access the subretinal space, small supra nasal conjuctival peritomy was performed; the conjuctiva and tenon were separated from beneath the sclera. A 90% partial thickness sclera incision was made at a distance of 7–8 mm from the limbus. Before transplantation, confirmation of cellular viability was performed with trypan blue exclusion dye. A total of 50,000–80,000 cells in 1.5 μL of DMEM/F12 were injected into the subretinal space using a pulled fine glass micropipette. The fundus and elevated retina were visualized with a 35° lens. The conjunctiva was sutured with Vicryl 7.0. Starting 2 days before transplantation, all groups of rabbits in transplanted/control and sham groups received daily injections of cyclosporine-A (20 mg/kg, s.c., Sandimmune; Novartis Pharmaceuticals) [36] for a total of 4 weeks to prevent rejection.

Electroretinography

Rabbits were dark-adapted for at least 4–5 h, anesthetized with ketamine and xylazine, and prepared for recording electroretinography (ERG). Pupils were dilated with Midrax (Tropicamid) 5 min before ERG recording. A gold ring electrode embedded in a contact lens was placed on a drop of 2% methyl cellulose on the cornea. Ground and reference copper electrodes were placed in the forehead and lateral conthus, respectively. Three different test flash intensities were sent and responses collected from each rabbit. ERGs were carried out under scotopic, phototopic, and Max conditions. ERG signals were amplified 5,000, filtered between low-pass filter 30 Hz–10 kHz and high-pass filter 0.02–10 kHz.

Sacrifice and cryosection

After ERG analysis, all rabbits of the sham and control/transplanted groups were sacrificed. Rabbit eye globes were dissected and postfixed at 4°C and then cryoprotected with 30% phosphate-buffered sucrose at 4°C for 96 h. Samples were longitudinally cryosectioned at 10 μm and stored at −20°C until staining.

Immunocytofluorescence and immunohistofluorescence staining

Immunocytofluorescence staining was performed as a standard protocol using the antibodies listed in Table 1. Labeled cells were visualized with either FITC- or TRITC-conjugated secondary antibodies, and nuclei were stained with DAPI (Sigma, D8417). A fluorescence microscope (Olympus BX51, Japan) equipped with an Olympus DP70 camera was used to evaluate the number of positive cells labeled with retinal cell markers out of approximately 500 DAPI nuclei-labeled cells.

Table 1.

Antibodies Used in This Study

	Species	Clonality	Source
Primary Ab
Chx10	Goat	Polyclonal	Santa Cruz, sc-21690
Gfap	Mouse	Monoclonal	Chemicon, Mab3402
Map2	Mouse	Monoclonal	Life span, LS-C8665
Otx2	Rabbit	Polyclonal	Sigma-Aldrich, HPA000633
Pax6	Mouse	Monoclonal	Chemicon, MAB5554
Six3	Mouse	Monoclonal	Santa Cruz, sc-49114
Zo-1	Mouse	Monoclonal	Invitrogen,330100
Rx	Rabbit	Polyclonal	LifeSpan LS-C53650-50
Cytokeratin18	Mouse	Monoclonal	Chemicon, MAB3234
Lim-1	Rabbit	Polyclonal	Abcam, ab-14554
HNu	Rabbit	Polyclonal	Abcam, ab-66601
S-Opsin	Rabbit	Polyclonal	Abcam, ab-65681
Rhodopsin	Mouse	Monoclonal	Santacruse, sc-56472
Secondary Ab
FITC anti-rabbit IgG	Goat	Polyclonal	Sigma-Aldrich, F1262
FITC anti-mouse IgG	Goat	Polyclonal	Chemicon, AP124F
FITC anti-goat IgG	Rabbit	Polyclonal	Chemicon, AP106F
TRITC anti-mouse	Goat	Polyclonal	Sigma-Aldrich, T7782

For immunohistofluorescence staining, sections were rehydrated in 0.1 M PBS and permeabilized with 0.4% Triton X-100 in PBS. After blocking nonspecific sites with 10 mg/mL bovine serum albumin, sections were incubated overnight with primary antibodies. Antibodies implemented for immunohistofluorescence are presented in Table 1.

For survival studies, we utilized anti-HNu and PHK-26. Normal IgG from the species of origin of the primary antibodies served as negative controls. After washing with PBS, the cells were incubated with the appropriate FITC- and TRITC-conjugated secondary antibodies for 1 h at 37°C. Sections were washed 3 times with PBS and counterstained with DAPI. The cover slips were mounted onto glass slides with Entellan. The same fluorescence microscope and photography was used to photograph positive cells that were labeled with different cell markers.

Reverse transcription–polymerase chain reaction and real-time reverse transcription–polymerase chain reaction for gene expression analysis

Total RNA was extracted from cultured cells using the QIAprep Spin Miniprep kit (QIAGEN, 27106) according to the manufacturer's protocol. Prior to reverse transcription (RT), RNA samples were digested with DNase I (Fermentas, EN0521) to remove contaminating genomic DNA. Standard RT was performed using 2 μg total RNA, oligo (dT) 18, and the Revert AidTM H Minus First Strand cDNA Synthesis kit (Fermentas, K1622) according to the manufacturer's instructions. Primer sequences, annealing temperature(s), cycles, and the lengths of amplified products are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertonline.com/scd). Amplification conditions were as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing for 45 sec, extension for 45 sec at 72°C, and a final polymerization at 72°C for 10 min. Polymerase chain reactions (PCRs) were performed in triplicate. PCR products were analyzed by gel electrophoresis on 1.7% or 2% agarose, stained with ethidium bromide (10 μg/mL), visualized, and photographed on a UV transilluminator (Uvidoc). CNS RNA of a 20-week-old human embryo was used as a positive control for RT-PCR analysis.

Real-time RT-PCR was performed using a Thermal Cycler Rotor gene 6000 (Corbett) with 10 μl SYBR Green PCR Master Mix (Takara), 5 pM of each primer, and 50 ng cDNA for each reaction in a final volume of 20 μl. Relative gene expression was analyzed using the comparative Ct method (2^−ΔΔCt) [37]. All samples were normalized to levels of GAPDH, which was used as the loading control. All measurements were performed in triplicate. Real-time specific primer pairs (Supplementary Table S2) were designed by Beacon software (Version 7.2).

Western blot analysis

TRI reagent (Sigma-Aldrich) was used to extract protein from induced cells. Protein concentration was determined with a Bradford assay (Bio-Rad) kit. The prepared samples were electrophoresed on 12% SDS-PAGE gels (Bio-Rad, Munich, Germany) and then transferred to PVDF membranes (Bio-Rad, Munich, Germany). Membranes were blocked overnight in 10% skim milk (Merck) and incubated with primary different antibodies including S-Opsin (1:2000) and β-Tubulin (1:2500; Sigma-Aldrich, T5293; housekeeping gene or internal control) for 90 min. The blots were washed and then incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:8000) for 1 h. Bands were visualized after further washes using an ECL advanced western blot detection kit (GE Healthcare).

Statistical analysis

All values are stated as means±standard error of mean (SEM). Data were analyzed by 1-way ANOVA followed by LSD post hoc, with a significance threshold of P<0.05.

Results

Generation of RP cells from hESCs

The undifferentiated feeder-free hESCs were maintained in the presence of a high concentration of bFGF (100 ng/mL) on Matrigel (Fig. 1B). Then, the cells were differentiated into RPs, using a published protocol [32], without embryoid body (EB) formation in adherent conditions. The hESCs were treated with noggin, Dkk-1, and IGF-1 for 2 days. Next, the cells were treated in RD medium for an additional 2 weeks. Consequently, the neuroepithelium and NTs emerged from hESCs (Fig. 1B). At day 16, NTs were mechanically isolated under a phase-contrast microscope, replated on laminin/ornithine-coated plates, and cultured in RD medium for 1 day. RT-PCR analysis of differentiated cells at day 17 (= 2+14+1 days) showed that the cells lost expression of pluripotency genes, Oct4 and Nanog (Fig. 1C), and acquired expression of eye field genes (Fig. 1C, D). The efficiency of retinal determination was tested by analyzing the expression of several key eye-field transcription factors at the protein level (Six3 at day 9 and Rx, Pax6, Otx2, and Chx10 at day 17).

During early eye development, Six3 is first expressed in the undifferentiated neuroepithelium and then its expression is downregulated in the differentiating neural retina and postmitotic photoreceptors. We first examined the most rostral marker, Six3 (Fig. 1E, F). Quantification of immunostaining revealed that 83.8%±1.7% of differentiated cells were positive for Six3 (Fig. 1F). The immunostaining for Six3 was negative at day 17 (data not shown). For specification of Six3 rostral CNS, the transcription factor Rx (an early bona fide marker for the eye field) is needed and immunostaining results revealed that around 79%±1.6% of cells express this marker (Fig. 1E, F). Additionally, coexpression of Rx with Pax6 characterized the neural retinal lineage and 78.3%±1.8% of the cells expressed Pax6 (Fig. 1E, F). In the neural retina, Otx2 is expressed in postmitotic anterior neuroblast and acquires the ability to differentiate to various cell types. A total of 86.2%±1.6% of the differentiated cells expressed Otx2 (Fig. 1E, F). In early eye development, during optic vesicle and cup formation, the expression of Chx10 in the inner layer of the optic cup becomes gradually apparent. Thus, Chx10, considered as the earliest specific marker for RPs, was observed following the expression of Otx2 (Fig. 1E). Quantitative analysis of the cultures showed that 90.53%±1.3% of the cells were positive for Chx10 using immunofluorescence staining (Fig. 1F). The expression of Crx, a photoreceptor marker, was confirmed by RT-PCR (Fig. 1C).

Therefore, a large fraction of the hESCs in cultures differentiated to RPs when maintained under RD conditions.

Coculture of RPs with RPE and/or Shh factor

To improve the generation of neural photoreceptors from hESC-derived RPs, the RPs were cultured in the presence and absence of Shh and/or RPE in RD medium (see later).

Before the coculture of RPs with rabbit RPE, we evaluated the preserved identity and viability of cultured RPE after 14 days. The RPE cells maintained pigmented hexagonal morphology and expressed cytokeratin 18, an intermediate protein, ZO-1, the marker of tight junction (Fig. 2A), and natriuretic peptid receptor (NPR-A), a rabbit RPE marker [38] (Fig. 2B). In addition, MTS assessment showed the maintenance of RPE viability in the in vitro condition (Fig. 2C). Therefore, RPE maintained their viability and identity during culture.

FIG. 2.

Isolated retinal pigmented epithelium (RPE) in vitro and experimental design. (A) Isolated RPE after 14 days of in vitro culture in RD medium showed hexagonal shape with expression of tight junction protein ZO-1 and intermediate cytokeratin-18 protein. (B) The expression of specific protein NRP-A. (C) MTS assay demonstrated survival of RPE. (D) Experimental design. At day 17, hESC-derived RPs were cocultured for 14 days in 4 groups (C: without treatment/control, R: RPE only, S: Shh only, and R+S: RPE+Shh). Scale bars: bright field and CK-18, 50 μm and ZO-1, 100 μm. Color images available online at www.liebertonline.com/scd

At day 17, RPs were cocultured in 4 groups (C: control/without treatment; R: RPE only; S: Shh only; R+S: RPE+Shh; Fig. 2D). After 14 days (day 31), some of the differentiated cells showed typical neuronal morphology with extended neurites (Fig. 3A) and further differentiation was achieved by an additional 10 days of culturing in NBM in all groups (day 41; Fig. 3B). RT-PCR analysis of the differentiated cells at day 41 revealed expression of the following photoreceptor markers in all groups: Recoverin, a later photoreceptor marker, Nrl and Nr2e3, rod photoreceptor transcription factors, Rhodopsin, a rod photosensitive pigment, and S-Opsin, a cone photoreceptor marker (Fig. 3). The differentiated cells also expressed Pax6, which is characteristic of ganglion and amacrine cells, and PKCα and Chx10, bipolar cell markers (Fig. 3C). Quantitative RT-PCR and western blot demonstrated that S-Opsin significantly increased (P<0.05) in the S group (Fig. 3D, E). At day 41, we further quantitatively analyzed expressions of Gli and Hes1, 2 mediators downregulated by Shh in the differentiation of RPs to both rod and cone cells. The results revealed significant reduction in expression of both factors upon removal of Shh and further culture in NBM (Fig. 3F). No significant difference was seen in expressions of Nr2e3 and Rhodopsin in all groups as indicated by qRT-PCR (Fig. 3H, I). However, Nrl was expressed more in the RPE coculture (Fig. 3G). Assessment of differentiated cells by immunofluorescence staining revealed that they were positive for Rhodopsin and S-Opsin (Fig. 4 and Supplementary Fig. S1) in addition to Lim1/MAP2, GFAP, and Chx10/MAP2 for horizontal, muller, and bipolar cell markers, respectively (Fig. 4). These markers have been previously described in retinal neurons of various species, including the human retina.

FIG. 3.

Transcript analyses of RP-derived retinal cells under different treatments at day 41. Representative phase-contrast micrographs of differentiating retinal cells at day 31 (A) and 41 (B) in Shh group. Scale bar: 100 μm. (C) RT-PCR analysis demonstrated stepwise acquisition of rod and cone markers of photoreceptors, Pkc-α and Chx10 (bipolar markers), and Pax6 (ganglionic marker). (D) Quantitative RT-PCR and western blot (E) demonstrate that S-Opsin significantly increased in the S group. (F) Quantitative RT-PCR shows downregulation of Gli and Hes1, 2 mediators of Shh in differentiation of RPs to both rod and cone cells. (G) Quantitative RT-PCR showed more expression of Nrl in RPE coculture. However, there was no significant difference in expression of Nr2e3 (H) and Rhodopsin (I) in all groups. *P<0.05. Color images available online at www.liebertonline.com/scd

FIG. 4.

Representative immunofluorescence staining of RP-derived retinal cells in Shh group at day 41. The retinal cells expressed Rhodopsin and S-Opsin, photoreceptor markers. Cells colabeled with horizontal cell marker Lim1 (green) and the neuronal marker Map2 (red) are shown. Immunostaining showed presence of glial cell with GFAP marker and bipolar Chx10 cells coexpressed Map2. Nuclei were counterstained with DAPI. Color images available online at www.liebertonline.com/scd

In vivo transplantation

Next, to evaluate whether retinal cells derived from hESCs improve the function of damaged retina, we transplanted retinal cells derived from the Shh group, which expressed more S-Opsin into the NaIO₃-injected rabbit model of RPE degeneration. Cyclosporine A was injected daily to preclude rejection of the human cells by the host. The cells were dissociated and immediately transplanted into the subretinal space of adult animals. Examination of histological sections of the retina was conducted in 4 groups as described earlier. Seven days after injection, the outer nuclear layer was disorganized and the numbers of RPE and photoreceptor cell nuclei were decreased (Fig. 5A). Transplantation success was microscopically indicated by the presence of a retinal bleb in the fundus of the eye immediately after injection.

FIG. 5.

Histology and electroretinography of NaIO₃-injured rabbit model. (A) H&E staining of paraffin-embedded retinal cross sections of normal, control, and sham rabbits, the latter were injected with NaIO₃ (50 mg/kg) at 1 week following injury. Over time, the RPE monolayer was destroyed and the layers of the neural retina were altered. (B) B-wave amplitudes following light flashes in different animal groups. N: normal/wild-type albino (n=6); S: injured with NaIO₃ injection and subsequent DMEM/F12 subretinal injection (n=6); T: transplant group that received retinal cells from the Shh treated group; C: control group, which was injected with only NaIO₃ for the eye of the same animal that had received the transplanted hESC-derived retinal cells (n=4). Eyes transplanted with hESC-derived retinal cells showed higher B-wave amplitudes compared with control eyes. *P<0.05, error bars represent SEM. Color images available online at www.liebertonline.com/scd

After a survival period of 4 weeks, ERG analysis of the animals showed that scotopic (dark-adapted), photopic (light-adapted), and Max B-wave peak amplitudes were reduced in control groups, which received NaIO₃ in comparison with normal animals (Fig. 5B and Supplementary Fig. S2). Four weeks after cell transplantation, a small light response was observed in comparison with the control group, which was unresponsive. Additionally, the scotopic, photopic, and Max B-wave peak amplitudes increased in the cell-transplanted animals when compared with the control and sham groups under similar excitation conditions (B-wave amplitude of scotopic: 10.6±1.7, photopic: 8.9±1.2, and Max: 13.4±1.3 μV in the cell-transplanted group compared with scotopic: 5.4±0.8, photopic: 2.8±0.4, and Max: 9.4±1.4 μV in the control group; P<0.05; Fig. 5B). ERG results showed that the mean B-wave amplitude following light flash in sham surgery was not significantly different from the control group after 4 weeks (scotopic: 4.4±0.7, photopic: 4.1±0.9, and Max: 8.7±0.8 μV in the sham group; Fig. 5B).

To evaluate transplanted cell survival, migration, and fate, rabbits were euthanized and their eyes removed, fixed, and sectioned after 4 weeks. The transplanted retinal cells were identified by lipophilic red dye (PKH26) labeling or immunofluorescence for a selective marker of human cell (HNu) and other photoreceptor markers. We found that HNu colabeled with PKH hESC-derived retinal cells survived in the subretinal space and migrated into the outer and inner nuclear layers and ganglionic cell layer of the retina (Fig. 6A–C) over the 4-week period. Therefore, histological analysis following ERG testing confirmed that the animals that demonstrated a response to light had a successful transplant. Of note, in 2 of the 6 rabbits we could not observe any PKH- or HNu-positive cells in their injected eyes perhaps because of apoptosis; these 2 rabbits did not present detectable ERG and were therefore excluded from analysis.

FIG. 6.

Tracing of transplanted cells in retinal sections at 4 weeks after cell transplantation. (A–C) PKH-prelabeled hESC-derived retinal cells costained with HNu/DAPI have migrated into the outer nuclear layer (ONL) (A) from the subretinal space. Some of these cells migrated to inner nuclear layer (INL) (B) and ganglionic layers. The migrated cells are shown by arrows. (C) Double-labeled transplanted cells with Rhodopsin (D) and S-Opsin (E) with PKH-26 showed integration of transplanted cells to ONL and their fate. Scale bars in A–E: 200 μm. Color images available online at www.liebertonline.com/scd

To determine the phenotype of PKH-positive cells, immunoflourescence was performed for Rhodopsin and S-Opsin. Examination of sections revealed that the majority of the transplanted cells were observed in the outer nuclear layer and expressed Rhodopsin and S-Opsin markers for rod and cone cells, respectively (Fig. 6D, E).

Therefore, a proportion of the transplanted hESC-derived retinal cells are light responsive and may transmit this response to the bipolar cells.

As undifferentiated hESCs can give rise to teratomas, we also assessed all eyes for evidence of teratoma formation. However, no teratomas were observed in any of the rabbits that received transplanted cells during our study. It is probable that if teratomas were to arise, they would be derived from undifferentiated hESCs. It is, however, improbable that any of the cells remained undifferentiated after 41 days of retina-directed cell culture prior to transplantation.

Discussion

In concordance with results of Lamba et al. [32], we have shown that hESCs can differentiate efficiently into RPs using defined media supplemented with a specific combination of growth factors, which included noggin, a BMP antagonist, Dkk, a Wnt antagonist, and IGF-1. The differentiation was induced as an adherent monolayer culture that highly (over 80%) expressed molecular features of RPs, such as Six3 at the early stage and Crx, Rx, Pax6, Otx2, and Chx10 at later stage. In contrast to the protocol of Lamba et al. [32], which used EBs at the initial step, we maintained hESCs in the adherent system on a matrigel matrix without a feeder layer. Because the differentiation process through the EBs is spontaneous and somewhat stochastic, the formation of EBs leads to the formation of numerous cell types originating from all 3 germ layers [39]. Our findings indicate efficient production of RPs of relatively high purity. Moreover, the adherent culture protocol facilitates visualization of the process of neural conversion and NT formation. Additionally, many cells located inside the EBs are in an apoptotic status, indicating that most ESCs die instead of differentiating and producing healthy ESC-derived cells.

Considering the fact that both RPE/Shh are required for retinal development [21 –29], and recently, as Yue et al. [10] have shown that coculturing of monkey ESCs with ESC-derived RPEs is a useful and efficient method for inducing photoreceptors, we evaluated whether RPE and/or Shh affect the differentiation of hESC-derived RPs in vitro, using a coculture system. Our results have shown that isolated RPE maintained their features, intercellular connections, intact tight junctions, intracellular microfilaments, and morphologic characteristics, which is important for their function. After RP maturation in all groups, the marker expressions of photoreceptors, ganglion cells, amacrine cells, bipolar cells, and horizontal cells were observed. Although we observed a significant increased expression of Nrl in the RPE group, the expressions of Nr2e3 and Rhodopsin were not significantly different between the groups. Additionally, RPE did not improve the expression of a cone photoreceptor marker, S-Opsin, but it enhanced in the presence of Shh only. This may be related to limited coculture time or the lack of a suitable time window for the coculture of RPE with RPs to induce mature photoreceptor differentiation, or further factors such as retinoic acid might be required to promote rod formation [10]. On the other hand, in retina, RPE sheet is closely attached to photoreceptor cells or their precursors and, therefore, more significant improvement could be gained when hESC-derived RPs are directly cultured onto RPE sheet.

In the presence of Shh, expressions of Gli and Hes1 decreased. It has been shown that removal or inhibition of Hes1 mainly leads to formation of cone cells [40] and further factors are required to deviate the default pathway and promote rod formation [41].

Previously, mouse photoreceptors have been shown to incorporate into adult retinas following transplantation into the subretinal space [5]. Therefore, to test whether hESC-derived retinal cells would similarly incorporate into the retinas and improve functionality of NaIO₃-injured retinal model of adult rabbits, we injected the Shh-treated retinal cells into the subretinal space in albino-type adult rabbits and analyzed them 4 weeks later. We found that transplanted hESC-derived retinal cells can be integrated into adult host retina and can restore a small but significant B-wave, whereas sham injections or control did not. It was demonstrated that human retinal cells derived from either hESC or hiPSCs integrate following transplantation and restore visual function in animal models [17,18]. The recognition of donor cells labeled with PKH was confirmed by expression of HNu. Immunohistochemical analysis revealed integration of these cells to the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglionic layer. Further, grafted cells expressed Rhodopsin and S-Opsin. The restoration of an ERG response correlates with the efficiency of transplantation. The transplanted retinal cells rescued visual function by the increased significant B-wave when compared with the control group. Interestingly, functional restoration of rabbits was detected in both dark and light responses. The mechanism of this improvement remains to be determined; however, it may be due to the attendance of a subpopulation of precursor or mature cells that survived, migrated, and differentiated to photoreceptors, thus improving visual function. A previous report has shown that the outer limiting membrane is a barrier within the adult retina to prevent migration of donor cells. Communication between host and graft tissues has been shown to occur through interruption of outer limiting membrane and laminar organization [42]. Correspondingly, local disruption probably facilitates migration of donor cells [43].

Taken together, as seen with other studies, our results indicated generation of retinal fate from adherent feeder-free hESCs. Following transplantation of Shh-committed retinal cells, a number of cells incorporated into the retina, differentiated, and effected a significant improvement in visual restoration to the rabbits. This provides a promising approach for replacement of cells in retinal diseases such as pigmentosa and age-related macular degeneration.

Footnotes

Acknowledgments

This study was funded by a grant from Royan Institute and Iranian Council of Stem Cell Technology.

Author Disclosure Statement

None of the authors has any conflict of interest to disclose and all authors support submission to this journal.

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Supplementary Material

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Differentiation of Human Embryonic Stem Cell–Derived Retinal Progenitors into Retinal Cells by Sonic Hedgehog and/or Retinal Pigmented Epithelium and Transplantation into the Subretinal Space of Sodium Iodate–Injected Rabbits

Abstract

Introduction

Materials and Methods

Culture of hESCs

Differentiation to RPs

Animals

Isolation and culturing of rabbit RPE

MTS assay

Experimental design

Preparation of cells for transplantation

Surgical procedure, experimental grouping, and transplantation

Sodium iodate–induced model of RPE degeneration

Experimental grouping and transplantation

Electroretinography

Sacrifice and cryosection

Immunocytofluorescence and immunohistofluorescence staining

Reverse transcription–polymerase chain reaction and real-time reverse transcription–polymerase chain reaction for gene expression analysis

Western blot analysis

Statistical analysis

Results

Generation of RP cells from hESCs

Coculture of RPs with RPE and/or Shh factor

In vivo transplantation

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Supplementary Material