Differentiation of Induced Pluripotent Stem Cells of Swine into Rod Photoreceptors and Their Integration into the Retina

Abstract

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO⁺ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.

Photoreceptor differentiation, Swine retina, Induced pluripotent stem cells, Retinal transplantation

Introduction

The outer nuclear layer (ONL) of the retina consists of cell bodies of rod and cone photoreceptors, which convert light signals to electrical potential that is transmitted to bipolar cells in the inner nuclear layer [1]. This signal in turn is sent to ganglion cells that transmit the signal to the visual cortex. A regenerative pathway is effective in restoring damaged retina in lower vertebrates [2, 3], but in higher vertebrates there is neither a comparable pathway with the ability to restore visual function following either retinal injury or disease nor have the adult stem cells been isolated from the retina. Therefore, therapeutic approaches to retinal damage and disease in higher vertebrates have focused on cell transplantation using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) generated from skin fibroblasts by viral transduction of stem cell specification genes POU5F1 (also known as OCT4), SOX2, KLF4, and c-MYC [4–13]. ESCs derived from mice, humans, and monkeys have been used successfully to generate cells expressing markers of retinal progenitors in culture [6–10], and iPSCs derived from mouse and human skin fibroblasts can also differentiate to generate photoreceptor-like progenitors in culture [8, 9, 11, 13].

The mouse is an attractive model for cell transplant studies because of the variety of genetic mutants available, and accordingly most retinal transplant studies have used rodents. Transplanted photoreceptors from neonatal mice have been shown to engraft into the damaged mouse retina, and some of these transplanted cells go on to generate outer segments [14], which consist of membranous disks containing visual pigment that are projected from the cell surface and reflect functional morphology [1]. In these studies, proliferating retinal progenitors did not functionally engraft into the mouse retina, demonstrating that differentiated photoreceptors may be necessary for successful cell transplantation. Differentiated ESCs have also been transplanted into the mouse retina, and these cells integrate and some of them generate outer segments, but by contrast differentiated iPSCs or retinal progenitor cells have thus far failed to generate outer segments following retinal transplantation [10]. Transplantation of differentiated ESCs into mouse models of rod photoreceptor loss has led to cell integration into the ONL and a modest increase in electrophysiological response to light measured by electroretinography (ERG) has been reported, but no outer segments have been detected [3–5, 9–11]. These studies in mice demonstrate the feasibility of retinal cell transplantation therapy for human retinal disease, but the mouse retina is rod-dominant, lacks a macula, and is thus not an ideal anatomical and physiological model of human retinal disease. As with humans, the swine retina contains a cone-dominant central visual streak analogous to the macula with rods enriched in the peripheral retina [15]. Thus, the swine retina is a much closer anatomical and physiological match to the human retina. Use of the swine retina as an experimental model for stem cell transplantation has been hampered by the fact that ESCs have not been isolated from pig [16, 17], and attempts have not been made to differentiate iPSCs of swine into photoreceptor lineages. However, retinal progenitors derived from embryos have been injected into the swine subretinal space, and, importantly, they have been shown to integrate and survive [18, 19], thus demonstrating the feasibility of stem cell transplantation into the swine retina. Here, we have used iPSCs of swine derived from fetal fibroblasts as a source of rod photoreceptor lineage cells for transplantation into a swine model of rod photoreceptor loss. We show that these cells can differentiate into photoreceptors in culture, and following transplantation into a model of swine rod photoreceptor loss, the cells integrate into the ONL and can generate outer segment-like projections. These results provide a foundation for future studies of retinal stem cell transplantation in a swine model.

Materials and Methods

Culture of iPSCs of Swine

The ID6 iPSC line of swine has been described previously [20]. A colony of uniform appearance at passage 24 resembling a typical human ESC colony was mechanically broken up [20] and passaged 1:10 on a feeder layer of irradiated mouse embryonic fibroblasts in media containing Dulbecco's modified Eagle medium (DMEM)/F12, 20% knockout serum replacer, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1% nonessential amino acids, and 4 ng/ml fibroblast growth factor 2 (FGF2) (Invitrogen, Carlsbad, CA, www.invitrogen.com).

Photoreceptor Differentiation of iPSCs of Swine

The differentiation protocol of iPSC photoreceptor of swine was modified from that of Lamba et al. [9, 10] for human ESCs. iPSCs at passages 28, 40, and 43 were used for photoreceptor differentiation with similar results. For photoreceptor differentiation, embryoid bodies were allowed to form by dissociating iPSCs into small clumps with 1 mg/ml type IV collagenase, and the resulting embryoid bodies were cultured in 100 mm ultra-low attachment plates (VWR, West Chester, PA, www.vwrsp.com) in medium containing DMEM/F12, 10% knockout serum replacer, neuronal culture supplements N2 and B27 (Invitrogen), 1 ng/ml Dickkopf homolog 1 (DKK1) (R&D, Oxford, UK, www.rndsystems.com), 1 ng/ml noggin (R&D), and 1 ng/ml insulin-like growth factor-1 (IGF1) (R&D) for 3 days. Embryoid bodies were then transferred to poly-D-lysine coated plates with undiluted Matrigel (BD), Matrigel diluted 1:10 or 1:20, or plates coated with a mixture of laminin (8 μg/cm²) and fibronectin (2 μg/cm²), after which they were cultured for 18 days in medium containing 10 ng/ml DKK1, 10 ng/ml noggin, 10 ng/ml IGF1, and 5 ng/ml human recombinant FGF2 (Invitrogen). Medium was changed daily. Retinoic acid (1 μM) and taurine (100 μM) (Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich.com) were added as indicated.

Primary Culture of Rod Photoreceptors

For primary culture of rod photoreceptors, eyes from adult pigs were obtained from the local slaughterhouse and kept in CO₂ Independent Medium (Invitrogen) on ice before dissection. The protocol for harvesting photoreceptors with attached outer segments has been described previously [21]. The following procedures were performed under dim red light. The anterior segment of the eye was removed, the neural retina was isolated from the eye cup, and the tissue is digested with activated papain (Sigma-Aldrich) at 37°C for 20 minutes. The papain solution was removed and enzymatic activity is inhibited by adding media containing 2% fetal bovine serum to the retina for 5 minutes. This media was then removed, and retinal cell culture medium containing neurobasal-A-medium, B27, and L-glutamine supplements (all from Invitrogen) was added. The digested tissue was pipetted gently several times with a P1000 Pipetman, and large tissue fragments were allowed to settle for a further 5 minutes. Disassociated photoreceptors, which remained in suspension, were transferred to tissue chamber slides coated with poly-D-lysine and Matrigel diluted 1:20. Two days later, the cells were examined for expression of various antigens by immunostaining.

Immunostaining

Cells or swine retinas were fixed with 4% paraformaldehyde for 15 minutes, and frozen sections of swine retina were dried at 37°C for 15 minutes. Cells and tissue sections were then washed with phosphate buffered saline (PBS) and blocking solution consisting of 0.8% bovine serum albumin, 4% goat serum, and 0.1% Tween-20 in PBS was added for 1 hour at 25°C. The samples were then incubated either overnight at 4°C or 1 hour at 25°C with primary antibody reagents in blocking solution. After primary antibodies had been removed and the samples washed, secondary antibodies were applied for 1 hour at 25°C. The primary antibodies used were: goat anti-POU5F1 (Santa Cruz, Santa Cruz, CA, www.scbt.com; 1:100), mouse anti-beta3 tubulin (anti-TUBB3) (Millipore, 1:500), rabbit anti-recoverin (anti-RCVRN) (Millipore, Billerica, MA, www.millipore.com; 1:1,500), mouse anti-rhodopsin (anti-RHO) (Millipore, 1:300), rabbit anti-neural retina leucine zipper protein (anti-NRL) (a generous gift from Anand Swaroop, National Eye Institute, Bethesda, MD, 1:1,000) [22], rabbit anti-protein kinase C-α (anti-PRKCA) (Sigma, 1:15,000), rabbit anti-rod outer segment-specific membrane protein 1 (anti-ROM1) (Sigma, 1:400), rabbit anti-red-green opsin (opsin long wavelength opsin 1 [OPN1LW]/opsin medium wavelength opsin 1 [OPN1MW]) (Millipore 1:500), rat anti-ABCG2 (Abcam, Cambridge, MA, www.abcam.com; 1:200). Bound antibodies were visualized with either Alexa fluor 488-conjugated (Invitrogen 1:500) or Alexa Fluor 568-conjugated (Invitrogen, 1:500) secondary antibodies. Nuclei were counterstained with DAPI, and images captured with a Zeiss inverted fluorescence microscope (Axiovert 200). The immunoreactivity of each antibody was confirmed by immunostaining swine retinal tissue as a positive control, and as a negative control, primary antibody was omitted (Supporting Information Fig. 1).

Statistical Analysis

Analysis of variance between groups with Bonferroni's post hoc test was used for the determination of statistical significance among treatment groups, as indicated.

Real-Time PCR Analysis

RNA was extracted from the cultures using the RNAeasy kit (Qiagen) and reverse transcribed with the Superscript III RT-PCR kit (Invitrogen). SYBR Green real-time PCR was performed by using a Stratagene Mx3000P Real-Time PCR system [23]. The complete coding sequence for swine genes was obtained from the European Molecular Biology Laboratory and National Center for Biotechnology Information Nucleotide Sequence Database. The POU5F1 mRNA forward primer was 5′-CGAAGCTGGACAAGGAGAAG-3′, and the reverse primer 5′-GCTGAACACCTTCCCAAAGA-3′ (product 176 bp). The RHO forward primer was 5′-CTTCCCC ATCAACTTCCTCA-3′ and the reverse primer was 5′-ACCACC ACGTACCGTTCAAT-3′ (product 264 bp). The cone-arrestin (ARR3) forward primer was 5′-AACGGCAAGCTCTCCATCTA-3′, and the reverse primer was 5′-CAGATCTTTGCGGAA TGTCA-3′ (product 195 bp). The RCVRN forward primer was 5′-GGGCTTTCTCCCTCTACGAC and the reverse primer 5′-CA TCGTCCTTCTTCCCAAAG (183 bp). The retinol binding protein 3 (RBP3) forward primer was 5′-GGCCAAGATAGCAGTC AAGC and the reverse primer 5′-CTCGAGCACGTTAGTGTG GA (product 244 bp). The NANOG forward primer was 5′-TT CCTTCCTCCATGGATCTG and the reverse primer 5′-ATCTG CTGGAGGCTGAGGTA (product 214 bp). The SOX2 forward primer was 5′-GCCCTGCAGTACAACTCCAT and the reverse primer 5′-GCTGATCATGTCCCGTAGGT (product 216 bp). The β-actin (ACTB) mRNA forward primer was 5′-GCCAACCGTGA GAAGATGAC-3′, and the reverse primer 5′-GAGTCCATCACG ATGCCAGT-3′ (126 bp product).

Quantification of iPSC Differentiation

Cells expressing differentiation markers were counted and compared with the total number of cells found in each field, as determined by DAPI nuclear staining. At least 2,000 cells were examined in each experiment. All sets of experiments were performed at least three times. Results are reported as means ± SE.

Lentiviral Infection

RBP3-green fluorescence protein (GFP) lentiviral particles were a gift from Thomas A. Reh, University of Washington [9, 10]. Differentiated iPSCs were infected with the RBP3-GFP lentivirus as described [9, 10].

Subretinal Transplantation

All animal protocols were approved by the University of Louisville Institutional Animal Care and Use. Domestic pigs were obtained at 6 weeks of age (12–16 kg) from Oak Hill Genetics (Ewing, IL). Iodoacetic acid was dissolved in normal saline, and pigs were administered 12 mg/kg intravenously via a catheter placed in the ear vein. Four days later, animals were sedated with Telzol (2.0–8.8 mg/kg) and maintained under general anesthesia with 1.5%–2% isoflurane mixed with oxygen. Intravenous access was achieved by placement of a 21-gauge catheter in an ear vein. Pupils were dilated and the ability to focus was inhibited with topical applications of 2.5% phenylephrine hydrochloride and 1% tropicamide. A three-port, 20 gauge pars plana vitrectomy was performed using a suction of 150 mmHg and a cutting rate of 600 oscillations per minute [24]. A posterior vitreous detachment was made by suction over the optic disc and posterior retina, and a neurosensory retinal detachment (i.e., a bleb) at the visual streak was created by injecting 50 μl of BSS Plus (Alcon) into the subretinal space using a 39-gauge cannula. Differentiated iPSCs (2 × 10⁶ in 0.1 ml) were injected into the bleb.

Histological Evaluation

Pigs were euthanized 3 weeks after cell transplant with Beuthanasia (1 ml/5 kg) administered through an ear vein catheter. Eyes were enucleated, and retinas were isolated and fixed by immersion in 4% paraformaldehyde. A strip of retina that extended from the dorsal to the ventral margin of the eyecup was bisected at the optic disc along the horizontal plane. The dorsal and ventral halves were bisected again along the horizontal plane. Each of the four pieces was notched on its dorsal edge to preserve orientation. Pieces were dehydrated and embedded in the polyvinyl alcohol, polyethylene glycol-based optimal cutting temperature cutting reagent. Tissues were oriented and cut to produce vertical sections along the dorsal to ventral axis. Frozen sections (14 μm) were cut for immunostaining using a cryostat.

Results

Differentiation of iPSCs of Swine into Photoreceptors

iPSCs from swine (ID6) were created by lentiviral transduction of cDNAs for POU5F1, KLF4, SOX2, and c-MYC into swine fetal fibroblasts and maintained on a standard medium for human ESCs containing FGF2 [20]. Although these cells were derived from a pig expressing a GFP transgene [20], colonies resembling fully reprogrammed ESCs at passage 28 had silenced GFP expression (Supporting Information Fig. 1). Such silencing of a GFP transgene has previously been used as an important criterion for selection of fully reprogrammed human and mouse iPSCs [25]. A phase image of iPSC colonies is shown in Figure 1A.

1

Photoreceptor differentiation protocol for induced pluripotent stem cells (iPSCs) from swine. (A): Phase image of iPSC colonies (arrows) of swine cultured on an irradiated fibroblast feeder layer. (B): Phase image of embryoid bodies in floating culture. (C): Three days after embryoid bodies were allowed to adhere to matrix, cells had migrated out onto the substratum. (D): Phase image showing cells at day 21 of the differentiation protocol. (E): Immunostaining of an iPSC colony for POU5F1. (F): Immunostaining of an iPSC colony for ABCG2. (G): Real-time PCR showing loss of stem cell mRNAs and induction of photoreceptor mRNAs during iPSC differentiation. Results were normalized to β-actin (ACTB) mRNA. (H): Immunostaining for beta3 tubulin following differentiation. (I, J): Immunostaining for rhodopsin (RHO) following differentiation. Note the two distinct cell morphologies of RHO⁺ cells shown by white and red arrows in panel (J) (also see Fig. 4). Scale bars = 50 μm. Abbreviations: ARR3, cone-arrestin; DAPI, 4′,6-diamidino-2-phenylindole; iPSC, induced pluripotent stem cells; RBP3, retinol binding protein 3; RCVRN, recoverin; RHO, rhodopsin; TUBB3, beta3 tubulin.

We used a two-step protocol for photoreceptor differentiation of the iPSCs of swine involving initial production of embryoid bodies (Fig. 1B) and subsequent outgrowth on extracellular matrix (Fig. 1C, 1D). Embryoid bodies in suspension on nonadherent plates were cultured with the WNT inhibitor DKK1, the bone morphogenetic protein inhibitor noggin, and IGF1 for times ranging from 3–7 days. Then, the embryoid bodies were transferred to plates coated with poly-D-lysine and different concentrations of either Matrigel or laminin/fibronectin. Culture was then continued for three more weeks with a 10-fold increase in the concentrations of DKK1, noggin, IGF1, and supplemental FGF2 in the medium. The iPSCs express POU5F1 in the nucleus and the multidrug resistance transporter ABCG2, which is expressed on stem cells and cancer stem cells, on their surface (Fig. 1E, 1F), and they also express mRNAs for POU5F1, NANOG, and SOX2 (Fig. 1G). Silencing of stem cell specification genes is a hallmark of ESC and iPSC differentiation, and 3 days of culture as embryoid bodies led to downregulation of POU5F1, NANOG, and SOX2 mRNAs (Fig. 1G; Supporting Information Fig. 2A, 2B) implying effective differentiation, which is important to avoid the potential risk of teratoma formation following cell transplantation. Although previous studies suggested that supplementation of the medium with retinoic acid and taurine promotes differentiation of rod precursor cells from cultures of either ESCs or iPSCs [6, 8, 13], these reagents did not appear to accelerate either loss of POU5F1 mRNA expression or the process of differentiation in the iPSCs of swine (Supporting Information Fig. 2A, 2F). Following differentiation, cells were immunostained for the general neuronal marker TUBB3. Approximately 40% of the cells showed neuronal morphology and were TUBB3-positive (TUBB3⁺) (Figs. 1H, 3), suggesting that many of the cells had become committed to a neuronal lineage.

RHO is a terminal marker of rod photoreceptor differentiation required for conversion of light to electrical signals. While earlier markers of rod photoreceptors such as NRL, RCVRN, and photoreceptor-specific RBP3 (also known as IRBP1) can be efficiently induced during differentiation of mouse, human, and monkey ESCs and iPSCs, few RHO⁺ cells have been reported [6, 8, 13]. We found that mRNAs for RCVRN, RBP3, and RHO as well as the cone marker, ARR3, were induced following the differentiation protocol (Fig. 1G), and approximately 6% of the cells were RHO⁺ by immunostaining (Figs. 1I, 1J, 3). These RHO⁺ cells showed two distinctly different morphologies. Some of the cells resembled TUBB3-expressing cells with elongated cell bodies and RHO distributed evenly throughout the cytoplasm, whereas other cells displayed rounded cell bodies with RHO concentrated in outer segment-like projections extending from the cell surface (Figs. 1H, 1J, 4A–4G below). Three days of embryoid body culture in the absence of retinoic acid and taurine generated the greatest number of RHO⁺ cells (Supporting Information Fig. 2C–2F), and these conditions were used for all subsequent differentiation assays.

Differentiating iPSCs of Swine Express Early Markers of the Rod Photoreceptor Lineage and a Marker of Rod Bipolar Cells

Differentiated iPSCs of swine were stained for the transcription factor NRL, which drives commitment to the rod lineage, and RCVRN, which is an additional marker of the early photoreceptor lineage (Fig. 2A–2D). All RHO⁺ cells coexpressed NRL and RCVRN, but a high percentage of RHO⁻ cells also expressed these two markers (Fig. 3), suggesting that the differentiation protocol was generating neuronal cells at early as well as later stages of rod photoreceptor lineage.

2

Rhodopsin (RHO⁺) cells derived from induced pluripotent stem cells coexpress early photoreceptor lineage markers neural retina leucine zipper protein (NRL) and recoverin (RCVRN). In addition, differentiated cells expressing the rod bipolar lineage marker protein kinase C-α (PRKCA) is also present. (A1–B5): Cells double immunostained with RHO and NRL. (C1–D5): Cells double immunostained for RHO and RCVRN. (E1–F4): Cells immunostained for PRKCA. Scale bars = 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NRL, neural retina leucine zipper protein; PRKCA, protein kinase C-α; RCVRN, recoverin; RHO, Rhodopsin.

3

Quantification of cells expressing markers of retinal differentiation. At least 2,000 cells were examined in each experiment and results are an average of three independent experiments. The total cell number present in a field was determined by DAPI nuclear staining. Abbreviations: NRL, neural retina leucine zipper protein; PRKCA, protein kinase C-α; RCVRN, recoverin; RHO, Rhodopsin; TUBB3, beta3 tubulin.

Rod photoreceptors synapse with rod bipolar cells to transmit visual signals to ganglion cells [26]. Such rod bipolar cells are one of the last lineages to appear during retinal development in vivo and they are marked by expression of PRKCA [27]. Approximately 2.5% of the differentiated cells are immunostained for PRKCA (Figs. 2E, 2F, 3), suggesting that the differentiation protocol also generates cells resembling rod bipolar cells.

Culture on Matrigel is Required for Efficient Generation of Cells Resembling Primary Cultures of Rods

Following embryoid body formation, we examined the effect of culturing the cells on different matrices. Culture on laminin/fibronectin led to the highest percentage of RHO⁺ cells (Fig. 4A). However, most of these RHO⁺ cells on laminin/fibronectin displayed elongated cell bodies resembling TUBB3⁺ cells seen in Figure 1H, and RHO was distributed evenly in the cytoplasm of these cells (Fig. 4A, 4B). By contrast, most of the RHO⁺ cells on Matrigel had rounded cell bodies with thin axonal projections. RHO and ROM1 were concentrated in outer segment-like projections extending from the body of these cells (Fig. 4A, 4C, 4E). We then compared the morphology of the iPSC-derived RHO⁺ cells with primary cultures of swine rod photoreceptors generated by an isolation protocol that conserves outer segments [21]. The morphology of the rod primary cultures was similar to that of the differentiated iPSCs cultured on Matrigel. In addition, RHO and ROM1 were concentrated in outer segment extensions resembling those seen on the cells cultured on Matrigel (Fig. 4D, 4F). Thus, even though culture on laminin/fibronectin led to a higher number of RHO⁺ cells, culture on Matrigel appears to be important in directing transition of RHO⁺ cells to a morphology with outer segment-like processes (Fig. 4G).

4

Adherent culture on Matrigel enhances the number of differentiating induced pluripotent stem cells (iPSCs) resembling primary cultures of rod photoreceptors. (A): Quantification of rhodopsin (RHO⁺) cells generated on different matrices. L/F is laminin/fibronectin and M is Matrigel. Phase images of cells are shown below. p < .05 for all values compares with undiluted Matrigel. (B1–B4): Differentiated iPSCs plated on laminin/fibronectin and immunostained for RHO. Note the cells with elongated cell bodies that immunostain uniformly for RHO. (C1–C4): Differentiated iPSCs plated on Matrigel and immunostained for RHO. Note that RHO is concentrated in projections from the main cell body. (D1–D4): Primary culture of swine retinal cells immunostained for RHO. Note that RHO is concentrated in outer segments extending from the rounded cell bodies, and that the cell morphology resembles that of the differentiated iPSCs plated on Matrigel in panels (C1–C4). (E1–E4): Differentiated iPSCs plated on Matrigel and immunostained with the rod outer segment marker, rod outer segment-specific membrane protein 1 (ROM1). Note that ROM1 colocalizes with RHO in projections from the rounded cell bodies. (F1–F4): Primary culture of swine retinal cells immunostained for ROM1. Note that, like RHO, ROM1 is concentrated in outer segments projected from the rounded cell bodies and that these cells resemble those in panels (E1–E4). Scale bars = 20 μm. (G): Quantification of RHO⁺ iPSCs morphologically resembling primary culture rod photoreceptors generated on different matrices. p < .05 for L/F when compared with M and M 1:10. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; L/F, laminin/fibronectin; M, Matrigel; RHO, rhodopsin; ROM1, rod outer segment-specific membrane protein 1.

Iodoacetic Acid Treatment Leads to Loss of Rod Photoreceptors in the Swine ONL

Iodoacetic acid blocks glycolysis and is thus toxic to neurons, which depend on this pathway [28–30]. Six-week-old pigs were injected intravenously with 12 mg/kg of iodoacetic acid, and 3 weeks later retinas were removed and sectioned for immunostaining with rod and cone markers. Immunostaining for RHO was lost following iodoacetic acid treatment, demonstrating a loss of rod photoreceptors, whereas immunostaining with an antibody against cone-specific red-green, which recognizes both OPN1LW and OPN1MW, was relatively unaffected by the treatment (Fig. 5A–5D). These results demonstrate that rod photoreceptors are damaged in the swine retina by iodoacetic acid.

5

Iodoacetic acid treatment leads to selective loss of rod photoreceptors in the swine retina. (A–B): Sections of retina from a 6-week-old pig immunostained for rod (rhodopsin; RHO) and cone (red-green opsin) markers. (C–D): Sections of retina from a 6-week-old pig, 3 weeks after iodoacetic acid treatment. Immunostaining for RHO and red-green opsin is shown. Scale bars = 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; INL, inner nuclear layer; ONL, outer nuclear layer; OPN1LW, opsin long wavelength opsin 1.

Expression of RBP3-GFP in Differentiating iPSCs of Swine

To mark cells committed to photoreceptor lineage, differentiated iPSCs of swine were infected with a lentivirus vector incorporating the promoter region of RPB3 driving expression of GFP [9, 10]. Following infection, approximately 44% of the cells expressed GFP (Fig. 6A, 6B), roughly similar to the number of neuronal cells positive for TUBB3 and slightly higher than the number expressing NRL (Fig. 3). Although RBP3 is a marker of differentiating photoreceptors, it is maintained in the adult retina [9, 10], and we reasoned that GFP expression from this promoter would be a useful marker to follow iPSC-derived rod precursor cells after transplantation into the eye.

6

Integration of transplanted induced pluripotent stem cells (iPSCs) from swine into the outer nuclear layer. (A–B): Retinol binding protein 3 (RBP3)-green fluorescence protein (GFP) expression in differentiated iPSCs. Differentiated iPSCs plated on Matrigel were infected with a lentivirus containing the photoreceptor-specific RBP3 gene promoter driving expression of GFP [9, 10]. The boxed region in panel A is shown at higher magnification in panel B. Phase images are shown. (C): Fundas photograph of a 6-week-old swine retina. The injection site is shown with a box. “D” indicates the optic disc, and “S” indicates the visual streak. Scale bar = 2,000 μm. (D): Differentiated iPSCs were injected 4 days after iodoacetic treatment, and then 3 weeks later the retina was removed. A fluorescent image of GFP⁺ cells in the boxed area of panel C is shown in a flatmount of the retina. (E, F): Sections of retina from the region of transplanted iPSCs were immunostained for rhodopsin (RHO) and GFP. (E1 and F1), a Nomarski image; (E2 and F2), DAPI staining; (E3 and F3), RHO staining; (E4 and F4), GFP image; (E5 and F5), merged DAPI, RHO, and GFP image; and (E6 and F6), merged DAPI, RHO, GFP, and Nomarski image. Arrows indicate outer segment projections. Scale bars = 50 μm (A, B, E, and F). Abbreviations: D, optic disc; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescence protein; INL, inner nuclear layer; ONL, outer nuclear layer; RHO, rhodopsin; S, visual streak.

Integration of RHO⁺ Transplanted Cells into the Swine ONL

Four days after iodoacetic acid treatment, 1 × 10⁶ differentiated iPSCs were injected subretinally into one eye (Fig. 6C), whereas DMEM/F12 medium was injected into the other control eye. After 3 weeks, the animals were sacrificed and their retinas removed. GFP⁺ cells were evident at the injection site in a flatmount of the retina (Fig. 6D). The injection site was then sectioned for immunostaining. GFP⁺/RHO⁺ transplanted cells had integrated into the ONL in this region, and some of these transplanted cells had outer segment-like projections (Fig. 6E, 6F). Integrated RHO⁺ cells were counted in six 100,000 μm² sections distributed throughout the injection site, which was approximately the size of the optic disk (Fig. 6C). The disk in the pig is 2 × 4 mm² and approximately 8 × 10⁶ μm². Assuming these six sections are representative of cell integration throughout the injection site, 10,414 ± 3,051 injected RHO⁺ cells integrated into the retina. This corresponds to approximately 1% of the injected cells. A previous study found that approximately 3,000 integrated cells (approximately 4% of the injected cells) in the mouse retina after injection of ESCs differentiated into photoreceptors and approximately 25% of these integrated cells expressed RHO [10].

Discussion

Here, we have presented a two-step differentiation protocol for photoreceptor generation from iPSCs of swine that involves suspension culture as embryoid bodies followed by adherent culture on an extracellular matrix. An early event following conversion to embryoid bodies was loss of expression of the pluripotency marker POU5F1. A high percentage of the differentiating cells acquired a neuronal morphology and expressed the general neuronal marker TUBB3, and the majority of such cells also expressed the rod-specification transcription factor NRL and the early photoreceptor marker RCVRN, indicating that most of these neuronal cells were committed to the rod lineage. However, only approximately 6% of the cells were positive for the terminal rod marker RHO. Together, these results suggested that cells at various stages of rod differentiation are present in the cultures, and the cells posed little risk of generating teratomas if injected into animals.

We noted two distinct morphologies of RHO⁺ cells in culture. Some cells had elongated cell bodies characteristic of neurons and RHO was distributed evenly throughout the cytoplasm, whereas other cells had rounded cell bodies with thin axonal projections and RHO and ROM1 were concentrated into outer segment-like extensions from the cell bodies. Because this latter morphology resembled primary cultures of rod photoreceptors, we suggest that following RHO expression, the cells can undergo a morphological change where RHO and ROM1 become concentrated in outer segments. Furthermore, this morphological transition is enhanced by culture of the cells on Matrigel during the differentiation process.

Treatment of pigs with iodoacetic acid led to loss of rod photoreceptors, and this was accompanied by a diminished dark-adapted ERG (Supporting Information Fig. 3). Subretinal injection of the differentiated iPSCs after iodoacetic acid treatment led to integration of the cells into the retina. While RHO⁺ transplanted cells were evident in all of the retinal layers, they were most concentrated in the ONL and some of the transplanted cells in the ONL had generated outer segments. It is of note that outer segment generation has yet to be detected in cells transplanted into mouse models of rod photoreceptor damage [10]. However, despite evidence of rod outer segment generation and thus transition to functional morphology, transplantation of these cells did not lead to a significant change in ERG in the swine retina (Supporting Information Fig. 3). However, it is of note that the pig retina is much larger than the mouse, and thus the injection site in these studies only represents a small region of the swine retina (Fig. 6C).

The mouse is an important model for stem cell transplant studies due to the variety of genetic models of retina disease available, but the ability to extend such experiments to the pig, because of the anatomical similarity between the pig and human retina, is likely to provide an additional model that ensures the technology is developed safely and efficiently before it attempts at human transplantation. As an initial step toward a swine model for retinal stem cell transplant, we have developed an efficient protocol for differentiation of iPSCs of swine into the rod photoreceptor lineage and shown that these cells are capable of integrating into the ONL of retinas depleted of rod photoreceptors.

Conclusion

These results demonstrate that swine iPSCs can differentiate into photoreceptors in culture and can integrate into the damaged retina. The studies provide a foundation for experiments using the pig as a model for retinal stem cell transplantation.

Disclosure of Potential Conflict of Interests

The authors indicate no potential conflicts of interest.

Acknowledgments

We thank the gift of retinol binding protein 3-green fluorescence protein lentivirus from T.A. Reh and the neural retina leucine zipper protein antibody from A. Swaroop. These studies were supported in part by American Health Assistance Foundation, NIH Grants (P20 RR018733 and EY015636), Research to Prevent Blindness, and The Commonwealth of Kentucky Research Challenge. The generation of induced pluripotent stem cells from swine was supported by grants from NIH (HD 21896) and the Missouri Life Sciences Research Board (09-1018).

Author notes

Author contributions: L.Z.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; W.W.: conception and design, collection and assembly of data, data analysis and interpretation, final approval of manuscript; Y.L.: performed real-time PCR analysis on induced pluripotent stem cells; J.F.d.C.: performed electroretinography measurements; T.E., B. P.V.L.T., and R.M.R.: provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript; H.K. and D.D.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

Disclosure of potential conflicts of interest is found at the end of this article.

First published online in STEM CELLS  EXPRESS April 13, 2011.

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