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.

Fig 1

Photoreceptor differentiation protocol for swine iPSC. (A): Phase image of swine iPSC colonies (arrows) 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 beta-actin (ACTB) mRNA. (H): Immunostaining for TUBB3 following differentiation. (I–J): Immunostaining for 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.

Fig 2

RHO⁺ cells derived from iPSC co-express early photoreceptor lineage markers NRL and RCVRN. In addition differentiated cells expressing the rod bipolar lineage marker PRKCA are also present. (A1–B4): Cells double immunostained with RHO and NRL. (C1–D4): Cells double immunostained for RHO and RCVRN. (E1–F4): Cells immunostained for PRKCA. Scale bars = 50 μm.

Fig. 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.

Fig 4

Adherent culture on Matrigel enhances the number of differentiating iPSC resembling primary cultures of rod photoreceptors. (A): Quantification of RHO⁺ cells generated on different matrices. “L/F” laminin/fibronection, “M” Matrigel. Phase images of cells are shown below. P<0.05 for all values compare to undiluted Matrigel. (B1–B4): Differentiated iPSC plated on laminin/fibronection and immunostained for RHO. Note cells with elongated cell bodies that immunostain uniformly for RHO. (C1–C4): Differentiated iPSC cells 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 iPSC plated on Matrigel in panels C1–C4. (E1–E4): Differentiated iPSC plated on Matrigel and immunostained with the rod outer segment marker 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⁺ iPSC morphologically resembling primary culture rod photoreceptors generated on different matrices. P<0.05 for L/F compared to M and M 1:10.

Fig. 5

Iodoacetic acid treatment leads to selective loss of rod photoreceptors in the swine retina. (A–B): Sections of retina from a six week old pig immunostained for rod (RHO) and cone (red-green opsin) markers. (C–D): Sections of retina from a six week old pig three weeks after iodoacetic acid treatment. Immunostaining for RHO and red-green opsin is shown. Scale bars = 50 μm.

Fig 6

Integration of transplanted swine iPSC into the ONL. (A–B): RBP3-GFP expression in differentiated iPSC. Differentiated iPSC plated on Matrigel were infected with a lentivirus containing the photoreceptor-specific RBP3 gene promoter driving expression of GFP [–10]. The boxed region in panel A is shown at higher magnification in panel B. Phase images are shown. (C): Fundas photograph of a six week old swine retina. The injection site is shown with a box. “D”, indicate the optic disc, and “S” indicates the visual streak. The scale bar is 2000 microns. (D): Differentiated iPSC 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 iPSC were immunostained for RHO and GFP. In panels E and F, 1 is a Nomarski image, 2 is DAPI staining, 3 is RHO staining, 4 is a GFP image, 5 is merged DAPI, RHO and GFP image, and 6 is the merged DAPI, RHO, GFP and Nomarski image. Arrows indicate outer segment projections. Scale bars in panels A, B, E and F = 50 μm.