Skip to main content
Download PDF

Endoplasmic reticulum stress and rhodopsin accumulation in an organoid model of Retinitis Pigmentosa carrying a RHO pathogenic variant

Stem Cell Research & Therapy volume 16, Article number: 71 (2025) Cite this article

Abstract

Background

Retinitis Pigmentosa (RP) is the most prevalent inherited retinal dystrophy, with more than 120 causative genes. Among them, RHO was the first photoreceptor gene described to harbor mutations responsible for RP. RHO pathogenic variants usually induce a dominant negative effect in which the accumulation of misfolded rhodopsin protein leads to ER stress, autophagy and lastly rod photoreceptor death.

Methods

We differentiated photoreceptor precursors and retinal organoids from an iPSC line of a patient carrying the Pro215Leu mutation in RHO gene. Both cell models were analyzed to determine their maturation, the expression and localization of RHO mRNA and the rhodopsin protein and the activation of autophagy or ER pathways.

Results

The Pro215Leu mutation causes rhodopsin accumulation in the soma of rod photoreceptor precursors along with a faster recycling by the proteasome. In both precursors and retinal organoids, we observed autophagy defects and late endoplasmic reticulum stress through CHOP increase.

Conclusions

Unraveling the molecular pathophysiology of these mutations is key for understanding the basis of the disease and design proper gene and cell therapies for its treatment.

Background

Retinitis Pigmentosa (RP) is one of the most prevalent hereditary rod-cone dystrophy, a degenerative disease characterized by the irreversible loss of the photoreceptor cells in the periphery of the retina. Its prevalence is approximately one in 4000, with around 1.5 million individuals affected worldwide [1], and accounting for around 23% of all inherited retinal dystrophies (IRD) [2].

RP is characterized by a progressive loss of peripheral vision leading to “tunnel vision”. The initial symptoms are impaired dark adaptation and the development of nyctalopia or night blindness. As photoreceptor (PR) loss proceeds, there is a loss of pigmentation from the retinal pigment epithelium (RPE), and a build-up of intraretinal melanin deposits [3]. The classic features include attenuated retinal blood vessels, intraretinal pigmentation, waxy pallor of the optic disc, and hyperfluorescent rings on fundus autofluorescence (FAF) [4]. Central visual acuity is often preserved until the end stages of RP.

Autosomal dominant RP (adRP) accounts for 20–30% of all RP cases, with mutations in the RHO gene being a common cause along other prevalent genes including PRPF3 and RP1 [5, 6]. While around 30 genes are responsible for adRP, more than 90 are responsible for the autosomal recessive and X-linked counterparts, with USH2A, RPGR and RPE65 having a higher prevalence [4].

RHO gene is located in the 3q22.1 cytogenetic position and is composed of 5 exons [3]. It encodes for the 39 kDa protein rhodopsin, which is predominantly present in rods [5]. Rhodopsin is the archetypal G-protein-coupled receptor (GPCR) with seven transmembrane α-helices [7]. Upon a light stimulus, the covalently bound ligand of rhodopsin, 11-cis retinal, isomerizes to all-trans retinal and activates the receptor. This signal is amplified by the G-protein transducin [8] and activates the cGMP phosphodiesterase (PDE) which hydrolyzes cGMP to GMP. This leads to closure of the cGMP-gated cation channels situated in the plasma membrane of the photoreceptor outer segment (POS) and induces hyperpolarization of the cell [9].

Models for the study of retinal dystrophies have been usually relegated to the use of animal models in mechanistic and preclinical studies [10]. These models have differences in retinal structure and life rhythm, limiting its use. Recently, patient-specific induced pluripotent stem cells (iPSC) have provided a very promising cell source for disease modeling, as they can differentiate into many primary tissues such as the retina. RPE and photoreceptor precursors (PhRPs) have been widely used in the past years [11] but the use of retinal organoids (RO) has become the most widely used model due to their ability to form proper retinal stratification with apical-basal polarity [12] and even possess light responses [13].

Here, we generated PhRP and RO from iPSC derived from a patient affected by autosomal dominant RP and analyzed the effect of its RHO mutation in the development and viability of those models.

Methods

iPSC passage and handling

Donor-provided skin fibroblasts were reprogrammed into pluripotent stem cells (iPSC) as already described [14]. iPS cells were maintained in complete StemFlex Media on Geltrex (Thermofisher Scientific) coated plates. Cells were split on a weekly basis at 1:5 – 1:10 dilutions. 10 µM Rock Inhibitor (EMD Millipore-Merck Group, Bedford, Massachussetts, US) was added after thawing the cells to promote survival. Two wild-type (WT) cell lines (Supplementary Fig. 1) and one line from an RP patient (Cell line: FRIMOi005-A—Patient: Fi24/01) were used. All cell lines were periodically tested during the assays to determine that pluripotency was maintained, and that there were no genetic aberrations. Karyotype was performed at passage 3 to 6 on ten G-banded metaphase cells at 400 band resolution (Reference Lab). Gene analysis by Whole Exome Sequencing (WES) of a panel of the most prevalent RP genes (128 genes) for both Wild-type and RP patient iPSC lines confirmed that there were no pathogenic variants in the control lines and no de novo pathogenic variants. Putative contamination by Mycoplasma was tested with a Mycoplasma Gel Detection Kit (Biotools S&M, Madrid, Spain).

Differentiation of iPSC into photoreceptor precursors

PhRP cells were obtained as described in Barnea-Cramer et.al [15] from low passage iPSC. Briefly, iPSCs were directly induced to differentiation to neural retina for the first 19 days and then they were collected and plated to form neural spheres in a suspension culture (Nunclon Delta low adhesion plates, Thermofisher Scientific). After 3 days, neural spheres were re-plated on matrigel coated surface. The neural spheres were rapidly attached and cultured until day 90 with media without noggin. Finally, cells were further cultured from day 90 to 104 in medium containing retinoic acid and DAPT (Sigma Aldrich, St. Louis, MO), brain-derived neurotrophic factor (BDNF) (Thermofisher Scientific, Waltham, MA) and ciliary neurotrophic factor (CNTF) (StemCell Technologies, Vancouver, Canada) to became rod-like photoreceptors. Sanger sequencing was used to determine that RHO P215L cell line was keeping the specific RHO mutation after iPSC-PhRP differentiation.

Differentiation of iPSC into retinal organoids

Organoids were differentiated from iPS cells using the method described in Gonzalez-Cordero et.al [16]. In brief, cells were grown to 90% confluency and then removed of fibroblast growth factor (FGF) for 2 days, followed by a neural induction period of up to 7 weeks. By 3 or 4 weeks of differentiation optic vesicle-like structures bearing retinal neuroepithelium started to form and between weeks 4 and 7 this neuroepithelium was manually dissected, together with some small amounts of RPE. These vesicles where then grown in suspension culture in the presence of fetal bovine serum (FBS) and taurine (Thermofisher Scientific, Waltham, MA), and retinoic acid (Sigma Aldrich, St. Louis, MO). This late retinal differentiation was carried until week 33, where mature photoreceptors with a clear outer segment were present in the outer layer of the organoid.

Differentiation of iPSC into retinal pigment epithelium

RPE cells were obtained as described in Regent et al. [17] from low passage iPSC. Briefly, cells were incubated in Basal Media (DMEM/F12, 1% Pen/Strep, 1% N2 media supplement, 1% B27 media supplement, 1% non-essential aminoacids (NEAA), and 10% KnockOut Serum Replacement (KSR) with an extra 10% KSR, 50 µM β-Mercaptoethanol, and 10 mM Nicotinamide for days 1–7. Nicotinamide was replaced by 100 ng/mL Activin A for days 8–14 and 3 µM CHIR99021 for days 15–42. After that period of time, cells were passed in two-steps using TrypLe (Thermofisher Scientific) for 15 min to remove undifferentiated cells, which have lower adherence to the flask, and up to 45 min to detach the RPE cells. The cells were cultured for two more weeks in RPE media (DMEM/F12, 4% KSR, 50 µM β-Mercaptoethanol, 1% NEAA) and after two passages were checked for specific RPE markers.

RT-PCR and qPCR

To isolate RNA and synthesize cDNA, iPSC-PhPR cells were lifted with TrypLe (Thermofisher Scientific, Waltham, MA) and rinsed in PBS. RO were pooled in groups of 3–4 individual and similar organoids. Cells were lysed using the SuperScript IV Cells direct Synthesis Kit (Thermofisher Scientific, Waltham, MA) and RT-PCR was performed following Kit instructions. cDNA yield from iPSC-RPE cells was determined using a Qubit 3.0 fluorometer. All gene expression assays were performed with TaqMan fluorescent probes (Thermofisher Scientific, Waltham, MA) paired with FAM or VIC fluorochromes. Forty cycles of PCR using 5–40 ng of input cDNA were performed on an Applied Biosystems QuantStudio 3 qPCR instrument using TaqMan gene expression master mix (Thermofisher Scientific, Waltham, MA) in quadruplicates. GAPDH and ACTB were used as a housekeeping gene.

Immunofluorescence

Immunofluorescence was performed in 12-well Geltrex-coated plates or 8-well Ibidi µ-slides (Ibidi GmbH, Planegg/Martinsried, Germany) with confluent iPSC-PhRP cells. Cells were rinsed 3 times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min., permeabilized with 1% Triton X-100 for 15 min and blocked with FBS 20% + 0.1% Triton X-100 for 1 h. For RO, organoids where rinsed very carefully with PBS and fixed with PFA 4% for 15 min and then dehydrated in 30% sucrose O/N. The next day sucrose was replaced with OCT mounting media (VWR International, Leuven, Belgium) and snap frozen into molds. 14uM cryosections were obtained and mounted into Poly-L-Lysine slides. Every section was rinsed with PBS, permeabilized with 1% triton X-100 for 15 min and blocked with FBS 20% + 0.1% Triton X-100 for 1 h. Primary antibody incubation was performed O/N with 1% FBS at 4 °C. Primary antibodies were tagged with an anti-mouse Alexa-488, anti-mouse Alexa-555, anti-rabbit Alexa-488 or anti-rabbit Alexa-568 secondary antibody (Invitrogen, Waltham, MA) for 1 h at RT. Cell nuclei were stained with DAPI (Thermofisher Scientific, Waltham, MA) for 10 min. A complete list of primary antibodies is included in Supplementary Fig. 4.

Images were obtained either with a ZEISS Axio Vert.A1 or a Zeiss LSM980 confocal microscope (Carl Zeiss Sports Optics, Jena, Germany) and processed and quantified by ImageJ software.

Western blotting

One p6 well of PhPR cells were lysed with Pierce RIPA buffer (Thermofisher Scientific, Waltham, MA) plus Halt Protease Inhibitor (Thermofisher Scientific, Waltham, MA), incubated at 95 °C with loading buffer and β-Mercaptoethanol and loaded in an acrilamide gel (Bio-Rad Laboratories, Hercules, CA). Western Blot was performed with the MiniProteanTetraCell System (Bio-Rad Laboratories, Hercules, CA) following manufacturer instructions and the PVDF membranes were incubated with membrane blocking solution (Life Technologies—Thermofisher Scientific, Waltham, MA) and incubated with primary antibody or anti-tubulin (11224-1AP) rabbit polyclonal (ProteinTech Group, Rosemont, IL) antibody for 6 h at RT or O/N at 4 °C and with goat-anti-rabbit-HRP or goat-anti mouse-HRP IgG secondary antibodies (Invitrogen, Waltham, MA) for 2 h at RT. A complete list of primary antibodies is included in Supplementary Fig. 4. Full-length blots/gels are presented in Supplementary Fig. 5.

Statistics

All experiments were performed at least three times from different biological replicates. Each biological replicate is an iPSC clone differentiated to PhRP or RO from a low cell passage. Results were analyzed with GraphPad Prism. All graphs are normalized, show the standard error of the mean (SEM) and statistical significance is determined using paired t-student tests, One-way or Two-way ANOVA statistical analysis with Tukey’s multiple comparison tests.

Results

Phenotype of the patient carrying a dominant RHO mutation

A 35-year-old subject was diagnosed with non-syndromic Retinitis Pigmentosa (RP) and sensorial exotropia. Autofluorescence, retinography and OCT images showed diffuse pigment spicules in the median periphery along with RPE alteration in the macula indicating rod photoreceptor death (Fig. 1A). The patient carried a RHO pathogenic variant at c.644C > T causing a missense mutation at position 215 (p.Pro215Leu) (Fig. 1B). We generated iPS cells from patient’s fibroblasts [14] and differentiated them into photoreceptor precursors (PhRP), retinal organoids (RO) and retinal pigment epithelium (RPE) (Fig. 1C). RPE are an important part of the retina, their main function being the digestion of the photoreceptor outer segments [18] and we were able to generate mature RPE cells from our patient and the healthy controls (Suppl. Figure 2A-B) but they don’t express RHO (Suppl. Figure 2C). For this reason, we didn’t perform further experiments with this cell model.

Fig. 1
figure 1

A Retinography, autofluorescence retinography and optical coherence tomography (OCT), respectively, of the right eye (OD) and left eye (OS) of Fi24/01 patient. B Sanger sequencing of the c.644 C > T variant in gene RHO of a healthy control and RHO P215L cell line. C Schematic representation of the several processes of iPSC differentiation into retinal cells (Created with Biorender.com)

Full size image

iPSC-derived PhRPs show specific photoreceptor markers

PhRP are immature cells that show specific photoreceptor maturation markers although without generating a complete POS. The process of differentiation is shown in Fig. 2A and involves a total of 104 days. PhRP differentiated from the RP patient (RHO P215L) and two controls (Wild-Type 1 and Wild-Type 2) were able to express all photoreceptor early markers: CRX, OTX2, NRL and RECOVERIN. They also expressed the more mature markers ABCA4 and Arrestin3 and we observed no differences between RHO P215L and control groups (Fig. 2B). Furthermore, they were not expressing the outer segment proteins Opsin L/M, Opsin S or USH2A and neither bipolar markers like Islet1 (data not shown). These PhRP cells expressed the protein Rhodopsin (Fig. 3A), suggesting a rod-dominant population, as already described [15].

Fig. 2
figure 2

A Schematic representation of the differentiation process of iPSC into PhRP and brightfield images of cell cultures. B Immunofluorescence images of wild-type and RHO P215L PhRP expressing precursor markers CRX, NRL, Otx2 and Recoverin and mature photoreceptor markers ABCA4 and Arrestin3

Full size image
Fig. 3
figure 3

A Rhodopsin expression by immunofluorescence in Wild-Type 1, Wild-Type 2 and RHO P215L PhRP cells. Yellow arrows mark rhodopsin accumulation spots. B Rhodopsin colocalization with TGN46 and calreticulin by immunofluorescence. C RHO mRNA expression by Real Time RT-qPCR of WT and RHO P215L PhRP cells. D mRNA expression of several differentiation markers by Real Time RT-qPCR. All qPCR experiments were run with triplicates for every condition and at least three independent experiments were performed. Ordinary One-way ANOVA with Tukey’s multiple comparisons test were used to analyze significance. E Rhodopsin expression by Western Blot and adjusted band intensity (n = 3) compared to the housekeeping marker tubulin. Ordinary One-way ANOVA with Tukey’s multiple comparisons test were used to analyze significance. ns = non-significant *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001

Full size image

Rhodopsin accumulation in PhRPs outside of the Golgi or the ER and increased mRNA and protein expression

Mutations in RHO gene associated with adRP have been classified into seven main classes based on the observed or predicted biochemical and cellular characteristics. Variant P215L, specifically, has been predicted to be in Class 2: Misfolding, ER retention and instability [3, 19], although the particular mechanism has never been demonstrated. We observed in PhRP cells, that rhodopsin localization seems to be perinuclear and cytoplasmatic, with some expression in the cell protrusions. Interestingly, the progenitors from patient RHO P215L showed spikes of accumulated rhodopsin inside the cell cytoplasm (Fig. 3A). This accumulation was localized also in the cell protrusions but was not colocalizing neither with Golgi (TGN46) or the endoplasmic reticulum (Calreticulin) (Fig. 3B).

We also quantified mRNA expression of rhodopsin, several photoreceptor precursor genes and rod and cone-specific genes, and found that RHO P215L PhRP-cells were expressing higher levels of RHO mRNA (Fig. 3C) but normal levels of mRNA from other rod-specific genes like NRL (Fig. 3D). Interestingly, the mature rod marker NR2E3 and the mature PR marker RCVRN were lowered in our patient PhRP compared to controls while there was a marked increase in cone-specific genes (CRX, ARR3 and CNGB3), which were very low or non-expressed in the control lines (Fig. 3D). Finally, rhodopsin protein expression was quantified by Western Blot, showing a statistically significant increase in the patient’s-derived model (Fig. 3E).

iPSC-derived PhRP from the RP patient show increased rhodopsin degradation and increase of autophagy and ER stress markers ATG5 and CHOP

It has been described that the processes that lead to cell death in the photoreceptor cells of RP patients are linked to ER stress and authophagy [20]. To determine the pathological extent of the rhodopsin increase and accumulation in our model we firstly investigated the speed of rhodopsin degradation in PhRP by incubating them at different time points with the protein synthesis inhibitor Cycloheximide (CHX). Results showed that the speed of rhodopsin degradation by the proteasome was increased in RHO P215L PhRP (Fig. 4A). Furthermore, among several ER stress, autophagy and apoptosis markers we observed an increase of DDIT3, ATG5 and ATG7 mRNA and a decrease on PMAIP and HSPA5 (Fig. 4B). This increase was also seen in protein expression of CHOP (the DDIT3 translation product) by Western Blot (Fig. 4C) and by increased nuclear expression by immunofluorescence (Fig. 4D). Another marker of autophagy, SQSTM1 (also known as p62) showed increased mRNA expression and increased protein expression in RHO P215L cells (Fig. 4B–C). The number of ATG5 puncta was also increased in RHO P215L PhRP (Fig. 4D). Other markers (Noxa protein, Grp78, BID or LC3) showed no differences between the patient line and controls (Fig. 4B–D and Suppl. Figure 3A).

Fig. 4
figure 4

A Western blot showing rhodopsin expression in control (Wild-Type 1) and RHO P215L PhRP after exposition with cicloheximide (CHX) (50 µg/mL) for different time points (n = 3). B mRNA expression by Real Time RT-qPCR of several apoptosis, ER stress and autophagy markers. All qPCR experiments were run with triplicates for every condition and at least three independent experiments were performed. Unpaired t-student test was performed to analyze significance between time point 0h and time point 6h: WT1 p = 0.2685; RHO P215L p = 0.0129. C Expression of Rhodopsin, CHOP,LC3A/B and SQSTM1 proteins in PhRP by Western Blot analysis and adjusted band intensity of CHOP and SQSTM1 related to tubulin expression (n = 4). LC3A vs LC3B ratio showed non-significant differences between groups. D Immunofluorescence expression of LC3A/B, Noxa, CHOP and ATG5 in PhRP. LC3A/B and ATG5 puncta were counted in several replicates (n = 10). Yellow arrows mark CHOP accumulation in the cell nuclei and ATG5 puncta. Ordinary One-way ANOVA with Tukey’s multiple comparisons test were used to analyze significance. ns = non-significant *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001

Full size image

Organoids derived from RP patient mature correctly but express different levels of cone and rod photoreceptor markers and show rhodopsin mislocalization

The PhRP model that we used for the previous experiments is a rod-dominant cell model, where the photoreceptor cells were not mature enough to generate a proper POS. To create a more physiologic model, we generated retinal organoids (RO) from the iPSC of RHO P215L and control cell lines. These organoids are formed from the induction of iPSC with Proneural media to obtain neuroretinas which are then manually dissected and cultured in p96 low attachment plates until full maturation at week 33 (Fig. 5A). At w33, RO from both RHO P215L patient and controls show expression of cone and rod markers CRX and NRL, we can distinguish between photoreceptor cells (Arrestin 3) and neural cells (CRALBP), and also distinguish between cone and rode populations by the maturation markers Rhodopsin and Opsin L/M found in the POS of the photoreceptors (Fig. 5B). Rhodopsin expression seems to be increased in the soma of RHO P215L RO (Fig. 5B, C and Suppl. Figure 3B), although this accumulation is not localized in the ER of the rod photoreceptors, marked with Calreticulin (Fig. 5C). Other markers were also properly expressed (Suppl. Figure 3C).

Fig. 5
figure 5

A Schematic representation of the differentiation process of iPSC into RO and brightfield images of individual organoids at different maturation times. B Immunofluorescence images of wild-type and RHO P215L RO expressing CRX, NRL, CRALBP, Arrestin 3, Rhodopsin and Opsin M/L maturation markers. C Immunofluorescence images of wild-type and RHO P215L RO expressing Rhodopsin and calreticulin markers. D Relative mRNA expression by Real Time RT-qPCR of genes involved in RO differentiation. All qPCR experiments were run with triplicates for every condition and at least three independent experiments were performed. Ordinary One-way ANOVA with Tukey’s multiple comparisons test were used to analyze significance. ns = non-significant *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001

Full size image

Compared to Wild-Type RO, organoids from RHO P215L show normal mRNA expression of several immature photoreceptor genes (NRL, AIPL1 and RCVRN) with some markers (NR2E3, CNGB3 and CRX) being increased. On the other side, rod and cone opsin-related genes (RHO, OPN1LW and ARR3) were significantly decreased (Fig. 5D).

Retinal organoids from RHO P215L patient show increased ER stress and autophagy markers

Expression of the ER stress, autophagy and apoptosis markers was also determined in the RO. CHOP expression was clearly increased and localized in the nuclei of RHO P215L RO and the number of ATG5 puncta was significantly superior (Fig. 6A). The mRNA expression of DDIT3, LC3 and ATG7 was also increased, with a more mildly increase on the expression of ATG5 and PMAIP mRNA (Fig. 6B).

Fig. 6
figure 6

A Immunofluorescent images of wild-type and RHO P215L RO expressing CHOP and ATG5 markers. The yellow frame is a zoomed image of ATG5 fluorescence in RHO P215L. Number of ATG puncta per image were quantified on independent organoids (n = 5). B Relative mRNA expression of autophagy and ER stress markers by Real Time RT-qPCR in RO from wild-type and RHO P215L. All qPCR experiments were run in triplicates for every condition and at least three independent experiments were performed. Ordinary One-way ANOVA with Tukey’s multiple comparisons test were used to analyze significance. ns = non-significant *p < 0.05 **p < 0.01 ***p < 0.001 ****p < 0.0001

Full size image

Discussion

Pathogenic variants in the gene RHO have been associated with both dominant and recessive inheritance in RP, although the latter are relatively uncommon. While the genotype–phenotype classification is complicated, on a molecular level RHO pathogenic variants are typically comprised in gain-of-function or dominant-negative [3]. They can also be classified based on their studied chemical and cellular characteristics [3, 21, 22]. Some mutations are related to the capacity of the protein to be translated and transported correctly (alter post Golgi trafficking and POS targeting, induce misfolding, ER retention and instability, show disrupted vesicular traffic and endocytosis, alter post-translational modifications or reduce stability), while others are more related to the signal transduction (altered transducing activation, constitutive activation or dimerization deficiency). While most of the classification of RHO variants has been investigated using overexpression in cell culture model systems like COS-1 and HEK293 cells, we used PhRP and RO differentiated from our patient iPSCs as a more physiological model to determine the effect of the P215L mutation.

In this work, we show that rhodopsin accumulation and mislocalization in photoreceptor cells might be the initiating cause for cell apoptosis. PhRP, although they don’t have a differentiated POS express rhodopsin in considerable quantities, which seems to be accumulated in different spots of the cell cytoplasm. Normally, rhodopsin is translocated into the ER of rod photoreceptors and trafficked to the POS via the Golgi apparatus. During this process, rhodopsin is glycosylated and palmitoylated in different residues, being these modifications essential for the expression, localization and activity of the protein [23, 24]. Several studies have shown that misfolded rhodopsin is retained in the ER, being the P23H mutation the most common of those examples [25,26,27]. As a consequence of this sequestering, the protein is not transported to the cell membrane and is instead degraded by the ubiquitin–proteasome system [28]. Actually, it has been demonstrated that co-expression of adRP-linked opsin folding-deficient mutants along with wild-type opsin resulted in an enhanced degradation through proteasome and steady-state ubiquitination of both mutant and WT opsins [29], showing a clear dominant negative effect that can also be happening with the P215L mutation. But in our assays we didn’t see rhodopsin accumulation neither in ER or Golgi using the specific markers TGN46 and calreticulin in combination with a rhodopsin antibody. This may be because previous in vitro models don’t properly mimic the rhodopsin biogenesis and trafficking of rod photoreceptor cells. It is worth noting that in vivo studies with mice have shown similar results as ours, where the protein didn’t accumulate in the rod photoreceptor cells ER but instead disrupted rod photoreceptor disks [30]. We also observed a decrease in the expression of rod-related genes with an increase of cone-related genes, probably indicating a lower rod proportion regardless of the increased rhodopsin expression. We were able to determine that the ratio of rhodopsin degradation by the proteasome was faster in iPSC-derived PhRP cells of our patient, a process already described with overexpressed R135W mutant rhodopsin [20].

In patients with adRP carrying RHO mutations, the process of cell death is relatively slow, spanning from the 3rd decade of life onward with the initial photoreceptor apoptosis and reaching severe visual impairment and blindness from the 5th decade of life onward [31]. This is a challenge to model using iPSC-derived cells, as the effects of rhodopsin accumulation/misfolding and photoreceptor death might take several years to appear. Retinal organoids are more complex models that include both photoreceptor cells with mature POS and neural cells like bipolar, amacrine and ganglionar cells. These models are more similar to human retina and are more useful for the research of the molecular mechanisms of IRDs, although their development takes from 25 to 33 weeks. They lack non-neural cells such vasculature and microglia and thereby they still do not represent the whole complexities of the disease.

In patient-derived RO we were able to see that differentiation and rod and cone maturation was correct all the way to week 33 and that rhodopsin was correctly expressed in the POS of rods but also there was an increased localization of the protein in the soma of patient’s RO. In the same fashion of what we observed in PhRP, there was no accumulation of rhodopsin in the ER and, surprisingly, we observed a decrease of the mRNA expression of POS-related genes in comparison to other maturation genes. It is worth noting that the differences we see in rhodopsin levels and other specific markers is heavily dependent on the model used. While PhRP differentiation takes less time and generates a rod predominant cell population without POS, RO differentiation is way longer and generates an intermixed population of mature rods and cones in the external layers along with other populations of neural cells. We hypothesize that, considering those cells populations, the PhRP cell culture from RHO P215L patient shifts to generate a population with less rods than the control counterparts. In RO, the increase of autophagy and the ER stress that starts during development generates a less mature population of PR. Sadly, we were unable to confirm an increase of rhodopsin protein and a faster recycling by the proteasome using retinal organoids. It would be interesting to see if the phenotype is maintained considering that mature rods express much more rhodopsin protein, which is stored in the POS.

Some models of RO carrying RHO mutations have been described in the literature: 100 day-old organoids carrying a P171L mutation in RHO showed rhodopsin accumulation in the ER and ER stress-induced apoptosis in rods [32] while 300 day-old RO carrying mono-allelic copy number variation (CNV) mutations in RHO showed photoreceptor dysgenesis with stunted POS, increased RHO mRNA expression and elevated levels and mislocalization of rhodopsin protein within the cell body of rod photoreceptors [33]. An organoid model with the R135W class 3 mutation showed several significant defects in rod photoreceptors: among them, they observed a reduction in rhodopsin protein and mislocalization at day 270. Curiously, they observed an increase on this rhodopsin expression on earlier (D120) organoids, in a similar fashion of what we can see in our 104 day-old PhRP [34]. With the information reported on those studies, it is still not clear if the accumulation of rhodopsin in the ER or in the cell soma is dependent on the cell model, on the differentiation time or it is mutation-specific, and needs further investigation. Other RO models of RP have shown rhodopsin mislocalization even without carrying RHO mutations and otherwise presenting pathogenic variants in the GTPase regulator RPGR [35].

In retinal diseases, early signs of cell damage might appear in the form of autophagy or ER stress [36]. Rhodopsin misfolding and ER retention has been shown to cause ER stress through activating the unfolded protein response (UPR). BiP (binding immunoglobulin protein; encoded by the gene HSPA5) is the hallmark of this event, and has been shown that is increased in several rhodopsin mutants [37,38,39]. Additionally, the increased BiP mRNA levels seen in a P23H mouse model start to decrease when degeneration advances and this change is accompanied by the induction of pro-apoptotic factors, such as CHOP [37]. CHOP has been usually found as a hallmark of the later stages of retinal degeneration [40, 41] but it has been demonstrated that its ablation does not prevent the death of rod photoreceptors [42, 43]. We clearly see this increase in CHOP expression in both our models, which is accompanied by an increase in ATG5 and SQSTM1 and also several other autophagy markers in RO at week 33. Increased autophagy has been described as the late event that comes after ER stress and before cell death in rods with misfolded rhodopsin protein [44], and inhibiting autophagy has been shown to reduce retinal degeneration caused by protein misfolding of the RHO P23H variant [45]. The Atg12-Atg5-Atg16 complex is involved in the maturation of the autophagosomes, along with LC3 and regulated by Atg7 [46], while p62/SQSTM1 acts as an autophagy substrate which helps deliver ubiquitinated proteins to the proteasome for degradation [47]. While we observed an increase of ATG5 and ATG7 in the RHO P215L which would be induced by the necessity of clearance of misfolded rhodopsin, the accumulation of SQSTM1 protein would indicate that the autophagy in our cell model is impaired at a further point of the autophagy process. While our PhRP just show early signs of autophagy defects, RO seem to be more affected as they are mature cell models and have a longer culture time (33 weeks). In Age-related Macular Degeneration (AMD) models, proteasome inhibition in RPE cells caused the accumulation of p62 aggregates, and the silencing of p62 caused the suppression of autophagy [48]. Conditional deletion of Atg5 and Atg7 in mouse RPE induced accumulation of SQSTM1 and markers of oxidative damage, along with a predisposition for AMD [49]. Some studies described that other molecules, like PERK; which attenuates the IRE1/XBP1 pathway, are necessary for the selective autophagy in the ER and would be a good target for the treatment of patients with RHO-associated RP [50]. The mechanisms of autophagy and proteasomal degradation in RP and other retinal diseases should be further studied: some theories support that autophagy is decreased in RP and others that autophagy increases photoreceptor cell death in RP [51]. Probably the overall process changes with the progression of the disease and the changes in the retina vulnerability to ROS and oxidative stress. While signs of cell death are not clearly visible in the RO models that we described, longer culture of these RO could lead to a better mimetization of the late disease phenotype.

Nowadays RP is the most common rod-cone dystrophy, and its genetic variability with more than 120 causative genes and over 5000 mutations (HGMD, 2024 [52]) makes it a difficult disease to be targeted by novel therapies like gene therapy. The knowledge of the molecular mechanisms underlying the pathogenesis of Retinitis Pigmentosa is key for finding an appropriate treatment. For example, the use of gene augmentation is very useful in those retinal dystrophies where pathogenic variants induce a reduction of the protein, whether it is by mRNA degradation or protein misfolding and degradation [53]. This is what happens in patients with Leber Congenital Amaurosis (LCA) caused by RPE65 mutations, in which Luxturna® a virus-associated gene augmentation therapy, has shown incredible results [54]. It won’t be the case in adRP with dominant-negative mutations, like those with rhodopsin protein misfolding, in which the approach would need to block or decrease the expression of the mutant protein by pharmacological means or gene editing therapy like CRISPR/Cas9 [55,56,57]. Cell therapy is the other hope for the treatment of these diseases, and big efforts are being done to achieve replacement of the dead cells in the retina using retinal organoids [58,59,60] with already the first assays of allogeneic iPSC-derived retinal organoid sheet transplantation taken place on RP patients [61].

Conclusions

Retinitis Pigmentosa continues to be the most prevalent inherited retinal dystrophy in the world. The research and development of effective therapies for this incurable disease has been delayed by its clinical and genetic heterogeneity, with more than a hundred different causative genes. We described for the first time the mechanisms underlying a class 2 mutation using photoreceptor precursors and retinal organoids from patient-derived stem cells. These results will be key in the comprehension of the genotype–phenotype correlations of Retinitis Pigmentosa, and the design and generation of personalized therapies for the treatment of these dystrophies.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

adRP:

Autosomal dominant RP

BDNF:

Brain-derived neurotrophic factor

CHX:

Cicloheximide

CTNF:

Ciliary neurotrophic factor

CNV:

Copy-number variation

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

FGF:

Fibroblast growth factor

GPCR:

G-protein coupled receptor

iPSC:

Induced pluripotent stem cell

IRD:

Inherited retinal dystrophies

LCA:

Leber Congenital Amaurosis

OCT:

Optical coherence tomography

PDE:

Phosphodiesterase

PhRP:

Photoreceptor precursor

POS:

Photoreceptor outer segment

PR:

Photoreceptor

RO:

Retinal organoid

RP:

Retinitis Pigmentosa

RPE:

Retinal pigment epithelium

UPR:

Unfolded protein response

References

  1. Sharon D, Kimchi A, Rivolta C. OR2W3 sequence variants are unlikely to cause inherited retinal diseases. Ophthalmic Genet. 2016;37(4):366–8.

    Article  PubMed  CAS  Google Scholar 

  2. Hanany M, Rivolta C, Sharon D. Worldwide carrier frequency and genetic prevalence of autosomal recessive inherited retinal diseases. Proc Natl Acad Sci U S A. 2020;117(5):2710–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Athanasiou D, Aguila M, Bellingham J, Li W, McCulley C, Reeves PJ, et al. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy. Prog Retin Eye Res. 2018;62:1–23.

    Article  PubMed  CAS  Google Scholar 

  4. Tsang SH, Sharma T. Retinitis pigmentosa (non-syndromic). Adv Exp Med Biol. 2018;1085:125–30.

    Article  PubMed  Google Scholar 

  5. Tsang SH, Sharma T. Autosomal dominant retinitis pigmentosa. Adv Exp Med Biol. 2018;1085:69–77.

    Article  PubMed  Google Scholar 

  6. Riera M, Abad-Morales V, Navarro R, Ruiz-Nogales S, Méndez-Vendrell P, Corcostegui B, et al. Expanding the retinal phenotype of RP1: from retinitis pigmentosa to a novel and singular macular dystrophy . Br J Ophthalmol. 2019;bjophthalmol-2018-313672.

  7. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science. 2000;289(5480):739–45.

    Article  PubMed  CAS  Google Scholar 

  8. Gao Y, Hu H, Ramachandran S, Erickson JW, Cerione RA, Skiniotis G. Structures of the rhodopsin-transducin complex: insights into G-protein activation. Mol Cell. 2019;75(4):781-790.e3.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Asano T, Kawamura S, Tachibanaki S. Transducin activates cGMP phosphodiesterase by trapping inhibitory γ subunit freed reversibly from the catalytic subunit in solution. Sci Rep. 2019;9(1):7245.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Gao M-L, Lei X-L, Han F, He K-W, Jin S-Q, Zhang Y-Y, et al. Patient-specific retinal organoids recapitulate disease features of late-onset retinitis pigmentosa. Front cell Dev Biol. 2020;8:128.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Navinés-Ferrer A, Ruiz-Nogales S, Navarro R, Pomares E. Impaired bestrophin channel activity in an iPSC-RPE model of best vitelliform macular dystrophy (BVMD) from an early onset patient carrying the P77S dominant mutation. Int J Mol Sci. 2022;23(13).

  12. Hasegawa Y, Takata N, Okuda S, Kawada M, Eiraku M, Sasai Y. Emergence of dorsal-ventral polarity in ESC-derived retinal tissue. Development. 2016;143(21):3895–906.

    Article  PubMed  CAS  Google Scholar 

  13. Zhong X, Gutierrez C, Xue T, Hampton C, Vergara MN, Cao L-H, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun. 2014;5:4047.

    Article  PubMed  CAS  Google Scholar 

  14. Domingo-Prim J, Riera M, Burés-Jelstrup A, Corcostegui B, Pomares E. Establishment of an induced pluripotent stem cell line (FRIMOi005-A) derived from a retinitis pigmentosa patient carrying a dominant mutation in RHO gene. Stem Cell Res. 2019;38: 101468.

    Article  PubMed  CAS  Google Scholar 

  15. Barnea-Cramer AO, Wang W, Lu S-J, Singh MS, Luo C, Huo H, et al. Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice. Sci Rep. 2016;6:29784.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Gonzalez-Cordero A, Kruczek K, Naeem A, Fernando M, Kloc M, Ribeiro J, et al. Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors. Stem Cell Rep. 2017;9(3):820–37.

    Article  Google Scholar 

  17. Regent F, Morizur L, Lesueur L, Habeler W, Plancheron A, Ben M’Barek K, et al. Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions. Sci Rep. 2019;9(1).

  18. Lakkaraju A, Umapathy A, Tan LX, Daniele L, Philp NJ, Boesze-Battaglia K, et al. The cell biology of the retinal pigment epithelium. Prog Retin Eye Res. 2020;100846.

  19. Mallory DP, Gutierrez E, Pinkevitch M, Klinginsmith C, Comar WD, Roushar FJ, et al. The retinitis pigmentosa-linked mutations in transmembrane helix 5 of rhodopsin disrupt cellular trafficking regardless of oligomerization state. Biochemistry. 2018;57(35):5188–201.

    Article  PubMed  CAS  Google Scholar 

  20. Yu Y, Xia X, Li H, Zhang Y, Zhou X, Jiang H. A new rhodopsin R135W mutation induces endoplasmic reticulum stress and apoptosis in retinal pigment epithelial cells. J Cell Physiol. 2019;234(8):14100–8.

    Article  PubMed  CAS  Google Scholar 

  21. Kaushal S, Khorana HG. Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochemistry. 1994;33(20):6121–8.

    Article  PubMed  CAS  Google Scholar 

  22. Sung CH, Schneider BG, Agarwal N, Papermaster DS, Nathans J. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991;88(19):8840–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Murray AR, Vuong L, Brobst D, Fliesler SJ, Peachey NS, Gorbatyuk MS, et al. Glycosylation of rhodopsin is necessary for its stability and incorporation into photoreceptor outer segment discs. Hum Mol Genet. 2015;24(10):2709–23.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Wang Z, Wen X-H, Ablonczy Z, Crouch RK, Makino CL, Lem J. Enhanced shutoff of phototransduction in transgenic mice expressing palmitoylation-deficient rhodopsin. J Biol Chem. 2005;280(26):24293–300.

    Article  PubMed  CAS  Google Scholar 

  25. Tam BM, Moritz OL. Characterization of rhodopsin P23H-induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa. Investig Ophthalmol Vis Sci. 2006;47(8):3234–41.

    Article  Google Scholar 

  26. Noorwez SM, Malhotra R, McDowell JH, Smith KA, Krebs MP, Kaushal S. Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H. J Biol Chem. 2004;279(16):16278–84.

    Article  PubMed  CAS  Google Scholar 

  27. Noorwez SM, Kuksa V, Imanishi Y, Zhu L, Filipek S, Palczewski K, et al. Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J Biol Chem. 2003;278(16):14442–50.

    Article  PubMed  CAS  Google Scholar 

  28. Illing ME, Rajan RS, Bence NF, Kopito RR. A rhodopsin mutant linked to autosomal dominant retinitis pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome system. J Biol Chem. 2002;277(37):34150–60.

    Article  PubMed  CAS  Google Scholar 

  29. Rajan RS, Kopito RR. Suppression of wild-type rhodopsin maturation by mutants linked to autosomal dominant retinitis pigmentosa. J Biol Chem. 2005;280(2):1284–91.

    Article  PubMed  CAS  Google Scholar 

  30. Sakami S, Maeda T, Bereta G, Okano K, Golczak M, Sumaroka A, et al. Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations. J Biol Chem. 2011;286(12):10551–67.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Nguyen X-T-A, Talib M, van Cauwenbergh C, van Schooneveld MJ, Fiocco M, Wijnholds J, et al. Clinical characteristics and natural history of RHO-associated retinitis pigmentosa: a long-term follow-up study. Retina. 2021;41(1):213–23.

    Article  PubMed  Google Scholar 

  32. Yang Q, Li J, Zeng S, Li Z, Liu X, Li J, et al. Retinal Organoid Models Show Heterozygous Rhodopsin Mutation Favors Endoplasmic Reticulum Stress-Induced Apoptosis in Rods. Stem Cells Dev. 2023;32(21–22):681–92.

    Article  PubMed  CAS  Google Scholar 

  33. Kandoi S, Martinez C, Chen KX, Reddy LVK, Mehine M, Mansfield BC, et al. Disease modeling and pharmacological rescue of autosomal dominant Retinitis Pigmentosa associated with RHO copy number variation. medRxiv: the preprint server for health sciences. United States; 2023.

  34. Lin X, Liu Z-L, Zhang X, Wang W, Huang Z-Q, Sun S-N, et al. Modeling autosomal dominant retinitis pigmentosa by using patient-specific retinal organoids with a class-3 RHO mutation. Exp Eye Res. 2024;241: 109856.

    Article  PubMed  CAS  Google Scholar 

  35. Ma C, Jin K, Jin Z-B. Generation of human patient iPSC-derived retinal organoids to model retinitis pigmentosa. J Vis Exp. 2022;(184).

  36. Song J-Y, Fan B, Che L, Pan Y-R, Zhang S-M, Wang Y, et al. Suppressing endoplasmic reticulum stress-related autophagy attenuates retinal light injury. Aging (Albany NY). 2020;12(16):16579–96.

    Article  PubMed  CAS  Google Scholar 

  37. Lin JH, Li H, Yasumura D, Cohen HR, Zhang C, Panning B, et al. IRE1 signaling affects cell fate during the unfolded protein response. Science. 2007;318(5852):944–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Mendes CS, Levet C, Chatelain G, Dourlen P, Fouillet A, Dichtel-Danjoy M-L, et al. ER stress protects from retinal degeneration. EMBO J. 2009;28(9):1296–307.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Griciuc A, Aron L, Roux MJ, Klein R, Giangrande A, Ueffing M. Inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila. PLoS Genet. 2010;6(8).

  40. Kroeger H, Messah C, Ahern K, Gee J, Joseph V, Matthes MT, et al. Induction of endoplasmic reticulum stress genes, BiP and chop, in genetic and environmental models of retinal degeneration. Investig Ophthalmol Vis Sci. 2012;53(12):7590–9.

    Article  CAS  Google Scholar 

  41. Lenox AR, Bhootada Y, Gorbatyuk O, Fullard R, Gorbatyuk M. Unfolded protein response is activated in aged retinas. Neurosci Lett. 2015;609:30–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Chiang W-C, Joseph V, Yasumura D, Matthes MT, Lewin AS, Gorbatyuk MS, et al. Ablation of Chop transiently enhances photoreceptor survival but does not prevent retinal degeneration in transgenic mice expressing human P23H rhodopsin. Adv Exp Med Biol. 2016;854:185–91.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Choudhury S, Nashine S, Bhootada Y, Kunte MM, Gorbatyuk O, Lewin AS, et al. Modulation of the rate of retinal degeneration in T17M RHO mice by reprogramming the unfolded protein response. Adv Exp Med Biol. 2014;801:455–62.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Kunte MM, Choudhury S, Manheim JF, Shinde VM, Miura M, Chiodo VA, et al. ER stress is involved in T17M rhodopsin-induced retinal degeneration. Investig Ophthalmol Vis Sci. 2012;53(7):3792–800.

    Article  CAS  Google Scholar 

  45. Yao J, Qiu Y, Frontera E, Jia L, Khan NW, Klionsky DJ, et al. Inhibiting autophagy reduces retinal degeneration caused by protein misfolding. Autophagy. 2018;14(7):1226–38.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Mitter SK, Rao HV, Qi X, Cai J, Sugrue A, Dunn WAJ, et al. Autophagy in the retina: a potential role in age-related macular degeneration. Adv Exp Med Biol. 2012;723:83–90.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Liu WJ, Ye L, Huang WF, Guo LJ, Xu ZG, Wu HL, et al. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett. 2016;21:29.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Viiri J, Hyttinen JMT, Ryhänen T, Rilla K, Paimela T, Kuusisto E, et al. p62/sequestosome 1 as a regulator of proteasome inhibitor-induced autophagy in human retinal pigment epithelial cells. Mol Vis. 2010;16:1399–414.

    PubMed  PubMed Central  CAS  Google Scholar 

  49. Zhang Y, Cross SD, Stanton JB, Marmorstein AD, Le YZ, Marmorstein LY. Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of Atg5 or Atg7. Mol Vis. 2017;23:228–41.

    PubMed  PubMed Central  CAS  Google Scholar 

  50. Zhao N, Li N, Wang T. PERK prevents rhodopsin degradation during retinitis pigmentosa by inhibiting IRE1-induced autophagy. J Cell Biol. 2023;222(5).

  51. Moreno M-L, Mérida S, Bosch-Morell F, Miranda M, Villar VM. Autophagy dysfunction and oxidative stress, two related mechanisms implicated in retinitis pigmentosa. Front Physiol. 2018;9:1008.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NST, et al. Human gene mutation database (HGMD): 2003 update. Hum Mutat. 2003;21(6):577–81.

    Article  PubMed  CAS  Google Scholar 

  53. Xi Z, Vats A, Sahel J-A, Chen Y, Byrne LC. Gene augmentation prevents retinal degeneration in a CRISPR/Cas9-based mouse model of PRPF31 retinitis pigmentosa. Nat Commun. 2022;13(1):7695.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Maguire AM, Bennett J, Aleman EM, Leroy BP, Aleman TS. Clinical perspective: treating RPE65-associated retinal dystrophy. Mol Ther. 2021;29(2):442–63.

    Article  PubMed  CAS  Google Scholar 

  55. McNamee SM, Chan NP, Akula M, Avola MO, Whalen M, Nystuen K, et al. Preclinical dose response study shows NR2E3 can attenuate retinal degeneration in the retinitis pigmentosa mouse model Rho(P23H+/)(). Gene Ther. 2024

  56. Burnight ER, Wiley LA, Mullin NK, Adur MK, Lang MJ, Cranston CM, et al. CRISPRi-mediated treatment of dominant rhodopsin-associated retinitis pigmentosa. Cris J. 2023;6(6):502–13.

    Article  CAS  Google Scholar 

  57. Du W, Li J, Tang X, Yu W, Zhao M. CRISPR/SaCas9-based gene editing rescues photoreceptor degeneration throughout a rhodopsin-associated autosomal dominant retinitis pigmentosa mouse model. Exp Biol Med (Maywood). 2023;248(20):1818–28.

    Article  PubMed  CAS  Google Scholar 

  58. Iwama Y, Nomaru H, Masuda T, Kawamura Y, Matsumura M, Murata Y, et al. Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies. Stem Cell Rep. 2023

  59. Cooke JA, Voigt AP, Collingwood MA, Stone NE, Whitmore SS, DeLuca AP, et al. Propensity of patient-derived iPSCs for retinal differentiation: implications for autologous cell replacement. Stem Cells Transl Med. 2023;12(6):365–78.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Watari K, Yamasaki S, Tu H-Y, Shikamura M, Kamei T, Adachi H, et al. Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy. Commun Biol. 2023;6(1):164.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Hirami Y, Mandai M, Sugita S, Maeda A, Maeda T, Yamamoto M, et al. Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa. Cell Stem Cell. 2023;30(12):1585-1596.e6.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank the patients for the participation in this study. We are indebted to the service of advanced optical microscopy UAT-Vall d’Hebron for technical support.

Artificial Intelligence (AI)

The authors declare that they have not used AI-generated work in this manuscript.

Funding

This work was supported by a private donation (grant number Fi-201401) to Fundació de Recerca de l'Institut de Microcirurgia Ocular Barcelona, Spain.

Author information

Authors and Affiliations

  1. Departament de Genètica, IMO Grupo Miranza, Barcelona, Spain

    Arnau Navinés-Ferrer & Esther Pomares

Authors
  1. Arnau Navinés-Ferrer
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Esther Pomares
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

ANF: Conception and design, acquisition, analysis and interpretation of data. Manuscript preparation and revision. EP: Conception and design. Manuscript revision, project administration and funding. Both authors have read and agreed to the submitted version of the manuscript.

Corresponding author

Correspondence to Esther Pomares.

Ethics declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Institut de Microcirurgia Ocular (Approved Project: “Generación de células iPS a partir de biopsias de piel de pacientes afectados de distrofias de la retina”—“Generation of iPS cells from skin biopsies of retinal dystrophy patients”. Protocol code: 170505_117. Date of approval: 2 of June—2017).

Consent for publication

Informed consent was obtained from all subjects involved in the study.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Navinés-Ferrer, A., Pomares, E. Endoplasmic reticulum stress and rhodopsin accumulation in an organoid model of Retinitis Pigmentosa carrying a RHO pathogenic variant. Stem Cell Res Ther 16, 71 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04199-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04199-4

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512