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Molecular pathology of Usher 1B patient-derived retinal organoids at single cell resolution

Summary Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL+ rods and apoptotic signaling pathways in VIM+ Müller cells, pointing to the defensive mechanisms that mitigate photoreceptor cell death. This first human model for USH1B-RP provides a representation of patient retina in vivo relevant for development of therapeutic strategies.


INTRODUCTION
Usher syndrome (USH) is an autosomal-recessive hereditary disease that causes sensorineural hearing loss, vestibular dysfunction, and retinitis pigmentosa (RP). It is the most common hereditary disease that causes deaf-blindness, affecting 1 to 4 in 25,000 newborns (Parker and Bitner-Glindzicz, 2015). Three major USH clinical types (I-III) have been identified. USH1 is the most severe causing profound congenital hearing loss, RP beginning in the first decade of life, and balance issues. RP is characterized by progressive loss of rod photoreceptors in the retina, resulting in night blindness and tunnel vision, followed by cone photoreceptor loss, affecting central vision and eventually leading to legal blindness (Bonnet and El-Amraoui, 2012). MYO7A gene mutation (USH1B subtype) accounts for 40%-55% of all USH1 cases, with >2,000 diseasecausing variants reported (Whatley et al., 2020). Although hearing loss can be effectively compensated for with cochlear implants, no treatments are currently available for USH1B-RP.
MYO7A is a class 7 myosin protein with an N-terminal motor domain and distinct properties . Like other members of the myosin superfamily, it binds actin filaments triggering ATP hydrolysis to energise its translocation. No faithful animal or human cell models of USH1B-associated RP are available to date. Myo7a mutant mouse models, such as shaker1, recapitulate hearing loss and vestibular dysfunction but not the USH1B retinal phenotype (Gibson et al., 1995). Studies using shaker1 identified rhodopsin mislocalization and showed MYO7A in the connecting cilium along the axonemal actin, where it facilitates rhodopsin transport from the photoreceptor inner segment (IS) to the outer segment (OS) (Liu et al., 1999). The same model showed the absence of MYO7A disrupted melanosome translocation to the apical processes of retinal pigment epithelium (RPE) cells and OS phagocytosis (Gibbs et al., 2003). Soni et al. (2005) demonstrated disrupted interaction of MYO7A with lysosomes in a human RPE cell line. In addition, contrasting findings of RPE resistance (Lopes et al., 2011) and rod vulnerability (Peng et al., 2011) to light exposure were reported in shaker1.
Subsequent studies in humans, non-human primates, and amphibians showed MYO7A and other USH1 proteins form a linkage between the OS and calyceal processes (CP), the actin filament-filled extensions sprouting from the IS to surround the base of OS (Sahly et al., 2012). Knockout of protocadherin-15 (USH1F) and cadherin-23 (USH1D) in frogs compromised the CP structural support, causing distortion of the OS shape (Schietroma et al., 2017). Notably, Myo7a knockout frogs acquired developmental defects hampering further study. The lack of CP in the mouse retina plus other differences compared with the human eye, such as eye size, high rod-to-cone ratio, and the absence of a macula could explain the absence of a USH1B retinal phenotype in mouse models. A study in dogs also failed to observe retinal phenotypes (Webb et al., 2019), while vestibular dysfunction and deafness were present. Pigs with MYO7A mutation showed balance difficulties and suspected hearing loss, but retinal phenotypes were not studied (Derks et al., 2021). Like the frog model, piglets with a homozygous nonsense MYO7A mutation died soon after birth.
In addition to the lack of faithful animal models, there is a wider lack of understanding of the molecular pathways leading to photoreceptor cell death that are triggered by the disease-causing mutations. Modeling USH1B with patient-derived induced pluripotent stem cells (iPSCs) offers an alternative route with potential to understand the early consequences of MYO7A mutation in human retinal cells. Tang et al. (2016) reported the first human USH1B cochlear hair cell model derived from iPSCs of a patient suffering sensorineural hearing loss (non-syndromic USH1B), but retinal cells have not previously been studied. Rod photoreceptor cells are highly metabolically active requiring high oxygen consumption. In RP, elevated oxidative stress is thought to accelerate rod photoreceptor cell loss and secondary cone photoreceptor death. The sensitivity of photopigments to light stress and the high turnover of OS contribute to the production and accumulation of reactive oxygen species (ROS), leading to lipid, protein and DNA damage in animal models (Punzo et al., 2012;Gallenga et al., 2021). In addition, antioxidants slow photoreceptor cell death in animal models (Komeima et al., 2006). Increased markers of oxidative stress, including elevation of endoplasmic reticulum (ER) stress markers, have been reported in non-USH1 RP patient iPSC-derived retinal cells grown in two-dimensional (2D) cultures (Jin et al., 2011(Jin et al., , 2012Yoshida et al., 2014) or in organoids (Tucker et al., 2013).
Here, we report the first three-dimensional (3D) retinal organoid model of USH1B generated from patient-derived iPSCs. The iPSC-derived organoids did not show evidence of cell degeneration in culture at fetal retina-equivalent stage, consistent with the onset of USH1B in childhood. However, bulk RNA sequencing (RNA-seq) and Gene Ontology (GO) enrichment analysis revealed increased aberrantly expressed genes in patient-derived cells over time. Single-cell RNA sequencing (scRNA-seq) analysis of 35 week organoids identified adaptive responses to stress specifically in USH1B rods, and enrichment for apoptotic signaling pathways in USH1B Müller cells. Together these findings indicate the presence of a mutation-induced molecular pathology in patient-derived retinal cells and provide evidence of increased adaptive responses in patient cells that maintain cellular homeostasis and prevent photoceptor cell death in the in vitro organoid system.
Generation of 3D retinal organoids from control and USH1B iPSCs USH1B and control iPSCs were differentiated to 3D retinal organoids in vitro using a published protocol (Cuevas et al., 2021;Gonzalez-Cordero et al., 2017) (Figure 1A). By 21 weeks, organoids contained stratified neuroepithelium with multiple cell layers that were characterized using IHC ( Figure 1B). Photoreceptors in the outer nuclear layer (ONL) stained for RCVRN. The inner nuclear layer (INL) stained for VSX2, marking bipolar cells (BCs), and the ganglion cell layer (GCL) was identified by BRN3A. Müller cells  Table S2 for full protein names). Representative examples shown from N > 3 differentiations per iPSC line, n > 3 organoids per differentiation. RPE, retinal pigment epithelium; NE, neural epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; OLM, outer limiting membrane. (D) Transmission electron micrographs show the presence of photoreceptor subcellular structures and separating photoreceptor cell body and segments in both control and patient 21 week retinal organoids. N = 2 differentiations per iPSC line, n > 2 organoids per differentiation.

Absence of RP degenerative features in USH1B retinal organoids
To determine if USH1B organoids reproduce degenerative phenotypes in patients, first, we compared the size of USH1B and control organoids from 7 to 19 weeks. Using 7 weeks as a baseline we found that although control and USH1B organoid size changed at variable rates, no USH1B-specific abnormality was observed ( Figure 2A). To assess whether USH1B organoids lost photoreceptors to degeneration by 21 weeks, we determined the level of photoreceptor marker gene expression and quantified the thickness of the ONL (where photoreceptor nuclei reside). No significant differences were observed in expression of CRX, RCVRN, NRL, ARR3 and RHO ( Figure 2B). Similarly, we found no aberrant ONL thinning in USH1B organoids, suggesting excessive photoreceptor death was not present ( Figures 2C and 2C 0 ).
Reactive gliosis is a secondary event to photoreceptor loss in RP, characterized by overexpression of glial fibrillary acidic protein (GFAP) and the disruption of OLM integrity. It was reported in iPSC-derived retinal organoids from RP patients harboring RPGR mutation (Deng et al., 2018) and in an Rpgr À/À mouse model (Megaw et al., 2017). We quantified GFAP protein levels using western blot and distribution using IHC of 21 week organoids (Figures 2E and 2E 0 ). One patient line (USH1B.3) showed a significantly higher level of GFAP. However, IHC revealed restricted expression of GFAP within inner retina (at the pseudo-GCL and occasionally INL), resembling the pattern in healthy mouse retina (Hippert et al., 2021), in both control and USH1B organoids (including USH1B.3). Overall, USH1B patient organoids did not display marked characteristics of degeneration, photoreceptor cell death, oxidative stress or reactive gliosis.
RNA sequencing analysis showed resemblance of latestage retinal organoids to fetal retina To investigate the development of retinal organoids and the ''closeness'' of their global transcriptomes to native retina, we performed bulk RNA sequencing analysis on control and USH1B iPSCs (day 0), early-stage (7 weeks), mid-stage Figure 2. Comparison of USH1B and control retinal organoids to identify USH1B-associated degenerative features (A) Top: bright-field images shows organoid growth; images from Control.1. Changes in organoid size over time relative to 7 weeks. Independent-samples t test was performed at each time point. *p < 0.001, control > USH1B when in blue, USH1B > control when in red. (B) qRT-PCR analysis of photoreceptor marker expression by 21 week patient and control organoids. One-way ANOVA with Dunnett correction. Fold change relative to GAPDH expression and normalized to Control.3. N = 3, n = 2-3. (C and C 0 ) Quantification of ONL thickness in 21 week USH1B and control organoids. Representative images show one field of view. IHC for ARR3 defined the ONL. N = 2, n = 2. Data points are measurements from one field of view. Independent-samples t test. (14 weeks), and late-stage (28 weeks) organoids, and human 20 weeks post-conception fetal and adult retina ( Figure 3A). Principal-component analysis (PCA) revealed close correlation of organoids according to their respective time of differentiation ( Figure 3B). Day 0 iPSCs clustered furthest from the fetal and adult retina along PC1, followed by 7 and 14 weeks, indicating the progression of retinal differentiation. All 28 weeks, organoids clustered closer to fetal than to adult retina ( Figure 3C). Pluripotency gene expression showed a drastic downregulation in 7, 14, and 28 weeks organoids compared with day 0 iPSCs, indicating loss of the pluripotency state ( Figure 3D). The expression of welldefined photoreceptor markers was upregulated starting at 14 weeks and further elevated at 28 weeks to resemble the level of human retina ( Figure 3E).
A gradual increase of expression was also observed in marker genes of other retinal cell types, including BCs, horizontal cells (HCs), ACs, and MCs, in the organoids (Figure 3F). Decreased RBPMS expression over time signaled diminishing RGC number, consistent with the trajectory in human retina and other retinal organoid studies (Cowan et al., 2020). Varying levels of BEST1 and RPE65 were due to the sporadic presence of RPE. Analysis of MYO7A expression at different stages showed levels increased concurrently with retinal maturation but were significantly lower than human retina ( Figure 3G).
To more accurately map the 28 weeks organoids to an in vivo developmental stage, we compared the 28 weeks organoids with a published bulk RNA-seq dataset of human embryonic eye, fetal, and infant retina (Table S3) (Mellough et al., 2019). Figure S3 shows that the 28 weeks organoids were more mature than embryonic eye and were similar to fetal/infant retina with respect to retinal cell marker expression. MYO7A expression showed a similar upward trend across in vivo developmental stages ( Figure 3H), with higher levels in 28 weeks organoids compared with embryonic eyes. As difference in MYO7A mRNA level was not apparent between USH1B and control organoids, we sought to investi-gate MYO7A protein levels in order to validate the predicted effect of MYO7A mutations on protein synthesis. Immunoblotting of 21 weeks organoids (Figures 3I and 3I 0 ) with an antibody (DSHB 138-1) that targets amino acids 927-1203 of MYO7A should detect products from both alleles (if translated; p.Asp75His, p.Arg2024*) of USH1B.2 and USH1B.3 but only one allele (splice site mutation c.133-2A>G) of USH1B.1 ( Figure S3). Residual MYO7A expression (fold change 0.30 ± 0.05) was detected in USH1B.2 and USH1B.3 by the p.Asp75His allele, while mRNA products from the p.Arg2024* allele were not detected suggesting nonsense-mediated mRNA decay. The USH1B.1 allele did not give rise to detectable protein.
To summarize, we showed that USH1B and control organoids developed and matured to acquire global transcriptomes at 28 weeks, largely similar to fetal retina. Profiles of expression of retinal cell markers were similar to adult retina suggesting completion of retinal histogenesis. Considering the onset of USH1B in childhood, the similarity to fetal retina is consistent with the absence of signs of photoreceptor cell degeneration in the USH1B organoids.

RNA-seq analysis revealed dysregulated GO terms in mid-and late-stage USH1B retinal organoids
To identify mutation-induced transcriptomic defects in USH1B organoids, we performed differential expression (DE) analysis comparing the transcriptomes of control and USH1B organoids (control versus USH1B). A total of 803, 1,866, 3,999, and 9,184 differentially expressed genes (DEGs) (adjusted p value < 0.05) were identified at day 0, 7 weeks, 14 weeks, and 28 weeks, respectively ( Figure 4A; Table S4), suggesting that control and USH1B organoids became more different over time, coinciding with the rise of MYO7A expression. Indeed, PCA and Pearson's correlation coefficient showed clear separation of control and USH1B 28 week organoids ( Figures 4B and 4C). Considering RP is progressive, we hypothesised that aberrant transcriptional patterns, if present, should persist, if not increase, at later time points. Therefore, to investigate the onset of molecular pathology associated with MYO7A mutation preceding degeneration, we performed GO enrichment analysis on DEGs shared across (1) all four time points (110 DEGs), (2) 7 weeks onward (280 DEGs), (3) 14 week onward (1,579 DEGs), and (4) unique DEGs for 28 weeks (6,491 DEGs), hereafter referred to as shared DEGs-1 to DEGs-3 and 28 weeks unique DEGs ( Figure 4A; Table S4). The top five DEGs for each group are shown in Figure 4D (Table S4 indicates links of these top five genes to retina and/or neurodegeneration).
No GO enrichment (q < 0.05) was identified in shared DEGs-1 and shared DEGs-2 (Table S4). Shared DEGs-3 showed enrichment for biological process terms chiefly relating to mitochondria functions, Wnt signaling pathway, protein localization, and cell cycle ( Figure 4E). DEGs unique to 28 weeks were enriched for protein ubiquitination, RNA splicing, cellular transport, and organelle localization ( Figure 4F; Table S4). Both the latter are relevant to the current understandings of MYO7A function. Consistently, GO enrichment analysis of molecular function terms identified similar findings as shown in Table S4. Ubiquitination is a critical pathway controlling proteosome homeostasis and regulating degradation of damaged or misfolded proteins, and its dysfunction is associated with a range of neurodegenerative diseases that affect the retina (Campello et al., 2013). Figure S4 shows the set of genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) ubiquitin-mediated proteolysis pathway that are dysregulated in the 28 weeks organoids.
In summary, through bulk RNA-seq analysis, we identified dysregulated genes in USH1B organoids, many of which have implications in retinal dystrophy or other neurodegenerative diseases. These aberrant transcriptomic patterns represent clues for the dissection of USH1B disease mechanisms and serve as potential early disease biomarkers.
Single-cell RNA sequencing showed the expression of Usher genes at a single-cell level We next sought to elucidate the site of action of MYO7A by establishing a cell atlas of USH1B organoids. The 35 weeks USH1B and control organoids were subjected to single-cell RNA sequencing. The 35 weeks time point was chosen because a recent study showed that organoids acquired a stable ''developed state'' between 30 and 38 weeks and did not mature further (Cowan et al., 2020). The RNA of 19,399 and 20,535 single cells from control and USH1B, respectively, was sequenced. Dimension reduction of the 39,934 single-cell transcriptomes with uniform manifold approximation and projection (UMAP) identified 25 clusters, encompassing 7 major retinal cell types, including rods, cones, BCs, ACs, HCs, MCs, and RPE ( Figure 5A), assigned on the basis of the expression of well-defined markers ( Figure 5B, Table S5). Further analyses showed the presence of cell subtypes, including rod subtypes differentiated by the expression of MYO9A, and BC and HC subtypes (Yi et al., 2021) (Figure S5). Each of the 25 clusters was well represented in the six organoid samples ( Figure 5C).
Next, we mapped the expression of all USH-causative genes to organoids ( Figure 5D). For USH1 genes, MYO7A, USH1G, USH1C, and CDH23 were highly expressed by MCs (cluster 1), while CIB2 was ubiquitously expressed and PCDH15 specific to photoreceptors. For USH2 genes, high USH2A expression was detected in photoreceptors, while ADGRV1 and WHRN were detected in all cell types. Last, USH3 gene CLRN1 was highly expressed in MCs and HARS was ubiquitously expressed. To further determine identities of cells expressing MYO7A, we performed dimension reduction on a total of 756 MYO7A-expressing cells ( Figures 5E and 5F; Table S5). Four distinct cell clusters were identified expressing top markers indicative of MCs, BCs, rods, and cones ( Figure 5G; Table S5). Despite previous MYO7A studies showing exclusive expression by RPE and photoreceptors, in particular rods, the expression of MYO7A by MCs reported in this study is consistent with a recent scRNA-seq analysis of human retina (Cowan et al., 2020). These findings indicate MYO7A could play roles in other cell types, which are undocumented and could contribute to pathology.
Single-cell DE analysis identified early signs of degeneration specific to rods and upregulation of apoptotic pathways in MC Finally, we sought to identify molecular defects specific to individual retinal cell types. Degeneration of rods is the primary clinical event in RP, hence we examined directly if rods show evidence of altered transcriptomes. NRL is an essential rod-specific transcription factor. We compared the transcriptomes of 3,072 and 2,325 NRL-expressing (NRL + ) rods from control and USH1B organoids, respectively. All cells were found in clusters 0, 2 and 4, previously identified as rods ( Figure 6A; Table S5). DE analysis identified 207 DEGs (Table S5; genes with links to retina and/or neurodegeneration are indicated). GO enrichment analysis identified biological process terms associated with cellular responses to chemical and oxidative stress as well as hydrogen peroxide metabolic process (Figures 6B and 6C; Table S5). Genes encoding pro-apoptotic factor (BNIP3), antioxidant enzymes (PRDX1, PRDX2, and PRDX5), and free radical scavenging enzyme (SOD1) were overexpressed in USH1B NRL + cells, suggesting ongoing adaptive responses to cell stress ( Figure 6C). The same events were not detected when analyzing 3,013 ARR3 + (cone arrestin) cones, suggesting that molecular pathology was limited to rods (Figures S6A-S6C; Table S5).
In view of the new interest in MCs as a MYO7A-expressing cell type, we performed the same analysis on a total of 5,207 vimentin (VIM + ) MCs (2,392 control and 2,815 USH1B) (Figures 6D-6F; Table S5). DE analysis identified 255 DEGs, which were enriched for biological process terms for regulation of apoptotic signaling pathways (Table S5). Last, we analyzed 2,277 BCs identified by expression of GRIK1 (1,136 cells) and PCP2 (1,141 cells) (Figures S6D-S6F). A total of 157 DEGs were identified, which were not enriched in either apoptotic-or stress response-related GO terms (Table S5). We noted that the abundant presence of ribosomal genes and ribosomerelated GO terms in the ARR3 + and PCP2 + /GRIK1 + cell-specific GO enrichment analyses might represent technical artifacts previously reported as common to scRNA-seq (Nowakowski et al., 2017).
In summary, we identified stress responses in rods, but not cones, consistent with clinical observations that the former is first to degenerate in USH1B-RP. Analysis of VIM + MCs showed USH1B MC exhibited apoptotic pathway-related transcription activities. These results suggest our organoid model reproduces a pre-death mutation-induced state of rod photoreceptors in vitro. It is also instrumental for further investigation into the vulnerability of MC.

DISCUSSION
By coupling iPSC technology and in vitro retinal differentiation protocols, we established the first retinal organoid disease model for USH1B. Bulk RNA-seq analysis revealed the transcriptomes of USH1B patient organoids were progressively distinct from controls after extended time in culture, coinciding with the gradual rise of MYO7A expression. Dysregulation of genes associated with mitochondrial function were followed by genes enriched for protein ubiquitination, RNA splicing, and cellular transport functions.
Elevated cellular responses to stress were detected exclusively in USH1B rods, suggesting organoids were modeling a molecular pathology underlying early RP manifestations. This is the first such analysis reported. By identifying mutation-induced altered transcriptomes in patient-derived retinal cells that predispose rods to cell stress, this study provides the first clues into molecular mechanisms underlying USH1B-RP.
Limitations of USH1B patient-derived 3D retinal organoid model Recent studies comparing retinal organoids with human retina suggested in vitro differentiation recapitulates the rate of retina development in vivo (Cowan et al., 2020). Consistently, bulk and scRNA-seq as well as mapping of the 11 USH genes to specific retinal cell types demonstrated the faithfulness of our organoid model as a surrogate for USH1B disease modeling. Despite these similarities, the level of MYO7A expression in 28 weeks control organoids was lower than that of the adult retina. This may be due to a reduction or absence of cellular activities involving MYO7A in the organoids. For example, the absence of juxtaposition of RPE to photoreceptors precludes OS phagocytosis. In addition, immature electrophysiological responses commonly reported in retinal organoids suggest attenuated phototransduction, possibly due to reduced protein trafficking and lack of the visual cycle (Hallam et al., 2018). Last, a lack of development of CP could abolish the need for USH1 protein linkage formation between OS and CP. The lack of retinal vasculature, blood supply and resident microglia in the organoid model also limits modeling of mutation-induced inflammatory responses, which may be important for understanding the notable identification of dysregulation of genes associated with neutrophil activation pathways in the USH1B rods and MC in this study (Table S5). In summary, although organoids provide valuable new insights into retinal disease pathology, further improvement on complexity and functionality of the model could improve representation of disease conditions in vivo.  Table S5.

Retinal cell types expressing MYO7A
Previous studies of USH1B focused entirely on the role of MYO7A in rods and the RPE on the basis of the clinical phenotype and distribution pattern of MYO7A in animal models. Analysis of MYO7A + cells in this study identified the majority of them as MCs, followed by BCs, rods, and cones. This raises the question of why, when MYO7A is mutated, rods are the first cell type to degenerate, which is typical of all RP clinically. Significantly, our scRNA-seq data showed heightened defensive responses against stress exclusively by rods, modeling their clinical susceptibility.
In addition, we found activation of apoptotic signaling pathways in USH1B patient MCs. MC death has not been widely studied or considered a primary event in RP. However, OCT examination of USH1B patient retina identified increased OLM visibility likely attributed to MC response to nearby photoreceptor cell stress or death (Jacobson et al., 2009). The same study also attributes OPL and INL thickening in USH1B patient retina to MC hypertrophy or hyperreactivity. Interrogating the role of MYO7A in the various cell types in which it is expressed would be important to further understand USH1B disease mechanism. To date we have not been able to confirm MYO7A protein localization in MCs using IHC with three different antibodies (Table S2) (data not shown), suggesting posttranscriptional control mechanisms and/or low or transient protein.
Dysregulated GO terms over time: Potential disease markers of mutation-induced state Over time in culture, USH1B organoids displayed increased numbers of DEGs associated with the ubiquitin-proteosome system (UPS), which is known to function in response to stress. Notably we found TOPORS and UBR1 dysregulated in the USH1B organoids (GO:0010498; Figure 4F; Table S4); mutation of the former is a cause of autosomal dominant RP (Chakarova et al., 2007), and the latter promotes rhodopsin protein degradation in the OS via the UPS during retinal inflammation . Altered protein trafficking of rhodopsin with a P23H mutation overwhelms the proteasome machinery due to UPS inefficiency in degrading the variant, leading to malfunction in various cellular pathways (Illing et al., 2002). The 28 weeks DEGs were also enriched for RNA splicing (GO:0008380). More than 25% of retinal disease-causing mutations alter splicing patterns (Bacchi et al., 2014). Mutation of the ubiquitously expressed pre-mRNA processing factors (PRPFs) accounts for 15% of autosomal-dominant RP (Wang et al., 2019). Several members of PRPFs (PRPF18, PRPF31 and PRPF39) were dysregulated in the USH1B organoids (Table S4). A recent study showed that USH1G (SANS) interacts with PRPFs to facilitate the transport of small nuclear ribonucleoprotein during pre-RNA splicing (Yildirim et al., 2021). PRPF31 mutation causes autosomal-dominant RP (RP11) and adversely affects primary cilia formation in photoreceptors and RPE function (Buskin et al., 2018). Last, with regard to cellular transport, KIF3A and KIF3B (a RP gene) along with other members of the kinesin family (GO:0048193) as well as components of the intraflagellar transport system (GO:0030705), including IFT20 were dysregulated in USH1B organoids.

Antioxidant enzymes differentially expressed between the control and patient retinal cells
Single-cell analysis of USH1B patient cells showed elevation of defensive cellular responses exclusively in rods. One interpretation of these findings is that patient cells were more sensitive to cellular stress compared with controls. Antioxidant enzymes including members from the peroxiredoxins (PRDXs) and superoxide dismutase (SOD) families were upregulated in rods. PRDX isoforms have previously been reported in different retinal cell types and organelles (Chidlow et al., 2016) and implicated in neurodegenerative and retinal diseases (Zheng et al., 2012;Kubo et al., 2009;Kim et al., 2001). However, PRDXs are not known to be expressed in rods under normal physiological conditions, and the absence of H 2 O 2 scavenging PRDXs may contribute to susceptibility of rods to degeneration. Sod1 À/À mouse retina showed mitochondrial degeneration in both INL and ONL and AMD-like phenotypes (Hashizume et al., 2008). BNIP3 was another notable dysregulated defensive gene identified as part of the cellular response to oxidative stress in NRL + rods in patient organoids (GO:0034599; Figure 6C). BNIP3 encodes a pro-apoptotic factor that interacts with anti-apoptotic proteins. A HIF-1a/BNIP3 pathway is neuroprotective against inner retinal neurodegeneration (Kunimi et al., 2021), whereas overexpression of BNIP3/ FUNDC1 signaling reversed cell stress and death caused by HIF-1a inhibition in an hypoxic retinal model .
Cellular responses in rods against stress: One-hit model A cumulative damage model is often used to describe cell death in neurodegenerative disorders, whereby gene mutation causes the accumulation of secondary damage in cells, increasing the likelihood of a cell dying over time. In contrast, the one-hit model described by Clarke et al. (2000) proposes that the risk of a cell's dying remains constant. Cells harboring gene mutations acquire a nearly normal homeostatic state called the mutant steady state (MSS), whereby a critical, but not lethal, alteration of gene activity that regulates pre-death reactions is present. Initiation of cell death only occurs when rare and random catastrophic events cause a surge of pre-death molecules to pass a critical cell-death threshold.
The altered transcriptomes of USH1B cells are consistent with an MSS being established in organoids. The enrichment of cell death-related pathways and cellular stress pathways (pre-death markers) may reflect the deployment of stress-mitigating enzymes to maintain the MSS and suppress cells from reaching the cell-death threshold. We speculate that the ample supply of nutrients in vitro and minimal exposure to light damage may limit the occurrence of catastrophic events leading to significant levels of cell death. The introduction of various stressors that an individual would normally experience in life, including exposure to natural light, free radicals, and reactive oxygen species, into the organoid system may better mimic the aging USH1B degenerative disease state.

EXPERIMENTAL PROCEDURES
Additional details are provided in the supplemental information.

iPSC generation
The study was approved by the National Research Ethics Committee London-Dulwich (11/LO/1243). Fibroblasts from USH1B patients and controls were reprogrammed to iPSC using Sendai virus. Details of mutations are given in Figure S1.

Retinal organoid differentiation and analysis
iPSCs were grown to 90% confluency before switching to embryoid body (EB) medium on day 0, neural induction medium (NIM) on day 2, followed by retinal differentiation medium (RDM) containing various supplements (RDM1-3) for retinal maturation and long-term culture of organoids. EB, NIM, RDM, and RDM1-3 are described in Table S1. Organoids were analyzed using RT-PCR, transmission electron microscopy (TEM), and western blot as described in supplemental information.

Immunohistochemistry
Retinal organoids were fixed with 4% paraformaldehyde (PFA) solution and cryosections of frozen organoids analyzed using immunohistochemistry. Primary and secondary antibodies are listed in the Table S2.
Bulk RNA sequencing RNA (100 ng) from organoids was processed using the KAPA mRNA HyperÀPrep Kit (KK8580; Roche) and libraries sequenced on a S1 flow cell on a NovaSeq 6000 system (Illumina). FASTQ files from 20 million reads per sample were aligned to the human genome UCSC hg38 using RNA-STAR 2.5.2b. Differential expression analysis was performed using limma-voom with cut-off of adjusted p value < 0.05 for statistical significance. Package clusterProfiler (version 3.0.4) was used for GO enrichment analysis.

Single-cell RNA sequencing
Single-cell RNA libraries were generated using the Chromium Single Cell 3ʹ Reagent Kits version 3 (10X Genomics) and analyzed on an Illumina sequencing platform with 10X Genomics Cell Ranger software to generate digital gene expression matrices. Data from 39,934 cells were analyzed using Seurat. The ''FindMarkers'' function was used to identify differentially expressed genes between control and patient cells globally or in a cell/cluster-specific manner.

Statistical analysis
Data were analyzed and graphs created using GraphPad Prism (version 9.2.0) or Microsoft Excel (version 2110). One-way ANOVA with Dunnett correction was performed on data from at least three independent experiments, represented as mean ± SD, unless stated otherwise. A p value < 0.05 or an adjusted p value < 0.05 was considered to indicate statistical significance. Graphical abstract and schematics were created in BioRender.com.

SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/ 10.1016/j.stemcr.2022.09.006.   Figure S1. A) Brightfield images show iPSCs maintained compact cellular morphology with well-defined edges, high nucleus-to-cytoplasm ratio (left and middle columns) and expressed pluripotency marker, NANOG (right column). B) Table shows (from first column) nomenclature of all iPSCs used in this study, their original names and source facility where iPSC were generated, passage number when Single Nucleotide Polymorphism (SNP) array analysis was performed, and all passages used in this study. SNP array data available upon request. C) Connecting cilium (expressing cilia marker, ARL13B; basal body marker, PCN) was present in photoreceptors. ARL13B also marked the photoreceptor outer segment. ARR3, cone marker. Observed in >3 organoids per iPSC line. D) Immunostaining of 28wks retinal organoids for outer segment markers. PRPH2, rod outer segment; GNAT2, cone outer segment. Observed in >3 organoids per iPSC line. Figure 3 and experimental procedure. A) Heatmap shows expression of photoreceptor markers comparing data from human developing eye (GEO accession: GSE98370, Mellough et al, 2019; encompassing three developmental stages: human embryonic eye (CS14-16), fetal (9 -17wks), infant (74pcw) and RNA-seq data from 28wks USH1B and control organoids (this study). B) Expression of markers of other retinal cell types in human developing eye with USH1B and control organoids (this study). C) Schematic of MYO7A shows anti-MYO7A antibody (DSHB, 138-1) binding site used in Western blot shown in Figure 3I-I'. MYO7A contains an N-terminal motor domain, where ATP-and actin-binding sites reside and is connected to a neck region composed of 5 isoleucine-glutamine (IQ) motifs followed by a complex tail region, which binds to cargos or other proteins to form a complex. The tail region begins with a stable single α-helix (SAH) followed by two MyTH4 (myosin tail homology 4)-FERM (four-point1, ezrin, radixin and myosin) regions (MF1 and MF2) sandwiching a SRC homology 3 (SH3) domain (Kabahuma et al., 2021, Heissler and. Figure 4F. Highlighted in red are the 40 genes in the KEGG ubiquitin mediated proteolysis pathway that were dysregulated in the 28wks USH1B organoids (Benjamini p-value 4.4 x 10 -39 ; generated using david-d.ncifcrf.gov) Figure S5 The presence of retinal cell subtypes in 35wks retinal organoids. Related to Figure 5. A) Rod photoreceptor clusters (0, 2 and 6) were indicated by RHO, NRL and NR2E3 expression. B) Two rod subtypes described by Yi et al. (2021) that can be differentiated by MYO9A expression. Cluster 2 expressed high level of MYO9A compared to cluster 0 and 4. C) Heatmap shows the expression, by cluster 0, 2 and 6, of the most distinctive genes comparing human MYO9A + and MYO9Arods identified by Yi et al. (2021). D) GRIK1, GRM6,VSX1,VSX2 and PRKCA expression identified bipolar cell clusters: 7,8,13,14 and 15;GRM6 and GRIK1 expression were indicative of ON-and OFF-bipolar cells, respectively. E) More bipolar cell subtypes can be differentiated by expression of markers described by Yi et al. (2021). F) Horizontal cell clusters were indicated by the expression of CNTNAP2, CALB1, ONECUT1 and ONECUT2: 9 and 21. G) Heatmap shows the expression, by cluster 9 and 21, of the most distinctive genes comparing human H1 and H2 horizontal cells identified by Yi et al. (2021).      Figure S1. iPSC cultures displayed a normal growth rate and morphology and maintained pluripotency marker expression. Single-nucleotide polymorphism (SNP) karyotype analysis of the iPSC lines and passage numbers used are given in Figure S2. SNP microarray analysis was performed using  the Table S2.

Quantification of organoid size and thickness of outer nuclear layer (ONL)
Organoid size was quantified from Fiji image analysis using 7wks organoid size as baseline because at 6wks, adherent organoids were micro-dissected and transferred to 3D suspension culture and at 7wks, medium was supplemented with fetal bovine serum, after which an increase in organoid size was observed. ONL thickness was quantified using Fiji with the areas measured guided by ARR3 (cone arrestin, cone photoreceptor) or RCVRN (recoverin, panphotoreceptor) IHC plus DAPI counterstain in retinal cryosections.

RT-PCR and qRT-PCR
RNA extraction was performed using miRNeasy micro kit (Qiagen, 217084) and cDNA was synthesised using miScript II RT kit (Qiagen, 218160). RNA was quantified using Thermo poly-adenylated mRNAs were isolated, chemically fragmented and primed with random hexamers, before generation of strand-specific first strand cDNA using reverse transcriptase in the presence of actinomycin D. Quality of libraries generated was accessed using High Sensitivity Agilent DNA 1000 assay (Agilent, 5067-4626)) on Agilent TapeStation 4200, before being enzymatically normalised using a Normalase assay (Swift BioSciences, 66096).
Libraries were sequenced on a S1 flow cell on a NovaSeq 6000 system (Illumina, San Diego, US) at 0.8 nM using v1.5 SBS chemistry and a 50bp paired-end recipe. Approximately 20 million reads per sample were generated. Data was demultiplexed and FASTQ files generated using Illumina's bcl2fast Conversion Software v2.20. FASTQ files were then tagged with the UMI read (with UMITools) and aligned to the human genome UCSC hg38 using RNA-STAR 2.5.2b. Aligned reads were UMI deduplicated using JE-Suite (1.2.1) and reads per transcript were counted by FeatureCounts to produce a digital output of gene expression. EdgeR was used to generate counts per million (CPM), to be used for filtering lowly expressed genes.
Differential expression analysis was performed using limma-voom with cut-off of adjusted p value < 0.05 for statistical significance. Package clusterProfiler (version 3.0.4) was used for GO enrichment analysis.

Single-cell RNA sequencing
Single cell RNA libraries were generated using the Chromium Single Cell 3ʹ Reagent Kits v3 (10X Genomics, CA, USA). The cells were counted using a trypan blue exclusion count assay with a Bio-Rad Automated Cell Counter TC10 and diluted for loading onto the Chromium Controller. Loading was performed to target capture of~8,000 Gel Bead-In-EMulsions (GEMs) per sample for downstream analysis, and samples were processed through the Chromium Controller following the standard manufacturer's specifications. The sequencing libraries were evaluated for quality on the Agilent TapeStation (Agilent Technologies, USA), and quantified by using a Qubit 2.0 Fluorometer (Invitrogen) and pooled libraries were quantified using qPCR (Applied Biosystems) prior to loading onto an Illumina sequencing platform. The samples were sequenced at a configuration compatible with the recommended guidelines as outlined by 10X Genomics. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and demultiplexed using the 10X Genomics' cellranger mkfastq command.
Subsequent UMI and cell barcode de-convolution along with mapping to the respective genome was performed using 10X Genomics' cellranger software package to generate the final digital gene expression matrices.
Data was analysed using the Seurat package. Only cells expressing more than 200 genes and fewer than 8000 and with mitochondrial gene percentages less than 30% were kept for further analysis. In total, 19,399 and 20,535 single cells from two independent controls and from three independent USH1B patients, respectively, remained (control.1 replicate1, 6,607 cells; control.1 replicate 2, 6,315 cells; control.3, 6,477 cells; USH1B.1, 6,653; USH1B.2, 6,983 cells and USH1B.3, 6,899 cells). A total of 39934 cells were integrated with the Seurat standard protocol and the effect of mitochondrial genes were regressed out using "ScaleData" function. The top markers for each cluster were identified using the "FindConservedMarkers" function. Cell identity assignment was performed based on well-defined retinal markers and two recent single-cell sequencing studies of human retina. Full list of top marker genes used for cell assignment can be found in Table S. The "FindMarkers" function was used to identify differentially expressed genes between control and patient cells globally or in a cell/clusterspecific manner.

Data availability
RNA-seq data have been deposited in ArrayExpress under accession numbers E_MTAB-