Keywords: Human embryonic stem cells, Induced pluripotent stem cells, Retina, Organoids, Tissue engineering

hESC Culture and Differentiation

One hESC line (H9, WiCell) one neonatal iPSC line (Neo1, male38,39) and one adult iPSC line (AD3, from a 37‐year‐old female40) were adapted from MEF‐supported to feeder‐free culture conditions on 6 well plates coated with growth factor‐reduced Matrigel basement membrane matrix (GFRM; Corning, New York) in TeSR1 (Stem Cell Technologies, Vancouver, Canada) defined medium. Cells were fed daily and passaged every 5 days at a ratio of up to 1:10 using 0.02% versene EDTA (Lonza, Basel, Switzerland). Cells were then differentiated by initiating EB formation, followed by long‐term culture (up to 150 days). EBs were generated either by mechanical, enzymatic, or dissociation–reaggregation approaches (Fig.1). Each method was tested in the presence or absence of ROCK inhibitor (Y‐27632, Chemdea, NJ) for the first 48 hours of differentiation, during the early phase of EB formation. All cultures were tested in parallel under either stationary (static, “St”) or shaking (“Sh”) conditions throughout differentiation. Shaking cultures were achieved by placing cells on an orbital shaker (30 rpm, Stuart) housed inside the incubator for the entire period of differentiation, or in the case of EBs generated via the dissociation–reaggregation method, from day 12 onward. Biological replicates were performed in triplicate (n ≥ 3) for all experimental conditions tested.

Immunocytochemistry and TEM

Organoids were fixed over days 35–150 (5–21 weeks) of differentiation and immunocytochemistry performed on cryostat sections as previously described3,8. Sections were reacted against a panel of retinal, lens, and corneal‐specific antibodies, listed in Supporting Information TableS1. At least five structures from each time point across each experimental group were immunostained. Images were obtained using a Zeiss Axio Imager.Z1 microscope with ApoTome.2 accessory equipment and AxioVision or Zen software. For TEM tissues were fixed in glutaraldehyde and processed by the Electron Microscopy Research Service at Newcastle University. Human embryonic and fetal samples were provided by the Human Developmental Biology Resource (http://hdbr.org) under ethics permission (09/H0906/21).

Array‐Based Gene Expression Analysis

RNA was extracted from retinal organoids (RNeasy Plus, QIAGEN, Manchester, U.K.), cDNA synthesized (Reverse Transcription Master Mix, Fluidigm, San Francisco, CA) and preamplified (PreAmp Master Mix, Fluidigm). Preamplified cDNA and TaqMan Assays (Thermo Fisher Scientific, MA) were loaded onto a primed 96.96 integrated fluidic circuit (Fluidigm) using a HX controller (Fluidigm). qPCR and data collection was then performed using a Biomark HD (Fluidigm). For TaqMan assays used, see Supporting Information TableS2. CT values were calculated using the Fludigm Real‐time Analysis software and the expression values extracted using HTQPCR bioconductor package version 1.30. To assess the success of the experiment, the number of genes detected per cell along with the total expression levels was analyzed. Samples were filtered if the total raw expression value was less than 500, or fewer than 60 genes were, or no housekeeping genes were expressed. The filtered data was normalized with the normalizeCtData() function in the HTQPCR package using the housekeeping genes. Technical replicates were condensed by taking the mean expression for each gene. Principal component analysis (PCA) of all samples was used to visualize the distance between all samples. The data was subset to look specifically at samples at week 5 and from day 120 and above. PCA was performed to visualize the samples at both of these time points.

The Wilcoxon test was used to look for significant differences between embryonic/fetal retinal and retinal organoids generated from hESCs and hiPSCs with the different methods for samples after 5 weeks of differentiation.p values were adjusted using Bonferroni correction and an adjustedp value threshold of <.05 was taken as significant. The total numbers of genes that were significantly different from the reference samples, and the numbers of 32 marker genes known to be expressed in rod, cone, and photoreceptors were calculated and plotted. The similarity in expression between human embryonic/fetal retina and retinal organoids generated from hESCs and hiPSCs differentiated in either static or shaking conditions at day 120 and above was compared. The mean expression for each of the 32 marker genes human embryonic/fetal samples was calculated and compared with individual samples by computing the Pearson correlation coefficient. These values were then plotted as a boxplot to visualize the similarities between each set of samples and the human embryonic/fetal samples. The Wilcoxon test with Bonferroni multiple test correction was applied to the data compare expression of samples in either static or shaking conditions and human embryonic/fetal retina samples. Known marker genes with a significant adjustedp‐value of below .05 were plotted as a heatmap.

Electrophysiology

Twenty‐four hours prior to electrophysiological recordings, 9‐cis‐retinal (10 nM; Sigma–Aldrich, U.K.) was added to the incubation medium. Organoids were transferred to 34°C artificial cerebrospinal fluid (aCSF) containing the following (in mM): 118 NaCl, 25 NaHCO₃, 1 NaH₂ PO₄, 3 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, 0.5l‐glutamine, and 0.01 9‐cis‐retinal. Organoids were opened longitudinally and placed, with the presumed RGC layer facing down on the electrodes, onto the 4,096 channel multielectrode array (MEA), flattened with a translucent polyester membrane filter (Sterlitech Corp., Kent, WA). The organoids were allowed to settle for at least 2 hours. Recordings were performed on the BioCam4096 MEA platform with BioChips 4096S+ (3Brain GmbH, Lanquart, Switzerland), integrating 4,096 square microelectrodes in a 64 × 64 array configuration.

Light stimuli were projected as described previously42. Broad white (high photopic) light pulses (WLP, 200 ms, 217 μW/cm² irradiance, 1 Hz) were flashed for 5 minutes onto the organoids following recording spontaneous activity in the dark for 5 minutes. We also used sustained broad blue light stimulation (SBL, 2 minutes darkness, 2 minutes SBL, 2 minutes darkness), same irradiance as WLP), to evoke responses from intrinsically photosensitive RGCs (ipRGCs). The drugs cGMP (8‐bromoguanosine 3′,5′‐cyclic monophosphate, Sigma–Aldrich, MO) and GABA (Tocris, U.K.) were puffed in the recording chamber (final concentrations: 100 μM and 125 μM, respectively) and activity was recorded continuously for 4 minutes, starting at 2 minutes before the puff.

To reliably extract spikes from the raw traces we used a quantile‐based event detection43. Single‐unit spikes were sorted using an automated spike sorting method for dense, large‐scale recordings44. Statistical significance (unpairedt test) and firing rate analyses were evaluated by using MATLAB (Mathworks, MA) and Prism (GraphPad, CA). RGCs were considered responsive if they show at least 25% increase or decrease in spiking activity during 30 seconds after WLP onset compared with a similar time window before the light was turned on. For each cell, all spikes occurring during these two time windows were counted and the mean % change (±SEM) in activity between windows was calculated. All RGCs respond to WLP. To single out ipRGCs, we performed additional analysis of their responses to SBL, taking into account that photoreceptor‐driven responses are relatively transient, and therefore stop after a while, whereas intrinsic light responses in ipRGCs are sustained (and generally have a slower onset). Hence, we classified RGCs as ipRGCs if they still exhibited significantly higher firing rate 30–60 seconds after the onset of the SBL stimulus. All other RGCs were analyzed for the WLP protocol (photoreceptor‐driven responses).

Early Stages of Embryoid Body Formation from hESCs and hiPSCs Using Differing Protocols

Proliferative cultures were processed to initiate EB formation using either a mechanical (M), enzymatic (E), or dissociation–reaggregation approach (V9; Fig.1). Cultures collected by mechanical means appeared as square‐shaped colony fragments at the onset of differentiation (Fig.2A,2D), whereas enzymatically collected fragments had softer, more phase‐bright edges (Fig.2G,2J). After 48 hours, small (<150 μm in diameter) EBs had formed within all mechanical cultures (Fig.2B,2C,2E,2F), with residual cell debris observed in the media under these conditions. ROCK inhibition of mechanical cultures by supplementation with Y‐27632 (M&Y, both St and Sh) gave rise to EBs which appeared as spherical formations of neuroepithelium (Fig.2C,2F, arrows). Fewer EBs were observed in shaking cultures at this time point (Fig.2E,2F). In comparison to mechanical cultures, EBs formed via the enzymatic method measured <200 μm in diameter after 48 hours (Fig.2H,2I,2K,2L), and very little cell debris was observed. The addition of Y‐27632 to enzymatic cultures resulted in larger (up to 350 μm), less phase‐bright EBs, which appeared to have small rosette formations in the interior (Fig.2I,2L). In general, EBs cultured in the absence of Y‐27632 appeared homogenous and phase‐bright during the first 48 hours, whereas ROCKi resulted in early organization of EBs into neuroepithelial‐like formations (either in vesicular or rosette formations). Dissociation–reaggregation of cells into 96 well plates (“V9,” 9,000 cells per well) allowed almost immediate aggregation of cells at the bottom of the well, surrounded by some cellular debris, forming EBs of 400 μm diameter (Fig.2M). V9 EBs were consistently larger (diameter increased to >600 μm) if generated in the presence of Y‐27632 (Fig.2N,2O). The effect of ROCKi was additive; EBs increased in size further if the concentration of Y‐27632 applied to cultures during the first 48 hours was doubled (Supporting Information Fig.S1). The diameter of EBs generated enzymatically without ROCK inhibition was <200 μm, whereas the addition of 10 μM Y‐27632 increased EB diameter to 200–350 μm, and treatment with 20 μM Y‐27632 increases EB diameter to >400 μm.

Neuroepithelial Formation Under Differing Methods

Retinal organoids exhibit a typical morphology that is clearly identifiable during in vitro culture, namely, phase‐bright spherical EBs with layers of thick peripheral neuroepithelium (Supporting Information Fig.S2). This is better exemplified following sectioning on week 5, where these layers can be clearly identified as inner and outer neuroblastic layers (Supporting Information Fig.S3A, S3B). The overall morphology of organoids within cultures after week 5 varied greatly depending on the method of EB formation used at the onset of differentiation (Supporting Information Fig.S2A–S2I). Of note, shaking mechanical cultures gave rise to organoids with significantly flattened edges (Supporting Information Fig.S2A) consisting of one dense peripheral layer (Supporting Information Fig.S3C, S3D), whereas 96‐well plate‐generated cultures gave rise to organoids which were folded and could become highly convoluted (Supporting Information Fig.S2G, S2H) with a main body containing neural rosettes (Supporting Information Fig.S3E–S3G, asterisks). The generation of organoids was particularly difficult under enzymatic shaking culture conditions with ROCKi (E&Y, Sh); all cell lines failed to generate retinal organoids using this approach. Furthermore, the hESC line did not respond well to retinal differentiation under any dissociation–reaggregation conditions tested, forming instead EBs which became folded and developed fine extensions growing from the main body, an effect which was more pronounced under shaking conditions (Supporting Information Figs. S2G, S3E–S3G, arrows). The Neo1 hiPSC line was unable to form retinal organoids in V9 cultures, whereas increasing the initial seeding density to 12,000 cells per well (V12) permitted retinal organoid formation under stationary culture conditions only (Supporting Information Figs. S2H, S3G). The AD3 hiPSC line in general showed a greater ability to self‐organize into retinal neuroepithelium under all conditions tested, with the exception of the E&Y, Sh approach. Furthermore, the overall morphology of organoids in a culture could be altered significantly by whether these were shaken or not, as in the case of H9's generated in 96 well plates, or whether cells were reaggregated in a U‐shaped or V‐shaped well, as in the case of AD3s (Supporting Information Fig.S4). Neuroepithelium was observed in organoids generated from hESCs using both U‐shaped and V‐shaped 96 well plates if kept under stationary conditions (Supporting Information Fig.S4A, S4B), but not under shaking conditions (Supporting Information Fig.S4E, S4F). Using the AD3 hiPSC line, U‐shaped wells gave rise to organoids with clearly identifiable retinal neuroepithelium under both stationary and shaking conditions (Supporting Information Fig.S4C, S4G), whereas those generated using V‐shaped wells displayed thinner, more convoluted neuroepithelium (Supporting Information Fig.S4D, S4H). Although retinal organoids were able to form at different frequencies under almost all conditions tested within 5 weeks, these were most consistently achieved using a mechanical stationary approach (Supporting Information Fig.S2A–S2C, top row and Supporting Information Fig.S3A, S3B). Together, these observations show cell line‐specific and method‐associated differences in the ability of hESC and hiPSC to form phase‐bright spherical organoids with layers of thick peripheral neuroepithelium.

Gene Expression Analysis Reveals Cell Line‐Specific Differences to Generate Retinal Organoids at Week 5 of Differentiation

To investigate potential differences between cell lines and methods used, a Taqman qRT‐PCR based array was compiled (Supporting Information TableS2) including key marker genes characterizing various retinal cell types. Retinal organoids generated from the pluripotent stem cells lines including hESC‐H9 (annotated as H), adult fibroblast derived hiPSC‐AD3 (annotated as A), and neonatal fibroblasts derived hiPSC‐Neo1 (annotated as N) were compared with eight samples of embryonic eyes obtained from 4.6 to 8 postconception week (PCW; annotated as hEye), 21 samples of fetal neural retina encompassing 8–18 PCW (annotated as hRet), and undifferentiated hESCs (annotated as hESC). Different methods of retinal organoid formation were compared under static (St) and shaking (Sh) conditions.

PCA analysis of gene expression data indicated that at week 5, the retinal organoid clustered more strongly by cell type (Fig.3A) than by method (Fig.3B). The Wilcoxon test was carried out to identify genes that were differentially expressed between week 5 retinal organoids from all three pluripotent stem cell lines (Supporting Information TableS3 and Fig.3C). This analysis indicated line specific differences; however, no significant differences in gene expression were observed between different methods used for retinal organoid formation at this time point (Fig.3B). Addition of ROCKi or static versus shaking conditions had no impact on gene expression at this stage (Fig.3B).

To investigate whether the expression of key retinal markers at week 5 retinal organoids was closer to embryonic or fetal retina, differentially expressed genes between each EB generation method (all cell lines pooled together) and hEye and hRet samples were identified (Fig.4A,4B and Supporting Information TableS4). This analysis indicated that across the methods, there were less differentially expressed genes when week 5 retinal organoids samples when compared with hEye (Fig.4A) than to hRet samples (Fig.4B), suggesting that at this stage week 5 organoids are closer in expression profile to the embryonic eye. One of the methods, namely mechanical under static conditions (M_St) showed the lowest number of differentially expressed genes when compared with both hEye and hRet (fetal eye) samples (Supporting Information TableS4 and Fig.4C). Overall, the retinal organoids made with enzymatic, mechanical or 96v shaped well plates with Y26732 inhibitor under static conditions, showed the least significant changes when compared with hEye samples (Fig.4A), suggesting that for week 5 of differentiation these could be the best methods for acquiring a gene expression pattern that mimics eye development between 4.6 and 8 PCW.

Comparison of retinal organoids samples at week 5 of differentiation to hRet samples showed a larger number of genes to be differentially expressed, as expected, due to differences in developmental times. Nonetheless, the genes that were downregulated between week 5 retinal organoids and fetal retina samples belonged to mature cone and rod markers (OPN1MW, OPN1LW, GNAT1), bipolar cells (GRM6, CABP5), cilia marker (RP1) and melanopsin‐photosensitive ganglion cells (OPN4), which develop later during retinogenesis45 and suggest that longer differentiation is needed to achieve appropriate expression of mature retinal cell markers (Supporting Information TableS4 and Fig.4D). Notwithstanding, retinal organoids generated via mechanical means with and without Y26732 under static conditions showed the least differences in gene expression patterns when compared with hRet samples, again indicating they may be the most appropriate for achieving a transcriptional profile akin to neural retina.

Following this transcriptional analysis, we performed immunohistochemistry (IHC) with retinal cell markers (Figs.5,6) which showed that at this stage, the highest frequency of developing laminated retinal tissue across all cell lines tested was observed in cultures derived using the “M&Y_St” method (78.5% of EBs in hESCs, 94.4% of EBs in AD3, and 12.5% of Neo1 EBs; Figs.5A–5E,6C–6F). Within these organoids, retinal progenitor cells immunopositive for VSX2 and SOX2, as well as photoreceptors stained with OTX2 and Recoverin were observed at the apical aspect of developing neuroepithelial tissue (Figs.5A–5C,6A,6F). Developing retinal ganglion cells with Smi32‐immunopositive processes46,47 (Figs.5D,6B) and RBPMS‐immunopositive nuclei48 (Fig.6B) were located at the basal aspect. Horizontal cells detected by cellular immunoreactivity for Ap2α were also present (Fig.6D). Furthermore, there was evidence of synaptogenesis in the developing outer nuclear layer (ONL) and outer plexiform layer as indicated by synaptophysin immunoreactivity (Figs.5E,6C) and beginning of Müller glia formation as shown by vimentin and CRALBP (RLBP1) staining throughout the retinal organoids (Fig.6E,6F). Mechanical stationary cultures initiated in the absence of Y‐27632 were also able to develop into retinal organoids with laminated retina containing Recoverin‐positive photoreceptors, however, these cultures gave a lower overall yield (in the best case, 73.3% of EBs sampled at week 5 in AD3‐hiPSC‐derived cultures).

In summary, our transcriptional and IHC analysis suggests the mechanical method of EB formation supplemented with Y27632 for the first 48 hours of differentiation and accompanied by stationary organoid maintenance conditions (M&Y_St) generate the best retinal organoids, which contain all key retinal cell types with gene expression that is closer to developing fetal retina.

Mechanical EB Generation and Supplementation of Culture Media with Y27632 Under Static Conditions Generates Retinal Organoids Containing Electrophysiologically Responsive Photoreceptors

The stability of retinal organoids was monitored by bright phase and fluorescence microscopy until the 19th–21st week of differentiation. Retinal organoids generated with the mechanical and 96 V‐shaped plates from all three cell lines survived past the 19th week of differentiation under both static and shaking conditions. Retinal organoids made from both hiPSC lines under the enzymatic conditions with the shaking or static methods degraded before the 19th week; however, organoids made with this method from hESC under static conditions were able to develop past this time point and were included in the qRT‐PCR array analysis for this later differentiation time point (Supporting Information TableS2). PCA analysis of gene expression data indicated that retinal organoids collected during the 19th–21st week of differentiation clustered more strongly by the condition (static or shaking) than by cell line (Fig.7A). To understand whether organoids generated with the static or shaking conditions were more similar to embryonic eye (hEye) or human fetal retinal samples (hRet), Pearson correlation between the organoid samples, hEye, hESC samples, and hRet was calculated based on the mean expression of all 96 marker genes included in the qRT‐PCR array. This analysis (Fig.7B) shows that retinal organoids generated under static conditions are much more similar to hRet samples than those generated under shaking conditions, indicating an advanced stage of maturation. This was further corroborated by comparison of gene expression profiles between retinal organoids generated under static and shaking conditions and hRet samples, which showed a higher expression of photoreceptor precursor markers (for exampleCRX, NRL, Supporting Information TableS5 and Fig.7C) in retinal organoids generated under shaking when compared with those from stationary. Collectively, these data indicate that shaking conditions are more likely to enhance the generation of photoreceptor precursors; however, the overall expression profile of retinal organoids cultured under static conditions is closer to fetal/adult neural retina samples, suggesting that the latter represents a useful method for generating retinal organoid samples that mimic more closely the developing human retina.

These findings were corroborated by the IHC analysis, which showed that static cultures (generated via mechanical and 96 well plate methods) across all cell lines contained impressive areas of laminated retina with aligned Recoverin‐immunopositive photoreceptors and some RPE by week 12 of differentiation. In contrast, shaking cultures by week 13 consistently displayed large disorganized patches of nonaligned immature photoreceptors with apically positioned Smi32 processes, however, the overall internal architecture was disorganized. By week 17, large clusters of photoreceptors were observed in the statically maintained cultures, some of which formed rosettes, whereas others were located at the apical aspect of laminated retinal neuroepithelium neighboring basally positioned retinal ganglion cells (data not shown). The most advanced retinal neuroepithelium with photoreceptors developing with opposing polarity to retinal ganglion cells, was observed in cultures generated using the M&Y_St method. Photoreceptors in the ONL of hESC‐derived and hiPSC‐derived retinal organoids derived using the M&Y_St approach developed mature morphological features and were immunoreactive for mature markers (Supporting Information Fig.S5). In addition to being immunopositive for the pan‐photoreceptor marker Recoverin (Supporting Information Fig.S5A, S5D), by week 21 of differentiation photoreceptors were also immunopositive for cone‐specific Opsin Blue, Bassoon (Supporting Information Fig.S5B), and rod‐specific Gαt1 (Supporting Information Fig.S5C). Transmission electron microscopy of “M&Y_St” retinal organoids on week 24 of differentiation revealed radially aligned photoreceptors displaying mitochondrial‐rich inner segments (Supporting Information Fig.S5E–S5H). A connecting cilium extended from each inner segment, and this was connected to rudimentary outer segments (Supporting Information Fig.S5E–S5I). Observation of developing outer segments at high magnification revealed the appearance of membrane discs which were stacked on top of each other (Supporting Information Fig.S5J, S5L), with some outer segments appearing disorganized (Supporting Information Fig.S5I, S5K). Depending on orientation, the edge of developing membrane discs was observed to be capped by circular membrane structures (most clearly visible in Supporting Information Fig.S5I, S5J).

To investigate the function of laminated retinal epithelium generated under static conditions, in vitro extracellular large‐scale high‐density multielectrode recordings (BioCam, 3brain AG, Switzerland) of retinal ganglion cells (RGCs) were carried out, as described in our previous study26, on samples generated from “M&Y_St” conditions. Many presumed RGCs at week 21 of differentiation exhibited at least 25% increase (37/164 RGCs) or decrease (40/164 RGCs) in spiking activity when exposed to a pulsed high intensity stimulus (WLP, Supporting Information Fig.S6A, S6B). These response types could come from premature ON and OFF RGCs. To ensure that the responses being observed were indeed photoreceptor driven, we used sustained blue light (SBL) to separate intrinsically photosensitive RGCs (ipRGCs) from photoreceptor‐driven RGCs and found many activated ipRGCs (48/164 RGCs) within organoids (Supporting Information Fig.S6C). Puffing 8‐br‐cGMP, a membrane permeable analog of cGMP, in the recording chamber triggers Na + influx and depolarises photoreceptors, thus mimicking the dark current. RGCs responding to the 8‐br‐cGMP puff with increased spiking activity (14/164 RGCs) are thus presumed photoreceptor‐driven OFF RGCs (Supporting Information Fig.S6D), whereas those exhibiting a decrease in activity (8/164 RGCs) after the puff are presumed photoreceptor‐driven ON RGCs (Supporting Information Fig.S6E). GABA signaling emerges early during central nervous system (CNS) development, it is then depolarising because of the developmentally regulated low expression of chloride exporters49. Therefore, GABA can even induce spiking (16/164 RGCs, Supporting Information Fig.S6F) or reduce (12/164 RGCs, Supporting Information Fig.S6G) in RGCs. Hence, responses to GABA puffs are indicative of emerging functional neural networks.

Collectively, our transcriptional and immunohistochemical analysis suggest that the method used for organoid formation and maintenance plays an important role in the generation of retinal organoids and their maturity. Importantly, this integrated analysis indicates the mechanical method of EB formation, supplemented with Y27632 for the first 48 hours of differentiation and linked to stationary maintenance conditions to be the most optimal for generating consistently laminated retinal organoids that are electrophysiologically responsive and closer to developing human fetal retina in gene expression profile.

• 1.Hirami Y, Osakada F, Takahashi K et al. Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci Lett2009;458:126–131. [DOI] [PubMed] [Google Scholar]
• 2.Lamba DA, Karl MO, Ware CB et al. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci USA2006;103:12769–12774. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 3.Mellough CB, Sernagor E, Moreno‐Gimeno I et al. Efficient stage‐specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells. Stem Cells2012;30:673–686. [DOI] [PubMed] [Google Scholar]
• 4.Osakada F, Ikeda H, Mandai M et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol2008;26:215–224. [DOI] [PubMed] [Google Scholar]
• 5.Osakada F, Jin ZB, Hirami Y et al. In vitro differentiation of retinal cells from human pluripotent stem cells by small‐molecule induction. J Cell Sci2009;122:3169–3179. [DOI] [PubMed] [Google Scholar]
• 6.Meyer JS, Shearer RL, Capowski EE et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci USA2009;106:16698–16703. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Collin J, Mellough CB, Dorgau B et al. Using zinc finger nuclease technology to generate CRX‐reporter human embryonic stem cells as a tool to identify and study the emergence of photoreceptors precursors during pluripotent stem cell differentiation. Stem Cells2016;34:311–321. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Mellough CB, Collin J, Khazim M et al. IGF‐1 signaling plays an important role in the formation of three‐dimensional laminated neural retina and other ocular structures from human embryonic stem cells. Stem Cells2015;33:2416–2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 9.Phillips MJ, Wallace KA, Dickerson SJ et al. Blood‐derived human iPS cells generate optic vesicle‐like structures with the capacity to form retinal laminae and develop synapses. Invest Ophthalmol Vis Sci2012;53:2007–2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Eiraku M, Takata N, Ishibashi H et al. Self‐organizing optic‐cup morphogenesis in three‐dimensional culture. Nature2011;472:51–56. [DOI] [PubMed] [Google Scholar]
• 11.Nakano T, Ando S, Takata N et al. Self‐formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell2012;10:771–785. [DOI] [PubMed] [Google Scholar]
• 12.Hiler D, Chen X, Hazen J et al. Quantification of retinogenesis in 3D cultures reveals epigenetic memory and higher efficiency in iPSCs derived from rod photoreceptors. Cell Stem Cell2015;17:101–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Kaewkhaw R, Kaya KD, Brooks M et al. Transcriptome dynamics of developing photoreceptors in three‐dimensional retina cultures recapitulates temporal sequence of human cone and rod differentiation revealing cell surface markers and gene networks. Stem Cells2015;33:3504–3518. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Kuwahara A, Ozone C, Nakano T et al. Generation of a ciliary margin‐like stem cell niche from self‐organizing human retinal tissue. Nat Commun2015;6:6286. [DOI] [PubMed] [Google Scholar]
• 15.Reichman S, Terray A, Slembrouck A et al. From confluent human iPS cells to self‐forming neural retina and retinal pigmented epithelium. Proc Natl Acad Sci USA2014;111:8518–8523. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Volkner M, Zschätzsch M, Rostovskaya M et al. Retinal organoids from pluripotent stem cells efficiently recapitulate retinogenesis. Stem Cell Rep2016;6:525–538. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.Wang XB, Xiong K, Lin C et al. New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue. Biomaterials2015;53:40–49. [DOI] [PubMed] [Google Scholar]
• 18.Zhong X, Gutierrez C, Xue T et al. Generation of three‐dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun2014;5:4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 19.Zhou S, Flamier A, Abdouh M et al. Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFβ and Wnt signaling. Development2015;142:3294–3306. [DOI] [PubMed] [Google Scholar]
• 20.Hayashi R, Ishikawa Y, Sasamoto Y et al. Co‐ordinated ocular development from human iPS cells and recovery of corneal function. Nature2016;531:376–380. [DOI] [PubMed] [Google Scholar]
• 21.Jin ZB, Okamoto S, Osakada F et al. Modeling retinal degeneration using patient‐specific induced pluripotent stem cells. PLoS One2011;6:e17084. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Meyer JS, Howden SE, Wallace KA et al. Optic vesicle‐like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells2011;29:1206–1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Deng WL, Gao ML, Lei XL et al. Gene correction reverses ciliopathy and photoreceptor loss in IPSC‐derived retinal organoids from retinitis pigmentosa patients. Stem Cell Rep2018;10:1267–1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 24.Lowe A, Harris R, Bhansali P et al. Intercellular adhesion‐dependent cell survival and rock‐regulated actomyosin‐driven forces mediate self‐formation of a retinal organoid. Stem Cell Rep2016;6:743–756. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Parfitt DA, Lane A, Ramsden CM et al. Identification and correction of mechanisms underlying inherited blindness in human iPSC‐derived optic cups. Cell Stem Cell2016;18:769–781. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Hallam D, Hilqen G, Dorgau B et al. Human‐induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient‐dependent efficiency. Stem Cells2018;36:1535–1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 27.Phillips MJ, Perez ET, Martin JM et al. Modeling human retinal development with patient‐specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2. Stem Cells2014;32:1480–1492. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 28.Tucker BA, Mullins RF, Streb LM et al. Patient‐specific iPSC‐derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. eLife2013;2:e00824. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Buskin A, Zhu L, Chichagova V et al. Splicing factor PRPF31 retinitis pigmentosa (RP11) is caused by disrupted alternative splicing programmes for genes implicated in pre‐mRNA splicing, cellular adhesion and ciliogenesis. Nature Commun2018;9:4234. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Ohgushi M, Matsumura M, Eiraku M et al. Molecular pathway and cell state responsible for dissociation‐induced apoptosis in human pluripotent stem cells. Cell Stem Cell2010;7:225–239. [DOI] [PubMed] [Google Scholar]
• 31.Watanabe K, Ueno M, Kamiya D et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol2007;25:681–686. [DOI] [PubMed] [Google Scholar]
• 32.Chen G, Hou Z, Gulbranson DR et al. Actin‐myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell2010;7:240–248. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Yanai A, Laver C, Joe AW et al. Efficient production of photoreceptor precursor cells from human embryonic stem cells. Methods Mol Biol2016;1307:357–369. [DOI] [PubMed] [Google Scholar]
• 34.Shirai H, Mandai M, Matsushita K et al. Transplantation of human embryonic stem cell‐derived retinal tissue in two primate models of retinal degeneration. Proc Natl Acad Sci USA2016;113:E81–E90. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.Compagnucci C, Baressi S, Petrini S et al. Rho kinase inhibition is essential during in vitro neurogenesis and promotes phenotypic rescue of human induced pluripotent stem cell‐derived neurons with oligophrenin‐1 loss of function. Stem Cells Translational Medicine2016;5:860–869. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Kamishibahara Y, Kawaguchi H, Shimizu N. Rho kinase inhibitor Y‐27632 promotes neuronal differentiation in mouse embryonic stem cells via phosphatidylinositol 3‐kinase. Neurosci Lett2016;615:44–49. [DOI] [PubMed] [Google Scholar]
• 37.Nakamura M, Kamishibahara Y, Kitazawa A et al. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons. Cytotechnology2016;68:409–417. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 38.Chunbo Yang YX, Min Y, Lee D et al. Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis. Hum Mol Genet2017;26:3031–3045. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 39.Jiang Y, Habibollah S, Tilgner K et al. An induced pluripotent stem cell model of hypoplastic left heart syndrome (HLHS) reveals multiple expression and functional differences in HLHS‐derived cardiac myocytes. Stem Cells Translational Medicine2014;3:416–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 40.Melguizo‐Sanchis D, Xu Y, Taheem D et al. iPSC modeling of severe aplastic anemia reveals impaired differentiation and telomere shortening in blood progenitors. Cell Death Dis2018;9:128. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 41.Eiraku M, Watanabe K, Matsuo‐Takasaki M et al. Self‐organized formation of polarized cortical tissues from escs and its active manipulation by extrinsic signals. Cell Stem Cell2008;3:519–532. [DOI] [PubMed] [Google Scholar]
• 42.Hilgen G, Pirmoradian S, Pamplona D et al. Pan‐retinal characterisation of light responses from ganglion cells in the developing mouse retina. Sci Rep2017;7:42330. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 43.Muthmann JO, Amin H, Sernagor E et al. Spike detection for large neural populations using high density multielectrode arrays. Front Neuroinform2015;9:28. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 44.Hilgen G, Sorbaro M, Pirmoradian S et al. Unsupervised spike sorting for large‐scale, high‐density multielectrode arrays. Cell Rep2017;18:2521–2532. [DOI] [PubMed] [Google Scholar]
• 45.Mellough CB, Bauer R, Collin J, An integrated transcriptional analysis of the developing human retina. Development2019;146, pii: dev169474 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 46.Lin B, Wang SW, Masland RH. Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts. Neuron2004;43:475–485. [DOI] [PubMed] [Google Scholar]
• 47.Coombs J, van der List D, Wang GY et al. Morphological properties of mouse retinal ganglion cells. Neuroscience2006;140:123–136. [DOI] [PubMed] [Google Scholar]
• 48.Rodriguez AR, de Sevilla Müller LP, Brecha NC. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina. J Comp Neurol2014;522:1411–1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 49.Ben‐Ari Y. Excitatory actions of gaba during development: The nature of the nurture. Nat Rev Neurosci2002;3:728–739. [DOI] [PubMed] [Google Scholar]
• 50.Ohgushi M, Minaguchi M, Sasai Y. Rho‐signaling‐directed YAP/TAZ activity underlies the long‐term survival and expansion of human embryonic stem cells. Cell Stem Cell2015;17:448–461. [DOI] [PubMed] [Google Scholar]
• 51.Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell2005;121:451–463. [DOI] [PubMed] [Google Scholar]
• 52.Heynen SR, Meneau I, Caprara C et al. CDC42 is required for tissue lamination and cell survival in the mouse retina. PLoS One2013;8:e53806. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 53.Compagnucci C, Piermarini E, Sferra A et al. Cytoskeletal dynamics during in vitro neurogenesis of induced pluripotent stem cells (iPSCs). Mol Cell Neurosci2016;77:113–124. [DOI] [PubMed] [Google Scholar]
• 54.Lamas NJ, Johnson‐Kerner B, Roybon L et al. Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell‐derived cultures. PLoS One2014;9:e110324. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 55.Chaddah R, Arntfield M, Runciman S et al. Clonal neural stem cells from human embryonic stem cell colonies. J Neurosci2012;32:7771–7781. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 56.Kim K, Ossipova O, Sokol SY. Neural crest specification by inhibition of the ROCK/Myosin II pathway. Stem Cells2015;33:674–685. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 57.Rungsiwiwut R, Manolertthewan C, Numchaisrika P et al. The ROCK inhibitor Y‐26732 enhances the survival and proliferation of human embryonic stem cell‐derived neural progenitor cells upon dissociation. Cells Tissues Organs2013;198:127–138. [DOI] [PubMed] [Google Scholar]
• 58.Del Debbio CB, Santos MF, Yan CY et al. Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo. Invest Ophthalmol Vis Sci2014;55:2631–2641. [DOI] [PubMed] [Google Scholar]
• 59.Fontainhas AM, Townes‐Anderson E. RhoA and its role in synaptic structural plasticity of isolated salamander photoreceptors. Invest Ophthalmol Vis Sci2008;49:4177–4187. [DOI] [PubMed] [Google Scholar]

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