Genetically complex ocular neuropathies, such as glaucoma, are a major cause of visual impairment worldwide. There is a growing need to generate suitable human representative in vitro and in vivo models, as there is no effective treatment available once damage has occurred. Retinal organoids are increasingly being used for experimental gene therapy, stem cell replacement therapy and small molecule therapy. There are multiple protocols for the development of retinal organoids available, however, one potential drawback of the current methods is that the organoids can take between 6 weeks and 12 months on average to develop and mature, depending on the specific cell type wanted. Here, we describe and characterise a protocol focused on the generation of retinal ganglion cells within an accelerated four week timeframe without any external small molecules or growth factors. Subsequent long term cultures yield fully differentiated organoids displaying all major retinal cell types. RPE, Horizontal, Amacrine and Photoreceptors cells were generated using external factors to maintain lamination.
Worldwide, an estimated 253 million people are blind or have some form of a visual impairment[
Since the introduction of human induced pluripotent stem cells (hiPSCs) in 2007[
To successfully model diseases in a whole system environment, every cell type needs to be present within these organoids, and proper cell development and maturation cannot be rushed. A drawback of organoid research is the large amount of time that is needed to produce these cells in high quantities needed for disease modelling and preclinical pharmacological testing. The retina contains on average 3.5 million pigmented epithelial cells[
The generation of retinal organoids can be divided into three stages. Embryoid body formation is the first stage of in vitro retinal organogenesis, in which dissociated ESCs/iPSCs differentiate into free-floating embryoid bodies (EBs). Secondly, neural enrichment takes place, whereby whole EBs are plated and allowed to grow out their epithelial-fate cells, while forming neuron enriched centres. Finally, organoids develop by mechanically lifting off the neural centres and allowing them to fold and form in a free-floating environment into spherical organoids.
Culture methods and protocols vary between groups, but a common theme is a similar order of successive developmental stages along the retinal lineage, specified by specific cell marker expression. In general, immature retinal pigmented epithelial cell markers are expressed first, around day 35 of differentiation, while mature markers are not found until after 50–60 days of differentiation. Mature ganglion cells have been found to appear after around 40 to 50 days of culturing, and photoreceptors expressing immature and mature markers around day 70 and day 100 respectively[
Zhu and coworkers were previously able to reduce these prolonged iPSC-RPE culture and differentiation times by using a 3D matrigel culture model, initiating a direct differentiation toward RPE cells[
Table 1 A comparison of popular retinal differentiation protocols against our MG/FF combination protocol, showing the expression timeline of specific cell markers used to confirm the generation of general retinal organoids, retinal ganglion cells (RGCs), retinal pigmented epithelium (RPE) and photoreceptors (PR).
Cell focus Method Method adapted from IHC confirmation PCR Confirmation References Year Stage Culture Days Marker Day Marker Day General SFEBq Nakano et al. PAX6 24 2015 Embryoid bodies Floating 0–18 CHX10 24 Neurospheres Floating 18+ MITF 24 (low) PKCa 24 BRN3 50 Crx 50 NRL 150 General Embryoid bodies Floating 0-6 Keller et al. PAX6 10 PAX6 6 2009 Rosettes Adherent 6–16 RAX 10 RAX 6 Neurospheres Floating 16+ CHX10 30 MITF 16 MITF 30 CHX10 23 ZO-1 30 RPE65 40 CRX 60 OPSIN 70 General Embryoid bodies Floating 0-6 Meyer et al. PAX6 20 PAX6 20 2011 Rosettes Adherent 6-16 CHX10 20–25 BIII 20 Neurospheres Floating 16+ ISL1 20–25 BRN3 30 Seperation of Retinal spheres Floating 20-25 ZO-1 40 CRX 30 MITF 40 MITF 40 BRN3 80 RPE65 40 PKCa 80 BEST1 40 CRX 80 NRL 60 RCVRN 80 NRL 80 General SFEBq PAX6 20 PAX6 10 2009 Embryoid Bodies Floating 0-21 RAX 20 MITF 10 Neurospheres Floating 21+ CHX10 20 RAX 20 PRs Adherent 90 MITF 35 CHX10 20 ZO-1 100 RPE65 120 RHO 140 General Embryoid Bodies Adherent 0-28 PAX6 14 LHX2 14 2017 Neurospheres Floating 28+ RAX 14 MITF 14 LHX2 14 RAX 14 MITF 14 PAX6 14 CHX10 14 VSX2 14 BRN3 21 CRX 14 CRX 21 BRN3 21 RCVRN 42 RCVRN 35 General SFEBq Nakano et al. RAX 7 VSX2 10 2016 Embryoid Bodies Floating 0-18 PAX6 7 RAX 10 Neurospheres Floating 18-41 LHX2 7 ISL1 10 MITF 10 ATOH7 10 VSX2 10 BRN3 10 BRN3 41 RCVRN 10 CRX 15 PAX6 15 NRL 18 General Embryoid Bodies Matrigel 0-4 Ohlemacher et al. and Zhu et al. RAX 4 This study 2019 Rosettes Adherent 4-11 PAX6 4 Neurospheres Floating 11+ SOX2 4 ISL1 28 HuC/D 28 PKCa 28 ZO-1 56 RCVRN 91 GFAP 96 RBPMS 96 RHO 168 General Embryoid Bodies Floating 0-7 Meyer et al. PAX6 8 PAX6 4 2014 Rosettes Adherent 7-28 RAX 12 LHX2 4 Neurospheres Floating 28+ MITF 14 RAX 8 VSX2 14 HuC/D 35 BRN3 35 RCVRN 63 RHO 119 General/PR SFEBq PAX6 18 2012 Embryoid Bodies Floating 0-18 CHX10 18 Neurospheres Floating 18-126 MITF 18 PKCa 18 BRN3 30 RCVRN 43 NRL 126 RHO 126 RGC Embryoid Bodies Floating 0-4 HuC/D 25 RAX 3 2016 RGCs Adherent 4-30 BIII 25 ISL1 3 BRN3a 45 ATOH7 3 PAX6 3 BRN3B 3 MITF 8 RPE65 11 RGC Embryoid Bodies Floating 0-4 MATH5 20 RAX 4 2018 Rosettes Adherent 4-8 BRN3B 40 LHX2 4 RGCs Adherent 8-40 ISL1 40 CHX10 12 MATH5 12 CRX 12 (low) MITF 12 BRN3B 19 RGC Rosettes Adherent 0-14 RAX 14 2014 Neurospheres Floating 14-40 PAX6 14 ZO-1 14 BRN3 40 RGC SFEBq Nakano et al. RAX 24 RAX 6 2015 Neurospheres Floating 0-26/29 PAX6 24 PAX6 6 RGCs Adherent 26/29+ MATH5 34 CRX 6 BRN3 34 CHX10 6 CRX 34 PKCa 18 MITF 18 BRN3 24 MATH5 24 RGC/RPE Embryoid bodies Floating 0–7 Meyer et al. PAX6 20 PAX6 20 2015 Rosettes Adherent 7-16 CHX10 30 RAX 20 Neurospheres Floating 16+ BRN3 40 CHX10 30 Seperation of Retinal spheres Floating 20-25 RCVRN 50 BRN3 40 HuC/D 70 ISL1 50 ISL1 70 HuC/D 50 Floating RBPMS 70 RPE Embryoid bodies Floating 0–7 Pax6 13 2014 Rosettes Adherent 7–16 MITF 13 Neurospheres Floating 16+ VSX2 15 ZO-1 60 RPE Embryoid Bodies Matrigel 0-5 PAX6 3 PAX6 5 2013 RPE Adherent 5-25 ZO-1 1 (EB) RAX 5 RAX 5 CHX10 5 CHX10 15 MITF 10 PR/RPE Embryoid Bodies Floating 0-30 Rho 45 PAX6 15 2012 Neurospheres Adherent 30-60 OPN1SW 45 RPE65 30 RPE65 45 CHX10 45 ZO-1 45 CRX 60 RCVRN 60 PR SFEBq Nakano et al. PAX6 37 2015 Embryoid Bodies Floating 0-12 BRN3b 37 Neurospheres Floating 12-90 CRX 37 RCVRN 37 PKCa 67 Arr 67 NRL 67 OPN1SW 90 PR Embryoid Bodies Floating 0-6 Meyer et al. PAX6 56 RAX 56 2018 Neurospheres Adherent 6+ RAX 56 VSX2 56 RCVRN 56 PAX6 56 CRX 56 RCVRN 56 BRN3B 140 CRX 56 MITF 56 PR SFEBq Nakano et al. BRN3 35 OPN1SW 12 2017 Embryoid Bodies Floating 0-10/12 RCVRN 45 VSX2 30 Neurospheres Floating 10/12+ RHO 160 RCVRN 30 CRX 65 NRL 100 ARR3 100
In this study, we combine the well-established free-floating culture protocol, referred to herein as FF (free-floating)[
Graph: Figure 1 From left to right: schematic diagram of the generation of retinal organoids over time. Initially, human stem cell clumps are cultured in a 3D environment by suspension in solidified matrigel drops (MG). During the next 4 days incubation, with medium changing gradually from stem cell maintenance medium (mTeSR1) to neural induction medium (NIM), organised embryoid bodies are formed. These are subsequently plated in an adhesive culture (2D) environment allowing the outgrowth of epithelial cells for a further 7 days to enrich the neural centres. These neural centres are scraped off on day 11 and cultured 3D in a floating suspension environment (FF) using retinal differentiation medium (RDM), where they form early retinal organoids by day 14. Organoids are kept in this environment until required, depending on the desired cell type being produced. To develop and maintain long term lamination, FBS and Taurine are added from day 37. After a week, Retinoic Acid (RA) is also added. At day 84, Triiodo-l-Thyronine (T3) is also added and Retinoic Acid concentrations are halved. By day 120, Retinoic Acid is removed completely to allow for rod and cone maturation, with FBS, Taurine and T3 still present. For retinal ganglion and photoreceptor cells, organoids are dissociated and plated on coated coverslips in complete BrainPhys medium to stimulate neuronal outgrowth at days 23 and 160 respectively. For retinal pigmented epithelium, organoids at developmental day 37 are dissociated and seeded on growth factor reduced matrigel coated inserts, where they are grown into a pigmented monolayer for 4 weeks, supplemented with Activin A.
The first stage of generating retinal organoids is to grow embryoid bodies (EBs). EBs are predominantly generated by breaking up stem cell clones into around 100 µm clusters and growing them in a 3D floating culture environment to generate spheres that contain a multitude of different early progenitor cells (FF). However, in our hands, this generally resulted in relatively unstructured and disorganised looking EBs (Fig. 2a). In contrast, our MG EBs encapsulated in a 3D matrigel drop looked, in general, much more structured and mature, almost like fully grown organoids rather than early stage EBs (Fig. 2b). In order to confirm that our EBs were more organised at this early stage without losing any neural potential, we performed IHC staining of EBs with the well-known early neural and retinal developmental markers SOX2, PAX6 and RAX. Comparisons between floating (Fig. 2c,d) and matrigel (Fig. 2e,f) derived EBs shows that similar amounts of SOX2, PAX6 and RAX are expressed in both methods. The results show that EBs grown encased in a 3D matrigel environment are more structurally organised when compared to their floating counterparts, without compromising their neural identity.
Graph: Figure 2 Differences between EBs grown in matrigel embedment (MG) culture vs floating suspension (FF) culture. (A,B) Under a light microscope, EBs grown using a floating culture technique (A) look morphologically unorganised when compared to EBs grown using matrigel 3D embedment culture technique (B) by day 4. Highly reproducible, this morphological difference (detailed in (A,B) inserts, scale bar = 400 µm) is clear to see throughout entire cultures. This is also evident when looking at cross sections using IHC. (C–F) Floating cultures generate a mass of cells, whereas the EBs cultured in matrigel drops possess a clear stratified layer of cells with a hollow centre, much like an already matured organoid, while also staying within the neural lineage. The acquisition of neural identity was confirmed in both the floating culture derived bodies (Ci-iii,Di-iii), as well as the matrigel derived bodies (Ei-iii,Fi-iii) using common neuronal markers SOX2 and PAX6 (both in green). The general retinal marker RAX (in red) was also confirmed in both sets of EBs.
To generate retinal organoids, we modified a previously published FF organoid protocol[
Graph: Figure 3 Confirmation of retinal organoid development. (Ai-ii) Whole organoids were observed to have clear portions of neural retina (black arrows) and pigmented cells (white arrows) as early as day 18 (Scale Bar = 400 µm). (Bi-iii) Cross sectioned IHC analysis showed the formation of a distinct ganglion cell layer, shown here using the ganglion marker ISL1 (in red). (Ci-iii) A separation of the inner ganglion layer (shown by ISL-1 in red), and the outer photoreceptor layer (shown by the common photoreceptor progenitor marker RCVRN, in green), was seen by day 45. (D–F) Quantitative RNA sequencing data of cell specific markers showed sequential development of retinal cell types in our organoids in line with other published protocols. (D) An average of multiple markers for each cell type show cell development over time. (E) Common pluripotent and early developmental markers showed expression with comparable results to previously published protocols (Table 1). (F) Retinal ganglion cell markers show peak expression between Day 32 and 63 before decreasing over time. Ganglion subtype markers were also shown to be present (FOXP2, FSTL4), as well as markers strongly associated with ISL1 and SNCG (EBF1, EBF3).
Fully differentiated retinal organoids characteristically have a laminated morphology, with ganglion cells found on the inner-most layer of the organoid, and photoreceptors located on the outside. To confirm this in our system, we sectioned and stained our MG/FF organoids at different developmental timepoints. By 32 days, a defined layer of ganglion cells has developed within the organoids, similar to an in vivo retina, that stain positive for ISL-1 and PAX6 (Fig. 3b). We observed a large difference between the strong PAX6 staining on the outer edge of the organoid and weak PAX6 staining in the inner layer (Fig. 3b). Close up analysis of the inner layer of ganglion cells show strong co-expression of both ISL-1 and PAX6 at day 32 (Supplementary Fig. S1). This advanced by day 45 to include the outer layer of photoreceptor progenitors (Fig. 3c). However, we observed that when using our MG/FF protocol, the organoids ultimately start to lose this structure around day 50–60 of differentiation. This could be offset by the addition of external factors such as Taurine, Retinoic Acid and Triiodo-l-Thyronine (T3) alongside FBS, a mixture commonly used to enhance long term layering and photoreceptor development[
In our MG/FF culture, the progression of the organoids through the different lineages was measured by the presence of different retinal developmental markers. Using RNA sequencing we analysed the presence and expression of multiple different cell type specific markers at a number of different timepoints. Figure 3d shows the average trend in expression for different sets of cell specific markers. We found that early retinal markers (n = 7) increase dramatically once differentiation is forced into the retinal lineage, before peaking at an early retinal organoid timepoint at day 25. RPE-specific markers (n = 5) increase steadily over the course of the differentiation, whilst RGC (n = 22) and horizontal cell (n = 7) specific markers increased in expression during the earlier cultures, reaching their peak at day 63 and subsiding during long term cultures. Amacrine markers (n = 8) also increase in expression during the early cultures before reaching a consistent level of expression from day 63 onwards. Finally, photoreceptor (n = 32), bipolar (n = 6) and Müller (n = 2) cell markers are not present during the earlier timepoints, but increase rapidly from day 63 onwards until the final timepoint at day 160.
We analysed a few commonly used markers in detail. We found high RNA expression of the known pluripotency markers NANOG and LIN28 in the initial stem cell and embryoid body stages, before decreasing once retinal differentiation started. Overlapping with the expression of these markers, the early neural and retinal progenitor markers PAX6 and RAX are expressed from day 11 onwards. Highly similar to the RAX expression pattern, the optic cup specific marker VSX2 increases from a low expression on Day 11 to a peak in expression on Day 25, remaining highly expressed throughout the culture. The ganglion specific markers ATOH7 and POU4F2 start to be expressed at day 16 and 25 respectively, peaking at days 32 and 63, before gradually decreasing in the later timepoints up until day 160. The presence of ganglion cells was also suggested by the expression of other commonly used markers such as ISL1, SNCG and HuC/D (In this case ELAVL4 refers to HuD), all increasing in expression during the early stages of organoid development, before peaking between days 32 and 63, and decreasing gradually up to day 160.
Interestingly, we also found expression of RGC subtype specific markers, such as FOXP2 and FSTL4. These are markers of F-type ganglion cells and ON/OFF ganglion cells respectively, and were also highly expressed between days 32–63. There was also expression of genes known to interact with ganglion markers ISL1 and SNCG[
Although our focus was to differentiate early ganglion cells for glaucoma disease modelling, we also aim to use our organoids for a variety of different disease models. Therefore we further analysed the cultures for the presence of RPE and other retinal cells. We observed areas of pigmentation within our organoids by 25 days of total differentiation (Fig. 4a). Pigmented organoids were subsequently dissociated into single cells and seeded on transwell inserts, to grow out and mature into an RPE monolayer (Fig. 4b) that strongly expresses the tight junction protein ZO-1, (Fig. 4c). RNA analysis of whole organoids indicates early expression of RPE progenitors such as MITF, which is present from day 4 onwards with the highest expression at day 25, before making way for the mature RPE marker RPE65, that appears from day 63 onwards (Fig. 4f).
Graph: Figure 4 Generation of RPE and other retinal cell types. (A) A subset of organoids developed visible pigmented areas by day 36. (B,Ci-iii) These were dissociated and grown as a monolayer that had the characteristic cobblestone appearance and ZO-1 tight junction presence (in red) of RPE. (Di-iii) According to our quantitative marker data, bipolar cells appeared in culture within 4 weeks. The presence of bipolar cells was confirmed by staining for PKCα (in green) at day 28. (Ei-iii) Dissociated organoids grew out Müller cells, which were confirmed by staining for GFAP (in red). (F) In addition to bipolar, horizontal, Müller and pigmented epithelial cells, amacrine cells were also found within the organoid, suggested by RNA sequencing data.
The horizontal marker PROX1 increases in expression gradually over the differentiation, with a significant increase between days 16 and 25, sustaining high expression after day 25 until the final timepoint at day 160. Similarly, the amacrine marker GAD1 exhibits low levels of expression until day 63 onwards. Bipolar cell markers PKCa and CaBP5 are expressed in early and late stage cultures respectively, with confirmation using IHC at day 28 (Fig. 4d). Müller cell markers GFAP and RLBP1 show no expression in the early timepoints, but steadily increase in expression from day 63 until the final timepoint at day 160, confirmed at day 96 by IHC staining with GFAP (Fig. 4e).
Generally, the last major retinal cell type to develop is the photoreceptor[
Graph: Figure 5 Generation of photoreceptors. (Ai-ii) Widefield images of plated photoreceptors at D168 (black arrow), obtained from dissociated organoids, show outgrowth that is morphologically different to plated ganglion cells (Shown in Fig. 6a) (Scale bar = 200 µm). (B) Quantitative RNA sequencing data of common photoreceptor progenitor markers CRX and RCVRN showed increasing expression after day 63, while the mature cone and rod markers ARR3, OPN1SW and NRL, RHO also increased over time, although not as dramatically. (Ci-iii) Expression of photoreceptor progenitor markers was seen with IHC staining of RCVRN (in red) with the general neuronal marker β-III Tubulin (in green). (Di-iii) More mature photoreceptors were stained using anti-rhodopsin antibody ROD (in red).
We initially observed that in our matrigel based protocol, withholding additional external factors meant that long term lamination was lost around day 50–60 onwards.
However, all cell types still develop within the organoid, while the morphology of the organoid turns into a sphere of rosettes. Within these rosettes photoreceptors develop and mature with other cell types appearing at the outer edges of the rosettes, in their corresponding layers. To rectify this loss of lamination, we added a mixture of FBS, Taurine, Retinoic Acid and T3, commonly used to enhance long term layering and photoreceptor development. Initially we copied a timeframe derived from previously published protocols, starting treatment around day 45[
Graph: Figure 6 Long term lamination of retinal organoids. Organoids were either 'treated' by the addition of the external factors FBS, Taurine, RA or T3, or left 'untreated' in standard RDM. A comparison between long term cultures of untreated (Ai–Aiii) and treated (Bi–Biii) retinal organoids showed significant differences. (Ai) Untreated organoids appear extremely dark and dense, and contain photoreceptor rosettes. (Bi) Treated organoids retain their golden outer layer, and develop photoreceptors outer segments (black arrows) (Scale bars = 400 µm). (Aii) Photoreceptors develop within defined rosettes on the outermost part of the untreated organoid, shown by staining with the photoreceptor progenitor marker, RCVRN (in red). (Bii) Treated organoids contain an outer layer of photoreceptors surrounding the organoid, shown by RCVRN (in red). (Aiii) Untreated organoids also do not develop Red-Green cones by Day 160, suggested by lack of RNA expression and IHC staining. (Biii) The addition of T3 in the treated organoid cultures stimulated the development of Red-Green cone photoreceptors. (Aiiii) Although having lost the lamination of the organoid and developing rosettes, untreated organoids did still develop other cells around the rosettes, such as horizontal cells, found at the bottom of the photoreceptors. (Biiii) Treated organoids retain an multi-layered appearance, with photoreceptors on the outer edge of the organoid, and other cells positioned below as found in the in vivo retina.
An important drawback of generating retinal ganglion cells for cell replacement treatments is the length of time needed to generate these cells and the great quantities that are needed. Most recently published protocols need between 40 and 50 days to generate retinal ganglion cells (see Lee et al.[
Graph: Figure 7 Confirmation of the presence of retinal ganglion cells. (A) Widefield images of ganglion cells observed in culture showed polar cell bodies and an outgrowth of long axons by 32 days of culture. (B) When calculating the efficiency of ganglion cell generation, we found approximately 30–50% of cells were positive for ganglion cell specific markers when compared to general DAPI staining. The early ganglion marker ATOH7 showed positive staining in over 50% of cells (n = 510), with SNCG, a marker highly present in mature RGCs alongside BRN3B, showing positive staining in approximately a third of cells (n = 1779). Other commonly used RGC markers, although not completely specific, showed similar levels of positive staining. ISL1 was present in approximately a third of total cells (n = 1251), with HuC/D staining observed in almost half of all cells (n = 746). (C) After dissociating organoids at day 23, we observed a consistent amount of RGC outgrowth after 5 days. (Di-iii, Ei-iii) IHC staining of early RGC cultures using multiple ganglion markers SNCG and ISL-1 (both in red) showed positive expression and neurite outgrowth within 4 weeks of differentiation, shown by βIII-Tubulin (in green). (Fi-iii, Gi-iii) Plated cells from timepoints after 90 days of culturing show expression of the ganglion specific markers ISL-1 and RBPMS (both in red) co-stained with the general nerual marker βIII-tubulin (in green). (Hi-iii): Later stage cultures at day 96 showed expression of the RGC and amacrine marker HuC/D (in green) alongside Müller cells, identified using GFAP (in red).
The generation of a rapid, reliable human model system to study retinal diseases has long been a challenging limitation in eye research. In the past, the most common methods were either to use immortal cell lines such as ARPE-19 or explants from freshly harvested human donor eyes. Neither of these are ideal, either due to different expression profiles for immortal cells, or short lifespans for explants. The generation of murine organoids in 2011[
The addition of specific inhibiting or promoting factors can direct retinal differentiation in vitro through the manipulation of certain signalling pathways. The Wnt, BMP, TGF-β and insulin signalling pathways are all responsible for sending developing organoids down the retinal lineage[
By using matrigel, we introduced an average concentration of IGF-1 of more than 1.5× the concentration used in previous cultures[
We used a range of specific cell type markers to compare our results with those of previously published studies (Table 1). The initial expression of certain developmental markers differed extensively across the many previously published studies. However in most cases, the early developmental markers PAX6 and RAX are simultaneously expressed first in culture, before early retinal-specific markers such as CHX10 and MITF join them. Our data shows PAX6, RAX, CHX10 and MITF expression by day 11, having slightly earlier expression than comparable studies[
In our study, retinal ganglion cells were present from an early point, with RNA sequencing showing robust ISL1 expression from day 11, ATOH7 expression from day 16 and the mature marker POU4F2 appearing by day 25, similar to data presented in other studies[
Apart from ganglion cells, we also generated and identified other retinal cell types in our organoids, such as RPE cells. Robust expression of early RPE markers such as MITF was shown within 11 days, and an RPE monolayer with tight junctions was confirmed by day 56, which mirrored other studies focused around generating RPE cells[
Our findings suggest that the addition of high doses of growth factors, such as IGF-1, in matrigel during the early stages of development may boost temporal retinal organoid differentiation compared to conventional floating body protocols[
We refrained from adding specific cell-promoting external factors such as Retinoic Acid or Taurine into the medium, which led to loss of accelerated development, lack of lamination and rosette formation over time. Nonetheless, we observed photoreceptors in comparable timeframes to previous studies[
Overall, our study presents a rapid and reproducible protocol to generate versatile retinal organoids for disease modelling in a shorter timeframe. An initial boost using a 3D matrigel culture containing high concentrations of growth factors leads to early differentiation of EBs. These present with a more structured and organised morphology compared to floating culture EBs, and result in a shorter timeframe for generation of retinal ganglion cells. Due to the lack of additional growth factors or small molecules in the organoid culture, we provide a versatile culture method that can be used to reliably model and study a number of retinal diseases. Common additions over time can be used to produce and maintain organoid lamination and develop fully differentiated organoids, including photoreceptor outer segments.
The H1 hESCs (WiCell) were maintained on hESC-qualified matrigel (Corning) coated 6 well plates with mTeSR1 medium (STEMCELL Technologies), passaging twice a week using gentle cell dissociation reagent (STEMCELL Technologies). Organoid formation followed the rough timeline and stage specification published previously[
Briefly, stem cell clones were dissociated into small clumps (around 100 µm in diameter) using 0.5 mM EDTA in PBS, and then suspended in undiluted matrigel (Corning). The matrigel suspension was then plated with 8 × 25 µl individual drops per 35 mm well, totally encasing the stem cell clumps in a matrigel microenvironment. After gelling for 10 min at 37 °C, 3 ml per well was added of a 3:1 ratio of mTeSR1 and neural induction medium (NIM), consisting of DMEM/F12 (-L-Glutamine) (1:1), N2 supplement, non-essential amino acids, heparin (2 µg/ml), PenStrep and GlutaMAX. Day of matrigel encasement was annotated as day 0, with the medium being changed on day 1, (1:1 ratio), day 2 (1:3 ratio) and day 3 (full NIM), as shown previously[
To generate individual neural retinal cell types, whole organoids were dissociated using StemPro Accutase (Thermofisher) following the previously published protocol[
For whole organoid immunohistochemistry (IHC) analysis, EBs and organoids were fixed for 25 and 40 min respectively using 4% Paraformaldehyde (PFA) in PBS, before being mounted in O.C.T Tissue-Tek (Sakura Finetek) on dry ice, and sectioned using a microtome at 10 µm thickness. For dissociated organoids, cells were fixed in 4% PFA for 20 min. Fixation was followed by 3 washes with PBS for 5 min before blocking for an hour at room temperature with 10% donkey serum and 0.2% Triton X-100 in PBS for permeabilisation. Primary antibodies (Supplementary Table S1) were applied overnight in 0.1% Triton X-100, 5% donkey serum in PBS at 4 °C. Following 3 washes of 5 min with PBS, the secondary antibodies were applied for an hour at room temperature in 0.1% Triton X-100, 5% donkey serum mix and DAPI. After washes with PBS and water slides were dried for an hour at room temperature in the dark. Coverslips were mounted onto slides using ProLong Gold Antifade Mountant (Thermofisher).
RNA sequencing was performed as previously described[
This research was carried out under the Marie Skłodowska-Curie Horizon 2020 Innovative Training Networks program, Project ID 675033. I would like to thank Céline Koster for her input regarding 3D matrigel culturing, and I would also like to thank Dr. Jason Meyer, Dr. Sarah Ohlemacher, Dr. Clarisse Fligor and Dr. Kirstin VanderWall at IUPUI, IN, USA for their help and guidance when learning about organoid culturing.
P.E.W., A.L.M.A. and J.B.B. performed experiments, P.E.W., A.L.M.A., N.M.J. and A.A.B.B. wrote the main manuscript text, and all authors reviewed the manuscript.
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
The authors declare no competing interests.
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By Philip E. Wagstaff; Anneloor L. M. A. ten Asbroek; Jacoline B. ten Brink; Nomdo M. Jansonius and Arthur A. B. Bergen
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