Retinal cell 3D culture

Abstract

The present invention relates to methods for obtaining a retinal progenitor cell and related products from stem cells. The present invention also relates to retinal progenitor cells that may be produced by said method and the uses of said retinal progenitor cells, such as further differentiation of said retinal progenitor cells into retinal pigment epithelium (RPE) cells and retinal organoids. The invention also relates to a suspension cell culture comprising a retinal progenitor cell or a cluster thereof obtained by the method. The invention also relates to a suspension cell culture comprising a retinal progenitor cell or a cluster thereof obtained by the method. In addition, the invention refers to CD133 as marker for identification / enrichment / isolation of photoreceptor cells.

Description

RETINAL CELL 3D CULTURETECHNICAL FIELD

[0001] The present invention relates to methods for obtaining a retinal progenitor cell and related products from stem cells The present invention also relates to retinal progenitor cells that may be produced by said method and the uses of said retinal progenitor cells, such as further differentiation of said retinal progenitor cells into retinal pigment epithelium (RPE) cells and retinal organoids. The invention also relates to a suspension cell culture comprising a retinal progenitor cell or a cluster thereof obtained by the method. In addition, the invention refers to CD133 as marked for identification / enrichment / isolation of photoreceptor cells.BACKGROUND OF THE INVENTION

[0002] The retina is a light-sensitive layer at the back of the eye, which captures incoming photons and transforms them into electrical and chemical signals that travel along the optic nerve to the brain, enabling perception of the world in images During embryonic development, retinal progenitor cells give rise to a range of diverse retinal cell types, which eventually form a highly structured and layered adult retina The neural part of the retina is comprised of several different neuronal cell types, including photoreceptors (rods and cones) and is supported by an outer layer of retinal pigment epithelium (RPE) cells.

[0003] RPE cells form a densely packed monolayer and constitute, together with Bruch’s membrane, the blood-retinal barrier, which separates the retina from the choroid RPE cells are highly pigmented and able to absorb stray photons, which are not captured by photoreceptors. In addition, RPE cells maintain photoreceptor cell health by phagocytosing the tips of the photoreceptor outer segments, thereby removing and recycling toxic components of the visual cycle Furthermore, RPE cells secret growth factors, such as VEGF, and transport ions and metabolic products from the subretinal space to the choroid, whilst taking up vital nutrients, such as glucose and fatty acids, from the blood stream to provide nutrients for the neuronal retina.

[0004] Given these multiple roles, disruption of RPE cell integrity or function leads to a plethora of retinal diseases, such as age-related macular degeneration (AMD), Best vitelliform macular dystrophy and Stargardt’s disease. Here, the malfunction and subsequent loss of RPE cells precedes photoreceptor cell loss and subsequent blindness in patients. In addition, certain types of retinitis pigmentosa can also cause RPE cell loss, either before or after photoreceptor degeneration. Since RPE cells play such a vital role in retinal health and their loss is a cause for many retinal diseases, much interest exists to better understand the disease mechanisms that ultimately lead to these blinding diseases.

[0005] The neuronal retina is a complex, layered structure where the highly specialized neurons called photoreceptors convert light into a neuronal signal, which is passed to the optic nerve and into the brain. The capture of photons by rods and cones and the subsequent phototransduction cycle generates high levels of reactive oxygen species, as well as toxic byproducts, such as all-trans retinal. The underlying RPE cells support photoreceptor health through phagocytosis of photoreceptor outer segments, as well as recycling of visual cycle components. Disruptions of this tightly regulated phototransduction cascade lead to severe blinding diseases Inherited retinal diseases often affect important photoreceptor genes without any treatment options

[0006] Currently, there is a lack of suitable in vitro models to understand disease processes, such as retina-related diseases, as well as to develop suitable drug treatments for retinal degenerations. In vitromodels, such as immortalized RPE cell lines, for example ARPE19 cells, exist, but their suitability to study and fully understand relevant disease pathways is limited due to their lack of RPE cell specific morphology and function. Very strikingly, those traditional in vitro systems lack cellular pigmentation and cobblestone morphology, which are both hallmarks of RPE cells in vivo. iPSC-RPE cells have filled this void by faithfully replicating morphology, gene and protein expression, as well as functionality of RPE cells in vivo. Similarly, photoreceptors can only be derived by differentiation of iPSCs into 3D retinal organoids, mimicking human developmental timelines. However, the differentiation of iPSC RPE and iPSC-derived retinal organoids is laborious. For example, through the requirement of manual dissecting of cells that have differentiated towards RPE cells from a monolayer of non-RPE cells and subsequent passaging of these cells to obtain a pure cell population. Similarly, manual dissection of neuroepithelia, which will be further differentiated into retinal organoids, is a common approach to generate photoreceptors These approaches are labour intensive and result in unpredictable differentiation outcomes, also in terms of homogeneity of the obtained iPSC RPE and / or retinal organoid cell populations, due to the high level of manual manipulation required Likewise, the number of obtainable IPSC RPE and / or photoreceptor cells is limited, and the process does not lend itself to up-scaling procedures To utilize iPSC RPE and iPSC-derived cells from retinal organoids, such as photoreceptors, as a superior cell model, in high throughput drug screening campaigns or cell replacement therapies, large numbers of reproducibly differentiated RPE, as well as photoreceptor cell batches, which are cryopreservable and require a minimum of manual and / or technical manipulation are vital

[0007] It is therefore desirable to develop novel methods for rapidly, efficiently, and reproducibly obtaining large numbers of RPE and / or photoreceptor cells. In particular, it would be desirable to be able to use human iPSCs to generate patient-derived and, thus, more physiologically relevant RPE and photoreceptor cells

[0008] Such RPE and / or photoreceptor cells may have utility in treating and / or preventing various pathological conditions, as well as in research settings such as for screening therapeutics.SUMMARY OF THE INVENTION

[0009] The present invention refers to a highly efficient and robust method of differentiating stem cells into retinal progenitor cells, which can be further differentiated into all types of retinal cells The present invention in particular covers the differentiation into (i) an RPE monolayer composed of a single cell type, the RPE cells and (ii) a retinal organoid, a 3D simplified model of the retina, where morphology, development, structural complexity, and some key functions (production of outer segments, light sensibility) are recapitulated.

[0010] In particular, the invention refers to a method comprising the steps: culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies (also denoted as step b herein); and culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates (also denoted as step c herein)

[0011] 3D suspension cultures during differentiation of the stem cells to eye-field aggregates allows the cells to form floating homogenous spheres throughout the differentiation process. This makes the process efficient, scalable, and robust. Thereby a homogenous composition of differentiated cells can be achieved at the different differentiation stages.In particular, the inventors could show that by the method of the invention high quality eye field cell aggregates as well as RPEs can be obtained. This is reflected by the homogenous population of cells expressing high levels of OXT2 and PAX6.Moreover, homogenous compositions of retinal organoids can be achieved by the method of the invention. That means that the suspension of retinal organoids retinal organoids is homogenous in terms of the type of the cell aggregates contained therein, i.e. it contains only retinal organoids and no other cell aggregates with a different differentiation fate.

[0012] The present invention relates to a new method for rapidly and efficiently obtaining RPE cells and retinal organoids from induced pluripotent stem cells (iPSCs) using a 3D culture protocol. The RPE cells and retinal organoids are obtained from iPSCs initially grown as suspension clusters. For RPE differentiation, these are replated on transwells at the stage of eyefield specification, while for retinal organoids, the cell clusters are continuously cultured in suspension. Advantageously, the 3D culture protocol is highly efficient with the potential to upscale for generating any major cell type in the eye, i e. terminally differentiated cells of the retina (such as retinal pigment epithelial cells and photoreceptors), by subsequently differentiating human iPSCs The invention also relates to cells produced by the new method, as well as cells expanded and / or differentiated from cells produced by the new method and uses of these cells The cells may be produced directly or indirectly with the new method.

[0013] The method of the invention is robust, efficient and cost saving and allows the generation of a large quantity of cryopreservable iPSC-derived RPE and photoreceptor cells These iPSC-RPE and / or photoreceptor cells may be banked, for example as cryostocks. Thus, a large number of iPSC-derived RPE and / or photoreceptor cells can be obtained and banked. Cryostability of frozen stocks allows for efficient upscaling and up-scaled production enables cost-effective high throughput screening (HTS), in particular for retinal toxicity and / or for drug discovery and / or target regulation, as well as retinal cell replacement approaches.

[0014] With the system described herein, starting at ddO with a 6-well plate with a growth area of 57,6cm2 (12 mio cells) about 2 to 3 million (mio) iPSC per well can be produced that give rise to a final RPE cell production in about 63 days of around 2 billion RPE cells or more, whereas in the 3D embedding system an equivalent 100mm dish with a growth area of 56,7cm2 and 12 mio iPS cells, approximately only 208 mio RPE cells can be produced.

[0015] A single full time equivalent (FTE) skilled person is capable to robustly produce 2,280 billion RPE cells or more from one batch of 22 mio iPSC. Using the 3D embedding system, starting with a similar number of iPSCs, one FTE can produce 730 mio RPE cells in one batch, albeit with high differences in differentiation outcomes.

[0016] Therefore, overall the 3D suspension differentiation protocol of the present invention is superior to classical 2D and 3D embedding protocols in at least the following aspects: (i) the 3D suspension iPSC- derived RPE cells can be more reliably produced and successful differentiation outcomes can be predicted very early on; (ii) only limited manual manipulation of cells is necessary, and (iii) potential to upscale iPSC- derived RPE production to enable high-throughput drug discovery campaigns, as well as cell therapy efforts.

[0017] Thus, the invention provides for the first time a way to differentiate iPSC-derived RPE cells from an initial suspension culture and to subsequent cultivation in 2D format. The iPSC-RPE cells acquire typical RPE cell morphology and protein expression and show, for instance, high expression of RPE cell markers Their strong gene and / or protein expression profile for important eye field specification markers early in the 3D-suspension culture provides a reliable prediction of a successful differentiation process. Furthermore, the newly developed 3D protocol enables cost efficient production of large amounts of functional iPSC- derived RPE cells and retinal organoids. In addition, or alternatively, the newly developed 3D protocol enables cost efficient production of large amounts of functional cryopreservable iPSC-derived RPE cells and / or cryostocks from cells of retinal lineage, for example through batch production. Thus, the invention allows for storage of RPE cells and / or cells of retinal lineage and enables high throughput screening, e.g , for drug discovery and / or transcriptomic profiling of drug candidates, including miniaturization into wells, forinstance, of 384 plates or their equivalent. In addition, the RPE cells and / or cells of retinal lineage produced according to the invention will have utility in treating and / or preventing various pathological conditions.

[0018] More specifically, the application discloses the following aspects:

[0019] A first aspect refers to a method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies; and(c) culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates.

[0020] Typically, in any one of steps (a), (b) and (c) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel In one specific embodiment, the 3D suspension culture is carried out in ultra-low attachment cell culture plates.

[0021] Preferably, in step (b) and / or step (c) shear stress is applied to the 3D suspension culture. The inventors found that the application of shear stress improves quality of the cell aggregates, in particular improves homogenous development of the cell aggregates Shear stress can for example be applied by agitating the 3D suspension culture. Hence, some embodiments, step (b) and / or step (c) may occur under agitation e.g. at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm.

[0022] The embryoid bodies obtained in step (b) comprise cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1 In some embodiments, the embryoid bodies comprise at least 60%, at least 70%, at least 80%, at least 90% cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1 .Typically, the embryoid bodies obtained in step (b) have a uniform shape and / or a diameter of about 150 pm. In one embodiment, the embryoid bodies comprise cells that are positive for one or more pluripotency markers as described above and are further positive for at least one eye field marker, such as OTX2 and / or PAX6

[0023] Another aspect refers to the eye-field cell aggregates obtained by the method as described herein. The eye-field cell aggregates may comprise cells expressing at least one of the eye field transcription factors PAX6, RAX, SIX3, LHX2 and OTX2. In one embodiment the eye-field cell aggregates comprise cells expressing PAX6 and OTX2 In one embodiment the eye-field cell aggregates comprise cells expressing RAX, PAX6 and OTX2. In specific embodiments, the eye-field cell aggregates comprise at least 50%, preferably at least 60%, more preferably at least 80% cells expressing PAX6 and OTX2

[0024] In specific embodiments, the single cell suspension of stem cells of step (a) is provided by the following steps:(a1 ) culturing stem cells in adherent culture; and(a2) dissociating the stem cells to obtain single cell suspension.

[0025] Step (a1 ) may last 3 to 7 days 3 to 6 days, more preferably 3 to 5 days, most preferably 4 to 5 days; Step (b) may last 1 to 6 days, preferably 2 to 5 days, more preferably 3 to 5 days, even more preferably 4 days. Step (c) may last 3 to 7 days, preferably 3 to 6 days, more preferably 4 to 5 days, most preferably 4 days. Typically, in step (a1 ) the stem cells are cultured until 70-80% confluency. In one embodiment, the stem cells express SOX2, Nanog, Oct4, SSEA1 and Tra1. In specific embodiments, at least 60%, at least 70%, at least 80%, at least 90% of the stem cells of step (a) express SOX2, Nanog, Oct4, SSEA1 and Tra1.

[0026] Typically, in step (b) a different medium is used as in step (c). In one embodiment, the medium in step (b) contains TGF-p and bFGF. In one embodiment no inhibitor selected from the group consisting of TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor and GSK3p inhibitor is used in steps (a), (b), (c), (d), (e), (f), (dd) and (ee). In one embodiment no inhibitor selected from the group consisting of TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor and GSK3p inhibitor is used throughout the protocol. In one embodiment no inhibitor selected from the group consisting of TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor, SMAD inhibitor, BMP inhibitor and GSK3p inhibitor is used throughout the protocol. In one embodiment no other inhibitor than ROCK inhibitor is used in throughout the protocol, i.e in steps (a), (b), (c), (d), (e), (f), (dd) and (ee). Typically, in step(b) a stem cell medium is used, optionally supplemented with a ROCK inhibitor. In one embodiment, the stem cell medium used in step (b) is supplemented with a ROCK inhibitor for a first period of step (b). Preferably, the first period of step (b) begins with the onset of the culturing of the single cell suspension and lasts for 2 to 48 h, preferably 12 to 36 h, most preferably 24 h. In one embodiment, in step (b) cells at a final concentration of 1 X 106cells / ml are seeded in ultra-low attachment cell culture plates. In a specific embodiment, in step (b) a total of 5,5 X 106cells were seeded per 1 ,6 cm2. In step (c) a medium comprising neurobasal medium may be used.

[0027] Another aspect of the invention refers to a method of differentiating stem cells into retinal organoids, comprising the steps (b) and (c), preferably (a), (b) and (c) as described herein, and further comprising the steps:(d) culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors;(e) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain early phase retinal organoids,(f) optionally culturing the early phase retinal organoids in a 3D suspension culture system to obtain late phase retinal organoids.

[0028] In one embodiment, any one of steps (d), (e) and (f) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel.

[0029] In one embodiment, the eye-field cell aggregates comprise at least 70%, preferably at least 80% cells expressing PAX6 and OTX2.

[0030] In one embodiment, the early phase retinal organoids obtained in step (e) are positive for at least one marker selected from the group of CRX, RECOVERIN and RXRy

[0031] In one embodiment culturing in step (d) comprises:(d1 ) culturing the eye-field cell aggregates in a medium comprising neurobasal medium; and(d2) culturing the eye-field cell aggregates obtained in step (d1 ) in a high glucose medium supplemented with retinoic acid.

[0032] Preferably, culturing in step (d2) occurs in a medium which does not contain neurobasal medium

[0033] In one embodiment, culturing in step (e) comprises:(e1 ) culturing in a high glucose medium supplemented with FBS and retinoic acid; and (e2) culturing in a high glucose medium supplemented with FBS, retinoic acid and lipids

[0034] Preferably, culturing in (e2) occurs in a medium having a lower retinoic acid concentration compared to the medium of (e1 ). In one embodiment, culturing in step (f) comprises culturing in a retinal maturation medium, without addition of small molecules. In a specific embodiment, culturing in (f) comprises culturing in a high glucose medium, preferably comprising at least 20 mM glucose, such as 25 mM glucose, supplemented with FBS and lipids. Preferably the medium of (f) does not contain retinoid acid supplementation.

[0035] Another aspect of the invention refers to a method for differentiating stem cells into retinal pigmented epithelium cells, comprising the steps (b) and (c), preferably (a), (b) and (c), as described herein and further comprising the steps:(dd) dissociating the eye-field cell aggregates to single cells;(ee) culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells.

[0036] In one embodiment, the eye-field cell aggregates comprise at least 50%, preferably at least 60% cells expressing PAX6 and OTX2. In one embodiment, in step (dd) and / or step (ee), the cells form a monolayer.

[0037] In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of SIX3, LHX2, RAX, EZRIN, CLDN19, MERTK, MITF, PMEL, RLBP1 , PAX6,RPE65, SERPINF1 , TYRP1 , VMD2, ZO-1, BEST 1 and ACTIN-F. In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of CLDN19, MERTK, MITF, PAX6, RPE65, SERPINF1, TYRP1 , VMD2, BEST 1 , EZRIN, ACTIN-F, PMEL and ZO-1 In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of MITF, PAX6, RPE65, TYRP1 , EZRIN, ACTIN-F, MERTK, PMEL and ZO-1. In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of MITF, PAX6, RPE65 and TYRP1

[0038] In one embodiment, the retinal pigmented epithelium cells have a functional capacity, for example phagocytic capacity transepithelial resistance (TER) and / or VEGF secretion.

[0039] In one embodiment, culturing in (ee) comprises culturing in a RPE differentiation medium which is optionally a serum-free medium, wherein said medium comprises at least one growth factor selected from agonists of the Activin A pathway

[0040] In one embodiment, culturing in (ee) comprises(eel ) culturing in a medium comprising neurobasal medium supplemented with a ROCK inhibitor; and (ee2) culturing in a RPE differentiation medium supplemented with at least one agonists of the Activin A pathway.

[0041] In one embodiment culturing in (ee) occurs in a culture vessel with an upper and lower chamber, optionally wherein said culture vessel is a transwell plate, optionally coated with at least two proteins, and preferably at least three proteins, selected from the group consisting of Laminin, Collagen IV, Entactin and Perlecan. Preferably, step (ee) lasts 10 to 200 days, 12 to 150 days, 15 to 100 days, 20 to 80 days, 30 to 70 days, 40 to 60 days.

[0042] The duration of the complete process starting from iPSC suspension culture to a late phase retinal organoid lasts about 100 to 140 days, such 105 to 130 days, even more preferably 110 to 125, most preferably 120 days These late phase retinal organoids (and the corresponding cell population obtained by dissociating the late phase retinal organoids) contain a high percentage of photoreceptor cells, including rods and cones. These late phase retinal organoids (and the corresponding cell population obtained by dissociating the late phase retinal organoids) are devoid of pluripotent cells and / or proliferating cells / and / or precursor cells and / or retinal precursor cells, and are at the desired committed but not fully mature stage, which is optimal for transplantation. The inventors established for the first time a highly reliable protocol that allows the differentiation to a homogenous cell population comprising differentiated photoreceptor cells, at the optimal stage for transplantation within this short time frame In particular the protocol allows for the first time the differentiation of iPSCs into a homogenous cell population comprising early opsin-expressing photoreceptors cells in a short time period, of about 100 to 140 days, such 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.

[0043] In one embodiment, the stem cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell and / or established stem cell line, preferably a pluripotent stem cell. Preferably, the stem cell is a mammalian stem cell; more preferably a human stem cell, most preferably a human induced pluripotent stem cell.

[0044] Typically, the method is carried out in the absence of a feeder cell.

[0045] Typically, an exogenous nucleic acid and / or exogenous protein is not introduced into the stem cells or differentiated cells derived thereof.

[0046] In one embodiment, the method further comprises the subsequent step of cryopreserving the organoids and / or cells obtained.

[0047] In one embodiment the method comprises steps of dissociation of the retinal organoid and sorting the cells obtained according to their cell fate

[0048] Another aspect relates to all cells and / or organoids obtained by the methods including all intermediates states thereof. One embodiment refers to the retinal organoid and / or the retinal pigmentedepithelium cells obtained by the method described herein. Another aspect refers to the retinal organoid as described herein for use in therapy, preferably transplantation therapy. In one embodiment, the retinal organoid is for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophy. Another aspect refers to the retinal organoid as described herein in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivo

[0049] Another aspect refers to the cells obtained from the retinal organoid as described herein. Another aspect refers to the cells obtained from the retinal organoid as described herein for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophy. Another aspect refers to the use of the cells obtained from the retinal organoid as described herein in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivo

[0050] Another aspect refers to the retinal pigmented epithelium cells as described herein for use in therapy, preferably transplantation therapy. In one embodiment, the retinal pigmented epithelium cells are for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophy Another embodiment refers to the use of the retinal pigmented epithelium cells as claimed herein, in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivo.

[0051] A further aspect relates to the efficient isolation of photoreceptor cells by a single marker CD133 for isolating photoreceptor cells. Contrary to the prior art teaching that CD133 cannot be used as single marker for photoreceptor cell isolation but only in combination with other biomarkers, such as CD73 (Lakowski et al , Stem Cells 2018 May 36(5): 709-722), the inventors found that CD133 as single marker can be successfully used for isolating photoreceptors with high yields

[0052] In particular, the inventors found that by using CD133 as single marker, the isolation of photoreceptor cells is highly efficient, e.g. leading to higher yields compared to sorting with CD73. In addition, since GMP antibodies are available for CD133 (compared to CD73, for which no GMP antibodies are available), the establishment of advanced therapy medicinal products (ATMP) production processes is much more efficient. In addition, the percentage of cones (important for color vision and detailed perception in daylight) in the enriched population is higher compared to sorting with CD73.BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic representation of an exemplary embodiment of the method for differentiating induced pluripotent stem cells (iPSCs) into retinal precursor clustered in eye field cell aggregates and further into RPE cells and retinal organoids according to the present invention using the differentiation protocol A common differentiation part up until differentiation day (dd) 8. B 2D differentiation into RPE cells up until dd63. C 3D differentiation into retinal organoids up until dd180

[0054] Figure 2 Detailed schematic representation of the differentiation procedure. A iPSCs and 3D suspension culture with differentiation days (dd), culturing format and medium B RPE cells from RPE precursor to RPE maturation with differentiation days (dd), culturing format and mediumC Retinal organoids from neuro-retinal precursors to late phase retinal organoids with differentiation days (dd), QC and medium of the earlier shown exemplary embodiment of the method for differentiating induced pluripotent stem cells (iPSCs) into retinal progenitor clustered in eye field cell aggregates and further into RPE cells and retinal organoids according to the present invention using the differentiation protocol.

[0055] Figure 3 Quality control and pluripotency assessment of iPSCs. A, B and C Exemplary embodiment of adherent iPSCs with phase contrast microscopy. Viability, quality and pluripotency capacity was determined based on morphology (large nucleus, with high nucleus:cytoplasmic ration, open chromatinand prominent nuclei), which was checked daily using phase contrast microscopy D, E and F Known pluripotency marker (SOX2, NANOG, OCT4, SSEA1 and TRA-1-60) were analysed using flow cytometry (FC).

[0056] Figure 4 Embryoid body formation and eye field specification. A and B Exemplary embodiment of brightfield imaging revealed embryoid body and eye field specification aggregates of differentiation at (A) day 3 (dd3) and at (B) day 8 (dd8) of the method. C, D, E and F Exemplary embodiment of flow cytometry (FC) analysis of the Evotec iPSC RPE differentiation method, showing that 93% of cells from the described method (B) are positive for PAX6 / OTX2 at day 8 (dd8). G and H Exemplary embodiment of maker for embryoid bodies and eye field cell aggregates as relative fold expression normalized to iPSCs.

[0057] Figure 5 Comparison of standard iPSC RPE differentiation (3D- embedding culture) and 2D- suspension culture (Evotec) shows the improved capacity with reliable high values for markers close to 80%. A Exemplary embodiment of FC analysis of the method compared to a standard iPSC RPE differentiation method, showing that 76,7% of cells from the standard protocol compared to 95% of cells from the described method (Evotec protocol) are positive for PAX6 / OTX2 at day 8 (dd8).B Comparison of standard iPSC RPE 3D- embedding culture (standard protocol) and 2D-suspension culture (Evotec protocol) shows reliable high values for markers PAX6 / OTX2 of approximately 80%, resulting in improved differentiation outcomes, as determined by RPE pigmentation capacity.

[0058] Figure 6 Development of RPE precursor morphology and pigmentation from dd 12 to dd28. A Exemplary embodiment of morphological development of a polygonal epithelial monolayer stained with phalloidin for Actin of RPE precursor dd9-16 and early RPE dd17-28 highlights increasing compactness and homogenous morphology of the RPE monolayer. B Exemplary embodiment for early RPE pigmentation development followed from dd12-28 assessed with brightfield imaging highlights increasing levels of pigmentation over the time of differentiation

[0059] Figure 7 Exemplary embodiment of qRT-PCR based expression of retinal precursor marker PAX6 (A) and RPE marker TYRP1 (B), MITF (C) and RPE65 (D) over the time course of the differentiation dd28, dd49 and dd63 Gene expression is normalized to the selected housekeeping genes GUSB, PPIA and RPL27

[0060] Figure 8 Comparison of RPE cell morphology and critical marker expression in maturing RPE cells between the standard protocol and Evotec’s protocol. A Exemplary embodiment of ZO-1 protein expression quantification on dd49 maturing RPE cells and dd63 mature RPE cells shows increased tight junction marker expression with Evotec’s protocol compared to the standard protocol B Exemplary embodiment of F-Actin protein expression quantification on dd49 maturing RPE cells and dd63 mature RPE cells shows increased compactness and honey-comb morphology with Evotec’s protocol compared to the standard protocol. C Exemplary embodiment of MITF protein expression quantification on dd49 maturing RPE cells and dd63 mature RPE cells shows increased expression of critical RPE markers using Evotec’s protocol compared to the standard protocol.

[0061] Figure 9 Exemplary embodiment of high content imaging of mature (dd63) iPSC-derived RPE cells reveal uniform expression of relevant RPE cell markers, such as MerTK, ZO-1 , EZRIN and PMEL-17 in RPE cells produced or producible by the method.

[0062] Figure 10 Functional characterization of mature RPE cells. A Phagocytosis kinetic of pHrodo labelled photoreceptor outer segments (POS). Comparison of internalization and degradation of POS on dd63 between Evotec’s iPSC-derived RPE cells compared to iPSC-RPE cells differentiated with the standard protocol shows earlier POS uptake and an increased degradation kinetic of cells generated with the Evotec protocol compared to cells from the standard protocol. B Exemplary embodiment of a VEGF ELISA shows polarized VEGF secretion of Evotec’s iPSC RPE cells on dd49 and dd63. C Exemplary embodiment of transepithelial resistance of Evotec’s iPSC RPE cells on dd49 and dd63 shows an increase of transepithelial resistance as RPE cells mature.

[0063] Figure 11 Optimization of Evotec’s protocol for neuro-retina differentiation A Exemplary embodiment of brightfield imaging showing differences between eye field aggregates (EFA) kept in M1 form d8 to d24 (left panel) and aggregates that were kept in media M4 (right panel). It is visible that aggregates fed with M4 developed an organized outer layer consistent with neuro-epithelial layer, which is absent in EFAs kept in M1. B and C Exemplary embodiment of measurement of the size of Eye-field aggregates over time (day 8 to day 25). Aggregates kept in M1 showed higher variability in size (B), while aggregates fed with M4 and M5 displayed a steady growth with more consistent sizing at each time point (C) (Mean, SD). D and E Exemplary embodiment of Immunohistochemistry using fluorescence microscopy, showing the difference in neuro-retinal precursors at day 25, in eye-field aggregates fed with 1 (D) compared to M4 (E), scale bar 200 pm In samples kept in M1 , VSX2 positive cells are rare (D). In samples fed with M4 almost all the cells in the most outer layer of the aggregate are positive for VSX2 (E) F Exemplary embodiment of gene expression, measured via qPCR, confirming the increased expression of VSX2 in EFAs fed with M4, compared to EFAs being fed with M1.

[0064] Figure 12 Regulation of early phase retinal organoid markers and induction of retinal ganglion cell development A Exemplary embodiment of gene expression, measured via qPCR, for neuro-retinal precursors markers, VSX2 and RAX, across different time points Both markers are not expressed in iPSCs and are upregulated by day 25 Peak expression occurs between day 50 and 60 followed by a decrease in expression, reflecting the restriction of VSX2 and RAX to specific cell types as part of retinal neuron maturation B Exemplary embodiment of Immunohistochemistry using fluorescence microscopy showing that, by day 100, stratification of retinal organoids increases, as more cell layers become evident within the retinal organoids (DAPI, top panels). VSX2 expression becomes restricted to a specific cell type and localizes to a more internal layer of the organoids (middle panels). The maturation of the organoids is further supported by the reduction in Ki67 positive cells, as on day 25 majority of the cells are positive for this proliferation marker and by day 100 most cells within the retinal organoid become non-proliferative. Scale bar 200pm for all images. C Exemplary embodiment of gene expression, measured via qPCR, for POI4F1 , a marker for mature retinal ganglion cells, showing that there is a peak of expression between day 50 and day 70 With eventual loss of expression of this marker at later time points D Exemplary embodiment of Immunohistochemistry using fluorescence microscopy confirming the present of retinal ganglion cells in the retinal organoids at day 70. Retinal ganglion cells were identified by staining for BRN3a (protein encoded by POU1 F4 gene), a marker for mature retinal ganglion cells, which co-localizes with pan neuron marker HuC / D, scale bar 200pm

[0065] Figure 13 Maturation of retinal organoids from day 100 to day 180. A Exemplary embodiment of brightfield imaging showing the morphological changes and maturation of retinal organoids across time. From day 25 to day 100 a neuro-epithelial layer (NEL) is visible. By day 120 retinal organoids become more stratified as more layers become better defined. On day 120 an outer nuclear layer-like layer, where mostly photoreceptor cells are found, can be identified. From day 150 onwards, further maturation can be seen by development of a visible brush-border, which corresponds to the area were photoreceptor specific structures, which are only present in mature cells and are essential for function, called inner and outer segments are located. This brush-border grows denser and elongates with time, as can be seen by day 180. B Exemplary embodiment of gene expression, measured via qPCR, for photoreceptor-specific genes, showing slight up regulation from day 50, followed by an increase and peaking between day 100 and 120. This corresponds to the peak of photoreceptor differentiation and identity commitment. After the peak expression, values decrease but the genes are still expressed as they are required for visual function. C Exemplary embodiment of Immunohistochemistry using fluorescence microscopy showing presence of photoreceptors and their localization, mainly, to the ONL-like layer of the organoid, at day 120 Presence of pan-photoreceptor markers (RECOVERIN and CRX) and early rod marker (NRL) in the ONL-like layer (top panel), recapitulating normal retinal development More mature markers (L / Mopsin and RHODOPSIN) canalso be seen and are mostly localized to the potential outer-segment-like area that is forming (bottom panel) and will eventually become visible in brightfield microscopy as a brush-border. D Exemplary embodiment of gene expression, measured via qPCR, for mature photoreceptor markers, required for visual functions These markers start being expressed only from day 70 or 100 and gene expression appears to reach a plateau by day 120 for all markers, except for RHODOPSIN, where a peak seems to be achieved only by day 150.

[0066] Figure 14 snRNA sequencing confirms presence of all relevant retinal cell types in retinal organoids. A Exemplary embodiment of transcriptomics data, via single nuclei RNA sequencing, at day 100. Rod photoreceptors and bipolar cells are the main cell populations. Cones can also be identified and categorized as M or S cones. All neuro retinal subtypes (bipolar, amacrine and ganglion cells, Muller glia) including a portion of RPE cells are also present The percentage of other cell types is 10% and all those are retinal cell types, such as Muller glia cells . B Exemplary embodiment of transcriptomics data, via single nuclei RNA sequencing, at day 120. Enrichment for photoreceptor cells (rods and cones) is shown, with rods and cones making up 49% of the cell population of the retinal organoids. As the percentage of photoreceptors increased from day 100 other interneurons and RPE cell proportions decrease The exception to this decrease are Muller glia cells, which double. This is expected and recapitulates in vivo retinal development. C Exemplary embodiment of transcriptomics data, via single nuclei RNA sequencing, at day 160. Clear further enrichment for photoreceptor cells (rods and cones) is shown with photoreceptor cells making up 60% of the total population The percentage of all other cell types decreases, with the exception of Muller glial cells. “Other” include inter alia horizontal cells and photoreceptor precursors. D Exemplary embodiment of transcriptomics data, comparing the present data, produced by single nuclei RNA sequencing, at day 120, with published single cell RNA sequencing data of retinal organoids, from a different cell line and protocol, at day 126 The present data showed good separation between rods and cones, which was not seen for the published data set. This is shown by specific cone markers, RXRy and MAP4, becoming restricted to cone photoreceptors in herein described organoids, while it is equally expressed in all photoreceptors, including rods, in the public data set. OPN1SW, a mature S cone marker, is expressed specifically in S cones in organoids produced by the protocol as described herein but absent in the age matching organoids from the public data set. As expected, NR2E3 expression is increased in rods derived from the organoids as described herein, while in the public data set it no differences between rods and cones was present. RHO, a mature rod marker, is already expressed in rods of the present organoids, but not expressed in the public data set at this time point E Exemplary embodiment of transcriptomics data, comparing the present data, produced by single nuclei RNA sequencing, at day 160, with published single cell RNA sequencing data of retinal organoids, from a different cell line and protocol, at day 168. The differences in maturation of photoreceptors are obvious, with mature markers (OPN1 MW, OPN1SW and RHO) being only expressed in present samples, being specific to the expected cell type (M cones, S cones and rods, respectively). Earlier rods markers (GNAT1 and NR2E3) are present in both data sets, showing that rod fate commitment has happened in the public data set, by day 168, but cells have not reached the same maturity as those from the protocol as described herein.

[0067] Figure 15 Characterization of day 3 population (embryoid bodies) regarding pluripotency markers A Exemplary embodiment of protein detection of pluripotency nuclear markers by flow cytometry In iPSCs stage, prior to dissociation and aggregation, -100% of cells remain positive for all markers By day 3 of aggregation, a decrease in the percentage of Nanog+ cells is detected This shows that surprisingly the aggregation itself, without any changes or additives to the media, is leading to the loss of pluripotency markers and that aggregating the cells under free floating, matrix free conditions leads to changes in the cells and promotes differentiation B Exemplary embodiment of the gene expression, detected by means of qPCR, of NANOG, OCT4 and SOX2 genes, normalized to the expression levels in iPSCs. There was a marked decreased in NANOG and OCT4 gene expression, already detectable by day 3 of aggregation, withcells at this stage expressing about 50% of lower values of these genes. By day 8 no expression of these genes was detected. Differences were also seen in the expression of SOX2, as this gene increased in expression, as can be detected in cells committed to a neuronal precursor fate, which strongly express SOX2.

[0068] Figure 16 Characterization of CD73 and CD133 in retinal organoids and its impact on sorting results at day 120 A Exemplary embodiment of gene expression, detected by qPCR, of PROM-1 , which encodes the CD133 protein, is expressed in iPSCs and remains expressed during differentiation. From day 25 to 70 there is an increase followed by an even more significant increase by day 100, which correlated with the time of differentiation of photoreceptor cells. NT5E, the gene that encodes CD73, is only expressed from day 70 onwards, when photoreceptors start to differentiate and its expression is lower than PROM1 and the increase rate is also lower B Exemplary embodiment of protein detection, by flow cytometry in dissociated organoids before (unsorted) and following sorting (sorted) for CD73 or CD133 as a selection marker. In the unsorted cell population the % of CD73 positive cells is lower than the % of CD133 cells. This is consistent with the gene expression data, as higher gene expression could reflect the presence of more positive cells C After calculating the number of cells in the positive fraction following cell sorting for each of the markers an increase in the number of isolated cells was detected, as CD133 selection led to a 1 ,5x increase of cells than using CD73 as a selection marker, while keeping a similar purity, as shown in C.D Exemplary embodiment of protein detection, by flow cytometry, in cells previously sorted for CD73.

[0069] Figure 17: Characterization of the population isolated from day 120 retinal organoids, using CD73 and CD133 as selective markers A Exemplary embodiment of transcriptomics data, via single nuclei RNA sequencing, at day 120, after dissociation of retinal organoids, staining with CD73 and magnetic sorting of the positive population. B Selection obtained when using CD133 as a selection marker. It is shown that this sorting method allows for enrichment of photoreceptors, both rods and cones Cone photoreceptors are mature enough to be further clustered as M or S cones. It is important to note that no pluripotent or proliferative cells were identified, showing that sorting for CD133 allowed the exclusion of such populations (pluripotent cells are already absent in the retinal organoids by this timepoint and a very small percentage of proliferating cells (less than 5%) was excluded by CD133 sorting) C and D quantification of the data displayed in A and B, showing high yields are achieved with CD133+ sorting. E Sorted cells were positive for the selection marker by immunostaining after seeding in 2D cultures. The identity of these cells as photoreceptor cells was confirmed by ICC for photoreceptor-specific markers CRX, in F, and RECOVEIN, in G For the population obtained by using CD133 as a selection marker (H) the results were similar, with cells preserving the sorting antibody and being positive for CRX (I) and RECOVERIN (J). Scale =20pmDETAILED DESCRIPTION OF THE INVENTION

[0070] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0071] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein

[0072] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0073] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0074] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the", this includes a plural of that noun unless something else is specifically stated Thus, for example, reference to “a stem cell” includes “stem cells”. The term “about” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0075] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0076] “Supplemented,” as used herein, refers to a composition, e.g., a medium comprising a supplemented component (e.g., ROCK inhibitor). For example, a medium “further supplemented” Rock inhibitor supplement, refers to the medium comprising ROCK inhibitor, and not to the act of introducing the ROCK inhibitor to the medium.

[0077] “High glucose medium” refers to a medium comprising at least 10 mM, preferably at least 12 mM, more preferably at least 15 mM, even more preferably at least 16, most preferably at least 17 mM glucose. The high glucose medium may comprise 10 to 50 mM, preferably 12 mM to 40 mM, even more preferably 15 mM to 30 mM For example, the glucose concentration in step (d2), (e1) and / or (e2) is 15 to 20 mM, may be 16 to 18 mM, about 17 mM, 17 5 mM, 18 mM glucose In another example, the glucose concentration in step (f) is 15 to 40 mM, preferably 20 to 30 mM, more preferably 22 to 27 mM, such as 25 mM.

[0078] The term “3D suspension culture” refers to a culture system in which cells forming 3D aggregates are floating in a cell culture medium In other words, the cells are cultured as floating aggregates in culture medium such that the cell aggregates are fully surrounded with medium. The 3D suspension culture may lack a (gel-like) matrix, such as a hydrogel structure, gel or matrix of proteinaceous of non-proeinaceous components The cells may not attach to the culture vessel and may not be adherent to the culture vessel, e.g. the cell may not form a layer or colonies of adherent cells. For example, the 3D suspension culture may be carried out in spinner flasks, bioreactors or in culture vessels containing an ultra-low attachment surface also termed non-cell adhesive surface, such as ultra-low attachment plates. The 3D suspension culture may be carried out in ultra-low attachment plates

[0079] The term “differentiation day”, also abbreviated herein as “dd” indicates the day after the onset of step b). Thus, ddO refers to the day on which the culturing of the single cell suspension of stem cells started. The days are then counted continuously throughout all steps of the differentiation protocol.

[0080] The term “the cells express one or more of the markers selected” or “the cell is positive for at least one marker from” means that the cells can be identified by measuring one or more of the markers selected from the groups consisting of the markers as indicated in this context. For example, when herein is described that “the retinal pigmented epithelium cells express one or more of the markers of MITF, PAX6, MERTK, VMD2, SERPINF-1, TYRP1, CLDN19 and RPE65” this means that the retinal pigmented epithelium cell may be identified by measuring expression of one or more of the makers identified from the group consisting of MITF, PAX6, MERTK, VMD2, SERPINF-1 , TYRP1 , CLDN19 and RPE65.

[0081] The term “marker” refers to a gene which can serve to determine the cell type by measuring its presence in the cell, in particular at RNA and / or protein level.

[0082] A first aspect of the invention refers to a method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies; and(c) culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates.

[0083] In some embodiments, the method is carried out in the absence of feeder cells.

[0084] Moreover, the method of the invention is very efficient, since it uses only a small set of regulatory molecules for differentiation. For example, the method does not use any of TGF-p inhibitor, TGF beta family signal transduction pathway inhibiting substance (e.g. Lefty, SB431542, A-83-01 , LDN193189 and Dorsomorphin), FGF8 inhibitor, AMPK inhibitor, GSK3f> inhibitor, a Sonic hedgehog signal transduction pathway activating substance (e.g. Shh, SAG and Purmorphamine), Wnt signal transduction pathway modulators CKI-7, D4476, IWR-1-endo and IWP-2, protein belonging to Wnt family, Wnt receptor, Wnt receptor agonist, BMP signal transduction pathway activating substance (such as BMP2, BMP4, BMP7 and GDF7). Thus, in one embodiment, except a ROCK inhibitor, no other regulatory inhibitor for differentiation is used throughout the method

[0085] In one embodiment, in each of step (c), step (d), step (e) and step (f) no TGFp and no bFGF may be usedof stem cells

[0086] The step of providing single cell suspension of stem cells is also referred herein as step (a) Whenever it is referred to step (a) herein, reference to the step of providing a single cell suspension of stem cells is made and vice versa

[0087] In step (a) a single cell suspension of stem cells is provided The step may contain the maintenance stem cells. A detailed protocol for maintenance of hiPSCs is described herein.

[0088] Stem cells can be undifferentiated or partially differentiated cells that can differentiate into various types of cells and / or are generally able to proliferate to produce more of the same stem cell in a process termed self-renewal The stem cell may be totipotent, which means that the stem cell can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable organism. The stem cell may be totipotent-like, meaning that it may be able to differentiate into embryonic and some extraembryonic cell types. The stem cell may be pluripotent, which means that the stem cell can differentiate into all intra-embryonic cells derived from any of the three germ layers (i.e., cells derived from endoderm, ectoderm and mesoderm) The stem cell may be multipotent, meaning that it can differentiate into a number of cell types, typically cell types of a closely related family of cells, such as blood cells. The stem cell may be oligopotent, meaning that it can differentiate into only a few cell types, and typically fewer cell types than multipotent stem cells. The stem cell may be unipotent, meaning that it can differentiate into only one cell type In one embodiment, the stem cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell and / or established stem cell line. Preferably, the stem cell is a pluripotent stem cell. One example of pluripotent stem cells are embryonic stem cells (ES cells). The invention as defined by the claims does not involve the destruction of human embryos. Preferably, ES cells are derived from mammals, more preferably from mice, rats, rabbits, guinea pigs, goats, pigs, cows, monkeys and humans. Human and murine or rat derived ES cells are preferred. Preferably the invention does not involve the use of human embryos for industrial or commercial purposes

[0089] Preferably the stem cell is a mammalian stem cell. In a more preferred embodiment, the stem cell is a human induced pluripotent stem cell As used herein, the term "iPS cell" or "induced pluripotent stem cell" is intended to indicate stem cell-like pluripotent cells which are derived from reprogrammed somatic cells (see for instance Takahashi K. et al. Cell 2007: 131 , 861-872 ; Yu J et al Science 2007:318, 1917-1920 ; Feng, B et al. Cell Stem Cell 2009:4, 301-312 ), preferably derived from a developed organism, such as adult fibroblasts.

[0090] As used herein, “stem cell” is also intended to encompass a progenitor cell, which is a cell that tends to differentiate into a specific type of cell but has been pushed to begin to differentiate into its target cell and may or may not be capable of self-renewal. The stem cell may be able to differentiate into at least one type of cell, at least two types of cells, at least 5 types of cells, at least 10 types of cells, at least 20types of cells, at least 50 types of cells or at least 100 types of cells. A population of stem cells (i.e. , a plurality of stem cells) may be used and may comprise only one of the types of stem cells described herein (i.e., a homogenous population of stem cells), or two or more of the types of stem cells described herein (i.e., a heterogeneous population of stem cells).

[0091] The stem cell may express one or more markers of pluripotency. For example, the stem cell may express one or more markers including Oct4, Nanog, SSEA-1 , SSEA-4, SOX2, TRA-1-60, STELLA and AP. The stem cell may have tri-germ layer differentiation capacity, meaning that the stem cell is able to differentiate into the cells of the ectodermal, endodermal and mesodermal lineages. The stem cell may be cultured and / or maintained as a colony of stem cells. Methods for culturing and / or maintaining suitable stem cells will be apparent to the skilled person.

[0092] The stem cell may be obtained from an isolated cell or tissue The isolated cell or tissue may be any suitable cell or tissue such as a stem cell as described herein, a progenitor cell, and / or a somatic cell, or a tissue containing such cells. Examples of somatic cells include blood cells (such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythroblasts, megakaryocytes, dendritic cells, T-cells, B- cells and NK-cells), neurons, neuroglial cells, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, fibroblasts, keratinocytes, melanocytes, endothelial cells, epithelial cells, including for example urinary or cheek cells, and adipocytes. A single isolated cell or a population of isolated cells may be used. A population of isolated cells may be made up of a single type of cell set out herein (i e , a homogenous starting cell population), or two or more of the types of cells described herein (i.e., a heterogeneous starting cell population). The stem cell may be free of artificially introduced and / or exogenous nucleic acid. Alternatively, or in addition, the stem cell may be free of artificially introduced and / or exogenous protein.

[0093] The isolated cell or tissue may be obtained from any suitable organism The isolated cell or tissue may be obtained from any suitable source such as a tissue sample, cord blood, peripheral blood and / or urine. By "isolated cell" or “isolated tissue” it is to be understood that the population of cells or tissue have been previously removed from the organism. The isolated tissue, stem cell or population of stem cells may be cultured, stored and / or manipulated ex vivo and / or in vitro using standard techniques known in the art, prior to being used in the method of the invention. Alternatively, the isolated stem cell or tissue may be used directly in the method of the invention.

[0094] Preferably, the method does not require the presence of a feeder cell A feeder cell may therefore be substantially absent or totally absent from the method A feeder cell is a cell upon which the isolated cell or tissue and resulting stem cell may be cultured on to support self-renewal and pluripotency Typically, a feeder cell supports pluripotency by secreting growth factors, cytokines and / or extracellular matrix proteins and are themselves unable to divide. A typical example of a feeder cell is a mouse embryonic fibroblast that has been mitotically inactivated.

[0095] In a preferable embodiment, the stem cell used in the method of the invention is an induced pluripotent stem cell. An induced pluripotent stem cell is a cell induced to have pluripotency by reprogramming a somatic cell by a known method. Specifically, a cell induced to have pluripotency may be obtained by reprogramming somatic cells such as fibroblast, skin cell, peripheral blood mononuclear cell and the like by the introduction of any combinations of a plurality of reprogramming factors selected from genes such as Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glisl , Nanog, Sall4, Lin28, Esrrb and the like. Examples of preferable combination of reprogramming factors can include: Oct3 / 4, Sox2, Klf4, and Myc (c- Myc or L-Myc); and Oct3 / 4, Sox2, Klf4, Lin28 and L-Myc. Other suitable combinations of reprogramming factors would be known to the skilled person. It is also possible to obtain and use established induced pluripotent stem cell lines. While the somatic cell used for obtaining induced pluripotent stem cell is not particularly limited, fibroblast, blood-lineage cell (e g., peripheral blood mononuclear cell or T cell, cordblood-derived cell) and the like can be used. As the fibroblast, those derived from dermis and the like can be used.

[0096] In one embodiment, step (a) comprises the following steps(a1 ) culturing stem cells in adherent culture; and(a2) dissociating the stem cells to obtain single cell suspension.

[0097] In step a1 ) culturing occurs in a medium which is suitable for maintaining the pluripotency of the stem cell. In one embodiment the stem cell medium contains bFGF and / or TGFp. The stem cell medium may contain bFGF and TGFp Typical components of a stem cell medium are DMEM / F12, L-Ascorbic acid, selenium, transferrin, NaHCo3, Insulin, FGF-2, TGFB1 . The stem cell medium may be TeSR, such as mTeSRTM1 (STEMCELL Technologies).

[0098] Typically, the stem cells are dissociated, when they reach 70% to 90% confluency If the stem cells have reached 70% to 90% confluency, they can either be passaged and cultured again to reach 70% to 90% confluency.

[0099] Typically, the stem cells are passaged for at most 7 times. Thus, in some embodiments, stem cells are passaged for at most 3, at most 4, at most 5, at most 6 times, at most 7 times

[0100] Typically, the duration after the last passage and dissociation of stem cells lasts 3 to 7 days preferably 3 to 6 days, more preferably 3 to 5 days, most preferably 4 to 5 days;

[0101] The single cell suspension of stem cells may be generated by detaching the cells from the wells and dissociating them into single cells Stem cell dissociation can be performed using any known procedures. These procedures include treatments with chelating agent (such as EDTA), enzymes (such as trypsin, collagenase), or the like, and operations such as mechanical dissociation (such as pipetting).

[0102] In one embodiment, the stem cell medium is removed from the iPSCs and the cells are washed, e g in buffer, such as BSA, followed by incubation with enzyme detachment medium having protease optionally with collagenolytic activity (e.g. Accutase by Invitrogen). Incubation may occur 1 to 10 minutes, such as 2 to 8 minutes, 3 to 6 minutes, preferably 5 minutes. Incubation is stopped by dilution e.g cell culture medium (e.g. DMEM / F-12, HEPES). Cells may be centrifuged and after discarding the supernatant resuspended in stem cell mediumFormation of embryoid bodies

[0103] The step of formation of embryoid bodies is also referred herein as step (b), or culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies. Whenever it is referred to step (b) herein, reference to the step of culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies is made and vice versa.

[0104] The single cell suspension of stem cells may be provided at a final concentration of preferably 1x104to 1x108, more preferably 1x105to 1x107cells, most preferably 1 x 10® cells / ml.

[0105] In one embodiment 5,5 x 106cells were provided in 5,5 ml of a 6 well ultra-low attachment plate, i.e. on a surface of 1 ,6 cm2.

[0106] In some embodiments, the 3D suspension culture may be carried out in spinner flasks, bioreactors or in culture vessels containing an ultra-low attachment surface also termed non-cell adhesive surface, such as ultra-low attachment plates. Preferably, the 3D suspension culture is carried out in ultra-low attachment plates. Accordingly, the 3D suspension culture may be free of a hydrogel structure, gel or matrix of proteinaceous of non-proteinaceous components.

[0107] In a preferred embodiment the cells in the 3D suspension culture is exposed to shear stress. This means that shear stress is applied to the cellular components of the 3D suspension culture, i.e. shear stress is applied to the single cell suspension and to the (forming) embryoid bodies. The shear stress can be carried out by agitation of the 3D suspension culture. Accordingly, step (b) may be carried out underagitation. The agitation may occur at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm. Preferably the agitation energy is chosen at a level which results in cell aggregates having a uniform shape and / or a diameter of about 150 pm More preferably, the agitation energy is at a level which results in cell aggregates having a uniform shape and a diameter of about 150 pm. The application of shear stress improves quality of the cell aggregates, in particular improves homogenous development of the cell aggregates, e g. including inter alia homogenous development of the cell aggregates in terms of shape size and cell fate.

[0108] Of note, step (b) of aggregate formation to obtain embryoid bodies is carried out in stem cell medium. Hence step (b) occurs in the absence of a neural induction medium. The differentiation to neuroectoderm with eye field specification by exchange of the stem cell medium to medium comprising neurobasal medium thus occurs after formation of embryoid bodies

[0109] Accordingly, in step b) culturing occurs in a medium which is suitable for maintaining the pluripotency of the stem cell. In one embodiment the stem cell medium contains bFGF and / or TGF0. The stem cell medium may contain bFGF and TGFp Typical components of a stem cell medium are DMEM / F12, L-Ascorbic acid, selenium, transferrin, NaHCo3, Insulin, FGF-2, TGFB1 The stem cell medium may be TeSR, such as mTeSRTMl (STEMCELL Technologies).

[0110] Step b) may last for at least 24 hours. In some embodiments step b) may last for 1 to 6 days, preferably 2 to 5 days, more preferably 3 to 4 days, most preferably 4 days.

[0111] In one embodiment, a Rock inhibitor may be added to the stem cell medium of step b) for a first period of step (b). In other words, at the onset of step (b) the cells may be incubated with a stem cell medium supplemented with a ROCK inhibitor The first period of step (b) begins with the onset of the culturing of the single cell suspension and lasts for 2 to 48h, preferably 12 to 36h, most preferably 24h. The ROCK inhibitor may in a concentration of 2 pM to 100 pM, preferably of 5 pM to 50 pM, more preferably of 10 pM.

[0112] Hence in one embodiment the method for differentiating stem cells into retinal cells, comprising the steps:(g) providing a single cell suspension of stem cells;(h) culturing the single cell suspension of stem cells in a 3D suspension culture with stem cell medium to obtain embryoid bodies; and(i) culturing the embryoid bodies in 3D suspension culture system in medium comprising neurobasal medium to obtain eye-field cell aggregates, wherein the 3D suspension culture system are ultra-low attachment plates.

[0113] In step b) culturing occurs in a medium which is suitable for maintaining the pluripotency of the stem cell. In one embodiment the stem cell medium contains bFGF and / or TGF . The stem cell medium may contain bFGF and TGFp Typical components of a stem cell medium are DMEM / F12, L-Ascorbic acid, selenium, transferrin, NaHCo3, Insulin, FGF-2, TGFB1 . The stem cell medium may be TeSR, such as mTeSR™1 (STEMCELL Technologies).

[0114] The term “embryoid body" as used herein (also denoted as embryonic body) is defined as three dimensional aggregate of pluripotent stem cells, that can be differentiated into any of the three germ layers (endoderm, ectoderm and mesoderm). The embryoid bodies are floating. This means, that the embryoid bodies are not adherent to the cell culture vessel (such as adherent colonies).

[0115] The pluripotency of the embryoid bodies may be confirmed by flow cytometry for known pluripotency markers, such as SOX2, Nanog, Oct4, SSEA1 and Tra1. In one embodiment the embryoid bodies comprise at least 60%, such as at least 70%, at least 80 % or at least 90% cells positive for at least one, at least two, at least three, at least four, at least five markers from SOX2, Nanog, Oct4, SSEA1 and Tra1. In one embodiment the embryoid bodies comprise at least 90% cells positive for at least one, at least two, at least three, at least four, at least five markers from SOX2, Nanog, Oct4, SSEA1 and Tra1.

[0116] In a preferred embodiment the embryoid bodies comprise at least 90 % cells positive for all five markers SOX2, Nanog, Oct4, SSEA1 and Tra1.

[0117] In one embodiment the embryoid bodies comprise cells that are positive for one or more pluripotency markers as described above and are further positive for at least one eye field marker, such as OTX2 and / or PAX6. This means that the embryoid bodies show an increased level of at least one eye field marker selected form OTX2 and PAX6 compared to iPSCs, i.e. the iPSC population they are derived from Typically, iPSCs are substantially negative, or express low amounts of at least one eye field marker such as OTX2 and PAX6. For example, if IPSCs express low amounts of OTX2 and / or PAX6, the embryoid bodies would show OTX2 and / or PAX6 amounts which are at least double, at least three times, at least four times as high as in iPSCs. Thus, the embryoid bodies positive for at least one eye field marker means, that the embryoid bodies express at least one eye field marker, such as OTX2 and PAX6 in substantial amounts The levels of OTX2 and / or PAX6 in embryoid bodies may be measured at the end of step (b), i.e. on d3.

[0118] The embryoid bodies may have a uniform shape and / or a diameter of about 50 to 300 pm, preferably of 100 to 200 pm, more preferably of 120 to 180 pm, most preferably of 150 pm. In a specific embodiment, the embryoid bodies have a uniform shape and a diameter of about 150 pm

[0119] Hence in one embodiment the invention refers to a method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture with stem cell medium to obtain embryoid bodies; and(c) culturing the embryoid bodies in 3D suspension culture system in medium comprising neurobasal medium to obtain eye-field cell aggregates, wherein steps (b) and (c) occur under agitation

[0120] Accordingly, one aspect of the invention refers to the embryoid bodies obtained in step (b), i.e. at day three (d3) and the cell population obtained by dissociating these embryoid bodies:

[0121] Hence, some embodiments refer to an embryoid body or a cell population derived from a dissociated embryoid body comprising at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing OTX2, at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Oct4, and at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Sox2

[0122] In some embodiments said embryoid bodies cell population derived therefrom comprises at least 20 % of the cells express Nanog.

[0123] Typically, compared to iPSCs, said embryoid bodies cell population derived therefrom expresses 0.1 to 1 fold, 0.1 to 0.5, preferably 0.1 to 0.3 fold amount of Nanog and / or 0.1 to 1 fold, 0.2 to 0.8, preferably 0.3 to 0.6 fold amount of Oct4.

[0124] In some embodiments, the embryoid bodies have a uniform shape and / or a diameter of about 50 to 300 pm, preferably of 100 to 200 |jm, more preferably of 120 to 180 pm, most preferably of 150 pm. field

[0125] The step of eye field specification is also referred herein as step (c) or culturing the embryoid bodies in 3D suspension culture to obtain eye field cell aggregates. Whenever it is referred to step (c) herein, reference to the step of culturing the embryoid bodies in 3D suspension culture to obtain eye field cell aggregates is made and vice versa

[0126] In step c) the embryoid bodies are cultured in 3D suspension culture system to obtain eye field cell aggregates. At the onset of step c) the medium of step b) is replaced with the medium of step c).

[0127] Eye field cell aggregates obtained by the method as decided herein (in particular steps a) to c) as described herein) are also encompassed. The term “eye field cell aggregates” refers to aggregates of retinal progenitor cells. The differentiation to eye-field aggregates may be confirmed by flow cytometry for known eye field transcription factors markers, such as PAX6, RAX, SIX3, LHX2 and OTX2. In one embodiment the eye field aggregates comprise at least 50%, 60%, such as at least 70%, at least 80 % or at least 90% cells positive for at least one, at least two, at least three, at least four, at least five markers fromPAX6, RAX, SIX3, LHX2 and OTX2. In one embodiment the eye field aggregates comprise at least 60% cells positive for at least one, at least two, at least three, at least four, at least five of the markers from PAX6, RAX, SIX3, LHX2 and OTX2. In one embodiment, the eye field aggregates express PAX6, OTX2 and RAX. In one embodiment the eye field aggregates comprise at least 50%, 60%, such as at least 70%, at least 80 % or at least 90%, preferably at least 60 % cells positive for at least one marker from PAX6 and OTX2. In a preferred embodiment the eye field aggregates comprise at least 50%, 60%, such as at least 70%, at least 80 % or at least 90%, preferably at least 60 % cells positive for PAX6 and OTX2. In one embodiment eye-field cell aggregates comprise at least 50%, preferably 60% cells expressing PAX6 and OTX2 if they are further differentiated to RPE cells and / or comprise at least 60% cells, preferably 70%, more preferably 80% cells expressing PAX6 and OTX2 if they are further differentiated into retinal organoids.

[0128] The eye field aggregates may be floating. This means, that the eye field aggregates are not adherent to the cell culture vessel (such as adherent colonies) Further, the eye field aggregates comprise cells, also termed, human retinal progenitor cells, that express eye field transcription factors Eye field transcription factors are factors that drive eye development.

[0129] Analysis of the respective cell markers can be carried out by the methods known to the skilled in the art, in particular by flow cytometry or qPCR. Preferably PAX6 and OXT2 are measured by flow cytometry

[0130] For analysis of the cell markers, a subset of aggregates is obtained from the culture and washed in buffer, such as PBS. The aggregates are dissociated by incubation with enzyme, pipetting, followed by stopping the enzyme reaction For flow cytometry cells are stained with the respective antibodies (e.g. targeting PAX6 and OTX2) and analyzed

[0131] Hence in one embodiment the invention refers to a method comprising the steps:- culturing a single cell suspension of stem cells in a 3D suspension culture with stem cell medium to obtain embryoid bodies; and- culturing the embryoid bodies in 3D suspension culture system in medium comprising neurobasal medium to obtain eye-field cell aggregates, wherein the eye-field cell aggregates comprise at least 50%, such as at least 60%, at least 80% cells expressing PAX6 and OTX2.

[0132] Hence in a particular preferred embodiment the invention refers to a method comprising the steps:- culturing a single cell suspension of stem cells in a 3D suspension culture under agitation with stem cell medium to obtain embryoid bodies; and- culturing the embryoid bodies in 3D suspension culture system under agitation in medium comprising neurobasal medium to obtain eye-field cell aggregates, wherein the eye-field cell aggregates comprise at least 50%, preferably at least 60%, more preferably at least 80% cells expressing PAX6 and OTX2.

[0133] In step c) culturing occurs in a medium which is suitable for differentiation into eye-field cell aggregates. In one embodiment a medium comprising neurobasal medium is used. Typically the medium comprising neurobasal medium comprises amino acids, inorganic salts, vitamins (such as biotin, DL Alpha Tocopherol Acetate, DL Alpha-Tecopherol, Vitamin A) proteins (such as BSA, catalase, human recombinant insulin, human transferrin , superoxide dismutase and other components (such as corticosterone, D- Galactose, Ehanolamine HCI, Glutathione, L-Carnitine HCI, Linoleic Acid, Linolenic Acid, Progesterone,Putrescine 2HCI, Sodium Selenite, T3 (triodo-l-thyronine) In one embodiment the medium comprising neurobasal medium may further comprise B-27 supplement, and N-2 supplement. In another embodiment the medium comprising neurobasal medium may further comprise B-27 supplement, N-2 supplement, 2- Mercaptoethanol and DMEM / F12. In a specific embodiment the medium comprising neurobasal medium may further comprise B-27 supplement, N-2 supplement, 2-Mercaptoethanol, DMEM / F12, sodium pyruvate and L-Glutamine.

[0134] Step c) may last for 3 to 7 days, preferably 3 to 6 days, more preferably 4 to 5 days, most preferably 4 days.

[0135] The culture medium may be changed in an interval commonly used by the skilled person, such as daily, every second day. In a preferred embodiment the medium is changed two days after the onset of step c).

[0136] Culturing in step c) occurs in 3D suspension culture.

[0137] In a preferred embodiment the cells in the 3D suspension culture are exposed to shear stress. This means that shear stress is applied to the cellular components of the 3D suspension culture, i.e. shear stress is applied to the embryoid bodies and (the forming) eye-field cell aggregates The shear stress can be carried out by agitation of the 3D suspension culture. Accordingly, step (c) may be carried out under agitation. The agitation may occur at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm. Preferably the agitation energy is chosen at a level which results in cell aggregates having a uniform shape and / or a diameter of about 150 pm More preferably, the agitation energy is at a level which results in cell aggregates having a uniform shape and a diameter of about 150 pm. The application of shear stress may improve quality of the cell aggregates, in particular shear stress may improve homogenous development of the cell aggregates.

[0138] The eye-field cell aggregates can be further differentiated into retinal cells In particular, the eyefield cell aggregates can be differentiated by two different protocols into the epithelial component of the retina: a monolayer composed of a single cell type, RPE cells, which will be cultured as a 2D adherent culture.Retinal organoids, that will remain as a 3D, stirred (exposed to shear stress due to shaking platform) suspension culture, comprising all cell types present in the neural retina will emerge, at different timepoints, recapitulating human development.RPE differentiation

[0139] The step of RPE differentiation refers to the differentiation of the cells which are obtained by dissociation of eye-field aggregates into single cells and culturing them in adherent culture to obtain retinal progenitor cells

[0140] Accordingly, one embodiment of the invention refers to the differentiation of stem cells into retinal pigmented epithelium cells. This method comprises the steps culturing a single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies and in addition the steps (dd) and (ee); and culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates In particular, the method comprises all the steps (a) to (c) and in addition the steps (dd) and (ee).

[0141] Accordingly, the method for differentiating stem cells into retinal pigmented epithelium cells comprises the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies;(c) culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates;(dd) dissociating the eye-field cell aggregates to single cells;(ee) culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epitheliumcells.

[0142] Moreover, one aspect of the invention relates to the differentiation of eye-field cell aggregates to retinal pigmented epithelium cells, comprising the steps (dd) and (ee). An alternative aspect of the invention relates to the differentiation of retinal progenitor cells to retinal pigmented epithelium cell, comprising step (ee).

[0143] In the following the specific steps for differentiating to retinal pigmented epithelium cells are described in more detail.

[0144] The step of dissociating the eye-field cell aggregates is also referred herein as step (dd) or dissociating the eye field cell aggregates to single cells Whenever it is referred to step (dd) herein, reference to the step dissociating the eye field cell aggregates to single cells is made and vice versa.

[0145] In one embodiment the eye-field cell aggregates used in step (dd) comprise at least 50%, preferably at least 60% expressing PAX6 and OXT2.

[0146] The eye-field aggregates may be dissociated by incubation with enzyme, pipetting, followed by stopping the enzyme reaction A cell strainer may be used to get rid of remaining aggregates. The skilled person is aware that seeding density depends on the transwell size and cell line used Usually, for 6 5 mm transwells 70-100k cells are seeded in 150 pl medium, for 24 mm transwells 400-600k cells are seeded in 800 pl M2 medium, lower chambers are filled with media. In one embodiment, the cells are seeded in the medium comprising neurobasal medium supplemented with a ROCK inhibitor. In one embodiment, the ROCK inhibitor is Y27632 The ROCK inhibitor may in a concentration of 2 pM to 100 pM, preferably of 5 pM to 50 pM, more preferably of 10 pM The cells may be cultured in a medium comprising neurobasal medium supplemented with a ROCK inhibitor for 2 to 48h, preferably 12 to 36h, most preferably 24h.

[0147] Accordingly, culturing in (ee) may comprise the steps(eel ) culturing in a medium comprising neurobasal medium supplemented with a ROCK inhibitor; and (ee2) culturing in a RPE differentiation medium.

[0148] The step “culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells” is also referred herein as step (ee). Whenever it is referred to step (ee) herein, reference to the step culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells is made and vice versa.

[0149] Culturing single cells obtained in step (dd) in adherent cell culture means that single cells are seeded at the onset of step (ee) which form a cell layer during culturing in step (ee).

[0150] In step (ee), the cells may be seeded on a culture vessel, e g a cell culture plate, comprising a upper and a lower chamber, such as a transwell plate. The cell culture may be carried out in a culture vessel which is coated with at least two proteins, and preferably at least three proteins selected from the group of Laminin, Collagen IV, Entactin and Perlecan. In one embodiment the culture vessel is coated with a mixture resembling the laminin / collagen IV-rich basement membrane extracellular environment. In one embodiment the culture vessel is coated with the solubilized basement membrane matrix secreted by Engelbreth-Holm- Swarm (EHS) mouse sarcoma cells (Matrigel, Corning Life Sciences).

[0151] Culturing in step (ee) occurs in a medium which is suitable for differentiating retinal progenitor cells into retinal pigmented epithelium cells.

[0152] In one embodiment culturing in step (ee) comprises culturing cells in a RPE differentiation medium. The RPE differentiation medium may contain at least one growth factor selected from agonists of the Activin A pathway. In one embodiment, the at least one growth factor selected from agonists of the Activin A pathway is activin, preferably human Activin A, more preferably recombinant human Activin A. The RPE differentiation medium may comprise glucose, amino acids, vitamins, transferrin, trace elements, insulin, collagen precursors, albumin preloaded with lipd, 2-Mercaptoethanol and Activin A. The RPE differentiation medium may be serum-free.

[0153] Culturing in step (ee) may last for at least 15 days, preferably at least 20 days, such as at least 30 days, at least 40 days at least 50 days. In some embodiments culturing in step (ee) lasts for 30 to 80 days, such as for 40 to 70 days, preferably for 45 to 65 days, more preferably for 50 to 60 days, most preferably for 53 to 57 days.

[0154] In one embodiment the cells are passaged at least once during culturing in (ee). In one embodiment the cells are passaged at least twice during culturing in (ee). In one embodiment the cells are passaged twice during culturing in (ee). Preferably cells were passaged in the early RPE stage (e g. dd16- 28, preferably dd28) and / or passaged in the maturing RPE stage (e.g dd28 to 63, preferably dd49).

[0155] Identity of RPE precursors may be confirmed with the appearance of a polygonal monolayer assessed, also termed cobblestone morphology. Appearance of a polygonal monolayer may be assessed for example by staining of actin with phalloidin Further, the identity of RPE precursors may be confirmed by determining the content of MITF and / or ZO-1 positive cells.

[0156] In one embodiment at least 50% of the cells were MITF positive, measured by FACS. In one embodiment, pigmentation of the cells appeared latest at dd20 to dd25, preferably at dd22. In one embodiment at least 50% of the cells were MITF positive and pigmentation of the cells appeared latest at dd20 to dd25, preferably at dd22.

[0157] In one embodiment, no selection of the cells occurs in the method of the invention. In one embodiment, no selection of the cells occurs in steps a) to e) or in the steps a) to f). In particular, during passaging in step (ee) no selection or enrichment of the cells occurs Selection as used herein encompasses sorting in order to enrich for a certain desired cell or aggregate type while discarding cell or aggregate types that are not desired.

[0158] In one embodiment VEGF secretion is monitored. VEGF secretion may be measured by ELISA. VEGF concentration may be measured at a later development stage (e g dd49 to dd63, such as dd62) compared and / or at an earlier development stage (e.g. dd35 to dd49, such as dd42) In one embodiment the RPE cells showed increased VEGF secretion, i.e. by showing a high VEGF concentration on a later development stage (e.g. dd49 to dd63, such as dd62) compared to an earlier development stage (e.g. dd35 to dd49, such as dd42) An interval of at least 5, preferably at least 10, more preferably of at least 15 days should be between the earlier measurement and the later measurement In one embodiment the RPE cells show polarisation, having a different VEGF secretion apical versus basolateral. For this the VEGF concentration in the upper well and the lower well of the transwell plate are compared.

[0159] In one embodiment, the retinal pigmented epithelium cells express one or more of the markers from the group of SIX3, LHX2, RAX, EZRIN, CLDN19, MERTK, MITF, PMEL, RLBP1, PAX6, RPE65, SERPINF1 , TYRP1 , VMD2, ZO-1 , BEST 1 and ACTIN-F. In one embodiment, the retinal pigmented epithelium cells express one or more markers of the group consisting of CLDN19, MERTK, MITF, PAX6, RPE65, SERPINF1 , TYRP1 , VMD2, BEST 1 , EZRIN , ACTIN-F, PMEL and ZO-1. In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of MITF, PAX6, RPE65, TYRP1 , EZRIN, ACTIN-F, MERTK, PMEL and ZO-1. In one embodiment, the retinal pigmented epithelium cells express one or more of the markers selected from the group of MITF, PAX6, RPE65 and TYRP1.

[0160] The measurement for these markers may be carried out in step (ee) at least one time, at least two times or at least three times (e.g. dd28, dd49, dd63). Measurement may be carried out by qPCR, for example by TaqMan, protein staining, for example ICC. For example, qPCR may be used to detect one or more of SIX3, LHX2, RAX, EZRIN, CLDN19, MERTK, MITF, PMEL, RLBP1 , PAX6, RPE65, SERPINF1 , TYRP1 , VMD2, and ZO-1. For example, ICC staining may be used to detect one or more of BEST 1, EZRIN, RPE65, MITF, MERTK, PMEL and ZO-1 F-Actin may be for example be detected by phalloidin staining.

[0161] In one embodiment the RPE cell layer exhibits at least one of the functions selected from transepithelial resistance (TER), VEGF secretion, phagocytosis Phagocytosis may be measured by the pHrodo assay. VEGF may be measured by ELISA essay.Organoid Differentiation

[0162] Organoid differentiation refers to culturing the eye-field aggregates obtained in step (c) further in suspension culture to obtain retinal organoids.

[0163] The differentiation into retinal organoids is undirected / not guided, as there is no exogenous direct modulation of signaling molecules. The early aggregates themselves, under favourable but not directed conditions, rely mainly on endogenous modulation of the necessary signal to differentiate - The initial embryoid bodies, under favourable conditions (glucose levels, FBS concentration, amino acids.. ), initiate neuro-retinal differentiation, without exogenous direct activation or blocking of any pathways.

[0164] Thus, in general terms, one embodiment of the invention refers to culturing a single cell suspension of stem cells in a 3D suspension culture with stem cell medium to obtain embryoid bodies; and culturing the embryoid bodies in 3D suspension culture system in a medium comprising neurobasal medium to obtain eye-field cell aggregates, culturing the eye-field cell aggregates in a 3D suspension culture system to obtain retinal organoids.

[0165] As set out in more detail below the step of culturing the eye-field cell aggregates in a 3D suspension culture system to obtain retinal organoids comprises the steps - culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors and - culturing the neuro- retinal precursors in a 3D suspension culture system to obtain retinal organoids;

[0166] More particularly, the step of culturing the eye-field cell aggregates in a 3D suspension culture system to obtain retinal organoids may comprise:(d) culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors;(e) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain early phase retinal organoids.(f) optionally culturing the early phase retinal organoids in a 3D suspension culture system to obtain late phase retinal organoids

[0167] In one embodiment step (d) occurs in the absence of a support matrix. In one embodiment, step (e) occurs in the absence of a support matrix. In one embodiment step (f) occurs in the absence of a support matrix. In one embodiment, any one of steps (d), (e) and (f) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel

[0168] In one embodiment the media used for the step of retinal organoid differentiation do not contain Vitamin A. Accordingly, the medium used in step (d), the medium used in step (e), the medium used in step (f) does not contain Vitamin A. Accordingly the medium used in step (d1 ), (d2), (e1 ), (e2) and or (e3) does not contain Vitamin A

[0169] Culturing in step (d) occurs in a medium comprising B-27 Supplement (without Vitamin A) and N-2 Supplement.

[0170] Culturing in step (d1 ) occurs in a medium comprising neurobasal medium, B-27 Supplement (without Vitamin A), N-2 Supplement. In a specific embodiment culturing in step (d 1 ) occurs in a medium comprising neurobasal medium, B-27 Supplement (without Vitamin A), N-2 Supplement and FBS. In one embodiment culturing in step (d1) occurs in a medium comprising neurobasal medium, DMEM / F12, glucose, B-27 Supplement (without Vitamin A), N-2 Supplement, 2-Mercaptoethanol, Sodium pyruvate and L- Glutamine, and FBS. In more specific embodiments culturing in step (d1 ) occurs in a medium comprising 30-50% neurobasal medium, 30 to 50% DMEM / F12, glucose, 0.4 % to 1 .6 % B-27 Supplement (50x; without Vitamin A), 0.1% to 1% N-2 (100x) Supplement, 0.01 to 0 1 % 2-Mercaptoethanol, 0 001% to 1 % Sodium pyruvate and L-Glutamine, and 5 % to 20% FBS. In a particular embodiment, culturing in step (1 ) occurs in a medium which comprises about 40 to 50% DEMEM / F12, high Glucose, GlutaMAX, 40 to 50% neurobasal Medium, 0.6 to 1.2% B-27 Supplement (50x; without vitamin A), 0.3% to 0.7% N-2 Supplement (100x), 0,05 to 0,15% 2-Mercaptoethanol, 0.001 % to 1 % Sodium pyruvate and L-Glutamine, and 8% to 15% FBS. A specific example of the medium, that could be used in step (d1 ) is described elsewhere herein as M4

[0171] In one embodiment culturing in step (d2) occurs in a medium comprising at least 10 mM, preferably at least 12 mM, more preferably at least 15 mM glucose. In one embodiment culturing in step (d2) occurs in a medium comprising 10 mM to 50 mM glucose, preferably 12 to 20 mM glucose, more preferably 15 to 25 mM glucose, even more preferably 17 to 18 mM glucose, such as 17 5 mM glucose

[0172] In one embodiment culturing in step (d2) occurs in a medium comprising retinoic acid. In one embodiment culturing in step (d2) occurs in a medium comprising 0.05 pM to 2 pM, preferably 0.1 pM to 1 pM, more preferably 0 2 pM to 0.7 pM, such as 0.5 pM retinoic acid.

[0173] In one embodiment culturing in step (d2) occurs in a medium B-27 Supplement (without Vitamin A), N-2 Supplement and glucose. In a specific embodiment culturing in step (d2) occurs in a medium comprising B-27 Supplement (without Vitamin A), N-2 Supplement, FBS and glucose. In a specific embodiment culturing in step (d1 ) occurs in a medium comprising B-27 Supplement (without Vitamin A), N-2 Supplement, FBS, retinoic acid and glucose In a specific embodiment culturing in step (d2) occurs in a medium comprising B-27 Supplement (without Vitamin A), N-2 Supplement, FBS, retinoic acid, Taurin and glucose. The content of B-27 Supplement (50 X) may be in the range of 02 % to 2%, preferably 0 4 to 1.6%, more preferably 0.6 to 1 .2%, such as 1%. The content of N-2 Supplement (100x) may be in the range of 0.1% to 2%, preferably 0 3% to 0 7%, such as 0 5% or 0 49% The content of 2-Mercaptoethanol may be in the range of 0 01 to 0.1 %, 0,05 to 0,15%, such as 1% or 0.098%. The content of FBS may be in range of 5 % to 20%, 8% to 15%, such as 10% or 9.8%. The content of Taurin may be in the range of 0.01 mM to 2 mM, 0.05 mM to 0.5 mM, 0.08 mM to 0.2 mM, such as 0 1 mM. The content of retinoic acid may be added in a range of 0 3 pM to 0 7 pM, preferably 0 5 pM

[0174] In a particular embodiment, culturing in step (d2) occurs in a medium comprising about 60 to 70% DMEM / F12, high Glucose, GlutaMAX, 0.6 to 1.6 % B-27 Supplement (50X, without Vitamin A), 0.6% to 1.4% N-2 Supplement (100x), Sodium pyruvate and L-Glutamine, 0.05 mM to 0.5 mM Taurin, 0.3 pM to 0.7 pM retinoic acid, 0,05 to 0,15% 2-Mercaptoethanol, and 8% to 15% FBS

[0175] In one embodiment, culturing in step (d2) occurs in a medium that is void of neurobasal medium. Accordingly, the application also discloses specific embodiments in which the above embodiments relating to culturing in step (d2) occur in a medium that does not contain neurobasal medium. The use of neurobasal medium in step (d 1 ) and omitting neurobasal medium in step (d2) leads to a decrease in the formation of undesired neuronal rosettes.

[0176] A specific example of the medium, that could be used in step (d2) is described elsewhere herein as M5.

[0177] In one embodiment, step (d) may last for 10 to 70 days, preferably 20 to 60 days, more preferably 30 to 50 days, even more preferably 35 to 45 days, most preferably 40 to 45 days.

[0178] In one embodiment, step (d1 ) may last for 5 to 30 days, preferably 10 to 25 days, more preferably 15 to 20 days.

[0179] In one embodiment, step (d2) may last for 5 to 60 days, preferably 10 to 40 days, more preferably 20 to 30 days, most preferably 23 to 27 days.

[0180] In one embodiment the neuro-retinal precursors obtained in step (d) express at least one of the markers selected from the group VSX2 and RAX. VSX2 may be detected by IHC. RAX and VSX2 may be detected by qPCR and IHC. In one embodiment the neuro-retinal precursors obtained in step (d) expressVSX2. In one embodiment at least 50%, preferably at least 60%, most preferably at least 70 % of the neuro- retinal precursors obtained in step (d) at least 50%, preferably at least 60%, most preferably at least 70% express VSX2. In one embodiment at day 25 at least 50%, preferably at least 60%, most preferably at least 70% of the neuro-retinal precursors obtained in step (d) at least 50%, preferably at least 60%, most preferably at least 70% express VSX2.

[0181] In one embodiment culturing in step (e) occurs in a medium comprising retinoic acid. In one embodiment culturing in step (e) occurs in a medium comprising 0.05 pM to 2 pM, preferably 0.1 pM to 1 pM, more preferably 0 2 pM to 0.7 pM, retinoic acid In one embodiment the retinoic acid concentration in step (e1 ) is higher than in step (e2).

[0182] In one embodiment culturing in step (e1 ) occurs in a medium comprising at least 5%, preferably at least 10%, more preferably at least 15%, most preferably at least 20% Advanced DMEM / F-12 In one embodiment culturing in step (e1 ) occurs in a medium comprising 5% to 50%, preferably 10% to 40%, more preferably 15% to 35%, most preferably 20% to 30% Advanced DMEM / F-12.

[0183] In one embodiment culturing in step (e1 ) occurs in a medium comprising at least 10 mM, preferably at least 12 mM, more preferably at least 15 mM glucose In one embodiment culturing in step (d2) occurs in a medium comprising 10 mM to 50 mM glucose, preferably 12 to 20 mM glucose, more preferably 15 to 25 mM glucose, even more preferably 17 to 18 mM glucose, such as 17.5 mM glucose.

[0184] In one embodiment culturing in step (e1 ) occurs in a medium comprising DMEM / F12, glucose, Advanced DMEM / F-12 and B-27 Supplement (without Vitamin A)

[0185] In one embodiment culturing in step (e1 ) occurs in a medium comprising retinoic acid. In one embodiment culturing in step (e1 ) occurs in a medium comprising 0.05 pM to 2 pM, preferably 0.1 pM to 1 pM, more preferably 0 2 pM to 0.7 pM, such as 0.5 pM retinoic acid.

[0186] In one embodiment culturing in step (e1 ) occurs in a medium comprising DMEM / F12, glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), Taurin and retinoic acid.

[0187] In a particular embodiment, culturing in step (e1 ) occurs in a medium comprises D-MEM / F12, high Glucose, GlutaMAX, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), N-2 Supplement, Antibiotic Antimycotic, Sodium pyruvate and L-Glutamine, Taurin, retinoic acid, and FBS

[0188] In a particular embodiment, culturing in step (e1 ) occurs in a medium comprises about 60 to 70 % DEMEM / F12, high Glucose, GlutaMAX, about 20 to 30% Advanced DMEM / F-12, 0.4 to 1 .6 % B-27 Supplement (50X; without Vitamin A), 0.3% to 0.7% N-2 Supplement (1 OOx), about 0.5 to 2% Antibiotic Antimycotic, 0 001% to 1% Sodium pyruvate and L-Glutamine, 0 05 mM to 0 5 mM Taurin, 0 1 pM to 1 pM retinoic acid, and 8% to 15% FBS.

[0189] In a particular embodiment, culturing in step (e1 ) occurs in a medium comprises about 60 to 70% DMEM / F12, high Glucose, GlutaMAX, about 20 to 30% Advanced DMEM / F-12, 0.6 to 1.2% B-27 Supplement (50x; without Vitamin A), 0 3% to 0.70 N-2 Supplement (1 OOx), about 0.5 to 2 % Antibiotic Antimycotic, 0.001% to 1% Sodium pyruvate and L-Glutamine, 0 08 mM to 0.2 mM Taurin, 0 3 pM to 0.7 pM retinoic acid, and 9% to 10% FBS.

[0190] A specific example of the medium, that could be used in step (e 1 ) is described elsewhere herein as M6.

[0191] In one embodiment culturing in step (e2) occurs in a medium comprising at least 10%, preferably at least 20%, more preferably at least 30%, most preferably at least 40% Advanced DMEM / F-12 In one embodiment culturing in step (e2) occurs in a medium comprising at least 5% to 50%, preferably at least 10% to 40%, more preferably at least 15% to 35%, most preferably at least 20% to 30% Advanced DMEM / F-12.

[0192] In one embodiment culturing in step (e2) occurs in a medium comprising at least 10 mM, preferably at least 12 mM, more preferably at least 15 mM glucose. In one embodiment culturing in step (d2) occurs ina medium comprising 10 mM to 50 mM glucose, preferably 12 to 20 mM glucose, more preferably 15 to 25 mM glucose, even more preferably 17 to 18 mM glucose, such as 17.5 mM glucose.

[0193] In one embodiment culturing in step (e2) occurs in a medium comprising DMEM / F12, glucose, Advanced DMEM / F-12 and B-27 Supplement (without Vitamin A). In one embodiment culturing in step (e2) occurs in a medium comprising DMEM / F12, glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A) and N-2 Supplement.

[0194] In one embodiment culturing in step (e2) occurs in a medium comprising DMEM / F12, glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), N-2 Supplement, Taurin and retinoic acid.

[0195] In a particular embodiment, culturing in step (e2) occurs in a medium which comprises DMEM / F12, high Glucose, GlutaMAX, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), N-2 Supplement, Antibiotic Antimycotic, Sodium pyruvate and L-Glutamine, Taurin, retinoic acid, Lipid mixture and FBS

[0196] In a particular embodiment, culturing in step (e2) occurs in a medium which comprises about 40 to 50% DMEM / F12, high Glucose, GlutaMAX, about 40 to 50% Advanced DMEM / F-12, 1 to 4 % B-27 Supplement (50x; without vitamin A), 0.2% to 2 N-2 Supplement (1 OOx), about 0.5 to 2% Antibiotic Antimycotic, 0 001% to 1% Sodium pyruvate and L-Glutamine, 0 05 mM to 0 5 mM Taurin, 0 1 pM to 1 pM retinoic acid, and 8% to 15% FBS.

[0197] In a particular embodiment, culturing in step (e2) occurs in a medium which comprises about 40 to 50% DMEM / F12, high Glucose, GlutaMAX, about 40 to 50% Advanced DMEM / F-12, 1 .6 to 3 % B-27 Supplement (50x; without Vitamin A), 0 8% to 1 2% N-2 Supplement (1 OOx), about 0 5 to 2% Antibiotic Antimycotic, 0.001% to 1% Sodium pyruvate and L-Glutamine, 0 08 mM to 0.2 mM Taurin, 0 2 pM to 0.3 pM retinoic acid and 9% to 10% FBS

[0198] In one embodiment culturing in step (e2) occurs in a medium comprising retinoic acid. In one embodiment culturing in step (e2) occurs in a medium comprising 0 05 pM to 2 pM, preferably 0 1 pM to 1 pM, more preferably 0 2 pM to 0.3 pM, such as 0.25 pM retinoic acid.

[0199] In one embodiment culturing in step (e2) occurs in a medium comprising retinoic acid and lipids.

[0200] A specific example of the medium, that could be used in step (e2) is described elsewhere herein asM7

[0201] The term “lipids” refers to a lipid mixture. The lipid mixture may comprise cholesterol, methyl esters, polyoxyethylenesorbitan monooleate D-a-tocopherol acetate.

[0202] In some embodiments culturing in step (d2) and (e) occurs in a medium comprising retinoic acid. In one embodiment culturing in step (d2), (e 1 ) and (e2) occurs in a medium comprising retinoic acid In one embodiment culturing in step (e2) occurs in medium having a lower retinoic acid concentration compared to the medium of (e1 ). Preferably, the retinoic acid concentration in (e2) is half of the retinoic acid concentration in (e1 ). The inventors surprisingly found that the addition of retinoic acid promotes rod differentiation and cone maturation without restraining the cone fate differentiation. In one embodiment culturing in step (f) occurs in a medium which does not contain retinoic acid.

[0203] In one embodiment, the early phase retinal organoids obtained in step (e) are positive for at least one, preferably at least two, more preferably at least 3, even more preferably at least 4, such as at least 5, at least 6 markers, selected from the group of CRX, RECOVERIN, RXRy, NLR, POU4F1 , Brn3a, AP2a and ProxT In one embodiment, the early phase retinal organoids obtained in step (e) are positive for all markers selected from the group of CRX, RECOVERIN, RXRy, NLR, POU4F1 , Brn3a, AP2a and Proxl . CRX, RECOVERIN, RXRy, NLR, POU4F1 , Brn3a, AP2a and Proxl may be detected by IHC.

[0204] In one embodiment, the early phase retinal organoids obtained in step (e) are positive for at least one marker, preferably at least two, more preferably at least 3 markers selected from the group of CRX, RECOVERIN, RXRy and NLR. In one embodiment, the early phase retinal organoids obtained in step (e) are positive for at least one marker selected from the group of CRX, RECOVERIN and RXRy.

[0205] In one embodiment CRX is expressed in early phase retinal organoids on day 50, on day 60 and on day 70 or later. CRX may be detected by IHC. In one embodiment RECOVERIN is expressed in early phase retinal organoids on day 70 and later

[0206] The term “early phase retinal organoids” refers to a cell aggregate comprising retinal ganglion cells, interneurons, such as amacrine an horizonal cells and photoreceptor precursors.

[0207] In one embodiment, the early phase retinal organoids obtained in step (e) comprise photoreceptor precursors and comprise at least one cell type selected from the group of mature retinal ganglion cells(RGCs), horizontal cells and amacrine cells.

[0208] The term “retinal ganglion cell” is also termed “Ganglion cell" herein.

[0209] In one embodiment, the early phase retinal organoids obtained in step (e) comprise at least one cell type selected from the group of photoreceptor precursors, mature RGCs, horizontal cells and amacrine cells.

[0210] In one embodiment, the early phase retinal organoids obtained in step (e) comprise all the cell types from the group of photoreceptor precursors, mature RGCs, horizontal cells and amacrine cells. In one embodiment, the early phase retinal organoids obtained in step (e) comprise all cell types from the group of photoreceptors, mature RGCs and horizontal cells.

[0211] Photoreceptor precursors may express CRX. Photoreceptor precursors may be early post-mitotic cells which express CRX. The photoreceptor precursors may be committed to a photoreceptor fate but may not have yet committed to a sub-type specification (e g cone or rod photoreceptors)

[0212] In one embodiment the RXRy positive cells may be localized substantially at the bottom of the most outer layer of the organoid.

[0213] In one embodiment, step (e) may last for 20 to 80 days, preferably 30 to 70 days, more preferably 40 to 60 days, even more preferably 45 to 55 days, most preferably 42to 48 days

[0214] In one embodiment, step (e1 ) may last for 5 to 40 days, preferably 10 to 30 days, more preferably 15 to 25 days, most preferably 18 to 22 days.

[0215] In one embodiments, step (e2) may last for 5 to 60 days, preferably 10 to 50 days, more preferably 20 to 40 days, even more preferably, 25 to 35 days, most preferably 27 to 33 days

[0216] In one embodiment culturing in step (f) occurs in a medium comprising at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% Advanced DMEM / F-12. In one embodiment culturing in step (f) occurs in a medium comprising at least 70% to 98%, preferably at least 80% to 95%, more preferably at least 85% to 95%, most preferably at least 87% to 93% Advanced DMEM / F-12.

[0217] In one embodiment culturing in step (f) occurs in a medium comprising at least 70% to 98%, preferably at least 80% to 95%, more preferably at least 85% to 95%, most preferably at least 87% to 93% Advanced DMEM / F-12, 1 to 3% FBS

[0218] In one embodiment culturing in step (f) occurs in a medium comprising at least 10 mM, preferably at least 20 mM, more preferably at least 25 mM glucose.

[0219] In one embodiment culturing in step (f) occurs in a medium comprising 10 mM to 50 mM glucose, preferably 15 to 30 mM glucose, more preferably 20 to 30 mM glucose, even more preferably 23 to 28 mM glucose, such as 25 mM glucose.

[0220] In one embodiment culturing in step (f) occurs in a medium comprising Advanced DMEM / F-12 and B-27 Supplement (without Vitamin A). In one embodiment culturing in step (f) occurs in a medium comprising glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A) and N-2 Supplement

[0221] In one embodiment culturing in step (f) occurs in a medium comprising glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), N-2 Supplement and Taurin.

[0222] In a particular embodiment, culturing in step (f) occurs in a medium comprising glucose, Advanced DMEM / F-12, B-27 Supplement (without Vitamin A), N-2 Supplement, Antibiotic Antimycotic, Taurin, Lipid mixture and FBS.

[0223] In one embodiment culturing in step (f) occurs in a medium comprising 0.5% to 5% FBS, preferably 1% to 3% FBS.

[0224] In a particular embodiment, culturing in step (f) occurs in a medium comprising glucose, Advanced DMEM / F-12, A specific example of the medium, that could be used in step (f) is described elsewhere herein as M8.

[0225] In one embodiment, the late phase retinal organoids obtained in step (f) comprise at least one cell type selected from the group of photoreceptors and Muller glia cells.

[0226] In one embodiment, the late phase retinal organoids obtained in step (f) comprise at least one cell type selected from the group of photoreceptors, mature RGCs, horizontal cells, amacrine cells, bipolar cells and Muller glia cells

[0227] In one embodiment, the late phase retinal organoids obtained in step (f) about 20 to 60% photoreceptors, 0 5 to 15% mature RGCs, 0 5 to 15% horizontal cells, 0 5 to 15% amacrine cells, 0 5 to 20 % bipolar cells and 0.5 to 15% Muller glia cells. In one embodiment, photoreceptors of the late phase retinal organoids obtained in step (f) comprise about 20 to 60%, preferably about 30 to 50%, more preferably 35 to 45% rod photoreceptors and 5 to 40%, preferably 10 to 30%, more preferably 15 to 25% cone photoreceptors In one embodiment, photoreceptors of the late phase retinal organoids obtained in step (f) comprise rods, S cones and M cones. More specifically, in one embodiment, photoreceptors of the late phase retinal organoids obtained in step (f) comprise about 10% to 50% rods, 1% to 15 % S cones and 1% to 20% M cones In one embodiment, the late phase retinal organoids obtained in step (f) comprise less than 5%, preferably, less than 3%, more preferably less than 2%, most preferably less than 1% dividing cells

[0228] In one embodiment, the late phase retinal organoids obtained in step (f) are positive for all markers selected from the group of CRX, RECOVERIN, RXRy and NLR.

[0229] In one embodiment, the late phase retinal organoids obtained in step (f) are positive for all markers selected from the group of CRX, RECOVERIN, RXRy, NLR, POU4F1 , Brn3a, AP2a, Proxl, RHODOPSIN, L / M-OPSIN, S-OPSIN and PERIPHERIN2.

[0230] In one embodiment, the late phase retinal organoids obtained in step (f) are positive for at least one, preferably at least 2, more preferably at least 3, even more preferably at least 4 of the markers selected from the group of RHODOPSIN, L / M-OPSIN, S-OPSIN and PERIPHERIN2

[0231] In one embodiment, the late phase retinal organoids obtained in step (f) are positive for at least one, preferably at least 2, more preferably at least 3, even more preferably at least 4 of the markers selected from the group of CRX, RECOVERIN, RXRy, NLR, POU4F1, Brn3a, AP2a, Proxl , RHODOPSIN, L / M- OPSIN, S-OPSIN and PERIPHERIN2.

[0232] In one embodiment, the late phase retinal organoids obtained in step (f) are positive for all markers selected from the group of RHODOPSIN, L / M-OPSIN, S-OPSIN and PERIPHERIN2.

[0233] The term “late phase retinal organoids” refers to refers to a cell aggregate comprising photoreceptor cells and Muller glia cells.

[0234] In one embodiment, the photoreceptors express at least one, at least two, at least three of the markers selected from the group of ARR3, Rhodopsin, S-Opsin L / M Opsin, Peripherin2. In one embodiment, the photoreceptors express all of the markers selected from the group of ARR3, Rhodopsin, S- Opsin L / M Opsin, Peripherin2.

[0235] In one embodiment, the photoreceptors express RECOVERIN and CRX co-localizing to all NRL+ or RXRy+ cells.

[0236] In one embodiment photoreceptor cells may express at least one marker selected from the group of CRX, RECOVERIN, RHO, OPNI SW and OPN1 MW.

[0237] In a particular embodiment, photoreceptor cells may express at least one marker selected from the group of CRX, RECOVERIN, RHO, OPN1SW, OPN1 MW, NLR, ARR3, RYRy and MPP4

[0238] In one embodiment rods may express at least one at least one marker selected from the group of RHO and NRL.

[0239] In one embodiment cones may express at least one at least one marker selected from the group of OPN1SW, OPN1 MW, ARR3 and RYRy.

[0240] In one embodiment S cones may express OPN1 SW.

[0241] In one embodiment M cones may express OPN1 MW.

[0242] In one embodiment, the bipolar cells may express at least one at least one marker selected from the group of VSX1 , VSX2, PKC-a.

[0243] In one embodiment the horizontal cells may express at least one at least one marker selected from the group of PROX1 and ONECUT1.

[0244] In one embodiment the Ganglion cells may express at least one at least one marker selected from the group of PUF4F1 , HuC / D, SLC17A6.

[0245] In one embodiment, the late phase retinal organoids obtained in step (f) express S-opsin In one embodiment, the late phase retinal organoids obtained in step (f) express S-opsin colocalizing to RXRy+.

[0246] In one embodiment in step (f) at least 5%, preferably at least 10% preferably at least 15% of cells of the late phase retinal organoids express CD73.

[0247] In one embodiment in step (f) 5% to 50%, preferably 10 to 40%, more preferably 15 to 25% of the cells of the late phase retinal organoid express CD73.

[0248] In one embodiment, the Muller glia cells may express at least one at least one marker selected from the group of SLC1A3 and RLBP1.

[0249] The Muller glia cells may be determined by expression of RLBP1 (CRALBP)

[0250] Step (f) may last at least 1 , at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80 days. Step (f) may be at 80, at most 100, at most 110, at most 120, at most 150, at most 200, at most 300 days

[0251] The term “retinal organoid” refers to an in vitro 3D model of the neural retina The retinal organoid does not comprise the immune system or the vasculature of the retina. The retinal organoid recapitulates the human development of the neural retina and thus comprises all neural cell types which are present in the human neural retina at a certain developmental stage in the corresponding developmental stage of the retinal organoid Hence the retinal organoid recapitulates morphology, development, structural complexity, and some key functions, such as the production of the outer segments and light sensibility as the human retina.

[0252] In one embodiment, homogenous compositions of retinal organoids can be achieved by the method of the invention. That means that throughout the protocol a homogenous composition of cell aggregates that are at the same development stage is achieved. That means that the suspension of retinal organoids is homogenous in terms of the types of the cell aggregates contained therein, i.e. it contains only retinal organoids and no other cell aggregates with a different differentiation fate. More specifically, in step (d) a homogenous composition of neuro-retinal precursors is obtained; in step (e) a homogenous suspension of early phase retinal organoids is obtained; in step (f) a homogenous suspension of late phase retinal organoids is obtained.

[0253] In one embodiment, the method further comprises the subsequent step of dissociation of the retinal organoid and sorting the cells obtained according to their cell fate. In other words, in one embodiment the method further comprising the subsequent steps (g) dissociation of the retinal organoid and (h) sorting the cells obtained according to their cell fate. Thereby in step (h) a population of sorted retinal cells is obtained. More specifically, the population obtained in step (h) is enriched for photoreceptor cells Accordingly, in one embodiment, the population of sorted retinal organoid cells comprises at least 60 %, preferably at least 70%, more preferably at least 80 %, even more preferably at least 85 %, most preferably at least 90 % photoreceptor cells. In one specific embodiment, the population of sorted retinal organoid cells comprises at least 5 %, preferably at least 8 %, more preferably at least 10 %, even more preferably at least 20 %, most preferably at least 30 % early opsin-expressing photoreceptor cells.

[0254] The term "early opsin-expressing photoreceptors" refers to a developmental stage of photoreceptor cells in the retina that begin to express opsins, which are light-sensitive proteins essential for vision. These photoreceptors are in the initial phase of differentiation and, although they may not yet fully function as mature photoreceptors, they start to express opsins at this early stage.

[0255] Typically, the retinal organoid dissociated in step (g) is a late stage retinal organoid, which was differentiated according to steps (a) to (f) for 100 to 160 days, preferably 110 to 150 days, more preferably 115 to 130 days, most preferably 120 days In other words, the total duration of step (a) to step (f) is to steps (a) to (f) retinal organoids.

[0256] In one embodiment, the cells expressing CD133 on the cell surface are identified as photoreceptor cells and isolated.

[0257] The use of late-stage retinal organoids, i e retinal organoids differentiated for 100 to 160 days, preferably 110 to 150 days, more preferably 115 to 130 days, most preferably 120 days is advantageous for the use of CD 133 as selection marker for photoreceptor cells At this stage the retinal organoids do not contain pluripotent cells , precursor cells and retinal precursors, that are not desired but might be detected by CD133 Proliferating cells are excluded by the CD133 sorting process In accordance with the embodiments, the population of sorted retinal organoid cells comprises substantially no pluripotent cells and / or nor precursor cells and / or nor retinal precursors and / or no proliferative cells.

[0258] Thus in one embodiment, the late stage retinal organoids only contain fate committed retinal cells. This is advantageous, since the cell population is defined and will not change its cell type composition after transplantation into the human body.

[0259] The term "precursor cell" refers to an undifferentiated cell that has the potential to develop into a variety of specialized cell types. These cells are typically in an early developmental stage and have not yet committed to a specific lineage Precursor cells have the capacity for differentiation into one or more distinct types of mature cells, depending on the signals and environment they encounter

[0260] The term "retinal precursors" refers to a developmental stage of retinal cells where early, undifferentiated cells retain the potential to differentiate into any type of neuroretinal cell These include photoreceptor cells (rods and cones), bipolar cells, and amacrine cells Retinal precursors are present in the early stages of retinal development before they undergo fate specification to become distinct cell types.

[0261] The term "fate-committed retinal cell" refers to a retinal cell that has already undergone a process of differentiation and is now committed to a specific cell type within the retina These cells have progressed beyond the precursor or retinal precursor stage and are destined to become a specific subtype, such as a photoreceptor, bipolar cell, or amacrine cell. The term "fate-committed retinal cell" includes “photoreceptor precursors” which can differentiate to different photoreceptor subtypes (i.e. cones or rods) however not to different retinal cell subtype (such as bipolar cell or amacrine cell).

[0262] Accordingly, it is not necessary to use further negative or positive selection markers to enrich high yields of photoreceptor cells. Accordingly, in one embodiment, no further maker besides CD133 is used for cell fate sorting. In particular in step (h) CD73 is not used as selection marker.

[0263] The term "photoreceptor cells" refers to rods and cones which are specialized cells, found in the retina of the eye that are responsible for detecting light and converting it into electrical signals that can be interpreted by the brain. Rod cells are primarily responsible for vision in low-light conditions, while cone cells are involved in color vision and detail perception in brighter light conditions. The term cones M-cones (medium wavelengths cones sensitive to light of about 530 nm), S-cones (short wavelengths conessensitive to light of about 420 nm) and L- cones (long wavelengths cones sensitive to light of about 650 nm).

[0264] In one embodiment in step (h) rods and cones are enriched, in particular rods, M-cones and S- cones.

[0265] Accordingly, the population of sorted retinal organoid cells may comprise at least 13 %, preferably at least 14% of cones and / or at least 70% rods, preferably at least 75% rods.

[0266] Thus, in one embodiment, the population of sorted retinal organoid cells comprises at least 70 % rods, preferably at least 75% rods, at least 13 % of cones, preferably at least 14 % cones.

[0267] In specific embodiments, the population of sorted retinal organoid cells comprises at least 4% S cones and at least 8 % M cones.

[0268] In one embodiment, the population of sorted retinal organoid cells comprises at least 80% cells, preferably 85 % cells are expressing CD133.

[0269] In a further embodiment, the population of sorted retinal organoid cells comprises cells expressing NRL.

[0270] Surprisingly, the inventors found that using the dissociated late-stage retinal organoids the yield photoreceptor cells in CD133+ sorted cell population is higher compared to the yield of a CD73+ cells sorted cell population. Accordingly, in one embodiment the number of photoreceptor cells in CD133+ sorted cell population is at least 1 times higher, such as at least 1.1 times, 1.2 times higher, 1.5 times higher, compared to the yield of a CD73+ cells sorted cell population Hence, one aspect of the invention refers to the use of CD133 as marker for isolating photoreceptor cells from population of retinal cells.

[0271] Accordingly, a related aspect of the invention refers to a method for identifying photoreceptor cells in a population of retinal cells, comprising the steps of i) determining whether or not the cell in the population express CD133 on the cell surface, and ii) identifying a cell as a photoreceptor cell if is CD133 positive.

[0272] Another aspect of the invention refers to a method of isolating photoreceptor cells in a population of cells, comprising the steps of i) determining whether a cell in the population expresses CD133 on the cell surface, ii) identifying a cell as a photoreceptor cell if the cell expresses CD133 on the cell surface, Hi) isolating the cell identified as photoreceptor cell.

[0273] In one embodiment, the cell population of retinal cells is obtained from dispersed retinal organoids. As already described above, it is advantageous to us late stage retinal organoids Such late stag retinal organoids are free of pluripotent cells and / or retinal progenitor cells and / or progenitor cells which are not desired and which might be also enriched CD133 sorting. Accordingly in one embodiment, the retinal organoids are late phase retinal organoids. Typically, the differentiation of the retinal organoids lasts 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days. In one embodiment, the differentiated according to steps (a) to (f) lasts for 100 to 160 days, preferably 110 to 150 days, more preferably 115 to 130 days, most preferably 120 days.

[0274] CD133 is used for identification and enrichment of photoreceptor cells. In other words, cells expressing CD133 on the cell surface are identified as photoreceptor cells. As described above, the inventors surprisingly found that CD133 can be used for enrichment of photoreceptor cells as single selection marker. Thus, in one embodiment no further positive marker and / or negative marker is used. Accordingly, in one embodiment CD133 is the single marker used for isolating photoreceptor cells. In particular, CD73 is not used for isolating photoreceptor cells.

[0275] Typically, an antibody binding to surface expressed CD133 is used for isolation of the photoreceptor cells.

[0276] For example, fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS) may be used for isolation of the photoreceptor cells

[0277] In some embodiments, the photoreceptor cells have not been genetically modified to aid the isolation of the photoreceptor cells

[0278] The use of CD133 as a selection marker for photoreceptor cells and the respective method described above allow for the reliable enrichment of photoreceptors in high yields.

[0279] Accordingly, the cell population obtained by CD133 sorting as described above may comprise at least 13 %, preferably at least 14% cones and / or at least 70% rods, preferably at least 75% rods.

[0280] In some embodiments the sorted cell population comprises 70 % rods, preferably at least 75% rods, at least 13 % cones, preferably at least 14 % cones. In some embodiments the cell population may comprise at least 4% S cones and / or M cones. Typically, the sorted cell population comprises (substantially) no pluriportent cells and or (substantially) no proliferative cells. In some embodiments, the sorted cell population comprises at least 80% cells, preferably 85 % cells, that are expressing CD133 In some embodiments, the sorted cell population comprises at least 80% cells, at least 90% cells, at least 95% cells expressing NRL.

[0281] Surprisingly, the inventors found that using the dissociated late stage retinal organoids the yield of photoreceptor cells in the CD133+ sorted cell population is higher compared to the yield of a cell population sorted for CD73+ cells . Accordingly, in one embodiment the number of photoreceptor cells in CD133+ sorted cell population is at least 1 times higher, such as at least 1.1 times, 1.2 times higher, 1.5 times higher, compared to the yield of a CD73+ cells sorted cell population.

[0282] Accordingly, a further aspect refers to a retinal cell population comprising at least 70 % rods, preferably at least 75% rods, and at least 13 % of cones, preferably at least 14 % cones.

[0283] In one embodiment, the retinal cell population comprises at least 4% S cones. In one embodiment, the retinal cell population comprises at least 8 % M cones In one embodiment, the retinal cell population comprises substantially no pluripotent cells In one embodiment, the retinal cell population comprises substantially no proliferative cells In one embodiment, the retinal cell population comprises at least 80%, preferably 85 %, cells expressing CD133 at the cell surface. In one embodiment, the retinal cell population comprises cells expressing NRL In one embodiment, the retinal cell population cells are obtained from retinal organoids cultured for 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days, i.e. late phase retinal organoids. In other words, the retinal organoids were grown from single cell culture within 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days The embodiments describing the late phase retinal organoids obtained in step (f) mentioned above, also apply to the retinal cell population as defined above.

[0284] Another aspect refers to a pharmaceutical composition comprising the retinal cell population as described above and a pharmaceutical acceptable carrier.

[0285] The application further comprises the following embodiments:Embodiment 1. A method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies; and(c) culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregatesEmbodiment 2. The method according to embodiment 1 , wherein any one of steps (a), (b) and (c) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel.Embodiment 3. The method according to embodiment 1 or embodiment 2, wherein in step (b) and / or step (c) shear stress is applied to the 3D suspension culture.Embodiment 4. The method of any one of the preceding embodiments, wherein step (b) and / or step (c) occur under agitation.Embodiment 5. The method according to embodiment 4, wherein agitation occurs at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm.Embodiment 6. The method according to any one of the preceding embodiments, wherein the 3D suspension culture is carried out in ultra-low attachment cell culture plates.Embodiment 7. The method according to any one of the preceding embodiments, wherein the embryoid bodies comprise cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1Embodiment 8. The method according to any one of the preceding embodiments, wherein the embryoid bodies comprise at least 60%, at least 70%, at least 80%, at least 90% cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1Embodiment 9. The method according to any one of the preceding embodiments, wherein the embryoid bodies have a uniform shape and / or a diameter of about 150 pm.Embodiment 10. The method according to any one of the preceding embodiments, wherein the eye-field cell aggregates comprise cells expressing at least one of the eye field transcription factors PAX6, RAX, SIX3, LHX2 and OTX2Embodiment 11 . The method according to any one of the preceding embodiments, wherein the eye-field cell aggregates comprise cells expressing PAX6 and OTX2.Embodiment 12 The method according to any one of the preceding embodiments, wherein the eye-field cell aggregates comprise at least 50%, preferably at least 60%, more preferably at least 80% cells expressing PAX6 and OTX2Embodiment 13 The method according to any one of the preceding embodiments, wherein the single cell suspension of stem cells of step (a) is provided by the following steps: (a1 ) culturing stem cells in adherent culture; and(a2) dissociating the stem cells to obtain single cell suspensionEmbodiment 14. The method according to embodiment 13, wherein step (a1 ) lasts 3 to 7 days preferably 3 to 6 days, more preferably 3 to 5 days, most preferably 4 to 5 days;Embodiment 15. The method according to any one of the preceding embodiments, wherein step (b) lasts 1 to 6 days, preferably 2 to 5 days, more preferably 3 to 5 days, even more preferably 4 days;Embodiment 16. The method according to any one of the preceding embodiments, wherein step (c) lasts 3 to 7 days, preferably 3 to 6 days, more preferably 4 to 5 days, most preferably 4 days.Embodiment 17. The method according to embodiments 13 to 16, wherein in step (a1 ) the stem cells are cultured until 70-80% confluency.Embodiment 18. The method according to any one of the preceding embodiments, wherein the stem cells express SOX2, Nanog, Oct4, SSEA1 and Tra1.Embodiment 19. The method according to any one of the preceding embodiments, wherein at least 60%, at least 70%, at least 80%, at least 90% of the stem cells of step (a) express SOX2, Nanog, Oct4, SSEA1 and Tra1.Embodiment 20. The method according to any one of the preceding embodiments, wherein in step (b) a different medium is used as in step (c).Embodiment 21 The method according to any one of the preceding embodiments, wherein the medium in step (b) contains TGF-p and bFGF.Embodiment 22. The method according to any one of the preceding embodiments, wherein no TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor or GSK3p inhibitor is used, preferably no TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor, SMAD inhibitor, BMP inhibitor and GSK3p inhibitor is used.Embodiment 23. The method according to any one of the preceding embodiments, wherein in step (b) a stem cell medium is usedEmbodiment 24. The method according to any one of the preceding embodiments, wherein in step (c) a medium comprising neurobasal medium is usedEmbodiment 25. The method according to any one of the preceding embodiments, wherein the stem cell medium used in step (b) is supplemented with a ROCK inhibitor.Embodiment 26 The method according to any one of the preceding embodiments, wherein the stem cell medium used in step (b) is supplemented with a ROCK inhibitor for a first period of step (b).Embodiment 27. The method according to any one of the preceding embodiments, wherein the first period of step (b) begins with the onset of the culturing of the single cell suspension and lasts for 2 to 48h, preferably 12 to 36 h, most preferably 24 h.Embodiment 28. The method according to any one of the preceding embodiments, wherein in step (b) cells at a final concentration of 1 X 106cells / ml were seeded in ultra-low attachment cell culture plates.Embodiment 29. The method according to any one of the preceding embodiments, wherein in step (b) a total of 5,5 X 106cells were seeded per 9,6Embodiment 30. A method of differentiating stem cells into retinal organoids, comprising the steps of embodiments 1 to 19 and further comprising the steps:(d) culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors;(e) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain early phase retinal organoids.Embodiment 31 . The method of embodiment 30, further comprising the step:(f) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain late phase retinal organoids.Embodiment 32. The method of embodiment 30 and 31 , wherein any one of steps (d), (e) and (f) occurs in the in the absence of a support matrix, preferably in the absence of a proteinaceous gel.Embodiment 33. The method according to any one of embodiments 30 to 32, wherein the eye-field cell aggregates used in step (d) comprise at least 70%, preferably at least 80% cells expressing PAX6 and OTX2.Embodiment 34. The method of embodiments 30 to 33, wherein the early phase retinal organoids obtained in step (e) are positive for at least one marker selected from the group of CRX, RECOVERIN and RXRyEmbodiment 35. The method according to any one of embodiments 30 to 34, wherein culturing in step (d) comprises:(d1 ) culturing the eye-field cell aggregates in a medium comprising neurobasal medium [M4]; and(d2) culturing the eye-field cell aggregates obtained in step (d1 ) in a high glucose medium comprising 17,5mM], supplemented with retinoic acid [M5],Embodiment 36 The method according to embodiment 35, wherein culturing in (d2) occurs in a medium which does not contain neurobasal medium.Embodiment 37. The method according to any one of embodiments 30 to 36, wherein culturing in step (e) comprises:(e1 ) culturing in a high glucose supplemented with FBS and retinoic acid; and(e2) culturing in a high glucose medium supplemented with FBS and retinoic acid.Embodiment 38 The method according to embodiment 37, wherein culturing in (e1 ) occurs in a high glucose medium supplemented with FBS and 0.5 pM retinoic acidEmbodiment 39. The method according to embodiment 37 or 38, wherein culturing in (e2) occurs in a high glucose medium supplemented with FBS and 0 25 pM retinoic acidEmbodiment 40. The method according to any one of embodiments 37 to 39 Wherein culturing in (e2) occurs in a medium supplemented with FBS, retinoic acid and lipids.Embodiment 41 . The method according to any one of embodiments 31 to 40, wherein culturing in step (f) comprises culturing in a retinal maturation medium, without addition of small molecules.Embodiment 42. The method according to any one of embodiments 31 to 41 , wherein culturing in (f) comprises culturing in a high glucose medium supplemented with FBS and lipids.Embodiment 43. The method according to any one of embodiments 31 to 42, wherein culturing in (f) occurs without retinoid acid supplementation.Embodiment 44. A method for differentiating stem cells into retinal pigmented epithelium cells, comprising the steps of embodiments 1 to 19 and further comprising the steps:(dd) dissociating the eye-field cell aggregates to single cells;(ee) culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells.Embodiment 45. The method according to embodiment 44, wherein the eye-field cell aggregates used in step (dd) comprise at least 50%, preferably at least 60% expressing PAX6 and OTX2Embodiment 46. The method according to embodiment 44 or 45, wherein in step (dd) and / or step (ee) the cells form a monolayerEmbodiment 47. The method according any one of embodiments 44 to 46, wherein the retinal pigmented epithelium cells express one or more of the markers selected from the group of MITF, PAX6, MERTK, VMD2, SERPINF-1, TYRP1, CLDN19 and RPE65.Embodiment 48. The method according to any one of embodiments 44 to 47, wherein the retinal pigmented epithelium cells express one or more of the genes selected from the group of MITF, PAX6, RPE65 and TYRP1Embodiment 49. The method according to any one of embodiments 44 to 48, wherein the retinal pigmented epithelium cells have at least one of the functions selected form the group of transepithelial resistance (TER), VEGF secretion, phagocytosisEmbodiment 50. The method according any one of embodiments 44 to 49, wherein culturing in (ee) comprises culturing in a RPE differentiation medium which is optionally a serum-free medium, wherein said medium comprises at least one growth factor selected from agonists of the Activin A pathwayEmbodiment 51 . The method according to any one of embodiments 44 to 50, wherein culturing in (ee) comprises(eel ) culturing in a medium comprising neurobasal medium supplemented with a ROCK inhibitor; and (ee2) culturing in a RPE differentiation medium supplemented with at least one agonists of the Activin A pathway.Embodiment 52 Method according to any one of embodiments 44 to 51 , wherein culturing in (ee) occurs in a culture vessel with an upper and lower chamber, optionally wherein said culture vessel is a transwell plate.Embodiment 53. The method according to any one of embodiments 44 to 52, wherein step (ee) lasts 10 to 200 days, 12 to 150 days, 15 to 100 days, 20 to 80 days, 30 to 70 days, 40 to 60 daysEmbodiment 54. The method according to any one of embodiments 44 to 53, wherein the culturing in step (ee) is carried out in a culture vessel coated with at least two proteins, and preferably at least three proteins, selected from the group of Laminin, Collagen IV, Entactin and Perlecan.Embodiment 55. The method according to any one of the preceding embodiments, wherein the stem cell is selected from the group of a pluripotent stem cell, an induced pluripotent stem cell and / or established stem cell line.Embodiment 56. The method according to any one of the preceding embodiments, wherein the stem cell is a pluripotent stem cell.Embodiment 57. The method according to any one of the preceding embodiments, wherein the stem cell is a mammalian stem cell; preferably a human stem cell.Embodiment 58. The method according to any one of the preceding embodiments, wherein the stem cell is a human induced pluripotent stem cell.Embodiment 59. The method according to any one of the preceding embodiments, wherein the method is carried out in the absence of a feeder cell.Embodiment 60. The method according to any one of the preceding embodiments, wherein exogenous nucleic acid and / or exogenous protein is not introduced into the stem cells or differentiated cells derived thereofEmbodiment 61 . The method according to any one of embodiments 1 to 43 and 55 to 60, further comprising the subsequent steps (g) dissociation of the retinal organoid and(h)sorting the cells obtained according to their cell fateEmbodiment 62. The method according to embodiment 61 , wherein in step (h) a population of sorted retinal cells is obtained.Embodiment 63. The method according to any one of embodiments 1 to 43 and 55 to 62, wherein the total duration of step (a) to step (f) is 100 to 160 days, preferably 110 to 150 days, more preferably 115 to 130 days, most preferably 120 days.Embodiment 64. The method according to any one of embodiments 61 to 63, wherein in step (h) the cells expressing CD133 on the cell surface are identified as photoreceptor cells and isolated.Embodiment 65 The method according to any one of embodiments 61 to 64, wherein in step (h) no further marker is used for cell fate sorting.Embodiment 66. The method according to any one of embodiments 61 to 65, wherein in step (h) the cells are sorted for cell fate not by CD73Embodiment 67. The method according to any one of embodiments 61 to 66, wherein the population obtained in step (h) is enriched for photoreceptor cellsEmbodiment 68. The method according to any one of embodiments 61 to 67, wherein the population of sorted retinal organoid cells comprises at least 60 %, preferably at least 70 %, more preferably at least 80 %, even more preferably at least 85 %, most preferably at least 90 % photoreceptor cells.Embodiment 69. The method according to any one of embodiments 61 to 68, wherein the population of sorted retinal organoid cells comprises at least 5 %, preferably at least 8 %, more preferably at least 10 %, even more preferably at least 20 %, most preferably at least 30 % early opsin-expressing photoreceptor cells.Embodiment 70. The method according to any one of embodiments 61 to 69, wherein the population of sorted retinal organoid cells comprises at least 13 %, preferably at least 14% cones.Embodiment 71. The method according to any one of embodiments 61 to 70, wherein the population of sorted retinal organoid cells comprises at least 70% rods, preferably at least 75% rods.Embodiment 72. The method according to any one of embodiments 61 to 71 , wherein the population of sorted retinal organoid cells comprises at least 70 % rods, preferably at least 75% rods, at least 13 % cones, preferably at least 14 % cones.Embodiment 73. The method according to any one of embodiments 61 to 72, wherein the population of sorted retinal organoid cells comprises at least 4% S cones.Embodiment 74 The method according to any one of embodiments 61 to 73, wherein the population of sorted retinal organoid cells comprises at least 8 % M cones.Embodiment 75. The method according to any one of embodiments 61 to 74, wherein the population of sorted retinal organoid cells comprises substantially no pluripotent cellsEmbodiment 76. The method according to any one of embodiments 61 to 75, wherein the population of sorted retinal organoid cells comprises substantially no proliferative cells.Embodiment 77. The method according to any one of embodiments 61 to 76, wherein the population of sorted retinal organoid cells comprises at least 80% cells, preferably 85 % cells, are expressing CD133Embodiment 78 The method according to any one of embodiments 61 to 77, wherein the population of sorted retinal organoid cells comprises cells expressing NRL.Embodiment 79. The method according to any one of embodiments 61 to 78, wherein the cells are obtained from retinal organoids cultured for 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 80. The method according to any one of embodiments 61 to 99, wherein the retinal organoids were grown from single cell culture within 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 81. The method according to any one of the previous embodiments, further comprising the subsequent step of cryopreserving the cells or organoids obtained.Embodiment 82. The method according any one of claims 1 to 29, wherein a homogenous population of eyefield cell aggregates is obtained.Embodiment 83. The method according to any one of claims 30 to 43, wherein a homogenous population of retinal organoids is obtainedEmbodiment 84. The method according to any one of claims 43 to 54, wherein a homogenous population of RPE cells is obtained.Embodiment 85. The retinal organoid obtained by the method of embodiment 30 to 43 and 55 to 60.Embodiment 86. Cells obtained from the retinal organoid according to embodiment 85, or obtained by the method of embodiments 61 to 81 .Embodiment 87. The retinal pigmented epithelium cells obtained by the method of embodiments 43 to 54.Embodiment 88. The eye-field cell aggregates obtained by the method according to any of the embodiments 1 to 29.Embodiment 89. The retinal organoid of embodiment 85 or the cells according to embodiment 86 for use in therapy preferably transplantation therapy.Embodiment 90. The retinal organoid for use of embodiment 89, wherein the retinal organoid is for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophy.Embodiment 91. Use of the retinal organoid of embodiment 85 in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivo.Embodiment 92 The cells according to embodiment 86 for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophy.Embodiment 93 Use of the cells according to embodiment 86 in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivo.Embodiment 94. The retinal pigmented epithelium cells of embodiment 87 for use in therapy preferably transplantation therapyEmbodiment95. The retinal pigmented epithelium cells for use of embodiment 94, wherein the retinal pigmented epithelium cells are for use in treating or preventing retinal diseases comprising age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or retinal pigment epithelium hypertrophyEmbodiment 96. Use of the retinal pigmented epithelium cells of embodiment 87, in screening the toxicity and / or activity of a test compound, optionally wherein screening is carried out in vitro or ex vivoEmbodiment 97. Use of CD133 as marker for isolating photoreceptor cells from a population of retinal cells.Embodiment 98. Use according to embodiment 97, wherein the cell population of retinal cells is obtained from dispersed retinal organoidsEmbodiment 99. Use according to embodiment 97 or 98, wherein cells expressing CD133 on the cell surface are identified as photoreceptor cellsEmbodiment 100. Use according to any one of embodiments 97 to 99, wherein no further positive marker is used.Embodiment 101. Use according to any one of embodiments 97 to 100, wherein no further negative marker is used.Embodiment 102. Use according to any one of embodiments 97 to 101 , wherein CD133 is the single marker used for isolating photoreceptor cellsEmbodiment 103. Use according to any one of embodiments 97 to 102, wherein CD73 is not used for isolating photoreceptor cells.Embodiment 104. Use according to any one of embodiments 97 to 103, wherein an antibody binding to surface expressed CD133 is used for isolation of the photoreceptor cellsEmbodiment 105. Use according to any one of embodiments 97 to 104, wherein fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS) is used for isolation of the photoreceptor cellsEmbodiment 106. Use according to any one of embodiments 97 to 105, wherein the photoreceptor cells have not been genetically modified to aid the isolation of the photoreceptor cellsEmbodiment 107 Use according to any one of embodiments 98 to 106, wherein the retinal organoids are late phase retinal organoidsEmbodiment 108. Use according to embodiment according to any one of embodiments 98 to 107, wherein the differentiation of the retinal organoids lasts 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 109. A method for identifying photoreceptor cells in a population of retinal cells, comprising the steps of i) determining whether or not the cell in the population express CD133 on the cell surface, and ii) identifying a cell as a photoreceptor cell if it is CD133 positiveEmbodiment 110 A method of isolating photoreceptor cells in a population of cells, comprising the steps of i) determining whether a cell in the population expresses CD133 on the cell surface, ii) identifying a cell as a photoreceptor cell if the cell expresses CD133 on the cell surface, Hi) isolating the cell identified as photoreceptor cell.Embodiment 111. Method according to embodiments 109 and 110, wherein no further positive marker is used.Embodiment 112. Method according to any one of embodiments 109 to 111, wherein no further negative marker is used.Embodiment 113. Method according to any one of embodiments 109 to 112, wherein CD133 is used as a single marker.Embodiment 114. Method according to any one of embodiments 109 to 113, wherein CD73 is not used.Embodiment 115. Method according to any one of embodiments 109 to 114, wherein FACS or MACS is used for isolation of the photoreceptor cells.Embodiment 116. Method according to any one of embodiments 109 to 115, wherein the photoreceptor cells have not been genetically modified to aid the isolationEmbodiment 117. Method according to any one of embodiments 109 to 116, wherein an antibody binding to surface expressed CD133 is used.Embodiment 118. Method according to any one of embodiments 109 to 117, wherein the retinal organoids are late phase retinal organoids.Embodiment 119 Method according to any one of embodiments 109 to 118, wherein the population obtained in step iii) comprises at least 60 %, preferably at least 70 %, more preferably at least 80 %, even more preferably at least 85 %, most preferably at least 90 % photoreceptor cellsEmbodiment 120 Method according to any one of embodiments 109 to 119, wherein the population obtained in step iii) comprises at least 5 %, preferably at least 8 %, more preferably at least 10 %, even more preferably at least 20 %, most preferably at least 30 % early opsin-expressing photoreceptor cells.Embodiment 121 The method according to any one of embodiments 109 to 120, wherein the population obtained in step iii) comprises at least 13 %, preferably at least 14% cones.Embodiment 122. The method according to any one of embodiments 109 to 121 , wherein the population obtained in step iii) comprises at least 70% rods, preferably at least 75% rodsEmbodiment 123. The method according to any one of embodiments 109 to 122, wherein the population obtained in step iii) comprises at least 70 % rods and at least 13 % cones, preferably at least 75% rods and at least 14% conesEmbodiment 124. The method according to any one of embodiments 109 to 123, wherein the population obtained in step iii) comprises at least 4% S cones.Embodiment 125. The method according to any one of embodiments 109 to 124, wherein the population obtained in step iii) comprises at least 8 % M cones.Embodiment 126. The method according to any one of embodiments 109 to 125, wherein the population obtained in step iii) comprises substantially no pluripotent cells.Embodiment 127. The method according to any one of embodiments 109 to 126, wherein the population obtained in step iii) comprises substantially no proliferative cells.Embodiment 128. The method according to any one of embodiments 109 to 127, wherein the population obtained in step iii) comprises at least 80% cells, preferably 85 % cells, are expressing CD133.Embodiment 129. The method according to any one of embodiments 109 to 128, wherein the population obtained in step iii) comprises cells expressing NRL.Embodiment 130. The method according to any one of embodiments 109 to 129, wherein the population of cells was obtained from dissociated retinal organoids.Embodiment 131. The method according to any one of embodiments 109 to 130, wherein the retinal organoids were cultured for 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 132. The method according to any one of embodiments 109 to 131 , wherein the retinal organoids were grown from single cell culture within 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 133. Retinal cell population comprising at least 70 % rods, preferably at least 75% rods, and at least 13 % cones, preferably at least 14 % cones.Embodiment 134 Retinal cell population according to embodiment 133, wherein the retinal cell population comprises at least 4% S cones.Embodiment 135. Retinal cell population according to embodiment 133 or 134, wherein the retinal cell population comprises at least 8 % M conesEmbodiment 136. Retinal cell population according to any one of embodiments 133 to 135, wherein the retinal cell population comprises substantially no pluripotent cells.Embodiment 137. Retinal cell population according to any one of embodiments 133 to 136, wherein the retinal cell population comprises substantially no proliferative cells.Embodiment 138 Retinal cell population according to any one of embodiments 133 to 137, wherein the retinal cell population comprises at least 80%, preferably 85 %, for express CD133.Embodiment 139. Retinal cell population according to any one of embodiments 133 to 138, wherein the retinal cell population is obtained by isolation with the selection marker CD133 as single selection markerEmbodiment 140. Retinal cell population according to any one of embodiments 133 to 139, wherein the retinal cell population comprises cells expressing NRL.Embodiment 141. Retinal cell population according to any one of embodiments 133 to 140, wherein the cells are obtained from retinal organoids cultured for 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 142. Retinal cell population according to any one of embodiments 133 to 141, wherein the retinal organoids were grown from single cell culture within 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days.Embodiment 143. Retinal cell population according to any one of embodiments 133 to 141 , comprising at least 5 %, preferably at least 8 %, more preferably at least 10 %, even more preferably at least 20 %, most preferably at least 30 % early opsin-expressing photoreceptor cellsEmbodiment 144. Pharmaceutical composition comprising the retinal cell population according to claims 133 to 143 and an pharmaceutical acceptable carrier.Embodiment 145. Cell population comprising at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing OTX2, at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Oct4, and at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Sox2.Embodiment 146. Cell population according to any one of embodiments 145, wherein the cell population is derived from a dissociated embryoid bodyEmbodiment 147. Cell population according to any one of embodiments 145 or 146, wherein at least 20 % of the cells express Nanog.Embodiment 148. Cell population according to any one of embodiments 145 to 147, wherein the cell population expresses 0.1 to 1 fold, 0.1 to 0.5, preferably 0.1 to 0.3 fold amount of Nanog compared to iPSCsEmbodiment 149 Cell population according to any one of embodiments 145 to 148, wherein the cell population expresses 0.1 to 1 fold, 0.2 to 0.8, preferably 0.3 to 0.6 fold amount of Oct4+ compared to iPSCs.Embodiment 150. Cell population according to any one of embodiments 145 to 149, wherein the cell population expresses 1 to 4 fold, 2 to 3 5, preferably 2 5 to 3 fold amount of Sox2 compared to iPSCsEmbodiment 152. Cell population according to any one of embodiments 145 to 150, wherein the cell population forms an embryoid body.Embodiment 153. Cell population according to embodiment 152, wherein the embryoid bodies have a uniform shape and / or a diameter of about 50 to 300 pm, preferably of 100 to 200 pm, more preferably of 120 to 180 pm, most preferably of 150 pm.The application further comprises the following items:1. A method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryonic bodies; and(c) culturing the embryonic bodies in 3D suspension culture system to obtain eye-field cell aggregates.2. The method according to item 1 , wherein any one of steps (a), (b) and (c) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel, optionally wherein the 3D suspension culture is carried out in ultra-low attachment cell culture plates.3. The method according to item 1 or item 2, wherein in step (b) and / or step (c) shear stress is applied to the 3D suspension culture, optionally wherein step (b) and / or step (c) occur under agitation, optionally wherein agitation occurs at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm.4. The method according to any one of the preceding items, wherein the stem cell is a human induced pluripotent stem cell and wherein an exogenous nucleic acid and / or an exogenous protein is not introduced into the stem cells or differentiated cells derived thereof.5. The method according to any one of the preceding items, wherein the embryonic bodies comprise at least 60%, at least 70%, at least 80%, at least 90% cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1 and optionally wherein the embryonic bodies have a uniform shape and / or a diameter of about 150 pm.6. The method according to any one of the preceding items, wherein the eye-field cell aggregates comprise at least 50%, preferably at least 60%, more preferably at least 80% cells expressing PAX6 and OTX2.7. The method according to any one of the preceding items, wherein the single cell suspension of stem cells of step (a) is provided by the following steps:(a1 ) culturing stem cells in adherent culture; and(a2) dissociating the stem cells to obtain single cell suspension8. The method according to any one of the preceding items, wherein no TGF-[3 inhibitor, FGF8 inhibitor, AMPK inhibitor or GSK3p inhibitor is used.9. A method of differentiating stem cells into retinal organoids, comprising the steps of items 1 to 9 and further comprising the steps:(d) culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors;(e) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain early phase retinal organoids, and optionally(f) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain late phase retinal organoids, wherein any one of steps (d), (e) and (f) occurs in the in the absence of a support matrix, preferably in the absence of a proteinaceous gel10. The method according to any one of item 9, wherein the eye-field cell aggregates used in step (d) comprise at least 70%, preferably at least 80% cells expressing PAX6 and OTX2 and / or wherein the early phase retinal organoids obtained in step (e) are positive for at least one marker selected from the group of CRX, RECOVERIN and RXRy.11. The method according to any one of item 9 or 10, wherein culturing in step (d) comprises:(d1 ) culturing the eye-field cell aggregates in a medium comprising neurobasal medium; and(d2) culturing the eye-field cell aggregates obtained in step (d1 ) in a high glucose medium not containing neurobasal medium, supplemented with retinoic acid, and wherein culturing in step (e) comprises:(e1 ) culturing in a high glucose supplemented with FBS and retinoic acid; and(e2) culturing in a high glucose medium supplemented with FBS and retinoic acid.12. The method according to item 11 , wherein culturing in (e1) occurs in a high glucose medium supplemented with FBS and 0.5pM retinoic acid and wherein culturing in (e2) occurs in a high glucose medium supplemented with FBS and 0.25 pM retinoic acid, optionally containing lipids.13. A method for differentiating stem cells into retinal pigmented epithelium cells, comprising the steps of items 1 to 8 and further comprising the steps:(dd) dissociating the eye-field cell aggregates to single cells;(ee) culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells, and wherein culturing in (ee) comprises(eel ) culturing in a medium comprising neurobasal medium supplemented with a ROCK inhibitor; and(ee2) culturing in a RPE differentiation medium having supplemented with at least one agonists of the Activin A pathway.14. The method according to item 13, wherein the retinal pigmented epithelium cells express one or more of the genes selected from the group of MITF, PAX6 and OTX2.15. The method according any one of items 13 and 14, wherein culturing in (ee) comprises culturing in a RPE differentiation medium which is optionally a serum-free medium, wherein said medium comprises at least one growth factor selected from agonists of the Activin A pathway.16. The eye-field cell aggregates obtained by the method according to any one of items 1 to 817. The retinal organoid obtained by the method according to any one of items 9 to 12.18. Cells obtained from the retinal organoid according to item 17.19. The retinal pigmented epithelium cells obtained by the method of items 13 to 15.EXAMPLES

[0286] A general, schematic overview of the method is given in Figure 1 , while a detailed schematic representation of the procedure is given in Figure 2Human induced pluripotent stem cells (hiPSC) maintenance:

[0287] In an exemplary embodiment step (a) as defined herein is carried out as follows:Each well of a 6-well plate (6-WP) (Corning, 3516) was coated with hESC qualified Matrigel (Corning, 354277), by incubating 1 mL / well of 1 :25 hESC qualified Matrigel in DMEM / F-12, HEPES (Gibco, 31330-038), for 15 minutes at 37°C After coating, the matrigel solution was aspirated and cells were seeded in mTeSRI media (Stem Cell Technologies, 85850) Cells were cultured as a monolayer (2D adherent cultures), at 37 °C and 5% CO2 and fed with 2mL / well of mTeSRI daily, until reaching 70-90% confluency, at which time cell were passaged or used for embryonic body-formation (starting of differentiation).

[0288] Viability, quality, and pluripotency capacity of iPSCs was determined based on morphology (large nucleus, with high nucleus:cytoplasma ratio, open chromatin, and prominent nuclei), which was checked daily using a phase contrast microscopy (Figure 2A-C) and by flow cytometry (FC), for known pluripotency markers (SOX2, Nanog, Oct4, SSEA1 and Tra1 ) (Figure 2D-F), performed once per each thawed vial.Formation of embryoid bodies (EBs)

[0289] In an exemplary embodiment step (b) as defined herein is carried out as follows: Differentiation was initiated by the formation of EBs, once iPSCs reached 70-90% confluency, herein referred as differentiation day zero (ddO). Three to four wells of a 6-WP were used per desired well of a 6-WP of EBs. On ddO,representative phase contrast pictures of iPSC cultures were taken (Figure 3A). iPSCs were detached from the wells and dissociated into single cells. This was achieved by aspirating the mTeSR media, washing once with 1 mL of PBS and incubating the cells with 0,5mL Accutase (Sigma, A6964) for 5 minutes at 37°C The detached cells were collected from the wells into a 15ml falcon tube. Accutase was diluted by adding 2mL / well of DMEM / F-12, HEPES (Gibco, 31330-038). Tubes containing the single cell suspension were centrifuged at 300 rpm ( 17xg) for 5 minutes at room temperature (RT). The supernatant was discarded, and cells were resuspended in 1 mL of mTeSRI media (Stem Cell Technologies, 85850) per millimetre of cell pellet Final cell concentration was determined by cell counting using a Neubeauer chamber. The single cell suspension was added to a 6-well, ultra-low attachment plate (Corning, 3471 ), at a final concentration of 1 x 106 cells / mL, in 5,5 mL / well of mTeSRI media containing 10 pM ROCK inhibitor Y27632 (Absource Diagnostics GmbH , S1049-10MG ) (media also denoted as M0 herein) The plates were then placed in the incubator at 37 °C and 5% CO2, on a shaking platform at 100 rpm. After 24 h, dd1 , iPSCs aggregated, forming uniform EB aggregates. Their morphology was evaluated using a brightfield microscope and media was changed, by removing and adding fresh 5,5mL of mTeSRI media (no ROCK inhibitor added). The plate with EBs was then returned to the shaking platform, in the incubator, under the same conditions described previously EBs, were fed as described, daily for 2 more days (until dd4). Brightfield microscope pictures and daily evaluation of the morphology was also performed daily during this period (Figure 4A, C, D and F). field

[0290] In an exemplary embodiment step (c) as defined in the claims is carried out as follows:

[0291] On dd4, after visual inspection, medium was changed from M0 to M1 .. Eye- field aggregates were cultured on the shaking platform under the same condition previously stated. On dd6 medium was changed, with removing of media in culture and addition of fresh 5,5mL / well of M1. Visual inspection and brightfield microscope picture were taken daily during the eye field induction period (dd4 to dd8) (Figure 4B)

[0292] On dd8, successful eye field specification was confirmed by flow cytometry for PAX6 and OTX2, markers of neuroectoderm (Figure 4E and F) For this quality control (QC), a small portion of presumptive eye-field cell aggregates were collected into a 15 mL falcon and centrifuged at 300 rpm (17xg) for 1 minute. Collection was done randomly by pipetting some of the free floating aggregates. The supernatant was discarded, and aggregates were washed with 2 ml PBS'Mg / 'cl. Presumptive eye-field cell aggregates were then dissociated into single cells for staining and flow cytometry analysis using TrypLE™ Express Enzyme (ThermoFisher, 12605028) dilutes 1 :6 in DPBS'MgACI. After washing PBS was aspirated and 1 mL of prewarmed TrypLE™ Express Enzyme solution was added per millimeter of space occupied by the presumptive neuroectoderm aggregates at the bottom of the falcon (typically 1 mL per falcon). DNase (Sigma, 10104159001 ) was added to the dissociation solution at a 20 pl / ml final concentration Next, presumptive neuroectoderm aggregates were incubated, in the dissociation solution, at 37 °C in a water bath for 10 minutes. The falcon tubes were flicked at least once during this period After the incubation, manual dissociation was performed by pipetting samples with p1000 pipette 2 to 4 times. To stop the enzymatic reaction, Soybean Trypsin inhibitor (Sigma, T6522) was added to the cell suspension, at a final concentration of 1 mg / ml Cell suspension was then centrifuged at 1000 rpm (188xg) for 2 min at RT, supernatant was discarded, and cells resuspended in fresh M1 media, 1 mL per millimeter of cell pellet Cell number was determined by cell counting using a Neubauer chamber and 0.2 x 10scells were transferred to a round bottom plate for antibody staining A total of 2 wells per differentiation were plated (one for flow cytometry staining control and the other for PAX6 / OTX2 staining).

[0293] For PAX / OXT2 staining cells were resuspended in 100 pl Fix / Perm solution and incubated for 30 min at 4 °C in the dark. After removal of fixation solution ells were resuspended in 100 pl antibody-mix (PAX6 / OXT2) and incubated for 30 min at 4 °C in the dark. Afterwards, cells were centrifuged at 300xg for 5min, supernatant was aspirated, and cells were washed with 100 pl Perm Buffer. Cells were again centrifuged at 300xg for 5 min, supernatant was discarded, and the cells were resuspended in 200 pl FC- Buffer. Analysis was performed using MASCQuant Analyzer 10 using FloJo software.

[0294] If the percentage of double positive cells (PAX6 and OTX2 positives) were superior to 50%, in particular superior to 70 % (e.g. in case of preparation of retinal organoids), the differentiation passed the QC threshold the eye-field cell aggregates were kept for further differentiation into either- Retinal pigmented epithelium (RPE) differentiation- Retinal organoid (RO) differentiation

[0295] If 50% of the threshold was not achieved cysts were discarded. Upregulation of SIX3, LHX2, OTX2, PAX6, RAX and SOX1 can be confirmed by qPCR (Figure 4G and H).Retinal organoid differentiationRetinal organoid differentiation: Differentiation of Neuro-retinal precursors

[0296] In an exemplary embodiment step (d) as defined herein is carried out as follows

[0297] From dd8 (±1 ) onwards, the aggregates that were kept in suspension for retinal organoid differentiation will be referred as “retinal organoids” (RO). ROs were fed every Monday, Wednesday and Friday, as follows: media was aspirated with a glass pipette and 5 mL of fresh M4 media was added to each well Visual inspection and brightfield microscope pictures were taken on a weekly basis (Figure 11 )

[0298] On dd25 organoids from 1 well were split (i.e. distributed) over 3 wells, to ensure nutrient requirements were met and cultures remained healthy, while sticking to the feeding schedule previously described. M4 media was replaced with M5 media, to promote commitment to neuro-retinal precursors cell fate M5 is supplemented with 0 5 pM retinoic acid. Samples were collected for QC: Flow cytometry and IHC for VSX2 (5 to 10 samples collected for either flow cytometry or histology), and qPCR for RAX and VSX2 (3 samples were collected, individually, and treated as technical replicates) (Figure 12A-C).Retinal organaid differentiation: Differentiation of RGCs, amacrine, horizontal and photoreceptor precursor cells

[0299] In an exemplary embodiment step (e) as defined herein is carried out as follows

[0300] From dd50 to dd70 samples were fed with M6 media supplemented with 0 5 pM retinoic acid, which was added fresh to the medium prior to each feeding.

[0301] Upregulation of CRX, by qPCR, between d50 and d70 was seen (Figure 13B) CRX protein was also detected by IHC, with rare cells being present at dd50 and the number increasing by dd70 By dd70 RECOVERIN and RXRy genes also becomes upregulated and can be detected by IHC, with RECOVERIN co-localizing with all CRX positive cells (both are pan-photoreceptor markers, being present in rods and cones) (Figure 13C) and RXRy appear only in a portion of the photoreceptor cells, as this marker is only present in cone precursors and absent in rods.

[0302] At dd70 retinal organoids start being fed with M7 medium supplemented with 0 25 pM retinoic acid as previously described. The feeding schedule remained the same as previously described.

[0303] In addition to photoreceptor differentiation, between dd50 and dd70, other cell types are differentiated. At dd70 we find mature RGCs, as shown by the expression of the POU4F1 gene and detectable Brn3a protein, characteristic of mature RGCs, withing the most internal layer of the organoids (Figure 12C and D). The RCGs are the first fully committed retinal cell type to differentiate from the neuro retinal precursors, which replicates human development in vivo.

[0304] In a similar timeframe, horizontal cells and some amacrine cells also appear in the retinal organoid. The presence of post-mitotic amacrine and horizontal cells was demonstrated by IHC for AP2a and Proxl , respectively.

[0305] Lamination is detected in histologically cut samples (Figure 13A). Rosette-like structure can also be present in the organoids, when a portion of the cells reorganize on it on small niche, with photoreceptors facing the rosette lumen. Those structures are not detrimental to the efficient differentiation of all the neuro- retinal cell types, and the presence in some inner areas of each organoid is not unlikely during this stageRetinal organoid differentiation: Maturation of photoreceptor cells and Muller glia cell differentiation (F)

[0306] In an exemplary embodiment step (f) as defined herein is carried out as follows.

[0307] By dd 100, the end of stage 6 RO / start of stage 7 RO, the expression of all the mentioned photoreceptor genes increases dramatically, and protein level can be detected by IHC, with RECOVERIN and CRX co-localizing to all NRL+ or RXRy+ cells, showing at this stage cells have committed to a specific photoreceptor sub-type, rod or cone photoreceptors.

[0308] From dd100 onwards, retinal organoids were fed with medium M8, according to the schedule previously described.

[0309] S-opsin can be detected in IHC by d100 (Figure 13C), showing maturing of cone photoreceptors, that start to further mature into S or L / M cones. This marker co-localizes with RXRy+ nuclei, confirming its specificity to cones. It is also seen mainly at the most outer layer of the organoid, according to the expected photoreceptor patterning. No outer segment-like structures were visible at d100, meaning that opsin protein is being produced but, the cell region where it needs to be transported to still developing. Up regulation and IHC presence of other mature proteins, involved in the visual cycle, such as ARRESTIN3 and PERIPHERIN2 were also detected by dd100 (Figure 13D). Flow cytometry was used to establish the % of CD73 positive cells. CD73 has been shown to, in the context of the retina, be specific to photoreceptors. CD73 is present in both rods and cones in early stages of maturation, becoming later restricted to rod photoreceptors At dd100 CD73 was expressed -20% of all cell withing the retinal organoid These were shown to be both rods and cones, as demonstrated by qPCR data of CD73 positively sorted cells, that showed expression of all opsin types

[0310] The remaining retinal cell type, Muller glial cells, was also detectable by dd100. Upregulation of RLBP1 was corroborated by IHC for CRALBP, showing presence of these markers in cells that extended across all layers of the organoid, resembling typical Muller glial cell morphology, as seen in in vivo retinas. The intensity and area of CRABP staining increased with time, as expected.

[0311] By dd120 Outer segment formation is visible in over 10% of the retinal organoid (Figure 13A) Such structures can be identified in vitro, under the brightfield microscope, and also by IHC In IHC QC, it was also confirmed that opsins (Rhodopsin, S-opsin and L / M-opsin) were present in these elongated structures, that extended beyond the most outer nuclear layer of the organoid. The % of organoid that showed present of Outer-segment-like structures increased with time, reaching 80% or more by dd180 (the last point of QC collection)

[0312] The % of CD73 positive cells also increased with time, reaching -50% by dd120.

[0313] Transcriptomic analysis (single nuclei RNA sequencing) corroborated all the previously mentioned data, as well as the highly reproducibility of the protocol, as minimal variance between biological replicates was observed. snRNAseq showed the presence of all retinal cell types, with a clear enrichment for photoreceptor cells (-40% rods and -20% cones at dd 160) (Figure 14A-C) These cells expressed opsins and other mature markers (markers related to phototransduction). All other retinal cell types were identifiedin a range of 12 to 1%. At dd150 the % of proliferating cells was under 1 %, further confirming the maturity of the retinal organoids.

[0314] Organoids were kept in culture until dd180, and samples were collected for IHC and qPCR, as previously mentioned.

[0315] In comparison to published data from Cowan et al (Cowan et al. 2020, Cell 182, 1623-1640) the herein disclosed retinal organoids mature faster in the differentiation process than with current state-of-the- art retinal differentiation methods. Comparing transcriptomics data at dd120 of the herein disclosed retinal organoids with published data at dd126, a clear and prominent induction of rod and cone markers is observed in the samples described herein, which is absent in retinal organoids from Cowan et al. (Figure 14D). The herein disclosed retinal organoids show strong upregulation of rod markers, such as NR2E3 and RHO, as well as upregulation of cone markers, such as OPN1 SW and MAP4 As described previously and shown in Figure 13B, RXRy is a photoreceptor marker, which gets a) restricted to cones and b) downregulated over time of retinal development This is clearly replicated in the herein disclosed retinal organoids, but not in the public data set. Comparing transcriptomics data from the retinal organoids at dd160 as described herein with the public data set at dd168, RHO upregulation in the data set as described herein is more pronounced that in the public data set, as well as the induction of cone specific markers, such as OPN1SW (Figure 14E). Also of note is that it is not possible to distinguish between cone subtypes, i e. M- and S-cones, due to the immaturity of the cells.Retinal organoid dissociation (G) and photoreceptor enrichment (H)

[0316] Between day 100 and day 120 retinal organoids were pipetted, using a wide bore pipette tip, into conical tubes. The organoids were washed twice with PBS Retinal organoids were dissociated using the papain-based Neurosphere Dissociation kit (M iltenyiBiotec, 130-095-943). All solutions were prepared according to manufacturer’s instructions and pre-warmed to 37°C. After addition of dissociation solution retinal organoids were incubated in papain solution for 10 minutes at 37°C, with gentle agitation. Retinal organoids were mechanically dissociated, by pipetting vigorously with a p1000 pipette until a homogeneous solution is obtained. The cell solution is passed in a per wet (with media) 40pM cell strainer and the enzymatic dissociation is stopped by dilution with cell culture media. Cell suspension is centrifuged at 300g for 10 minutes and the supernatant is removed. Cells are resuspended in 1 mL / mm of autoMACS Rising Solution / BSA (MiltenyiBiotec, 130-091-222) and counted.

[0317] The cell suspension was centrifuged at 300g for 10 minutes and the pellet was resuspend in 100|jL of autoMACS Rising Solution per 1 million cells. 2pL antibody (CD73 or CD133) / million cells was added and incubated and washed with autoMACS and resuspend in 80pL autoMACS Rising Solution per 10 million cells. Antibodies used: CD133 / 1 Antibody, anti-human, APC conjugated, clone AC133, Miltenyi 130-113- 106, CD73 Antibody, anti-human, REAfinity™ APC conjugated, clone REA804, Miltenyi 130-111-909

[0318] MicroBeads (Miltenyibiotec), were added, incubated and further processed according to manufacturer’s instructions The cell solution was passed through the selection column (placed in the sorting magnet) according to manufacturer’s instructions and the positive cells selected.RPE differentiation

[0319] When at least 60% of cells were double positive for PAX6 and OTX2, cells were used for seeding onto transwells The protocol as described herein performed better to induce high levels of PAX / OTX2 expressing cells than a published iPSC RPE differentiation protocol from Zhu et al. (PLoS One. 2013; 8(1 ):e54552; Figure 5A). Transwells were coated with growth factor reduced (GFR) matrigel at least 1 hourat RT. First, eye field aggregates were dissociated into single cells. To get rid of remaining cell aggregates, a 30 pm cell strainer was used. Single cell suspension was counted and supplemented with 5 pM ROCK inhibitor Y27632 GFR matrigel was aspirated. Cells were seeded onto transwells. Seeding density depends on the transwell size and cell line used. Usually, for 6.5 mm transwells 70-100k cells are seeded in 150 pl M2 medium, for 24 mm transwells 400-600k cells are seeded in 800 pl M2 medium, lower chambers are filled with M2 mediaRPE differentiation: RPE precursor DD9-16

[0320] On dd9, after 24 hours, upper transwells were washed once with M3 media by aspirating M2 and adding M3 media. Then, media is removed from lower and upper chamber and filled with M3 media containing 10 ng / ml Activin A. Media changes are performed every three days . RPE precursor cells were differentiated until dd28 on transwells and quality controlled for correct RPE induction Identity of RPE precursors was confirmed with the appearance of a polygonal monolayer assessed with the staining of actin with phalloidin, MITF and ZO-1 on dd12 and dd16. In addition, the MITF positive cells were analysed with FC If MITF values were above 50% the differentiation process was continued., and the RPE precursor cells are named early RPE from this timepoint onwardsRPE differentiation: early RPE DD17-28

[0321] The early RPE cells (dd 17) were further differentiated and quality controlled for polygonal epithelial monolayer (cobblestone morphology) using phalloidin staining to visualize the Actin on dd20 / dd24 and dd28 (Figure 6A). In this stage of the differentiation pigmentation appeared between dd16 and dd22 (Figure 6B). Here, protocol described herein delivered more consistent results than a start-of-the art published RPE differentiation protocol from Zhu et al The consistency and reliable generation of high PAX6 / OTX2 levels in cells at dd8 lead to predictable pigmentation outcomes in the protocol as described herein, while we observe a lot of variance using the published protocol (Figure 5B).

[0322] On dd28 early RPE were passaged while no manual manipulation / selection or harvesting was performed. For passaging, M3 media was removed and cells were washed with T rypsin-EDTA (TE) solution. For a 6.5 mm transwell washing step 100 pi I were used for the upper chamber and 400 pl for the lower chamber. For a 24 mm transwell washing step 1000 pl were used for the upper and for the lower chamber. Washing solution was aspirated from the upper chamber and refilled with fresh TE solution (100 pl for 6.5 mm transwells, 1000 pl for 24 mm transwells) and incubated for 10 min at 37 °C. During the incubation a fresh solution of 10 mg / ml Soybean Trypsin Inhibitor (STI) was prepared in RPE medium (500 pl per 6 5 mm transwell, 1500 pl per 24 mm transwell). After finishing TE incubation time, cells were detached and dissociated by pipetting inside the transwell until no visible clumps remained. Detached cells were collected in the prepared M3 media STI solution and centrifuged at 1000 rpm (180xg) for 2 min at RT Supernatant was discarded and the cell pellet was resuspended in M3 medium containing 10 ng / ml Activin A. Cells were filtered using a 30 pm cell straining to remove remaining cell aggregates. Cells were counted and seeded again onto GFR matrigel coated transwells (cell amounts are depending on the cell line used, common seeding densities for 6.5 mm transwells are 200k - 400k and for 24 mm transwells 2-4 million cells). RPE media containing 10 ng / ml Activin A was changed every three days.RPE differentiation: RPE maturation DD 29-63

[0323] On dd49 a second passage was performed with no manual selection. This second passage was done in the same way as on dd28 but seeding was performed into 384-well plates (seeding density dependson the cell lines used, usually between 25k - 40k cells per well) For quality control, 6 5 mm transwells were used at dd49 and dd64.

[0324] To follow the developing polygonal epithelium monolayer development the transepithelial resistance (TER) was measured using three 6.5 mm transwells (see TER measurement) and supernatant of three upper and corresponding lower chambers was collected and stored at -80 °C for VEGF ELISA (see VEGF ELISA). These functional assessments show polarized VEGF and an increase in TER, as RPE cells mature (Figure 10B and C). Two transwells were used for trizol sample for qPCR to control for increasing mRNA expression of RPE markers: PAX6, TYRP1, MITF and RPE65 on dd28 / dd49 / dd63 (Figure 7). Here we compared again the protocol as described herein with the published protocol from Zhu et al and found slightly increased expression of MITF across all time points, but otherwise similar gene expression profiles. Additionally, transwells werefixed with 4% PFA and used for ZCH , Phalloidin and MITF staining (Figure 8A- C). Quantification revealed that RPE cells differentiated with the Evotec protocol not only expressed higher protein levels of MITF, as expected from the RNA expression data, but also expressed more ZO1 and F- Actin, markers for tight junctions and overall cell maturity. Only final differentiated cells (dd63 of fresh RPE cells and dd63 of thawed RPE cells) are quality controlled for RPE marker staining (Figure 9) and assessment of phagocytic capacity (Figure 10A).

[0325] The cryopreservation of RPE in the maturation state is possible for RPE cells on dd42 to generate a large stock of cells. For this purpose, the cells were detached from transwells according to the passaging protocol After filtering and counting the RPE cells were centrifugated at 300xg for 5 min at RT The cell pellet was resuspended in Cryostor CS10 at a final concentration of 20 x 106 cells / ml Vials of 2ml, 1 ml and 500|jl were frozen and stored in liquid nitrogen tanks until further use. Thawing of cells was performed in a 37 °C water bath until a small ice piece was left. Cells were then resuspended and transferred to a tube containing M2 media and centrifuged for 2 min at 300xg at RT Supernatant was discarded and the cell pellet resuspended in M2 media with the addition of 20|jg / ml DNase. Cells were counted and diluted in appropriate amounts of M2 media containing additional DNase as above. Cells were then seeded onto 384- well plates (usual seeding density of 20k - 40k ce lls / we 11 ) or 6.5 mm transwells (usual seeding density of 300k - 600k cells / transwell) coated with GFR matrigel or GFR Geltrex One day after seeding medium was changed to M3 medium containing only. Media changes were performed every other day for 384-well plates and every three days for transwells. After a cultivation time of up to three weeks, RPE cells were ready to use for QC and assay development.RPE differentiation Quality control assaysVEGF ELISA:

[0326] A commercially available ELISA (Thermo Fisher Scientific, Cat. No KHG0112) was used according to manufacturer’s instructions Mean values of all three samples from the upper or lower chamber of each time point were calculated.TER measurements:

[0327] To confirm the well-established polygonal epithelium monolayer a transepithelial resistance (TER) measurement was done using EVOM2 device on 6,5 mm transwells. The chopstick electrode of this system was adjusted by using fresh M3 medium at RT. For a blank value, 6.5 mm transwells coated with GFR matrigel and containing M3 medium were used. At least three 6 5 mm transwells were used in differentiation state 5 at dd49 and dd63 by placing the chopstick electrode into both chambers and waiting for the TERvalue to stabilise. Each transwell was measured three times and the mean values calculated Final TER values were obtained by subtracting blank values from transwell TER values containing RPE cells. qPCR RPE marker analysis:

[0328] For qPCR analysis transwells of fresh RPE cells at dd28, dd49 and dd63 were used. To obtain trizol samples for qPCR, M3 medium of two 6.5 mm transwell was removed and washed with 1x DPBS-Mg / - Cl. Washing solution was removed and 200 l trizol added for 5 min at RT to both upper chambers of two 6.5 mm transwells. Cells were rinsed from the transwells and the trizol cell solution was transferred to a 2 ml tube and stored at -20 °C until use qPCR was performed using the TaqMan method for the selected marker genes, such as BEST1 , CLDN19, EZR, MERTK, MITF, OTX2, PAX6, PMEL, POU5F1 , RLBP1 , RPE65, SERPINF1 , TYRP1 , and VMD2.Mature RPE marker ICC of384-well plates:

[0329] Fresh RPE cells at dd63 were used for mature RPE marker staining on 384-well plates. Plates were fixed with 4% PFA for 10 min at RT by removing RPE medium and adding fixation solution to each well. After 10 min, fixation solution was removed and cells were blocked by adding 1x DPBS-Mg / -CI containing 1% BSA and 0.3% triton X-100 (blocking buffer) for 1 hour at RT. Then, blocking buffer was discarded, prepared primary antibodies (containing primary antibodies against BEST1 , MERTK, EZR, ZO-1 , MITF, RPE65, PMEL) or prelabelled phalloidin in blocking buffer were added to the wells and incubated over night at 4°C in the dark On the next day, wells were quickly washed three times by removing solutions by adding 1x DPBS-MG / -CI. Final washing solution was discarded and secondary antibody-mix solutions were prepared in 1x DPBS-Mg / -CI and added for 2 hours at RT in the dark. After 2 hours, secondary antibody solution was discarded and the wells were washed with 1x DPBS-Mg / -CI 3 times Afterwards nuclei were stained with Hoechst 33342 diluted 1:2000 in 1x DPBS-Mg / -CI for 30 min at RT in the dark. Plates were sealed and imaged in with an Operetta using a 60x objective. Proper localisation of all markers was evaluated for a good quality of RPE cells.Phalloidin ICC of trans wells:

[0330] To evaluate morphological changes, Phalloidin and ZO1 staining of 6 5 mm transwells from dd49 and dd63 was performed. Transwells were fixed with 4% PFA for 10 min at RT by removing M3 media and adding fixation solution to the upper chamber After 10 min, fixation solution was removed and cells were blocked by adding 1x DPBS-Mg / -CI containing 1% BSA and 0.3% triton X-100 (blocking buffer) for 1 hour at RT Then, blocking buffer was discarded, prepared antibody-mix (containing AF488-labeled phalloidin or primary ZO1 antibody) in blocking buffer was added to the upper chamber and incubated over night at 4°C in the dark. On the next day, upper chambers were quickly washed three times by removing solutions and adding 1x DPBS-MG / -CI containing 0.3% triton X-100. Final washing solution was discarded and secondary antibody mix for ZO1 prepared in washing solution, added, and incubated for 2 hours at RT in the dark. After 2 hours, secondary antibody solution was discarded and the upper chamber was washed three times with 1x DPBS-MG / -CI. Afterward nuclei were stained with Hoechst 33342 diluted 1 :2000 in 1x DPBS-Mg / -CI for 30 min at RT in the dark. Finally, staining solution was removed and cells were washed three times with 1x DPBS-Mg / -CI. Filters were cut out with a scalpel and mounted onto glass slides using Aqua-Poly / Mount. After drying overnight, filters were imaged in an automated slide scanner.RPE phagocytic capacity assay:

[0331] Photoreceptor outer segments (POS) were isolated from pig eyes.

[0332] For pHrodo phagocytosis assay: POS were labelled with pHrodo iFL Green STP ester (ThermoFisher Scientific, Cat. No. P36013) in 0.1 M sodium bicarbonate buffer (pH 8.5) for 30 min at RT on an overhead shaker. Labelled POS were centrifuged at 3600xg for 5 min at 4 °C and the supernatant discarded POS were resuspended in 1x DPBS+Mg / +CI containing 1% BSA and incubated on the overhead shaker for 15 min at RT in the dark. POS were washed once with 1x DPBS+Mg / +CI. POS were washed twice using Fluorobrite DMEM. Between all washing steps, POS were centrifuged for 5 min at 3600xg at 4 °C. Final washing solution was discarded and POS particles were resuspended in Fluorobrite DMEM

[0333] To evaluate the phagocytic capacity of RPE cells, final differentiated cells dd63 on 384-well plates were used. M3 medium was removed and 40 pl of ligands (MFGE8 and ProteinS) in Flurorbrite DMEM (vehicle control of 0.1% DMSO) were added to the wells Afterwards, 10 pl of final POS solution was added to each ligand condition. Live imaging was done with pHrodo-labelled POS for 24 hours with pictures taken every hour and then fixed Plates were fixed with 4% PFA for 10 min at RT, washed with 1x DPBS-Mg / -CI and nuclei were stained with Hoechst 33342 diluted 1 :1000 in 1x DPBS-Mg / -CI for 45 min at RT in the darkPlates were sealed and imaged with an Operetta using a 10x objective for nuclei imaging Analysis of nuclei and spot counts, spot area, and spot intensity was performed.AbbreviationsAMD - age-related macular degeneration BPCs - bipolar cellsCRX - cone-rod homeobox proteinDAPI - 4',6-diamidino-2-phenylindole dd - differentiation dayEB - embryonic bodyEFTFs - eye field transcription factorsESC - embryonic stem cellFBS - foetal bovine serumFC - flow cytometryFGF - fibroblast growth factorFM - Freezing MediumGCL - ganglion cell layer h - humanIHC - immune histochemistryICC - immune cyctochemistry iPSCs - induced pluripotent stem cells m - mouse mL - millilitrePOS - photoreceptor outer segmentsPBS - phosphate-buffered salineQC - quality controlRA - retinoic acidRec - RecoverinRGCs - Retinal ganglion cellsRPCs - Retinal progenitor cellsRPE - Retinal pigmented epitheliumRO - Retinal organoids RT - room temperatureWP - well plate

Claims

CLAIMS1. Method of isolating photoreceptor cells in a population of cells, comprising the steps of i) determining whether a cell in the population expresses CD133 on the cell surface, ii) identifying a cell as a photoreceptor cell if the cell expresses CD133 on the cell surface,Hi) isolating the cell identified as photoreceptor cell, wherein CD133 is the single marker used for identifying photoreceptor cells2. Method according to claim 1 , wherein CD73 is not used as marker.

3. Method according to claim 1 or 2, wherein FACS or MACS is used for isolation of the photoreceptor cells.

4. Method according to any one of the preceding claims, wherein an antibody binding to surface expressed CD133 is used.

5. Method according to any one of the preceding claims, wherein the population of cells is obtained from a dissociated retinal organoids, wherein the retinal organoids are late phase retinal organoids.

6. Method according to any one of the preceding claims, wherein the population obtained in step Hi) comprises at least 60 %, preferably at least 70 %, more preferably at least 80 %, even more preferably at least 85 %, most preferably at least 90 % photoreceptor cells.

7. Method according to any one of the preceding claims, wherein the population obtained in step Hi) comprises at least 5 %, preferably at least 8 %, more preferably at least 10 %, even more preferably at least 20 %, most preferably at least 30 % early opsin-expressing photoreceptor cells8. The method according to one of the preceding claims, wherein the population obtained in step iii) comprises at least 13 %, preferably at least 14% cones9. Method according to any one of the preceding claims, wherein the population obtained in step iii) comprises at least 70% rods, preferably at least 75% rods.10 Method according to any one of the preceding claims, wherein the population obtained in step iii) comprises at least 4% S cones and / or at least 8 % M cones.

11. Method according to any one of the preceding claims, wherein the population obtained in step iii) comprises substantially no pluripotent cells and or substantially no proliferative cells12. Method according to any one of the preceding claims, wherein the population obtained in step iii) comprises at least 80% cells, preferably 85 % cells expressing CD133.

13. Method according to any one of the preceding claims, wherein the population of cells was obtained from dissociated retinal organoids.

14. Method according to any one of the preceding claims, wherein the retinal organoids were cultured for 90 to 150 days, preferably 100 to 140 days, more preferably 105 to 130 days, even more preferably 110 to 125, most preferably 120 days15. A method for differentiating stem cells into retinal cells, comprising the steps:(a) providing a single cell suspension of stem cells;(b) culturing the single cell suspension of stem cells in a 3D suspension culture to obtain embryoid bodies; and(c) culturing the embryoid bodies in 3D suspension culture system to obtain eye-field cell aggregates.

16. The method according to claim 1 , wherein any one of steps (a), (b) and (c) occurs in the absence of a support matrix, preferably in the absence of a proteinaceous gel, optionally wherein the 3D suspension culture is carried out in ultra-low attachment cell culture plates17. The method according to claim 1 or claim 2, wherein in step (b) and / or step (c) shear stress is applied to the 3D suspension culture, optionally wherein step (b) and / or step (c) occur under agitation, optionally wherein agitation occurs at 50 to 200 rpm, preferably at 80 to 120 rpm, more preferably at 100 rpm18. The method according to any one of claims 15 to 17, wherein step (b) lasts 1 to 6 days, preferably 2 to 5 days, more preferably 3 to 5 days, even more preferably 4 days;19. The method according to any one of claim 15 to 18 , wherein step (c) lasts 3 to 7 days, preferably 3 to 6 days, more preferably 4 to 5 days, most preferably 4 days.20 The method according to any one claims 15 to 19, wherein the stem cell is a human induced pluripotent stem cell and wherein an exogenous nucleic acid and / or an exogenous protein is not introduced into the stem cells or differentiated cells derived thereof21 The method according to any one of claims 15 to 20, wherein the embryoid bodies comprise at least 60%, at least 70%, at least 80%, at least 90% cells expressing SOX2, Nanog, Oct4, SSEA1 and Tra1 and optionally wherein the embryoid bodies have a uniform shape and / or a diameter of about 150 pm.22 The method according to any one of claims 15 to 21 , wherein the eye-field cell aggregates comprise at least 50%, preferably at least 60%, more preferably at least 80% cells expressing PAX6 and OTX2.

23. The method according to any one of claims 15 to 22, wherein the single cell suspension of stem cells of step (a) is provided by the following steps:(a1 ) culturing stem cells in adherent culture; and(a2) dissociating the stem cells to obtain single cell suspension24. The method according to any one of claims 15 to 23, wherein no TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor or GSK3f> inhibitor is used.

25. The method according to any one of claims 15 to 24, wherein no TGF-p inhibitor, FGF8 inhibitor, AMPK inhibitor, SMAD inhibitor, BMP inhibitor and GSK3p inhibitor is used.

26. The method according to any one of claims 15 to 24, wherein the cells obtained in step (b) comprise at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing OTX2, at least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Oct4, andat least 90%, preferably at least 93 %, more preferably at least 95 % cells expressing Sox2.

27. A method of differentiating stem cells into retinal organoids, comprising the steps defined in claims 15 to 26 and further comprising the steps:(d) culturing the eye-field cell aggregates in a 3D suspension culture system to obtain neuro-retinal precursors;(e) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain early phase retinal organoids, and optionally(f) culturing the neuro-retinal precursors in a 3D suspension culture system to obtain late phase retinal organoids, wherein any one of steps (d ), (e) and (f) occurs in the in the absence of a support matrix, preferably in the absence of a proteinaceous gel28. The method according to any one of claim 27, wherein the eye-field cell aggregates used in step (d) comprise at least 70%, preferably at least 80% cells expressing PAX6 and OTX2 and / or wherein the early phase retinal organoids obtained in step (e) are positive for at least one marker selected from the group of CRX, RECOVERIN and RXRy.

29. The method according to any one of claim 27 or 28, wherein culturing in step (d) comprises:(d1 ) culturing the eye-field cell aggregates in a medium comprising neurobasal medium; and(d2) culturing the eye-field cell aggregates obtained in step (d1 ) in a high glucose medium not containing neurobasal medium, supplemented with retinoic acid, and wherein culturing in step (e) comprises:(e1 ) culturing in a high glucose supplemented with FBS and retinoic acid; and(e2) culturing in a high glucose medium supplemented with FBS and retinoic acid.

30. The method according to claim 29, wherein culturing in (e1 ) occurs in a high glucose medium supplemented with FBS and 0 5pM retinoic acid and wherein culturing in (e2) occurs in a high glucose medium supplemented with FBS and 0.25 M retinoic acid, optionally containing lipids.

31. A method for differentiating stem cells into retinal pigmented epithelium cells, comprising the steps of claims 15 to 26 and further comprising the steps:(dd) dissociating the eye-field cell aggregates to single cells;(ee) culturing single cells obtained in step (dd) in adherent cell culture to obtain retinal pigmented epithelium cells, and wherein culturing in (ee) comprises(eel ) culturing in a medium comprising neurobasal medium supplemented with a ROCK inhibitor; and(ee2) culturing in a RPE differentiation medium having supplemented with at least one agonists of the Activin A pathway.

32. The method according to claim 31 , wherein the retinal pigmented epithelium cells express one or more of the genes selected from the group of MITF, PAX6 and OTX2.

33. The method according any one of claims 31 and 32, wherein culturing in (ee) comprises culturing in a RPE differentiation medium which is optionally a serum-free medium, wherein said medium comprises at least one growth factor selected from agonists of the Activin A pathway.

34. The eye-field cell aggregates obtained by the method according to any one of claims 15 to 26.

35. The retinal organoid obtained by the method according to any one of claims 27 to 30.

36. Cells obtained from the retinal organoid according to claim 35.

37. The retinal pigmented epithelium cells obtained by the method of claims 31 to 33.