Single lumen neuroepithelial cyst derived organoids

Abstract

Described herein are processes of producing single lumen epiblast cysts, of producing further differentiated epiblasts cysts such as neuroepithelial cysts, and of producing organoids, such as CNS organoids, as well as kits for performing these processes. Further described are epiblast cysts, further differentiated epiblast cysts such as neuroepithelial cysts, and organoids such as CNS organoids, as well as their uses.

Description

[0001] P28241 PCOO 28.08.2025

[0002] 1 / 119

[0003] Single Lumen Neuroepithelial Cyst Derived Organoids

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to processes of producing single lumen epiblast cysts, of producing further differentiated epiblasts cysts such as neuroepithelial cysts, and of producing organoids, such as central nervous system (CNS) organoids, as well as kits for performing these processes. The disclosure further relates to epiblast cysts, further differentiated epiblast cysts such as neuroepithelial cysts, and organoids such as CNS organoids, as well as their uses.

[0006] BACKGROUND OF THE INVENTION

[0007] Human brain tissue is very poorly accessible for research. Therefore, there is a high demand to build physiologically relevant in vitro models of the brain to study development, homeostasis and disease. Over the last years, human brain organoids generated from human and mouse pluripotent stem cell lines (PSCs) have become the center of attention in the establishment of models that can mimic the development and disorders of the human brain.

[0008] Different methods are known to access human brain models, such as organoids. For example, brain organoids can be generated using undirected protocols where stem cells differentiate spontaneously to resemble a variety of neural structures with variable regional identities. In order to generate defined regions in the brain, protocols have been optimized with developmental signaling pathway treatments to guide the differentiation in a particular region of interest such as forebrain, ventral forebrain, hypothalamus, hippocampus, thalamus, midbrain, cerebellum, retina and choroid plexus.

[0009] Although brain organoids provide an insight in human specific early development and neurological function in health and disease, they remain rudimentary and have multiple limitations. Current brain organoids fail to recapitulate several structural, molecular and functional aspects of the developing brain. For example, most organoid models develop multiple units of neuroepithelium at an early stage in differentiation which does not recapitulate the overall architecture of the developing brain and leads to an improper cytoarchitecture, big size and development of a necrotic core over time. There have been studies focusing on improving the organoid architecture and P28241 PCOO 28.08.2025

[0010] 2 / 119 longer-term culture by providing nutrients and oxygen but these methods require manual slicing and culturing of the organoids making them very low throughput.

[0011] In addition, brain organoids also show a rudimentary cytoarchitecture seen in developing brain with stem cells in the ventricular zone and neurons in the cortical plate, however this cytoarchitecture dissolves over time and does not mimic the complex neuronal organization present in the brain.

[0012] In addition, even though there have been advances in generating various regional brain organoids, the initial culture condition and starting differentiation parameters vary greatly in these protocols. For a systematic approach to study the development and disease of the brain, it is important that different regional organoids can be generated with common starting culture conditions by simply manipulating the developmental signaling supporting the formation of different regions. There have not been many efforts to develop generalized protocols that can be modified to generate different regions of the brain from the same starting conditions.

[0013] Another caveat of current brain organoid is the low throughput and high level of heterogeneity. The brain organoid field has been dominated by PSCs derived embryoid body (EBs) based methods to derive neuroectoderm followed by neuroepithelium differentiation. EBs tend to be a heterogeneous group of cells leading to an inherent variability in the system early on and fail to mimic the early organization of cells in the embryo.

[0014] Finally, although methods for generation of neuroepithelial cysts from human PSCs have been reported previously, they display several drawbacks. For example, they require either mechanical isolation of single lumen neuroepithelium, manual cutting of 2D sheet of cells, specific matrices, embedding methods and dual SMAD inhibition to induce neuroectoderm, or they display only limited fate establishment to epiblast stage. Thus, these methods are very limited, for example because they are not amenable to high throughput or automation, and because they often suffer from labor intensity, poor reproducibility and poor robustness.

[0015] SUMMARY OF THE DISCLOSURE

[0016] It is the general object of the present disclosure to advance the state of the art in the field of human biology and modelling of development and disease in vitro, in particular organoid engineering. The study of human brain development and associated disorders is significantly hampered by the limited accessibility of native tissue. CNS organoids have emerged as a promising P28241 PCOO 28.08.2025

[0017] 3 / 119 tool to bridge this gap, offering an in vitro model that partially mimics the biology of the tissues of the human central nervous system. CNS organoids derived from pluripotent stem cells are often generated using free floating aggregates of stem cells (embryoid bodies), which spontaneously give rise to non-reproducible neuroepithelial units and variable cell fate patterns. These units further differentiate to generate a disorganized and heterogeneous population of neurons and glial cells that often do not recapitulate the correct cytoarchitecture and cell diversity. The present disclosure seeks to improve the cytoarchitecture of CNS organoids by improving the early phase of differentiation to more closely mimic the morphology observed in the developing embryo. To do that a method was developed to efficiently generate single-lumen epiblast cysts which can be further differentiated into neuroepithelial cysts. By initiating CNS organoid generation from a neuroepithelium cyst rather than an embryoid body, the presented approach allows establishment of an appropriate cytoarchitecture, enables robust and targeted differentiation into distinct brain regions, and facilitates sustained neuronal growth.

[0018] Against this background, the present disclosure aims in particular to advance the state of the art in the field of organoid engineering. In at least some embodiments, the present disclosure aims to provide a process of producing epiblast cysts and neuroepithelial cysts in a robust and reproducible manner, preferably amenable to high-throughput applications. For example, in at least some embodiments, a process is provided for producing epiblast cysts having a single lumen. Preferably, the process is robust, reliable and reproducible and allows single lumen emergence with high fidelity. Finally, in at least some embodiments, the process provides access to highly versatile neuroepithelial cysts which can be further differentiated into a broad range of different organoids.

[0019] The general object is achieved by the subject-matter of the independent claims. Further favorable embodiments follow from the dependent claims and the overall disclosure. The following summary is provided to introduce the reader to the more detailed discussion to follow. It is not intended to limit the disclosure to certain embodiments.

[0020] The present disclosure provides, among other aspects and embodiments, a process of producing epiblast cysts that can be further differentiated into (preferably single lumen) neuroepithelial cysts followed by brain region specific brain organoids. The process allows producing highly homogeneous organoids in a controlled fashion. It allows generating a single lumen neuroepithelium organoid model to promote proper initial architecture, to permit directed specification to different CNS regions and to allow long-term neural growth. These single lumen neuroepithelium organoids mimic anterior neural tube tissue and are patternable upon morphogen treatment. The P28241 PCOO 28.08.2025

[0021] 4 / 119 method also allows simple and scalable generation of these organoids for further improvement. Selected brain regions that may be modelled include, among others, forebrain, midbrain, hindbrain or spinal cord organoids, such as cortex, choroid plexus, retina, cerebellum or optic cup organoids.

[0022] A first aspect of the present disclosure relates to a process of producing epiblast cysts. The process comprises the step of a) seeding stem cells in microwells at a density of less than 500 cells per microwell. The process further comprises the step of b) culturing the seeded cells of step a) in the respective microwells for a first culture period in the presence of a first culture medium to obtain in each microwell a first cell population forming an epiblast cyst.

[0023] Preferably, the epiblast cysts produced in the process are single lumen epiblast cysts.

[0024] In some embodiments, in step a) the stem cells are seeded in the microwells at a density from 1 to 15, preferably from 1 to 14, cells per microwell.

[0025] In some embodiments, the first culture medium and / or the second culture medium is supplemented with an extracellular matrix gel, preferably in a non-solid state, after aggregation of the stem cells in the respective microwells.

[0026] Further, the present disclosure provides a process of producing neuroepithelial cysts. The process comprises the step of a) seeding pluripotent stem cells in microwells at a density from 1 to 15 cells per microwell, wherein each microwell has a diameter from 100 pm to 1’600 pm. The process further comprises the step of b) culturing the seeded cells of a) in the respective microwells for a first culture period (preferably lasting from 1 to 3 days) in the presence of a first culture medium, thereby obtaining in each microwell a first cell population forming an epiblast cyst. The process further comprises the step of c) culturing the first cell populations obtained in step b) for a second culture period (preferably lasting at least 4 days) in the presence of a second culture medium comprising a neural differentiation agent, thereby obtaining a plurality of second cell populations each comprising neuroepithelial cysts. Preferably, from no later than day 6 after seeding onwards, the second culture medium is supplemented with an extracellular matrix gel.

[0027] In some embodiments, the first culture medium and / or the second culture medium is supplemented with the extracellular matrix gel at a time point falling in the time period ranging from 1 hour after seeding to day 8 after seeding, preferably on day 4 after seeding. In some embodiments, the first culture medium and / or the second culture medium is supplemented with the extracellular matrix gel within the first 6 days after seeding, preferably on or after day 2 after seeding but before or on day 6 after seeding. Preferably, the first culture medium and / or the second culture P28241 PCOO 28.08.2025

[0028] 5 / 119 medium is supplemented with the extracellular matrix gel on or after day 3 after seeding but before or on day 5 after seeding.

[0029] In some embodiments, the first culture period lasts from 1 to 4 days, preferably from 1 to 3 days, more preferably for 2 days, after seeding.

[0030] In some embodiments, during step b), each microwell is filled with a volume of the first culture medium ranging from 1 pL to 400 pL, preferably from 5 pL to 150 pL, more preferably from 10 pL to 100 pL.

[0031] In some embodiments, each microwell has a non-adherent inner surface.

[0032] In some embodiments, each microwell comprises a hydrogel, preferably a polyethylene glycol (PEG) hydrogel, and / or wherein each microwell comprises a modified plastic that imparts nonadherent properties to the microwell and / or wherein at least a section of the inner surface of each microwell has a non-adherent coating.

[0033] In some embodiments, the microwells each have a microwell volume of less than 1’000 pL, preferably from 5 pL to 500 pL, more preferably from 20 pL to 300 pL.

[0034] In some embodiments, each microwell has a diameter from 10 pm to 4’000 pm, preferably 100 pm to 2’000 pm, more preferably from 100 pm to 1’500 pm. In some embodiments, each microwell has a diameter from 100 pm to 1’000 pm, e.g. from 200 pm to 1’000 pm, preferably from 300 pm to 500 pm. Particularly preferable is a diameter of 300 pm.

[0035] In some embodiments, a sidewall of each microwell is inclined such that a cross-sectional open area of the respective microwell being at least partially delimited by the inclined sidewall decreases towards a bottom of the respective microwell.

[0036] In some embodiments, in each microwell, a transition from the sidewall to the base of the respective microwell is at least partially, preferably fully, rounded.

[0037] In some embodiments, each microwell is U-shaped or V-shaped in a cross-section parallel to the vertical direction.

[0038] In some embodiments, a ratio between a depth and a diameter of each microwell ranges from 3:1 to 0.8: 1.0, preferably from 1.5: 1.0 to 1.0: 1.0.

[0039] In some embodiments, in step a), the stem cells are seeded in the microwells such that each microwell contains less than 10 cells per pL of microwell volume, preferably less than 5 cells per pL of microwell volume, more preferably less than 3 cells per pL of microwell volume, even more P28241 PCOO 28.08.2025

[0040] 6 / 119 preferably less than 2 cells per pL of microwell volume. In some embodiments, in step a), the stem cells are seeded in the microwells such that each microwell contains less than 100 cells per pL of microwell volume, preferably less than 15 cells per pL of microwell volume.

[0041] In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are mammalian, preferably human or murine pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0042] In some embodiments, the density of cells per microwell, the first culture period and the first culture medium are chosen such that at least 30%, preferably at least 40%, more preferably at least 50%, of all viable epiblast cysts obtained in step b) have a single lumen.

[0043] Further provided is a process of producing further differentiated epiblast cysts (preferably neuroepithelial cysts), comprising producing epiblast cysts (preferably single lumen epiblast cysts) according to the process of any of the embodiments disclosed herein, including but not limited to any of the embodiments described in the context of the first aspect. The process of producing further differentiated epiblast cysts further comprises the step of: c) culturing the first cell populations forming epiblast cysts obtained in step b) for a second culture period in the presence of a second culture medium comprising a second differentiation agent to obtain a plurality of second cell populations each comprising further differentiated epiblast cysts, preferably neuroepithelial cysts.

[0044] In some embodiments, the second differentiation agent comprises or consists of a neuroepithelial differentiation agent, such that the plurality of second cell populations each form a neuroepithelial cyst.

[0045] In some embodiments, on day 10 after seeding, the neuroepithelial cysts express one or more of the neuroepithelial markers selected from the group consisting of: PAX6, NES, VIM and N cad- herin.

[0046] In some embodiments, on day 10 after seeding, the neuroepithelial cysts express one or more of the anterior neuroepithelial markers selected from the group consisting of: OTX2, LHX5, SIX3, LHX2, HESX1 , FEZF1 and RAX, preferably selected from the group consisting of: SIX3, OTX2 and LHX2.

[0047] In some embodiments, the epiblast cysts and / or the further differentiated epiblast cysts are patternable to at least three different CNS regions, preferably to at least five different CNS regions, P28241 PCOO 28.08.2025

[0048] 7 / 119 through treatment with an appropriate morphogen. In some embodiments, the first cell populations and / or the second cell populations can be differentiated towards at least three different regions selected from the following regions: forebrain region, ventral region, midbrain region, hindbrain region, spinal cord, roof plate region, floor plate region and neural crest region.

[0049] Further provided is a process of producing a plurality of organoids, comprising producing further differentiated epiblast cysts according to the process of any of the embodiments disclosed herein, including but not limited to any of the embodiments described in the context of the first aspect. The process of producing a plurality of organoids further comprises the step of: d) culturing the second cell populations in a third culture medium comprising a third differentiation agent to obtain the plurality of organoids.

[0050] In some embodiments, the second differentiation agent and / or the third differentiation agent comprises or consists of a central nervous system differentiation agent, such that the organoids obtained are central nervous system organoids, preferably brain organoids.

[0051] In some embodiments, the CNS organoid is a target regional CNS organoid selected from one of the following: forebrain, midbrain, hindbrain or spinal cord organoid, preferably cortex, choroid plexus or retina organoid. In some embodiments, the CNS organoid is an optic cup organoid or a cerebellum organoid.

[0052] Further provided is a process for generating forebrain organoids, comprising performing the process of producing a plurality of organoids as described herein, wherein in step d), the second cell populations are treated with a BMP pathway inhibitor, a SMAD signaling inhibitor and / or a Wnt pathway inhibitor, preferably with Dorsomorphin and / or SB-431542. In some embodiments, on day 30 after seeding, a single neuroepithelial unit is formed, which preferably expresses apical marker N cadherin.

[0053] Further provided is a process for generating a choroid plexus organoid, comprising performing the process of producing a plurality of organoids as described herein, wherein in step d), the second cell populations are treated with pulsed delivery of a Wnt agonist, preferably CHIR-99021 , for a period of 1 to 10 days, preferably 2 to 8 days, most preferably 3 to 6 days, followed by culture in a neuronal growth medium.

[0054] Further provided is a process for generating a retinal organoid, comprising performing the process of producing a plurality of organoids as described herein, wherein in step d), matrigel is removed from the second cell populations, followed by long term maturation culture. P28241 PCOO 28.08.2025

[0055] 8 / 119

[0056] In some embodiments, at least one of steps b), c) and d) comprises a morphogen treatment.

[0057] Further provided is an automated process for producing a library of CNS organoids according to any one of the embodiments described herein, wherein step a) comprises the sub-steps of: a.i) filling a first seed volume from a seed stock cell suspension into each of a plurality of at least 100 microwells; a.ii) determining for at least some of the microwells a density difference between a target density corresponding to the density of the previous embodiments and an initial density of the stem cells in the respective microwells after step a.i); and a.iii) if the density difference determined for a given microwell exceeds a density difference threshold, adjusting the cell density in the respective microwell such that it reaches the target density.

[0058] In some embodiments, in step d), at least three different third differentiation agents are used for each of at least three different groups of second cell populations, such that the generated library of CNS organoids includes at least three different regional CNS organoids.

[0059] The present disclosure further relates to epiblast cysts produced by any of the embodiments of the process of producing epiblast cysts as disclosed herein. Typically, at least 30% (e.g. at least 40%, such as at least 50%) of the epiblast cysts produced have a single lumen.

[0060] The present disclosure further relates to a use of the epiblast cysts in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets, and cell therapy.

[0061] The present disclosure further relates to further differentiated epiblast cysts, preferably neuroepithelial cysts, produced by any of the embodiments of the process of producing further differentiated epiblast cysts as disclosed herein.

[0062] The present disclosure further relates to a use of the further differentiated epiblast cysts in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets and cell therapy.

[0063] The present disclosure further relates to organoids, preferably CNS organoids, produced by any of the embodiments of the process of producing a plurality of organoids as disclosed herein. P28241 PCOO 28.08.2025

[0064] 9 / 119

[0065] The present disclosure further relates to a use of the organoids in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets and cell therapy.

[0066] The present disclosure further relates to a kit comprising one or more components useful for performing any of the embodiments of the processes disclosed herein.

[0067] In some embodiments, the kit comprises one or more of the following components:

[0068] - at least one micropatterned substrate, preferably comprising a plurality of microwells each having a non-adherent inner surface;

[0069] - the first culture medium, optionally comprising one or more differentiation agents;

[0070] - the second culture medium, optionally comprising one or more differentiation agents;

[0071] - the third culture medium, optionally comprising one or more differentiation agents;

[0072] - one or more morphogens;

[0073] - one or more markers, preferably a pluripotency marker and / or a CNS marker, such as a neuroepithelial marker;

[0074] - an extracellular matrix gel, preferably matrigel, RTM, laminin, cultrex, hyaluronic acid, or a mixture thereof.

[0075] - a manual containing instructions how to perform the method of any one of the embodiments described herein;

[0076] - stem cells;

[0077] - a seeding device for facilitating seeding the stem cells into the microwells at a density of less than 500 cells per microwell;

[0078] - a third culture location, preferably a bottom plate, a bioreactor, or a culture vessel, such as a cell culture plate or a cell culture dish.

[0079] It is to be understood that both the foregoing general description and the following detailed description present embodiments, and are intended to provide an overview or framework for understanding the nature and character of the disclosure. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the disclosure. The description of preferred embodiments is P28241 PCOO 28.08.2025

[0080] 10 / 119 not intended to limit the disclosure to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the disclosure.

[0081] INCORPORATION BY REFERENCE

[0082] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

[0083] BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The present disclosure will be more fully understood from the detailed description given herein below and the accompanying drawings, which should not be considered limiting to the disclosure described in the appended claims.

[0085] Fig. 1 relates to single lumen neuroepithelial cysts derived from human pluripotent stem cells. Fig. 1 A illustrates generation of epiblast cyst and further differentiation into neuroepithelial cysts in microwells. Fig. 1 B shows time course brightfield (uppermost panel), ZO1-GFP (middle panel) and SOX2-GFP (lowermost panel) images showing progression of differentiation and lumen formation in neuroepithelial cyst. Fig. 10 shows quantification of cyst diameter measured using SOX2-GFP signal over time. Fig. 1 D shows quantification of cyst roundness measured using SOX2-GFP signal over time. Fig. 1 E shows quantification of lumen size measured using ZO1-GFP signal over time. Fig. 1 F shows a brightfield image of cyst after transfer to a dish; Fig. 1G shows immunofluorescence analysis showing expression of SOX2 and E cadherin on day 4 and day 6 and appearance of N cadherin on day 12. Fig. 1 H shows immunofluorescence antibody staining for phosphor histone 3 to show dividing cells at the apical surface of the cyst; Fig. 11 shows representative images at day 6 of differentiation and quantification of single lumen neuroepithelial cyst formation in three independent pluripotent stem cells lines including WTC-11 iPSCs (GM25256), PGP1 iP- SCs (GM23338) and H9 ESCs. P28241 PCOO 28.08.2025

[0086] 11 / 119

[0087] Fig. 2 relates to single lumen neuroepithelial cysts derived from human pluripotent stem cells. Fig. 2A shows representative images showing effect of initial seeding density on morphology of organoids on day 16. Organoids were generated using unguided brain organoid differentiation protocol. Fig. 2B shows formation of single lumen cysts in microwells of different sizes. Fig. 2C shows representative images of neuroepithelial cyst formation in Eplasia (Corning) and Aggrewell (Stem cell technologies) cell culture plate formats. Fig. 2D shows titration of Matrigel concentration for neuroepithelial cyst formation (day 8). Fig. 2E shows representative images show individual SOX2-GFP cells at day 0 and growth pattern on day 6 of differentiation. Fig. 2F shows quantification of effect of seeding cell number on cyst size on day 6. Fig. 2G shows quantification of effect of seeding cell number on lumen number on day 6. Fig. 2H shows reaggregation of neuroepithelial cells after 2 days of dissociation and plating. Fig. 2I shows single lumen neuroepithelial cysts generated with mouse embryonic stem cells.

[0088] Fig. 3 relates to experiments demonstrating that neuroepithelial cysts acquire default anterior fate and can be patterned. Fig. 3A shows a LIMAP plot showing single cell transcriptomes on day 10 of differentiation highlighting two major identified clusters. Figs. 3B-C show feature plots and dotplot showing expression pattern of cell cycle, neuroepithelial, anterior and posterior markers. Scale indicates log-fold change. Fig. 3D shows Immunostaining showing expression of neuroepithelial markers PAX6 and SOX2, anterior markers OTX2, and apical polarity marker NCAD on day 15 of differentiation. Fig. 3E shows a schematic showing morphogen treatment plan. Fig. 3F shows a LIMAP plot showing cell clusters identified in the single cell transcriptomes. Fig. 3G shows a LIMAP plot showing single cell transcriptomes upon morphogen treatment. Fig. 3H shows Dotplot showing expression of marker genes from different cell types and effect of morphogen treatment on cell differentiation. Fig. 3I shows Change in morphology and cell fate in response to Wnt, Retinoic acid and BMP signaling agonist

[0089] Fig. 4 relates to experiments showing long term differentiation of neuroepithelial cysts into forebrain organoids. Fig. 4A shows a schematic showing current approaches to generate regional brain organoids (also see Table 1). Compared to these approaches, we propose to use neuroepithelial cyst to diversify into different brain regions, keeping the starting culture and architecture uniform. Fig. 4B shows an immunohistochemistry P28241 PCOO 28.08.2025

[0090] 12 / 119 analysis of 30-day forebrain organoid generated from neuroepithelial cyst showing expression of cortical marker FOXG1 and apical polarity marker NCAD. Fig. 40 shows an immunohistochemistry analysis of 60-day forebrain organoid showing cortical and neuron markers. Fig. 4D shows a LIMAP showing cell clusters identified in the single cell transcriptomes. Fig. 4E shows a dotplot showing expression of marker genes from different cell types in identified clusters. Fig. 4F shows feature plots expression pattern of cell cycle (MKI67), outer radial glial cells, (HOPX), progenitor cells (PAX6), intermediate progenitors (EOMES), cortical neurons (BCL11 B, SATB2), interneurons (GAD2) anterior and posterior markers. Scale indicates log-fold change. Fig. 4G shows feature plots expression pattern of retinal progenitor cells (CRX, VSX2). Scale indicates log-fold change.

[0091] RGCs - radial glial cells, oRGs - outer radial glial cells, I PCs - intermediate progenitors, I Ns - interneurons, RPCs - retinal progenitor cells.

[0092] Fig. 5 Relates to experiments showing long term differentiation of neuroepithelial cysts into choroid plexus organoids. Fig. 5A shows representative brightfield images of 60-day old choroid epithelium organoid cultures. Immunohistochemistry analysis showing expression of choroid epithelium markers (TTR, AQP1 , OTX2) and polarity marker PODXL1 in 60-day old choroid plexus organoid generated from neuroepithelial cyst. Fig. 5B shows Hematoxylin and Eosin staining of 60 day old organoid. Fig. 5C shows a LIMAP showing cell clusters identified in the single cell transcriptomes. Fig. 5D shows feature plots expression pattern of choroid epithelium markers (TTR, TJP1 , AQP1 , CLIC6, HTR2C). Scale indicates log-fold change. Fig. 5E shows a dotplot showing expression of marker genes from different cell types in identified clusters. Fig. 5F shows a feature plots expression pattern of epithelial cells (KRT18) and neurons (MAP2, NCAM1). Scale indicates log-fold change.

[0093] Fig. 6 Relates to experiments showing long term differentiation of neuroepithelial cysts into retinal organoids. Fig. 6A shows representative brightfield images of 60-day old retinal organoid cultures generated from neuroepithelium cysts. Immunohistochemistry analysis showing expression of neural retina markers (CRX, PAX6, OTX2), cell division marker KI67 and progenitor marker SOX2. Fig. 6B shows a LIMAP showing cell clusters identified in the single cell transcriptomes of 25 week old organoids. Fig. 6C shows feature plots expression pattern of rods (NRL), cones (ARR3), Horizontal cells (ONECUT1), bipolar cells (GRIK1), Amacrine cells (FILIP1 L), GANGLIAN CELLS P28241 PCOO 28.08.2025

[0094] 13 / 119

[0095] (RBPMS), Muller cells (RLBP1), retinal progenitor cells (VSX2, SOX2). Scale indicates log-fold change. Fig. 6D shows a Dotplot showing expression of marker genes from different cell types in identified clusters.

[0096] Fig. 7 Effect of microwell size on single-lumen neuroepithelial cyst formation; Fig. 7(a): Brightfield microscopy images showing the formation of neuroepithelial cysts at different well diameters (300, 500, 800, 1000, 1500 pm), in II bottom ultra-low attachment 96 well plate (ll-ULA) and Il-bottom PEG coated 96 well plate (ll-PEG) at day 4 and day 8; Fig. 7(b): Quantification of the percentage of single-lumen neuroepithelial cysts formed across different well diameters and 96 well plates.

[0097] Fig. 8 Directed differentiation of forebrain, midbrain, and hindbrain regionalized organoids from neuroepithelial cysts. Fig. 8(a): Schematic illustration of the directed differentiation protocol for generating forebrain, midbrain, and hindbrain organoids from neuroepithelial cysts. The growth factors used at each differentiation stage are indicated. Fig. 8(b): Brightfield microscopy images showing the morphological development of forebrain, midbrain, and hindbrain organoids from day 8 to day 60. Fig. 8(c): Immunofluorescence staining of forebrain organoids at day 30 and day 60, showing expression of forebrain markers including FOXG1 (telencephalic neural progenitors and neurons), TBR2 (intermediate progenitor cells) and CTIP2 (deep-layer cortical projection neurons). Fig. 8(d): Immunofluorescence staining of midbrain organoids at day 20 and day 60, showing expression of FOXA2 (midbrain floor plate progenitors), LMX1A (midbrain dopaminergic neuron progenitors), NLIRR1 and TH (dopaminergic neurons). Fig. 8(e): Immunofluorescence staining of hindbrain organoids at day 30 and day 90 showing expression of ATOH1 (rhombic lip progenitors), PAX2 (GABAergic interneurons), SKOR2 (Purkinje cells).

[0098] Fig. 9 Generation and characterization of retinal organoids. Fig. 9(a): Brightfield microscopy images showing the morphological development of neuroepithelial cyst-derived retinal organoids at different time points. A magnified view of the week 30 organoid is shown on the right, highlighting distinct mature photoreceptor cells. Fig. 9(b): Immunofluorescence staining of retinal organoids at day 60, showing the expression of early retinal progenitor markers (VSX2, PAX6). Fig. 9(c): Immunofluorescence staining of retinal organoids at day 120, showing the expression of various retinal cell type markers, including photoreceptor progenitor cells (CRX), photoreceptors (OPN1SW and NRL), bipolar cells (PKC-a), Amacrine cells (AP2a), Muller glia (SOX9). Insets P28241 PCOO 28.08.2025

[0099] 14 / 119 show magnified views of specific regions. Fig. 9(d): Immunofluorescence staining of retinal organoids on day 120, demonstrating the expression and localization of NRL (outer nuclear layer) and SOX9 (inner nuclear layer). Fig. 9(e): A magnified view of a specific region is shown on the right. Fig. 9(f): Heatmaps illustrating the spatial distribution of SOX9 and NRL positive cells as a function of distance from the organoid edge, suggesting the formation of distinct retinal layers. Color intensity indicates cell count. Fig. 9(g): Immunofluorescence staining of NRL in multiple retinal organoids from four independent replicates, demonstrating reproducibility in photoreceptor development. Fig. 9(h): Immunofluorescence staining of retinal organoids at week 25, showing the mature expression of various retinal cell type markers, including ARR3 (cones), NRL (rods), RHO (rod opsin), PRKCa (bipolar cells), and Opsin (photoreceptors).

[0100] Fig. 10 Development and characterization of human choroid plexus organoids. Fig. 10(a): Brightfield microscopy images showing the morphological development of choroid plexus organoids from day 10 to day 190. Fig. 10(b): Immunofluorescence staining of choroid plexus organoids at day 60, demonstrating the expression of TTR and AQP1 (transporters), CLAUDIN5 (tight junctions) and OTX2. Fig. 10(c): Immunofluorescence staining of choroid plexus organoids at day 60, demonstrating the apical basal polarity: ZO1 (tight junctions), COLIV (basement membrane), AR13B (Cilia) and FOXJ1 (ciliated cells). Fig. 10(d): Hematoxylin and eosin staining of choroid plexus organoids showing gross morphology of the "apical out" and "apical in" organoids. Fig. 10(e): Immunofluorescence staining of choroid plexus organoids at day 60, showing the localization of apical and basolateral markers in "apical out" and "apical in" organoids. Fig. 10(f): Quantification of the cell density in choroid plexus organoids in both "apical out" and "apical in" organoids. Each dot represents an individual organoid, and bars indicate the mean with standard deviation. Fig. 10(g): Transmission electron microscopy image of a choroid plexus organoid at day 60, showing typical features of choroid plexus epithelium, including microvilli (MV), cilia (C), and extracellular vesicles (EV), multivesicular body (MVB), tight junction (TJ), glycogen (G), mitochondria (M), dark cells (DC), light cells (LC). Fig. 10(h): Venn diagram showing the shared protein components identified in cerebrospinal fluid (CSF) from various sources: choroid plexus organoids (oCSF), human embryonic CSF (heCSF), CSF from choroid plexus organoids generated by a published protocol (poCSF), and human adult CSF (haCSF). Fig. 10(i): Heatmap showing the expression of proteins P28241 PCOO 28.08.2025

[0101] 15 / 119 known to be secreted from choroid plexus epithelial cells in media control and oCSF. Color intensity represents relative expression. Fig. 10(j): Heatmap showing the relative expression of selected proteins, known to be present in human adult CSF, within choroid plexus organoids secretome. Fig. 10(k): Gene Ontology enrichment analysis of proteins identified in the choroid plexus organoids -derived CSF, categorized by cellular component, biological process, and molecular function.

[0102] Fig. 11 Generation of single-lumen neuroepithelial cysts from hPSCs. Fig. 11(a): Quantification of cyst size measured using SOX2-GFP signal over time, n = 3 independent experiments. Fig. 11(b): Quantification of lumen size measured using ZO1-GFP signal over time, n = 3 independent experiments. Fig. 11 (c): Quantification of cyst roundness measured using SOX2-GFP signal over time, n = 3 independent experiments.

[0103] Fig. 12 Generation of single-lumen neuroepithelial cysts from hPSCs. Fig. 12(a): Representative brightfield images of neuroepithelial cyst formation in PEG microwell plates (Gri3D, Sunbioscience), ultra low attachment microwells (Elplasia, Corning and Ag- greWell, StemCell Technologies) cell culture plate formats on day 6. Fig. 12(b): Representative brightfield images of transferred NECs from different culture plate formats on day 8. Fig. 12(c): Quantification of the diameter of cysts cultured in different plate formats (n>50 cysts per condition).

[0104] Fig. 13 Generation of single-lumen neuroepithelial cysts from hPSCs. Fig. 13(a): Immunofluorescence antibody staining for E-cadherin (ECAD), N-cadherin (NCAD), and SOX2 on day 6 and day 12 of differentiation. Fig. 13(b): Immunofluorescence staining for neuroepithelial markers on day 14 of differentiation. Fig. 13(c): Representative images on day 6 of differentiation of four independent pluripotent stem cells lines including WTC-11 iPSCs (GM25256), PGP1 iPSCs (GM23338), H9 ESCs and CRMi003-A iP- SCs.

[0105] Fig. 14 Generation of single-lumen neuroepithelial cysts from hPSCs. Fig. 14(a): Scatter plot showing the distribution of cell number settling into microwells in the well plate on day 0 for three cell lines. Fig. 14(b): Boxplot showing the relationship between the distance of cysts from the center of the well and the cyst category [no cyst (E), single (S) or multiple (M) lumen] on day 6 in three cell lines. Fig. 14(c): Boxplot showing the distribution of initial seeding cell numbers in microwells that formed no cyst (E), single (S) or multiple (M) lumen cysts on day 6 in three cell lines. P28241 PCOO 28.08.2025

[0106] 16 / 119

[0107] Fig. 15 (a) Live imaging snapshots of neuroepithelial cyst derived from a mix of ZO1-GFP

[0108] WTC-11 and CAAX-RFP WTC-11 iPS cells. Arrow indicates the dividing cells at the apical surface exhibiting interkinetic nuclear migration, (b) Time-lapse confocal microscopy images of a mosaic cyst containing CAAX-RFP- and ZO1-GFP-expressing WTC-11 cells stained with a live nuclear dye, showing interkinetic nuclear migration (arrowheads indicate nuclear movement over At = 0.5 h). Inset shows a magnified view of the apical constriction.

[0109] Fig. 16 Regional specification of neuroepithelial cysts from a default anterior state, (a) LIMAP visualization of single cells from day 10 cysts, colored according to their inferred cell cycle phases (G1 , S, G2M). (b) Feature plots overlaid on the LIMAP shown in (a), (c) Schematic overview of the morphogen treatment and downstream analysis (d) LIMAP embedding of the integrated single-cell transcriptomes from all treatment conditions, with cells colored by cell type annotation derived from Braun et al. (Emelie Braun et al. Comprehensive cell atlas of the first-trimester developing human brain. Sci- ence382,eadf1226(2023)) NE - neuroepithelium, NC - neural crest, PSC - pluripotent stem cell, (e) LIMAP embedding as in (d) with cells colored according to the different morphogen treatment groups, (f) Representative immunofluorescence images showing the morphological changes and expression of SOX2 and N cadherin. Nuclei are counterstained with DAPI. (g) Dot plot showing the expression levels and percentage of expressing cells for selected regional identity marker genes across the different cell type annotations, (h) Hierarchical clustering of transcriptomics data and bar graph showing the proportion of different cell types under various differentiation conditions, (i) Violin plots illustrating the dorsal-ventral patterning score of NECs under treatment conditions, (j) Violin plots illustrating the anterior-posterior patterning score of NECs under treatment conditions. Immunofluorescence analysis of NECs (k) after anterior- izing morphogen treatment for DAPI (nucleus) and FOXG1 (dorsal forebrain) (I) after SAG treatment with ventral marker NKX2-1 (m) after CHIR treatment with neuroepithelial marker PAX6 and neural crest cells marked by SOX10. (n) after RA treatment with HOX genes (o) after BMP4 treatment with PAX6, SOX2, neuroepithelial apical marker NCAD, non-neural epithelial marker ECAD.

[0110] Fig. 17 Regional specification of neuroepithelial cysts from a default anterior state, (a) Feature plots overlaid on a LIMAP embedding of single cells from day 10 cysts, displaying the distribution of the percentage of ribosomal proteins (RP%) and the number of P28241 PCOO 28.08.2025

[0111] 17 / 119 detected genes (#Genes) per cell, indicating cell quality, (b) Brightfield (left) and fluorescence (middle) microscopy images showing SOX2-GFP-positive cells seeded in microwells following dissociation of day 10 cysts. The inset (right) displays the reorganisation of dissociated cells within a microwell into cystic neuroepithelial structures that retain SOX2-GFP expression, (c) Brightfield images illustrating the morphology of NECs after 7 days of differentiation under various morphogen treatment conditions, (d) Dot plot showing the expression levels and percentage of expressing cells for selected regional identity marker genes across the different morphogen treatment groups, (e) LIMAP plots highlighting the expression of selected genes, (f) Representative immunofluorescence images showing the morphological changes and expression of PAX6 and N cadherin. Nuclei are counterstained with DAPI. (g) Immunofluorescence images showing FOXG1 and OTX2 expression after treatment with CHIR. (h) BMP4 treated cyst stained for FOXG1 and OTX2. (i) LIMAP visualization of single cells from day BMP4 treated cysts, colored for cell type annotation, (j) Feature plots overlaid on the LIMAP showing expression of different cell types, (k) Dot plot showing the expression levels of selected cell type marker genes in identified cell clusters. (I) Immunofluorescence images showing FOXG1 and OTX2 expression after treatment with FGF2 and FGF8b. (m) Immunofluorescence images of hPSC aggregates stained for NANOG and SOX2 after AA treatment.

[0112] Fig. 18 Generation of regionally specified brain organoids from neuroepithelial cysts, (a) Schematic diagram illustrating the patterned differentiation strategy to generate forebrain, midbrain, and hindbrain organoids from NECs using specific combinations of growth factors, (b) Immunohistochemistry analysis of a 30-day forebrain organoid section derived from a neuroepithelial cyst, demonstrating the expression of the cortical progenitor marker FOXG1 and the apical polarity marker NCAD. Nuclei are counterstained with DAPI. (c) Immunohistochemistry analysis of a 60-day forebrain organoid, revealing distinct populations of cortical progenitors (SOX2, FOXG1), intermediate progenitors (TBR2), and neurons (CTIP2, MAP2). Magnified views show organisation of progenitors (SOX2, TBR2) and neurons (CTIP2) in the developing organoids. (d) Immunofluorescence images of a day 20 midbrain organoid section stained with DAPI and midbrain progenitors marked by FOXA2, LMX1a and NKX6-1. (e) Immunofluorescence images of a day 60 midbrain organoid section stained with DAPI, TH (dopaminergic neurons), and FOXA2, LMX1A, NLIRR1 (dopaminergic neuron precursors) and GABAergic neuron marker GAD67. (f) Quantification of TH and GAD67 P28241 PCOO 28.08.2025

[0113] 18 / 119 positive neurons on day 60 ( ## organoids from 2 independent replicates) (g) Immunofluorescence images of a day 30 hindbrain organoid section stained with DAPI, ATOH1 , PAX6 and HOXC9 (h) Immunofluorescence images of a day 60 hindbrain organoid section stained with progenitors (PAX6, ATOH1), GABAergic neurons (GAD67), SKOR2 (Purkinje cell) and posterior marker (HOXC9). (i) Quantification of HOXC9 and SKOR2 on day 60 ( ## organoids from 2 independent replicates).

[0114] Fig. 19 Generation of regionally specified brain organoids from neuroepithelial cysts, (a) Hematoxylin and Eosin (H&E) staining and Immunofluorescence analysis of NECsshow- ing the expression of the cortical marker PAX6, apical polarity marker NCAD, basement membrane marker COLIV, and nuclei (DAPI). Dissolved Matrigel was removed on day 10. (b) Brightfield images showing the morphological development of forebrain organoids over time, (e) Brightfield images showing the morphological development of midbrain organoids from NECs over time, (f) Representative immunofluorescence images of day 20 midbrain organoids cultured without (Midbrain) or with matrigel (Mid- brain+ECM) stained for neuroepithelial marker SOX2 and apical marker NCAD. (g) Representative immunofluorescence images of day 20 midbrain organoids cultured without (Midbrain) or with matrigel (Midbrain+ECM) stained for FOXA2, LMX1A and NKX6-1. (h) Representative immunofluorescence images of day 60 midbrain organoids cultured without matrigel stained for midbrain markers, (i) Representative immunofluorescence images of day 60 midbrain organoids cultured with matrigel stained for midbrain markers, (j) Quantification of midbrain markers on day 60 (## organoids from 2 independent replicates).

[0115] Fig. 20 Retinal organoid generation from neuroepithelial cysts, (a) Schematic illustrating the differentiation protocol from NECs to retinal organoids using sequential growth factor addition, (b) Immunofluorescence images of a day 60 retinal organoid section stained with DAPI (nuclei) and antibodies against retinal progenitor markers PAX6, VSX2 and SOX9, and photoreceptor progenitor marker CRX (c) Immunofluorescence staining of retinal organoids at day 120, showing the expression of various retinal cell type markers, including photoreceptor progenitor cells (CRX), photoreceptors (OPN1SW, NRL, ARR3), bipolar cells (PKC-a), Amacrine cells (AP2A), Muller glia (SOX9). Insets show magnified views of specific regions, (d) Immunofluorescence staining of retinal organoids on day 120, demonstrating the expression and localization of NRL (outer nuclear layer) and SOX9 (inner nuclear layer), (e) Magnified view of the highlighted region P28241 PCOO 28.08.2025

[0116] 19 / 119 from (d), showing and counted cells (boxed) and the concentric bands used for spatial analysis (f) Heatmaps illustrating the spatial distribution of SOX9 and NRL positive cells as a function of distance from the organoid edge, suggesting the formation of distinct retinal layers. Color intensity indicates cell count. (n= 4 independent experiments, 146 organoids measured) (g) Scatter plot showing overall distribution of NRL and SOX2 positive cells measured in (h) at the organoid edge. (n=4 independent experiments) (h) Immunofluorescence staining of retinal organoids at week 25, showing the mature expression of various retinal cell type markers, including ARR3 (cones), NRL (rods), RHO (rod opsin), PRKCa (bipolar cells), and Opsin (photoreceptors), (i) UMAP visualization of single cells from week 25 retinal organoids, colored by major retinal cell types (MG: Muller glia, IPC: Intermediate Progenitor Cells, AC: Amacrine Cells, HC: Horizontal Cells, BC: Bipolar Cells, Rods, Cones, ECM: Extracellular Matrix). (j) Dot plot showing the expression levels and percentage of expressing cells for selected marker genes across the identified cell types, (k) Heatmap displaying tran- scriptomic similarity between the cell types identified in the organoids of the present disclosure and previously defined cell types in published retinal organoids (Wahle et al Nat Biotechnol. 2023 Dec; 41(12):1765-1775). (I) Violin plots showing tran- scriptomic similarity scores of organoids generated in this study to organoids across different time points from a published time-course retinal organoid dataset (Wahle et al Nat Biotechnol. 2023 Dec; 41 (12): 1765-1775).

[0117] Fig. 21 Generation of hindbrain organoids from neuroepithelial cysts, (a) Brightfield images showing the morphological development of hindbrain organoids from NECs over time, (b) Immunofluorescence image showing expression of NCAD and SOX2 on day 20 of differentiation, (c) Representative immunofluorescence images of day 30 hindbrain organoids stained for PAX6, ATOH1 and HOXC9. (d) Quantification of hindbrain markers on day 20 (## organoids from 2 independent replicates), (e) Representative immunofluorescence images of day 60 midbrain organoids stained for hindbrain markers, (f) Quantification of hindbrain markers on day 60 ( ## organoids from 2 independent replicates).

[0118] Fig. 22 Retinal organoid generation from neuroepithelial cysts, (a) Brightfield images showing the morphological development of neural retina organoids over an extended culture period, from day 8 to week 30). Inset shows a magnified view of the retinal organoid periphery. Arrowhead marks photoreceptor outer segments (b) Representative low P28241 PCOO 28.08.2025

[0119] 20 / 119 magnification brightfield images illustrating selection of retinal organoids based on morphology. Dark and small organoids similar to ones marked by white stars were selected out. (c) Line graph showing the change in organoid diameter over time during differentiation. For each time point 2-4 replicates were quantified for each time point, (d) Representative confocal fluorescence microscopy images of retinal organoids from 4 independent replicates in a tissue microarray at day 120. Images show the spatial distribution of the rod photoreceptor marker NRL and nuclei (DAPI). (e) Feature plots showing expression of selected marker genes from cell types identified in figure 4h.

[0120] DETAILED DESCRIPTION

[0121] Reference will now be made in detail to certain embodiments, examples of which are illustrated in the accompanying drawings, in which some, but not all features are shown. Indeed, embodiments disclosed herein may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Whenever possible, like reference numbers will be used to refer to like components or parts.

[0122] Definitions

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

[0124] In describing the embodiments and claiming the disclosure, the following terminology will be used in accordance with the definitions set out below.

[0125] Single lumen epiblast cyst, as used herein, means a structure of cells resembling the epiblast cells in a developing embryo, arranged to enclose a single lumen. The cells may for example be described to be arranged in an epithelial form with a single lumen. In typical embodiments, the processes described herein effectively induce single lumen emergence in at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40% of all epiblast cysts P28241 PCOO 28.08.2025

[0126] 21 / 119

[0127] (respectively neuroepithelial cysts). For example, in typical embodiments, at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40% of all first cell populations forming an epiblast cyst display a single lumen epiblast cyst (respectively neuroepithelial cyst). In some embodiments, at a given point in time, the ratio between the number of epiblast cysts (respectively neuroepithelial cysts) having a single lumen relative to the number of epiblast cysts (respectively neuroepithelial cysts) having two or more lumens is at least 1.0: 1.0, preferably at least 1.1 : 1.0, more preferably at least 1.2: 1.0, even more preferably at least 1.3: 1.0, even more preferably at least 1.4: 1.0, even more preferably 1.5: 1.0, even more preferably at least 1.6: 1.0, even more preferably at least 1.7: 1.0. In some embodiments, said ratio may be at least 1.8: 1.0, such as at least 1.9: 1.0, preferably at least 2.0:1.0. Depending on the application, the number of lumens may be determined at different time points. For example, in some embodiments, the number of lumens (i.e. e.g. whether there is a single lumen or multiple lumens) is determined at any time point in the time period from day 1 after seeding to day 12 after seeding. In some embodiments, the number of lumens is determined at any time point in the time period from day 4 after seeding to day 6 after seeding, such as on day 6 after seeding. In other embodiments, the number of lumens is determined at any time point in the time period from day 1 after seeding to day 3 after seeding, such as on day 2 after seeding. In other embodiments, the number of lumens is determined at any time point in the time period from day 4 after seeding to day 12 after seeding. Different methods may be used to determine whether an epiblast cyst has a single lumen. For example, in some embodiments, imaging-based methods are used. For example, in some embodiments, bright-field imaging of the cells is used. In some embodiments, a reporter cell line marking any of the apically expressed proteins is used. In some embodiments, the cell lines WTC-11 iPSCs (GM25256), PGP1 iPSCs (GM23338) and H9 ESCs may for example be used to determine the number of lumens.

[0128] As used herein, the term “epiblast cyst” means a structure of cells resembling the epiblast cells in a developing embryo, arranged to enclose at least one lumen. The cells may for example be described to be arranged in an epithelial form with at least one lumen. The cyst may e.g. be identifiable as such by imaging, e.g. using immunostaining. Epiblast cells are pluripotent cells and are capable of differentiating, under appropriate conditions, into all three embryonic germ layers, ectoderm, mesoderm and endoderm.

[0129] As used herein, the term “neuroepithelial cyst” means a structure of cells resembling the neuroepithelial cells in a developing embryo, arranged to enclose at least one lumen. For example, P28241 PCOO 28.08.2025

[0130] 22 / 119 the cells resembling the neuroepithelial cells may be described to form a cyst. The cyst may e.g. be identifiable as such by imaging, e.g. using immunostaining.

[0131] As used herein, “organoid” means a 2D or 3D multicellular tissue construct derived from stem cells. Typically, the organoid is a 3D multicellular tissue construct.

[0132] As used herein, the term “pluripotent stem cell” (PSC) means a cell capable of continued selfrenewal and of capable, under appropriate conditions, of differentiating into cells of all three germ layers. Examples of PSCs (hPSCs) include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). As used herein, “iPS cells” refer to cells that are substantially genetically identical to their respective differentiated somatic cell of origin and display characteristics similar to higher potency cells, such as ES cells, as described herein. The cells can be obtained by reprogramming non-pluripotent (e.g., multipotent or somatic) cells. Induced pluripotent stem cells exhibit morphological properties (e.g., round shape, large nucleoli and scant cytoplasm) and growth properties (e.g., doubling time of about seventeen to eighteen hours) akin to ESCs. In addition, iPS cells express pluripotent cell-specific markers. Induced pluripotent stem cells, however, are not immediately derived from embryos. As used herein, “not immediately derived from embryos” means that the starting cell type for producing iPS cells is a non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a postnatal individual. In some embodiments, the pluripotent stem cells used in the present disclosure may e.g. be selected from one or more of the following: WTC11 (GM25256, wildtype) human induced pluripotent stem cell line, PGP1 (GM23338, wildtype) human iPSC line, CRMi (CRMi003- A, wildtype) and H9 (WA09, wildtype) human embryonic stem cell line.

[0133] As used herein, “pluripotency” means a cell's ability to differentiate into cells of all three germ layers.

[0134] As used herein, “neural stem cell” (NSC) refers to a multipotent stem cell that is PAX6+ and / or Sox2+, and is capable of differentiating into neurons or glia of the CNS or peripheral nervous system (PNS).

[0135] “Supplemented,” as used herein, refers to a composition, e.g., a medium comprising a supplemented component (e.g., retinoic acid, FGF). For example, a medium “further supplemented” with component A (where A could be a hypothetical compound, structure or composition), refers to the medium comprising A, and not to the act of introducing the A to the medium.

[0136] As used herein, “effective amount” means an amount of an agent sufficient to evoke a specified cellular effect according to the present disclosure. P28241 PCOO 28.08.2025

[0137] 23 / 119

[0138] "Differentiation agent", as used herein, refers to an agent (e.g. a biological, chemical, or biochemical compound or composition) capable of promoting and supporting differentiation of stem cells towards a particular lineage. For example, “neural differentiation agent,” as used herein, refers to a differentiation agent capable of promoting and supporting differentiation of pluripotent stem cells towards a neural lineage, e.g., towards neuroectoderm and neuroepithelium. A medium comprising at least one neural differentiation agent may be described as “neural differentiation medium”. Accordingly, “neural differentiation medium,” as used herein, refers to a medium capable of promoting and supporting differentiation of pluripotent stem cells towards a neural lineage, e.g., towards neuroectoderm and neuroepithelium. In some embodiments, the neural differentiation agent is a neuronal differentiation agent.

[0139] As used herein, “morphogen” means a substance (e.g. a compound, a growth factor, a recombinant protein) that is able to activate or inhibit a biological signaling pathway and can impart pattern formation through non-uniform distribution.

[0140] As used herein, “serum-free” means that a medium does not contain serum or serum replacement, or that it contains essentially no serum or serum replacement. For example, an essentially serum-free medium can contain less than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% serum, wherein the culturing capacity of the medium is still observed.

[0141] As used herein, “substantially free of” means that a culture medium or other composition or solution is free or nearly free of a particular component. For example, “substantially free of A” (where A could be a hypothetical compound, structure or composition) means no A is added to a cell culture medium above and beyond any A present in the medium. Alternatively, “substantially free of A” means a final concentration of A of less than or equal to 0.1 mg / L.

[0142] As used herein, “viability” means the state of being viable. Viability can be assessed using different methods. For example, viable pluripotent cells typically do not stain with the membrane im- permeant dye propidium iodide. Short term viability relates to the first 24 hours after plating the cells in culture.

[0143] Pyramidal, as used herein, is defined as a solid with triangular lateral faces and a polygonal base. In some embodiments, the polygonal base is a triangular base or rectangular (e.g. square) base, preferably a rectangular base, more preferably a square base.

[0144] As used in the present disclosure, the expression “at least from day X after seeding until day Y after seeding” refers to a time interval that commences at the latest on day X (including day X itself), and that ends at the earliest on day Y (including day Y itself). Thus, the time interval may P28241 PCOO 28.08.2025

[0145] 24 / 119 e.g. commence on a day prior to day X and / or may end on a day later than day Y. As such, the expression describes a minimum time interval. In some embodiments, the respective time interval may start on (and including) day X and may end on (and including) day Y.

[0146] As used herein, the expression that a given cell population is “treated with an agent X at a concentration of Y” at a given day Z means that on day Z, the respective agent X is present in the culture medium in which the given cell population is in on day Z, at the given concentration Y. For example, when cell population W is in a given medium on day Z, and the cell population is said to be treated with agent X at concentration Y on day Z, then on day Z, it is ensured that the concentration of agent X in the medium is Y. This may e.g. involve changing and / or refreshing the medium, e.g. by replacing the medium. It is understood, therefore, that the concentration Y refers to a concentration of agent X in the medium.

[0147] When a numerical range for a given parameter having a particular unit is provided herein in the format “from X to Y [unit]”, i.e. where the unit is mentioned once at the end of the range, this means that the provided unit refers to both X and Y. For example, where it is specified “from 0.1 to 5 vol.-%”, this means from 0.1 vol.-% to 5 vol.-%. As another example, where it is specified “from 400 to 500 pm”, this means from (and including) 400 pm up to (and including) 500 pm. Thus, where the unit includes an order of magnitude, and where the unit and the order of magnitude are indicated once at the end, the unit and also the order of magnitude of the unit refer to both the first-mentioned (typically lower) value and to the second-mentioned (typically higher) value given, unless it is explicitly provided otherwise.

[0148] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage or a physical dimension such as length, width, or diameter, is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified value or amount, as such variations are appropriate to perform the disclosed methods.

[0149] Process

[0150] In general, the present disclosure provides a process for seeding stem cells and subsequently culturing the seeded stem cells. The stem cells may be further differentiated to varying degrees to provide a variety of different tissue types or organoid types. Furthermore, a morphogen treat- P28241 PCOO 28.08.2025

[0151] 25 / 119 merit may optionally be included as well. Depending on the application, more or less differentiation may be desired. Accordingly, the process may be performed to different degrees of differentiation and patterning.

[0152] A first aspect of the present disclosure relates to a process of producing epiblast cysts. The process comprises the step of a) seeding stem cells in microwells at a density of less than 500 cells per microwell. The process further comprises the step of b) culturing the seeded cells of step a) in the respective microwells for a first culture period in the presence of a first culture medium to obtain in each microwell a first cell population forming an epiblast cyst.

[0153] A second aspect of the present disclosure relates to a process of producing further differentiated epiblast cysts, comprising producing epiblast cysts according to the process of the first aspect or any of its embodiments described herein, and further comprising the step of: c) culturing the first cell populations forming epiblast cysts obtained in step b) for a second culture period in the presence of a second culture medium comprising a second differentiation agent to obtain a plurality of second cell populations each comprising further differentiated epiblast cysts. In some embodiments, the further differentiated epiblast cysts are neuroepithelial cysts.

[0154] A third aspect of the present disclosure relates to a process of producing neuroepithelial cysts. Said process comprises the step of a) seeding pluripotent stem cells in microwells at a density from 1 to 15 cells per microwell, wherein each microwell preferably has a diameter from 100 pm to 1’600 pm. The process further comprises the step of b) culturing the seeded cells of a) in the respective microwells for a first culture period (which preferably lasts from 1 to 3 days) in the presence of a first culture medium, thereby obtaining in each microwell a first cell population forming an epiblast cyst; and c) culturing the first cell populations obtained in step b) for a second culture period lasting at least 4 days in the presence of a second culture medium comprising a neural differentiation agent, thereby obtaining a plurality of second cell populations each comprising neuroepithelial cysts. Preferably, from no later than day 6 after seeding onwards, the second culture medium is supplemented with an extracellular matrix gel.

[0155] The third aspect is an advantageous realization of the process of the first and second aspect of the disclosure, and it specifically relates to neuroepithelial cysts. Consequently, all embodiments disclosed herein in the context of the first or second aspect also relate to the third aspect, and vice versa.

[0156] A fourth aspect of the present disclosure relates to a process of producing a plurality of organoids, comprising producing further differentiated epiblast cysts according to the process of the second P28241 PCOO 28.08.2025

[0157] 26 / 119 aspect or any of its embodiments described herein, and further comprising the step of: d) culturing the second cell populations in a third culture medium comprising a third differentiation agent to obtain the plurality of organoids.

[0158] In an embodiment, the process of the fourth aspect is a process of producing a plurality of organoids, comprising producing neuroepithelial cysts according to the process of the third aspect or any of its embodiments described herein, and further comprising the step of: d) culturing the second cell populations for a third culture period in a third culture medium comprising a third differentiation agent, thereby obtaining the plurality of organoids.

[0159] It is understood that the processes of these four aspects are interconnected in that the process of the second aspect makes use of the epiblast cysts produced in the process of the first aspect, the process of the third aspect is a specific preferred realization of the process of the first and second aspect, and the process of the fourth aspect makes use of the further differentiated epiblast cysts produced in the process of the second aspect respectively the neuroepithelial cysts produced in the process of the third aspect. As a result, the processes of these four aspects share common embodiments, and the embodiments described herein generally refer to all four aspects, unless specifically stated otherwise or unless the context clearly dictates otherwise.

[0160] The processes described herein, or at least selected steps of the processes, may be performed in different settings. In some embodiments, the processes are performed ex vivo, for example in vitro. In some embodiments, at least the first process step is performed ex vivo, for example in vitro. In some embodiments, all process steps are performed ex vivo, for example in vitro.

[0161] In some embodiments, the stem cells may or may not derive from a patient. In some embodiments, the stem cells or further differentiated forms of the stem cells, such as the epiblast cyst, the further differentiated epiblast cysts (e.g. the neuroepithelial cysts), or the organoids (e.g. the brain organoids), are not transferred back into the patient.

[0162] Density

[0163] Depending on the application, the stem cells may be seeded in the microwells at different densities. Further, different specifications of the densities may be envisioned.

[0164] As used herein, density of cells per microwell refers to the number of cells present in each microwell for aggregation. It is understood that the density of the cells in the microwells changes over time. Thus, the specification that the stem cells are seeded in the microwells at a certain P28241 PCOO 28.08.2025

[0165] 27 / 119 density in step a) typically refers specifically to the time point at which the stem cells are seeded. In some embodiments, the density may refer to a time point falling within a time period ranging from the moment of seeding to 5 hours after seeding, preferably from the moment of seeding to 3 hours after seeding, more preferably from the moment of seeding to 1 hour after seeding, even more preferably from the moment of seeding to 30 minutes of seeding. In some embodiments, the density may also be determined at a later point in time. Optionally, extrapolation may be used to determine the density of the cells at the time point of seeding.

[0166] In some embodiments, the stem cells are seeded in the microwells at a density of less than 100 cells per microwell. For example, in some embodiments, the stem cells are seeded in the microwells at a density from 1 to 20, preferably from 1 to 15, more preferably from 1 to 14 (e.g. from

[0167] I to 13), cells per microwell. In some embodiments, the stem cells are seeded in the microwells at a density of 2 or more cells per microwell, such as 10 or more cells per microwell. In some embodiments, the stem cells are seeded in the microwells at a density from 8 to 12 cells per microwell.

[0168] In some embodiments, the stem cells are seeded in the microwells at a density from 3 to 20, preferably from 3 to 15, even more preferably from 3 to 14, e.g. from 3 to 13, cells per microwell. In some embodiments, the stem cells are seeded in the microwells at a density from 5 to 15, such as from 5 to 14, such as from 5 to 13, or from 10 to 15, such as from 10 to 14, such as from 10 to 13, cells per microwell.

[0169] In some embodiments, the stem cells are seeded in step a) in the respective microwells at a density of at least 5, 6, 7, or 8 cells per microwell.

[0170] In some embodiments, the stem cells are seeded in step a) in the respective microwells at a density of up to and including 15, 14, 13 or 12 cells per microwell.

[0171] In some embodiments, the stem cells are seeded in step a) in the respective microwells at a density from 1 to 15, such as from 2 to 14, such as from 3 to 14, such as from 4 to 14 cells per microwell. In some embodiments, the stem cells may be seeded at a density from 5 to 15, such as from 6 to 14, such as from 7 to 13, such as from 8 to 12 cells per microwell.

[0172] It is understood that the range defined by the expression “from x to y” includes both the value x, the value y, and all values in between. Thus, for example, from 10 to 15 cells includes 10 cells,

[0173] I I cells, 12 cells, 13 cells, 14 cells and 15 cells. P28241 PCOO 28.08.2025

[0174] 28 / 119

[0175] Depending on the application, different densities may be used for different cell lines. To provide non-limiting examples, in some embodiments, the stem cells are WTC SOX2-GFP stem cells and are seeded in the microwells at a density of up to 10 cells per microwell. In some embodiments, the stem cells are WTC wildtype stem cells and are seeded in the microwells at a density of up to 15 cells per microwell. In some embodiments, the stem cells are H9 stem cells and are seeded in the microwells at a density of up to 15 cells per microwell. In some embodiments, the stem cells are PGP1 stem cells and are seeded in the microwells at a density of up to 15 cells per microwell. It is understood that the expression “up to”, in this context, includes the respective limit of the range. For example, “up to 10 cells” includes 10 cells, 9 cells, 8 cells, 7 cells, 6 cells, 5 cells, 4 cells, 3 cells, 2 cells and 1 cell.

[0176] In some embodiments, the stem cells are seeded in the microwells such that each microwell contains less than 10 cells per pL of microwell volume. For example, in some embodiments, the stem cells are seeded in the microwells such that each microwell contains less 5 cells per pL of microwell volume, more preferably less than 3 cells per pL of microwell volume, even more preferably less than 2 cells per pL of microwell volume. The microwell volume as used herein is defined as the total volume of a given microwell. In other words, the microwell volume is the maximum volume that the microwell can receive. In particular, the microwell volume refers to an operational volume which during operation is intended to contain a previously seeded cell aggregate during aggregation. Thus, the microwell volume typically does not include any adjacent volumes (e.g. supply channels or supply headspace volume) which are fluidically interconnected with the operational volume e.g. to supply media or nutrients to the microwells, but which are not intended to contain the cell aggregate. Furthermore, in embodiments in which a plurality of microwells are arranged in a corresponding well, the microwell volume is limited to the volume of said microwell and does not include the remaining (typically overhead) volume of the rest of the well in which the microwell is arranged. The remaining (typically overhead) volume of the rest of the well is typically a volume of the well that is shared for all of the plurality of microwells arranged in the respective well.

[0177] The stem cells may be seeded in the respective microwells, e.g. in step a), using standard laboratory equipment and methodology. For example, in some embodiments, the stem cells are seeded in the respective microwells by pipetting, e.g. manual or automated pipetting. In some embodiments, the process or at least parts of the process or parts of the process steps are performed by automation, for example by a laboratory robot. In some embodiments, for example, in step a), the stem cells are seeded in the respective microwells by an automated pipetting system. P28241 PCOO 28.08.2025

[0178] 29 / 119

[0179] Microwells

[0180] As used herein, a microwell is a small, individual cavity or chamber used for culturing and analyzing cells, tissues, and organoids. Different microwell formats may be used. For example, the microwell can be a well of a microplate, wherein the well of the microplate typically has a diameter of less than 3’000 pm, preferably less than 2’000 pm, in particular less than 1’600 pm. Preferably, the well of the microplate has a diameter from 200 pm to 1’000 pm. In some embodiments, the microwells are part of a culturing device. For example, the culturing device may comprise at least one cavity and said cavity may comprise a plurality of microwells. In some embodiments, the culturing device comprises a plurality of cavities and each of the plurality of cavities comprises a plurality of microwells.

[0181] Different microwell formats are commercially available and may be used in the processes described in the present disclosure. They may have different cross-sectional profiles. For example, both U-shaped and V-shaped microwells are compatible with the processes.

[0182] Non-limiting examples of commercially available microwell plates that may be used include e.g. Gri3D®96 from Sunbiosciences, Elplasia (Corning, 4442) and Aggrewell (Stem cell technologies, 34415, 34811). Preferably, a microwell plate with a plurality of microwells is used.

[0183] In some embodiments, a microwell plate may be used as described in US 2010 / 0068793 A1 , titled “DEVICES AND METHODS FOR PRODUCTION OF CELL AGGREGATES”, which is incorporated herein by reference in its entirety. In particular, those sections describing the microwells in said document are incorporated herein by reference.

[0184] In some embodiments, a microwell plate may be used as described in US 2011 / 0086375 A1 , titled “DEVICES AND METHODS FOR PRODUCTION OF CELL AGGREGATES”, which is incorporated herein by reference in its entirety. In particular, those sections describing the microwells in said document are incorporated herein by reference.

[0185] In some embodiments, a microwell plate may be used as described in CA 2972057 C, titled “MICROSTRUCTURED THIN HYDROGEL FILMS”, which is incorporated herein by reference in its entirety. In particular, those sections describing the microwells in said document are incorporated herein by reference.

[0186] In some embodiments, stem cells are cultured under non-adherent culture conditions. Therefore, the cells bind to each other instead of a culturing vessel (e.g. microwell walls such as microwell sidewalls) to form aggregates of the cells. P28241 PCOO 28.08.2025

[0187] 30 / 119

[0188] The non-adherent culture conditions may in particular be applied during step a), but they may also further be applied during at least some of the subsequent steps. For example, in some embodiments, during steps b) and c), the seeded cells respectively the first cell populations are cultured under non-adherent culture conditions. Preferably, step c) is also performed under nonadherent culture conditions.

[0189] Non-adherent culture conditions ensure that the aggregates do not attach to a culture vessel wall (e.g. microwell wall). For example, they may comprise a hydrogel or a non-adherent coating, forcing cells into a suspended state. Such non-adherent culture vessels (e.g. microwells) are known in the art.

[0190] In some embodiments, each microwell has a non-adherent inner surface. In some embodiments, the microwells each comprise an inner surface that is resistant to cell adherence or cell attachment. In some embodiments, the inner surface of the microwells are essentially free of regions or structures that promote cell attachment or cell adherence.

[0191] Preferably, the microwells have a non-adherent inner surface. In some embodiments, each microwell comprises a hydrogel, preferably a polyethylene glycol (PEG) hydrogel. Alternatively or in combination, in some embodiments, each microwell may comprise a modified plastic that imparts non-adherent properties to the microwell. For example, in some embodiments, at least a section or region of the inner surface of each microwell has a non-adherent coating. For example, in some embodiments, the inner surface of each microwell may have a non-adherent coating material. It is understood that, when a coating material is present, the coating material is typically arranged such that it is exposed to the cell culture condition that may be received by the respective microwell plate. In some embodiments, at least 5%, preferably at least 20%, more preferably at least 50%, of the inner surface of each microwell may be coated by the non-adherent coating material. In some embodiments, at least 70%, such as at least 90%, or even essentially 100%, of the inner surface of each microwell may be coated by the non-adherent coating material. Depending on the application, different non-adherent coating materials may be used. For example, in some embodiments, the non-adherent coating material is based on a hydrophilic polymer, such as a synthetic hydrophilic polymer. For example, the non-adherent coating material in some embodiments comprises or consists of one or more of the following: poly(ethylene glycol), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly(ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate), poly (hydroxy ethyl methacrylate), or mixtures thereof. P28241 PCOO 28.08.2025

[0192] 31 / 119

[0193] Many non-adherent microwells are commercially available. For example, the non-adherent coating may comprise a covalently bound hydrogel layer selected from Corning™ ultralow attachment range, Thermo Fisher™ Nunclon Sephera range, or polyHEMA (poly-2-hdroxylethyl methacrylate).

[0194] In some embodiments, the microwells are made in a hydrogel layer. For example, in some embodiments, the hydrogel layer is based on synthetic hydrophilic polymers or naturally derived components or hybrids of synthetic polymers and naturally derived components. In some embodiments, the synthetic hydrophilic polymer is selected from the group consisting of: poly(ethylene glycol), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, poly(ethylene oxide), polypropylene oxide, polyethylene glycol, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyhydroxy ethyl acrylate), poly (hydroxy ethyl methacrylate), or mixtures thereof.

[0195] In some embodiments, the hydrogel is prepared by mixing and cross-linking of at least two precursor components using a chemical reaction, wherein the first precursor component comprises n nucleophilic groups and the second precursor component comprises m electrophilic groups, wherein n and m are at least two and the sum n+m is at least five, and wherein the crosslinking is preferably conducted between

[0196] - a multi-arm-PEG macromer, preferably a four-arm-PEG macromer, end- functionalized with nucleophilic, preferably thiol-groups, with

[0197] - a multi-arm-PEG macromer, preferably an eight-arm-PEG macromer, end-functionalized with electrophilic, preferably vinylsulfone-groups at appropriate concentrations and conditions such as to allow for the crosslinked hydrogel layer to exhibit a shear modulus between 0.1 and 100 kPa.

[0198] Depending on the application, the microwells may have different shapes, sizes and structures. In some embodiments, the shape, size and structure of the microwells are chosen such that they do not promote cell adherence. For example, cell adherence may be essentially prevented.

[0199] In some embodiments, each microwell comprises an opening, a sidewall and a base arranged in a vertical direction opposite the base. The sidewall and the base define the volume of the respective microwell. The opening defines an opening plane. Each microwell further comprises an axis extending perpendicularly to the opening plane of the respective microwell.

[0200] The opening plane is typically perpendicular to the sidewall. The volume denoted as “microwell volume”, as used herein, typically does not extend outwardly beyond the opening of the respective P28241 PCOO 28.08.2025

[0201] 32 / 119 microwell. Thus, for example, in embodiments in which a plurality of microwells are arranged in a corresponding well, the opening of the microwell may, for example, separate the microwell volume from an overhead volume of the rest of the well which connects all microwells arranged in the well with each other.

[0202] In some embodiments, at least a component of the sidewall extends inwardly towards the axis of the respective microwell. Thereby, for a microwell of given dimensions, if 2 or more cells are deposited in the microwell, they will be forced into contact with one another, e.g. via gravity or centrifugation.

[0203] In some embodiments, the entire sidewall may extend inwardly towards the axis of the respective microwell. In some embodiments, only a component (e.g. a section or a region) of the sidewall extends inwardly towards the axis of the respective microwell. For example, in some embodiments, an upper component of the sidewall adjacent to the microwell opening may extend essentially vertically, while a lower component of the sidewall opposite the microwell opening may extend inwardly towards the axis of the respective microwell.

[0204] In some embodiments, the component of the sidewall that extends inwardly towards the axis of the respective microwell has a substantially constant slope. In other embodiments, the component of the sidewall that extends inwardly towards the axis of the respective microwell has a varying slope. For example, the slope may increase or decrease towards the base of the microwell. It is also possible to combine these embodiments. Thus, for example, one section of the sidewall may extend inwardly towards the axis at an essentially constant slope, and another section of the sidewall may extend inwards towards the axis at a varying slope, e.g. with a slope that increases or decreases towards the base of the microwell.

[0205] In some embodiments, the sidewall of each microwell is inclined such that a cross-sectional open area of the respective microwell being at least partially delimited by the inclined sidewall decreases towards the bottom of the respective microwell.

[0206] Depending on the application, the microwells may have different shapes. In some embodiments, each microwell is U-shaped or V-shaped in a cross-section parallel to the vertical direction. For example, in those embodiments in which the microwells are V-shaped, the base may in some embodiments be seen as a point on which the sidewalls converge. In those embodiments in which the microwells are U-shaped, for example, the base may in some embodiments be seen as a curved base plane. It is understood that in these case, the transition between the curved base plane and the sidewalls may be fluent. For example, the sidewalls may in these embodiments for P28241 PCOO 28.08.2025

[0207] 33 / 119 example be seen to have an upper component that may e.g. be essentially vertical and a lower component that may e.g. converge towards the axis of the respective microwell.

[0208] In some embodiments, in each microwell, a transition from the respective sidewall to the respective base is at least partially, preferably fully, rounded.

[0209] Depending on the application, the sidewall of the microwells may have different shapes. For example, in some embodiments, the sidewall of each microwell may have an essentially smooth profile that may e.g. be devoid of edges. In some embodiments, the sidewall may comprise one or more edges. For example, in some embodiments, the sidewalls each have a cylindrical shape. In some embodiments, at least a component of the sidewall of each microwell is conical, frustro- conical, pyramidal, frustro-pyramidal, or frustro-spherical.

[0210] In some embodiments, at least a component of the sidewall is at an angle of less than 90°, preferably from about 20° to about 80°, with respect to the opening plane of the respective microwell. In some embodiments, the angle is from 40° to 70°, preferably from 50° to 60°, more preferably about 54.7°.

[0211] In some embodiments, the microwells each have a microwell volume of less than 1’000 pL. In some embodiments, the microwells each have a microwell volume of 0.1 pL or more, preferably of 0.2 pL or more. In some embodiments, the microwells each have a microwell volume from 0.1 pL to 500 pL, more preferably from 0.2 pL to 300 pL. It is understood that the microwell volume is the maximum volume of liquid that a microwell can receive.

[0212] In some embodiments, during step b), each microwell is filled with a volume of the first culture medium ranging from 1 pL to 400 pL, preferably from 5 pL to 150 pL, more preferably from 10 pL to 100 pL. It is understood that the volume of the first culture medium does not exceed the volume of the respective microwell. In some embodiments, the volume of the first culture medium does not exceed 80%, particularly 70%, of the volume of the respective microwell.

[0213] In some embodiments, each microwell has a diameter from 1 pm to 4’000 pm, such as from 10 pm to 4’000 pm, preferably from 100 pm to 2’000 pm, more preferably from 100 pm to 1’500 pm. In some embodiments, each microwell has a diameter from 100 pm to 1’000 pm, e.g. from 200 pm to 1’000 pm. In some embodiments, each microwell has a diameter from 300 pm to 500 pm. Particularly preferable is a diameter of 300 pm or 500 pm.

[0214] In some embodiments, each microwell has a diameter of at least 100 pm, such as at least 200 pm, such as at least 300 pm, such as at least 400 pm. P28241 PCOO 28.08.2025

[0215] 34 / 119

[0216] In some embodiments, each microwell has a diameter of no more than 1’600 pm, such as no more than 1’400 pm, such as no more than 1’200 pm, such as no more than 1’000 pm, e.g. no more than 800 pm.

[0217] In some embodiments, each microwell has a diameter from 100 pm to 1’000 pm, such as from 200 pm to 800 pm, such as from 300 pm to 600 pm, particularly from 400 pm to 600 pm. For example, each microwell may have a diameter from 400 pm to 500 pm.

[0218] It is noted that the term “diameter” is not limited to circular microwell openings, but also includes e.g. rectilinear microwell openings. For example, in some embodiments, the diameter of a microwell may be understood to refer to a maximum distance, parallel to the opening plane, between two opposite points of the inner wall of the respective microwell. In some embodiments, the diameter of a microwell may be understood to refer to a maximum width of the respective microwell at the opening plane of the respective microwell.

[0219] In some embodiments, each microwell has a microwell height from 1 pm to 3’000 pm. In some embodiments, each microwell has a microwell height of less than 2’500 pm, e.g. less than 2’000 pm. The height of a given microwell may be defined as the distance along the vertical axis from the opening to the base. It may also be labelled as microwell depth.

[0220] In some embodiments, a ratio between the depth and the diameter of each microwell ranges from 3: 1 to 0.8: 1.0, preferably from 1.5: 1.0 to 1.0: 1 .0.

[0221] Preferably, the stem cells are seeded in the microwells in step a) such that direct contact with the microwell sidewall is avoided. In some embodiments, in step a), the stem cells are seeded in the microwells such that they have a minimum distance from a sidewall of the respective microwell of at least 10%, preferably of at least 20%, more preferably of at least 30%, with respect to a diameter of the respective microwell. Alternatively or in combination, in step a) the stem cells are seeded in the respective microwells such that they are positioned centrally inside the respective microwells. For example, the stem cells may be seeded in the respective microwells such that a distance to the sidewall of the respective microwell is maximized.

[0222] In some embodiments, in step a), the stem cells are seeded in the microwells such that they have a minimum distance from a nearest sidewall of the respective microwell of at least 10%, preferably of at least 20%, more preferably of at least 30%, of the diameter of the respective microwell. For example, if the diameter of the microwell is x, then the minimum distance could be 10% of x, i.e. 0.1x. P28241 PCOO 28.08.2025

[0223] 35 / 119

[0224] Depending on the application, multiple microwells may be used. For example, 25 or more microwells may be used. For example, in some embodiments, 500 or more microwells are used.

[0225] Depending on the application, a microwell plate may be used and carried through one or multiple of the process steps. For example, in some embodiments, the stem cells are kept in the same microwell throughout steps a) through c) (inclusive) or even throughout steps a) through d) (inclusive). In some embodiments, the stem cells are transferred from the microwells in which they were seeded in step a) to a different substrate.

[0226] In some embodiment, the cells are kept in their respective microwells throughout steps b) and c). Thus, in some embodiments, in step c) the first cell populations forming epiblast cysts obtained in step b) are cultured in their respective microwells to obtain in each microwell a second cell population.

[0227] In some embodiments, the cells are also kept in their respective microwells throughout steps b), c) and d). In other embodiments, the cells are transferred from their respective microwells to another substrate. For example, in some embodiments, the second cell populations obtained in step c) are transferred from their respective microwells to a further culture location where they are cultured in the third culture medium. The third culture location may e.g. be a bioreactor, a culture vessel or a bottom plate, preferably a culture vessel. The culture vessel may for example comprise a cell culture plate and / or a cell culture dish. Preferably, the third culture location may have a non-adherent surface. In some embodiments, during the step of culturing the neuroepithelial cysts in the respective culture location, the respective culture location (e.g. the bottom plate or culture vessel) is moved on a shaker, preferably an orbital shaker at 50-200 rpm, particularly at 100 rpm.

[0228] In typical embodiments, the seeded pluripotent stem cells are not encapsulated in a microgel, in particular not in an agarose microgel.

[0229] Single Lumen

[0230] The processes of the present disclosure allow to obtain epiblast cysts and further differentiated forms thereof having a single lumen. In some embodiments, at least 30%, preferably at least 40%, more preferably at least 50%, of all viable epiblast cysts obtained in step b) have a single lumen. For example, the density of cells per microwell, the first culture period and the first culture medium may be chosen such that at least 30%, preferably at least 40%, more preferably at least 50% (in some embodiments at least 60%, such as at least 80%), of all viable epiblast cysts obtained in P28241 PCOO 28.08.2025

[0231] 36 / 119 step b) have a single lumen. The ratio of those viable epiblast cysts that have a single lumen is generally provided by the number of viable epiblast cysts having a single lumen divided by the total number of viable epiblast cysts. It is understood that this ratio may be determined at different time points. In some embodiments, the ratio is determined 4 days after seeding or later. In some embodiments, the ratio is determined at a time point falling within the time period from 4 days after seeding to 6 days after seeding. As the skilled person understands, the ratio may be determined in many different ways and using different methods. In some embodiments, it involves extrapolation from measurements of a sub-set of all wells. In some embodiments, the ratio is determined using an imaging-based method.

[0232] Culture Media, Culture Periods, Differentiation Agents and Morphogens

[0233] Different culture media and culture periods may be used in different embodiments. For example, commercially available culture media known to the skilled person may be used. The culture media may comprise one or more different components. The component may e.g. be selected from one or more of the following: a basal medium, a buffer system, glutamine, serum (or a serum alternative), one or more growth factors, or additional supplements. For example, the additional supplements may include small molecules to increase cell survival.

[0234] Different culture media may be used for different steps in the processes described herein. Certain components of the different culture media may be the same, e.g. the base medium and / or certain supplements or differentiation agents. When reference is made in the following in general to a “culture medium”, this is to be understood as an embodiment of all three culture media independently of each other, unless it is stated otherwise or unless the context clearly dictates otherwise. For example, a hypothetical embodiment according to which “the culture media may comprise component A” (where component A is a hypothetical component), is to be understood as encompassing all of the following embodiments: embodiments in which all culture media comprise component A, embodiments in which any two of the three culture media comprise component A (i.e. embodiments in which the first and the second culture media comprise component A, embodiments in which the first and the third culture media comprise component A and embodiments in which the second and third culture media comprise component A), and embodiments in which any one of the three culture media comprise component A (i.e. embodiments in which the first culture medium comprises component A, embodiments in which the second culture medium comprises component A, and embodiments in which the third culture medium comprises component A). P28241 PCOO 28.08.2025

[0235] 37 / 119

[0236] In some embodiments, the processes described herein may be used in the context of CNS organoids. In some embodiments, the culture media may be neural culture media, such as neural differentiation media.

[0237] In some embodiments, the second culture medium and the third culture medium each independently comprise at least one differentiation agent. Independently, in this context, means that the differentiation agent respectively the differentiation agents of the second culture medium is respectively are chosen independently of the differentiation agent respectively differentiation agents of the third culture medium. As an example, the second culture medium may comprise at least one second differentiation agent and the third culture medium may comprise at least one third differentiation agent. In some embodiments, the first culture medium comprises at least one first differentiation agent. In some embodiments, the first culture medium is essentially free of differentiation agents. For example, the first culture medium may be essentially free of differentiation agents within the time period from seeding of the stem cells until day 2 after seeding, particularly from seeding until day 1 after seeding.

[0238] In some embodiments, the culture media are serum-free. In some embodiments, the culture media contain serum. In some embodiments, the culture media are xeno-free.

[0239] In some embodiments, the culture media comprise or consist of one or more of the following commercially available media: mTeSRplus, mTESR, Nutristem, E8 medium, PluriSTEM, TeSR medium, basal medium supplemented with N2 and B27 supplements, or basal medium supplemented with FGF2. For example, in some embodiments, the culture media may comprise at least 50 vol.-%, preferably at least 70 vol.-%, more preferably at least 90 vol.-%, even more preferably at least 95 vol.-%, of one or more of the commercially available media listed in the previous sentence.

[0240] In some embodiments, the culture media comprise one or more cell survival enhancers, such as small molecules to support cell survival. These small molecules may be selected from one or more of the following: Rock inhibitor (Y-27632), chroman 1 , emricasan, polyamines, trans-ISRIB.

[0241] As the skilled person knows, Chroman 1 is commercially available. For example, Chroman 1 as used herein may be obtained from Medchem Express, HY- 15392. Its SMILES code is O=C([C@@H]1COC2=CC=C(OC)C=C2C1)NC3=CC=C(C4=CNN=C4)C=C3OCCN(C)C.

[0242] As the skilled person knows, Y-27632 (i.e. Y-27632 dihydrochloride) is commercially available. For example, it may be obtained from Tocris, 1254. Its chemical name is trans-4-[(1R)-1-Ami- noethyl]- / V-4-pyridinylcyclohexanecarboxamide dihydrochloride. P28241 PCOO 28.08.2025

[0243] 38 / 119

[0244] In some embodiments, the culture media may comprise one or more further additional supplements. For example, in some embodiments, the culture media comprise one or more of the following: a BMP pathway inhibitor (preferably Dorsomorphin), a SMAD signaling inhibitor (preferably SB-431542), and a Wnt pathway activator (preferably CHIR-99021). These supplements may in some embodiments e.g. be comprised in the first culture medium, in the second culture medium and / or in the third culture medium.

[0245] In some embodiments, the culture media are supplemented with an extracellular matrix gel, preferably in a non-solid state. For example, in some embodiments, the extracellular matrix gel comprises or consists of a basement membrane. In some embodiment, the extracellular matrix gel comprises or consists of one or more of the following: matrigel, RTM, laminin, cultrex, hyaluronic acid, or a mixture thereof. For example, the extracellular matrix gel may comprise or consist of matrigel.

[0246] As the skilled person knows and as used herein, matrigel is a basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0247] As the skilled person knows and as used herein, Cultrex is a reduced grwoth factor basement membrane extract, type 2. It is for example available from R&D Systems, 3533-005-02. Cultrex Basement Membrane Extract (BME) is a soluble form of basement membrane purified from En- gelbreth-Holm-Swarm (EHS) tumor. Cultrex BME gels at 37 °C to form a reconstituted basement membrane. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan.

[0248] Depending on the application, the stem cells may be differentiated towards or into different cell lineages. Accordingly, the culture media may in some embodiments comprise one or more differentiation agents. In some embodiments, the first culture medium comprises one or more first differentiation agents. In some embodiments, the second culture medium comprises one or more second differentiation agents. In some embodiments, the third culture medium comprises one or more third differentiation agents. The distinction between first differentiation agent, second differentiation agent and third differentiation agent is primarily terminological, in order to clarify which culture medium comprises which differentiation agent(s). It is understood that the first differentiation agent(s), the second differentiation agent(s) and the third differentiation agent(s) may or may not be identical or partly identical to each other. For example, the second culture medium may in some embodiments comprise the same differentiation agent(s), or at least some of the same differentiation agents, as the third culture medium. In other embodiments, the second culture medium comprises different differentiation agents than the third culture medium. P28241 PCOO 28.08.2025

[0249] 39 / 119

[0250] In some embodiments, the processes of the present disclosure are aimed at differentiation of the stem cells towards a particular target group of cell lineages. For example, in some embodiments, the processes may be aimed at generating central nervous system (CNS) organoids or progenitors thereof.

[0251] In some embodiments, the second differentiation agent comprises or consists of a central nervous system differentiation agent, such that the organoids obtained are central nervous system organoids, preferably brain organoids. Alternatively or in combination, in some embodiments, the third differentiation agent comprises or consists of a central nervous system differentiation agent, such that the organoids obtained are central nervous system organoids, preferably brain organoids. In some embodiments, the CNS organoid is a target regional CNS organoid selected from one of the following: forebrain, midbrain, hindbrain or spinal cord organoid, preferably cortex, choroid plexus or retina organoid. In some embodiments, the CNS organoid is an optic cup organoid or a cerebellum organoid.

[0252] In some embodiments, at least one of the differentiation agents comprises a neural differentiation agent. For example, in some embodiments, the second differentiation agent and / or the third differentiation agent comprises a neural differentiation agent. In some embodiments, the second differentiation agent comprises or consists of a neuroepithelial differentiation agent, such that the plurality of second cell populations each form a neuroepithelial cyst.

[0253] Depending on the application, different markers may be used to characterize the epiblast cyst, the further differentiated epiblast cyst (e.g. the neuroepithelial cyst) and the CNS organoids. For example, in some embodiments, one or more of the following markers may be used for the neuroepithelial cyst: SOX2, PAX6, NESTIN, VIMENTIN, NCAD, ZO1 , COLIV, Laminin, OTX2, SIX3, FEZF1 , LHX2, HES1 , KI67. In some embodiments, one or more of the following markers may be used for forebrain organoids: SOX2, MAP2, FOXG1 , CALR, KI67, CTIP2, TBR2. In some embodiments, one or more of the following markers may be used for retina organoids: PAX6, KI67, SOX2, CRX, OTX2. In some embodiments, one or more of the following markers may be used for choroid plexus organoids: PODXL1 , TTR, AQP1.

[0254] In some embodiments, on day 10 after seeding, the neuroepithelial cysts express one or more of the following neuroepithelial markers: PAX6, NES, VIM and N cadherin.

[0255] In some embodiments, on day 10 after seeding, the neuroepithelial cysts express one or more of the following anterior neuroepithelial markers: SIX3, OTX2 and LHX2. P28241 PCOO 28.08.2025

[0256] 40 / 119

[0257] Depending on the application, it may be desirable to control the differentiation profile over time. In some embodiments, it may be desirable to arrive at more or less differentiated cell populations at a given point in time. In some embodiments, on day 10 after seeding, the neuroepithelial cysts express essentially none of the following posterior markers: GBX2

[0258] The expression (or lack thereof) of a number of cell type-associated markers can be used to characterize the differentiation of the stem cells (e.g. the hPSCs) over the course of the methods described herein. For example, the expression of some markers associated with pluripotency in hPSCs decline over the course of differentiation of the hPSCs into further differentiated cells. Such pluripotency markers include Oct4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Suitable markers (at the RNA or protein level) for neural stem cells and neural differentiation include, but are not limited to, PAX6, SOX2, Nestin, N-Cadherin, and SOX1.

[0259] Any appropriate method can be used to detect expression of biological markers characteristic of cell types described herein. For example, the presence or absence of one or more biological markers can be detected using, for example, RNA sequencing, immunohistochemistry, polymerase chain reaction, qRT-PCR, or other technique that detects or measures gene expression. Quantitative methods for evaluating expression of markers at the protein level in cell populations are also known in the art. For example, flow cytometry is typically used to determine the fraction of cells in a given cell population that express (or do not express) a protein marker of interest. As described in the Examples section below, differentiation of human pluripotent stem cells into epiblast cysts and further differentiated cell populations according to methods of the present disclosure can be confirmed based on the absence of non-neural cell types such as and the presence of cells having neural identity. Differentiated cell identity is also associated with downregulation of pluripotency markers (relative to human ES cells or induced pluripotent stem cells).

[0260] In some embodiments, any of the processes described herein may include at least one morphogen treatment step. Typically, the morphogen may be applied such that it is non-uniformly distributed.

[0261] In some embodiments, at least one of steps b) and c) comprises a morphogen treatment, wherein the morphogen treatment preferably includes treatment with one or more of the following:

[0262] - Wnt agonist, preferably CHIR-99021 ;

[0263] - sonic hedgehog agonist, more preferably SAG;

[0264] - treatment with a Notch agonist, preferably DAPT ; P28241 PCOO 28.08.2025

[0265] 41 / 119

[0266] - Retinoic acid agonist, preferably retinoic acid;

[0267] - BMP signaling agonist, preferably BMP4;

[0268] - a FGF signaling agonist, preferably FGF2 and FGF8;

[0269] - Wnt antagonist, preferably IWP2;

[0270] - a BMP signaling antagonist, preferably LDN193189;

[0271] - TGF beta signaling antagonist, preferably SB431542;

[0272] - Nodal signaling agonist, preferably Activin A.

[0273] In some embodiments, the epiblast cysts and / or the further differentiated epiblast cysts are patternable to at least three different CNS regions, preferably to at least five different CNS regions, through treatment with an appropriate morphogen. For example, in some embodiments, the epiblast cysts and / or the further differentiated epiblast cysts are patternable to at least the following CNS regions: forebrain, midbrain, hindbrain or spinal cord organoid, preferably cortex, choroid plexus or retina organoid. In some embodiments, the CNS organoid is an optic cup organoid or a cerebellum organoid.

[0274] Producing Neuroepithelial Cysts

[0275] During step b), the seeded cells are cultured in the presence of a first culture medium. Many different culture media can be used for this stem, including conventional stem cell maintenance media or, in general, culture media that are able to sustain and maintain stem cells, in particular pluripotent stem cells.

[0276] Preferably, the first culture medium is a stem cell maintenance medium that is configured to keep the seeded cells (preferably the seeded pluripotent stem cells) in an undifferentiated state.

[0277] In some embodiments, the first culture medium comprises a stem cell maintenance medium. The stem cell maintenance medium may for example be selected from the group consisting of mTeSRplus, mTeSR 1 , Essential 8, StemFlex, TeSR-E8, and a mixture thereof. In some embodiments, the first culture medium comprises or consists of mTeSRplus.

[0278] As the skilled person knows, mTeSRplus is commercially available. For example, it may be obtained from Stemcell Technologies, 100-0276. It is a cGMP, stabilized feeder-free maintenance P28241 PCOO 28.08.2025

[0279] 42 / 119 medium for human ES and iPS cells. Its preparation is e.g. described on: https: / / cdn.stem- cell.com / media / files / pis / 10000007758-PIS_03.pdf

[0280] In some embodiments, the first culture medium comprises a stem cell maintenance medium. The stem cell maintenance medium may for example be selected from the group consisting of mTeSRplus, mTeSR 1 , Essential 8, StemFlex, TeSR-E8, and a mixture thereof. Preferably, the first culture medium comprises mTeSRplus. For example, each microwell may comprise mTeSRplus in an amount from 20 pl to 100 pl per microwell.

[0281] Preferably, the pluripotent stem cells are seeded in the microwells in mTeSRplus (preferably supplemented with CEPT cocktail), preferably in 20 pl to 100 pl mTeSRplus. In some embodiments, additional mTeSR plus (e.g. in an amount from 50 pl to 200 pl per microwell) is added to the first culture medium within 30 minutes and 3 hours after seeding, preferably 1 hour after seeding.

[0282] In some embodiments, mTeSR or mTeSR plus is added to the first cell culture medium between 30 minutes and 3 hours after seeding, preferably within 30 minutes and 90 minutes after seeding. Preferably, from 50 pl to 150 pl, particularly from 80 pl to 120 pl, more particularly 100 pl, mTeSR or mTeSR plus are added to the first cell culture medium.

[0283] In some embodiments, the first culture medium comprises a rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g. Chroman 19). Optionally, the first culture medium may additionally comprise a pan-caspase inhibitor (e.g. emricasan). Furthermore, the first culture medium may additionally comprise a polyamine supplement (i.e. a lyophilized mixture of putrescine, spermine and spermidine). Furthermore, the first culture medium may additionally comprise an integrated stress response (ISR) inhibitor (e.g. trans-ISRIB, i.e. / V, / V'-trans-1 ,4-cyclohex- anediylbis[2-(4-chlorophenoxy)acetamide]).

[0284] In some embodiments, the first culture medium comprises a CEPT cocktail. As the skilled person knows, CEPT cocktail refers to a combination of Chroman 1 , Emricasan, Polyamines, and Trans-ISRIB. CEPT cocktail is a commonly used cell culture supplement that is commercially available and has e.g. been described in Nat Methods 2021 May;18(5):528-541. It is commercially available, e.g. from: Polyamine (Sigma, P8483), Chromanl (Medchem Express, HY-15392), Emricasan (Selleckchem, S7775), trans-ISRIB (Thermo Fisher scientific, 5284). The CEPT cocktail may be used for promoting the viability and fitness of self-renewing pluripotent stem cells.

[0285] In some embodiments, the stem cells are seeded in the microwells in mTeSR plus supplemented with CEPT cocktail (e.g. with a volumetric ratio between mTeSR and CEPT cocktail of 1 :1000). P28241 PCOO 28.08.2025

[0286] 43 / 119

[0287] In some embodiments, mTeSR supplemented with CEPT cocktail is added to the first culture medium within 30 minutes and 3 hours after seeding, preferably 1 hour after seeding.

[0288] In some embodiments, the first culture medium comprises or consists of mTeSR plus supplemented with CEPT cocktail. For example, a volumetric ratio between mTeSR and CEPT cocktail may be from 1 :700 to 1 :1300, particularly 1 :1000. In these embodiments, the first culture period may for example last from 1 to 4 days, preferably from 1 to 3 days, more preferably for 2 days, after seeding.

[0289] In some embodiments, the first culture medium comprises a rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor. As the skilled person knows, ROCK inhibitors are known, commercially available supplements to reduce apoptosis and promote cell survival. For example, the ROCK inhibitor may be Chroman 1 or Y-27632, preferably Chroman 1.

[0290] In some embodiments, in step d), the second cell populations are treated with a BMP pathway inhibitor, preferably Dorsomorphin, a SMAD signaling inhibitor, preferably SB-431542, and / or a Wnt pathway activator, preferably CHIR-99021.

[0291] In some embodiments, in step d), the second cell populations are treated with pulsed delivery of a Wnt agonist, preferably CHIR-99021 , for a period of 1 to 10 days, preferably 2 to 8 days, most preferably 3 to 6 days, followed by culture in a neuronal growth medium.

[0292] In some embodiments, in step d), matrigel is removed from the second cell populations, followed by long term maturation culture.

[0293] In some embodiments, the first culture medium is supplemented with an extracellular matrix gel, preferably in a non-solid state, after aggregation of the stem cells in the respective microwells. For example, in some embodiments, the first culture medium is in each microwell supplemented with the extracellular matrix gel. In the context of the embodiments of this paragraph, unlike the general definition, supplemented is to be understood to refer to the act of introducing (e.g. adding) the extracellular matrix gel, and typically does not merely refer to the state of the first culture medium. In other words, typically, in these embodiments, the extracellular matrix gel is added to the first culture medium after aggregation of the stem cells. Thus, in some embodiments, the amount of extracellular matrix gel in the first culture medium is higher after supplementation than before supplementation. In some embodiments, the first culture medium is essentially free of extracellular matrix gel before aggregation. Essentially free, in this context, means that before supplementation, the extracellular matrix gel comprises less than 1 vol.-%, preferably less than 0.1 vol.-%, more preferably less than 0.01 vol.-%, even more preferably less than 0.001 vol.-%, even P28241 PCOO 28.08.2025

[0294] 44 / 119 more preferably less than 0.0001 vol.-%, even more preferably less than 0.00001 vol.-%, even more preferably less than 0.000001 vol.-%, of the first culture medium.

[0295] In some embodiments, the first culture medium is supplemented with the extracellular matrix gel at a time point falling in the time period ranging from 1 hour after seeding to day 8 after seeding, preferably on day 4 after seeding. In some embodiments, the first culture medium is supplemented with the extracellular matrix gel at a concentration of at least 1 vol.-% with respect to the total volume of the first culture medium. In some embodiments, the first culture medium is supplemented with the extracellular matrix gel at a concentration ranging from 1 vol.-% to 30 vol.-%, preferably from 1 vol.-% to 5 vol.-%, with respect to the total volume of the first culture medium.

[0296] Generally, when the concentration of a component of a culture medium (e.g. extracellular matrix gel such as Matrigel) is provided herein as vol.-%, this is relative to a total volume of the respective culture medium.

[0297] In some embodiments, the first culture period lasts from 1 to 4 days (e.g. 1 day, 2 days, 3 days, or 4 days). In some embodiments, the first culture period lasts for 2 days. In some embodiments, the first culture period lasts for 35 hours to 65 hours.

[0298] In some embodiments, the first culture period lasts for 2 days, such that the second culture period commences on day 2 after seeding.

[0299] In some embodiments, the second culture medium is supplemented with an extracellular matrix gel, preferably in a non-solid state, after aggregation of the stem cells in the respective microwells. For example, in some embodiments, the first culture medium is in each microwell supplemented with the extracellular matrix gel. In some embodiments, the extracellular matrix gel comprises or consists of matrigel, cultrex, laminin, hyaluronic acid, or a mixture thereof. In some embodiments, the second culture medium is supplemented with an extracellular matrix gel in an amount ranging from 1 vol.-% to 20 vol.-%, preferably from 1 vol.-% to 10 vol.-%, more preferably 3 vol.-% to 7 vol.-%, even more preferably 5 vol.-%, with respect to the total volume of the first culture medium.

[0300] In some embodiments, the second culture medium comprises or consists of a neural induction medium. As used herein, a neural induction medium is a culture medium capable of directing stem cells (in particular pluripotent stem cells) to become neural progenitor cells (NPCs) or neural stem cells (NSCs). Preferably, the neural induction medium comprises N2 supplement.

[0301] In some embodiments, the second culture period lasts at least 3 days, preferably from 3 to 15 days (e.g. 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 P28241 PCOO 28.08.2025

[0302] 45 / 119 days, 14 days, 15 days, inclusive). In some embodiments, the second culture period lasts at least 4 days, preferably at least 5 days. In some embodiments the second culture period lasts from 4 days to 10 days, preferably from 6 days to 8 days. In some embodiments, the second culture period lasts at least 6 days.

[0303] In some embodiments, the second culture period ends on day 8, 9 or 10 after seeding

[0304] In some embodiments, the second culture medium comprises basal medium with N2 supplement. In some embodiments, the second culture medium comprises a BMP pathway inhibitor, preferably Dorsomorphin, a SMAD signaling inhibitor, preferably SB-431542.

[0305] In some embodiments, the second culture medium is supplemented with the extracellular matrix gel at a concentration of at least 1 vol.-%, preferably from 1 to 20 vol.-%, more preferably from 2 vol.-% to 10 vol.%, even more preferably from 3 vol.-% to 5 vol.-%, with respect to a total volume of the second culture medium. In these embodiments, it is particularly advantageous to use Mat- rigel as extracellular matrix gel.

[0306] The inventors have found that the time of addition of the extracellular matrix gel (which is preferably Matrigel) during the second culture medium may be optimized to improve the reliability and reproducibility of the process and the quality of the cysts obtained. Preferably the second culture medium is supplemented with the extracellular matrix gel from no later than day 5 after seeding onwards, in particular from no later than day 4 after seeding onwards. In advantageous embodiments, the second culture medium is supplemented with the extracellular matrix gel from day 4 after seeding onwards. In the embodiments described in this paragraph, the extracellular matrix gel preferably comprises or consists of Matrigel.

[0307] As used herein, the expression „from day 4 after seeding onwards" includes day 4 after seeding. Thus, when it is specified that the second culture medium is supplemented with Matrigel from day 4 after seeding onwards, this means that the second culture medium is comprised by (e.g. added to) the second culture medium on day 4 after seeding and onwards, until the second culture medium is changed. For example, where the second culture medium is changed on day 7 after seeding to another medium that is free of Matrigel, and where the second culture medium is refreshed e.g. every second day during the second culture period, this would mean that Matrigel is added to the second culture medium on day 4 after seeding and on day 6 after seeding, but no longer on day 8 after seeding because in this example, the medium has been changed on day 7 to another medium that is free of Matrigel. In other examples in which the medium is changed to another medium that also contains Matrigel, Matrigel would be added further. P28241 PCOO 28.08.2025

[0308] 46 / 119

[0309] In some embodiments, the second culture medium is supplemented with the extracellular matrix gel until the end of the second culture period. For example, the second culture medium may be supplemented with the extracellular matrix gel at the latest from day 6 after seeding until the end of the second culture period, e.g. at the latest from day 5 after seeding until the end of the second culture period, preferably at the latest from day 4 after seeding until the end of the second culture period. In these embodiments, it is preferable that the second culture medium is refreshed at least once every 3 days, preferably at least once every 2 days.

[0310] In some embodiments, the second culture medium and optionally also the third culture medium are supplemented with the extracellular matrix gel at least until day 9 after seeding, preferably at least until day 10 after seeding. For example, in some embodiments, the second culture medium and optionally also the third culture medium are supplemented with the extracellular matrix gel from day 4 after seeding until at least day 10 after seeding. These embodiments can be advantageous for later differentiation into different organoid types, in particular for forebrain organoids.

[0311] In some embodiments, the second culture medium is supplemented with the extracellular matrix gel from day 4 after seeding onwards until at least day 6 after seeding, preferably until termination of the second culture period.

[0312] The second culture medium may comprise a neural differentiation agent, i.e. a differentiation agent capable of promoting and supporting differentiation of pluripotent stem cells towards a neural lineage, e.g., towards neuroectoderm and neuroepithelium. Preferably, the neural differentiation agent is capable of promoting and supporting differentiation of pluripotent stem cells towards a neuroepithelium lineage.

[0313] In some embodiments, the neural differentiation agent may comprise or consist of N2 supplement. N2 supplement is commercially available and is diluted into a base medium at 1 :100. In some embodiments, the second culture medium comprises or consists of N2 medium.

[0314] In some embodiments, the N2 supplement is present in the second culture medium in an amount sufficient to differentiate the pluripotent stem cells to exit pluripotency in an amount of at least 50%, preferably at least 80%, of the pluripotent stem cells within 8 days of starting induction with the N2 supplement.

[0315] In some embodiments, the N2 supplement is present in the second culture medium in an amount sufficient to differentiate the pluripotent stem cells to neural lineages in an amount of at least 50%, preferably at least 80%, of the pluripotent stem cells within 8 days of starting induction with the N2 supplement. P28241 PCOO 28.08.2025

[0316] 47 / 119

[0317] In some embodiments, from day 4 after seeding onwards, the second culture medium comprises or consists of N2 medium supplemented with extracellular matrix gel, preferably at a concentration from 1 to 20 vol.-% (e.g. from 1 to 10 vol.-%), with respect to a total volume of the second culture medium. The extracellular matrix gel is preferably Matrigel in these embodiments.

[0318] N2 medium, as used herein, is a basal medium comprising N2 supplement. It may e.g. comprise any basal medium that is able to support the growth and proliferation of neural stem cells. For example, the basal cell culture medium may comprise DMEM / F12 medium, which is preferably supplemented as described herein in further detail.

[0319] N2 supplement is a serum-free, chemically defined supplement based on Bottenstein’s N2 formulation (Bottenstein, J.E. (1985) Cell Culture in the Neurosciences, Bottenstein, J.E. and Harvey, A.L., editors, p. 3, Plenum Press: New York and London).

[0320] In some embodiments, the N2 medium comprises at least: Dulbecco’s modified eagle medium (DMEM) / F12 and N2 supplement (e.g. at a concentration from 0.1 vol.-% to 5 vol.-%, e.g. from 0.5 vol.-% to 2.0 vol.-%, such as 1 vol.-% with respect to a total volume of the second culture medium). Preferably, the N2 medium may comprise: Dulbecco’s modified eagle medium (DMEM) / F12, MEM-NEAA, N2 supplement (e.g. at a concentration from 0.1 vol.-% to 5 vol.-%, e.g. from 0.5 vol.-% to 2.0 vol.-%, such as 1 vol.-% with respect to a total volume of the second culture medium) and one or more of: glutamine and glutamax.

[0321] In some embodiments, the N2 medium comprises: Dulbecco’s modified eagle medium (DMEM) / F12, MEM-NEAA, N2 supplement (e.g. at a concentration from 0.1 vol.-% to 5 vol.-%, e.g. from 0.5 vol.-% to 2.0 vol.-%, such as 1 vol.-% with respect to a total volume of the second culture medium), heparin and one or more of: glutamine and glutamax. Preferably, in the embodiments described in the previous sentence, the N2 culture medium may further comprise HEPES and may optionally further comprise one or more of the following: pencilling and streptomycin.

[0322] As a non-limiting example, in some embodiments, the N2 medium may for example be the “homemade” N2 medium as used in the examples. In some embodiments, the N2 medium comprises or consists of: DMEM / F12 HEPES glutamine (e.g. available from Therma Fisher Scientific, 11330057), Penicillin and Streptomycin (e.g. available from Thermo Fisher, P0781), N2 supplement (e.g. available from Thermo Fisher, 17502048), minimum essential medium (MEM) non- essential amino acids (e.g. available from Thermo Fisher, 11140035) and Heparin (e.g. available from Sigma-Aldrich, H3149-25KU). P28241 PCOO 28.08.2025

[0323] 48 / 119

[0324] An illustrative, non-limiting example of a possible composition of an N2 medium that may e.g. be used in the processes of the present disclosure is provided in the following table:

[0325] In some embodiments, the second culture medium is refreshed at least once every 3 days, pref- erably at least once every 2 days, during the second culture period.

[0326] It is understood that as used in the present disclosure, when it is said that a given medium is refreshed, this relates to the entire culture medium, including any supplements, factors, additives, etc. that were previously contained therein. For example, where the second culture medium comprises on day 4 after seeding N2 medium and Matrigel at given concentrations and volumes, and where this medium was specified to be refreshed e.g. on day 6 after seeding, then this means that on day 6 after seeding the medium remaining in the microwells is replaced by a fresh mixture of N2 medium and Matrigel having the same concentrations and volumes as previously administered on day 4 after seeding.

[0327] When a given medium is replaced by another medium (or refreshed by fresh medium), the me- dium exchange may for example involve first removing (at least a portion of) the earlier medium, followed by filling in the new or fresh medium. As the skilled person knows, care may be taken not to interfere with the cells during medium exchange. As such, it may be advantageous to only remove only a portion of the earlier medium rather than the entire earlier medium. Optionally, the process of removal of a portion of earlier medium and filling in new or fresh medium may be P28241 PCOO 28.08.2025

[0328] 49 / 119 repeated once or twice or even more times to essentially dilute down any remaining earlier or old medium.

[0329] In some embodiments, every second day during the second culture period, starting with a second day after initiation of step b), at least 50 vol.-% of the second culture medium is removed from the respective microwells and fresh N2 medium is subsequently added to the respective microwells.

[0330] In some embodiments, on day 4 after seeding, 100-200 pl, preferably 150 pl, of the second culture medium is removed from the respective microwells and 50-150 pl, preferably 100 pl, fresh N2 medium is subsequently added to the respective microwells. Furthermore, preferably, on day 6 after seeding, 50-150 pl, preferably 100 pl, of the second culture medium is removed from the respective microwells and 50-150 pl, preferably 100 pl, fresh N2 medium is subsequently added to the respective microwells. Furthermore, preferably, on day 8 after seeding, 100-200 pl, preferably 150 pl, of the second culture medium is removed from the respective microwells and 50-150 pl, preferably 100 pl, fresh N2 medium is subsequently added to the respective microwells.

[0331] Typically, the cell culture medium is changed between the first and second culture periods. Thus, for example, between the first culture period and the second culture period, the first culture medium may be replaced with the second culture medium.

[0332] Preferably, during the second culture period of step c), the first cell populations are cultured in the respective microwells in which they were previously cultured in step b). In other words, the cells are typically not removed from the respective microwells during or between steps b) and c).

[0333] In some embodiments, a first portion of extracellular matrix gel (e.g. Matrigel) is added to the second culture medium between 50 hours and 170 hours after seeding, preferably between 70 hours and 140 hours after seeding.

[0334] In some embodiments, a second portion of extracellular matrix gel (e.g. Matrigel) is added to the second culture medium between 24 hours and 72 hours, preferably between 38 hours and 58 hours, after addition of the first portion of extracellular matrix gel. Depending on the application, before addition of the second portion of the extracellular matrix gel, at least some of the second culture medium may be removed and discarded. In other words, the second culture medium may essentially be refreshed.

[0335] In some embodiments, the first culture medium is supplemented with the extracellular matrix gel at a concentration of at least 1 vol.-% with respect to the total volume of the first culture medium. P28241 PCOO 28.08.2025

[0336] 50 / 119

[0337] In a particularly advantageous embodiment of the process of the third aspect of the present disclosure, the process of producing neuroepithelial cysts comprises the steps of: a) seeding the pluripotent stem cells in the microwells at a density from 5 to 13 cells per microwell, wherein each microwell has a diameter from 300 pm to 800 pm and a non-adherent inner surface; b) culturing the seeded cells of a) in the respective microwells for the first culture period lasting 2 days in the presence of a first culture medium comprising a stem cell maintenance medium and further comprising a ROCK inhibitor and / or a CEPT cocktail, thereby obtaining in each microwell a first cell population forming an epiblast cyst; and c) culturing the first cell populations obtained in step b) in the respective microwells for the second culture period lasting at least 5 days in the presence of the second culture medium comprising N2 medium, thereby obtaining a plurality of second cell populations each comprising neuroepithelial cysts; wherein from day 4 after seeding onwards, the second culture medium is supplemented with Matrigel at a concentration of from 1 to 20 vol.-%.

[0338] Producing Organoids

[0339] In typical embodiments, between steps c) and d) the second cell populations are transferred from the microwells to a cell culture plate or a cell culture dish. Thereby, the second cell populations are cultured in the cell culture plate or in the cell culture dish during at least a portion of the third culture period. Preferably, the cell culture plate or the cell culture dish has a diameter from 3 cm to 12 cm, preferably from 6 cm to 9 cm.

[0340] In preferred embodiments, the cell culture dish is an ultra-low attachment cell culture dish. For example, it may be an ultra low attachment 6 cm or 9 cm dish (Corning, LSBio).

[0341] Depending on the application, different numbers of cysts may be transferred. In some embodiments, from 300 to 1’000, preferably from 500 to 600, neuroepithelial cysts are transferred to each cell culture plate respectively each cell culture dish.

[0342] In some embodiments, the third culture period lasts at least 3 days, preferably at least 10 days, more preferably from 10 days to 150 days, such as from 10 days to 100 days. P28241 PCOO 28.08.2025

[0343] 51 / 119

[0344] Preferably, during at least the first 10 days of the third culture period, the second cell populations are cultured under non-adherent culture conditions. These embodiments further improve the quality of the organoids obtained.

[0345] In preferred embodiments, the third culture period commences on day 8 or day 9 or day 10 or day 11 after seeding. Preferably, the third culture period commences on day 8 or day 9 or day 10 after seeding. The exact starting point may depend on the type of organoid envisioned.

[0346] Preferably, the third culture medium comprises N2 supplement. For example, the third culture medium may comprise N2 supplement for at least the first 10 days of the third culture period, preferably for at least the first 20 days of the third culture period, or even throughout the entire third culture period.

[0347] In some embodiments, during at least the first 5 days of the third culture period, preferably during at least the first 8 days of the third culture period, the third culture medium comprises B27 supplement. B27 supplement is a commonly used medium supplement for neural cells and has been commercially available for more than 25 years. It is a complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids. A protocol for "home-made" medium B27 supplement is, for example, available from https: / / hannalabweb.weizmann.ac.il / wp-content / up- loads / 2016 / 02 / HANNA-LAB-B22- B27-PROTOCOL-V3.pdf. This "home-made" B27 supplement contains the 21 components as discussed below, and 100gr BSA Fraction V IgG free Fatty Acid Poor (Invitrogen 30036578 x 1 unit) assembled in Neurobasal medium (Invitrogen 21103-049 x 2 units). For 800 ml of B27 the 21 components - that can, for example, be obtained from Sigma- Aldrich - are: 1) Catalase - (store at -20 as is) C40-100MG X 1 unit 2) Glutathione reduced - (store at +4 as is) G6013-5G X 1 unit 3) # Human Insulin - (store as is -20, store aliquots at -80) 91077C- 250MG X 1 unit 4) Superoxide Dismutase - (store at -20 as is) S5395-75KU X 4 units 5) Human Holo-Transferin - (store at +4 as is) T0665-100MG X 2 units 6) T3 - (store as is -20, store aliquots at -80) T6397-100MG X 1 unit 7) L-carnitine - (store at RT as is) C0283-1 G X 1 unit 8) Ethanolamine - (store at RT as is) E9508-100ML X 1 unit 9) D+-galactose - (store at RT as is) G0625- 100G X 1 unit 10) Putrescine - (store at RT as is) P5780-5G X 1 unit 11) Sodium selenite - (store at RT as is, aliquots at -80) S9133-1 MG X 1 unit 12) Corticosterone - (store at RT as is, aliquots at -80) C2505-500MG X 1 unit 13) Linoleic acid - (store as is -20, store aliquots at -80) L1012- 100MG X 1 unit 14) Linolenic acid - (store as is -20, store aliquots at -80) L2376-500MG X 1 unit 15) Progesterone - (store at RT, store aliquots at -80) P8783-1 G X 1 unit 16) Retinol acetate - (store at RT, store aliquots at -80) R7882-1 G X 1 unit 17) DL-alpha tocopherol (vit E) (store @+4 as is, store aliquots at -80) T3251-5G X 1 unit 18) DL-alpha tocopherol acetate (store @RT as is, P28241 PCOO 28.08.2025

[0348] 52 / 119 store aliquots at -80) T3001-10G X 1 unit 19) Oleic acid - (store as is -20, store aliquots at -80) 01383-1 G X 1 unit 20) Pipecolic acid- (store as is -20, store aliquots at -80) P2519- 100MG X 1 unit 21) Biotin - (store at +4 as is) B4639-100MG X 1 unit.

[0349] In some embodiments, during at least the first 5 days of the third culture period, preferably during at least the first 8 days of the third culture period, the third culture medium comprises IDM-A medium.

[0350] IDM-A medium, as used herein, may for example be the IDM-A medium as described in Giandomenico et al, Nat Protoc. 2020 Dec 16; 16(2):579-602, which is incorporated by reference herein in its entirety. As a non-limiting example, in some embodiments, the IDM-A medium may for example be the “homemade” IDM-A medium as used in the examples. In some embodiments, the IDM-A medium may comprise or consist of 50% DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057), 50% Neurobasal (Thermo Fisher, 21103049), Penicillin / Streptomy- cin (Thermo Fisher, P0781), N2 supplement (Thermo Fisher, 17502048), B27 without vitamin A (Thermo Fisher, 12587010), Glutamax (Thermo Fisher, 35050061), MEM NEAA (Thermo Fisher, 11140035), Insulin (Millipore, I9278).

[0351] An illustrative, non-limiting example of a possible composition of an IDM-A medium that may e.g. be used in the processes of the present disclosure is provided in the following table: P28241 PCOO 28.08.2025

[0352] 53 / 119

[0353] Typically, the B27 in IDM-A medium is free of vitamin A. In some embodiments, the IDM-A medium is free of vitamin A.

[0354] It was found to be advantageous to supplement the third culture medium with an extracellular matrix gel, at least initially. In some embodiments, during at least the first 5 days of the third culture period, preferably during at least the first 8 days of the third culture period, the third culture medium is supplemented with an extracellular matrix gel, preferably Matrigel. For example, the third culture medium may be supplemented with the extracellular matrix gel at a concentration from 0.1 vol.-% to 5 vol.-% extracellular matrix gel, with respect to a total volume of the third culture medium.

[0355] In some embodiments, from no earlier than day 16 after seeding onwards, the IDM-A medium is replaced with IDM+A medium.

[0356] IDM+A medium, as used herein, is preferably the IDM+A medium as described in Giandomenico et al, Nat Protoc. 2020 Dec 16; 16(2):579-602, which is incorporated by reference herein in its entirety.

[0357] In some embodiments, IDM+A medium comprises or consists of a mixture of IDM-A medium and vitamin A. For example, IDM+A medium may be identical to IDM-A, with the only difference that IDM+A medium comprises B27 with vitamin A (Thermo Fisher, 17504044) instead of B27 without vitamin A (Thermo Fisher, 12587010), and that IDM+A medium comprises ascorbic acid and Na bicarbonate.

[0358] As a non-limiting example, in some embodiments, the IDM+A medium may for example be the “homemade” IDM+A medium as used in the examples. Thus, the IDM+A medium may comprise or consist of 50% DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057), 50% Neu- P28241 PCOO 28.08.2025

[0359] 54 / 119 robasal (Thermo Fisher, 21103049), Penicillin / Streptomycin (Thermo Fisher, P0781), N2 supplement (Thermo Fisher, 17502048), B27 with vitamin A (Thermo Fisher, 17504044), Glutamax (Thermo Fisher, 35050061), MEM NEAA (Thermo Fisher, 11140035), Insulin (Millipore, I9278).

[0360] An illustrative, non-limiting example of a possible composition of an IDM+A medium that may e.g. be used in the processes of the present disclosure is provided in the following table:

[0361] In some embodiments, during the third culture period, the second cell populations are cultured on a shaker. For example, the second cell populations may be cultured on an orbital shaker with a rotation orbit diameter of 25 mm and at a speed of 64 rpm. P28241 PCOO 28.08.2025

[0362] 55 / 119

[0363] Forebrain Organoids

[0364] In some embodiments, the process of the present disclosure is used to make forebrain organoids. Optimized conditions have been developed for generating forebrain organoids, which are described in this section.

[0365] In some embodiments of the process of the fourth aspect of the present disclosure, a process for generating forebrain organoids is provided. Said process comprises performing the process according to any one of the embodiments disclosed herein, in particular including but not limiting to the embodiment of the fourth aspect disclosed herein. Further, in step d), the second cell populations are treated with a TGF beta pathway inhibitor, a Wnt pathway inhibitor and extracellular matrix gel for a period of at least two days during the third culture period.

[0366] The TFG beta pathway inhibitor preferably comprises or consists of SB 431542.

[0367] As the skilled person knows, SB 431542 is known and commercially available. For example, it may be obtained from Tocris, 1614. Its chemical name is 4-[4-(1 ,3-benzodioxol-5-yl)-5-(2-pyridi- nyl)-1 / 7-imidazol-2-yl]benzamide.

[0368] The Wnt pathway inhibitor preferably comprises or consists of IWR1.

[0369] As the skilled person knows, IWR-1 is widely known and commercially available. For example, it may be obtained from Sigma-Aldrich, 681669. It has e.g. been described in Chen, B., et al. 2009. Nature Chem. Biol.5, 100.

[0370] The extracellular matrix gel comprises or consists of Matrigel or Cultrex.

[0371] In some embodiments, at least from day 8 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with SB 431542 and with the extracellular matrix gel. Preferably in the embodiments described in the previous sentence, the second cell populations are treated with SB 431542 at a concentration from 5 to 15 pM, and with the extracellular matrix gel (preferably Cultrex) at a concentration from 0.1 vol.-% to 5 vol.-% extracellular matrix gel.

[0372] In some embodiments, at least from day 8 after seeding until day 10 after seeding, the second cell populations are treated with IWR1 , preferably at a concentration from 1 pM to 5 pM IWR1.

[0373] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with IWR1 or CHIR99021 , P28241 PCOO 28.08.2025

[0374] 56 / 119 preferably at a concentration from 1 pM to 5 pM IWR1 respectively from 0.1 pM to 2 pM CHIR99021.

[0375] As the skilled person knows, CHIR99021 is known and commercially available. For example, it may be obtained from Millipore, 361571. Its chemical name is 6-(2-(4-(2,4-Dichlorophenyl)-5-(4- methyl-1 H-imidazol-2-yl)-pyrimidin-2-ylamino)ethyl-amino)-nicotinonitrile.

[0376] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the third culture medium comprises or consists of IDM-A medium supplemented with SB 431542, extracellular matrix gel, preferably Cultrex, and one of the following: IWR1 or CHIR99021.

[0377] In some embodiments, at least from day 8 after seeding until day 10 after seeding, the third culture medium comprises or consists of IDM-A medium supplemented with SB 431542, IWR1 and extracellular matrix gel, preferably Cultrex.

[0378] In preferred embodiments, at least from day 8 after seeding until day 14 after seeding, preferably until day 16 after seeding, the third culture medium is refreshed at least once every 3 days, preferably at least once every 2 days.

[0379] In some embodiments, at least from day 6 after seeding until day 8 after seeding, the first cell populations are treated with SB 431542, IWR1 and extracellular matrix gel, preferably at a concentration from 5 pM to 15 pM SB 431542, from 1 pM to 5 pM IWR1 and from 1 vol.-% to 10 vol. - % extracellular matrix gel, preferably Matrigel.

[0380] In some embodiments, from day 6 after seeding until day 8 after seeding, the first culture medium comprises or consists of N2 medium supplemented with SB 431542, IWR1 and extracellular matrix gel, preferably Matrigel.

[0381] In some embodiments, at least from day 16 after seeding until day 25 after seeding, preferably until day 30 after seeding, the second cell populations are cultured in IDM-A medium. Preferably, from day 16 after seeding until at least day 30 after seeding, the third culture medium is refreshed at least once every 3 days, preferably at least once every 2 days.

[0382] In some embodiments, at least from day 30 after seeding onwards, the second cell populations are cultured in IDM+A medium supplemented with an extracellular matrix gel, preferably Cultrex. For example, from day 30 after seeding onwards, the IDM+A medium may be supplemented with from 0.1 vol.-% to 2 vol.-% Cultrex with respect to a total volume of the third culture medium. P28241 PCOO 28.08.2025

[0383] 57 / 119

[0384] In some embodiments, the second culture period lasts until day 8 after seeding and the third culture period commences on day 8 after seeding.

[0385] Preferably, on day 8 after seeding, the second cell populations are transferred from the microwells to a cell culture plate or a cell culture dish.

[0386] In some embodiments, the third culture medium comprises from day 8 after seeding until day 10 after seeding: IDM-A medium supplemented with SB 431542, IWR1 and Cultrex; wherein on day 10 after seeding, the supplemented IDM-A medium is replaced by IDM-A medium supplemented with SB 431542, Cultrex and at least one or CHIR99021 and IWR1 , such that from day 10 until day 16 after seeding, the third culture medium comprises IDM-A medium supplemented with SB 431542, Cultrex and at least one or CHIR99021 and IWR1 ; wherein on day 16 after seeding, the supplemented IDM-A medium is replaced by IDM-A medium, such that from day 16 until day 30 after seeding, the third culture medium comprises IDM-A medium; wherein on day 30 after seeding, the IDM-A medium is replaced by IDM+A medium supplemented with Cultrex, such that from day 30 onwards, the third culture medium comprises IDM+A medium supplemented with Cultrex.

[0387] In some embodiments, the third culture period lasts for at least 30 days.

[0388] Midbrain Organoids

[0389] In some embodiments, the process of the present disclosure is used to make midbrain organoids. Optimized conditions have been developed for generating midbrain organoids, which are described in this section.

[0390] In some embodiments of the process of the fourth aspect of the present disclosure, a process for generating midbrain organoids is provided. Said process comprises performing the process according to any one of the embodiments disclosed herein, in particular including but not limiting to the embodiment of the fourth aspect disclosed herein. Further, in step d), the second cell populations are treated with a Wnt pathway activator, Sonic Hedgehog pathway activator, and a fibroblast growth factor for a period of at least four days during the third culture period.

[0391] Preferably, the Hedgehog pathway activator comprises or consists of SAG.

[0392] As the skilled person knows, SAG is a hedgehog pathway activator, widely known and commercially available. For example, it may be obtained from Stem Cell Technologies, 73412. Its chemical name is 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]- benzo[b]thiophene-2-carboxamide. P28241 PCOO 28.08.2025

[0393] 58 / 119

[0394] Preferably, the Wnt pathway activator comprises or consists of CHIR99021.

[0395] Preferably, the fibroblast growth factor comprises or consists of FGF8b. It may for example be obtained from Peprotech, 100-25.

[0396] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably from day 8 after seeding until day 16 after seeding, the second cell populations are treated with CHIR99021 , SAG and FGF8b, preferably at a concentration from 1 pM to 2 pM CHIR99021 , from 0.1 pM to 1.0 pM SAG, from 50 ng / ml to 150 ng / ml FGF8b.

[0397] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably from day 8 after seeding until day 16 after seeding, the third culture medium comprises or consists of IDM-A medium supplemented with CHIR99021 , SAG, FGF8b and an extracellular matrix gel, preferably Matrigel.

[0398] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably from day 8 after seeding until day 16 after seeding, the third culture medium comprises an extracellular matrix gel, preferably Matrigel, at a concentration from 0.1 vol.-% to 5 vol.-%.

[0399] In some embodiments, from day 8 after seeding until day 16 after seeding, the third culture medium was refreshed at least once every 3 days, preferably at least once every 2 days.

[0400] In some embodiments, on day 14 after seeding or later, preferably on day 16 after seeding, the third culture medium is switched to IDM+A and subsequently refreshed at least once every 4 days.

[0401] Hindbrain Organoids

[0402] In some embodiments, the process of the present disclosure is used to make hindbrain organoids. Optimized conditions have been developed for generating hindbrain organoids, which are described in this section.

[0403] In some embodiments of the process of the fourth aspect of the present disclosure, a process for generating hindbrain organoids is provided. Said process comprises performing the process according to any one of the embodiments disclosed herein, in particular including but not limiting to the embodiment of the fourth aspect disclosed herein. Further, in step d), the second cell populations are treated with insulin, FGF2 and EGF for a period of at least two days.

[0404] As the skilled person knows, FGF2 is commercially available. For example, it may be obtained from Peprotech, 100-18B. P28241 PCOO 28.08.2025

[0405] 59 / 119

[0406] As the skilled person knows, EGF is commercially available. For example, it may be obtained from R&D, 236-EG-200. Its Uniprot accession number is P01133.

[0407] In some embodiments, at least from day 6 after seeding until day 18 after seeding, preferably at least until day 22 after seeding, the first cell populations respectively the second cell populations are treated with SB431542, Dorsomorphin, CHIR99021 , insulin, FGF2 and EGF, preferably at a concentration from 5 pM to 15 pM SB431542, from 1.0 pM to 4.0 pM Dorsomorphin, from 0.5 pM to 2.5 pM CHIR99021 , from 4 pg / ml to 10 pg / ml insulin, from 15 ng / mL to 25 ng / mL FGF2 and from 15 ng / mL to 25 ng / mL EGF.

[0408] As the skilled person knows, Dorsomorphin is widely known and commercially available, e.g. from Sigma-Aldrich, P5499. Its SMILES string is C1CCN(CC1)CCOc2ccc(cc2)-c3cnc4c(cnn4c3)- c5ccncc5.

[0409] In some embodiments, from day 6 after seeding until day 8 after seeding, the first cell populations are further treated with an extracellular matrix gel, preferably Matrigel, at a concentration from 1 vol.-% to 10 vol.-%.

[0410] In some embodiments, from day 8 after seeding until day 14 after seeding, the second cell populations are treated with FGF8b, preferably at a concentration from 50 ng / ml to 150 ng / ml.

[0411] In some embodiments, from day 14 until at least day 20, preferably until day 22, after seeding, the second cell populations are treated with FGF19, preferably at a concentration from 50 ng / ml to 150 ng / ml.

[0412] In some embodiments, from day 6 after seeding until day 8 after seeding, the second culture medium comprises or consists of N2 medium supplemented with SB431542, Dorsomorphin, CHIR99021 , insulin, FGF2, EGF and extracellular matrix gel, preferably Matrigel.

[0413] In some embodiments, from day 8 after seeding until day 14 after seeding, the third culture medium comprises or consists of IDM-A supplemented with SB431542, Dorsomorphin, CHIR99021 , insulin, FGF2, EGF and FGF8b.

[0414] In some embodiments, from day 14 after seeding until day 20 after seeding, preferably until day 22 after seeding, the third culture medium comprises or consists of IDM-A supplemented with SB431542, Dorsomorphin, CHIR99021 , insulin, FGF2, EGF and FGF19. P28241 PCOO 28.08.2025

[0415] 60 / 119

[0416] In some embodiments, from day 8 after seeding until day 22 after seeding, the third culture medium was refreshed at least once every 3 days, preferably at least once every 2 days. For example, in some embodiments, at least from day 6 after seeding until day 18 after seeding, the first cell populations respectively the second cell populations are cultured in N2 medium supplemented with from 5 pM to 15 pM SB431542, from 1.0 pM to 4.0 pM Dorsomorphin, from 0.5 pM to 2.5 pM CHIR99021 , from 4 pg / ml to 10 pg / ml insulin, from 15 ng / mL to 25 ng / mL FGF2 and from 15 ng / mL to 25 ng / mL EGF. In some embodiments, from day 6 until day 8 after seeding, the N2 medium is further supplemented with from 1 vol.-% to 10 vol.-% extracellular matrix gel, preferably Matrigel. Alternatively or in combination, from day 8 until day 14 after seeding, the N2 medium is further supplemented with from 50 ng / ml to 150 ng / ml FGF8b. Alternatively or in combination, from day 14 until day 22 after seeding, the N2 medium is additionally supplemented with from 50 ng / ml to 150 ng / ml FGF19.

[0417] In some embodiments, at least from day 22 after seeding until day 28 after seeding, preferably at least until day 30 after seeding, the third culture medium comprises or consists of IDM-A. Thus, for example, on day 22, the medium may be switched to IDM-A.

[0418] In some embodiments, at least from day 32 after seeding until day 50 after seeding, the third culture medium may comprise or consist of IDM-A medium supplemented with SDF1a and T3, preferably at a concentration from 50 ng / ml to 150 ng / ml SDF1a and from 0.1 ng / ml to 1.0 ng / ml T3.

[0419] In some embodiments, at the latest on day 70 after seeding, the third culture medium comprises or consists of IDM-A medium.

[0420] In some embodiments, the third culture medium is replaced with fresh medium at least once every 3 days, preferably at least once every 2 days during the third culture period.

[0421] Retinal Organoids

[0422] In some embodiments, the process of the present disclosure is used to make retinal organoids. Optimized conditions have been developed for generating retinal organoids, which are described in this section.

[0423] In some embodiments of the process of the fourth aspect of the present disclosure, a process for generating retinal organoids is provided. Said process comprises performing the process according to any one of the embodiments disclosed herein, in particular including but not limiting to the embodiment of the fourth aspect disclosed herein. Further, in step d), the second cell populations P28241 PCOO 28.08.2025

[0424] 61 / 119 are treated with a BMP pathway inhibitor, a TGF-beta pathway inhibitor, and a Wnt pathway inhibitor for a period of at least four days during the third culture period, and afterwards with BMP4 for a period of at least six days during the third culture period.

[0425] As the skilled person knows, BMP4 is widely known and commercially available, e.g. from Pepro- tech, 120-05ET.

[0426] Preferably, the BMP pathway inhibitor comprises or consists of Dorsomorphin.

[0427] Preferably, the TGF-beta pathway inhibitor comprises or consists of SB 431542.

[0428] Preferably, the Wnt pathway inhibitor comprises or consists of IWR1.

[0429] In some embodiments, at least from day 6 after seeding until day 10 after seeding, preferably until day 12 after seeding, the first cell populations respectively the second cell populations are treated with Dorsomorphin, SB 431542 and IWR1 , preferably at a concentration from 5 pM to 15 pM SB 431542, from 2 pM to 4 pM IWR1 and from 1.0 pM to 4.0 pM Dorsomorphin.

[0430] In some embodiments, at least from day 6 after seeding until day 10 after seeding, preferably until day 12 after seeding, the first second culture medium respectively the third culture medium comprises or consists of N2 medium supplemented with SB 431542, IWR1 and Dorsomorphin.

[0431] In some embodiments, the second culture period lasts until day 8 after seeding and the third culture period commences on day 8 after seeding.

[0432] In some embodiments, from day 8 after seeding until day 24 after seeding, the third culture medium is refreshed at least once every 3 days, preferably at least once every 2 days.

[0433] In some embodiments, on day 11 or 12 or 13 after seeding, preferably on day 12 after seeding, the treatment with Dorsomorphin, SB 431542 and IWR1 is stopped and the treatment of the second cell populations with BMP4 commences.

[0434] In some embodiments, at least from day 12 after seeding until day 22 after seeding, preferably until day 24 after seeding, the second cell populations are treated with BMP4, preferably at a concentration from 25 ng / ml to 75 ng / ml.

[0435] In some embodiments, at least from day 12 until day 22 after seeding, preferably until day 24 after seeding, the third culture medium comprises or consists of IDM-A medium supplemented with BMP4. P28241 PCOO 28.08.2025

[0436] 62 / 119

[0437] In some embodiments, at least from day 50 after seeding until week 9 after seeding, preferably at least from day 40 after seeding until week 10 after seeding, the second cell populations are cultured in retina differentiation medium 1 (RDM1).

[0438] In some embodiments, RDM1 medium comprises or consists of: DM EM, high glucose, GlutaMAX, F-12 Nutrient Mixture (Ham), B27 without vitamin A, NEAA Solution, Fetal Bovine Serum (ES cell qualified, ES FBS), Sodium Pyruvate, Penicillin / Streptomycin, Taurine. As a non-limiting example, in some embodiments, the RDM1 medium may for example be the “homemade” RDM1 medium as used in the examples.

[0439] An illustrative, non-limiting example of a possible composition of an RDM1 medium that may e.g. be used in the processes of the present disclosure is provided in the following table:

[0440] As the skilled person knows, GlutaMAX is a dipeptide, L-alanyl-L-glutamine, which is more stable in aqueous solutions than L-glutamine, and which does not spontaneously degrade. P28241 PCOO 28.08.2025

[0441] 63 / 119

[0442] In some embodiments, at least from week 11 after seeding until week 13 after seeding, preferably from 10 after seeding until week 14 after seeding, the second cell populations are cultured in RDM2 medium.

[0443] In some embodiments, RDM2 medium comprises or consists of: DM EM, high glucose, GlutaMAX, F-12 Nutrient Mixture (Ham), B27 without vitamin A, NEAA Solution, Fetal Bovine Serum, ES cell qualified (ES FBS), Sodium Pyruvate, Penicillin / Streptomycin, Taurine, Retinoic acid. As a nonlimiting example, in some embodiments, the RDM2 medium may for example be the “homemade” RDM2 medium as used in the examples.

[0444] An illustrative, non-limiting example of a possible composition of an RDM2 medium that may e.g. be used in the processes of the present disclosure is provided in the following table:

[0445] In some embodiments, in week 13 or 14 or 15 after seeding, the second cell populations are transferred to separate wells of a well plate.

[0446] In some embodiments, at least from week 14 after seeding onwards, the second cell populations are cultured in RDM3. P28241 PCOO 28.08.2025

[0447] 64 / 119

[0448] In some embodiments, RDM3 medium comprises or consists of: DM EM, high glucose, GlutaMAX, F-12 Nutrient Mixture (Ham), N2 supplement, NEAA Solution, ES FBS, Sodium Pyruvate, Peni- cillin / Streptomycin, Albumax, Taurine, Retinoic acid. As a non-limiting example, in some embodiments, the RDM3 medium may for example be the “homemade” RDM3 medium as used in the examples.

[0449] An illustrative, non-limiting example of a possible composition of an RDM3 medium that may e.g. be used in the processes of the present disclosure is provided in the following table:

[0450] As the skilled person knows, Albumax is a lipid-rich bovine serum albumin for cell culture. As the skilled person knows, NEAA Solution is a MEM non-essential amino acids solution used as a growth supplement for cell culture. P28241 PCOO 28.08.2025

[0451] 65 / 119

[0452] Choroid Plexus Organoids

[0453] In some embodiments, the process of the present disclosure is used to make choroid plexus organoids. Optimized conditions have been developed for generating choroid plexus organoids, which are described in this section.

[0454] In some embodiments of the process of the fourth aspect of the present disclosure, a process for generating choroid plexus organoids is provided. Said process comprises performing the process according to any one of the embodiments disclosed herein, in particular including but not limiting to the embodiment of the fourth aspect disclosed herein. Further, in step d), the second cell populations are treated with CHIR99021 for a period of at least four days during the third culture period.

[0455] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with CHIR99021 , preferably at a concentration from 1 pM to 5 pM.

[0456] In some embodiments, at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with SB431542, preferably at a concentration from 1 pM to 5 pM.

[0457] In some embodiments, from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the third culture medium comprises or consists of IDM+A supplemented with CHIR99021 and SB431542.

[0458] In some embodiments, from day 10 until day 16 after seeding, the third culture medium is refreshed at least once every 3 days, preferably at least once every 2 days.

[0459] In some embodiments, on day 8 after seeding until day 10 after seeding, the first cell populations are cultured in IDM+A medium, preferably in a cell culture plate or a cell culture dish.

[0460] In some embodiments, from day 16 after seeding onwards, the second cell populations are cultured in IDM+A medium, preferably in the absence of SB431542 and CHIR99021.

[0461] In some embodiments, from day 16 after seeding onwards, the medium is replaced with fresh medium at least once every 4 days. P28241 PCOO 28.08.2025

[0462] 66 / 119

[0463] Stem Cells

[0464] Depending on the application, different stem cells may be used. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are multipotent stem cells. In some embodiments, the stem cells are mammalian stem cells, such as human stem cells. For example, in some embodiments the stem cells are mammalian pluripotent stem cells, preferably human or murine pluripotent stem cells.

[0465] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0466] In some embodiments, the pluripotent stem cells are cultured to confluence before step a). For example, the pluripotent stem cells may be cultured to confluence in mTeSR Plus medium before step a).

[0467] In some embodiments, the pluripotent stem cells are cultured to confluence before step a) on Geltrex-coated culture plates (Thermo Fischer, A1413201).

[0468] In some embodiments, the culturing to confluence of the pluripotent stem cells comprises culturing the pluripotent stem cells in mTeSR Plus medium supplemented with a ROCK inhibitor, preferably Y-27632 dihydrochloride, and subsequently culturing the pluripotent stem cells in mTeSR Plus medium without supplements.

[0469] In some embodiments, the pluripotent stem cells are dissociated into single cell suspension, preferably using Accutase (Corning, 25-058-CI), before seeding the pluripotent stem cells in step a).

[0470] In some embodiments, the pluripotent stem cells are dissociated before being seeded, preferably using Accutase.

[0471] Automation

[0472] The process of the present disclosure may in some embodiments be automated. The present disclosure relates in some embodiments to an automated process for produced a library of CNS organoids according to any one of the embodiments described herein.

[0473] In some embodiments, step a) comprises the sub-steps of: a.i) filling a first seed volume from a seed stock cell suspension into each of a plurality microwells (preferably at least 100 microwells, more preferably at least 1’000 microwells); P28241 PCOO 28.08.2025

[0474] 67 / 119 a.ii) determining for at least some of the microwells a density difference between a target density corresponding to the density of the previous embodiments and an initial density of the stem cells in the respective microwells after step a.i); and a.iii) if the density difference determined for a given microwell exceeds a density difference threshold, adjusting the cell density in the respective microwell such that it reaches the target density.

[0475] In some embodiments, in step a.ii), the density difference is measured for only a selection of microwells. The density difference may then be determined for the remaining, non-selected microwells e.g. by extrapolation.

[0476] It is understood that in the automated process, either some of the steps or all of the steps may be performed in an automated fashion. For example, in some embodiments, the filling of the first seed volume is performed manually.

[0477] In some embodiments, in step d), at least three different third differentiation agents are used for each of at least three different groups of second cell populations, such that the generated library of CNS organoids includes at least three different regional CNS organoids.

[0478] In a further aspect the present disclosure relates to a library of CNS organoids produced by any of the embodiments of the automated process described herein.

[0479] Epiblast cysts, neuroepithelial cysts and organoids

[0480] A fifth aspect of the present disclosure relates to epiblast cysts produced by the process of any of the embodiments of the process of the first aspect disclosed herein.

[0481] A sixth aspect of the present disclosure relates to further differentiated epiblast cysts (preferably neuroepithelial cysts) produced by any of the embodiments of the process of the second aspect or the third aspect (preferably for neuroepithelial cysts) disclosed herein. Preferably, in some embodiments, the further differentiated epiblast cysts are single lumen neuroepithelial cysts.

[0482] In some embodiments, the neuroepithelial cyst expresses at least three, preferably at least four, more preferably at least five, even more preferably at least six, markers selected from the group consisting of: MKI67, UBE2C, CDK1 , SOX2, SOX1 , SOX3, PAX6, LHX2, FZD5, NOTCH1 , NES, VIM, HES1 , FABP7, SOX10, OTX2, LHX5, SIX3, LHX2, HESX1 , FEZF1 , EMX2, RAX, NCAM1 , S100B, DCX, OCLN, CDH11 , CDH15, CDH2, CDH23, CDH3, CDH4, CTNNBL1 , CLDN10, P28241 PCOO 28.08.2025

[0483] 68 / 119

[0484] CLDN12, CLDN3, CLDN4, CLDN5, CLDN6, GJA3, GJC1 , JAM3, TJP1 , TJP2, TJP3, PCDH18, COLIV, ZO1 , Laminin. In some embodiments, the neuroepithelial cyst expresses at least three, preferably at least four, more preferably at least five, even more preferably at least six, markers selected from the group consisting of: SOX2, PAX6, NESTIN, VIM ENTIN, NCAD, ZO1 , COLIV, Laminin, OTX2, SIX3, FEZF1 , LHX2, HES1 , and KI67.

[0485] A seventh aspect of the present disclosure relates to organoids produced by any of the embodiments of the process of the fourth aspect disclosed herein. Preferably, in some embodiments, the organoids are CNS organoids, such as brain organoids.

[0486] In some embodiments, the organoids produced are forebrain organoids. In some embodiments, the forebrain organoids express at least three, preferably at least four, more preferably at least five, even more preferably at least six, markers selected from the group consisting of: MKI67, SATB2, INHBA, FRMD4B, BCL11 B, CRYM, TLE4, GAD2, DLX2, HOPX, TNC, LGALS3, GFAP, VIM, PAX6, ASPM, NR2F1 , FOXP2, RORB, ASCL1 , EOMES, PPP1 R17, TMEM158, STMN2, MAPT, LRRC7, MYT1 L, ANKS1 B, NRXN1 , MAP2, NFIB, NFIA, DCX, PDE4D, NEUROD6, GLIS3, GLI3, TUBB2B, NEUROD2, MEF2C, NECAB1 , POU3F2, TBR1 , FOXG1 , SLC17A7, SOX2, CALR. In some embodiments, the forebrain organoids express at least three, preferably at least four, more preferably at least five, markers selected from the group consisting of: SOX2, MAP2, FOXG1 , CALR, KI67, CTIP2 (BCL11 B), and TBR2 (EOMES).

[0487] In some embodiments, the organoids produced are retina organoids. In some embodiments, the retina organoids express at least three, preferably at least four, more preferably at least five, even more preferably at least six, markers selected from the group consisting of: PAX6, KI67, SOX2, CRX, OTX2, GNGT1 , RHO, NRL, ARR3, OPN1 LW, GNAT2, LHX1 , ONECUT1 , PRKCA, GRIK1, PCP2, FILIP1 L, GJD2, TFAP2A, GAD1 , SLC17A6, RBPMS, RPE65, APOE, PRDM1 , RXRG, RLBP1 , MITF, RCVRN, DCX, NRXN3, RP1 , DCLK1 , NFIB, PRPH2, PDC, SLC24A2, AIPL1 , IMPG2, CRYBG3, MYO3B, NR2E3, PDE6H, ROM1 , UNC119, USH2A, HES1 , ROM1 , EPHA7, ROBO2. In some embodiments, the retina organoids express at least three, preferably at least four, markers selected from the group consisting of: PAX6, KI67, SOX2, CRX and OTX2.

[0488] In some embodiments, the organoids produced are choroid plexus organoids. In some embodiments, the choroid plexus organoids express at least three, preferably at least four, more preferably at least five, even more preferably at least six, markers selected from the group consisting of: TTR, OTX2, SULF1 , HTR2C, CLIC6, AQP1, MSX1 , PLTP, LMX1A, ITGB5, KRT18, FOLR1 , IGFBP7, PRLP, BMP7, BMP4, TGFBI, VEGFB, KCNJ13, SLC4A10, SLC40A1 , SLC31A1 , LC4A5, SLC2A3, SLC39A12, SLC12A2, SLC16A10, SLC2A1 , SLC6A20, SLC5A6, SLC41A1 , P28241 PCOO 28.08.2025

[0489] 69 / 119

[0490] ABCA4, MEIG1 , F0XJ1 , DYNLRB2, PIFO, SHISA8, CCDC67, CCNO, IGF1 , IGFBP2, OGN, SPARC, FBN1 , BGN, CLU, SFRP1 , APOE, PSAP, AEBP1 , CST3, ENPP2, ACE, GPC3, NID2, APP, B2M, CDH11 , CDH15, CDH2, CDH23, CDH3, CDH7, CTNNBL1 , CLDN1 , CLDN2, CLDN10, CLDN11 , CLDN12, CLDN18, CLDN19, CLDN23, CLDN3, CLDN4, CLDN5, GJC1 , JAM3, TJP2, TJP3, PCDH18, LAMA3, LAMA4, OCLN. In some embodiments, the choroid plexus organoids express at least one, preferably at least two, markers selected from the group consisting of: PODXL1 , TTR, AQP1 and OTX2.

[0491] In some embodiments, the choroid plexus organoids have apical-out polarity. For example, an apical surface of at least a portion of the organoids (e.g. at least 50% of all viable organoids, preferably at least 75% of all viable organoids, e.g. at least 90% of all viable organoids) is faced away from a core of the respective core thereof.

[0492] As used herein, “apical-out” refers to an organoid wherein the apical surface of the organoid is in direct contact with the external environment (i.e. a cell culture medium). For example, the organoids may comprise a lumen, and a basolateral side thereof may be in direct contact with the lumen.

[0493] In some embodiments, the choroid plexus organoids have a cell density of at least 2500 cells / mm2, preferably from 3000 cells / mm2to 8000 cells / mm2, more preferably from 4000 cells / mm2to 6000 cells / mm2.

[0494] The present disclosure (hereinbefore and hereinafter) is discussed in the context of different aspects and embodiments to facilitate understanding of the disclosure. However, the present document is to be understood as a unified disclosure. In particular, although some embodiments are discussed in the context of a particular aspect, they are nevertheless to be understood generally as embodiments of the present disclosure and, as such, generally also extend to and apply to other aspects of the present disclosure, unless it is clearly specified otherwise or unless the context dictates otherwise. For example, embodiments discussed in the context of the process of the first aspect are also embodiments of the fifth aspect (among other aspects), unless it is clearly specified otherwise or unless the context dictates otherwise. Similarly, embodiments discussed in the context of the process of the second or third aspect are also embodiments of the sixth aspect (among other aspects), and embodiments discussed in the context of the process of the fourth aspect are also embodiments of the seventh aspect (among other aspects), unless it is clearly specified otherwise or unless the context dictates otherwise. P28241 PCOO 28.08.2025

[0495] 70 / 119

[0496] Kits

[0497] An eighth aspect of the present disclosure relates to kits. For example, the kits may be kits for producing single lumen epiblast cysts. The kits may also be kits for producing neuroepithelial cysts. The kits may also be kits for producing organoids, such as CNS organoids, preferably brain organoids.

[0498] In some embodiments, the kit comprises one or more components useful for performing the process of any one of the embodiments disclosed herein. For example, a kit for producing organoids may comprise one or more components useful for performing the process of any one of the embodiments of the process of the fourth aspect of the present disclosure.

[0499] In some embodiments, the kit comprises one or more of the following components:

[0500] - at least one micropatterned substrate, preferably comprising a plurality of microwells each having a non-adherent inner surface;

[0501] - the first culture medium, optionally comprising one or more differentiation agents (such as one or more of the differentiation agents disclosed herein);

[0502] - the second culture medium, optionally comprising one or more differentiation agents (such as one or more of the differentiation agents disclosed herein);

[0503] - the third culture medium, optionally comprising one or more differentiation agents (such as one or more of the differentiation agents disclosed herein);

[0504] - one or more morphogens (such as one or more of the morphogens disclosed herein);

[0505] - one or more markers, preferably a pluripotency marker and / or a CNS marker, such as a neuroepithelial marker;

[0506] - an extracellular matrix gel, preferably matrigel, RTM, laminin, cultrex, hyaluronic acid, or a mixture thereof.

[0507] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein;

[0508] - stem cells;

[0509] - a seeding device for facilitating seeding the stem cells into the microwells at a density of less than 500 cells per microwell, preferably of less than 100 cells per microwell, more preferably of less than 20 cells per microwell, more preferably of 15 or less cells per microwell, even more preferably of from 2 to 15 cells per microwells, even more preferably of from 2 to 14 cells per microwells; P28241 PCOO 28.08.2025

[0510] 71 / 119

[0511] - a third culture location, preferably a bottom plate, a bioreactor, or a culture vessel, such as a cell culture plate or a cell culture dish.

[0512] Components of the kit may in some embodiments include micropatterned substrates. For example, components of the kit may include a multiwall plate comprising a plurality of microwells.

[0513] In some embodiments, the kit comprises a manual containing instructions how to perform the method of any of the embodiments described herein.

[0514] The present disclosure further provides a kit for producing neuroepithelial cysts and / or organoids, particularly brain organoids, which kit comprises N2 medium and an extracellular matrix gel (preferably Matrigel). Preferably, the kit further comprises one or more of the following:

[0515] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein, in particular in the context of the third aspect of the disclosure;

[0516] - IDM-A medium

[0517] - IDM+A medium;

[0518] - a microwell plate.

[0519] The present disclosure further provides a kit for producing forebrain organoids, which kit comprises N2 medium, an extracellular matrix gel (preferably Matrigel), SB431542 and IWR1. Preferably, the kit further comprises one or more of the following:

[0520] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein for producing forebrain organoids, in particular in the context of the third aspect of the disclosure;

[0521] - Cultrex

[0522] - CHIR99021

[0523] - IDM-A medium

[0524] - IDM+A medium;

[0525] - a microwell plate.

[0526] The present disclosure further provides a kit for producing midbrain organoids, which kit comprises N2 medium, an extracellular matrix gel (preferably Matrigel), CHIR99021 , SAG, FGF8b. Preferably, the kit further comprises one or more of the following: P28241 PCOO 28.08.2025

[0527] 72 / 119

[0528] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein for producing midbrain organoids, in particular in the context of the third aspect of the disclosure;

[0529] - I DM-A medium;

[0530] - I DM+A medium;

[0531] - a microwell plate.

[0532] The present disclosure further provides a kit for producing hindbrain organoids, which kit comprises N2 medium, an extracellular matrix gel (preferably Matrigel), SB431542, Dorsomorphin, CHIR, insulin, FGF2, EGF, FGF8b, FGF19. Preferably, the kit further comprises one or more of the following:

[0533] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein for producing hindbrain organoids, in particular in the context of the third aspect of the disclosure;

[0534] - I DM-A;

[0535] - SDF1a;

[0536] - T3;

[0537] - a microwell plate.

[0538] The present disclosure further provides a kit for producing retinal organoids, which kit comprises N2 medium, an extracellular matrix gel (preferably Matrigel), Dorsomorphin, SB431542, IWR1 , BMP4. Preferably, the kit further comprises one or more of the following:

[0539] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein for producing retinal organoids, in particular in the context of the third aspect of the disclosure;

[0540] - IDM-A medium

[0541] - RDM1 medium

[0542] - RDM2 medium;

[0543] - RDM3 medium;

[0544] - a microwell plate. P28241 PCOO 28.08.2025

[0545] 73 / 119

[0546] The present disclosure further provides a kit for producing choroid plexus organoids, which kit comprises N2 medium, an extracellular matrix gel (preferably Matrigel), CHIR99021. Preferably, the kit further comprises one or more of the following:

[0547] - a manual containing instructions how to perform the method of any one of the embodiments or aspects disclosed herein for producing choroid plexus organoids, in particular in the context of the third aspect of the disclosure;

[0548] - I DM+A medium;

[0549] - SB431542;

[0550] - a microwell plate.

[0551] Uses

[0552] The epiblast cysts described herein or further differentiated forms thereof (such as further differentiated epiblasts cysts such as neuroepithelial cysts, or organoids, such as CNS organoids) may be used in different applications. For example, they may be used in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets, and cell therapy.

[0553] In some embodiments, in these applications, iPS cells are used in the process of the present disclosure, which iPS cells derive from a particular subject. For example, the present disclosure may be used to identify genetic factors and epigenetic influences in said subject that contribute to variable effects of a known or unknown drug on CNS development or CNS differentiation. Subject-specific somatic cells for reprogramming into induced pluripotent stem cells can be obtained or isolated from a target tissue of interest by biopsy or other tissue sampling methods. Such applications may e.g. be used for patient-specific treatments and for producing personalized drugs.

[0554] Further provided herein is the use of the neuroepithelial cysts produced by the process according to any one of the embodiments disclosed herein, for generating organoids (in particular CNS organoids, such as brain organoids) by further differentiation of the neuroepithelial cysts. For example, the neuroepithelial cysts may be produced by the process according to any one of the embodiments disclosed herein in the context of the second or third aspect of the disclosure.

[0555] A further aspect of the present disclosure provides a method comprising the steps of: generating a plurality of organoids according to any one of the embodiments disclosed herein (including but not limited to any of the embodiments disclosed herein in the context of the fourth aspect of the P28241 PCOO 28.08.2025

[0556] 74 / 119 present disclosure); and treating at least some of the generated organoids of the plurality of organoids with a substance, such as a biologically active substance. The substance may for example be selected from one or more of the following:

[0557] - One or more substances to be tested (e.g. drug candidates);

[0558] - One or more substances inducing a condition of interest or a disease of interest (e.g. a condition to be studied or modelled, or a disease to be studied or modelled);

[0559] - One or more active pharmaceutical ingredients (e.g. active pharmaceutical ingredients to be tested or screened or modelled);

[0560] - One or more pharmaceutical compositions, which may optionally comprise an active pharmaceutical ingredient.

[0561] In some embodiments, the plurality of organoids is treated with a library of substances, e.g. in a high content screening application.

[0562] Further provided herein is a method of preparing a cellular product, a graft or an implant, the method comprising the steps of generating a plurality of organoids according to any one of the embodiments disclosed herein (including but not limited to any of the embodiments disclosed herein in the context of the fourth aspect of the present disclosure); and combining at least some of the generated organoids of the plurality of organoids to create the cellular product. Preferably, the at least some organoids are further combined with a cellular product formulation to create the cellular product.

[0563] Examples

[0564] In the following, non-limiting examples of the present disclosure are provided:

[0565] A1. Example relates to a process of producing single lumen epiblast cysts, comprising: a) seeding stem cells in microwells at a density of less than 500 cells per microwell; b) culturing the seeded cells of a) in the respective microwells for a first culture period in the presence of a first culture medium to obtain in each microwell a first cell population forming an epiblast cyst.

[0566] A2. Example relates to a process according to example 1 , wherein in step a) the stem cells are seeded in the microwells at a density of less than 100, preferably from 1 to 20, more preferably from 1 to 15, even more preferably from 1 to 14, cells per microwell P28241 PCOO 28.08.2025

[0567] 75 / 119

[0568] A3. Example relates to the process according to any one of the previous examples, wherein the first culture medium is supplemented with an extracellular matrix gel, preferably in a non-solid state, after aggregation of the stem cells in the respective microwells.

[0569] A4. Example relates to the process according to example 3, wherein the first culture medium is supplemented with the extracellular matrix gel at a time point falling in the time period ranging from 1 hour after seeding to day 8 after seeding, preferably on day 4 after seeding.

[0570] A5. Example relates to the process according to example 3 or 4, wherein the first culture medium is supplemented with the extracellular matrix gel in an amount of at least 1 vol.-% with respect to the total volume of the first culture medium.

[0571] A6. Example relates to the process according to any one of the previous examples, wherein the first culture period lasts from 1 to 4 days, preferably from 1 to 3 days, more preferably for 2 days, after seeding.

[0572] A7. Example relates to the process according to any of the previous examples, wherein during step b), each microwell is filled with a volume of the first culture medium ranging from 1 pL to 400 pL, preferably from 5 pL to 150 pL, more preferably from 10 pL to 100 pL.

[0573] A8. Example relates to the process according to any one of the previous examples, wherein each microwell has a non-adherent inner surface.

[0574] A9. Example relates to the process according to any one of the previous examples, wherein each microwell comprises a hydrogel, preferably a polyethylene glycol (PEG) hydrogel, and / or wherein each microwell comprises a modified plastic that imparts non-adherent properties to the microwell and / or wherein at least a section of the inner surface of each microwell has a non-adherent coating.

[0575] A10. Example relates to the process according to any one of the previous examples, wherein the microwells each have a microwell volume of less than 1’000 pL, preferably from 5 pL to 500 pL, more preferably from 20 pL to 300 pL.

[0576] A11. Example relates to the process according to any one of the previous examples, wherein each microwell has a diameter from 10 pm to 4’000 pm, preferably 100 pm to 2’000 pm, more preferably from 300 pm to 500 pm.

[0577] A12. Example relates to the process according to any one of the previous examples, wherein a sidewall of each microwell is inclined such that a cross-sectional open area of the respective P28241 PCOO 28.08.2025

[0578] 76 / 119 microwell being at least partially delimited by the inclined sidewall decreases towards a bottom of the respective microwell.

[0579] A13. Example relates to the process according to any one of the previous examples, wherein in each microwell, a transition from the sidewall to the base of the respective microwell is at least partially, preferably fully, rounded.

[0580] A14. Example relates to the process according to any one of the previous examples, wherein each microwell is U-shaped or V-shaped in a cross-section parallel to the vertical direction.

[0581] A15. Example relates to the process according to any one of the previous examples, wherein a ratio between a depth and a diameter of each microwell ranges from 3:1 to 0.8: 1.0, preferably from 1.5: 1.0 to 1.0: 1.0.

[0582] A16. Example relates to the process according to any of the previous examples, wherein in step a), the stem cells are seeded in the microwells such that each microwell contains less than 100 cells per pL of microwell volume, preferably less than 15 cells per pL of microwell volume, more preferably less than 10 cells per pL of microwell volume, even more preferably less than 5 cells per pL of microwell volume, even more preferably less than 3 cells per pL of microwell volume, even more preferably less than 2 cells per pL of microwell volume.

[0583] A17. Example relates to the process according to any one of the previous examples, wherein the stem cells are pluripotent stem cells.

[0584] A18. Example relates to the process according to example 17, wherein the pluripotent stem cells are mammalian, preferably human or murine pluripotent stem cells.

[0585] A19. Example relates to the process according to example 17 or 18, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[0586] A20. Example relates to the process according to any one of the previous examples, wherein the density of cells per microwell, the first culture period and the first culture medium are chosen such that at least 30%, preferably at least 40%, more preferably at least 50%, of all viable epiblast cysts obtained in step b) have a single lumen.

[0587] A21. Example relates to the process of producing further differentiated epiblast cysts, comprising producing single lumen epiblast cysts according to the process of any one of the previous examples, and further comprising the step of: P28241 PCOO 28.08.2025

[0588] 77 / 119 c) culturing the first cell populations forming epiblast cysts obtained in step b) for a second culture period in the presence of a second culture medium comprising a second differentiation agent to obtain a plurality of second cell populations each comprising further differentiated epiblast cysts, preferably neuroepithelial cysts.

[0589] A22. Example relates to the process according to example 21 , wherein the second differentiation agent comprises or consists of a neuroepithelial differentiation agent, such that the plurality of second cell populations each form a neuroepithelial cyst.

[0590] A23. Example relates to the process according to example 21 or 22, wherein on day 10 after seeding, the neuroepithelial cysts express one or more of the neuroepithelial markers selected from the group consisting of: PAX6, NES, VIM and N cadherin.

[0591] A24. Example relates to the process according to any one of examples 21-23, wherein on day 10 after seeding, the neuroepithelial cysts express one or more of the anterior neuroepithelial markers selected from the group consisting of: OTX2, LHX5, SIX3, LHX2, HESX1 , FEZF1 and RAX, preferably selected from the group consisting of: SIX3, OTX2 and LHX2.

[0592] A25. Example relates to the process according to any one of examples 21-24, wherein the epiblast cysts and / or the further differentiated epiblast cysts are patternable to at least three different CNS regions, preferably to at least five different CNS regions, through treatment with an appropriate morphogen.

[0593] A26. Example relates to the process of producing a plurality of organoids, comprising producing further differentiated epiblast cysts according to the process of any one of examples 21-25, and further comprising the step of: d) culturing the second cell populations in a third culture medium comprising a third differentiation agent to obtain the plurality of organoids.

[0594] A27. Example relates to the process according to example 26, wherein the second differentiation agent and / or the third differentiation agent comprises or consists of a central nervous system differentiation agent, such that the organoids obtained are central nervous system organoids, preferably brain organoids.

[0595] A28. Example relates to the process according to example 27, wherein the CNS organoid is a target regional CNS organoid selected from one of the following: forebrain, midbrain, hindbrain or spinal cord organoid, preferably cortex, choroid plexus or retina organoid. P28241 PCOO 28.08.2025

[0596] 78 / 119

[0597] A29. Example relates to the process for generating forebrain organoids, comprising performing the process according to any one of examples 26-28, wherein in step d), the second cell populations are treated with a BMP pathway inhibitor, a SMAD signaling inhibitor and / or a Wnt pathway inhibitor, preferably with Dorsomorphin and / or SB-431542.

[0598] A30. Example relates to the process for generating a choroid plexus organoid, comprising performing the process according to any one of examples 26-28, wherein in step d), the second cell populations are treated with pulsed delivery of a Wnt agonist, preferably CHIR-99021 , for a period of 1 to 10 days, preferably 2 to 8 days, most preferably 3 to 6 days, followed by culture in a neuronal growth medium.

[0599] A31. Example relates to the process for generating a retinal organoid, comprising performing the process according to any one of examples 26-28, wherein in step d), matrigel is removed from the second cell populations, followed by long term maturation culture.

[0600] A32. Example relates to the process according to any one of examples 21-31 , wherein at least one of steps b), c) and d) comprises a morphogen treatment.

[0601] A33. Example relates to the Automated process for producing a library of CNS organoids according to any one of the previous examples, wherein step a) comprises the sub-steps of: a.i) filling a first seed volume from a seed stock cell suspension into each of a plurality of at least 100 microwells; a.ii) determining for at least some of the microwells a density difference between a target density corresponding to the density of the previous examples and an initial density of the stem cells in the respective microwells after step a.i); and a.iii) if the density difference determined for a given microwell exceeds a density difference threshold, adjusting the cell density in the respective microwell such that it reaches the target density.

[0602] A34. Example relates to the automated process according to example 33, wherein in step d), at least three different third differentiation agents are used for each of at least three different groups of second cell populations, such that the generated library of CNS organoids includes at least three different regional CNS organoids.

[0603] A35. Example relates to the epiblast cysts produced by the process of any one of examples 1-20. P28241 PCOO 28.08.2025

[0604] 79 / 119

[0605] A36. Example relates to the further differentiated epiblast cysts, preferably neuroepithelial cysts, produced by the process of any one of examples 21-25.

[0606] A37. Example relates to the organoids, preferably CNS organoids, produced by the process of any one of examples 26-32.

[0607] A38. Example relates to the use of the epiblast cysts of example 35 in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets, and cell therapy.

[0608] A39. Example relates to the use of the further differentiated epiblast cysts of example 36 in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets and cell therapy.

[0609] A40. Example relates to the use of the organoids of example 37 in one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets and cell therapy.

[0610] A41. Example relates to the kit comprising one or more components useful for performing the process of any one of examples 1-34.

[0611] A42. Example relates to the kit according to example 41 , wherein the kit comprises one or more of the following components: a. at least one micropatterned substrate, preferably comprising a plurality of microwells each having a non-adherent inner surface; b. the first culture medium, optionally comprising one or more differentia-tion agents; c. the second culture medium, optionally comprising one or more differen-tiation agents; d. the third culture medium, optionally comprising one or more differentia-tion agents; e. one or more morphogens; f. one or more markers, preferably a pluripotency marker and / or a CNS marker, such as a neuroepithelial marker; g. an extracellular matrix gel, preferably matrigel, RTM, laminin, cultrex, hyaluronic acid, or a mixture thereof. P28241 PCOO 28.08.2025

[0612] 80 / 119 h. a manual containing instructions how to perform the method of any one of examples 1 -34; i. stem cells; j. a seeding device for facilitating seeding the stem cells into the microwells at a density of less than 500 cells per microwell; k. a third culture location, preferably a bottom plate, a bioreactor, or a culture vessel, such as a cell culture plate or a cell culture dish.

[0613] EXPERIMENTAL EXAMPLES

[0614] Introduction

[0615] The following examples describe a refined and scalable method for generating neuroepithelial cysts (NECs) which can be sustained long-term to support differentiation into diverse neural tissues. Microwell technology was utilized to aggregate hPSCs in a high-throughput manner. These hPSCs spontaneously self-organize into apico-basally polarized neuroepithelium, forming cysts with a single lumen. These NECs intrinsically exhibit an anterior neural fate and demonstrate characteristic morphogenetic and fate changes in response to specific morphogen treatments. By initiating neural organoid generation directly from these NECs, the approach described herein advances the scalability and cytoarchitecture of neural organoids, enabling the recapitulation of the entire anterior-posterior axis of the developing brain. This includes the efficient generation of forebrain, midbrain, hindbrain, and neural retina structures. For example, the method allows for the efficient generation of retinal organoids directly from NECs, eliminating the need for manual scraping or excision, which greatly enhances throughput and scalability. Furthermore, all regionally specified organoids are derived using a common basal medium and culture condition, substantially streamlining the overall organoid culture workflow. Specifically, the cysts can be grown into regional neural organoids representing the forebrain, midbrain, hindbrain, and neural retina. In conclusion, an improved method to generate neural organoids is described, which improves the scalability and initial structural fidelity of these models, thereby enabling more accurate recapitulation of human brain development.

[0616] Example 1 : Materials and Methods iPSC culture P28241 PCOO 28.08.2025

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[0618] WTC (GM25256, male, wildtype) human iPSC, PGP1 (GM23338, male, wildtype) human iPSC and H9 (WA09, male, wildtype) human ESC lines were routinely cultured on Matrigel (Corning, 354263) or Geltrex (Thermo Fisher, A1413201) coated culture plates in mTeSR plus (Stemcell Technologies, 100-0276) at 37 °C and normoxia. Upon confluency, cultures were dissociated into single cells using Accutase (Corning, 25-058-CI). Cells were seeded on Matrigel / Geltrex coated culture plates in mTeSR plus supplemented with 10pM Y-27632 dihydrochloride (Tocris, 1254) at a density of 20, 000-25, 000 / cm2 for routine maintenance of iPSC and ESC lines. On the following day, medium was replaced with mTeSR plus only and medium was changed every day. Cultures became confluent every fourth day and were passaged as described above. For freezing, cultures were dissociated into single cells with Accutase and frozen in Stem cell banker Medium (AMSbio, 11924).

[0619] Differentiation of iPSCs into si lumen iPSCs / ESC cultures were dissociated into single cell suspension using Accutase. For generating epiblast cysts, cells were plated into different microwell plate types including PEG based microwell plates (Sunbiosciences, Gri3D®96) Eplasia (Corning, 4442) and Aggrewell (Stem cell technologies, 34415). For a typical experiment, cells were seeded in a 400 - 1000 pm microwell Sunbio Grid3D plate, at a density of 10-15 cells per microwell in mTeSR plus supplemented with 1 :1000 CEPT cocktail [Chen et al. Nature Methods 2021, 18, 528-541] [Polyamine Sigma P8483, Chro- man1 Medchem Express HY-15392, Emricasan Selleckchem S7775, trans-ISRIB Thermo Fisher scientific 5284], On day 2, medium was replaced with a N2-based medium [N2 medium - DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057 + Penicillin / Streptomycin (Thermo Fisher, P0781) + 1x N2 supplement (Thermo Fisher, 17502048) + 1x MEM NEAA (Thermo Fisher, 11140035), 1ug / ml Heparin (Sigma-Aldrich, H3149-25KU]. On day 4, medium was replaced by N2 medium supplemented with 5% Matrigel. Organoids were kept in the microwell plate until they reached the size of the microwell (usually until day 10) in N2 medium + 5% Matrigel, with medium changes every other day.

[0620] For further culture of the organoids they were moved to ultra low attachment 6 well plate (Corning, 3471) in N2B27 based medium [N2B27 medium - 50% DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057 + 50% Neurobasal (Thermo Fisher, 21103049) + Penicillin / Streptomycin (Thermo Fisher, P0781) + 0.5x N2 supplement (Thermo Fisher, 17502048) + 1x B27 (Thermo Fisher, 17504044) + Ix Glutamax (Thermo Fisher, 35050061) + 1x MEM NEAA (Thermo Fisher, 11140035) + 2.5ug / ml Insulin (Millipore, I9278)] and 1% matrigel. For long term culture, P28241 PCOO 28.08.2025

[0621] 82 / 119 the organoids were moved to 10cm dishes on an orbital shaker (100 rpm) in N2B27 medium containing B27 with vitamin A (Thermo Fisher, 17504044). Medium was replaced every third day. differentiation into classical brain

[0622] Forebrain organoids:

[0623] Neuroepithelial cysts were treated with 10 pM SB 431542 (Tocris, 1614) and 3uM IWR1 (Sigma- Aldrich, 681669) from day 8 to day 20. Around 500 single-lumen organoids are transferred to a low attachment 10 cm dish on day 10 and placed on an orbital shaker (64 rpm). From that day, the medium is replaced every other day with 10mL IDM-A medium with Matrigel. On day 30, medium was changed to IDM+A and kept long-term in static condition.

[0624] Retinal organoids:

[0625] Neuroepithelial cysts were treated with dual SMAD treatment (2.5 pM Dorsomorphin, (Sigma- Aldrich, P5499) and 10 pM SB 431542 (Tocris, 1614) from day 6 to day 12. Around 500 singlelumen organoids are transferred to a 6 cm dish on day 10 and placed on a small orbital shaker (100 rpm). From that day, the medium is replaced every other day with 5m L IDM-A medium without Matrigel and supplemented until day 12 with 2.5 pM Do and 10 pM SB. On day 45, organoids showing retinal morphology (i.e. shiny multilayered epithelium) are transferred to an ultra-low attachment 10 cm dish with retinal differentiation media and kept long-term in static condition.

[0626] Choroid epithelium organoids:

[0627] Neuroepithelial cysts were treated with CHIR99021 (Tocris, 4423) on day 13 to day 16 of differentiation. Around 150 single-lumen organoids are transferred to a 10 cm low attachment dish on day 10 and placed on a orbital shaker (64 rpm). From that day, the medium is replaced every third day with 10mL IDM-A medium without Matrigel.

[0628] Cyst culture in different plate formats

[0629] Single lumen neuroepithelial cysts were generated using various commercially available microwell plates. These included Elplasia plates (Corning, 4441), Aggrewell plates (Stemcell Technologies, 34411 and 34415) and PEG microwell plates (Sunbiosciences, Gri3D®96) with microwell diameters of 300 pm, 500 pm, 800 pm, 1000 pm, and 1500 pm, as well as 96-well II- bottom plates coated with polyethylene glycol (PEG) (Sunbiosciences) and ultra-low attachment (ULA) 96-well Il-bottom plates (SBio, MS-9096UZ). For each plate format, the cell suspension P28241 PCOO 28.08.2025

[0630] 83 / 119 density was adjusted to ensure a seeding density of 12 cells per microwell. Culture plates were handled according to the respective manufacturer's instructions. Subsequently, all cultures underwent the standard neuroepithelial cyst differentiation protocol of the present disclosure as previously described adjusted for medium volume as per manufacturer's instructions. The cysts were live imaged on day 6 and day 8 on Nikon Eclipse Ti inverted microscope.

[0631] Whole mount staining:

[0632] Organoids were collected from microwell plates in microfuge tubes, washed with 1X Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) for 2 hours at 4C. Samples were washed with PBS and stored in PBS supplemented with 0.2% Sodium Azide (Sigma- Aldrich, 71290) until used for immunostaining. Fixed samples were incubated in blocking solution [PBS supplemented with 3% donkey serum (Jackson ImmunoResearch, 017-000-121) and 0.1% Triton-X (Millipore Sigma, T8787)] for 1 hour at room temperature in microfuge tubes on a shaker. Samples were then incubated with primary antibodies diluted in blocking solution at 4°C overnight on a shaker. Next day, samples were washed 3 times with PBS for 5 minutes. Samples were then incubated with secondary antibodies and Hoechst 33342 (ThermoFisher Scientific, H3570) diluted in blocking solution at 4°C overnight on a shaker followed by 3 washes with PBS for 5 minutes each. Samples were stored in PBS until imaged. For clearing, PBS was removed and samples were submerged in Cubic mount [Lee et al. Scientific Reports 2016, 6, Article number: 18631] [-250g sucrose 50%w / v Sucrose, 25%w / v Urea, 25%w / v N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine] overnight at RT. Samples were stored at RT until imaged. Samples were transferred to glass bottom 96-well plates (Cellvis, P96-1.5H-N) and imaged using Nikon Tie2 microscope.

[0633] Sections - first protocol:

[0634] Samples were fixed as mentioned above and embedded into histogel (Epredia, HD-4000-012) molds followed by paraffin embedding. 5-10pm thick sections were cut and processed for depar- affinization and antigen retrieval followed by antibody staining. For staining, sections were incubated in blocking solution [PBS supplemented with 3% donkey serum (Jackson ImmunoResearch, 017-000-121) and 0.1 % Triton-X] for 1 hour at room temperature. Samples were then incubated with primary antibodies diluted in blocking solution at 4°C overnight. Next day, P28241 PCOO 28.08.2025

[0635] 84 / 119 samples were washed 3 times with PBS for 5 minutes. Samples were then incubated with secondary antibodies and Hoechst 33342 (ThermoFisher Scientific, H3570) diluted in blocking solution for 1 hour at room temperature followed by 3 washes with PBS for 5 minutes each. Cultured cell samples were stored in PBS until imaged. Slides were mounted using Fluoromount aqueous mounting medium (Sigma-Aldrich, F4680) and were stored at 4°C until analyzed. Samples were imaged using Nikon Tie2 microscope. A list of primary and secondary antibodies is provided in the Key Resource Table.

[0636] Sections - further protocols:

[0637] Samples were washed with 1X Phosphate Buffered Saline (PBS, Sigma-Aldrich, P5493) and fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) overnight at 4°C. Samples were washed with PBS and stored in PBS supplemented with 0.2% Sodium Azide (Sigma-Aldrich, 71290) until used for immunostaining. Fixed samples were embedded into histogel (Epredia, HD- 4000-012) wells generated using custom made molds specific to the size of the organoid type (Harter et al, High-throughput histopathology for complex in vitro models, BioRxiv, Version posted April 10, 2025). Samples were dehydrated and paraffin-embedded using an automated tissue processor (Leica HistoCore PEARL) and an embedding station (Medite TES99). Paraffin blocks were sectioned at 5 pm on a microtome (Thermo Microm HM355S microtome). Sections were mounted on slides (Fisher scientific, 11976299) and dried.

[0638] For automated staining, sections were subjected to multiplexed immunofluorescence using Opal dyes (Akoya Biosciences) on a Roche Ventana Discovery Ultra stainer following manufacturer’s instructions. For multiplexed immunofluorescence, slides underwent deparaffinization, heat-induced antigen retrieval, blocking, antibody incubation, Opal dye application, and antibody neu- tralization / denaturation steps between each staining cycle. Slides were mounted with ProLong™ Gold Antifade Mountant (Invitrogen P36930) and dried prior to imaging. Slides were imaged using a Vectra Polaris (Akoya Biosciences Perkin Elmer) at 20x magnification for all 7 colors (Opal 480, Opal 520, Opal 570, Opal 620, Opal 690 and Opal 780). Laser exposure and intensity settings were adjusted on multiple slides per staining panel. Channels were unmixed and images tiled with PhenoChart (v1.0.12) and inForm (v2.4). Raw images were saved as .qptiff and fused in HALO (Indica labs, v3.6.4134.396). Image analysis of multiplexed immunofluorescence images was performed with HALO.

[0639] For manual staining, after antigen retrieval sections were incubated in blocking solution [PBS supplemented with 3% donkey serum (Jackson ImmunoResearch, 017-000-121) and 0.1% Tri- ton-X] for 1 hour at room temperature. Samples were then incubated with primary antibodies P28241 PCOO 28.08.2025

[0640] 85 / 119 diluted in blocking solution at 4°C overnight. The next day, samples were washed 3 times with PBS for 5 minutes. Samples were then incubated with secondary antibodies and Hoechst 33342 (ThermoFisher Scientific, H3570) diluted in blocking solution for 1 hour at room temperature followed by 3 washes with PBS for 5 minutes each. Slides were mounted with Prolong™ Gold Antifade Mountant (Invitrogen P36930) and were stored at 4°C until imaged. Samples were imaged using Nikon Eclipse Ti inverted microscope or Olympus Slideview VS200 Universal Whole Slide Imaging Scanner. Image analysis was performed with HALO (Indica labs, v3.6.4134.396).

[0641] Live imaging of the single lumen organoids was performed using the Nikon Eclipse Ti inverted microscope with 10x magnification equipped with Hamamatsu ORCA-Fusion C14440-20UP camera and an incubation chamber at 37 °C, 5% CO2. Cells were imaged on day 4, 6, 8 and 10 of differentiation. Tiles images of each well were analyzed with Imaged 2.9.0 using built-in plugins. To determine organoids’ size over time, ten brightfield images were thresholded and segmented. The distance between the two parallel planes restricting the organoid, namely the Feret’s diameter, was measured on the detected particles. Similarly, lumen’s diameter was measured using ZO1-GFP reporter signal from 8 different wells imaged at day 4, 6, 8 and 10. Graphs were plotted using GraphPad Prism 9.4.1. Data was plotted in Python (v3.11.4) using Matplotlib (v3.8.3) and Seaborn (vO.13.2) within a Jupyter Notebook.

[0642] Quantification of neuroepithelial cyst classes [no cyst (empty), single lumen (SL) and mulit-lumen (ML)] was performed using Aivia imaging software (v14.1.9, Leica Microsystems) on brightfield images acquired with a Nikon Eclipse Ti inverted microscope. Initially, pixel classifiers were trained on a representative image from each well size through regional annotation of background, well features and cysts, and then applied to the acquired image set for each well size. Next, the obtained channels were input in Aivia’s built-in “Cell count” recipe with optimized detection parameters to generate a set of quantifiable objects with minimal background signal. Finally, extracted objects from one image per well size were used as an input to train object classifiers capable of distinguishing and quantifying single- and multilumen cysts. These trained object classifiers were applied on the corresponding well size images datasets, with class allocation iteratively refined to ensure classification accuracy. Data for microwell size was plotted in RStudio (v2024.12.0) using dplyr (v1 .1.4) and ggplot2(v3.5.2). Data from different iPSC lines was plotted in Python (v3.11.4) using Matplotlib (v3.8.3) and Seaborn (vO.13.2) within a Jupyter Notebook. P28241 PCOO 28.08.2025

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[0644] For regional organoids, marker quantification analysis was done using the HALO software v3.6.413. A classifier was trained for each replicate and timepoint using the Random Forest classifier, by drawing sample areas across organoid sections in order to detect organoids, their necrotic core and background glass. Necrotic cores were only separated for d60 samples as d20 samples did not show any signs of a deteriorating core. Nuclear and marker detection was run on the organoid classified area excluding necrotic cores and background using the CytoNuclear FL module v.2.0.12 for nuclear marker as well as Area Quantification FL c2.3.4 for cytosolic marker. For the CytoNuclear FL analysis of FOXA2, LMX1A, NURR1 and NKX6.1 , nuclei were detected using the DAPI stain. Minimum nuclei size for detection has been set to 9 pm2, a nuclear contrast threshold of 0.525 was applied and nuclear segmentation was applied. Minimum detection intensities were selected for each marker with the help of the Real-time Tuning feature to resemble the actual signal. Due to varying signal intensities across timepoints minimum detection intensities for markers had to be adjusted slightly to better match the sample’s fluorescent signal. For TH and GAD1 quantification, no cell detection was performed on the organoid section of the classifier. As before minimum detection intensities were set according to the apparent signal and a blur radius of 0.25 was applied. Both analyses were performed on individual organoids using the TMA core function. The data was exported, analyzed and visualized using R (version 4.4.1 (2024-06- 14)).

[0645] Retina, size over time:

[0646] For quantifying the nuclear layer in retinal organoids, NRL and SOX9 location analysis was done using the HALO software v3.6.413. A classifier was trained for each replicate image using the Random Forest classifier, by drawing sample areas across organoid sections in order to separate organoids from background glass. The classifier was set up to generate an annotation around the perimeter of the organoid section. Separate analyses were created for SOX9 and NRL for each replicate using the CytoNuclear FL module v.2.0.12. Cell detection parameters were set up to identify nuclear signals. Using the “Real-time Tuning” feature, values had to be adjusted slightly across replicates depending on the fluorescent intensities to accurately represent the apparent fluorescent signal. Before running the analysis, images were divided into smaller sections using the “Segment TMA (tissue mold array)” function. Subsequently, the analysis was performed on the individual TMA cores. The initial analysis was followed up by an infiltration analysis. The object data was exported and analyzed using R (v4.4.1). Briefly, based on the TMA core location unique Organoid IDs were generated and assigned to the respective data. The data was cleaned up by P28241 PCOO 28.08.2025

[0647] 87 / 119 removing N / A values, duplicate entries and applying organoid specific exclusions as well as reintroducing 0-value organoids. Exclusions were applied in cases of missing data for either of the markers. 0-value organoids were added for organoids that went through analysis but had no marker-positive cells and thus did not appear in the exported data. Cell counts were divided into 1 pm bands and plotted as a heatmap.

[0648] Live i for interkinetic nuclear

[0649] To visualize cell morphology and tissue organization, WTC-CAAX-RFP and WTC-ZO1-GFP iPSC lines were mixed at a ratio of 1 :100 before seeding into the microwell plates. Following the standard cyst differentiation protocol, neuroepithelial cysts were carefully transferred to glass-bottomed plates (Cellvis, P96-1.5H-N) for live imaging. Prior to imaging, cysts were stained with BioTracker 650 red nuclear dye ( Sigma-Aldrich, SCT119) according to the manufacturer's instructions. Live imaging was performed using a Nikon Eclipse Ti inverted microscope equipped with a 20x objective within a controlled incubation chamber maintained at 37 °C and 5% CO2.

[0650] Single cell analysis of differentiating iPSCs was performed using 10x Chromium (10x Genomics). Cells were differentiated as described above, on day 10 organoids were transferred to a micro- fuge tube and washed with PBS (Gibco, 14190) and dissociated into single cells using Papain based Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628). Dissociated cells were run through 30pm cell strainer (CellTrics, 04-0042-2316), spun at 300g for 5 minutes and resuspended in PBS + 0.1% bovine serum albumin (Gibco, 15260-037). Cell density was adjusted to 1 million cells / ml and 5000 cells were captured for RNA sequencing. Single cells were encapsulated using 10x Chromium and libraries were prepared according to manufacturer’s recommendations. Samples were sequenced on an Illumina NextSeq 500 using the NextSeq 75 High Output Kits using standard Illumina sequencing primers and 61 cycles for Readl , 14 cycles for Read2, 8 cycles each for IndexReadl and lndexRead2. Sequence FASTQ files were processed using Cellranger. Single cell transcriptomes were mapped to human (GRCh38 / hg19) reference transcriptomes. Single cell transcriptomic data was analyzed using Scanpy.

[0651] Further methods for single cell RNA sequencing:

[0652] For analyzing the day 10 neuroepithelial cysts, cells were differentiated as described above. On day 10, 250-300 cysts were transferred to a microfuge tube and washed with PBS (Gibco, 14190) and dissociated into single cells using Papain-based neural tissue dissociation kit (Miltenyi Biotec, 130-092-628). Dissociated cells were run through 30pm cell strainer (CellTrics, 04-0042-2316), P28241 PCOO 28.08.2025

[0653] 88 / 119 spun at 300g for 5 minutes and resuspended in PBS + 0.1 % bovine serum albumin (Gibco, 15260- 037). Cell density was adjusted to 1 million cells / ml and 5000 cells were captured for RNA sequencing using 10X Genomics Single Cell 3' v3.1 protocols with feature barcode technology.

[0654] For single cell analysis of the cyst after morphogen treatment, half the cultured cysts were transferred to a microfuge tube, washed with PBS, and dissociated into single cells using a Papainbased neural tissue dissociation kit. Dissociated cells were run through a 30-pm cell strainer, centrifuged at 300g for 5 minutes, and resuspended in PBS + 0.05% bovine serum albumin. Samples were counted, pooled, and barcoded using the 3' CellPlex Kit (10X Genomics, 1000261) according to the manufacturer’s protocol. Briefly, samples were washed with 10% FBS + PBS solution, centrifuged at 300g for 5 min at 4°C. Individual samples were resuspended in Cell Multiplexing Oligo, incubated at room temperature for 5 minutes, washed with 10% FBS + PBS, and centrifuged at 300g for 5 min at 4°C. Samples were resuspended, counted, and pooled in equal ratios for encapsulation using 10X Genomics Single Cell 3' v3.1 protocols with feature barcode technology.

[0655] For single cell analysis of regional organoids, 3 organoids were pooled, washed with PBS, and dissociated into single cells using a Papain-based neural tissue dissociation kit. The time for dissociation was optimised for the organoids type. Dissociated cells were run through a 30-pm cell strainer, centrifuged at 300g for 5 minutes, and resuspended in PBS + 0.1 % bovine serum albumin. Cell density was adjusted to 1 million cells / ml and 10000-20000 cells were captured for RNA sequencing using 10X Genomics Single Cell 3' v4 protocols with feature barcode technology.

[0656] For all experiments, libraries were prepared according to manufacturer’s recommendations (10X Genomics Single Cell 3' v3.1 or v4). Samples were sequenced on an Illumina NovaSeq6000 or NovaSeqX plus. Sequence FASTQ files were processed using Cellranger. Single cell transcriptomes were mapped to human (GRCh38 / hg19) reference transcriptomes.

[0657] Statistical Analysis

[0658] Statistical analyses were performed using GraphPad Prism (9.4.1.). Statistical parameters such as the value of n, mean, standard deviation (SD), p values, and the statistical tests used are reported in the figure legends. For hiPSC differentiation experiments, the “n” refers to the number of independent hiPSC differentiation experiments analyzed. P28241 PCOO 28.08.2025

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[0660] Example 2: Protocol

[0661] Steps of the protocol:

[0662] Step 1 : Dissociate iPSCs / ESC cultures single cell suspension using Accutase. For generating epiblast cysts, plate 10-15 cells per microwell into different microwell plate types including PEG based microwell plates (Sun Biosciences, Gri3D®96) Eplasia (Corning, 4442) and Aggrewell (Stem cell technologies, 34415). For a typical experiment, cells were seeded in a 400 - 1000 pm microwell Sunbio Grid3D plate, at a density of 10-15 cells per microwell in 50 pl mTeSR plus supplemented with 1 :1000 CEPT cocktail [Polyamine Sigma P8483, Chromanl Medchem Express HY-15392, Emricasan Selleckchem S7775, trans-ISRIB Thermo Fisher scientific 5284], After 1 hour, another 100ml of mTeSR supplemented with CEPT was added to each well.

[0663] Step 2: On day 2, remove 10OpI of old medium and replace with 200 pl of an N2-based medium [N2 medium - DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057) + Penicillin / Streptomycin (Thermo Fisher, P0781) + 1x N2 supplement (Thermo Fisher, 17502048) + 1x MEM NEAA (Thermo Fisher, 11140035) + 1 ug / ml Heparin (Sigma-Aldrich, H3149-25KU], Repeat once again to remove any remaining old medium. Remove 200 pl of medium and add 200 pl of N2 medium.

[0664] Step 3: On day 4, remove 200 pl of old medium and gently add 200pl of N2 medium supplemented with 5% Matrigel (Corning, 254230).

[0665] Step 4: On day 6, gently remove 200 pl of old medium and gently add 200pl of N2 medium supplemented with 5% Matrigel (Corning, 254230).

[0666] Step 5: On day 8, gently remove 200 pl of old medium and gently add 200pl of N2 medium supplemented with 5% Matrigel (Corning, 254230).

[0667] Step 6: Keep the cyst in the microwell plate until day 8 or day 10 after which they can be transferred to shaking culture for long term culture. For region specific differentiation, the cysts can be treated with suitable morphogens.

[0668] Step 7: For further culture of the organoids, transfer the cyst to ultra low attachment 90mm dish (SBio, MS-90900Z) in 15 ml of an N2B27 based medium [N2B27 medium - 50% DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057 + 50% Neurobasal (Thermo Fisher, 21103049) + Penicillin / Streptomycin (Thermo Fisher, P0781) + 0.5x N2 supplement (Thermo Fisher, 17502048) + 1x B27 (Thermo Fisher, 17504044) + 1x Glutamax (Thermo Fisher, 35050061) + 1x MEM NEAA (Thermo Fisher, 11140035) + 2.5ug / ml Insulin (Millipore, I9278)] P28241 PCOO 28.08.2025

[0669] 90 / 119 with or without 1% matrigel (Corning, 254230) on an orbital shaker. From here on, change the medium every other day.

[0670] The following tables 1 and 2 list markers that may be used to characterize the neuroepithelial cysts and different CNS organoids. Table 1 : List of selected markers and their expression

[0671] T able 2: List of markers to characterize the neuroepithelial cyst and different CNS brain organoids. P28241 PCOO 28.08.2025

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[0674] Example 3: Human pluripotent stem cells derived single lumen neuroepithelial cysts

[0675] To generate epiblast cyst in a robust, reproducible and high-throughput manner, previously described PEG hydrogel based microwell plates were utilized (Brandenberg et al. 2020, Nature Biomedical Engineering, Vol 4, September 2020, 863-874) (Figure 1A). Using the protocol described in example 2, the inventors were able to aggregate 10-15 human pluripotent stem cells (hPSCs) per microwell and generate single lumen epiblast cysts at day 2. To dynamically follow the differentiation of the cysts, epiblast and neuroepithelial marker SOX2 (tagged with GFP) and apical marker ZO1 (tagged with GFP) reporter iPSC lines were used. These cysts containing SOX2-GFP positive epiblast-like cells continued to grow and maintain apical polarity marked by ZO1-GFP over time (Figure 1 B). By day 10, the cyst grew to an average diameter of 179.77 pm containing a large lumen of an average size of 131.76 pm (Figure 1C-E). After day 10 of differentiation, the neuroepithelial cysts can be transferred to ultra low attachment bottom plates for long term culture.

[0676] The results of a further experiment of the growth in size of the cysts over time and of lumen diameter are shown in Figures 11A-11C. They show the increase in size and lumen diameter, while a high circularity is maintained.

[0677] Only varying the initial seeding cell number in a previously reported protocol (which protocol has been developed to later generate brain organoids in an unguided fashion from the neuroepithelial tissues obtained in the protocol, see Lancaster et al., Cerebral organoids model human brain development and microcephaly. Nature 501 , 373-379 (2013)), from standard 3000 cells per well to 1000, 100 or 20 cells, did not lead to an epiblast cyst and the organoids grew containing multiple neuroepithelial units (Figure 2A).

[0678] Other microwell based plate formats, for example, different microwell sizes in Gri3D plates from SunBiosciences, Elplasia plates (ULA bottom) from Corning or Aggrewell plates (ULA bottom) from Stem Cell Technologies were also compatible with generation of the single lumen cysts suggesting that this organized aggregation of pluripotent stem cells is independent on the material of the substrate (Figure 2B-C, Figures 12A-C). However, a size difference was observed in different plate formats (Supplementary Figs. 12A-C). The microwell plate format allows high throughput generation of cysts and is compatible with automation. P28241 PCOO 28.08.2025

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[0680] Additionally, a titration of Matrigel showed that a wide range of Matrigel concentration can support the neuroepithelial cyst formation (Figure 2D). An optimal balance for cyst formation was observed at 5% Matrigel, allowing for robust and uniform neuroepithelial structures.

[0681] Immunocytochemistry analysis of the cyst on day 4 and day 6 of differentiation confirmed the epiblast-like fate of the cells marked by expression of SOX2 and E-cadherin. By day 12, the cyst continued to express SOX2 and starts to express N-cadherin confirming the neuroepithelial fate of the cyst (Figure 1G). Dividing cells were consistently present on the apical surface in the neuroepithelial cyst suggesting the cells might be performing interkinetic nuclear migration and shifting to the apical surface to undergo cell division (Figure 1 H). The results of further experiments are shown in Figures 13(a)-(c). Specifically, immunofluorescence analysis of the cysts on day 6 confirmed the epiblast-like cell fate, marked by E-cadherin expression (Figure 13a). By day 12, the cysts switched the expression to N-cadherin, confirming their switch to the neural fate (Figure 13a). By day 14, the cysts maintained their structural integrity and expressed relevant neural markers such as SOX2, PAX6 and OTX2 (Figure 13b). The versatility of this platform was demonstrated by successful cyst formation from various hPSC lines, including WTC-11 iPSCs, PGP1 iPSCs, H9 ESCs, and CRMi003-A iPSCs, all exhibiting similar morphological development (Figure 13c).

[0682] To estimate the efficiency of single lumen neuroepithelial cyst formation, the frequency of failed aggregation (including lost cysts during medium changes), cysts containing single lumen and cysts containing more than 1 lumen were quantified. To avoid any effect of cell line specificity for the protocol, three independent PSC lines including WTC-11 iPSCs (GM25256), PGP1 iPSCs (GM23338) and H9 ESCs were utilized. Interestingly, all three cell lines were comparable in differentiation with efficiency of single lumen neuroepithelial cyst formation of 42, 54 and 55%, respectively (Figure 11).

[0683] To assess the reasons for multiple lumen formation in cysts, the cell distribution in microwells at the time of seeding and number of lumens formed in individual cysts after 6 days were quantified. The cyst size corresponded to the number of initial seeded cells in microwells (Figure 2E-G). Interestingly, seeding of more than 13 cells led mostly to multiple lumens in cyst, whereas smaller numbers mostly led to single lumen cysts (Figure 2H-I). This analysis confirmed that cell distribution in microwells at seeding affected the number of lumen present in the cysts.

[0684] The results of further experiments are shown in Figures 14(a)-(c). The relationship between initial cell number and distance from the center of the microwell was determined, observing that cell number increased with proximity to the edge of the well (Fig. 14(a)), which - without wishing to P28241 PCOO 28.08.2025

[0685] 94 / 119 be bound to a theory - could potentially be explained by the meniscus effect at the well edges which draws cells due to surface tension. Furthermore, the distance of cysts from the center correlated with formation of muti-lumen cyst suggesting an influence of initial cell number on subsequent phenotype (Fig. 14(b)). Analysis of cyst phenotype in relation to initial cell number indicated that larger initial cell numbers generally led to multi lumen formation in all three cell lines (Fig. 14(c)). In conclusion, regarding the optimal number of single-lumen cysts, it was found that using a range of 8-12 cells effectively balanced between empty wells and multi-lumen formation (Fig. 14(c)).

[0686] Dividing cells were consistently observed at the apical surface of the cyst, suggesting cells are dividing by interkinetic nuclear migration. To confirm that, the sparsely labelled WTC-11 CAAX- RFP reporter iPSCs hashed into the WTC-11 ZO1-GFP reporter iPSC line were tracked using live imaging. The nuclei in the dividing cells in the pseudostratified epithelium migrated to the apical surface during division, showing cells undergo interkinetic nuclear migration (Figure 15a, b). Post day 10, NECs could be easily transferred to ultra-low attachment plates for long-term culture (Figure 15).

[0687] To test whether the neuroepithelium in these cysts is able to self organize after dissociation, the cyst were dissociated at day 10 and reaggregated in microwell plates. Interestingly, the cells organized into small lumen containing neuroepithelial cyst within a few days (Figure 2H). This observation together with the single cell RNA seq analysis shows that cystic structures are made exclusively of neuroepithelial population with correct polarity in 3D. This opens the opportunity to engineer the neuroepithelial cells derived from these cysts to generate architecturally robust and uniform models.

[0688] In addition to the human induced pluripotent and embryonic stem cells, the culture approach was applied to mouse embryonic stem cells. This allowed to generate neuroepithelial cysts from mouse embryonic stem cells (Figure 2I) suggesting that the principles of neuroepithelial cyst formation in vitro did not depend on the species and this methodology would allow further development of brain organoids from pluripotent stem cells from different species.

[0689] Example 4: Neuroepithelial cyst acquire default anterior fate and can be patterned

[0690] To investigate the homogeneity and the default fate of the neuroepithelial cysts, single cell RNA sequencing was performed at day 10 of differentiation. Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) based embedding revealed two major clusters of cell P28241 PCOO 28.08.2025

[0691] 95 / 119 populations representing neuroepithelial cells and mitotic neuroepithelial cells (Figure 3A). Apart from cell cycle related genes (e.g. MKI67, CDK1 , LIBE2C), the two clusters shared a similar tran- scriptomics profile. The cells at this stage expressed typical neuroepithelial markers including PAX6, NES, VIM (Figure 3B, 3C). Interestingly, the cells also expressed anterior neuroepithelial markers such as SIX3, OTX2, LHX2 suggesting that they defaultly acquire an anterior fate during differentiation (Figure 3B, 3C). These observations were corroborated by antibody staining for neuroepithelial (PAX6, N cadherin) and anterior neural tube markers (OTX2) (Figure 3D). As expected, no expression posterior (GBX2) markers were detected in the single cell analysis (Figure 3C).

[0692] To assess whether neuroepithelial cysts can be patterned to other fates along the dorsal-ventral and anterior-posterior axis in neural tube, different signaling pathways known to be important for patterning during brain development were manipulated, namely - Wnt, BMP, TGFb, Activin, RA, Notch, Sonic hedgehog and FGF signaling. After 8 days of treatment the organoids were harvested and analyzed using single cell RNA sequencing (Figure 3E). It was observed that the cells differentiated to different fates along the dorsal-ventral and anterior-posterior axis (Figure 3F-H). In addition to the fate changes the cells showed varied morphology in response to the morphogens (Figure 3I).

[0693] Example 5: Long-term differentiation of neuroepithelial cysts into neural organoids

[0694] The neuroepithelial form in the cyst is amenable to patterning into different brain regions. This allows establishment of uniform protocols where one can start with the same starting culture and diversify into different brain regions (Figure 4A). It was also hypothesized that an initial improved architecture of the neuroepithelial cyst would advance the robustness, homogeneity and quality of region specific neural organoids compared to the current brain organoid models.

[0695] As a proof of principle, single lumen neuroepithelial cysts were differentiated into three different brain regions - forebrain, choroid plexus and retina. For differentiation into the forebrain, the early cysts were treated with BMP, SMAD and Wnt pathway inhibitors and further cultured in differentiation medium. Immunohistochemistry analysis was performed at day 30 and day 60 forebrain organoids which showed expression of dorsal forebrain marker (FOXG1) and a single neuroepithelial unit on day 30 marked by apical marker N cadherin (Figure 4B).

[0696] These single lumen organoids differentiated into more complex structures and became neuron rich overtime (Figure 4C). Single cell RNA sequencing was utilized to access the cell composition P28241 PCOO 28.08.2025

[0697] 96 / 119 of the 60-day forebrain organoids. Clusters expressing markers for cortical cell types including outer radial glial cells, (HOPX), progenitor cells (PAX6), intermediate progenitors (EOMES), cortical neurons (BCL11 B, SATB2) were identified. Furthermore, a small population of interneurons marked by GAD1 , DLX1 and DLX2 (Figure 4D-F) was identified. This analysis also revealed off target cell type, mostly of retinal fate, expressing retinal progenitor markers VSX and CRX (Figure 4G).

[0698] For differentiation into choroid plexus, a pulse of CHIR99021 was provided for 3 days followed by long term culture in the neuronal growth medium. After 60 days of culture, a distinct morphology appeared containing an epithelial cell network (Figure 5A-B). The expression of choroid plexus specific markers e.g. Transthyretin (TTR), a thyroxine and retinol transporter, and Aquaporin 1(AQP1), a selective water channel present on the apical surface of choroid plexus epithelial cells (Figure 5A) was confirmed.

[0699] Using single cell transcriptom ics, the gene expression profile of choroid epithelial organoids on day 60 was investigated. In corroboration with the immunostaining analysis, a big population of choroid epithelial cells expressing typical markers such as TTR, AQP1 , PRLR and CLIC6 (Figure 5C-E) was identified. Furthermore, a small population of unrelated epithelium (higher KRT18) was observed, suggesting presence of more differentiated epithelial cells. Additionally, a small cluster of neuronal cells expressing MAP2 and STMN2 was observed.

[0700] For retinal differentiation, the use of matrigel was removed and long term maturation culture was applied which yielded neural retina morphology seen in retinal organoids (Figure 6A). Immunohistochemistry analysis confirmed the presence of a laminar structure and expression of early retinal markers such as OTX2 and cone rod homeobox (CRX, expressed in photoreceptor cells / bi- polar cells) (Figure 6A). Upon long term differentiation, the organoids expressed typical retinal markers, identified by single cell RNA sequencing. Based on gene markers we found cell types only from retinal lineage and identified seven cell types present in human retina (Figure 6B-C).

[0701] These analyses confirm that neuroepithelial cysts can be differentiated into different brain regions to obtain more mature neural organoids.

[0702] Example 6: Optimized Protocols

[0703] In the following sections, optimized protocols are described which were used in particular in the subsequent examples 7-13. P28241 PCOO 28.08.2025

[0704] 97 / 119

[0705] Pluripotent Stem Cell Culture

[0706] WTC11 (GM25256, wildtype) human induced pluripotent stem cell (iPSC) line, PGP1 (GM23338, wildtype) human iPSC line, CRMi (CRMi003-A, wildtype) and H9 (WA09, wildtype) human embryonic stem cell (ESC) line were routinely cultured on Geltrex-coated culture plates (Thermo Fisher, A1413201) in mTeSR Plus medium (Stemcell Technologies, 100-0276) at 37°C under normoxic conditions. Upon reaching confluency, cultures were dissociated into single cells using Accutase (Corning, 25-058-CI). Cells were then seeded onto Geltrex-coated plates in mTeSR Plus supplemented with 10 M Y-27632 dihydrochloride (Tocris, 1254) at a density of 20, DOO- 25, 000 cells / cm2for routine maintenance of iPSC and ESC lines. The following day, the medium was replaced with mTeSR Plus without supplements, and medium changes occurred daily. Cultures typically reached confluency every four days and were passaged using the same dissociation and seeding protocol. For cryopreservation, cultures were dissociated into single cells using Accutase and frozen in Stem Cell Banker Medium (AMSbio, 11924). Cells were tested for mycoplasma contamination before first stocking. WTC11 (GM25256, wildtype), CAAX-RFP, ZO1-GFP and SOX2-GFP reporter cell lines were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research.

[0707] Differentiation of pluripotent stem cells into single lumen neuroepithelial cyst iPSCs / ESC cultures were dissociated into single cell suspension using Accutase. For generating neuroepithelial cysts, cells were plated into different microwell plate types including PEG based microwell plates (Sunbiosciences, Gri3D®96), Elplasia (Corning, 4442) and Aggrewell 400 (Stem cell technologies, 34415, 34811). For a typical experiment, cells were seeded in a 400 - 500 urn microwell Sunbio Grid3D plate, at a density of 10-15 cells per microwell in mTeSR plus supplemented with 1 :1000 CEPT cocktail (Chen et al Nat Methods. 2021 May; 18(5): 528-541) [Polyamine (Sigma, P8483), Chromanl (Medchem Express, HY-15392), Emricasan (Selleckchem, S7775), trans-ISRIB (Thermo Fisher scientific, 5284)]. On day 2, medium was replaced with a NIM medium (Giandomenico et al Nature Protocols 16, 579-602 (2021)) [N2 medium - DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057 + Penicillin / Streptomycin (Thermo Fisher, P0781) + 1x N2 supplement (Thermo Fisher, 17502048) + 1x MEM NEAA (Thermo Fisher, 11140035), 1 ug / ml Heparin (Sigma-Aldrich, H3149-25KU]. On day 4, medium was replaced by N2 medium supplemented with 5% Matrigel. Cysts were kept in the microwell plate until day 10 in N2 medium supplemented with 5% Matrigel, with medium changes every other day. P28241 PCOO 28.08.2025

[0708] 98 / 119

[0709] For long term culture, cysts were pipetted out of the microwells using a 1 ml pipette and then moved to ultra low attachment 6 cm or 9cm dish (Corning, LSBio) in IDM-A medium (Giandomenico et al Nature Protocols 16, 579-602 (2021)) [IDM-A medium - 50% DMEM / F12 HEPES glutamine (Thermo Fisher Scientific, 11330057 + 50% Neurobasal (Thermo Fisher, 21103049) + Pen- icillin / Streptomycin (Thermo Fisher, P0781) + 0.5x N2 supplement (Thermo Fisher, 17502048) + 1x B27 (Thermo Fisher, 12587010) + 1x Glutamax (Thermo Fisher, 35050061) + 1x MEM NEAA (Thermo Fisher, 11140035) + 2.5ug / ml Insulin (Millipore, I9278)] and 1 % matrigel. The cysts were cultured on an orbital shaker with a rotation orbit diameter of 25mm set at a speed of 64 rpm. At later time points the medium was switched to IDM+A medium (IDM-A medium containing 1x B27 with vitamin A (Thermo Fisher, 17504044) instead of 1x B27 without vitamin A (Thermo Fisher, 12587010)). Medium was replaced every second day till day 20. From day 20, the medium was changed twice a week.

[0710] Mouse cyst development

[0711] The mouse embryonic stem cell line SBR [##] was maintained under feeder-free conditions in 2i LIF medium. This medium consisted of high-glucose DM EM (Gibco, 61965026) supplemented with 1 % non-essential amino acids (Thermo Fisher, 11140035), 1% sodium pyruvate (Thermo Fisher, 11360039), 0.1 mM p-mercaptoethanol (Thermo Fisher, 31350010), 10% fetal bovine serum (FBS) (Thermo Fisher, 16141079), 1 ,000 U / ml LIF (Sigma, ESG1106), 3 pM CHI R99021 (Millipore, 361571), and 1 pM PD0325901 (Selleck Chemicals, S1036). Mouse ES cells were passaged every 2 days using Accutase (Gibco, cat. no. 12605010) for dissociation and seeded at a density of 30,000-40,000 cells per 6-well plate in MEF-conditioned medium supplemented with 2i LIF. All ES cell lines were routinely tested for mycoplasma contamination. For neuroepithelial cyst differentiation, 5-10 cells were seeded per microwell in 500 pm microwell plates (Grid3D, Sunbiosciences) in NIM supplemented with CEPT. Two days post-seeding, the medium was changed to NIM. The medium was refreshed every other day.

[0712] Differentiation of neuroepithelial cyst into brain organoids

[0713] Forebrain organoids

[0714] Neuroepithelial cysts were generated following the protocol previously described. On day 6, the cysts were treated with NIM supplemented with 10 pM SB 431542 (Tocris, 1614), 3 pM IWR1 P28241 PCOO 28.08.2025

[0715] 99 / 119

[0716] (Sigma-Aldrich, 681669) and 5% Matrigel. On day 8, 500-600 cysts were transferred from microwells to an ultra-low attachment 9 cm dish in IDM-A supplemented with 10 pM SB 431542, 3 pM IWR1 (Sigma-Aldrich, and 1% Cultrex (R&D systems, 3533-005-02). Optionally, 1 pM CHIR (Tocris, 4423) was added instead of IWR1 starting day 10. The medium was refreshed every other day, and cultures were maintained on an orbital shaker with a rotation orbit diameter of 25mm set at a speed of 64 rpm. From day 16, the medium was switched to IDM-A and the medium was changed every other day. From day 30 onwards, the medium was switched to IDM+A, supplemented with 1 % Cultrex, and the medium was refreshed twice a week.

[0717] Midbrain organoids

[0718] Neuroepithelial cysts were generated as previously described. On day 8, 250-300 cysts were transferred to an ultra-low attachment 9 cm dish. The culture medium was switched from NIM to IDM-A, supplemented with 1.5 pM CHIR (Tocris, 4423), 0.5 pM SAG (Stem Cell Technologies, 73412), 100 ng / ml FGF8b (Peprotech, 100-25) and 1 % matrigel. The medium was refreshed every other day, and cultures were maintained on an orbital shaker with a rotation orbit diameter of 25mm set at a speed of 64 rpm. The medium, supplemented with growth factors and extracellular matrix, was refreshed every other day until day 16. Starting on day 16, the medium was switched to IDM+A and replaced twice a week. From day 30 onwards, the medium was refreshed twice a week. For testing the effect of soluble ECM on organoid growth, 1% Matrigel was added to the cultures from day 8 to day 30.

[0719] Hindbrain organoids

[0720] Neuroepithelial cysts were generated as previously described. On day 6, the cysts were treated with NIM supplemented with 10 pM SB431542, 2.5 pM Dorsomorphin (Sigma-Aldrich, P5499), 1.5 pM CHIR, 7 pg / ml insulin (Sigma-Aldrich, I9278), 20ng / mL FGF2 (Peprotech, 100-18B), 20ng / mL EGF (R&D, 236-EG-200) and 5% Matrigel. On day 8, 500-600 cysts were transferred to an ultra-low attachment 9 cm dish containing IDM-A supplemented with 10 pM SB431542, 2.5 pM Dorsomorphin, 1.5 pM CHIR, 7 pg / ml insulin, 20ng / mL FGF2, 20ng / mL EGF and 100 ng / ml FGF8b (Peprotech, 100-25). The medium was refreshed every other day, and cultures were maintained on an orbital shaker with a rotation orbit diameter of 25mm set at a speed of 64 rpm. Beginning on day 14, the medium was switched to 10 pM SB431542, 2.5 pM Dorsomorphin, 1.5 pM CHIR, 7 pg / ml insulin, 20ng / mL FGF2 and 20ng / mL EGF and 100 ng / ml FGF19. The medium was refreshed every other day, and cultures were maintained on an orbital shaker. On day 22, the medium was switched to IDM-A and changed every other day until day 30. From day 30 to day 60, IDM-A was supplemented with 100 ng / ml SDF1a (Peprotech, 300-28A) and 0.5 ng / ml T3 P28241 PCOO 28.08.2025

[0721] 100 / 119

[0722] (Merck, T-074-1 ML) was refreshed two times a week. From day 60 onwards, the medium was switched to IDM-A.

[0723] Retinal organoids

[0724] Neuroepithelial cysts were generated as previously described. From day 6 to day 12, the cysts were treated with dual SMAD inhibition treatment with 2.5 pM Dorsomorphin (Sigma-Aldrich, P5499), 10 pM SB 431542 (Tocris, 1614) and 3 pM IWR1 (Sigma-Aldrich, 681669) in NIM. On day 8, 500-600 cysts were transferred to a 9 cm ultra-low attachment dish and cultured on an orbital shaker with a rotation orbit diameter of 25mm set at a speed of 64 rpm. Beginning on day 12, the medium was switched to IDM-A supplemented with 50 ng / ml BMP4 (Peprotech, 120- 05ET), and it was replaced every other day. On day 24, the medium was switched to IDM-A and refreshed every other day.

[0725] For long term maturation, the medium was transitioned to a series of differentiation media based on previously published protocols (Zhong et al Nat Commun. 2014 Jun 10 :5 :4047; Cowan et al Cell. 2020 Sep 17 ;182(6) : 1623-1640. e34 ; West et al, Stem Cell Report. 2022 Apr 12;17(4):775- 788)). Specifically, from day 40, cultures were maintained in retina differentiation medium 1 [RDM1 - DM EM high glucose, GlutaMAX, F-12 Nutrient Mixture (Ham), 1x B27 without vitamin A, 1x NEAA Solution, 10% ES FBS (Thermo Fisher, 16141079), 1x Sodium Pyruvate 100 mM, 1x Penicillin / Streptomycin, 1x Amphotericin B, freshly supplemented with 100 mM Taurine (Sigma, T8691-25G)] with medium changes every other day. From week 10 onwards, the medium was switched to RDM2 (RDM1 supplemented with 1 pM Retinoic acid (Sigma-Aldrich, R2625) and 100mM Taurine).

[0726] From week 14, organoids were transferred to an ultra-low attachment 24-well plate, with individual organoids in separate wells under static conditions to prevent fusion and mechanical stress, and the medium was switched to retina differentiation medium 3 [ RDM3 - DMEM, high glucose, GlutaMAX, F-12 Nutrient Mixture (Ham), 1x N2 supplement, 1x NEAA Solution, 10% ES FBS, 1x Sodium Pyruvate 100 mM, 1x Penicillin / Streptomycin, 200mg / ml Albumax (Thermo Fisher, 11021029), Amphotericin B, freshly supplemented with 0.5 pM Retinoic acid and 100mM Taurine], At this time point, organoids with a shiny neuroepithelial rim were selected under a stereo microscope to further culture. Organoids with dense morphology or small organoids with very small neuroepithelium perimeter were discarded. P28241 PCOO 28.08.2025

[0727] 101 / 119

[0728] Morphogen treatment experiment

[0729] For each condition, 500-600 neuroepithelial cysts on day 8 were transferred to one 6 cm dish in IDM-A supplemented with 3% Matrigel and the following individual morphogens: 3 pM CHIR (Tocris, 4423), 0.5 pM SAG (Stem cell Technologies, 73412), 2.5 pM DAPT (Stem cell Technologies, 72082), 1 pM Retinoic acid (Sigma-Aldrich, R2625-50MG), 10 ng / ml Recombinant Human BMP-4 (Peprotech, 120-05ET), 100 ng / ml Recombinant Human / Murine FGF-8b (Peprotech, IDO- 25), 2.5 pM IWP2 (Selleckchem, S7085), 0.5 pM LDN193189 (Stem cell Technologies, 72147), 10 pM SB 431542 (Tocris, 1614), 20 ng / ml Recombinant Human FGF-2 (Peprotech, 100-18B), 50 ng / ml Activin A (Peprotech, 120-14E-100UG) and untreated. The medium was changed every other day, and cysts were harvested on day 16 for single-cell RNA sequencing and immunostaining.

[0730] Reaggregation experiments

[0731] To evaluate the reaggregation capacity of differentiated neuroepithelial cysts, 500-600 cysts were harvested on day 10 of the standard differentiation protocol and dissociated into single cells using the Papain-based Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) according to the manufacturer's instructions. The resulting cell suspension was centrifuged at 300 g for 5 minutes, and the cell pellet was resuspended in NIM supplemented with CEPT. These cells were then seeded into 500 pm microwells at a density of 100 cells per microwell. The following day, the culture medium was replaced with NIM supplemented with 5% Matrigel. The medium was subsequently changed every other day. Reaggregating cells were imaged for SOX2-GFP expression and under brightfield illumination on day 6 post-seeding using Nikon Eclipse Ti inverted microscope.

[0732] Example 7: Role of Microwell Diameter

[0733] To assess the role of microwell diameter in single-lumen cyst generation, the inventors systematically varied the well diameters in which human induced pluripotent stem cells were cultured. Specifically, microwell diameters ranging from 300 pm to 1500 pm and Il-bottom PEG-coated and plastic ultra-low attachment (ULA) plates were tested. Brightfield microscopy on day 4 and day 8 (Figure 7a) revealed that smaller well diameters consistently promoted the formation of well-defined, single-lumen neuroepithelial cysts. Quantification of single-lumen neuroepithelial cysts further confirmed that well diameters of 300 pm and 500 pm yielded the highest percentage P28241 PCOO 28.08.2025

[0734] 102 / 119 of successful cyst formation (Figure 7b), indicating an optimal confinement for initial self-organization. 500 pm yielded both good results and provided enough space for subsequent cyst growth.

[0735] Example 8: Directing Differentiation into Regionalized Brain Organoids

[0736] Building upon successful neuroepithelial cyst formation, the inventors proceeded to direct their differentiation into regionalized brain organoids (Figure 8a). Morphological development of these organoids was monitored over 60 days, showing distinct growth patterns for forebrain, midbrain, and hindbrain organoids (Figure 8b). Immunofluorescence staining at various time points validated the regional identity and cellular composition of the organoids. Forebrain organoids, examined at day 30 and day 60, exhibited robust expression of FOXG1 , confirming the presence of telencephalic neural progenitors and neurons, fundamental to forebrain development. The detection of TBR2 and CTI P2 further indicated the generation of intermediate progenitor cells and deeplayer cortical projection neurons (Figure 8c). Midbrain organoids, assessed at day 20 and day 60, demonstrated efficient induction of the midbrain dopaminergic lineage. Early expression of FOXA2 marked the formation of midbrain floor plate progenitors, which subsequently co-ex- pressed LMX1A, indicative of committed midbrain dopaminergic neuron progenitors. By day 60, the definitive presence of NLIRR1 and TH (Tyrosine Hydroxylase) confirmed the successful maturation into functional dopaminergic neurons (Figure 8d). Finally, characterization of hindbrain organoids at day 30 and day 90 revealed the emergence of diverse hindbrain cell types. Immunostaining for ATOH1 identified rhombic lip progenitors, known to give rise to excitatory neurons of the cerebellum and brainstem. PAX2 expression marked GABAergic interneurons, including those critical for cerebellar circuitry, while SKOR2 presence indicated the differentiation of Purkinje cells, a unique and essential inhibitory neuron type of the cerebellum (Figure 8e). Collectively, these findings underscore the ability of this protocol to generate regionally specified brain organoids with appropriate cell type representation.

[0737] Example 9: Retinal Organoids

[0738] Beyond regional brain organoids, the inventors successfully generated and characterized retinal organoids, showcasing the versatility of the system of the present disclosure (Figure 9). The morphological development of these organoids was tracked till week 30, revealing progressive maturation and the emergence of distinct retinal structures (Figure 9a). Immunofluorescence staining at day 60 confirmed the early stages of retinal differentiation, evidenced by the expression of key retinal progenitor markers VSX2 and PAX6 (Figure 9b). As differentiation progressed, P28241 PCOO 28.08.2025

[0739] 103 / 119 by day 120, a diverse array of retinal cell types was identified (Figure 9c). CRX expression marked photoreceptor progenitor cells, while the presence of OPN1SW (S-opsin) and NRL indicated the development of cone and rod photoreceptors, respectively. Other neuronal cell types included PKCa-positive bipolar cells and AP2a-positive amacrine cells. Additionally, SOX9 expression confirmed the presence of Muller glia, the major glial support cells of the retina. (Figure 9c). Further analysis on day 120 highlighted the organized structure within the organoids, with NRL (rods) primarily localizing to regions resembling the outer nuclear layer and SOX9 (Muller glia) distributed in areas consistent with the inner nuclear layer (Figure 9d, e). Quantitative heatmaps illustrating the spatial distribution of SOX9 and NRL positive cells as a function of distance from the organoid edge further supported the formation of distinct retinal layers (Figure 9f). The reproducibility of photoreceptor development was demonstrated by consistent NRL expression across multiple retinal organoids from four independent replicates (Figure 9g). By week 25, the retinal organoids displayed markers indicative of more mature retinal cell types (Figure 9h). This included ARR3 (cone arrestin), confirming mature cones, and robust expression of NRL (rods) and RHO (rhodopsin), indicating the presence of mature rod photoreceptors. These findings collectively demonstrate the successful generation of highly organized retinal organoids containing key retinal cell types with advanced maturation.

[0740] Example 10: Choroid Plexus Organoids

[0741] The approach described herein was extended to generate human choroid plexus organoids (Figure 10). choroid plexus organoid development was tracked till day 190, showing their progressive morphological maturation (Figure 10a). Immunofluorescence staining at day 60 confirmed the presence of key choroid plexus epithelial cell (ChPEC) markers. Organoids expressed Transthyretin (TTR), the carrier protein for thyroid hormones and retinol and water transporter AQP1 (Aq- uaporin 1), both being crucial for choroid plexus function (Figure 10b). Epithelial barrier formation was confirmed by immunofluorescence analysis with tight junction markers ZO1 and CLAUDIN5 (Figure 10b, 9c). the organoid also exhibited a correct apical basal polarity marked by ZO1 delineating tight junctions, COLIV highlighted the basement membrane, and ARL13B and FOXJ1 marked motile cilia and ciliated cells, respectively, on the apical surface. In culture, organoids with an inflated morphology named ‘apical-in’ was rarely observed. Histological analysis using H&E staining revealed the gross morphology of the organoids (Figure 10d). Immunofluorescence staining confirmed the localization of apical and basolateral markers corresponding to these dif- P28241 PCOO 28.08.2025

[0742] 104 / 119 ferent orientations (Figure 10e). Quantification of cell density showed that cells are densely organized in the "apical out" organoids compared to "apical in" organoids (Figure 10f). High-reso- lution transmission electron microscopy at day 60 provided ultrastructural evidence of characteristic choroid plexus epithelium features, including numerous microvilli and cilia on the apical surface, tight junctions between cells, and intracellular organelles such as mitochondria and mul- tivesicular bodies. Also, the presence of both dark and light cells was observed (Figure 10g). To assess the functional output of the organoids, the secreted protein in the culture medium was analyzed. A significant overlap in identified protein components between organoid-derived CSF (oCSF), human embryonic CSF (heCSF), CSF from organoids generated by a published protocol (poCSF), and human adult CSF (haCSF), indicating shared protein profiles (Figure 10h) was observed. In addition, the inventors observed the expression of known protein that are secreted by ChPECs in the culture medium from the choroid plexus organoids (Figure 10i) and demonstrated that the organoids expressed proteins known to be present in human adult CSF (Figure 10j). Gene Ontology enrichment analysis of the top 500 identified proteins in organoid-derived CSF revealed cellular components specific to extracellular vesicle, focal adhesion and endoplasmic reticulum lumen and biological processes related to cell adhesion, ECM organization, axon guidance (Figure 10k). Collectively, these data confirm the successful generation of human choroid plexus organoids that faithfully recapitulate key morphological, molecular, and functional characteristics of the native choroid plexus.

[0743] Example 11 : Regional Specification of Neuroepithelial Cysts from a Default Anterior State

[0744] To comprehensively characterize the cellular composition of NECs, single-cell transcriptomics analysis was performed of NECs on day 10. This analysis revealed that NECs maintained a high degree of transcriptional homogeneity. The primary variations observed were predominantly attributed to cell cycle phases, ribosomal protein content and number of genes detected (Figure 16a, Figure 17a), indicating active proliferation and robust metabolic activity across the largely uniform neuroepithelial cells. This homogeneous population of cells expressed typical neuroepithelial markers, including Nestin (NES), cell cycle marker MKI67, and anterior neural markers OTX2 (Figure 16b). Additionally, the cells lack expression of posterioring markers such WLS suggesting that the cysts inherently acquire an anterior fate (Figure 16b). Given that proper cell fate acquisition also influences tissue architecture, the inventors investigated whether the pure neuroepithelium generated in the cyst could self-organize. The cysts were dissociated at day 10 and reaggregated them in microwell plates. Notably, within days, the cells reorganized into small, P28241 PCOO 28.08.2025

[0745] 105 / 119 lumen-containing NECs (Figure 17b). This observation, alongside the single-cell RNA-seq analysis of the cysts, confirmed that NECs were composed exclusively of a neuroepithelial population with correct polarity.

[0746] The inventors further investigated how various developmental signaling pathways influenced cell fate, morphogenesis and patterning within the cysts. Key developmental signaling pathways were modulated, including Wnt, BMP, retinoic acid (RA), Notch, Sonic hedgehog (Shh), FGF, TGFp, and Activin and performed single-cell RNA sequencing and immunofluorescence analysis (Figure 16c). Following 8 days of treatment, the cysts showed remarkable morphological responsiveness to various signaling pathway modulations (Figure 17c). Most cells in the cyst at this stage expressed neural progenitor marker SOX2 and the neuroepithelial units in the cyst maintain N-cad- herin expression (Figure 16f) . Non-treated cysts, as well as those exposed to anteriorizing factors such as inhibitors of Wnt (IWP2), TGFp (SB431542), BMP (LDN193189) and (DAPT), maintained a spherical cystic structure (Figure 16f, Figure 17c). In contrast, activation of Wnt (CHIR99021), BMP (BMP4), retinoic acid (RA), FGF (FGF2, FG8), and Nodal (Activin A) pathways induced a drastic alteration in tissue organization (Figure 16f, Figure 17c).

[0747] To determine cell fate specification within the differentiating cysts, the scRNA-seq data was projected onto a single-cell atlas of the developing human brain at 5 post-conception weeks (pew) (Bruan et al Science 2023 Oct 13; 382(6667):eadf1226)) using reference similarity spectrum analysis (He et al Nature 635, 690-698 (2024)). Uniform manifold approximation and projection (UMAP) embedding revealed neuroepithelial cell trajectories corresponding to prosencephalic, telencephalic, diencephalic, mesencephalic, and rhombencephalic populations, consistent with regionalization of the developing brain (Figure 16d- 16e). Additionally, non-neuroepithelial trajectories, including non-neuroepithelial cells, neural crest derivatives and pluripotent stem cell-like populations, were identified (Figure 16d-16e). To quantify the influence of morphogen conditions on dorso-ventral and antero posterior fate identity, DV and AP scores were calculated using a regularized linear model, trained on the radial glia of the human developing brain cell atlas. As anticipated inhibition of Wnt (IWP2), TGFp (SB431542), BMP (LDN193189), Notch (DAPT) and no treatment, consistently yielded telencephalic progenitors within the cysts (Figure 16g, 16h), which robustly expressed the forebrain marker FOXG1 (Figure 17d, 16e), confirming maintenance of a dorsal-anterior neural fate (Figure 16i, 16j). Interestingly, all these anteriorizing conditions also maintained a spherical cystic structure suggesting that by suppressing posteriorizing influences, the cells maintain a more uniform, less regionally specified state, resulting in a simple spherical morphology (Figure 16k). In response to Shh activation (SAG), the cysts differentiated P28241 PCOO 28.08.2025

[0748] 106 / 119 into a ventral-anterior fate (Figure 16g-16j) and expressed NKX2-1 (Figure 161, Figure 17d). Corroborating with anterior morphology, SAG treated cysts, also maintained a cystic morphology (Figure 161, Figure 17c).

[0749] Activation of Wnt (CHIR99021) and retinoic acid (RA) signaling promoted posterior tissue fates and led to a drastic morphology change (Figure 16f, 16j, Figure 17c). Activation of Wnt signaling led to differentiation into mesencephalic territory (OTX2, EN2) in addition to the formation of SOX10 expressing neural crest cells (Figure 16m, Figure 17g, Figure 17d). RA activation elongated the cysts and drove the cells into rhombencephalic fate with expression of CYP26A1 and HOX genes including HOXB1 and HOXB4 (Figure 16n, Figure 17d). In response to BMP activation (BMP4), the cysts generated a multi-layered tissue resembling morphogenesis described previously (Karzbrun et al Nature 599, 268-272 (2021)) (Figure 16o) and generated a mix of non- neural ectoderm and telencephalic progenitors (Figure 17d). The inner layer formed a SOX2, PAX6, FOXG1 and OTX2 positive anterior neuroepithelium lined apically by NCAD (Figure 16o, Figure 17h). The intermediate non-neural layer expressed SOX2, PAX6, OTX2 and E cadherin suggesting formation of surface ectoderm. Finally, the outermost E cadherin expressing cells formed the epidermal-like cell layer (Figure 16o). These observations were also confirmed by single cell transcriptomics data, showing the presence of 3 distinct populations (Figure 17i). The innermost layer (NE) expressed anterior neuroepithelial markers. The intermediate layer (SE) expressed genes related to surface ectoderm Interestingly, these cells also expressed olfactory epithelium markers such as DLX5, suggesting the surface ectoderm might also be anteriorly patterned (Figure 17k). The outermost non-neural epithelial layer expressed surface ectoderm genes in addition to the genes reported to be present in amniotic ectoderm such as GATA3, ISL1 , RSPO3 and LIFR (Figure 17k).

[0750] Activation of FGF (FGF2, FG8b) dissolved the cystic structure, led to rapid cell proliferation and gave rise to a mix populations of FOXG1 -expressing telecephalic progenitors, POLI5F1 -expressing pluripotent stem cells and posterior cells expressing CYP26A1 (Figure 16d, 16e, 161). Lastly, upon activation of Nodal signaling (Activin A) cysts showed POLI5F1 expression and largely maintained the pluripotent stem cell fate (Figure 17d, 16e, 16m) corroborating with no specific role of nodal signaling during early neural tube patterning.

[0751] Collectively, these analyses showed that upon morphogen treatment the cyst acquired regional identities along the dorsal-ventral and anterior-posterior axes of the developing brain. In addition to the change in cell fate, we observed significant morphological plasticity in response to signalling P28241 PCOO 28.08.2025

[0752] 107 / 119 pathway manipulation. Thus, NECs serve as a powerful model for understanding the sophisticated interplay that guides complex tissue formation.

[0753] Example 12: Generation of regionally specified brain tissues from neuroepithelial cysts

[0754] To assess the capacity of single-lumen NECs to grow long term and generate diverse brain regions from a uniform starting culture, the differentiation was directed into regional organoids. The inventors optimized timing and duration of morphogens known to be involved in brain patterning and have been used in published protocols for forebrain (Kadoshima Proc Natl Acad Sci U S A. 2013 Dec 10;110(50):20284-9; Velasco Nature. 2019 Jun ;570(7762) :523-527), midbrain (Qian et al., 2016, Amin Cell Stem Cell. 2024 Dec 5;31(12):1831-1846.e9) and hindbrain (Muguruma et al Cell Rep. 2015 Feb 3;10(4):537-50, Atamian et al Cell Stem Cell. 2024 Jan 4;31(19):39-51.e6), Amin Cell Stem Cell. 2024 Dec 5;31 (12):1831-1846.e9) organoids (Figure 18a).

[0755] To induce forebrain differentiation, NECs were exposed to anteriorizing factors, specifically the TGF-p inhibitor SB431542 and the Wnt pathway inhibitor IWR1 , followed by Wnt activator CHIR99021 treatment in addition to matrigel from day 8 to day 14 (Figure 18a, Figure 19b). The inclusion of soluble Matrigel (1%) during early days of differentiation was important for preserving apical-basal polarity throughout the differentiation process. Removal of soluble Matrigel at day 10 resulted in disruption of tissue organization over time. Up to day 14, cysts maintained typical apico-basal polarization with collagen and laminin expression on the basal side and N cadherin expression on the apical side. However, by day 20, immunocytochemical analysis revealed the presence of N-cadherin on both the inner and outer surfaces of the developing neuroepithelium, indicating a loss of polarized organization (Figure 19a). This observation underscores the essential role of the extracellular matrix in maintaining neuroepithelial polarity during brain organoid development. Immunohistochemical analysis of organoids at day 30 of differentiation confirmed the expression of the anterior-dorsal neuroepithelial marker FOXG1 , alongside a single neuroepithelial unit delineated by apical N-cadherin staining (Figure 18b). However, as the organoids continued to develop, the initial organized neuroepithelium transitioned to a more randomized tissue architecture, characterized by the emergence of multiple neuroepithelial units over time marked by N cadherin on the apical surface (Figure 18c). Consistent with forebrain differentiation, the organoids exhibited expression of canonical cortical markers, including PAX6, TBR2, and CTIP2, as well as neuronal markers MAP2 (Figure 18c). P28241 PCOO 28.08.2025

[0756] 108 / 119

[0757] To direct differentiation towards a midbrain fate, NECs were treated with midbrain-inducing factors, specifically Wnt activator CHIR99021 , Sonic hedgehog activator SAG, and FGF8 signaling ligand FGF8b, used in guided midbrain organoids as well as identified as midbrain inducing in a morphogen screen (Amin Cell Stem Cell. 2024 Dec 5;31 (12):1831-1846.e9) (Figure 18a). In response to these morphogens, the organoids underwent rapid morphological changes, developing a more complex structure with expanded epithelium than the forebrain organoids (Figure 19e). Immunofluorescence analysis at day 30 of differentiation revealed the expression of the ventral midbrain floor plate marker FOXA2, along with other ventral midbrain markers, including LMX1A and NKX6-1 (Figure 18d). To test the effect of ECM on midbrain organoids, the differentiation in presence or absence of soluble matrigel was performed. Matrigel addition led to larger neuroepithelium formation in organoids with even more complex morphology (Figure 19f). However, both conditions gave rise to midbrain fate (Figure 19g). On day 60 the organoids maintained the expression of FOXA2 and consisted of ventral midbrain progenitors marked by LMX1A, NLIRR1 , NKX6-1 and midbrain dopaminergic neurons marked by tyrosine hydroxylase (TH). The organoids also consisted of GABAergic neurons expressing GAD67 and progenitor marker NKX6-1 (Figure 18e-f). The expression of these markers was comparable between the with or without ECM conditions, however with ECM condition showed a higher variability (Figure 19h-j)

[0758] To induce hindbrain tissue differentiation, NECs were treated with dual SMAD inhibition using Dorsomorphin and SB431542, coupled with posteriorizing factors such as Wnt activator CHIR99021 , FGFs (FGF2, FGF8b, FGF19), EGF, and insulin (Figure 18a, Figure 21a). By day 30, the resulting organoids exhibited neuroepithelium positive for SOX2 with multiple neuroepithelial units (Figure 21 b). Interestingly the organoids consisted of PAX6 positive populations that were mutually exclusive from rhombic lip progenitor marker ATOH1 positive cells. In addition, the ATOH1 territory was also negative for posterior marker HOXC7 (Figure 18g, Figure 21c-d). This suggests that the organoids consisted of hindbrain cells coming from different rhombomeres, where cells from rhombomeres 1 and 2 give rise to rhombic lip and do not express HOX genes and more posterior rhombomeres express HOX genes. On day 60, immunohistochemical analysis revealed the presence of SKOR2-positive Purkinje cells juxtaposed to Atohl -positive germinal cells, GABAergic neurons and HOXC7 expressing posterior cells (Figure 18h-i, Figure 21e-f).

[0759] To profile the cell types generated in the regional organoids we performed single cell RNA sequencing of the 90-day old forebrain and 60-day old midbrain and hindbrain organoids was performed. P28241 PCOO 28.08.2025

[0760] 109 / 119

[0761] Example 13: Retinal organoid generation from neuroepithelial cysts

[0762] In the following, a scalable retinal organoid methodology is presented. Moreover, the retinal organoid differentiation protocol, starting from NECs, employs a culture setup analogous to those established for other brain regions (Figure 18a), adapted with retinal-specific growth factors to guide neural retina development.

[0763] NECs were differentiated into early eye field with the inhibition of TGFb (SB431542), BMP (Dor- somorphin) and Wnt signalling (IWR1) (Nakano et al Cell Stem Cell. 2012 Jun 14;10(6):771-785, Zhou et al Development (2015) 142 (19):3294-3306). Then the proliferation and maintenance of retinal progenitor cells in retinal organoids was induced through sequential addition of BMP4 (Kuwahara Nat Commun. 2015 Feb 19:6:6286) and long term culture media described in previous studies (Cowan et al Cell. 2020 Sep 17;182(6):1623-1640.e34) (Figure 20a). Brightfield imaging revealed the progressive morphological development of neural retina organoids over an extended culture period till week 30 (Figure 20a). Early in differentiation, organoids appeared as compact spheres with a bright neuroepithelium, which gradually increased in size and complexity, exhibiting photoreceptor outer segments like structures by week 30 (Figure 22a). On day 120, a morphology-based selection strategy was used to enrich for well-formed retinal organoids by removing too dense and smaller organoids from the culture (Figure 22b). Quantitative analysis of organoid diameter over time showed a consistent increase in organoid size during differentiation, reaching a plateau around 90 days (Figure 22c).

[0764] Immunofluorescence analysis at day 60 demonstrated the presence of early retinal progenitor markers within the developing organoids, including PAX6, VSX2, and SOX9, along with the photoreceptor marker progenitor CRX, indicating the initiation of retinal specification (Figure 20b). By day 120, the organoids displayed clear evidence of retinal cell type differentiation and laminar organization (Figure 20c). Expression of photoreceptor progenitor marker CRX, mature photoreceptor markers ARR3 and OPN1SW (cones) and NRL (rods), bipolar cell marker PKC-a, amacrine cell marker AP2a, and Muller glia marker SOX9 was observed. Further investigation into laminar organization at day 120 revealed distinct distributions of NRL-positive rod photoreceptors in the outer nuclear layer and SOX9-positive Muller glia and other inner nuclear layer cells (Figure 20d). To quantify the organisation of outer and inner nuclear layers across organoids generated in multiple independent replicates, the organoids were embedded in tissue micro arrays on day 120 and performed immunohistochemistry analysis for NRL and SOX9 (Figure 22d). NRL and SOX2 positive cell distribution along the organoid's perimeter was profiled, quantifying cellular localization within 100 pm inward from its periphery (Figure 20e). This analysis confirmed the P28241 PCOO 28.08.2025

[0765] 110 / 119 formation of distinct layers in most organoids, with NRL-positive cells predominantly located at a specific distance from the organoid edge, while SOX9-positive cells were enriched in a more inner region (Figure 20f). This layered arrangement was consistently observed across four independent experiments and over 150 organoids measured (Figure 20f, 20g).

[0766] At week 25, the retinal organoids demonstrated advanced maturation, as evidenced by the laminar spatial localization and expression of key markers for cone photoreceptors (ARR3), rods (NRL and RHO), bipolar cells (PRKC-a), and general photoreceptors (Opsin) (Figure 20h). To comprehensively delineate and validate the cellular diversity within these mature organoids, single-cell transcriptomic profiling was performed. LIMAP visualization of week 25 organoids revealed a rich array of major retinal cell types, including Muller glia (MG), Intermediate Progenitor Cells (IPC), Amacrine Cells (AC), Horizontal Cells (HC), Bipolar Cells (BC), Rods, and Cones (Figure 20i). Further analysis of marker gene expression unequivocally confirmed the identity of these populations (Figure 20j). For instance, rods exhibited high expression of NRL and NR2E3, while cones specifically expressed GNAT2 and 0PN1SW. Similarly, Muller glia were characterized by the expression of S0X9 and DCN (Figure 20j, Figure 22e). To benchmark the cellular repertoire and maturation trajectory of the organoids of the present disclosure against existing methodologies, a transcriptomic comparison was performed with published retinal organoid datasets (Wahle et al Nat Biotechnol. 2023 Dec; 41 (12): 1765-1775). Correlation analysis of gene expression profiles revealed a strong transcriptomic similarity between the organoid cell types in the model of the present disclosure and those identified in published retinal organoids (Figure 20k). Finally, an unbiased analysis of the organoids' maturation trajectory by comparing their transcriptomes across different organoid ages in the Wahle et al. dataset was performed. This analysis indicated that the cell types within the organoids of the present disclosure most closely resembled the transcriptomes of 6-month-old reference organoids (Figure 20I). In conclusion, these analyses confirmed that NEC-derived retinal organoids present a scalable retinal organoid methodology utilizing exclusive suspension culture, effectively overcoming the challenge of co-generating retinal pigmented epithelium.

Claims

P28241 PCOO 28.08.2025111 / 119PATENT CLAIMS1. Process of producing neuroepithelial cysts, the process comprising the steps of: a) seeding pluripotent stem cells in microwells at a density from 1 to 15 cells per microwell, wherein each microwell has a diameter from 100 pm to 1’600 pm; b) culturing the seeded cells of a) in the respective microwells for a first culture period lasting from 1 to 3 days in the presence of a first culture medium, thereby obtaining in each microwell a first cell population forming an epiblast cyst; and c) culturing the first cell populations obtained in step b) for a second culture period lasting at least 4 days in the presence of a second culture medium comprising a neural differentiation agent, thereby obtaining a plurality of second cell populations each comprising neuroepithelial cysts; wherein from no later than day 6 after seeding onwards, the second culture medium is supplemented with an extracellular matrix gel.

2. Process according to claim 1 , wherein in step a) the stem cells are seeded in the microwells at a density from 1 to 14, preferably from 1 to 13, more preferably from 8 to 12, cells per microwell.

3. Process according to any one of the previous claims, wherein from no later than day 5 after seeding onwards, preferably from day 4 after seeding onwards, the second culture medium is supplemented with Matrigel.

4. Process according to any one of the previous claims, wherein the second culture medium is supplemented with the extracellular matrix gel from no later than day 4 after seedingP28241 PCOO 28.08.2025112 / 119 onwards and preferably at a concentration from 1 vol.-% to 20 vol.-%, more preferably from 2 vol.-% to 10 vol.%, even more preferably from 3 vol.-% to 5 vol.-%.

5. Process according to any one of the previous claims, wherein each microwell comprises a hydrogel and / or wherein each microwell comprises a modified plastic that imparts non-ad- herent properties to the microwell and / or wherein at least a section of the inner surface of each microwell has a non-adherent coating.

6. Process according to any one of the previous claims, wherein the second culture medium is refreshed at least once every 3 days, more preferably at least once every 2 days, during the second culture period.

7. Process according to any one of the previous claims, wherein the first culture period lasts 2 days; and / or wherein the second culture period lasts at least 6 days.

8. Process according to any one of the previous claims, wherein each microwell has a diameter from 100 pm to 1’000 pm, preferably from 300 pm to 800 pm, more preferably from 400 pm to 500 pm.

9. Process according to any one of the previous claims, the process comprising the steps of: a) seeding the pluripotent stem cells in the microwells at a density from 5 to 13 cells per microwell, wherein each microwell has a diameter from 300 pm to 800 pm and a nonadherent inner surface; b) culturing the seeded cells of a) in the respective microwells under non-adherent culture conditions for the first culture period lasting 2 days in the presence of a first culture medium comprising a stem cell maintenance medium and preferably further comprising aP28241 PCOO 28.08.2025113 / 119ROCK inhibitor, thereby obtaining in each microwell a first cell population forming an epiblast cyst; and c) culturing the first cell populations obtained in step b) in the respective microwells under non-adherent culture conditions for the second culture period lasting at least 5 days in the presence of the second culture medium comprising a neural induction medium comprising N2 supplement, thereby obtaining a plurality of second cell populations each comprising neuroepithelial cysts; wherein from day 4 after seeding onwards, the second culture medium is supplemented with Matrigel at a concentration of from 1 vol.-% to 20 vol.-%.

10. Process of producing a plurality of organoids, comprising producing neuroepithelial cysts according to the process of any one of the previous claims, and further comprising the step of: d) culturing the second cell populations for a third culture period in a third culture medium comprising a third differentiation agent, thereby obtaining the plurality of organoids.

11. Process according to claim 10, wherein between steps c) and d) the second cell populations are transferred from the microwells to a cell culture plate or a cell culture dish, preferably to a ultra-low attachment cell culture dish.

12. Process according to any one of claims 10-11 , wherein the third culture medium comprises N2 supplement.

13. Process according to any one of claims 10-12, wherein during at least the first 5 days of the third culture period, preferably during at least the first 8 days of the third culture period,P28241 PCOO 28.08.2025114 / 119 the third culture medium is supplemented with an extracellular matrix gel, preferably Mat- rigel.

14. Process for generating forebrain organoids, comprising performing the process according to any one of claims 10-13, wherein in step d), the second cell populations are treated with a TGF beta pathway inhibitor, a Wnt pathway inhibitor and extracellular matrix gel for a period of at least two days during the third culture period.

15. Process according to claim 14, wherein the TGF beta pathway inhibitor comprises or consists of SB 431542; and / or wherein the Wnt pathway inhibitor comprises or consists of IWR1 ; and / or wherein the extracellular matrix gel comprises or consists of Matrigel or Cultrex.

16. Process according to any one of claims 14-15, wherein at least from day 8 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with SB 431542 and with the extracellular matrix gel, preferably at a concentration from 5 pM to 15 pM SB 431542 and from 0.1 vol.-% to 5 vol.-% extracellular matrix gel, preferably Cultrex.

17. Process according to any one of claims 14-16, wherein at least from day 8 after seeding until day 10 after seeding, the second cell populations are treated with IWR1 , preferably at a concentration from 1 pM to 5 pM IWR1 ; and wherein at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with IWR1 or CHIR99021 , preferably at a concentration from 1 pM to 5 pM IWR1 respectively from 0.1 pM to 2 pM CHIR99021.

18. Process according to any one of claims 14-17, wherein at least from day 6 after seeding until day 8 after seeding, the first cell populations are treated with SB 431542, IWR1 andP28241 PCOO 28.08.2025115 / 119 extracellular matrix gel, preferably at a concentration from 5 pM to 15 pM SB 431542, from 1 pM to 5 pM IWR1 and from 1 to 10 vol.-% extracellular matrix gel, preferably Matrigel.

19. Process for generating midbrain organoids, comprising performing the process according to any one of claims 10-13, wherein in step d), the second cell populations are treated with a Wnt pathway activator, a Sonic Hedgehog pathway activator, and a fibroblast growth factor for a period of at least four days during the third culture period.

20. Process according to claim 19, wherein the Hedgehog pathway activator comprises or consists of SAG; and / or wherein the Wnt pathway activator comprises or consists of CHIR99021 ; and / or wherein the fibroblast growth factor comprises or consists of FGF8b.21 . Process according to any one of claims 19-20, wherein at least from day 10 after seeding until day 14 after seeding, preferably from day 8 after seeding until day 16 after seeding, the second cell populations are treated with CHIR99021 , SAG and FGF8b, preferably at a concentration from 1 pM to 2 pM CHIR99021 , from 0.1 pM to 1.0 pM SAG, from 50 ng / ml to 150 ng / ml FGF8b.

22. Process according to any one of claims 19-21 , wherein at least from day 10 after seeding until day 14 after seeding, preferably from day 8 after seeding until day 16 after seeding, the third culture medium comprises an extracellular matrix gel, preferably Matrigel, at a concentration from 0.1 vol.-% to 5 vol.-%.

23. Process for generating hindbrain organoids, comprising performing the process according to any one of claims 10-13, wherein in step d), the second cell populations are treated with insulin, FGF2 and EGF for a period of at least two days.

24. Process according to claim 23, wherein at least from day 6 after seeding until day 18 after seeding, preferably at least until day 22 after seeding, the first cell populations respectivelyP28241 PCOO 28.08.2025116 / 119 the second cell populations are treated with SB431542, Dorsomorphin, CHIR99021 , insulin, FGF2 and EGF, preferably at a concentration from 5 pM to 15 pM SB431542, from 1.0 pM to 4.0 pM Dorsomorphin, from 0.5 pM to 2.5 pM CHIR99021 , from 4 pg / ml to 10 pg / ml insulin, from 15 ng / mL to 25 ng / mL FGF2 and from 15 ng / mL to 25 ng / mL EGF.

25. Process according to any one of claims 23-24, wherein from day 6 after seeding until day 8 after seeding, the first cell populations are further treated with an extracellular matrix gel, preferably Matrigel, at a concentration from 1 vol.-% to 10 vol.-%.

26. Process according to any one of claims 23-25, wherein from day 8 after seeding until day 14 after seeding, the second cell populations are treated with FGF8b, preferably at a concentration from 50 ng / ml to 150 ng / ml.

27. Process according to any one of claims 23-26, wherein from day 14 until at least day 20, preferably until day 22, after seeding, the second cell populations are treated with FGF19, preferably at a concentration from 50 ng / ml to 150 ng / ml.

28. Process for generating retinal organoids, comprising performing the process according to any one of claims 10-13, wherein in step d), the second cell populations are treated with a BMP pathway inhibitor, a TGF-beta pathway inhibitor, and a Wnt pathway inhibitor for a period of at least four days during the third culture period, and afterwards with BMP4 for a period of at least six days during the third culture period.

29. Process according to claim 28, wherein the BMP pathway inhibitor comprises or consists of Dorsomorphin; and / or wherein the TGF-beta pathway inhibitor comprises or consists of SB 431542; and / or wherein the Wnt pathway inhibitor comprises or consists of IWR1.

30. Process according to any one of claims 28-29, wherein at least from day 6 after seeding until day 10 after seeding, preferably until day 12 after seeding, the first cell populationsP28241 PCOO 28.08.2025117 / 119 respectively the second cell populations are treated with Dorsomorphin, SB 431542 and IWR1 , preferably at a concentration from 5 pM to 15 pM SB 431542, from 2 pM to 4 pM IWR1 and from 1.0 pM to 4.0 pM Dorsomorphin.31 . Process according to any one of claims 28-30, wherein at least from day 12 after seeding until day 22 after seeding, preferably until day 24 after seeding, the second cell populations are treated with BMP4, preferably at a concentration from 25 ng / ml to 75 ng / ml.

32. Process for generating choroid plexus organoids, comprising performing the process according to any one of claims 10-13, wherein in step d), the second cell populations are treated with CHIR99021 for a period of at least four days during the third culture period.

33. Process according to claim 32, wherein at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with CHIR99021 , preferably at a concentration from 1 pM to 5 pM.

34. Process according to claim 32 or 33, wherein at least from day 10 after seeding until day 14 after seeding, preferably until day 16 after seeding, the second cell populations are treated with SB431542, preferably at a concentration from 1 pM to 5 pM.

35. Neuroepithelial cysts produced by the process of any one of claims 1-9.

36. Organoids produced by the process of any one of claims 10-34.

37. Choroid plexus organoids produced by the process of any one of claims 32-34, wherein the choroid plexus organoids have apical-out polarity and / or wherein the choroid plexus organoids have a cell density of at least 2500 cells / mm2, preferably from 3000 cells / mm2to 8000 cells / mm2, more preferably from 4000 cells / mm2to 6000 cells / mm2.P28241 PCOO 28.08.2025118 / 11938. Use of the neuroepithelial cysts of claim 35 or of the organoids of claims 36 or 37 for one or more of the following applications: disease modelling, modelling of drug responses, development of new pharmaceutically active ingredients, high content screening, identification of biological targets and cell therapy.

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