WO2023121449A1

WO2023121449A1 - Central nervous system organoids

Info

Publication number: WO2023121449A1
Application number: PCT/NL2022/050739
Authority: WO
Inventors: Johannes Carolus Clevers; Benedetta Artegiani; Delilah Florentine Gertrude HENDRIKS
Original assignee: Koninklijke Nederlandse Akademie Van Wetenschappen; Prinses Maxima Centrum Voor Kinderoncologie B.V.
Priority date: 2021-12-21
Filing date: 2022-12-20
Publication date: 2023-06-29

Abstract

The present invention provides a CNS organoid capable of propagation by dissection and culture of the organoid. Further provided is a method of producing, propagating or inducing maturation of an organoid of the present invention. The present invention also provides a maturation medium and an expansion medium and a kit of parts comprising such media and optionally an organoid of the invention.

Description

Central Nervous System Organoids

Field of Invention

[0001] The present invention relates to CNS organoids, and to methods by which such CNS organoids may be produced. It also relates to methods of propagating CNS organoids and of inducing maturation of CNS organoids. The invention further relates to cell culture media for the expansion or maturation of CNS organoids.

Background

[0001] Brain development is a complex process that requires a careful balance of stem cell and progenitor expansion and their differentiation into neurons and glial cells. Radial glial stem cells lining on the ventricles - in the so-called ventricular zone (VZ) - divide to generate more differentiated progenitors, the intermediate progenitors, located on top of the VZ, the subventricular zone (SVZ). Neurons, generated from all the progenitors, migrate on top of germinal layers, and undergo highly regulated fate specification. Most of our knowledge is derived from studies in animal models. However, fully translating this information to human brain development is problematic, given the considerable changes that occurred during brain evolution, notably from rodents to primates. More complex organization, a strikingly expanded and folded neocortex, and higher abundance of specific cell types, such as the basal radial glia, are hallmarks of the human brain.

[0002] In the last decade, much progress has been made regarding generation of human brain organoids that more and more closely mimic the organization, regional and cellular specification as well as functionalities of the developing brain, alongside with improved reproducibility. The common ground of all these human brain organoid cultures is the starting material, i.e. pluripotent stem cells (PSCs), either induced or embryonic. The use of PSCs allows the generation of organoids by attempting to mimic the differentiation cascades that naturally occur during embryonal brain development. In fact, a plethora of published differentiation paradigms by using seemingly physiological molecular cues, can achieve the imprinting towards the proper germinal layer, the neuroectoderm, and then further maturation into structures containing neural stem cells, glial cells and neurons. Not guided or guided (patterned) induction and differentiation protocols also revealed successful in generating either organoids containing at the same time various brain identities or representing specific and discrete brain regions.

[0003] Human and mouse tissues showed the ability to directly grow into organoids from somatic stem cells upon the appropriate culture conditions that resemble the endogenous niche, without the need of cellular reprogramming. These tissue-derived human organoids are different compared to the PSC-derived organoids: they do not require specification but rather culture conditions that allow at the same time the expansion, the maintenance, and the generation of more differentiated cell types physiologically present and specified in the tissue origin. As such, they constitute self-sustained expandable culture systems showing a high degree of reproducibility across organoid lines. While others successfully generated organoids cultures from essentially all human organs, amongst which intestine, liver, pancreas, etc., comparable brain-derived systems do not exist.

[0004] While in PSCs-derived organoids single brain areas can be generated to a certain extent by using patterning factors, they do not capture the human brain diversity, which can naturally maintain the proper regional identities and tissue interaction, such as correct unidirectional cell migration.

[0005] Jacob, Fadi, et al. "A patient-derived glioblastoma organoid model and biobank recapitulates inter-and intra-tumoral heterogeneity." Cell 180.1 (2020): 188-204. discloses methods for generating patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. However, these GBOs consist of tumorous (unhealthy) tissue, limiting their applicability in the study of the central nervous system not in a disease state and for studying normal development.

Summary of Invention

[0006] In a first aspect, the invention provides a CNS organoid capable of propagation by dissection and culture of the organoid.

In a second aspect, the invention provides a CNS organoid consisting of foetal CNS cells that are TBXT-, COL1A1-, and LUM-.

In a third aspect, the invention also provides a CNS organoid consisting of foetal CNS cells that are SOX17-, HNF3B-, and GATA4-.

[0007] The features of the CNS organoids of the various aspects of the invention described herein are able to be combined with one another. For example, a CNS organoid capable of propagation in accordance with the first aspect of the invention may also express the markers characteristic of the second and/or third aspects of the invention. Similarly, a CNS organoid of the first, second, or third aspect of the invention may also have a structure or comprise cells expressing defined markers as set out elsewhere in the specification.

[0008] An organoid of the invention (in accordance with any of the aspects or embodiments described herein) is formed of cells of foetal origin. Accordingly, any such organoid of the invention comprises or consists of foetal cells. The organoids provided herein are directly derived from foetal tissue. This is in contrast to other organoids that are derived from embryonic stem cells (ESCs) and/or induced pluripotent stem cells (iPSCs). That is to say that the CNS organoids of the invention are not derived from ESCs and/or iPSCs. This allows for CNS organoids and methods of making such CNS organoids that do not require specification to region or cell identity unlike organoids derived from ESCs and/or iPSCs.

[0009] In a fourth aspect, the invention provides a method of producing a CNS organoid, the method comprising:

• mechanically dissecting an isolated sample of foetal CNS tissue to produce portions of foetal CNS tissue comprising foetal CNS cells that maintain cell-to-cell contacts; and

• culturing a portion of foetal CNS tissue in an expansion medium for a period until a CNS organoid is formed from the portion.

[0010] In a fifth aspect, the invention provides a method of propagating a CNS organoid, the method comprising:

• producing a CNS organoid by a method according to the fourth aspect of the invention;

• culturing the resultant organoid until it has a diameter of at least approximately 2 mm;

• mechanically dissecting the organoid to produce at least two organoid fractions; and

• culturing the organoid fractions in an expansion medium for a period until a CNS daughter organoid is formed from the fractions.

[0011] In a sixth aspect, the invention provides a method of inducing maturation of a CNS organoid, the method comprising:

• producing a CNS organoid by a method according to any embodiment of the fourth or fifth aspects of the invention;

• culturing the organoid in a maturation medium until the organoid is matured.

[0012] The methods of the fourth, fifth and sixth aspects of the invention can respectively be used for producing, propagating and maturing organoids in accordance with any of the aspects of the invention disclosed herein.

[0013] In a seventh aspect, the invention provides an expansion medium comprising EGF, FGF-10 and FGF-2.

[0014] The methods of the fourth or fifth aspects of the invention may be practiced using an expansion medium in accordance with the eighth aspect of the invention.

[0015] In an eighth aspect, the invention provides a maturation medium comprising basement membrane extract. [0016] The methods of the sixth aspect of the invention may be practiced using a maturation medium in accordance with the eighth aspect of the invention.

[0017] In a ninth aspect, the invention provides a kit comprising an expansion and/or maturation medium of the invention. Optionally the kit includes an organoid of the invention.

Brief Description of Figures

[0018] Figure 1 shows donor characteristics of FeBOs and marker expression in human fetal brain tissue, a, Overview of the gender and age of the different human fetal tissue donors used to derive FeBOs. b, Overview of the number of months in culture of different FeBO lines since line derivation. Arrowheads indicate that lines are actively expanding in culture, c, Examples of brightfield images of FeBOs derived from different donors, d, Representative confocal imaging of immunofluorescent stainings for SOX2, MKI67, TLIJ1, and PAX6 of corresponding human fetal brain tissue;

[0019] Figure 2 shows generation of human brain organoids through self-organization of human fetal brain tissue, a, Main steps for the establishment of the tissue-derived fetal human brain organoid (FeBOs) lines and corresponding representative brightfield images, b, Brightfield images of a FeBOs reassembly after splitting, c, Volume expansion of FeBOs from different donors in between splitting. Each dot represents a different independent FeBO. d, Estimated total biomass produced by the expansion of a FeBO line over culturing time, e, Quantification of the different cell type distribution (neural stem cells, proliferating cells and neurons) within the FeBOs. Each dot represents quantification in a different independent FeBO f, Representative confocal imaging of immunofluorescent stainings for the indicated markers at different magnifications depicting the cellular composition and distribution of the FeBOs;

[0020] Figure 3 shows characterization of FeBOs through viral infection, immunofluorescence, and transmission electron microscopy, a, Representative confocal imaging of immunofluorescent staining for SOX2 in FeBOs infected with a H2B::mCherry lentivirus at different days post infection. Note the gradual migration of infected cells towards the inside of the organoid, b, Representative confocal imaging of immunofluorescent stainings at different magnifications for HOPX and PAX6 (top) and HOPX and MKI67 (bottom) in FeBOs. Arrowheads point toward colocalization of HOPX and MKI67. c, Representative confocal imaging of immunofluorescent stainings of the apical marker ZO-1 in FeBOs. d, Representative transmission electron microscopy images of FeBOs demonstrating the presence of typical brain features. * indicates myelin sheets, g = golgi, m = mitochondria, N = nucleus, rER = ribosomal endoplasmic reticulum, nf = neurofilament, nt = neurite; [0021] Figure 4 shows FeBOs generated from different brain regions maintain cellular and molecular identity of the tissue of origin, a, Schematic showing the strategy for the derivation of regional FeBOs from fetal brain material, b, Brightfield images of FeBOs derived from the dorsal (top) and ventral (bottom) forebrain, c, Differences in relative expression (normalized to ACTB) of specific regional markers quantified by qPCR in dorsal (D) and ventral (V) forebrain- derived FeBOs. Each dot represents the average of a triplicate measurement from a different independent FeBO. d, Representative confocal imaging of immunofluorescent stainings for the indicated markers of ventral (top) and dorsal (bottom) forebrain-derived FeBOs at lower (left) and higher magnification, e, Level of selected transcript expression from bulk RNA-sequencing analysis of dorsal and ventral FeBOs, showing no expression of pluripotency (P) and mesenchymal (M) genes, and expression of markers associated with specific populations of the developing brain: apical progenitors (AP), intermediate progenitors (IP), dividing progenitors (D), migrating neurons (MN) and neurons (N). Each dot represents bulk-RNA sequencing normalized transcript count of an independent FeBO. f, Principal component plot (PCA) for the bulk-RNA sequencing of FeBOs derived from dorsal (green) or ventral (orange) forebrain tissue in expansion condition and FeBOs in maturation condition (see Figure 3). g, Hierarchical clustering analysis and heat-map depicting the z-score values for the significant (p-value <0.05) differentially expressed genes between ventral and dorsal forebrain derived- FeBOs. Each row represents the transcriptome of an independent FeBO. h, Barplots showing the estimated cell abundancy in dorsal (top) and ventral (bottom) FeBOs of different ages, as calculated by deconvolution of bulk-RNA transcriptomes. Each dot represents the transcriptome of an independent FeBO;

[0022] Figure 5 shows assignment of brain tissue region identity through marker expression analysis, a, Schematic of the expression of typical markers defining the different regions in the developing human fetal brain. On the right, markers used to define dorsal vs. ventral forebrain are depicted. T = telencephalon, D = diencephalon, M = midbrain, H = hindbrain, b, qPCR analysis of the expression of a panel of markers in different pieces of human fetal brain tissue subsequently used for FeBO line establishment. Note that tissue sample 5 does not have an assigned tissue identity due to lack of a typical expression profile and is therefore not used for FeBO generation;

[0023] Figure 6 shows marker expression during long-term culture of FeBOs and generation of fused telencephalic FeBOs. a, Representative confocal imaging of immunofluorescent stainings for SOX2, MKI67, and TLIJ1 in 2- and 6-month old FeBOs. Quantifications of the amount of positive cells per organoid area are presented. Each dot represents an individual FeBO. b, Representative confocal imaging of immunofluorescent stainings for DLX2 and PAX6 in 2- and 6-month old ventral and dorsal FeBOs. Quantifications of the amount of positive cells per organoid area are presented. Each dot represents an individual FeBO. c, Representative confocal imaging of immunofluorescent stainings for NKX2.1 , GAD65, and GAD67 in 2- and 6- 8-month old ventral forebrain-derived FeBOs. Quantification the amount of positive cells per organoid area are presented. Each dot represents an individual FeBO. d, Representative confocal imaging of immunofluorescent stainings for astrocytic marker GFAP and neuronal marker MAP2 in 8-month old FeBOs. e, A schematic representation of the experimental procedure to generate human fetal dorsal-ventral forebrain fusoids is shown. On the right, representative images of a fused ventral (infected with H2B::mCherry) and dorsal (infected with H2B::GFP) forebrain organoid taken at day 2, 7, and 11 post co-culturing. Note the migration of H2B::mCherry ventral forebrain-derived cells into the dorsal forebrain organoid area;

[0024] Figure 7 shows transcriptomic comparison between FeBOs derived from dorsal and ventral forebrain and the effects of long-term culture, a, Selected GO-terms on significantly upregulated genes in dorsal (left) and ventral (right) forebrain-derived FeBOs when comparing dorsal vs. ventral forebrain-derived FeBOs (log2FC < 1). b, Volcano plot on differentially expressed genes between ventral and dorsal forebrain-derived FeBOs (log2FC < 1). c, Heatmap depicting the transcriptome of dorsal forebrain-derived FeBOs over time in culture with hierarchical clustering only on genes. Note the highlighted cluster demonstrating gradual changes during long-term culture. Selected terms on genes selected from this cluster from the human gene atlas are depicted, d, Expression of selected genes from the highlighted cluster in c in dorsal forebrain-derived forebrain organoids during long-term culture. Each dot represents bulk-RNA sequencing normalized transcript count of an independent FeBO. e, Heatmap depicting the transcriptome of ventral forebrain-derived FeBOs over time in culture with hierarchical clustering only on genes. Note the highlighted cluster demonstrating gradual changes during long-term culture, f, Expression of selected genes from the highlighted cluster in e in dorsal forebrain-derived forebrain organoids during long-term culture. Each dot represents bulk-RNA sequencing normalized transcript count of an independent FeBO;

[0025] Figure 8 shows Transmission electron microscopy on matured FeBOs. a, Examples of typical subcellular structures in matured FeBOs. m = mitochondria, g = golgi apparatus, rER = ribosomal endoplasmic reticulum, b, (b-b”) Examples of cross-sections of axons presenting with neurofilaments (nf) and neurites (nt); (b’”) a top view of an axon is presented, c, (c-c’) Examples of synaptic vesicle-like (SV) structures in matured FeBOs. d, Examples of oligodendrocytic myelin sheath-like structures wrapped around axons in matured FeBOs. High magnifications of marked areas are presented in / and //. e, (e-e”) Examples of myelin sheathlike structures; [0026] Figure 9 shows Cellular and molecular maturation of FeBOs. a, Schematic showing that expansion culture conditions allow the long-term expansion of FeBOs that at any given time can be subjected to further maturation by indicated changes in the culture conditions, b, Barplot showing the changes in cell abundancy in expansion vs maturation culture conditions, c, Heatmaps showing the significant (pvalue<0.05) differentially expressed genes in maturation vs expansion for dorsal (left) and ventral (right) forebrain FeBOs. Upregulated (in pink) and downregulated (in black) markers are shown. Each column represents the transcriptome of an independent FeBO. d, Representative confocal imaging of immunofluorescence stainings of FeBOs in maturation conditions for different specific neuronal markers (SATB2, CTIP2, BRN2 and TLIJ1) and stem cell/proliferation markers (SOX2 and MKI67). e, Live imaging of electric activity in FeBO slices in maturation media. Confocal representative images of a time-course live imaging experiment of an FeBOs slice cultured in maturation media and incubated with the fluorescence calcium indicator Fluo-4 (top). Asterisks in the first frame indicate the position of cells displaying spontaneous calcium spikes as visualized by changes of fluorescence overtime (following frames). Examples of calcium recording in different cells as changes of fluorescence intensity in time (bottom). Interval of recording was 2 sec. f, Representative confocal imaging of immunofluorescent staining for MAP2 at different magnifications in matured dorsal forebrain FeBOs. g, Representative confocal imaging of immunofluorescent staining for DLX2 and OLIG2 (left) and GAD65 and NKX2-1 (right) at different magnifications in matured ventral forebrain FeBOs. h, Representative confocal imaging of immunofluorescent stainings for SATB2 and CTIP2 in FeBOs upon maturation at different timepoints in culture demonstrating distinct layering over time. Quantification of the percentage of distribution of SATB2+, CTIP2+, and SATB2+CTIP2+ cells relative to the total amount of positively stained cells per bins ranging from the edge to the center of the organoid is given per time point

[0027] Figure 10 shows characterization of matured FeBOs through genetic labeling and immunofluorescence, a, On the left, schematic representation of the experimental set-up to visualize single cells in FeBOs upon maturation. On the right, examples of different labeled neurons color coded by depth, b, Representative confocal imaging of immunofluorescent staining for GAD65 and NKX2.1 in ventral forebrain-derived FeBOs. Note the ring structure- 1 ike organized GAD65 expression that is observed in some organoids, c, Quantifications of marker expression per organoid area in FeBOs in expansion or maturation medium. Each dot represents an independent FeBO. d, Representative confocal imaging of immunofluorescent stainings for different neuronal markers in matured FeBOs;

[0028] Figure 11 shows cell type-specific marker expression in FeBOs in expansion and maturation medium, a, Expression of cell type-specific markers in expanding and matured FeBOs. Each dot represents bulk-RNA sequencing normalized transcript count of an independent FeBO. Dashed lines indicate expression levels observed in human fetal brain tissue, b, Analysis of cumulative expression of genes belonging to specific neuronal features (derived from GO-annotation) in expanding and matured FeBOs as well as in human fetal brain tissue. The sum of bulk-RNA sequencing normalized transcript counts of all genes belonging to each class is shown, c, Expression of selected genes involved in specific neuronal features derived from the classes in b in expanding and matured FeBOs. Each dot represents bulk-RNA sequencing normalized transcript count of an independent FeBO. Dashed lines indicate expression levels observed in human fetal brain tissue;

[0029] Figure 12 shows single-cell RNA sequencing analysis of ventral forebrain-derived FeBOs. a, LIMAP of ca. 25K cells derived from ventral forebrain FeBOs, showing initial clustering into 13 clusters b, Violin plots of several genes defining the different clusters as defined in main figure 4a. c, Cell type-specific signature analysis. Each signature is based on a specific set of genes derived from previous studies;

[0030] Figure 13 shows single cell sequencing reveals cellular composition of FeBOs. a, LIMAP plot of ca. 25K cells of ventral forebrain-derived FeBOs colored by annotated cell types, b, LIMAP plots displaying expression of cell type identity depicting progenitors (NESTIN), glycolytic radial glia (BNIP3), astrocytes (GFAP), dividing progenitors (PCNA, ASPM), oligodendrocyte precursors (OLIG2), neuronal precursors (ASCL1 , HMGB2), and interneurons (CALB2, DLX2, DLX5, GAD2) in ventral forebrain-derived FeBOs. c, Dot plot depicting expression of markers defining the different cell types in ventral forebrain-derived FeBOs. d, LIMAP plot of ca. 25K cells of dorsal forebrain-derived FeBOs colored by annotated cell types. e, LIMAP plots displaying expression of cell type identity depicting progenitors (HES5), glycolytic radial glia (BNIP3), astrocytes (AGT, GFAP), dividing progenitors (PCNA), and neurons (BCL11 B, DCX). f, Dot plot depicting expression of markers defining the different cell types in dorsal forebrain-derived FeBOs;

[0031] Figure 14 shows Genetic engineering of FeBOs enables interrogation of gene function, a, Schematic of CRISPR engineering of FeBO pieces by electroporation and a representative example of a brightfield and GFP fluorescence overlay image demonstrating transfected (GFP⁺ cells) 24 hours post electroporation (p.e.). b, Brightfield and GFP fluorescence overlay images of TP53'^/_ and control organoids depicting stably integrated GFP and enrichment of gene- modified GFP⁺ cells in TP53'^/_ organoids two months post electroporation, c, Quantification of the GFP fluorescence area within individual organoids (dots) derived from an initially transfected organoid (#1 , #2) in control and TP53'^/_ conditions. Note the presence of more organoids (dots) in TP53'^A organoids indicating more splitting events (enhanced growth), d, Representative confocal imaging of immunofluorescent stainings for SOX2, MKI67, and GFP in control and TP53'^/_ FeBOs. GFP signal indicates genome-edited cells, e, Schematic of the experimental setup to interrogate gene function in brain development using CRISPR. f, Representative example of a section of a control FeBO demonstrating localization of stably integrated GFP 8 days post electroporation (p.e.). g, Representative confocal imaging of immunofluorescent stainings for SOX2 and GFP in control and RAB3GAP2 sgRNA FeBOs. Arrowheads indicate overlapping GFP and SOX2 signal, h, Quantification of co-localization of specific progenitor, neuronal, and astrocyte markers in the GFP⁺ population in control and RAB3GAP2 sgRNA FeBOs. Each dot represents an individual organoid, i, Relative abundancy of stem cells and neurons in the GFP⁺ population in control and RAB3GAP2 sgRNA FeBOs. j, Representative confocal imaging of immunofluorescent staining for TUJI sgRNA FeBOs;

[0032] Figure 15 shows Characterization of CRISPR-engineered FeBOs. a, Schematic representation of the generation and use of TP53'^/_ FeBOs. b, Schematic of the sgRNA used to introduce TP53 mutations and an example of a genotype of a TP53'^/_ FeBO. c, On the left, sections of TP53'^A and control FeBOs. On the right, representative confocal imaging of immunofluorescent staining for MKI67 in TP53'^A FeBOs. Note the high overlap of MKI67 signal within the GFP⁺ population, d Representative confocal imaging of immunofluorescent staining for TP21 (TP53 target gene) in TP53'^/_ organoids. Note the minimal overlap between GFP⁺ cells and TP21 signal, e, Quantification of MKI67⁺ cells relative to the total amount of cells (top) and within the GFP⁺ population (bottom), f, Schematic of the proposed role of TP53 in human fetal brain development, g, Expression profile of RAB3GAP2 in the different cell types in FeBOs as determined through generating pseudobulk data computed from scRNA-sequencing data. Expression profile of RAB3GAP2 in expanding and matured FeBOs. Each dot represents bulk- RNA sequencing normalized transcript count of an independent FeBO. h, A max projection and a slice of an electroporated FeBO 2 days post electroporation (p.e.), demonstrating the majority of transfected (GFP⁺) cells to initially reside on the outside of the organoid, i, Schematic of the proposed role of RAB3GAP2 in neuronal differentiation during human fetal brain development;

[0033] Figure 16 shows establishment of CNS organoids from human fetal spine, a, Brightfield images of a fetal human spine tissue used for the establishment of human fetal tissue spine organoids and an example of a derived organoid, b, Relative expression (normalized to ACTB) of specific spine progenitor regional markers (left) and spine motor neuron markers (left) quantified by qPCR. Each dot represents the average of a triplicate measurement from a different independent human fetal tissue spine organoids, c, Representative confocal imaging of immunofluorescent stainings for the indicated markers the cellular composition and presence of different cell types (progenitors as well as neurons), d, Representative confocal imaging depicting sparse labelled cells (by electroporation of transposable RFP) and their colocalization with the motor neuron marker HB9; and

[0034] Figure 17 shows similarity of ECM-component expression of the CNS organoids of the actual fetal brain and specific secretion of ECM proteins, a, Heatmap showing the level of expression of multiple ECM components in the human fetal brain and in the fetal human brain- derived organoids (FeBOs). b, heatmap showing the results of secretome analysis from FeBOs and neurosphere, showing the specific and higher production of ECM component in the FeBOs. [0035] Figure 18 shows hierarchical clustering analysis and heat-map depicting the z-score values for gene markers of the different subareas of the human cortex in different cortex-derived FeBOs (each column represents an independent FeBO). On the left, the expression pattern of each gene across the different cortical areas as identified in human fetal brain tissue is displayed, where the presence of a box indicates enriched expression in that region.

[0036] Figure 19 shows a, a Correlation plot showing the primary tissue age resemblance of ventral forebrain FeBOs based on pseudobulk comparisons with the dataset on primary human ganglionic eminence tissues. MGE = medial ganglionic eminence, LGE = lateral ganglionic eminence, CGE = caudal ganglionic eminence, b, Correlation plot comparing organoid-tissue clusters with assigned cell identities between ventral forebrain FeBOs and primary tissue (GE 14GW). c, LIMAP representation showing the integration of expanded and matured ventral forebrain FeBO datasets with the dataset on primary ganglionic eminence tissue (14GW), showcasing identification of common clusters with cells derived from both organoids and tissue, d, Correlation plot comparing organoid-tissue clusters with assigned cell identities between dorsal forebrain FeBOs and primary tissue (cortex 14GW). e, LIMAP representation showing the integration of expanded and matured dorsal forebrain FeBO datasets with the dataset on primary cortex tissue (14GW), showcasing identification of common clusters with cells derived from both organoids and tissue.

Detailed Description

[0037] Each of the first, second and third aspects of the present invention relates to CNS organoids.

[0038] The CNS organoids of the invention are capable of propagation, as reflected in the definition of CNS organoids of the first aspect of the invention, which are characterised in that they are capable of propagation by dissection and culture of the organoid.

[0039] The CNS organoids of the present invention are the first reported to be able to undergo propagation such that a single organoid can give rise to a plurality of daughter organoids. This allows the development of organoid “lines”, within which each organoid is substantially identical to each other. As discussed further elsewhere in this specification, the ability to produce organoid lines in this manner offers advantages in terms of consistency as compared to previous.

[0040] In particular, propagation of the organoids of the invention may be achieved by dissection of the initial organoid and continued culturing of the dissected fractions. This property allows the CNS organoids of the invention to readily be distinguished from CNS organoids of the prior art. Furthermore, the ability to propagate the CNS organoids of the invention in this manner provides considerable advantages that have not previously been made available by CNS organoids of the prior art. The capacity for propagation allows very large quantities of CNS organoids to be produced from small samples of starting material. Indeed, the inventors have demonstrated an ability to achieve a volume of daughter organoids that representing a 20,000-fold increase compared to the volume of the original foetal CNS tissue from which the organoids are derived. This is important given the difficulties that can be associated with the procurement of foetal CNS tissue. The capacity for propagation also allows CNS organoid “lines” (analogous to cell lines produced by cell culture) to be prepared.

Individual organoids within a line will share the same source material and the same resulting characteristics with one another. The ability to produce large numbers of organoids that share the same properties offers notable advantages in terms of the reproducibility of results that can be achieved when using such organoids as an experimental model.

[0041] CNS organoids of the third aspect of the invention are characterised with respect to their constituent cells, which are derived from the neuroectoderm and consist of foetal CNS cells that are N-cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+.

[0042] The CNS organoids of the invention are self organizing, and preserve heterogeneity of cells. They can be produced from different regions of the CNS, and retain properties that are characteristic of these source regions. The organoids of the invention retain the cell to cell organisation found in native brain tissues, and, as with the brain, the migration of cells in CNS organoids of the invention is unidirectional. As the CNS organoids of the invention are not derived from ESCs or iPSCs they do not require specification to region or cell identity.

[0043] The CNS organoids of the invention can be matured at any desired time, and can undergo maturation much earlier than can organoids of the prior art.

[0044] The CNS organoids of the invention have proved to be capable of doubling in volume every 2 weeks. They are also capable of long term expansion and “life time” in culture (being capable of maintenance for more than a year. These properties combine to allow a 20000-fold increase from original tissue volume used for seeding to be obtained.

[0045] In addition to the CNS organoids of the invention themselves, the invention also provides methods by which such organoids may be produced, and methods by which the organoids may be propagated. The present disclosure is the first time that propagation of CNS organoids in this manner has been reported.

[0046] The invention also provides methods of inducing maturation of CNS organoids of the invention, as well as cell culture medium compositions that can be used in the production, maintenance, and maturation of the organoids disclosed.

[0047] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in- chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986)

[0049] Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. CNS organoids of the invention

[0050] The invention provides a number of different aspects that define CNS organoids. CNS organoids in accordance with the invention may be defined with respect to their structure, with respect to their biological activities, and with respect to their constituent cells. It will be appreciated that cell types referred to in connection with a particular aspect or embodiment of the invention may also be present in organoids in accordance with different aspects or embodiments of the invention.

[0051] A CNS organoid of the invention may be composed of foetal CNS tissue and comprise progenitor cells, proliferating cells, glial cells, and mature neuronal cells, and consist of cells solely derived from the neuroectoderm. As the CNS organoids of the invention are directly derived from foetal tissue they do not include and are not derived from ESCs or iPSCs.

[0052] The organoids of the invention may also usefully be defined with reference to the tissue from which they are derived, and with respect to whether or not they have undergone maturation or whether or not they have been subjected to gene editing.

Structures of CNS organoids of the invention

[0053] CNS organoids of the invention can be distinguished from those that have previously been described in the literature by virtue of their structure. The organoids of the invention have a characteristic stratified structure in which the outer layer of cells is enriched for progenitor cells. Below this layer a gradient of differentiation can be observed, with differentiation increasing further into the organoid

[0054] Accordingly, the invention provides a CNS organoid that this organisation observed in respect of organoids of the invention is the opposite of that found in previously reported CNS organoids derived by differentiation of pluripotent stem cells. In these prior art organoids, the more differentiated, neuronal cells are located in the external portion of the organoid, and less differentiated stem or progenitor cells towards the centre. Thus, the orientation observed in CNS organoids of the present invention allows them to be distinguished from those developed by other means.

[0055] The progenitor cells of the outer layer may be characterised as exhibiting low differentiation and high levels of proliferation.

[0056] The differentiated cells of the inner layer may be characterised as exhibiting elevated differentiation and low levels of proliferation. Suitably, the differentiated cells of the inner layer exhibit elevated differentiation to neuronal cell phenotypes and/or to glial cell phenotypes. [0057] Further considerations regarding cells that may be found in CNS organoids of the invention, and in particular layers of such organoids, are set out elsewhere in the specification. [0058] In a further aspect, the invention also provides a CNS organoid characterised by the presence of a folded outer morphology. The folded outer layer of CNS organoids in accordance with this aspect of the invention is not found in CNS organoids of the prior art. However, this characteristic arrangement more closely mirrors that found in brains that have undergone gyrification. Thus, as with the first aspect of the invention, this aspect offers advantages by virtue of providing CNS organoids that more closely resemble the natural structure of the brain. [0059] “First generation” CNS organoids of the invention (which is to say those produced directly from foetal material) comprise foetal extracellular matrix material, which is retained from the original foetal tissue sample source by virtue of the mechanical dissection of the tissue. Dissection in this manner avoids destruction of the ECM and disruption of cell to cell contacts within the portions of foetal tissue used as the starting material for the organoids.

[0060] As set out in the Examples, the inventors have also demonstrated that second (and subsequent) generation CNS organoids of the invention produced by propagation also comprise ECM that has characteristic features of foetal ECM. In particular, the ECM present in such organoids may be distinguished by expression levels of ECM-components similar to expression levels of the same components in foetal CNS tissue. For example, the CNS organoids may have similar or comparable levels of expression of glypicans, syndecans, glycosaminoglycans, and/or proteoglycans in comparison to wild type or control CNS. For example, the control or wild type CNS tissue is foetal CNS tissue. In some examples, the control or wild type CNS tissue is healthy foetal CNS. For example, in comparison to healthy foetal CNS tissue. For example, brain and/or spinal tissue. In some examples, the control or wild type tissue is human foetal CNS tissue, such as healthy foetal CNS tissue. For example, the second (and subsequent) generation CNS organoids of the invention may express one or more of: glypican 1 , glypican 2, glypican 3, glypican 4, glypican 5, glypican 6, CD44, chondroitin sulfate proteoglycan 4, Syndecan 1, Syndecan 2, Syndecan3, Syndecan 4, aggrecan, brevicam, neurocan, versican, agrin, heparan sulfate proteoglycan 2, Structural Maintenance Of Chromosomes 3, seriglycin, biglycan, Chondroadherin, Decorin, Fibronectin 1, Tenascin C, Laminin Subunit Alpha 4, and/or Laminin Subunit Alpha 5 at a level that is similar to an expression in a control or wild type CNS tissue as described herein.

[0061] Similar refers to a level of expression that differs from the control or wild type tissue by at most 50%, at most 40%, at most 30%, at most 20%, at most 10 %, at most 5%, at most 4%, at most 3%, at most2%, at most 1% for each one or more of the ECM-components described herein.

[0062] Thus, the second (and subsequent) generation CNS organoids of the invention may have an ECM-component expression profile similar to an ECM-component expression profile of a control or wild type CNS tissue as described herein. Methods of determining the expression profile of ECM-components are known and may include transcriptomic techniques such as DNA microarray analysis, and RNA-sequencing techniques.

[0063] In some examples, the second (and subsequent) generation CNS organoids of the invention may have an ECM-component secretome and/or proteome similar to control or wild type CNS tissue as described herein. For example, the CNS organoids may produce and/or secrete one or more of Pleiotrophin, Dystroglycan 1, Neurocan, Agrin, and/or Brevican. In some examples, the CNS organoids may produce and/or secrete one or more of Pleiotrophin, Dystroglycan 1, Neurocan, Agrin, and/or Brevican at a level that is similar to control or wild type CNS tissue as described herein. In some examples, the CNS organoids may produce and/or secrete one or more of Pleiotrophin, Dystroglycan 1 , Neurocan, Agrin, and/or Brevican at a level greater than a control culture or tissue, such as a neurosphere. “Neurosphere” refers to a cellular aggregate of neural stem cells and neuroprogenitor cells that form a floating sphere formed as a result of proliferation of the neural stem cells and neuroprogenitor cells in appropriate proliferation conditions. Neurospheres are not considered organoids but are 3- dimensional cultures of neurons and may also be referred to as neural spheriods or neuroaggregates. The cells of neurospheres are usually cultured in non-adherent plates and they cluster together, growing in suspension rather than on the base of the plate.

[0064] Similar refers to a level of production that differs from the control or wild type tissue by at most 50%, at most 40%, at most 30%, at most 20%, at most 10 %, at most 5%, at most 4%, at most 3%, at most2%, at most 1% for each one or more of the ECM-components described herein.

[0065] Similar refers to a level of secretion that differs from the control or wild type tissue by at most 50%, at most 40%, at most 30%, at most 20%, at most 10 %, at most 5%, at most 4%, at most 3%, at most2%, at most 1% for each one or more of the ECM-components described herein.

[0066] The term "proteome" refers to the entire set of proteins expressed by a genome, cell, tissue or organism at a given time. More specifically, it can refer to the entire set of proteins expressed in a given type of cell or organism under defined conditions at a given time. Proteomes can include, for example, protein variants by alternative splicing and I or post- translational modifications (such as glycosylation or phosphorylation) of genes. As such an ECM-component proteome refers to the production of proteins that form part of the ECM, for example the ECM components described herein. Proteomic analysis can be performed in many ways, and all known methods of proteomic analysis are contemplated herein. For example, methods include antibody-based methods and mass spectrometry methods (especially selective reaction monitoring). Furthermore, proteomic analysis not only provides qualitative and/or quantitative information about the protein itself, but if the protein has catalytic activity or other functional activity, the proteomic analysis can also include protein activity data. It should be noted. Exemplary techniques for conducting proteomic assays include US Pat. No. 7,473,532 and US Pat. No. 9,091 ,651.

[0067] The secretome is the set of proteins expressed by a cell, tissue, organoid or organism and secreted, for example into the extracellular space. The secretome of a specific tissue can be measured by mass spectroscopy methods. For example, serum or supernatant containing secreted proteins is digested with a protease and the proteins are separated by 2D gel electrophoresis or chromatographic methods. Each individual protein is then analysed by mass spectrometry and the peptide-mass fingerprint generated can be run through a database to identify the protein. Other example methods include, stable isotope labelling by amino acids in cell culture (SILAC) and antibody based methods such as antibody array methods. Prediction based methods may also be used.

Biological activities of CNS organoids of the invention

[0068] The CNS organoids of the invention are the first identified as being capable of propagation (sometimes referred to herein as “expanding” organoids) to produce new organoids. This capacity for propagation enables the production of organoid lines, as discussed elsewhere in this disclosure, which have the capability to improve the reproducibility and consistency of studies undertaken using such organoids as investigative tools.

[0069] The CNS organoids of the invention are also capable of undergoing maturation, associated with further differentiation of cells to lineages including neuronal and astrocyte phenotypes. Details of features that may be used in the characterisation of mature CNS organoids of the invention are considered under the relevant headings below.

Cell types making up CNS organoids of the invention

[0070] Suitably, the organoids of the invention may comprise a mixture of cells selected from the group consisting of: progenitor cells; proliferating cells; glial cells; neuronal cells; and migrating cells. Each of the cell types present in the organoids of the invention is derived from neuroectoderm present in the foetal tissue samples from which the CNS organoids are produced. Progenitor cells

[0071] The CNS organoids of the invention may comprise progenitor cells. Progenitor cells present in the CNS organoids of the invention (for example, in the outer layer of organoids in accordance with the first aspect of the invention) may include cells selected from the group consisting of: SOX2+ progenitor cells; neuronal stem cells; radial glial stem cells (which may suitably be selected from: glycolytic radial glial cells, ventricular radial glial cells, and outer radial glial cells, such as HOPX+ outer radial glial cells); apical progenitor cells; intermediate progenitor cells; and oligodendrocyte precursor cells.

[0072] Of the cells referred to above, apical progenitor cells, intermediate progenitor cells, and oligodendrocyte precursor cells may particularly be present in telencephalon organoids (and in the case of oligodendrocyte precursors, particularly in ventral telencephalon organoids of the invention). Radial glial cells present in a mature ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: SOX2; and/or ID2; and/or ID4; and/or HES1. Ventricular radial glial cells present in a mature ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: NESTIN; and/or HES5; and/or FABP7. Radial glial cells present in a mature dorsal telencephalic organoid of the present invention may be positive for PAX6. Outer radial glial cells present in a mature dorsal telencephalic organoid of the present invention may be positive for FAM107A.

[0073] Proliferating progenitor cells present in a ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: MKI67; and/or PCNA; and/or TOP2A.

[0074] Intermediate progenitor cells present in a mature dorsal telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: HES6; and/or BTG2; and/or TM EM 158; and/or ELAVL2; and/or GADD45G; and/or MFNG.

Proliferating cells

[0075] The CNS organoids of the invention may comprise proliferating cells. The proliferating cells may be identified by virtue of expressing markers selected from the group consisting of: MKI67 and/or CENPF and/or PCNA and/or TOP2A.

[0076] CENPF+ proliferating cells may particularly be present in telencephalon organoids of the invention. MKI67+ and/or PCNA+ and/or TOP2A+ cells may particularly be present in mature ventral telencephalon organoids of the invention. Glial cells

[0077] The CNS organoids of the invention may comprise glial cells. The glial cells present in CNS organoids of the invention may comprise astrocytes and/or oligodendrocytes.

[0078] Astrocytes present in a mature ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: MT2A; and/or AQP4; and/or AGT. Astrocytes present in a mature dorsal telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: AQP4; and/or AGT.

Oligodendrocytes present in a mature ventral telencephalic organoid of the present invention may be positive for SOX10.

Neuronal cells

[0079] CNS organoids of the invention may comprise neuronal cells, for example within the inner layer of organoids in accordance with the first aspect of the invention. The neuronal cells may be selected from the group consisting of: immature neuronal cells; and/or mature neuronal cells; and/or migrating neuronal cells; and/or GABAergic neuronal cells; and/or glutamatergic neuronal cells; and/or cortical neurons.

[0080] Suitably, neuronal cells present in a CNS organoid of the invention may comprise TLIJ1 and/or MAP2 positive cells.

[0081] In the case that immature neuronal cells are present, these may comprise DCX positive and/or TLIBB3 positive and/or TLIJ1 positive; and/or DLX2 positive and/or GAD65 positive cells. Immature neurons present in a mature ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: GAD2; and/or CALB2; and/or DLX5. Immature neurons present in a mature dorsal telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: DCX; and/or SOX11. [0082] Should mature neuronal cells be present, these may comprise SOX5, MEF2C and/or TLIBB3 positive cells (particularly in the case of telencephalon organoids)

[0083] In the case that migrating neuronal cells are present, these may comprise DCX and/or MEF2C positive cells.

[0084] GABAergic cells present in a mature ventral telencephalic organoid of the present invention may be positive for markers selected from the list consisting of: VGAT; and/or DLX6; and/or CALB2.

[0085] Cortical neurons present in a mature dorsal telencephalic organoid of the present invention may be positive for makers selected from the group consisting of: RND2; and/or POU3F2; and/or CUX1 ; and/or SOX5; and/or CLIM1 ; and/or SATB2; and/or BCL11 B (CTIP2) ; and/or MLLT11 ; and/or NFIX SOX4; and/or SOX11 ; and/or STMN2.

Migrating cells

[0086] CNS organoids of the invention may comprise migrating cells. Migrating cells may be identified as NRP1+, LINC5D+ and/or DCX positive cells.

[0087] Migrating cells present in the CNS organoids of the invention may comprise migrating neuronal cells, as considered above. As set out previously, such migrating neuronal cells may suitably comprise DCX+ and/or MEF2C positive cells.

Telencephalon organoids in accordance with the invention

[0088] As referred to above, the inventors have found that CNS organoids in accordance with the invention retain properties of the region of foetal CNS tissue from which they are derived. Thus, CNS organoids of the invention that are derived from foetal telencephalon tissue are able to maintain cell types and markers characteristic of this regional identity.

[0089] A telencephalon organoid in accordance with the present invention may comprise one or more types of progenitor cells selected from the group consisting of: apical progenitor cells; and intermediate progenitor cells.

[0090] A telencephalon organoid in accordance with the present invention may comprise proliferating cells that are MKI67+ and/or CENPF+.

[0091] A telencephalon organoid in accordance with the present invention may comprise one or more types of neuronal cells selected from the group consisting of: DCX+ cells and/or TLIJ1 + cells.

[0092] A telencephalon organoid in accordance with the present invention may comprise one or more types of migrating cells selected from the group consisting of: NRP1+ cells, LINC5D+ cells and/or DCX+ cells.

[0093] A telencephalon organoid in accordance with the present invention may comprise one or more types of mature neuronal cells selected from the group consisting of: SOX5+ cells, MEF2C+ cells and/or TLIBB3+ cells.

[0094] Telencephalon organoids of the invention may suitably be derived from foetal CNS tissue of the ventral telencephalon (also referred to as “ventral telencephalon organoids” in the context of the present invention) or may suitably be derived from foetal CNS tissue of the dorsal telencephalon (also referred to as “dorsal telencephalon organoids” in the context of the present invention). [0095] Ventral telencephalon organoids and dorsal ventral telencephalon organoids can both be matured, for example by means of the methods described herein.

[0096] As discussed further below, fused CNS organoids of the invention can be prepared, in which organoids derived from different regions of the brain are fused with one another. These fusions can give rise to organoids that further replicate the structure of the brain itself.

[0097] Ventral telencephalon organoids in accordance with the present invention may comprise one of more of the following groups of cells: DLX2, NKX2-1 , OTX2 and/or GSX2 positive cells; oligodendrocyte precursor cells; and immature neuronal cells comprising DLX2, and/or GAD65 positive cells.

[0098] Dorsal telencephalon organoids in accordance with the present invention may comprise EMX1 and/or PAX6 positive cells.

[0099] Cell types that may be found in mature telencephalon organoids (such as ventral mature telencephalon organoids, or mature dorsal telencephalon organoids) are described further elsewhere in the present disclosure.

Fused telencephalon organoids

[0100] Telencephalon organoids of the invention are able to combine with one another to produce a fused organoid. If the organoids are from different regions, then the fused organoid may comprise cells characteristic of both regions. Accordingly, the invention provides a CNS organoid (such as a CNS organoid according to any previous aspect of the invention) comprising a fusion of at least one dorsal telencephalon organoid and at least one ventral telencephalon organoid. Such fused telencephalon organoids are capable of mimicking the whole telencephalon.

[0101] Accordingly, the invention provides a method of producing a fused CNS organoid, the method comprising co-culturing a first CNS organoid and a second CNS organoid for a time sufficient for fusion of the first and second organoids to occur.

[0102] In a suitable embodiment of such a method the first and second CNS organoids are ventral telencephalic organoids and dorsal telencephalic organoids.

Maturation of CNS organoids, and mature organoids

[0103] CNS organoids of the invention are able to undergo maturation. Accordingly, in a suitable embodiment an organoid of the invention is a matured CNS organoid.

[0104] Maturation may be spontaneous, or may be induced (for example by a method in accordance with the sixth aspect of the invention). Maturation of organoids of the invention causes changes in the cells of both the outer layer and the inner layer, examples of which are discussed in more detail below.

Characteristics of mature organoids

[0105] In the outer layer, the number of progenitor cells present decreases with maturation, and a reduction in expression of markers of proliferation and of progenitor cells is observed. Maturation of an organoid of the present invention may be associated with a reduction of expression in the cells of the outer lay of: PCNA and/or MKI67 and/or GL13 and/or GSX2 and/or BLBP.

[0106] In the outer layer, the number of progenitor cells present decreases with maturation, and a reduction in expression of markers of proliferation and of progenitor cells is observed. Maturation of an organoid of the present invention may be associated with a reduction of expression in the cells of the outer lay of: PCNA and/or MKI67 and/or GL13 and/or GSX2 and/or BLBP.

[0107] In the inner layer, maturation is associated with an increase in differentiation of cells present. This may give rise to a change in the types of cells present, as well as in cellular expression of markers. These changes can be used to identify and characterise mature organoids of the invention.

Cell types and markers characteristic of mature organoids

[0108] Generally, a mature organoid of the invention may comprise one or more cell types independently selected from the group consisting of: cortical neurons; oligodendrocytes; oligodendrocytes precursor cells; radial glial cells; glycolytic radial glial cells; outer radial glial cells; ventricular radial glial cells; intermediate progenitor cells; GABAergic cells; glutamatergic neuronal cells; immature neuronal cells; and astrocytes.

[0109] Generally, a mature organoid of the invention may comprise one or more cell types independently selected from the group consisting of: cortical neurons; oligodendrocytes; oligodendrocytes precursor cells; radial glial cells; glycolytic radial glial cells; outer radial glial cells; ventricular radial glial cells; intermediate progenitor cells; GABAergic cells; glutamatergic neuronal cells; immature neuronal cells; and astrocytes.

[0110] Maturation is associated with an increase in markers characteristic of early neuronal specification and of mature neurons. Expression of transcription factors associated with initiation of neuronal differentiation, such as SOX11 and/or SOX4, may be increased among cells of the inner layer on maturation of a CNS organoid of the invention. Cell types and markers characteristic of mature dorsal telencephalic organoids [0111] Mature dorsal telencephalic organoids of the invention may exhibit increased expression of neuronal layer markers such as BRN2 and/or CTIP2 and/or CUX1 among cells of the inner layer.

[0112] A mature dorsal telencephalic organoid of the invention may comprise one or more cell types independently selected from the group consisting of: BRN2, CTIP2, CLIX1 , and/or SATB2 positive cells; cortical neurons (which may comprise RND2, POLI3F2, CLIX1 , SOX5, CLIM1 , SATB2, BCL11 B (CTIP2), SLC17A6, MLLT11 , NFIX SOX4, SOX11 and/or STMN2 positive cells); radial glial cells (which may comprise PAX6 positive cells); glycolytic radial glial cells; outer radial glial cells comprising FAM107A positive cells; intermediate progenitor cells comprising HES6, BTG2, TMEM158, ELAVL2, GADD45G and/or MFNG positive cells; glutamatergic neuronal cells; immature neuronal cells, comprising DCX and/or SOX11 positive cells; and/or astrocytes comprising AQP4 and/or AGT positive cells.

Cell types and markers characteristic of mature ventral telencephalic organoids

[0113] Mature ventral telencephalic organoids of the invention may exhibit expression of markers characteristic of interneuron progenitors, or GABAergic cell markers, such as DLX1/2/5/6 and/or CALB2 and/or GAD1 and/or GAD2 and/or VGAT, among cells of the inner layer.

[0114] A mature ventral telencephalic organoid of the invention may comprises one or more cell types independently selected from the group consisting of: PROX1 and/or GSX2 positive cells; oligodendrocytes comprising SOX10 positive cells; oligodendrocytes precursor cells; radial glial cells comprising SOX2, ID2, ID4 and/or HES1 positive cells; glycolytic radial glial cells; outer radial glial cells; ventricular radial glial cells, wherein the ventricular radial glial cells comprise NESTIN, HES5 and FABP7 positive cells; proliferating progenitor cells comprising MKI67, PCNA and TOP2A positive cells; GABAergic cells (which may comprise VGAT, DLX6, CALB2 positive cells); immature neuronal cells (which may comprise GAD2, CALB2, DLX5 positive cells); and/or astrocytes (which may comprise MT2A, AQP4, and AGT positive cells).

Cells markers characterizing CNS organoids of the invention

[0115] Organoids of the invention may be characterized with respect to certain cell types being positive or negative for a protein or other marker of interest.

[0116] For the purposes of the present disclosure, references to “positive” (or “+”) cells, or to cells “expressing” a protein or other marker, should be interpreted as encompassing both cases in which an expressed protein is detectable in respect of a cell, and cases in which increased expression of a recited gene (or gene encoding a recited protein) is detectable in respect of a cell.

[0117] In the case of a detectable protein, this may be confirmed by a suitable approach such as antibody labelling. In the case of increased expression of a gene, this may be confirmed by the presence of elevated levels of mRNA, which can be detected by any appropriate RNA analysis approach. Elevation of mRNA levels can be determined by normalization with respect to an appropriate control, such as a housekeeping gene.

[0118] References to cells being “negative” (or

should be construed with the same considerations in mind.

[0119] A further aspect of the invention provides a CNS organoid consisting of foetal CNS cells which are derived from the neuroectoderm , and are N-cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+. A CNS organoid in accordance with this aspect of the invention may consist of foetal CNS cells that are N-cadherin+ (CDH2+), Nestin+, and SOX2+.

[0120] These markers identify the cells of such CNS organoid as being derived from the neuroectoderm. Accordingly, the fact that an organoid of in accordance with this aspect of the invention consists of such cells demonstrates that the cells present are derived solely from the neuroectoderm. CNS organoids that have been known from the prior art are not solely derived from the neuroectoderm in this fashion, and hence such organoids contain at least some cells that are N-cadherin- (CDH2-) and/or Nestin- and/or SOX2-. Typically, prior art organoids will retain some cells that express markers indicative of pluripotency, or will include non- neuroectoderm-derived cell types produced on differentiation of pluripotent cells. The presence of these cells provides an indication that the organoid has been derived from cells from sources other than the neuroectoderm

[0121] CNS organoids in accordance with this aspect of the invention can be distinguished from those of the prior art in that these prior art organoids, produced via the differentiation of pluripotent stem cells (whether iPSCs or ESCs), contain cells that retain markers of pluripotency, such as KLF4 and NANOG. Since the CNS organoids of the present invention are produced from cells of the neuroectoderm, rather than pluripotent cells, they do not include cells expressing such markers. This difference may be used to characterise the CNS organoids of the present invention, and provides them with benefits as compared to organoids of the prior art, as the organoids of the invention more accurately reflect the constituents of the brain itself. As the CNS organoids of the invention are not derived from an do not include iPSCs or ESCs they do not require specification to region or cell identity. This differs from iPSC and ESC derived organoids which do require specification to become neuronal cells. [0122] In keeping with the above, the invention also provides a CNS organoid that does not contain cells expressing pluripotency markers. For example, such a CNS organoid may not contain cells that are KLF4+ and/or NANOG+. The invention also provides a CNS organoid that consists of cells that are negative for pluripotency markers. For example, such a CNS organoid may consist of cells that are KLF4- and/or NANOG-.

[0123] In a suitable embodiment, the N-cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+ foetal CNS cells comprise cell types selected from the group consisting of: progenitor cells and/or proliferating cells and/or glial cells and/or mature neuronal cells.

[0124] As referred to above, the second aspect of the invention provides a CNS organoid consisting of foetal CNS cells that are TBXT-, COL1A1-, and LUM-. The markers referred to in the second aspect of the invention are indicative of mesenchymal origin of cells, and may arise in CNS organoids of the prior art due to their derivation from pluripotent stem cells. The fact that the organoids of the second aspect of the invention lack these markers clearly demonstrates that their cells are not obtained or produced from these sources.

[0125] Similarly, the third aspect of the invention also provides a CNS organoid consisting of foetal CNS cells that are SOX17-, HNF3B-, and GATA4-. he markers referred to are indicative of endodermal origin of cells, and may arise in CNS organoids of the prior art due to the presence of non-neuroectodermal cells. The absence of such cells from the organoids of the third aspect of the invention demonstrates that they do not comprise cells from such alternative sources.

[0126] A further aspect of the invention provides a CNS organoid consisting of CNS cells that are E-Cadherin- (CDH1-), and EpCam-. Expression of these markers would indicate the presence of non-neural epithelial cells in an organoid, which is avoided by the organoids of the present invention, and their methods of production.

[0127] Suitably a CNS organoid of the invention may consist of foetal CNS cells that are:

• TBXT-, COL1A1-, LUM-, SOX17-, HNF3B-, and GATA4-; or

• TBXT-, COL1A1-, LUM-, SOX17-, E-cadherin- (CDH1-), and EpCam-; or

• HNF3B-, GATA4-, E-cadherin- (CDH1-), and EpCam-; or

• TBXT-, COL1A1-, LUM-, SOX17-, HNF3B-, GATA4-, E-cadherin- (CDH1-), and EpCam-.

[0128] In each of the cases considered above, the foetal CNS cells may additionally be N- cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+.

[0129] Progenitor cells found in the CNS organoids of the invention may express SOX2, and in the case of outer radial glial cells may express HOPX.

[0130] Proliferating cells found in the CNS organoids of the invention may express MKI67. [0131] Neuronal cells found in the CNS organoids of the invention may express TLIJ1 and/or MAP2. Immature neuronal cells found in the CNS organoids of the invention may express DCX and/or TLIBB3. Migrating neuronal cells found in the CNS organoids of the invention may express DCX and/or MEF2C.

[0132] Glial cells found in the CNS organoids of the invention may include astrocytes that express GFAP and/or S100P

[0133] Telencephalic tissue cells found in the CNS organoids of the invention may include proliferating cells that express CENPF; immature neuronal cells that express DCX and/or TLIJ1 ; migrating cells that express NRP1 and/or LINC5D and/or DCX; and mature neuronal cells that express SOX5 and/or MEF2C and/or TLIBB3.

[0134] Ventral telencephalic tissue cells found in the CNS organoids of the invention may express DLX2 and/or NKX2-1 and/or OTX2 and/or GSX2. Ventral telencephalic tissue cells found in the CNS organoids of the invention may include: oligodendrocyte precursor cells that express OLIG2; apical progenitor cells that express SOX2 and/or HES1 and/or NESTIN positive cells; and immature neuronal cells that express DLX2 and/or GAD65.

[0135] As described further in the Examples, the inventors have used principal component and clustering analysis to identify “clusters” that describe the majority of cells found in CNS organoids of the invention. This information may also be particularly helpful in providing groups of markers that can be used to characterize cells found in ventral and dorsal telencephalon organoids.

[0136] In ventral telencephalon organoids, cluster “C1” encompasses mature radial glia primed or transitioned to astrocyte differentiation, and so the markers described here can be used to characterize these cells. These cells exhibit the expression of S0X2, ID2, ID4, HES1 (which are radial glial markers), as well as MT2A, AQP4, and AGT (which are more typically astrocytic genes).

[0137] In ventral telencephalon organoids, cluster “C2” encompasses cells in transition from neurogenesis to gliogenesis, which potentially represent an intermediate state to astrocytes. The markers described here can be used to characterize cells of this sort. These cells exhibit expression of radial glial and astrocyte markers (although to a lower level than in C1) and expression of COUPTF1 and COUPTF2 (being the only cells present in the organoids that have been shown to express these markers).

[0138] In ventral telencephalon organoids, cluster “C3” encompasses glycolytic radial glia cells. These cells are characterized by the expression of radial glial genes, and also expressed high level of glycolytic genes and additional genes such as BNIP3. These cells express lower levels of NEST/N. This pattern of expression of these markers can be used to characterize cells of this sort. [0139] In ventral telencephalon organoids, cluster “C4” encompasses cells of the outer radial glia, and the more ventricular radial glia, that appear committed towards neuronal fate. The markers described here can be used to characterize cells of this sort. The outer radial glia particularly express progenitor and stem cell markers, such as HOPX, and PTPRZ1, and ventricular radial glia express NESTIN, HES5 and FABP7. The cells in this cluster also express neuronal markers, such as GAD43, suggesting their commitment towards neuronal fate.

[0140] In ventral telencephalon organoids, cluster “C5” encompasses actively cycling cells, and the markers set out here can be used to characterize such cells. The cells of C5 express typical markers of proliferating neural progenitors (such as MKI67, PCNA and TOP2A) and ventral telencephalic neuronal markers (such as PR0X1 and GSX2), as well as displaying the expression of already more committed neuronal cells (S0X4, S0X11).

[0141] In ventral telencephalon organoids, cluster “C6” encompasses interneuron cells, and the patter of marker expression can be used in characterizing such cells. In addition to some of the markers expressed in C5, cells of C6 clearly acquired the expression of early-stage interneuron genes (such as DLX2) and typical inhibitory GABAergic interneuron markers (e.g. GAD2, CALB2, DLX5) which are the main type of neurons being produced in the ventral telencephalon). Thus, these cells represent newborn interneurons. Cells displaying a higher expression of progenitor genes such as ASCL1, were observed to have lower expression of interneuron genes such as DLX2, and vice versa.

[0142] In ventral telencephalon organoids, cluster “C7” encompasses mature neurons, characterized by their expression of mature GABAergic interneurons markers (such as VGAT, DLX6, and CALB2), and lack of progenitor gene expression.

[0143] In ventral telencephalon organoids, cluster “C8” contained a mixture of precursors of both the neurogenic and oligodendrocyte lineages. These cells were characterized by expression of the progenitor marker ASCL1, (which in the ventral forebrain is also expressed by oligodendrocyte lineage precursors), and additional oligodendrocyte precursors markers (such as OLIG2, and OLIG1) as well as the more mature oligodendrocyte marker SOX10 and interneuron marker DLX2 in a mutually exclusive fashion.

[0144] In dorsal telencephalon organoids, cluster “C1” also encompasses mature radial glia primed or transitioned to astrocyte differentiation, with the cells expressing typical RG markers (S0X2, HES1, S0X9) and astrocytic markers (AQP4, AGT).

[0145] In dorsal telencephalon organoids, cluster “C2” again defines a glycolytic radial glial cluster.

[0146] In dorsal telencephalon organoids, cluster “C3” instead constituted a mixed cluster. While multiple cells expressed neurogenic progenitor (HES5, PAX6) and oRG markers (HOPX, PTPRZ1, FAM107A), many expressed some specific markers of migrating as well as cortical neurons (RND2, POU3F2, CUX1, SOX5, CLIM1, SATB2) and typical functional markers of glutamatergic neurons, like the glutamate transporters GRIA2, GRIN1, or glutamate synthetase GLUL. The gene signature t-SNE plot showed that in addition to clusters C5-C6 (CTIP2^+>), the glutamatergic neurons are scattered within cluster C3 (SATB2⁺).

[0147] In dorsal telencephalon organoids, cluster “C4” represents the actively proliferating neurogenic progenitors, expressing markers of committed neural cells (S0X4, POU3F2), alongside markers of intermediate progenitors {ELAVL2, TMEM158).

[0148] In dorsal telencephalon organoids, cluster “C5” represents newborn neurons, characterized by expression of newborn neurons marker DCX, S0X11 and the cortical neuronal markers BCL11B (CTIP2), MLLT11, NF IX and STMN2.

[0149] In dorsal telencephalon organoids the cells of cluster “C6” (which represents the mature neuronal cluster) also expressed BCL11B (CTIP2), MLLT11, NFIX and STMN2.

[0150] Clusters representing endothelial, ependymal, microglia or other immune cells were not identified, indicating that those cells are not part, and not necessary for the propagation, of CNS organoids of the present invention.

Uses of the CNS organoids of the invention

[0151] The CNS organoids of the invention may be used in drug discovery, disease modelling, and the modelling of CNS development.

[0152] Disease modelling using the CNS organoids of the invention may particularly focus cancers of the CNS, or developmental diseases of the CNS.

[0153] Suitable studies may investigate the role of certain genes or gene pathways (which can be manipulated in the organoids of the invention in a manner that cannot be undertaken using human brains in vivo), or the actions of putative modifying agents (for example, potentially active biological molecules or small molecule drugs).

[0154] CNS organoids of the invention may be co-cultured with other cells or tissues to investigate their interactions. Merely by way of example, the CNS organoids of the invention may be co-cultured with cancer cells (or cancer tissue samples) to produce fusion models, or may be cultured with cells of the immune system (including modified cells of the immune system, such as those used in adaptive immunotherapy) to investigate their impact on the CNS organoids (which may be manipulated to replicate brain cancers).

[0155] Methods by which CNS organoids of the invention can be modified for use in such methods are described below. Modified and gene edited organoids in accordance with the invention

[0156] The CNS organoids of the invention may be modified to generate models of diseases of tissues of the central nervous system in a manner that cannot readily be done in animal models (and particularly cannot be done in human subjects). Such modifications may involve the introduction of mutations known to be associated with diseases (or other disorders) of interest.

[0157] The modifications may be achieved by any suitable method known to those skilled in the art. Merely by way of example, suitable modifications may be undertaken using gene editing techniques (such as CRISPR), transposons, or viral over-expression. As set out in the Examples, the inventors have demonstrated that gene editing approaches, exemplified using CRISPR, are particularly useful in the modification of CNS organoids of the invention to provide models of CNS diseases.

[0158] Suitably a CNS organoid of the invention may be modified in a manner that provides a model of a neurodevelopmental disorder. Merely by way of example, the inventors have produced such a model by using a gene editing approach to introduce mutations into RAB3GAP2 and thereby impair its function.

[0159] A CNS organoid of the invention may be modified in a manner that provides a model for a cancer of the CNS. Merely by way of example, the inventors have illustrated this use of the CNS organoids of the invention by using gene editing to introduce mutations into TP53 within the cells of the organoid.

Methods of producing CNS organoids

[0160] The fourth aspect of the invention provides a method of producing a CNS organoid, the method comprising:

[0161] The CNS organoids of the present invention are derived from samples of foetal CNS tissue. The samples of foetal CNS tissue may be of human origin, or non-human origin (for example, of non-human primate origin). Foetal material may be obtained from sources in which a foetus will not go to term (for example, from foetuses that have been aborted or miscarried). [0162] For the purposes of the present disclosure, references to “mechanical dissection” should be taken as requiring that the sample of foetal CNS tissue is processed to yield the required portions without dissociation of foetal CNS cells within the sample. Suitably such processing is carried out without chemical digestion and without enzymatic digestion. [0163] Suitably, mechanical dissection is carried out in a manner that retains native foetal CNS ECM within the portions produced. Thus, in a suitable embodiment of the methods of the invention, the portions of foetal CNS tissue further comprise foetal CNS extracellular matrix (ECM).

[0164] In a suitable embodiment, the foetal CNS cells in the portions of foetal CNS tissue consist of neuroectoderm cells. The portion of CNS tissue may consist of foetal CNS cells that are N- cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+. Such embodiments are of particular relevance in the production of organoids in accordance with the third aspect of the invention.

[0165] Mechanical dissection may be undertaken such that the portions of foetal CNS tissue span the germinal and neuronal layers. The dissection may be undertaken such that the portions of foetal CNS tissue comprise cells that are SOX2⁺, PAX6⁺ and MKI67⁺ (indicative of proliferating progenitor cells), as well as cells that are TUIJ⁺ (indicative of neuronal cells).

[0166] In a suitable embodiment, the method is for producing a telencephalon organoid, and the portion of foetal CNS tissue is selected for as being FOXG1+.

[0167] In such an embodiment the method may be for producing a dorsal telencephalon organoid, and the portion of foetal CNS tissue is selected for as being EMX1+ and/or PAX6+. A dorsal telencephalon organoid may subsequently be characterised the presence of markers and cell types as discussed previously in the specification.

[0168] Alternatively, the method may be for producing a ventral telencephalon organoid, and the portion of foetal CNS tissue is selected for as being DLX2+ and/or NKX2-1+ and/or GSX2+. A ventral telencephalon organoid may subsequently be characterised the presence of markers and cell types as discussed previously in the specification.

[0169] In a suitable embodiment of a method of the invention, the portions of foetal CNS tissue are of between approximately 0.2 mm and approximately 3 mm diameter.

[0170] The inventors have found that portions of foetal CNS tissue between these sizes are well suited to culture, and also retain a quantity of foetal CNS ECM that promotes cell organization to promoting the development of CNS organoids in accordance with the first, second or third aspects of the invention.

[0171] In a suitable embodiment the sample of foetal CNS tissue is at a developmental stage between 6 gestational weeks (GW) and 24 GW. Suitably, the sample of foetal CNS tissue is at a developmental stage of between 12 GW and 15 GW (for example, between 12 to 14 GW or 13 to 15 GW). The Examples demonstrate the effectiveness of methods using such tissues, which represent the mid-neurogenesis period in human foetuses. Alternatively, the sample of foetal CNS tissue is at a developmental stage of between 6 GW and 8 GW. In another suitable embodiment, the sample of foetal CNS tissue is at a developmental stage of between 22 GW and 24 GW.

[0172] In a suitable embodiment the sample of foetal CNS tissue is a sample of healthy tissue. By “healthy” is meant that the sample of CNS tissue does not exhibit any signs or markers indicative of the presence of a disease. For example, the sample of CNS tissue may not exhibit any signs or markers indicative of the presence of an inherited disease. The sample of CNS tissue may not exhibit any signs or markers indicative of the presence of cancer. The CNS tissue may be a non-tumorous and/or non-cancerous tissue. In some embodiments, the tissue sample may be unhealthy tissue that is non-tumorous and/or non-cancerous tissue. For example, the sample may be tissue that is taken from a subject suffering from a disease other than cancer. For example, a genetic and/or CNS disease other than cancer. For example, the sample of CNS tissue may exhibit signs or markers indicative of the presence of a genetic or CNS disease but no markers or signs of cancer. For example, CNS diseases include Parkinson’s disease, degenerative brain disorders (such as Alzheimer's disease), Huntington's disease, apoplexy (stroke), epileptic conditions (for example, epilepsy), neuropsychiatric disease be (for example, depression, hot-tempered strongly fragrant Disease, schizophrenia), migraine and attention deficit/mostly dynamic obstacle (ADHD) and amyotrophic lateral sclerosis (ALS). Example genetic disorders include Alexander disease, Canavan disease, Krabbe disease, leukoencephalopathy with vanishing white matter, megalencephalic leukoencephalopathy with subcortical cysts, metachromatic leukodystrophy, X-linked adrenoleukodystrophy, ataxia, Charcot-Marie-Tooth disease, Pick’s disease, familial Creutzfeldt-Jakob disease, familial ALS, familial Alzheimer’s disease, familial dystonia including Dopa-responsive dystonia, fragile X and Fragile X associated Tremor Ataxia Syndrome, Huntington's disease, hereditary spastic paraplegia, lysosomal storage disorders, mitochondrial encephalomyopathies, mucopolysaccharidoses, neurofibromatosis, Tourette's syndrome, tuberous sclerosis, Von- Hippel-Lindau disease and Wilson’s disease. By using unhealthy tissue as described above (i.e. non-cancerous tissue) this may allow for the study of such diseases using CNS organoids of the invention.

[0173] In a suitable embodiment, the sample of foetal CNS tissue is a sample comprising or consisting of brain tissue. A method in accordance with this embodiment of the invention may be used in the production of a brain organoid.

[0174] In a suitable embodiment, the sample of foetal CNS tissue is a sample comprising or consisting of spinal cord tissue. A method in accordance with this embodiment of the invention may be used in the production of a spinal cord organoid. [0175] In a suitable embodiment of a method of producing a CNS organoid, the portions of foetal CNS tissue are transferred to separate vessels for culture until CNS organoids are formed.

[0176] Suitably, the portions of foetal CNS tissue may be cultured for a period of at least 3 days to allow organoid formation.

[0177] Suitably, the portions of foetal CNS tissue may be cultured for a period of up to 20 days to allow organoid formation. For example, the portions of foetal CNS tissue may be cultured for a period of up to 15 days to allow organoid formation.

[0178] A method in accordance with the fourth aspect of the invention may further comprise maintaining the organoid in expansion medium.

[0179] Suitably the organoid may be maintained in expansion medium for a period of at least a week. For example, the organoid may be maintained in expansion medium for a period of at least 2 weeks. In a suitable embodiment, the organoid is maintained in expansion medium for a period of 10 weeks or more, of 20 weeks or more, of 30 weeks or more, of 40 weeks or more, or of 50 weeks or more.

[0180]

[0181] Suitably, the organoid is maintained in expansion medium for a period of 1 year or more. For example, the organoid may be maintained in expansion medium for a period of 2 years or more.

[0182] In a suitable embodiment of a method of the invention, the organoid is maintained until it reaches a size of between approximately 4 mm and approximately 8 mm.

[0183] Suitably, the organoid is maintained until it stops growing in size.

[0184] An expansion medium may comprise at least one growth factor selected from the group consisting of: EGF, FGF-10 and FGF-2. A suitable expansion medium for use in the methods of the invention may be as defined elsewhere in the present disclosure.

[0185] Suitably, in a method of the invention in which an organoid is produced or maintained, the expansion medium is refreshed every 2 to 3 days.

Methods of propagating organoids of the invention

[0186] The second aspect of the invention relates to CNS organoids that have the novel and advantageous property that they are capable of propagation by dissection and culture of the organoid. Accordingly, in a fifth aspect, the invention provides a method of propagating a CNS organoid, the method comprising:

• producing a CNS organoid by a method according to any embodiment of the fourth aspect of the invention;

• culturing the resultant organoid until it has a diameter of at least approximately 2 mm; • mechanically dissecting the organoid to produce at least two organoid fractions; and

[0187] In a suitable embodiment of a method of propagating a CNS organoid, the organoid is cultured until it has a size of between approximately 2 mm and approximately 4 mm prior to mechanical dissection.

[0188] In a suitable embodiment of a method of propagating a CNS organoid according to this fifth aspect of the invention, the organoid is mechanically dissected to produce organoid fractions of at least 0.5 mm size. In a suitable embodiment, the organoid is mechanically dissected to produce organoid fractions of approximately 1 mm size.

[0189] In a suitable embodiment of a method of propagating a CNS organoid, the organoid fractions are transferred to separate vessels for culture until daughter organoids are formed. The separate vessels may be individual wells of a multiwell plate.

[0190] Suitably, the organoid fractions are cultured for a period of at least 3 days to allow daughter organoid formation. By way of example, the organoid fractions may be cultured for a period of up to 20 days to allow daughter organoid formation.

[0191] Suitably, the expansion medium for use in a method of propagating a CNS organoid in accordance with the invention comprises a growth factor selected from the group consisting of: EGF, FGF-10 and FGF-2. A suitable expansion medium for use in such methods may be as defined elsewhere in the present disclosure.

Methods of maturing CNS organoids

[0192] The properties and characteristics of mature organoids have been considered elsewhere in the present disclosure. As set out above, maturation of a CNS organoid of the invention may occur spontaneously or may be carried out intentionally.

[0193] In keeping with this, in its sixth aspect, the invention provides a method of inducing maturation of a CNS organoid, the method comprising:

[0194] Maturation may be confirmed as having taken place with reference to any of the features (for example characteristics cell types or markers) described elsewhere in the specification with reference to matured CNS organoids. Merely by way of example, a method of inducing maturation in accordance with the sixth aspect of the invention may comprise culturing a CNS organoid until extensive neurogenesis is observed in the organoid. Additionally, or alternatively, a method of inducing maturation in accordance with the sixth aspect of the invention may comprise culturing a CNS organoid until the presence of astrocytes is noted in the organoid.

[0195] In a suitable embodiment of a method of inducing maturation of a CNS organoid in accordance with the invention the maturation medium comprises basal membrane extract.

[0196] Suitably, the maturation medium is as defined below, in accordance with the eighth aspect of the invention.

[0197] A method of inducing maturation of a CNS organoid in accordance with the present invention, wherein the organoid has been maintained for a period of up to 10 weeks between production and culturing in the maturation medium. For example, in a suitable embodiment the organoid has been maintained for a period of up to 9 weeks, up to 8 weeks, up to 7 weeks, up to 6 week, or up to 5 weeks between production and culturing in the maturation medium. Suitably, the organoid has been maintained for a period of up to 4 weeks, up to 3 weeks, up to 2 weeks, or up to 1 week between production and culturing in the maturation medium.

[0198] In a suitable embodiment the invention provides a method of inducing maturation of a CNS organoid according to the invention, wherein the organoid has been maintained for a period of at least a month between production and culturing in the maturation medium. Suitably, in such an embodiment, the organoid may be maintained for a period of at least three months, at least six months, or at least a year between its production and culturing in the maturation medium.

Cell culture media in accordance with the invention

[0199] The inventors have developed a range of novel cell culture media that are well suited to use in the methods of the invention, and with the CNS organoid products of the invention.

[0200] In its seventh aspect, the invention provides an expansion medium comprising EGF, FGF-10 and FGF-2. EGF and FGF-10 may each be provided at a concentration of approximately 50 ng/mL in an expansion medium of the invention, while FGF-2 may be provided at a concentration of approximately 40 ng/mL. Suitable constituents of an expansion medium in accordance with the seventh aspect of the invention may be as follows:

• Neurobasal medium (supplemented with pen/strep), which may comprise approximately 50% of the cell culture medium by volume;

• Adv+++ (Advanced DMEM supplemented with pen/strep, Glutamax, and HEPES), which may comprise approximately 50% of the cell culture medium by volume;

• Non-essential amino acids;

• N2 supplement;

• B27 minus vitamin A;

• Primocin (optionally at a concentration of 50-100 ug/mL); • EGF (optionally at a concentration of 50 ng/mL);

• FGF-10 (optionally at a concentration of 50 ng/mL); and

• FGF-2 (optionally at a concentration of 40 ng/mL).

[0201] In its eighth aspect, the invention provides a maturation medium comprising basement membrane extract. The basement membrane extract may be provided at a concentration of approximately 0.5% in maturation medium of the invention. Without wishing to be bound by any hypothesis, the presence of the basement membrane extract may replicate the effects of the native ECM in the natural process of brain development. Suitable constituents of a maturation medium in accordance with the eighth aspect of the invention may be as follows:

• Non-essential amino acids;

• N2 supplement;

• B27 plus vitamin A;

• Primocin (optionally at a concentration of 50-100 ug/mL);

• Basement membrane extract (optionally at a concentration of 0.5%).

[0202] A maturation medium for use with ventral telencephalon CNS organoids in accordance with the present invention may have the constituents set out for the maturation medium above, and further comprise BDNF and GDNF (optionally both at a concentration of approximately 20 ng/mL).

[0203] A maturation medium for use with dorsal telencephalon CNS organoids in accordance with the present invention may have the constituents set out for the maturation medium above, and further comprise a GSK-3 inhibitor (such as CHIR-99021), which may optionally be provided at a concentration of approximately 3pM; BMP4 and BMP2 (optionally both at a concentration of approximately 20 ng/mL). Examples

Generation of brain organoids from healthy human fetal brain tissue.

[0204] Fresh fetal human brain tissue was obtained from different donors, of gestational week 13 to 15 (Fig. 1a), which corresponds to the mid-neurogenesis period. Importantly, the tissue was processed not by dissociating it into single cells, but by cutting it into pieces spanning the whole germinal as well as neuronal layers, in order to have a representation of the different cell types present in the brain at that specific stage, which includes various progenitors and proliferating SOX2⁺, PAX6⁺ and KI67⁺ cells, as well as neuronal TUIJ⁺ cells (Fig. 1a and Fig. 2d). The brain pieces were seeded in the culture media provided in Table 1 (which included the growth factors EGF, FGF-2, FGF-10) and cultured on an orbital shaker to facilitate the diffusion of nutrients.

TABLE 1

[0205] About 5 to 10 days later, it was observed that while some tissue pieces underwent cell death, alongside appeared the generation of tissue pieces with clearly defined borders and a 3D- structure, which grew bigger over time (organoids) (Fig. 2a). Those organoids could be picked, split, and underwent a rapid growth that typically led to generation of a stable line of tissue-derived organoids within 30 days from tissue isolation.

[0206] Single organoids acquired over time a more regular 3D shape, with folded borders (Fig. 2a). Upon splitting, single organoid pieces demonstrate the ability to reform and grow entire organoids (Fig. 2b). The expansion capacity of the organoids was assessed by measuring their size over time and it was found that, on average, organoids duplicate their volume each passage (every 2 to 3 weeks), and reach an average size of approximately 4-8 mm³ (Fig 2c). Importantly, the estimated total produced biomass from one donor over the culturing period was around 400,000 mm³, which increased the original tissue volume of ±20,000 fold (Fig. 2d). The robust capacity of brain tissue under the defined culture conditions to generate self-organizing organoids has been confirmed using tissue from 4 donors. These could be maintained in culture over a year, and lead to similar results in terms of growth and expansion (Fig. 2c and Fig. 1 b, c)). Immunofluorescence analysis on established organoids (ca. 2 months old) using common neurodevelopmental markers showed abundant expression of neural stem cell/progenitor markers SOX2 and PAX6, proliferating cell marker MKI67, the newborn and migrating neuron DCX and the pan-neuronal marker TUIJ (Fig 1 e,f), demonstrating the maintenance of cellular heterogeneity within the organoids, similarly to tissue (Fig. 1d). Some GFAP⁺ and S100p⁺ astrocytes were also observed, interspersed within DCX⁺ neurons (Fig. 2f). Importantly, sustained presence of HOPX⁺ cells was also observed, a marker for the outer radial glial cells (oRGs), specialized progenitors abundantly present in the brain of gyrencephalic species, including primates and human, and believed to be important for the size expansion and folding of the human brain (Fig 2f and Fig. 3b). Those different cell populations presented a clear distribution, with the SOX2⁺, PAX6⁺ or HOPX⁺ cells present mostly on the external portion of the organoid, and the neuronal marker-expressing cells more on the inside (Fig 2e).

[0207] Accordingly, MKI67⁺ expression was mostly located in the external layers, and colocalized with SOX27PAX6* or HOPX⁺ cells (Fig 2e,f). HOPX⁺ cells were positive for SOX2, PAX6 and often MKI67, and were also located on top of the single positive SOX2⁺/PAX6⁺ dense layer (Fig. 2f and Fig 3b), all hallmarks of oRGs.

[0208] Adherent junctions were maintained at the surface of the organoids, where the stem cell layers are located (Fig. 3c). Therefore, the organization of the human fetal brain derived- organoids (FeBOs) resembles the organization of the germinal layers during brain development. [0209] Transmission electron microscopy (TEM) analysis revealed cell biological features typical of neural tissue (Fig. 3d). Cell developmental dynamics in FeBOs was assessed using lentiviral infection to follow labelled cells over time. Given that the stem cells are positioned at the external part of the FeBOs and are therefore readily accessible, 1 day post infection (p.i.) infected cells were located on the outer side of the organoid in the SOX2⁺ layer (Fig. 3a). At later time points, more labelled cells were progressively located in the inner neuron-rich part of the organoids which were SOX2' (Supplementary Fig. 2a), consistent with ongoing neurogenesis taking place in the FeBOs from their NSCs. identities are an intrinsic tissue

and are maintained in

[0210] Different brain regions are characterized by different cellular heterogeneity, as well as distinct functions. Different parts of human fetal brain were isolated and separately cultured to generate lines of different origin (Fig 4a, b). To identify the origin of these cultures, qPCR analysis on tissue isolated prior to culturing was performed using a defined marker panel (Fig. 5a). Most of the tissue pieces expressed FOXG1 , a typical telencephalic marker (Fig. 5b), reflecting the abundancy of telencephalic tissue at that gestational interval. It was possible to reliably identified biopsies exclusively expressing EMX1 and PAX6, typical dorsal markers, and on the other hand, pieces positive for DLX2, NKX2-1 , and GSX2, ventral telencephalic markers (Fig. 5b).

[0211] After 2 months of culture, qPCR was used to analyze the expression of the same markers in organoids of these assigned dorsal and ventral telencephalic origin. Both ventral- and dorsal- forebrain derived organoids retained similar levels of expression of FOXG1 and SOX2. The cortex-specific PAX6 displayed higher expression in the dorsal organoids, while OTX2 as well as ventral telencephalic markers DLX2 and NKX2-1 were enriched in the ventral organoids (Fig. 4c). Immunofluorescence analysis on organoid sections confirmed the selective expression of key markers at protein level, and visualized the localization of those cell populations within the organoids (Fig. 5d and Fig. 6). MAP2⁺ neurons were located mostly in the inner part of the organoid with SOX2⁺ and PAX6⁺ stem cells located on the outer layer of the dorsal organoids, similarly to the distribution of the SOX2⁺ and NKX2.1⁺ ventral forebrain precursors cells in the ventral organoids. OLIG2⁺ oligodendrocyte precursors cells, typically abundantly present in the germinal layers of the ganglionic eminence, were detectable in the ventral forebrain organoids. The early interneuron marker DLX2⁺ and GAD65⁺ and GAD67⁺ interneuron markers were enriched in the ventral FeBOs. MKI67 was similarly expressed in the outside progenitor portion in both the different regional organoids (Fig. 4d and Fig. 6a). Abundancy and localization of the cell populations revealed to be substantially unchanged over culturing time (Fig. 6a,b,c).

[0212] Co-culture of dorsal and ventral FeBOs labelled with differently-colored lentiviruses prior to co-culturing, demonstrated the capacity of these organoids to fully assemble into fused organoids within 48 hrs thereby mimicking the whole telencephalon (Fig. 6d). Notably, unidirectional migration of cells from the ventral to the dorsal telencephalon was observed, suggestive of the resemblance of the interneuron migration naturally occurring during brain development from subpallium to the cortex.

[0213] Whole transcriptome analysis of ventral and dorsal FeBOs of different ages was performed to thoroughly characterize these cultures. Since FeBOs are generated directly from self-assembly of human fetal brain tissue, expression of pluripotency genes (e.g. KLF4 and NANOG) and mesenchymal-related genes (e.g. C0L1A 1, LUM and DCN) was extremely low (Fig. 4e), indicating the absence of non-neural cell types. The expression profiles of FeBOs were consistent with a developing brain profile (Fig. 7a) and showed robust enrichment of multiple genes expressed in the various cell populations present in the developing human brain (Fig. 4e and Fig 8a).

[0214] These included apical progenitors (e.g. S0X2, HES1, GLI3), intermediate neurogenic progenitors (e.g. BTG2, HES6), proliferating cells (e.g. MKI67, CENPF), newborn migrating neurons (e.g. NRP1, UNC5D, DCX), and neurons (e.g. S0X5, MEF2C and TUBB3) (Fig. 2e). Comparison of the dorsal vs. ventral forebrain organoids showed distinct clustering based on their region origin (Fig. 4f,g and Fig. 7b). Finally, the relative proportion of cell types in organoids of different ages from the bulk RNA sequencing was inferred and found virtually unchanged proportions over culturing (Fig. 4h), with about 30% of the cells being neurons. When the transcriptomes were inspected for changes over time in culture, it was noticed that, despite rather stable transcriptomes (Figure 7c, e), the expression of some neuronal markers was increased in older organoids (Fig. 7d and f). These includes typical genes connected with neuronal functions and maturation (e.g. the interneuron markers DLX1 and DLX2 as well as synaptic proteins PSD- 93, PSD-95, neurexins and neuroligins), but also astrocytic (S100/3) and oligodendrocytic (PDGFRA) genes, suggesting a certain degree of gradual maturation occurring in culture.

Expansion and maturation of fetal-derived brain organoids can be modulated by culture conditions [0215] Conditions to boost maturation of the differentiated cells was investigated. To this end, the growth factors present in the medium were withdrawn and instead supplemented amongst others basal membrane extract BME^R (Fig 9a and Tables 2 to 4).

TABLE 2

TABLE 3

[0216] Under those conditions, it was observed that the organoids arrested their growth.

Comparison of organoids maintained in expansion medium with organoids maintained for 10 days in maturation media highlighted a substantial transcriptomic switch (Fig. 9c), as well as in cell composition (Fig 9b). Various progenitors and proliferation markers, such as PCNA, MKI67, GLI3, GSX2, BLBP, showed reduced expression. [0217] On the contrary, markers for early neuronal specification and mature neurons were upregulated (Fig 9c and Fig.8a). These included transcription factors involved in initiating neuronal differentiation such as S0X11 and S0X4, as well as neuronal layer markers like BRN2, CTIP2 and CUX1 for the dorsal organoids, and interneurons progenitors and GABAergic cells markers such as DLX1/2/5/6, CALB2, GAD1, GAD2 and VGAT, for ventral organoids.

[0218] Furthermore, multiple genes associated with mature neuronal functions, e.g. synaptic components, neurotransmitter receptors and secretion, and neuron projections showed marked upregulation (Fig. 8b). In agreement, also on protein level a sharp decrease in the amount of proliferating MKI67⁺ cells as well as SOX2⁺ stem cells (Fig. 9d and Fig. 10a, b) was observed, as well as a change in their distribution, which appeared more scattered within the organoids, indicating a reduction of the germinal layers.

[0219] Dorsal organoids displayed sustained and diffused expression of the pan-neuronal marker TUI J, as well as newborn neuron marker DCX and layer-specific markers CTIP2, SATB2 and BRN2, which were indeed organized in a layered-fashion (Fig. 9d and Fig. 10a,b,c). Abundant neuronal processes stained by MAP2 were also evident (Fig. 9e).

[0220] Ventral organoids in maturation media displayed reduced expression the ventral progenitor markers NKX2-1 and OLIG2 and abundant presence of DLX2⁺ and GAD67⁺ interneurons (Fig. 9f), which in some organoids appeared more organized in bundles (Fig. 10c). In both type of organoids, abundant presence of astrocytes (GFAP/S100P ) was observed which showed a mature morphology and were interspersed within MAP2⁺ neurons (Figure 10b,c).

[0221] Fluorescent labelling of single cells by PiggyBac-mediated integration of GFP following electroporation, allowed to visualize cell morphology upon 10 days of maturation, which revealed the presence of cells with typical neuronal shape showing multiple processes and arborization (Fig. 10a). Ultrastructural analysis by TEM further proved the acquisition of mature features in the FeBOs such as elongated neuronal processes, synaptic vesicles, and oligodendrocytes that can myelinate axons (Fig. 11).

[0222] Live imaging of sliced organoids in the presence of the fluorescent, cell permeable calcium probe Fluora-4 provided functional validation that the neurons in FeBOs are electrophysiologically active. In fact, it was possible to record many cells across organoids with spontaneous and regular calcium spikes (Fig. 9g).

[0223] It was investigated whether there are differences in maturation related to the culture age of the organoids. While differences in the overall differentiation capacity (as assed by the expression of neuronal markers such as TUI J or DCX) were not observed, it was noticed that at early stage (1.5 months) differentiated organoids displayed almost complete overlap of the SATB2+ and CTIP2+ populations, similarly to what was observed in the tissue from which the organoids were derived and according to the developmental stage.

[0224] When maturation was induced later during culturing (2.5 and 5 months), the percentage of single SATB2+ and CTIP2+ increased at the expense of the double positive population, and their distribution resembled the layering occurring in human brain during development, again suggesting that a certain degree of maturation occurs spontaneously over time during expansion.

Single cell sequencing analysis reveals FeBOs cellular heterogeneity

[0225] To address in more detail the cellular heterogeneity of the human brain derived- organoids, single cell sequencing analysis of dorsal and ventral organoids was performed (ca. 5 month old) (See Methods). After pre-processing and filtering steps, two datasets of 20,000 and 20,000 cells for the dorsal and ventral FeBOs, respectively were retrieved.

[0226] Principal component and clustering analysis of ventral FeBOs revealed the existence of multiple distinct clusters (see Methods for detailed explanation) (Fig. 11a and 12a). Based on differential expression of specific cell markers (Fig 11 b), it was possible to assign the cells to 8 main cluster identities (c1 to c8) (Fig. 13a, b,c). Additionally, multiple gene groups were used to unbiasedly define cell type-specific signatures that were used to inspect the dataset (Fig. 12b).

[0227] As expected, all clusters belonged to the neuroectodermal lineage. C1 was characterized by the expression of radial glial markers (amongst which S0X2, ID2, ID4, HES1), but also of more typical astrocytic genes, for instance MT2A, AQP4, and AGT, suggesting that those cells are a more mature state of radial glia primed or transitioned to astrocyte differentiation.

[0228] Cells in C2 expressed radial glial and astrocyte markers although to a lower level than in c1 and were characterized by exclusive expression of COUPTF1 and COUPTF2, which have been previously shown to be important in an initial phase of transition from neurogenesis to gliogenesis. Therefore, they could represent an intermediate state to astrocytes.

[0229] Radial glial genes were also expressed by c3, which also expressed high level of glycolytic genes and additional genes such as BNIP3 that have been associated to previously described “glycolytic radial glia”, which, also in agreement to previous reports, expresses lower levels of NEST/N .

[0230] Progenitors and stem cell markers, amongst which HOPX, PTPRZ1, characteristic of the outer radial glia, and the more ventricular radial glia NESTIN, HES5 and FABP7 were enriched in c4, in which already also some more neuronal markers appeared (such as GAD43) which were therefore assigned as a cluster containing progenitors likely committed towards neuronal fate.

[0231] These cells transition towards the actively cycling cells of c5 that expressed typical markers of proliferating neural progenitors (such as MKI67, PCNA and TOP2A) and ventral telencephalic neuronal markers (PR0X1 and GSX2), as well as displaying the expression of already more committed neuronal cells (S0X4, S0X11).

[0232] Expression of some of these markers was retained in c6, which clearly in addition acquired the expression of early-stage interneuron genes, such as DLX2 and typical inhibitory GABAergic interneuron markers (the main type of neurons being produced in the ventral telencephalon (e.g. GAD2, CALB2, DLX5), thus representing newborn interneurons. It was noted that cells still displaying a higher expression of progenitor genes such as ASCL1, had lower expression of the interneuron genes such as DLX2, and vice versa (see t-SNE maps in Fig. 13b). [0233] The mature GABAergic interneurons markers (e.g. VGAT, DLX6, CALB2) were expressed in neuronal cluster c7, which did not display progenitor gene expression, and therefore represents mature neurons.

[0234] A very small cluster, c8, expressed the progenitor marker ASCL1, which in the ventral forebrain is also expressed by oligodendrocyte lineage precursors, and additional oligodendrocyte precursors markers (e.g. OLIG2, OLIG1) as well as the more mature oligodendrocyte marker SOX10 and interneuron marker DLX2 in a mutually exclusive fashion. Thus, c8 contained a mixture of precursors of both the neurogenic and oligodendrocyte lineages. [0235] Similar number of clusters were detected for the dorsal forebrain derived-organoids (Fig. 13d). For these organoids c1 expressing typical RG markers was also identified (S0X2, HES1, S0X9) and astrocytic markers (AQP4, AGT) and c2 being the glycolytic radial glial cluster.

[0236] C3 instead constituted a mixed cluster. While multiple cells expressed neurogenic progenitor (HES5, PAX6) and oRG markers (HOPX, PTPRZ1, FAM107A), many expressed some specific markers of migrating as well as cortical neurons (RND2, POU3F2, CUX1, S0X5, CLIM1, SATB2) and typical functional markers of glutamatergic neurons, like the glutamate transporters GRIA2, GRIN1, or glutamate synthetase GLUL. In fact, also the gene signature t-SNE plot showed that in addition to cluster c5-c6 (CTIP2⁺), the glutamatergic neurons are scattered within the c3 SATB2⁺) (Fig. 4 d-f).

[0237] C4 represents the actively proliferating neurogenic progenitors, expressing markers of committed neural cells (S0X4, POU3F2), alongside markers of intermediate progenitors (ELAVL2, TMEM158).

[0238] A cluster of newborn neurons was also detected, c5, characterized by expression of newborn neurons marker DCX, S0X11 and the cortical neuronal markers BCL11B (CTIP2), MLLT11, NFIX and STMN2, which were also present in the mature neuronal cluster c6.

[0239] Intermediate progenitor expression, the neuronal committed progenitors, were found both within c4 and c5, However, despite multiple intermediate progenitor markers being expressed (HES6, BTG2, TMEM158, ELAVL2, GADD45G, MFNG), some characteristic genes for this cell population (e.g. EOMES) were lowly expressed. As consequence, also some important genes for later steps of glutamatergic neuron development (NEUR0D2, NEUR0D6) were absent, suggesting that under the current culture conditions this population is disfavored. Clusters representing endothelial, ependymal, microglia or other immune cells were not identified, indicating that those cells are not part, and not necessary for the propagation, of FeBOs.

Gene-edited human fetal brain-derived organoids to address cancer- and development-related gene function.

[0240] Multiple genes have been found mutated in human brain developmental diseases as well as in cancer but for many of those the pathological mechanisms are still unexplored. This is for instance the case of TP53, whose function during brain development is still obscure, as well as its potential contribution to embryonic-derived brain cancers . To elucidate whether FeBOs could recapitulate developmental genetic defects and therefore be a crucial asset for human brain disease modelling, models of cancer-related genes (TP53) and neurodevelopmental disorders (RAB3GAP2) were established by adapting CRISPR-Cas9 to the FeBOs described above.

[0241] TP53 mutations were introduced by electroporation of whole organoids, which resulted in targeting of sparse cells at 24 hrs p.e. while concomitantly labeling genome-edited cells with a PiggyBac-based transposed GFP (Fig. 14a). During long-term culturing (/.e. 2 months), a sharp increase of the GFP⁺ TP53-targeted population was observed as compared to control organoids (targeted with a non-human sgRNA). With time the GFP⁺ cells overgrew the GFP' population (Fig 14b, c and Fig. 15a).

[0242] TP53 deficiency was confirmed by genotyping and functionally validated by decreased protein expression of the TP53 downstream target TP21 (Fig. 15c). TP53'^A FeBOs could be expanded and used for multiple downstream analysis (Fig. 15). In line with the overtaking TP53~ '' population over time, an increase of MKI67⁺ proliferating cells was observed, together with abundant presence of SOX2⁺ stem cells as compared to control organoids (Fig. 14c). Even when switched to maturation media, TP53' mutant cells failed to reduce the number of proliferating MKI67⁺ cells (Fig. 15d,e), which was maintained to a percentage normally found under expansion conditions. These results are consistent with a model in which TP53-deficient NSCs present impaired differentiation capacity and are less sensitive to growth factor withdrawal and differentiation signals (Fig. 15f).

[0243] Many neurodevelopmental disorders have been predominantly investigated in mouse studies. Mutations leading to impaired RAB3GAP2 activity have been found in patients with Warburg Micro syndrome, characterized by postnatal growth retardation and microencephaly, as well as its milder Martsoft phenotype, depending on the type and severity of the specific gene mutation. While mouse models have correlated impairments in the RAB18 pathway with neural migration and secretion of neurotransmitters, investigation into the developmental defects associated with the rare RAB3GAP2 mutations has not been accomplished. [0244] The expression of RAB3GAP2 in FeBOs trancriptomes was comparable to actual human tissue cortices expression which reduced in FeBOs cultured in maturation medium (Fig.15g). Similarly, it was found that RAB3GAP2 expression decreased along the cellular development trajectory as assessed by scRNA-sequencing analysis, indicative of having a role during the differentiation lineage in a cellular-step limiting fashion (Fig.15g).

[0245] A sgRNA targeting RAB3GAP2 together with Cas9 and a transposable GFP (as readout for genome-edited cells) was electroporated to address cellular effects upon introducing loss- of-function mutations (Fig. 14d,e). Cells targeted by electroporation are located at the outer border of the organoids where stem cell are located (Fig. 7j), which allows following their fate upon gene manipulation, conceptually analogous to the use of in utero electroporation in the developing mouse brain.

[0246] By immunostaining analysis 8 days p.e., a conspicuous decrease of SOX2⁺ stem cells (from ±20% to 10%) within the targeted GFP⁺ population was observed (Fig. 14f). In parallel, a reduction of PAX6⁺ progenitors and a concomitant increase of the neuronal MAP2⁺ and TUJ1 ⁺ populations (Fig. 14h,j) was seen. Thus, RAB3GAP2 mutations lead to an unbalanced fate of progenitor maintenance vs. neuronal differentiation (Fig. 14i, and Fig.15i).

[0247] Changes in the proportion of GFAP+ astrocytes was not observed (Fig 14h), excluding a switch towards gliogenesis. Finally, to evaluate if absence of RAB3GAP2 would affect the initiation of neurogenesis, the RAB3GAP2-targeted organoids were collected after a 5 day-pulse in maturation media (Fig. 14d). Quantification of the DCX⁺ newborn neurons again confirmed the increase of newly generated neurons upon RAB3GAP2 deficiency (Fig, 14j, k). This is consistent with a model in which RAB3GAP2 is important in progenitor cycling where loss leads to premature neuronal differentiation (Fig. 15i).

Discussion

[0248] This study demonstrates that human fetal brain tissue is able to self-organize in vitro - and repeatedly after each split- into a 3D organoid-layered structure in which less differentiated cells localize in contact with the mitogens present in the media, while a progressive less proliferative gradient is established towards the center of the organoid in which neurogenesis can be preserved, therefore creating a self-sustaining organized organoid system.

[0249] This process allows on the one hand to not have a clonal growth from single NSCs and on the other hand to maintain the cell-to-cell interaction as naturally established in the human brain tissue. Recently, the use of a decellularized human brain tissue derived-extracellular matrix, instead of non-neuronal matrices such as Matrigel, improved representation of cellular heterogeneity, functionality, and maturation of iPSC-derived brain organoids (Ann-na Cho, 2021 Nat Comm). This supports the hypothesis that the maintenance of tissue integrity and endogenous brain extracellular matrix in the FeBOs is essential to preserve the different stem and progenitor populations in a “ground-state”, ensuring long term-expansion and differentiation. [0250] Maintenance of tumor integrity was also key to establish 3D organoids from glioblastoma samples (Jacobs et al Cell 2018, Hubert Cancer research 2016). While for aggressive tumor tissue it is somehow not surprising to be proliferative in culture, this is not trivial for wild-type healthy tissue. Slices of developing human brain could be maintained for a few days ex utero and have been previously used as invaluable tools to study human brain development, but they are short lived and this strongly limits their application.

[0251] It is shown that pieces of human brain fetal tissue can turn into an expanding human brain culture with defined cellular organization, composition, and functional properties that can be preserved up to one year. This capacity of great expansion from a single piece of tissue translates into essentially not existing batch effects, currently a main limitation of iPSC/ESC brain systems, and given also the rather stable nature of the FeBOs over passages, allows the generation of “brain organoid lines”, rather than “brain organoid batches”. By developing directly from specified NSCs, the FeBOs contain only cells of brain origin, and never detected cells reminiscent of an undifferentiated embryonic state or from different developmental origin. Accordingly, cells from different lineages such as endothelial cells, microglia or other immune cells are also not present or preserved in the FeBOs, while all the differentiated brain cell types such as neurons, astrocytes and oligodendrocytes are present. Importantly, at any given point the FeBOs can be switched to conditions that promote further production and maturation of differentiated cells within a matter of days.

[0252] Gliogenesis occurs in later phases as compared to neurogenesis during brain development, and maturation processes, such as synapse development and myelination are delayed till after birth. In pluripotent stem cell-derived organoids, the generation of astrocyte and oligodendrocytes and the appearance of mature markers for these cell populations (e.g. GFAP, AGT, AQP4, SOX10) is therefore occurring late (can take up to 6 months) and can require the use of specific maturation protocols (Marton, Pasca 2019 Nature Neuroscience). In the selforganizing FeBOs, the presence of all type of differentiated cell types intermingled amongst each other and showing interactions such as for instance the achievement of certain degrees of myelination, can be boosted by a short maturation.

[0253] It is also demonstrated that different regions of the human fetal forebrain can be successfully cultured. This can be likely extended to other parts of the brain as well. Importantly, it is shown that the organoids can maintain and capture regional identity expression profiling and specific cellular subtypes over prolonged periods of time. [0254] Regional identities during brain development are established thanks to complex regulation of multiple morphogens and signaling gradients as well as by cell-autonomous mechanisms.

[0255] The results herein suggest that at the considered gestational period, stem cell fate is partially determined despite the use of the same medium for different regions. This opens exciting avenues for the use of the FeBOs in determining specification timing and potential as well as plasticity of germinal populations at various gestational ages.

[0256] The findings highlight the relevance of FeBOs models to human in vivo disease phenotypes. In fact, by genetic engineering the FBOs it can be hypothesized that concomitant depletion of neural progenitors and premature neuronal differentiation could be at the basis of the disease phenotype observed in the Warburg Micro and Martsolf patients.

Material and Methods

Tissue isolation, dissection, and processing for organoid culture

[0257] Human fetal brain tissue was obtained from Leiden University Medical Center from abortion material from healthy subjects, under ethical approval and informed consent. Four different donors were used in this study, ranging between 13 to 15 gestational weeks. Immediately after the termination of pregnancy, recognizable pieces of brain tissue were collected and washed in 0.9% (wt/vol) NaCI and kept on ice. Within 24 hrs, the tissue was extensively washed with cold Advanced DMEM/F12 (Thermo) containing 10 U/ml Penicillin-Streptomycin (Thermo). Evident necrotic areas were removed using micro-dissecting scissor (WPI). Single pieces where isolated and individually transferred into a well of a 12-well plate. Each piece was further processed and cut into smaller pieces (approximately 0.2-0.3 mm diameter) with a micro-dissecting scissor while avoiding extensive mincing of the tissue. A few of these small pieces were collected for RNA isolation for gPCR analysis using a panel of genes to define the origin of the tissue, and for fixation in 4% formaldehyde to use for further immunofluorescence analysis, while the rest was used for establishment of the organoid lines.

Organoid establishment and long-term culturing

[0258] Pieces of human fetal brain tissue obtained from the dissection procedure were seeded in 12-well plates (about 10-15 pieces/well). The optimized culturing medium contained a base medium of a 1 to 1 mixture of Advanced DMEM/F12 (Thermo) supplemented with 10 U/ml Penicillin-Streptomycin (Thermo), IX GlutaMax (Thermo), and 1X HEPES (Thermo) (Advanced DMEM +++) and Neurobasal medium (Thermo) plus 10 U/ml Penicillin-Streptomycin. Then, 1X B27 without VitaminA supplement (Thermo), 1X N2 supplement (Thermo), 1X MEM non-essential amino acid solution, 50 ng/ml FGF10 (Peprotech), 40 ng/ml FGF2 (Peprotech), 50 ng/ml EGF (Peprotech), and 100 pg/ml Primocin (Thermo) were added to the base medium to constitute the expansion medium. The expansion medium was refreshed every second day in initial culture, or every day if massive cell death was noticed. The organoids were cultured in an incubator at 37°C and 5% CO2 under constant rotation (80 rpm) using an orbital shaker. Between 5 and 10 days after seeding the pieces of tissue, formation of organoid-like structures with defined borders was observed. These structures were transferred in a new well of a 12 well plate using a blunt-cut P1000 pipette tip and allowed to grow further until they reached a size of approximately 2-4 mm diameter. Those initial organoids were split by cutting into smaller pieces using micro-dissecting scissors, washed with wash buffer (Advanced DMEM +++) and 2-3 pieces were transferred into a new well, and typically within 3-5 days new organoids were formed again. Over time the organoids acquired a more regular spherical morphology with folded edges. Regular splitting of the organoids was performed approximately every 2 to 4 weeks as described. Expansion medium was refreshed every second day. For further maturation, single organoids at any given point were transferred to a well of a 24-well plate and the expansion medium was completely removed by rinsing once with wash buffer. Maturation medium was prepared fresh by using the base medium supplemented with 1X N2 supplement (Thermo), 1X MEM non-essential amino acid solution, 100 pg/ml Primocin (Thermo), 1X B27 Supplement with VitaminA (Thermo), 20 ng/ml BDNF, 20 ng/ml GDNF (Peprotech), 100 pg/ml Primocin (Thermo), and 0.5% BME^R. Maturation medium was changed every second day and kept from 5 to 10 days depending on the application.

FBOs growth assessment

[0259] The growth of the organoids was measured by two independent means: increase of volume of single organoids in between passages; and by overall tissue expansion by considering the increase of volume/passage and the split ratio volume. After splitting, composite pictures of multiple organoids were taken at regular intervals using a DMi8 microscope (Leica). The images were analyzed by Imaged software, and volumes calculated by measuring the diameter size and using the formula 4-nrir2r3/3. The average increased volume across multiple organoids in between a split was then multiplied per the split ratios over time.

FBOs and tissue processing for immunofluorescence

[0260] For immunofluorescence analysis, organoids or human fetal brain tissue were fixed by incubation with 4% formaldehyde over night at 4°C. Samples were then washed 2-3 times with 1X PBS and kept at 4°C before further processing. Samples were embedded in 3% low melting agarose and cut using a slicing Vibratome (VT1200S, Leica) to obtain 40 Dm sections. Those were preserved in 1X PBS at 4°C or for long-term storage at -20 °C in freezing medium (50% 2X PBS, 25% Ethylene Glycol and 25% Glycerol). Stainings were performed on floating sections by an initial permeabilization and blocking incubation step of 2 hrs at room temperature in blocking buffer (1X PBS, 5% BSA, 0.02% Triton-X-100), and a 48 hrs primary antibody incubation at 4°C with the appropriate primary antibody at the indicated dilutions in incubation buffer (1X PBS, 2% BSA). Sections were washed 3 times with 1X PBS and incubated for 24 hrs at 4°C with appropriate secondary antibodies diluted 1 :1000 in incubation buffer. Sections were then incubated with DAPI (1 ptg/ .1) diluted in 1x PBS for 20 min at RT to counterstain the nuclei. Sections were washed 3 times with 1X PBS and transferred to 24-well glass-bottom plates (SensoPlate) using a thin brush and mounted using Immu-Mount mounting medium (Thermo) for further imaging.

Lentiviral infection of FBOs

[0261] Lentiviruses were produced, using pLV-H2B-mNeon-ires-Puro or pLV-H2B-mCherry- ires-Puro constructs. FBOs were infected by incubating each organoid with expansion medium containing concentrated lentiviruses (diluted 1 to 1000) in a well of a 24-well plate for 6 to 8 hrs. After incubation, virus-containing medium was removed and organoids were washed 3 times with wash buffer and then cultured in expansion medium. Infected FBOs were collected at different time points and processed for immunofluorescence staining as indicated above.

Generation of fusoids between dorsal and ventral forebrain FBOs

[0262] For the generation of dorsal and ventral forebrain fused FBOs, dorsal forebrain and ventral forebrain organoid pieces were first independently infected with H2B::mNeon and H2B::mCherry lentiviruses, respectively, as described above. 24 hrs after infection, dorsal and ventral FBOs were split by cutting each organoid into half and placing one piece of dorsal and one piece of ventral FBOs together into a well of a 24-well plate. Fusion of the organoids and cell migration was monitored by taking brightfield and fluorescence images at regular intervals (day 2, day 7, day 12 post co-coculture) with a DMi8 microscope (Leica).

Transmission electron microscopy (TEM) in FBOs

[0263] For TEM, FBOs in expansion and maturation medium were fixed overnight with PFA 4% and washed twice with PBS 1X solution. They were then placed on 3 mm diameter and 200 mm depth standard flat carriers for high-pressure freezing and immediately cryoimmobilized using a Leica EM high-pressure freezer (equivalent to the HPM10), and stored in liquid nitrogen until further use. They were freeze substituted in anhydrous acetone containing 2% osmium tetroxide and 0.1% uranyl acetate at -90°C for 72 h and warmed to room temperature at 5°C for 1 hour (EM AFS-2, Leica). The samples were kept for 2 h at 4°C and for 2 h more at room temperature. After several acetone rinses (4 X 15 min), samples were infiltrated with Epon resin for 2 days (acetone/resin, 3:1 for 3 h; 2:2 for 3 h; 3:1 overnight; pure resin for 6 h + overnight + 6 h + overnight + 3 h). Resin was polymerized at 60 C during 96 h. Ultrathin sections from the resin blocks were obtained using a Leica Ultracut UC6 ultramicrotome and mounted on formvar-coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate. Sections were observed in a Tecnai T12 Spirit equipped with an Eagle 4kx4k camera (FEI Company) and large electron microscopy overviews were collected using the principles and software described previously (Faas et al., 2012).

RNA extraction and qPCR

[0264] For preparation of RNA from FBOs and human fetal brain tissue, entire organoids (or pieces of tissue) were first washed once with 1X PBS, cut using micro-dissecting scissors and then collected in 1 ml TRIzol Reagent. The samples were fully lysed by extensive pipetting and then snap-frozen in liquid nitrogen. RNA was extracted using isopropanol precipitation using the manufacturer’s protocol. Extracted RNA was stored at -80 °C. For qPCR gene expression quantification, RNA concentration and purity was determined using a NanoDrop spectrophotometer. 250 ng of RNA were used as initial input for cDNA production using the SuperScript IV kit (Thermo). The cDNA reaction was diluted 1 to 10 and 2 pl of the diluted cDNA was used for each qPCR reaction. qPCR reactions were performed using iQSYBRGreen mix (Bio-rad). For each experiment and each organoid, technical triplicates were performed.

Bulk RNA sequencing, analysis and deconvolution

[0265] For bulk RNA sequencing, RNA was extracted and processed as described above from organoids derived from ventral and dorsal forebrain, in expansion and 10 days of maturation medium and from different donor and different ages in culture as indicated in the manuscript. RNA integrity was measured using the Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer and RNA concentrations were determined using the Qubit RNA HS Assay Kit. RIN values of RNA samples were typically 9.5-10 and only samples with RIN >8.5 were used for libraries preparation. RNA libraries were prepared using TruSeq Stranded mRNA polyA kit (Illumina) and paired-end (2x50 bp) sequenced on an Illumina Nextseq 2000. Library preparation and sequencing was performed by USEQ (Utrecht Sequencing Facily). Reads were mapped to the human GRCh38 genome assembly. From the dataset, firstly filtered out lowly expressed genes (>less than 20 transcript counts across all samples). Normalization and any differential gene expression analysis presented in this study was performed using the DESeq2 package in RStudio environment. Considered Iog2 fold changes and significance (p-values) are indicated throughout the paper. Calculation and visualization of principal components for bulk RNA sequencing was computed using the R package factoextra. Unsupervised hierarchical clustering was performed to classify 1) groups of genes having similar expression profiles across samples or time points and 2) groups of samples having similar gene expression. Gene Ontology analyses were performed using either EnrichR or Panther. Data visualization was obtained either using ggplot in Rstudio environment or GraphPad Prism.

[0266] CIBERSORTx was used for estimation of cell type abundancy from bulk RNA sequencing. Gene expression profiles for the different clusters obtained from the FBO single cell sequencing dataset were extracted as pseudobulk, by aggregating reads coming from cells belonging to the same cell type (as defined by clusters), after creating a SingleCellExperiment object and then using the aggregate. Matrix function form the Matrix.utils package. A gene list was obtained by combining the top expressed genes per the different cell types and this was used as input signature file to probe each RNA bulk sequencing sample for deconvolution in CIBERSORTx.

Single cell RNA sequencing and bioinformatic analysis

[0267] Ventral and dorsal forebrain derived organoids of 5 months of age in culture both in expansion or 5 days into maturation medium were cut into small pieces (0.2-0.4 mm) and washed 2-3 times with the wash buffer to remove possible cellular debris. The organoid pieces were then collected in a tube and washed 2 times with HBSS without calcium and magnesium (Thermo). Multiple organoids were pulled together and were dissociated using the Papain Neural Dissociation kit (Miltenyi), according to the manufacturer’s instruction. The second enzymatic step was supplemented with 10 pl of 100 U/pl of DNAse I (Roche) and was incubated for approximately 40 minutes with regular gently pipetting using blunt-cut P1000 pipette tips. The dissociated cells were washed twice with HBSS with calcium and magnesium (Thermo) and then twice with HBSS without calcium and magnesium. Centrifugation steps were performed at low speed (100 g) in a swinging bucket for 5 min. Viability was calculated and only samples with > 90% viable cells for the expansion condition and > 85% for the maturation condition were used. For preparation of dissociated cells in maturation condition, given the lower general viability, the Dead Cell Removal kit (Miltenyi) was used with MS columns after dissociation according to the manufacturer’s instructions. Live cells were manually counted using Trypan blue and resuspended in 1X PBS containing 0.04% BSA at an optimal concentration of 700-1200 cells/pl. Approximately 16,000 cells per sample (ventral expansion, ventral maturation, dorsal expansion and dorsal maturation) were loaded onto a Chromium Single Cell 3' chip and used for library preparation using a Chromium Next GEM Automated Single Cell 3’ Library and Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Each library was sequenced using the NovaSeq 6000 SP v1.5 (200 cycles) on a 2 x 150 bp. Mapping and UMI counting were performed using the Cell Ranger software (version 3.1.0). In general, >98% of barcodes and UMI were valid, >92% of reads could be mapped to the genome and >50% to the transcriptome. From dorsal expansion samples, 13,600 cells were revered with an average of 16,040 reads/cell; from dorsal maturation samples, 12,798 cells were revered with an average of 14,353 reads/cell; from ventral expansion samples, 16,422 cells were revered with an average of 13,633 reads/cell; from ventral maturation samples, 12,775 cells were revered with an average of 16,212 reads/cell. Bioinformatic analysis were performed using the R package Seurat 4.0. Libraries were combined using the merge function, and cells with less than 200 detected genes, more than 6000 genes and more than 20% of mitochondrial content were removed. Preprocessing of the data was performed according to the suggested analysis pipeline from Seurat developers. Briefly, gene expression normalization was performed by employing a global-scaling normalization method “LogNormalize” with the default scale factor of 10,000. FindVariableFeatures function was used to find the top 2000 most variables genes in the data set, using the standard selection method “vst” and prior to calculation of principal component analysis the dataset was scaled by applying linear transformation so that the average expression for each gene across cells was 0 and the variance across cells was 1. Dimensionality of the dataset was determined by using the function ElbolwPlot and chose 10 principal components with a 0.5 resolution to determine clusters with the functions “FindNeighbors” and “FindClusters”. Data were visualized by UMAP. For definition of cluster identity, known markers based on literature annotation were used and the dataset probed for these signatures using the AddModuleScore from Seurat. Based on this collective information, defined clusters were assigned to specific identities. Data visualization was obtained with Seurat plotting functions in RStudio environment.

Electroporation and CRISPR-Cas9 gene knock-out in FBOs

[0268] Plasmids expressing a specific sgRNA were obtained by cloning the sgRNA sequences for the different targets in the vector pSPgRNA (Addgene plasmid 47108) as described previously. A previously used targeting sequence for TP53 was used while for targeting of RAB3GAP2 gRNAs were designed using an online web-tool (www.atum.bio/eCommerce/cas9/input). A plasmid encoding both SpCas9 as well as mCherry for visualization of transfected cells (Addgene #66940) was co-transfected together with the gRNA-expressing plasmid. To be able to permanently fluorescently label the transfected cells, a two-plasmid transposon system was used (piggyBac transposase and a donor plasmid with terminal repeats bearing a cassette with CAG- EGFP Addgene Plasmid #40973) and co-transfected those with the gRNA and the Cas9-mCherry plasmids. Electroporation was performed. Briefly, the FBOs were cut into pieces of approximately 0.5-1 mm of diameter and were washed once with Opti-MEM without Phenol Red (Thermo). The pieces were incubated for 5-10 min in 200 pl of Opti-MEM containing 100 pg of the DNA mixture. Two to three organoid pieces were transferred in a 4 mm gap cuvette and electroporated with a NEPA21 electroporator using the following parameters: Poring pulse: Voltage = 175V, Pulse Length = 7.5, Pulse Interval = 50 msec, Number of Pulses = 2; Transfer pulse: Voltage = 20V, Pulse Length = 50 msec, Pulse Interval = 50 msec, Number of Pulses = 5. Immediately after electroporation, 1 ml of expansion medium was added to the cuvette to recover the organoid pieces for 15 min, after which the they were transferred back into a well of a 12-well plate, washed once with wash buffer and let to reform into organoids in expansion medium (1 piece/well). Genetically-engineered organoids were cultured as described for wild-type organoids with regular splitting. When indicated in the text, the mutant organoids were transferred to maturation medium and both mutant organoids in expansion or maturation medium were processed for further analysis. When the transfected population overgrew the not-transfected population as assessed by increase in fluorescence, part of the transfect organoids was cut and lysed using a lysis buffer described in and genomic DNA isolated by isopropanol precipitation. TP53 mutation was genotyped by Sanger sequencing of the PCR amplicon obtained using primers encompassing the gRNA targeted region . If necessary, genotypes were deconvoluted using the ICE v2 CRISPR tool (Hsiau bioRxiv). To visualize cell morphology, electroporation with the piggyBac transposase and the donor CAG-EGFP plasmid (Addgene) was performed to label sparse cells within the organoid. A week after electroporation, organoids were transferred to maturation medium. Whole organoids (10 days after switching to maturation medium), were imaged at a confocal microscope as previously described.

Confocal imaging, analysis, and quantification

[0269] Stained organoids (and tissue) slices and live whole genetically engineered organoids were imaged with a 20X objective on a Sp8 confocal microscope (Leica). Lif files of the merged images were processed and analyzed for measurement of mean fluorescence intensity and manually counting of positive cells for any given markers using Imaged software and Photoshop CS4. For quantification of cell distribution, regions of interest (ROIs) were defined to divide the organoids in bins and number of positive cells/mean fluorescence intensity for any given ROI was determined. Measurement of Calcium flux by live imaging

[0270] Live organoids cultured in expansion or 5 days of maturation medium were embedded in agarose and sliced to 100 .m-thick sections using a slicing Vibratome (VT1200S, Leica). These samples were then processed using the Fluo-4 Calcium Imaging Kit (Thermo) according to the manufacturer’s instruction with a few modifications. Slices were incubated for 15 min at 37°C followed by an incubation for 15 min at room temperature both in complete medium diluted 1 to 2 with 1X PBS and supplemented with 20 mM glucose. Slices were then washed once and maintained in medium supplemented with Neuro Backdrop Background Suppressor solution (dilution 1 :10) and immediately imaged on a confocal Sp8 microscope (Leica). For any defined position, images were acguired every 2 seconds for a total of 5 min in the 488 channel. Images over time (t) were seguentially assembled to create movies with Imaged software. To record calcium spikes, single cells were carefully outlined, and the position defined as ROI and intensity of fluorescence measured over the entire time series. The AF1/F0 trace for each ROI was calculated by dividing the fluorescence by the initial baseline fluorescence for each time point and record traces visualized for individual cells in GraphPad Prism.

[0271 ] Culturing of individual fragments of subareas of the human fetal cortex:

Along the rostro-caudal axis, the human cortex is subdivided into distinct specialized areas, with the prefrontal cortex (PFC) and primary visual (V1) cortex spanning the two poles. Single-cell seguencing studies have highlighted gene expression patterns that may be associated with cortical area patterning. Therefore, the expression signatures of these markers was evaluated in the different cortex-derived FeBO lines (each derived from a unigue cortex fragment). Distinct clustering between these lines was observed, with certain lines showing an enrichment of V1 markers (e.g. PENK, NPY, LHX2), while other lines showed enrichment for either PFC markers (e.g. CLMP, VSTM2L, CPNE8), or a temporal/parietal signature (e.g. WNT7B, LIX1 , NR2F1) (Figure 18). These data show that FeBOs may have the capacity to reflect specific subareas of the developing human brain. Altogether, the in vivo tissue “imprinting” appears thus sufficient to reflect the programmed regional fate in vitro as FeBO cultures.

Fetal brain organoids (FeBOs) possess similarity with actual primary human fetal brain tissue [0272] To compare the dataset with the single-cell seguencing profiles of primary human fetal brain tissues, the dataset from Bhaduri et al. (2021 , Nature 598, 200-204) was analyzed. Tissue similarity was judged by several means. Tissue resemblance of the FeBOs (derived from 14GW tissue) was first evaluated according to the full transcriptomic profiles (pseudobulks) of prenatal ganglionic eminence tissues ranging from 14 to 25 gestational weeks. Organoids-to-tissue correlation was strikingly high, which was most marked for the 14 to 18GWage range, and aligned well with the original tissue age from which FeBOs were derived (Figure 19A). In-depth comparisons with the GE 14 GW primary tissue dataset by analyzing cluster correlation (taken from Bhaduri etal.) was then performed. Strikingly, distinct clusters of specific cell types with each cluster comprising of both organoid and tissue clusters (Figure 19B) were seen. A distinct neuronal cluster comprised of 3 tissue clusters and 2 organoid clusters was observed, all highly correlating with each other. A second cluster comprised of cycling progenitors as well as newborn neurons was likewise composed of both organoid and tissue clusters. A defined cluster of radial glia stem cells originating from both tissue and FeBOs was also present. A more mixed identity cluster comprising of both OPCs and more stem/astrocytic cells was detected. Vasculature and microglia clusters were exclusively detected in tissue and clustered apart from all other clusters. These intermingled cluster correlations highlighted the faithful cell type identities as observed in primary tissue. Finally, integration of the tissue and FeBOs datasets show that similar cell type clusters were present (Figure 19C).

Tissue similarity for dorsal forebrain (cortex) FeBOs were also evaluated, using a published dataset on primary cortex tissue (Bhaduri et al.). Cortical tissue clusters at 14GW and dorsal forebrain FeBOs clusters having the same cell identity highly intermingled and resembled each other in correlation analysis (Figure 19D). Like ventral FeBOs, also the dorsal FeBOs single cells integrated well with its tissue counterpart, and the identified clusters in the integrated dataset show a significant representation of cells coming from both tissue and organoids (Figure 19E). Altogether these characterizations and comparisons highlighted that FeBOs can capture longterm maintenance of tissue similarity and cellular heterogeneity.

Claims

55 Claims

1. A CNS organoid capable of propagation by dissection and culture of the organoid.

2. A CNS organoid consisting of foetal CNS cells that are TBXT-, COL1 A1-, and LUM-.

3. A CNS organoid consisting of foetal CNS cells that are SOX17-, HNF3B-, and GATA4-.

4. A CNS organoid according to any preceding claim consisting of foetal CNS cells that are

TBXT-, COL1A1-, LUM-, SOX17-, HNF3B-, and GATA4-.

5. A CNS organoid consisting of foetal CNS cells that are N-cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+.

6. A CNS organoid according to any preceding claim, having a stratified structure comprising an outer layer of progenitor cells and an inner layer of differentiated cells.

7. A CNS organoid according to any preceding claim, comprising foetal extracellular matrix material.

8. A CNS organoid according to any preceding claim, wherein the CNS organoid comprises an ECM-component expression profile similar to an ECM-component expression profile of a control or wild-type CNS tissue; and/or comprises secretion levels of one or more ECM components greater than a control culture.

9. A CNS organoid according to any preceding claim, comprising progenitor cells selected from the group consisting of: SOX2+ progenitor cells; neuronal stem cells; radial glial stem cells (which may suitably be selected from: glycolytic radial glial cells, ventricular radial glial cells, and outer radial glial cells, such as HOPX+ outer radial glial cells); apical progenitor cells; intermediate progenitor cells; and oligodendrocyte precursor cells.

10. A CNS organoid according to any preceding claim, comprising proliferating cells expressing markers selected from the group consisting of: MKI67 and/or CENPF and/or PCNA and/or TOP2A. 56

11. A CNS organoid according to any preceding claim, comprising neuronal cells selected from the group consisting of: immature neuronal cells; and/or mature neuronal cells; and/or migrating neuronal cells; and/or GABAergic neuronal cells; and/or glutamatergic neuronal cells; and/or cortical neurons.

12. A CNS organoid according to any preceding claim, that is a ventral telencephalic organoid, or a dorsal telencephalic organoid.

13. A method of producing a CNS organoid, the method comprising:

•

14. A method according to claim 13, wherein the portions of foetal CNS tissue further comprise foetal CNS extracellular matrix.

15. A method according to claim 13 or claim 14, wherein foetal CNS cells in the portions of foetal CNS tissue consist of neuroectoderm cells.

16. A method according to claim 15, wherein foetal CNS cells in the portions of foetal CNS tissue consist of cells that are N-cadherin+ (CDH2+) and/or Nestin+ and/or SOX2+.

17. A method according to any of claims 13 to 16, wherein the sample of foetal CNS tissue is at a developmental stage between 6 gestational weeks (GW) and 24 GW.

18. A method of propagating a CNS organoid, the method comprising:

•

19. A method according to any of claim 13 to 18, wherein the expansion medium is as defined in claim 24. 57

20. A method according to claim 18 or claim 19, wherein the organoid is cultured until it has a size of between approximately 2 mm and approximately 4 mm prior to mechanical dissection.

21. A method according to any of claims 18 to 20, wherein the organoid is mechanically dissected to produce organoid fractions of approximately 1 mm size

22. A method of inducing maturation of a CNS organoid, the method comprising:

23. A method according to claim 22, wherein the maturation medium is as defined in claim

25.

24. An expansion medium comprising:

Neurobasal medium (supplemented with pen/strep);

Adv+++ (Advanced DMEM supplemented with pen/strep, Glutamax, and HEPES);

Non-essential amino acids;

N2 supplement;

B27 minus vitamin A;

Primocin (optionally at a concentration of 50-100 ug/mL);

EGF (optionally at a concentration of 50 ng/mL);

FGF-10 (optionally at a concentration of 50 ng/mL); and FGF-2 (optionally at a concentration of 40 ng/mL).

25. A maturation medium comprising:

Neurobasal medium (supplemented with pen/strep);

Adv+++ (Advanced DMEM supplemented with pen/strep, Glutamax, and HEPES);

Non-essential amino acids;

N2 supplement;

B27 plus vitamin A;

Primocin (optionally at a concentration of 50-100 ug/mL); and

Basement membrane extract (optionally at a concentration of 0.5%). 58

26. A kit of parts comprising an expansion medium according to claim 24 and/or a maturation medium according to claim 25 and, optionally, an organoid of any of claims 1 to 12.

27. An organoid formed by the method according to any one of claims 13 to 23.