Choroid plexus organoids and methods for producing the same

Abstract

Disclosed herein are methods and materials involving cultured choroid plexus organoids comprising: (a) epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium, plus other authentic features and markers.

Description

Field of the Invention

[0001] The present invention relates to choroid plexus organoids, in particular choroid plexus organoids comprising a tight epithelial barrier and capable of producing cerebrospinal fluid-like fluid. The present invention also relates to methods of producing choroid plexus organoids. Background Art

[0002] The choroid plexus is a highly conserved and understudied secretory tissue in the brain. This tissue expands in humans in proportion to the size of our brains and exhibits many important functions in the brain, such as forming a protective epithelial barrier and secreting cerebrospinal fluid (CSF)(1).

[0003] Anatomically, the choroid plexus is located in the lateral, third, and fourth ventricles that emerge in each corresponding cavity. The mature choroid plexus consists of a single cuboidal epithelium that surrounds a core of stromal tissue of mesenchymal origin and fenestrated capillaries. The epithelial cells are closely connected by tight junctions and are highly polarized, with the apical brush edge facing the ventricular cavity and the basal surface on the stromal side.

[0004] The highly polarized secretory epithelium of the choroid plexus displays many important functions in the brain, such as forming a protective epithelial barrier and secreting CSF.

[0005] CSF is important for maintaining physiological levels of nutrients in the brain, for the transport of signaling molecules and growth factors, and for its protective role in the regulation of intracranial pressure. CSF is a clear, colorless fluid that circulates through the ventricles and subarachnoid space and is partially reabsorbed into the blood circulation via the arachnoid granulations (2–4).

[0006] CSF is produced by active secretion of the choroid plexus epithelial cells, which maximizes the number of transporters and pumps in their apical brush borders; and by filtration of the blood allowed by the presence of tight junctions within the epithelial cells. The choroid plexus plays a key role in maintaining the rate of production and reabsorption of CSF. Disturbance of this balance can lead to pathologies such as hydrocephalus.

[0007] The production of CSF involves different mechanisms. First, Na + -K + The ATPase pump and cytosolic carbonic anhydrase generate an osmotic gradient composed mainly of Na +Secretion into the ventricular cavity is driven. This osmotic force drives the influx of water across the cell through the aquaporin channel. Secondly, ATP binding cassette (ABC) proteins and solute carrier transporters (SLC) allow the exchange of amino acids, nucleosides and small peptides with and without the need for ATP hydrolysis, respectively. Choroid plexus epithelial cells also express efflux transporters, such as MRP1 and MRP4. These pumps regulate the clearance of metabolites and prevent the entry of drugs and toxic compounds, thereby reducing their availability in CSF. Thirdly, the highly specialized and expanded secretory system of choroid plexus epithelial cells enables the secretion of more complex proteins and hormones. For example, thyroxine transporter (TTR), a carrier of thyroid hormone and retinol, is the most abundant protein synthesized and secreted by the choroid plexus (Li, X. and Buxbaum, JN (2011) Mol. Neurodegener. 6: 1-17; Richardson, SJ et al. (2015) Front. Neurosci. 9: 1-8; Johnson, BA et al. (2018) Fluids Barriers CNS 15: 22). TTR-mediated delivery of thyroid hormones to the CSF appears to play an important role in the regulation of brain development.

[0008] The tightly sealed epithelium of the choroid plexus is also called the blood-CSF barrier (B-CSF-B). The B-CSF-B is similar to the blood-brain barrier (BBB), preventing toxic substances or signals in the circulation from reaching the brain. However, unlike the BBB, the B-CSF-B is formed by a single cell type, the choroid plexus epithelial cells, and it directly faces the CSF rather than the brain parenchyma (5, 6). However, since CSF can freely enter the brain, the transport of molecules through the B-CSF-B is an alternative route to enter the brain.

[0009] Little is known about human choroid plexus development. The choroid plexus develops through a series of stages, from pseudostratified epithelium to columnar epithelium to a final stage of highly folded cuboidal epithelium with microvilli and basally located nuclei. In the dorsal telencephalon, around 7 weeks of gestation, a local BMP signaling gradient drives the development of the choroid plexus from the neuroepithelium, which begins to extend toward the ventricular cavity. By establishing a local threshold, BMP-secreted signals progressively define the choroid plexus epithelium and the adjacent cortical margin, which is a wingless gene-related (WNT) signaling center that develops the hippocampal primordium.

[0010] Choroid plexus patterning has been studied in vitro, and over the past decade, major advances in 3D culture systems such as brain organoids have led to fundamental discoveries in human developmental biology (7–12).

[0011] However, the induced choroid plexus cells generated to date have not been shown to exhibit the same complex structure as the in vivo choroid plexus, nor have they been shown to be functional in producing CSF.

[0012] Therefore, there is still a great need in the art to establish a functional model of the choroid plexus. The choroid plexus is an important entry point for drugs to enter the CNS; however, little is known about the regulation and permeability of B-CSF-B and how to use it as a tool to increase the availability of certain drugs in the brain. Studies of choroid plexus functional models may help to understand this aspect, especially for determining the ability of candidate drugs to cross B-CSF-B and / or the BBB. Summary of the invention

[0013] The inventors have established a protocol to generate choroid plexus organoids using a combination of signaling molecules.

[0014] The organoids (hereinafter referred to as "ChP organoids") have a selective epithelial barrier that secretes CSF-like fluid in self-contained compartments, much like the CSF of the ventricles. Characterization of the ChP organoids by single-cell RNA sequencing revealed the presence of epithelial and stromal ChP populations. Proteomic analysis of the organoid CSF-like fluid revealed human-specific developmental factors, as well as disease-related biomarkers. Finally, using NMR, the inventors showed that this in vitro barrier is as selective for small molecules as the in vivo barrier, and that the ChP-CSF organoids can predict the CNS permeability of new compounds.

[0015] Thus, the organoids provided by the inventors' protocol recapitulate the essential functions of the choroid plexus, namely secretion and formation of a tight epithelial barrier. The inventors determined that the organoids provided by their protocol reliably and reproducibly develop a choroid plexus epithelium with polarized cells that actively secrete a fluid within the organoid that has properties very similar to cerebrospinal fluid (CSF).

[0016] In addition, the inventors used histological and electron microscopic (EM) analysis to discover the presence of choroid plexus-specific water channels and transporters located on the apical brush edge of the choroid plexus organoid epithelium. These tissues were found to show tight junctions forming an epithelial barrier, and the inventors observed large, fluid-filled cysts protruding from the organoids, the contents of which were highly similar to human embryonic CSF by mass spectrometry.

[0017] The present invention provides a tool to study, for example, choroid plexus function, CSF production, and the penetration of compounds, peptides and serum-derived proteins into the human brain through the CSF.

[0018] Thus, in one aspect, the invention provides a choroid plexus organoid comprising: (a) an epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0019] In some embodiments, the choroid plexus organoid comprises an epithelium comprising a tight epithelial barrier. In some embodiments, the choroid plexus organoid comprises one or more cysts surrounded by the epithelium. In preferred embodiments, the choroid plexus organoid comprises an epithelium comprising a tight epithelial barrier, and one or more cysts surrounded by the epithelium.

[0020] In a preferred embodiment, the epithelium is a cuboidal epithelium.

[0021] In some embodiments, the choroid plexus organoid further comprises (c) a stromal cell population.

[0022] In some embodiments, the choroid plexus organoid has characteristics of a lateral ventricle.

[0023] In a preferred embodiment, the choroid plexus organoids are LUM+, DCN+, and DLK1+.

[0024] In some embodiments, the choroid plexus organoids are TTR+, MRP1+, Aqp1+, and / or ZO1+. In preferred embodiments, the choroid plexus organoids are TTR+, MRP1+, Aqp1+, and ZO1+.

[0025] In other embodiments, the choroid plexus organoids are TTR+, MRP1+, MRP4+, Aqp1+, and / or ZO1+. In other embodiments, the choroid plexus organoids are TTR+, MRP1+, MRP4+, Aqp1+, and ZO1+.

[0026] In some embodiments, the choroid plexus organoids comprise at least 50% TTR+ tissue. In some embodiments, the choroid plexus organoids comprise at least 60% TTR+ tissue. In some embodiments, the choroid plexus organoids comprise at least 70% TTR+ tissue. In some embodiments, the choroid plexus organoids comprise at least 80% TTR+ tissue. In some embodiments, the choroid plexus organoids comprise at least 90% TTR+ tissue.

[0027] In some embodiments, the epithelium is TTR+, MRP1+, Aqp1+, and / or ZO1+. In preferred embodiments, the epithelium is TTR+, MRP1+, Aqp1+, and ZO1+.

[0028] In some embodiments, the epithelium is MRP4+.

[0029] In some embodiments, TTR is enriched on the apical surface of the epithelium. In some embodiments, Aqpl is enriched on the apical surface of the epithelium. In some embodiments, ZO1 is enriched on the apical surface of the epithelium.

[0030] In some embodiments, TTR and Aqpl are enriched on the apical surface of the epithelium. In some embodiments, TTR, Aqpl and ZO1 are enriched on the apical surface of the epithelium.

[0031] In some embodiments, MRP1 is enriched on the apical and basal surfaces of the epithelium.

[0032] In some embodiments, MRP4 is enriched on the apical and basal surfaces of the epithelium.

[0033] Enrichment of a marker in a region (e.g., a cell) may mean that the marker is present in greater abundance in that region compared to other regions (e.g., all other regions). The marker may be substantially absent in other regions (e.g., all other regions).

[0034] In some embodiments, the choroid plexus organoid is FOXG1-.

[0035] In some embodiments, the epithelium comprises apical microvilli.

[0036] In some embodiments, the epithelium comprises a centrally located basal nucleus.

[0037] In some embodiments, the epithelium is apical-basal polarized.

[0038] In some embodiments, the epithelium is polarized and Aqp1 and ZO1 are expressed on the luminal side of the epithelium.

[0039] In some embodiments, LAT-1 is expressed on the apical surface of the epithelium.

[0040] In some embodiments, the epithelium is rich in specific transporters involved in nutrient transport, such as SLC23A2 (vitamin C) and / or SLC46A2 (folate). In some embodiments, the choroid plexus organoids are capable of secreting a fluid comprising one or more proteins selected from the group consisting of: thyroxine transporter (TTR), clusterin (CLU), apolipoprotein E (APOE), apolipoprotein A4 (APOA4), and basal membrane polysaccharide (LUM).

[0041] In some embodiments, the liquid comprises phospholipid transfer protein (PLTP).

[0042] In some embodiments, the liquid comprises alpha-fetoprotein (AFP).

[0043] In some embodiments, the choroid plexus organoid is capable of secreting a cerebrospinal fluid (CSF)-like fluid.

[0044] In some embodiments, the choroid plexus organoid is capable of secreting cerebrospinal fluid (CSF).

[0045] In preferred embodiments, the capsule contains a liquid. In preferred embodiments, the liquid contains one or more proteins selected from thyroxine transporter (TTR), clusterin (CLU), apolipoprotein E (APOE), apolipoprotein A4 (APOA4) and basement membrane polysaccharide (LUM). In some embodiments, the liquid is CSF.

[0046] In some embodiments, the liquid comprises alpha-fetoprotein (AFP).

[0047] In some embodiments, the fluid comprises developmental-specific CSF proteins, such as insulin-like growth factor 2 (IGF2), IGFBP7, and / or follistatin-like protein 1 (FSTL1).

[0048] In some embodiments, the choroid plexus organoid comprises less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% (v / v) forebrain neuronal tissue. In some embodiments, the choroid plexus organoid comprises essentially no forebrain neuronal tissue.

[0049] In some embodiments, the choroid plexus organoid comprises less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% (v / v) FOXG1+ tissue. In some embodiments, the choroid plexus organoid comprises essentially no FOXG1+ tissue.

[0050] In some embodiments, the choroid plexus organoid comprises an extracellular matrix (ECM).

[0051] In some embodiments, the choroid plexus organoids have primary cilia (e.g., with apically located microvilli), tight junctions between cells, multivesicular bodies, and extracellular vesicles.

[0052] In some embodiments, the choroid plexus organoid is a human or mouse organoid, preferably a human organoid.

[0053] In some embodiments, choroid plexus organoids are obtainable by the methods of the present invention.

[0054] In some embodiments, choroid plexus organoids are generated from stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

[0055] In another aspect, the present invention provides the use of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator for generating choroid plexus organoids, wherein the choroid plexus organoids comprise (a) an epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0056] Although there are previous reports using Bmp4 and / or CHIR (see (15) and (16)), these early publications did not generate ChP organoids with the authentic features described herein.

[0057] In another aspect, the invention provides a method for generating choroid plexus organoids, comprising contacting a population of neuroepithelial cells with a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, wherein the choroid plexus organoid comprises: (a) epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0058] The method can be carried out with or without microfolding of the fibers. No microfolding may be preferred.

[0059] In another aspect, the present invention provides a method for generating choroid plexus organoids, comprising generating a neuroepithelial cell population from a stem cell population, and culturing the neuroepithelial cell population in the presence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator.

[0060] In a preferred embodiment, the population of neuroepithelial cells is an aggregate of neuroepithelial cells.

[0061] In preferred embodiments, the choroid plexus organoid comprises: (a) epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0062] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells.

[0063] In some embodiments, less than 18 days (e.g., between about 8-12 days) after starting to culture a stem cell population, a neuroepithelial cell population is started to be cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 9-11 days after starting to culture a stem cell population, a neuroepithelial cell population is started to be cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 9.5-10.5 days after starting to culture a stem cell population, a neuroepithelial cell population is started to be cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 10 days after starting to culture a stem cell population, a neuroepithelial cell population is started to be cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator.

[0064] In another aspect, the present invention provides a method for producing a choroid plexus organoid, comprising the steps of:

[0065] (a) generating an embryoid body population by culturing a stem cell population;

[0066] (b) culturing the embryoid body population under conditions suitable for neural induction;

[0067] (c) embedding the product of step (b) in a three-dimensional matrix, preferably an extracellular matrix

[0068] (ECM);

[0069] (d) culturing the product of step (c) in the presence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, preferably wherein step (d)

[0070] Start about 8-12 days after step (a) begins.

[0071] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells.

[0072] In some embodiments, the culturing of step (a) lasts for about 4-6 days, preferably about 4.5-5.5 days, preferably about 5 days.

[0073] In a preferred embodiment, the culturing of step (b) is performed until the neuroepithelium is present. In some embodiments, the culturing of step (b) lasts for about 1-3 days, preferably about 1.5-2.5 days, preferably about 2 days.

[0074] In some embodiments, the ECM is a polymeric ECM. In some embodiments, the ECM is a polymeric ECM gel. In some embodiments, the ECM is Matrigel.

[0075] Thus, in some embodiments, step (c) is performed using Matrigel about 5-9 days, more preferably 6-8 days, and most preferably 7 days after initiation of culture.

[0076] In some embodiments, step (c) further comprises culturing the embedded embryoid bodies. In some embodiments, the culturing of step (c) lasts for about 2-4 days, preferably about 2.5-3.5 days, preferably about 3 days.

[0077] In some embodiments, step (d) is initiated about 9-11 days, preferably about 9.5-10.5 days, preferably about 10 days after initiating step (a).

[0078] In some embodiments, culturing in the presence of a WNT pathway activator and a BMP signaling pathway activator continues for about 6-8 days, preferably about 6.5-7.5 days, preferably about 7 days.

[0079] In a preferred embodiment, the culturing of step (d) comprises agitation.

[0080] In some embodiments, agitation is not applied during contact with the WNT pathway activator and the BMP signaling pathway activator. In some embodiments, agitation begins at about day 15.

[0081] In some embodiments, the method comprises feeding the culture with culture medium every 3-4 days beginning on day 15, optionally wherein ECM is added to the culture medium beginning on day 30. Preferably, the ECM is a soluble ECM - such as dissolved Matrigel.

[0082] In a preferred embodiment, the method is an in vitro method.

[0083] In some embodiments, the WNT pathway activator is selected from CHIR99021, Wnt1, Wnt2, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b, BML-284, 6-bromoindirubin-3'-oxime (BIO), WAY-316606, IQ1, QS11, SB-216763, LY2090314, DCA, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine and lithium.

[0084] In a preferred embodiment, the WNT pathway activator is CHIR99021.

[0085] In some embodiments, the BMP signaling pathway activator is selected from Bmp4, Bmp2, Bmp3, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, Bmp10, Bmp11, Bmp15, isoliquiritigenin, 4'-hydroxychalcone, apigenin, diosgenin, and ventromorphins (e.g., SJ000291942, SJ000063181, and / or SJ00037178).

[0086] In a preferred embodiment, the BMP signaling pathway activator is Bmp4.

[0087] In a preferred embodiment, the WNT pathway activator is CHIR99021 and the BMP signaling pathway activator is Bmp4.

[0088] In some embodiments, the method or use does not comprise contacting the cell with a WNT pathway inhibitor.

[0089] In some embodiments, the method or use does not include exposing the cells to high oxygen partial pressure, such as an oxygen partial pressure greater than the oxygen partial pressure in air (20%). In some embodiments, the method or use includes culturing only at an oxygen partial pressure less than or equal to 20%.

[0090] As explained above, as part of its selective barrier function, the ChP epithelium is also highly selective for small molecules, including therapeutic agents.The present invention has particular utility in the study and screening of CNS active drugs.

[0091] New drugs often fail before entering clinical trials due to lack of efficacy, inability to enter the CNS, or limited translatability between animal models (52).

[0092] The ChP organoids of the present invention have utility, for example, in preclinical testing and modeling of in vivo drug permeation into the CNS, which may have benefits compared to current standards for preclinical assays of CNS permeability, such as canine MDCK or Caco-2 cells, or artificial membrane assays based on phospholipid mixtures that lack transporters and carrier proteins (53).

[0093] In another aspect, the present invention provides a use of the choroid plexus organoid of the present invention for drug discovery screening or determining drug toxicity. The drug may be a CNS drug, such as a small molecule drug for neurodegenerative diseases such as Alzheimer's disease.

[0094] In some embodiments, the use is for testing the ability of a candidate agent to cross and / or selectively cross the blood-cerebrospinal fluid (CSF) barrier. In another aspect, the invention provides a method for testing the ability of a candidate agent to cross the blood-cerebrospinal fluid (CSF) barrier, comprising contacting a choroid plexus organoid of the invention with a candidate agent and determining whether the candidate agent crosses the epithelium. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1. Generation of choroid plexus organoids from human PSCs. (A) Protocol timeline showing different images of H9 organoid growth stages. BMP4 and the Wnt activator CHIR were added on day 10 of the protocol to promote dorsalization. Scale bar: 1000 μm. (B) Comparison of H1 treated choroid plexus (ChP) organoids with untreated (UT) forebrain organoids at day 55. Scale bar: 1 mm. (C) H&E stained sections of H1 forebrain and choroid plexus organoids at day 40. Scale bar: 500 μm. (D) Histological sections of E18.5 mouse embryonic brain (H&E stain), human fetal ChP at 15 weeks post-conception (Nissl stain, taken from Allen Brain Map http: / / portal.brain-map.org / ), and H1 human ChP organoids at day 40 (H&E stain). Scale bar: 500 μm, magnification: 50 μm. (E), (F) Representative confocal images of IMR90-4 iPSC-derived forebrain and ChP organoids stained for TTR (grey, anti-sheep Alexafluor 647), ZO1 (green, anti-mouse Alexafluor 488), and Sox2 (magenta, anti-rabbit Alexafluor 568) at day 28 (E) and day 39 (F). Blue nuclei are stained with DAPI. Scale bar: 100 μm. (G) Quantification of TTR-positive area as a percentage of total organoid area in n = 4 independent H9 batches (3-4 organoids were collected per batch at days 30, 40, and 48).

[0096] Figure 2. Organoids recapitulate histological features of the human choroid plexus. (A) Representative confocal images of H1 organoids stained for Krt18 (green, anti-mouse Alexafluor 488), 5HT-2C (magenta, anti-rabbit Alexafluor 568), PH3 (grey, anti-rat Alexafluor 647) and DAPI (blue) at days 30 and 46. Scale bar: 50 μm. (B) Confocal images of ChP epithelial and stromal tissues of organoids stained positive for TTR (magenta) and DLK1 (green, anti-rabbit Alexafluor 488). Scale bar: 100 μm. (C) UMAP showing the population structure identified by scRNAseq of all combined samples (teleencephalic organoids collected at day 55, ChP organoids collected at days 27, 46, and 53). (D) UMAP showing the progressive enrichment in the ChP population in treated organoids compared to telencephalic organoids. (E) UMAP showing the enrichment of genes involved in ChP development and maturation (CLIC6, HRT2C, MSX1, PAX6), cortical neuron marker DCX and matrix marker COL1A1 in the identified scRNAseq clusters. (F) Violin plots showing the expression levels of ChP immature / hem (OTX2, RSPO3, PAX6), mature (TTR, KRT18, CA2, NME5, KCNJ13, CA12) and matrix markers (LUM, DCN, DLK1) identified by scRNAseq analysis.

[0097] Figure 3. Choroid plexus organoids develop specialized epithelial features and closely recapitulate the genetic architecture of the tissue in vivo. (A) PCA of E18.5 mouse embryonic whole ChP and isolated ChP epithelial cells (EC) clustered with organoids by scRNA-seq. (B) Pearson correlation analysis of in vivo human samples clustered with organoids. (C) Color-coded heat map showing unbiased clustering of organoids with in vivo human samples. (D) Pearson correlation analysis of mouse, human, and organoid samples. (E) Violin plots showing expression levels of local ChP markers in the lateral ventricle (LV, LY6E), third ventricle (3V, INS), and fourth ventricle (4V, PENK). (F) Violin plots showing expression of DYNLRB2, FOXJ1, and AQP1 in organoid cell populations identified by scRNAseq. (G) Confocal images of H1 ChP organoids stained for CCDC67 (green, anti-rabbit Alexafluor 488), TTR (grey, anti-sheep Alexafuor 647), Foxj1 (magenta, anti-mouse Alexafluor 568), Arl13b (green, anti-rabbit Alexafluor 488) and DAPI (blue) at day 40 and day 46, respectively. Scale bars: 50 μm and 100 μm (magnification: 20 μm). (H) Confocal images of H1 ChP organoids stained positive for Aqp1 (magenta, anti-rabbit Alexafluor 568) and TTR (green, anti-sheep Alexafluor 488) at day 40, and in vivo ChPs from mouse embryos (E18.5), blue nuclei are stained with DAPI. Scale bar: 50 μm. (I) Electron microscopy image showing a large number of microvilli on the apical surface of the ChP epithelium from organoids. Arrows indicate cilia (C), tight junctions (TJ), multivesicular bodies (MVBs), and extracellular vesicles (Ex). Scale bar: 1 μm. (J) Violin plots showing the expression levels of APOE, CLU, and PLTP in organoid cell clusters identified by scRNAseq. (K) Representative confocal images of H1 organoids stained for lipid droplets (LipidTox, gray), TTR (magenta, anti-sheep Alexafluor 568), PH3 (green, anti-rat Alexafluor 488), and DAPI (blue) at days 30 and 46. Scale bar: 50 μm.

[0098] Figure 4. Choroid plexus organoids develop fluid-filled compartments and form tight barriers. (A) Violin plots showing expression levels of claudin 1, 3, and 5 in organoid cell clusters identified by scRNAseq. (B) UMAPs showing enrichment of tight junction proteins in different organoid populations and in immature and mature clusters of ChP. Visualization of tight junction proteins TJP1 and TJP2, occludens (OCLN), INADL, and MPDZ. (C) Representative images of human H1 ChP organoids stained positive for TTR (magenta, anti-sheep Alexafluor 568), claudin 1 (green, anti-rabbit Alexafluor 488), claudin 3 (green, anti-rabbit Alexafluor 488), claudin 4 (red, anti-mouse Alexafluor 568), and claudin 5 (green, anti-rabbit Alexafluor 488) at day 40. Blue nuclei are labeled with DAPI. Scale bar: 50 μm. (D) Representative bright field images of untreated forebrain organoids and ChP organoids developing fluid-filled compartments. Scale bar: 1 cm. Magnified bright field image of ChP organoid developing from contained fluid compartments. Scale bar: 1 mm. (E) Confocal image of the epithelium surrounding the fluid-filled compartments of the organoids stained positive for ZO1 (grey, anti-mouse Alexafluor 647), TTR (green, anti-sheep 488), Aqp1 (red, anti-rabbit Alexafluor 568). Nuclei are labeled blue with DAPI. Scale bar: 50 μm.

[0099] Figure 5. Human developmental proteins are secreted by choroid plexus organoids. (A) gProfileR analysis of proteins detected in iCSF from at least 2 organoid batches, showing significant (p<0.05) enrichment of GO classes cellular component (GO:CC), molecular function (GO:MF) and biological process (GO:BP), REAC, WP, HP databases. (B) Color-coded heat map showing the relative abundance (emPAI value) of proteins detected in organoid iCSF from at least two organoid batches (3 H9 batches, 1 H1 batch and 1 iPSC batch) and the corresponding relative abundance values ​​of the proteins in culture medium and in vivo CSF ​​sample pools, including human adult CSF (hCSF, telencephalic CSF, n=3 healthy donors, Caltag-Medsystem), bovine fetal CSF (bCSF, BioIVT) and embryonic mouse CSF (msCSF, E12.5-13.5). The values ​​shown are the average values ​​of proteins with emPAI≥1. (C) Violin plots of expression levels of secretory proteins IGFBP7 and SERPINF1 in organoid cell clusters identified by scRNAseq analysis. (D) UMAP showing the enrichment of secretory proteins RBP1, IGF2, NPC2, and specific transporters SLC23A2 and SLC46A1 identified by organoid scRNAseq in different cell populations. (E) Two color-coded heat maps (emPAI values): at the top, abundant proteins with emPAI ≥ 1 were detected only in human-derived samples (organoid iCSF and adult hCSF); at the bottom, abundant development-related proteins with emPAI ≥ 1 were detected only in organoid iCSF and embryonic or fetal in vivo CSF. (F) Immunoblot validation of IGF2 and TTR in total organoid lysates from 3 independent batches of H9 untreated and treated ChP organoids, culture media, and iCSF. (G) Representative confocal images of the ChP epithelium of organoids stained positive for IGF2 (green, anti-rabbit Alexafluor 488) and TTR (grey, anti-sheep Alexafluor 647). Nuclei are labeled in blue with DAPI. Scale bar: 100 μm. (H) Venn diagram of CSF proteins present in human early embryonic (Carnegie stage 20), adult, and ChP organoids.

[0100] Figure 6. Choroid plexus organoid model of human brain barrier permeability. (A) Relative quantification of NMR spectra of DOPA and L-DOPA in culture medium and iCSF. (B) Relative quantification of NMR spectra of bupropion, methotrexate and vincristine in culture medium and iCSF after 2 h of incubation. (C) Relative quantification of NMR spectra of sephin 1 in culture medium and iCSF after 2 h of incubation. (D) Representative images of the ChP epithelium of organoids stained positive for TTR (grey, anti-sheep Alexafluor 647), LAT1 (magenta, anti-mouse Alexafluor 568), Pgp (green, anti-rabbit Alexafluor 488), transporter MRP4 (green, anti-rabbit Alexafluor 488) and MRP1 (green, anti-rabbit Alexafluor488). Scale bar: 50 μm. Cell nuclei are labeled blue with DAPI. (E) In vivo CSF / plasma ratio (K) showing unbound drug p,uu,CSF ) and in vitro iCSF / medium ratio (K m,uu,iCSF ) 2 =0.9921; slope =1.004) pharmacokinetic model (Supplementary Table 5). It has been reported that in vivo CSF / plasma measurements of sephin 1 (red dots) in mice are much greater than those detected in human organoids (45). (F) Relative quantification of NMR spectra of BIA 10-2474 in culture medium and iCSF after 2 h of incubation. (G) Time course analysis of the ratio of BIA 10-2474 and bupropion in iCSF to culture medium at 2 h, 12 h, 24 h, and 72 h. (All drugs were tested in n = 3 independent experiments; Supplementary Table 2). (H) Schematic showing that ChP organoids can be used as a human in vitro preclinical model, which quantitatively predicts the permeability of unknown compounds and their kinetics over time.

[0101] Figure 7. (A) Confocal images of forebrain organoids whose curled, cubic ChP epithelium stains positive for markers TTR, ZO1, and Aqp1. Scale bar: 100 μm. (B) Comparison of treated ChP organoids compared to untreated forebrain organoids at day 14. Arrows point to elongated neuroepithelial sprouts in early treated ChP organoids. Scale bar: 1 mm. (C) Bright field images of different stages of the protocol using the fiber seeding method. Scale bar 1000 μm. (D) Representative confocal images of H9 ChP organoids generated using the circle method and the fiber method at days 30 and 34, respectively. Scale bar: 500 μm. (E) Representative confocal images of iPSC IMR90-4 organoids treated with BMP4 and CHIR at day 10 at day 35 and H1 organoids at day 40. ChP tissue in organoids was stained for TTR (green, anti-sheep Alexafluor 488), and nuclei were visualized with DAPI. Scale bar: 1000 μm (cropped image: 500 μm).

[0102] Figure 8. (A) Representative confocal images of H1 forebrain and ChP organoids stained for Sox2 (magenta, anti-rabbit Alexafluor 568), cortical intermediate progenitor marker Tbr2 (grey, anti-sheep Alexafluor 647), neuronal marker HuC / D (green, anti-mouse Alexafluor 488). Scale bar: 100 μm. (B) Representative confocal images of H1 ChP organoids stained for DCN (green, anti-mouse Alexafluor 488), LUM (magenta, anti-rabbit Alexafluor 568), ZO1 (grey, anti-mouse Alexafluor 647), TTR (grey, anti-sheep Alexafluor 647) and DAPI (blue). Scale bar: 100 μm.

[0103] Figure 9. (A) Representative bright field images of 4 independent batches of H1 untreated and BMP4+CHIR treated organoids (from top to bottom: 57, 54, 46, 40 days). (B) Time course series of images of H1 treated ChP organoids showing the development and expansion of the iCSF compartment from day 14 to day 50. Scale bar: 1000 μm. (C) Immunoblots of ChP epithelial transporters MRP1, LAT1, CLIC6, secreted protein IGF2, and loading control GAPDH from day 10 to day 31 ChP organoid lysates.

[0104] Figure 10 (A) Venn diagram of proteins with emPAI≥1 detected by mass spectrometry in at least one sample in culture medium, organoid iCSF, and in vivo CSF ​​samples. (B) Color-coded heat map of the same proteins in Figure 3E, showing all samples analyzed by mass spectrometry. Proteins with emPAI≥1 detected in at least two iCSF samples are shown. (C) Color-coded heat map of abundant proteins (emPAI≥1) excluded or missing from organoid iCSF but present in at least two in vivo samples. (D) Color-coded heat map showing abundant proteins (emPAI≥1) shared between culture medium and iCSF (average of five samples shown) of at least two organoid batches, and average values ​​of in vivo CSF ​​samples from adult CSF (hCSF), bovine fetal CSF (bCSF), and embryonic mouse CSF (msCSF, E12.5-13.5). (E) gProfileR analysis of proteins detected only in in vivo CSF ​​samples. (F) Histogram showing top categories with significant (p<0.05) enrichment from gProfileR analysis.

[0105] Figure 11. (A-Top) ChP epithelium of human organoids stained for TTR (grey, anti-sheep Alexafluor 647), claudin 2 (green, anti-rabbit Alexafluor 488), and APOE (magenta, anti-mouse Alexafluor 568). Scale bar: 50 μm. DAPI in blue marks the nuclei. (A-Bottom) ChP epithelium from human organoids stained for tight junction markers occludin (green, anti-rabbit Alexafluor 488), TTR (grey, anti-sheep Alexafluor 647), ZO1 (magenta, anti-mouse Alexafluor 568), and DAPI (blue). Scale bar: 50 μm. (B) Bright field image of ChP organoids with self-contained iCSF fluid isolated from culture medium incubated for 2 h with 647-Alexafluor labeled 10 kDa-dextran. (C) Fluorescence intensity measured in culture medium and iCSF after 2 h incubation with 70 kDa Oregon Green-dextran, 10 kDa Alexa 647-dextran and 3-5 kDa FITC-dextran. (D) NMR spectra of DOPA, L-DOPA and control (Ctrl) iCSF measured after 2 h incubation.

[0106] Figure 12. (A) Control samples (culture medium and iCSF) 1 H-NMR analysis (H2O / D2O). For clarity, the expanded spectra are reported. (B) Spectra of samples after incubation with L-DOPA (500 μM) and carbidopa (500 μM). 1H-NMR analysis (H2O / D2O). (C) Samples incubated with dopamine (500 μM) (500 μM) 1 H-NMR analysis (H2O / D2O). Arrows indicate peaks of interest.

[0107] Figure 13. (A) Samples (culture medium and iCSF) incubated with (A) Sephin 1 (500 μM), (B) vincristine (500 μM), and (C) methotrexate (500 μM). 1 H-NMR analysis (H2O / D2O). For clarity, an expansion of the spectrum is reported. Arrows indicate peaks of interest.

[0108] Figure 14. (A) Samples (culture medium and iCSF) incubated with bupropion (500 μM) 1 H-NMR analysis (H2O / D2O). For clarity, an expansion of the spectrum is reported. Arrows indicate peaks of interest.

[0109] Figure 15. (A) Samples (culture medium and iCSF) incubated with BIA10-2474 (500 μM) 1 H-NMR analysis (H2O / D2O). For clarity, an expansion of the spectrum is reported. Arrows indicate peaks of interest. DETAILED DESCRIPTION

[0110] As used herein, the terms "comprising", "comprises" and "comprised of" are synonymous with "including" or "includes"; or "containing" or "contains", and are inclusive or open-ended and do not exclude additional, unrecited components, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0111] Organoids

[0112] Organoids are three-dimensional (3D) in vitro cell cultures. Organoids can provide miniaturized and simplified versions of organs that incorporate some of the key features of organs. These in vitro culture systems can contain self-renewing stem cell populations that differentiate into multiple organ-specific cell types, whose spatial organization resembles that of an organ.

[0113] Choroid plexus

[0114] The choroid plexus is a highly conserved secretory tissue in the brain that displays many important functions, such as forming a protective epithelial barrier and secreting cerebrospinal fluid (CSF).

[0115] Anatomically, the choroid plexus is located in the lateral, third, and fourth ventricles that emerge in each corresponding cavity. The mature choroid plexus consists of a single cuboidal epithelium that surrounds a core of stromal tissue of mesenchymal origin and fenestrated capillaries. The epithelial cells are closely connected by tight junctions and are highly polarized, with the apical brush edge facing the ventricular cavity and the basal surface on the stromal side.

[0116] The production of CSF involves different mechanisms. First, Na + -K + The ATPase pump and cytosolic carbonic anhydrase generate an osmotic gradient composed mainly of Na + Secretion into the ventricular cavity drives. This osmotic force drives the water across the cell to flow in through the aquaporin channel. Secondly, ATP binding cassette (ABC) proteins and solute carrier transporters (SLC) allow the exchange of amino acids, nucleosides and small peptides with and without the need for ATP hydrolysis, respectively. Choroid plexus epithelial cells also express efflux transporters, such as MRP1 and MRP4 (Redzic, Z. (2011) Fluids Barriers CNS 8:3; Strazielle, N. and Ghersi-Egea, JF (2013) Mol. Pharm. 10: 1473-1491). These pumps regulate the clearance of metabolites and prevent the entry of drugs and toxic compounds, thereby reducing their availability in CSF. Third, the highly specialized and expanded secretory system of choroid plexus epithelial cells enables the secretion of more complex proteins and hormones. For example, thyroxine transporter (TTR), a carrier of thyroid hormone and retinol, is the most abundant protein synthesized and secreted by the choroid plexus (Li, X. and Buxbaum, JN (2011) Mol. Neurodegener. 6: 1-17; Richardson, SJ et al. (2015) Front. Neurosci. 9: 1-8; Johnson, BA et al. (2018) Fluids Barriers CNS 15: 22). TTR-mediated delivery of thyroid hormones to the CSF appears to play an important role in the regulation of brain development.

[0117] Choroid plexus tissue can be identified by the expression of choroid plexus markers and / or structural characteristics of the tissue.

[0118] The choroid plexus may be characterized by the expression of one or more markers selected from the group consisting of thyroxine transporter (TTR), MRP1, MRP4, aquaporin 1 (Aqp1), and zonula occludens (ZO1).

[0119] The choroid plexus may be characterized by the expression of markers that are localized or enriched in certain types of cells. For example, the choroid plexus may be characterized by the expression of one or more markers selected from TTR, MRP1, MRP4, Aqp1, and ZO1 in epithelial cells.

[0120] In addition, the markers can be localized or enriched in certain regions of certain cells. For example, TTR, Aqp1 and / or ZO1 can be enriched on the apical surface of the epithelium. MRP1 and / or MRP4 can be enriched on both the apical and basal surfaces of the epithelium.

[0121] The choroid plexus can be characterized by the absence or low expression of FOXG1.

[0122] The choroid plexus can be characterized by the presence of apical microvilli in the epithelium. The choroid plexus epithelium can contain centrally located basal nuclei.

[0123] The choroid plexus epithelium can be apical-basal polarized. For example, the epithelium can be polarized where Aqp1 and ZO1 are expressed on the luminal side of the epithelium.

[0124] Technicians can easily determine the presence, absence and location of such markers. For example, the marker can be identified by using an antibody specific to the marker for immunostaining, for example in conjunction with visualization techniques such as confocal microscopy. In addition, or alternatively, a specific oligonucleotide probe preferably attached to the marker nucleic acid can be used.

[0125] Tight epithelial barrier

[0126] A tight epithelial barrier can be formed by tight junctions between epithelial cells.

[0127] Tight junctions (also called occluded junctions or zonula occludens) may be formed by multiprotein complexes and may seal off the paracellular pathway and prevent the leakage of transported solutes and water.

[0128] The tightly sealed epithelium of the choroid plexus is also called the blood-CSF barrier (B-CSF-B). The B-CSF-B is similar to the blood-brain barrier (BBB), preventing toxic substances or signals in the circulation from reaching the brain. However, unlike the BBB, the B-CSF-B is formed by a single cell type, the choroid plexus epithelial cells, and it directly faces the CSF rather than the brain parenchyma. However, since CSF can freely enter the brain, the transport of molecules through the B-CSF-B is an alternative route to enter the brain.

[0129] Tight junctions such as those contained in tight epithelial barriers may be characterized by the expression of zonula occludens (ZO1).

[0130] Cerebrospinal fluid (CSF)

[0131] Cerebrospinal fluid (CSF) is a clear, colorless fluid that circulates through the ventricles and subarachnoid space and is then partially reabsorbed into the blood circulation through the arachnoid granulations.

[0132] CSF is important for maintaining physiological levels of nutrients in the brain, for the transport of signaling molecules and growth factors, and for its protective role in the regulation of intracranial pressure.

[0133] CSF is produced by active secretion of the choroid plexus epithelial cells, which maximizes the number of transporters and pumps in their apical brush borders; and by filtration of the blood allowed by the presence of tight junctions within the choroid plexus epithelial cells. The choroid plexus plays a key role in maintaining the rate of production and reabsorption of CSF. Disturbance of this balance can lead to pathologies such as hydrocephalus.

[0134] The CSF may be characterized by the presence of one or more markers selected from the group consisting of TTR, clusterin (CLU), apolipoprotein E (APOE), apolipoprotein A4 (APOA4), and lumigan (LUM).

[0135] CSF can be characterized by the presence of alpha-fetoprotein (AFP).

[0136] WNT pathway activators

[0137] The WNT pathway activator that can be used to produce the choroid plexus organoids of the present invention is not particularly limited as long as it can activate the WNT signaling pathway.

[0138] WNT pathway activators include, but are not limited to, agents such as CHIR99021, Wnt1, Wnt2, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b, BML-284, 6-bromoindirubin-3'-oxime (BIO), WAY-316606, IQ1, QS11, SB-216763, LY2090314, DCA, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, and lithium.

[0139] In preferred embodiments, the WNT pathway activator is CHIR99021 (also referred to herein as "CHIR").

[0140] CHIR99021 is an aminopyrimidine derivative that is a potent inhibitor of GSK3 and inhibits GSK3β (IC 50=6.7 nM) and GSK3α (IC 50 =10 nM) and acts as a WNT activator. CHIR99021 has the following structure:

[0141]

[0142] In some embodiments, CHIR99021 is used at a concentration of about 1-25, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3 μM. In some embodiments, CHIR99021 is used at a concentration of about 1-5 μM.

[0143] In some embodiments, CHIR99021 is used at a concentration of about 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 μM. In a preferred embodiment, CHIR99021 is used at a concentration of about 2-4 μM.

[0144] In some embodiments, CHIR99021 is used at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μM. In a preferred embodiment, CHIR99021 is used at a concentration of about 3 μM.

[0145] Bone morphogenetic protein (BMP) signaling pathway activators

[0146] The bone morphogenetic protein (BMP) signaling pathway activator that can be used to produce the choroid plexus organoid of the present invention is not particularly limited as long as it can activate the BMP signaling pathway.

[0147] BMP signaling pathway activators include, but are not limited to, agents such as Bmp4, Bmp2, Bmp3, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, Bmp10, Bmp11, Bmp15, isoliquiritigenin, 4'-hydroxychalcone, apigenin, diosgenin, and ventromorphins (e.g., SJ000291942, SJ000063181, and / or SJ00037178).

[0148] In a preferred embodiment, the BMP signaling pathway activator is Bmp4.

[0149] Bone morphogenetic protein 4 (Bmp4) is a member of the TGF-β protein superfamily. It is involved in bone and cartilage development, such as tooth and limb development, and fracture repair.

[0150] In human embryonic development, Bmp4 is an important signaling molecule that is involved in the early differentiation of the embryo and the establishment of the dorsal-ventral axis.

[0151] An exemplary amino acid sequence of human Bmp4 is the sequence deposited under NCBI Accession No. NP_001193.2.

[0152] An exemplary amino acid sequence of human Bmp4 is:

[0153] MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATLLQMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSFHHEEHLENIPGTSENSAFRFLFNLSSIPENEVISSSAELRLFREQVDQGPDWERGFHRINIYEVMKPPAEVVPGHLITRLLDTRL VHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTHLHQTRTHQGQHVRISRSLPQGSGNWAQLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNK NCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSAISMLYLDEYDKVVLKNYQEMVVEGCGCR

[0154] (SEQ ID NO:1)

[0155] In some embodiments, Bmp4 is used at a concentration of about 1-40, 5-35, 10-30, 15-25, 16-24, 17-23, 18-22, or 19-21 ng / ml. In some embodiments, Bmp4 is used at a concentration of about 19-21 ng / ml.

[0156] In some embodiments, Bmp4 is used at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 ng / ml. In a preferred embodiment, Bmp4 is used at a concentration of about 20 ng / ml.

[0157] Salt

[0158] The agents of the present invention (eg, WNT pathway activators and / or BMP signaling pathway activators) may exist as salts, particularly pharmaceutically acceptable salts, or as esters.

[0159] Pharmaceutically acceptable salts of the agents of the present invention include suitable acid addition salts or base salts thereof. A review of suitable pharmaceutically acceptable salts can be found in Berge et al. (1977) J. Pharm. Sci. 66: 1-19. Salts are formed, for example, with strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or hydrohalic acid; strong organic carboxylic acids, for example unsubstituted or substituted (e.g. substituted with halogen) alkanecarboxylic acids of 1 to 4 carbon atoms, such as acetic acid; saturated or unsaturated dicarboxylic acids, for example oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; hydroxycarboxylic acids, for example ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; amino acids, for example aspartic acid or glutamic acid; benzoic acid; or organic sulfonic acids, such as (C1-C4)-alkyl or arylsulfonic acids, which are unsubstituted or substituted (e.g. substituted with halogen), such as methanesulfonic acid or p-toluenesulfonic acid.

[0160] Enantiomers / Tautomers

[0161] The present invention also includes all appropriate enantiomers and tautomers of the medicament. The corresponding enantiomers and / or tautomers can be separated / prepared by methods known in the art.

[0162] Stereoisomers and geometric isomers

[0163] Some of the agents of the present invention may exist as stereoisomers and / or geometric isomers. For example, they may have one or more asymmetric and / or geometric centers and may therefore exist in two or more stereoisomers and / or geometric forms. The present invention contemplates the use of all individual stereoisomers and geometric isomers of these agents, and mixtures thereof. The terms used in the claims encompass these forms, as long as the form retains the appropriate functional activity (although not necessarily to the same extent).

[0164] The present invention also includes all suitable isotopic variants of the agent or a pharmaceutically acceptable salt thereof. An isotopic variant of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and its pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H. 3 H. 13 C. 14 C. 15 N. 17 O.18 O. 31 P. 32 P. 35 S. 18 F and 36 Certain isotopic variations of the pharmaceutical agent and its pharmaceutically acceptable salts, for example, those in which radioactive isotopes such as 3 H or 14 C, can be used for drug and / or substrate tissue distribution studies. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred because of their ease of preparation and detectability. In addition, isotopes (such as deuterium, i.e., 2 H) Substitution may result in certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances. Isotopic variations of the agents of the invention and their pharmaceutically acceptable salts can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

[0165] Solvate

[0166] The present invention also includes solvated forms of the agents of the present invention. The terms used in the claims encompass these forms.

[0167] Polymorph

[0168] The invention also relates to the agents of the invention in their various crystalline forms, polymorphic forms and (anhydrous) aqueous forms. It is well established in the pharmaceutical industry that chemical compounds can be isolated in any such form by slightly varying the methods of purification and or isolation from the solvents used to prepare such compounds synthetically.

[0169] Stem Cells

[0170] The choroid plexus organoids of the invention can be generated from stem cells (eg, in vitro). The choroid plexus organoids are not isolated from the brain of an animal, which facilitates the generation of tissue without the need to obtain a tissue sample from an animal.

[0171] Stem cells are cells that have the ability to differentiate into more specialized cells and can also divide to produce more stem cells.

[0172] As differentiation ability decreases, stem cells differentiate in the following order: multipotent, multipotent, unipotent. During the development of the organoids of the present disclosure, stem cells can differentiate, for example, from multipotent to multipotent neural stem cells, further to unipotent stem cells, and then to non-stem cell tissue cells. The tissue cells can be, for example, neuroepithelial cells.

[0173] Methods of the invention may comprise contacting a precursor cell population (eg, a neuroepithelial cell population) with a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, wherein the precursor cell population itself has been generated by differentiation of a stem cell population (eg, in vitro).

[0174] Thus, methods of the invention may include the step of generating a cell population (eg, in vitro) from a stem cell population.

[0175] Preferably, the stem cells of the present invention are pluripotent stem cells.

[0176] Pluripotent stem cells are stem cells that can reproduce indefinitely. Pluripotent stem cells cannot grow into a complete organism, but are able to generate cell types derived from all three germ layers, namely mesoderm, endoderm and ectoderm, and can differentiate into all cell types of the human body. These stem cells are expected to provide a single source of cells to replace cells affected by damage or disease.

[0177] Pluripotent stem cells can be generated by a variety of techniques, such as the generation of induced pluripotent stem cells or embryonic stem cells.

[0178] In some embodiments, the stem cells of the invention are induced pluripotent stem cells (iPSCs).

[0179] iPSCs are a type of pluripotent stem cell that can be generated directly from adult cells. Technicians can easily prepare iPSCs, for example, by introducing specific transcription factors into adult cells or contacting adult cells with a specific protein combination.

[0180] iPSCs are superior to embryonic stem cells because they overcome the need to use embryonic material and can be prepared from a subject into which they (or cells generated from them) are subsequently reintroduced. Such autologous cell transplants can overcome the risk of immune rejection of the transplanted material.

[0181] The stem cells of the present invention may be embryonic stem cells, particularly those produced without destroying an embryo.

[0182] Methods for producing pluripotent stem cells (such as mammalian embryonic stem cells) without destroying the embryo are known in the art. In particular, it has been shown that mouse and human embryonic stem cells can be produced from a single blastomere, while the embryo remains intact. For example, Chung, Y. et al. (2006) Nature 439:216-219 describes a method for preparing mouse embryonic stem cells from a single blastomere. The later progress of this procedure provides a method (Chung, Y. et al. (2008) Cell Stem Cell 2:113-117) that does not require co-culturing of blastomere cell lines with other ESCs.

[0183] In some embodiments, the stem cells of the present invention are mammalian stem cells, preferably human stem cells.

[0184] Methods for generating choroid plexus organoids

[0185] The choroid plexus organoids of the present invention can be generated using a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator.

[0186] In one aspect, the present invention provides the use of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator for generating choroid plexus organoids, wherein the choroid plexus organoids comprise (a) an epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0187] In another aspect, the invention provides a method for generating choroid plexus organoids, comprising contacting a population of neuroepithelial cells with a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, wherein the choroid plexus organoid comprises: (a) epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0188] In another aspect, the present invention provides a method for producing a choroid plexus organoid, comprising generating a neuroepithelial cell population from a stem cell population, and culturing the neuroepithelial cell population in the presence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator. In a preferred embodiment, the choroid plexus organoid comprises: (a) epithelium comprising a tight epithelial barrier; and / or (b) one or more cysts surrounded by the epithelium.

[0189] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are induced pluripotent stem cells.

[0190] In a preferred embodiment, the population of neuroepithelial cells is an aggregate of neuroepithelial cells.

[0191] In a preferred embodiment, the neuroepithelial cell population is embedded in a three-dimensional matrix, preferably an extracellular matrix (ECM), prior to contacting with the WNT pathway activator and the bone morphogenetic protein (BMP) signaling pathway activator.

[0192] In some embodiments, about 8-12 days after starting to culture the stem cell population, the neuroepithelial cell population is cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 9-11 days after starting to culture the stem cell population, the neuroepithelial cell population is cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 9.5-10.5 days after starting to culture the stem cell population, the neuroepithelial cell population is cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator. In some embodiments, about 10 days after starting to culture the stem cell population, the neuroepithelial cell population is cultured in the presence of a WNT pathway activator and a BMP signaling pathway activator.

[0193] In some embodiments, the method or use comprises stirring, preferably after contacting with the WNT pathway activator and the BMP signaling pathway activator. Stirring can be achieved using, for example, an oscillator (such as an orbital oscillator).

[0194] In another aspect, the present invention provides a method for producing a choroid plexus organoid, comprising the steps of:

[0195] (a) generating an embryoid body population by culturing a stem cell population;

[0196] (b) culturing the embryoid body population under conditions suitable for neural induction;

[0197] (c) embedding the product of step (b) in a three-dimensional matrix, preferably an extracellular matrix

[0198] (ECM);

[0199] (d) culturing the product of step (c) in the presence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, preferably wherein step (d) begins about 8-12 days after the start of step (a).

[0200] Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells that undergo spontaneous or directed differentiation.

[0201] The methods and uses of the present invention may comprise embedding cells or cell aggregates in a three-dimensional matrix, preferably an extracellular matrix (ECM).

[0202] In some embodiments, ECM is polymerized ECM. In some embodiments, ECM is polymerized ECM gel. Suitable three-dimensional matrix can include collagen. Preferably, the three-dimensional matrix includes ECM or any component thereof secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as laminin, collagen, type 4 collagen, entactin and optional other heparan sulfate proteoglycans, or any combination thereof. In some embodiments, ECM is matrigel. Matrigel is described in, for example, US4829000.

[0203] Typically, the three-dimensional matrix is ​​a three-dimensional structure of a biocompatible matrix. It preferably comprises collagen, gelatin, chitosan, hyaluronic acid, methylcellulose, laminin and / or alginate. The matrix can be a gel, in particular a hydrogel. Organic chemical hydrogels can comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with a large number of hydrophilic groups. Hydrogels contain a hydrophilic network of polymer chains, sometimes in the form of a colloidal gel in which water is used as a dispersion medium. Hydrogels are highly absorbent (they can contain more than 99% water) natural or synthetic polymers. Hydrogels also have a certain degree of flexibility, which is very similar to natural tissues because their water content is very high.

[0204] Generation of an embryoid body population can be performed by culturing a stem cell population in a suitable culture medium, referred to herein as embryoid body (EB) medium.

[0205] Exemplary EB culture medium comprises serum replacement formulation, fetal bovine serum, glutamine, non-essential amino acids, 2-mercaptoethanol and FGF (e.g., basic FGF) (preferably about 4 ng / ml bFGF). Preferably, the EB culture medium comprises a ROCK inhibitor. Preferably, the EB culture medium comprises a ROCK inhibitor only during the first 3 days of culturing stem cells.

[0206] A particularly suitable culture medium is the EB medium used in the Examples or the EB medium disclosed below.

[0207] Neural induction can be performed by culturing in a suitable medium (referred to herein as neural induction medium). The neural induction medium can be a depleted medium, such as a medium lacking serum or serum replacement. The lack of such nutrients required by non-neural tissues may result in the loss of other non-neural characteristics and purification towards neural characteristics.

[0208] An exemplary neural induction medium comprises N2 supplement (Price and Brewer (2001) Protocols for Neural Cell Culture 255-264; e.g., Invitrogen, catalog number 17502048), glutamine, non-essential amino acids, and heparin.

[0209] A particularly suitable culture medium is the neural induction medium used in the examples or the neural induction medium disclosed below.

[0210] In a preferred embodiment, individual embryoid bodies are embedded in individual three-dimensional matrix droplets.

[0211] Embedding in a three-dimensional matrix can be achieved by adding the embryoid bodies to a soluble matrix in a suitable container, followed by incubation under conditions suitable for matrix polymerization. For example, the embryoid bodies can be transferred one by one to a recessed paraffin film; after removing excess culture medium, a drop of matrix (e.g., Matrigel) can be added to each embryoid body; the mixture can then be incubated under conditions suitable for matrix polymerization (e.g., at 37° C. for about 20 min).

[0212] In a preferred embodiment, the embryoid bodies embedded in the three-dimensional matrix are cultured under conditions suitable for expansion. Expansion can be performed by culturing in a suitable culture medium (referred to herein as expansion medium).

[0213] An exemplary expansion medium comprises N2 supplement (Price and Brewer (2001) Protocols for Neural Cell Culture 255-264; e.g., Invitrogen, catalog number 17502048), B27 supplement lacking vitamin A (Price and Brewer (2001) Protocols for Neural Cell Culture 255-264; e.g., Invitrogen, catalog number 12587010), insulin, 2-mercaptoethanol, glutamine, and non-essential amino acids.

[0214] Particularly suitable culture media are the expansion media used in the examples or the expansion media disclosed below.

[0215] Cultivation in the presence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator and subsequently culturing in the absence of a WNT pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator can be performed in a suitable culture medium, which may be referred to herein as a maturation medium.

[0216] An exemplary maturation medium comprises N2 supplement (Price and Brewer (2001) Protocols for Neural Cell Culture 255-264; e.g., Invitrogen, Catalog No. 17502048), B27 supplement comprising vitamin A (Price and Brewer (2001) Protocols for Neural Cell Culture 255-264; e.g., Invitrogen, Catalog No. 17504044), insulin, 2-mercaptoethanol, glutamine, and non-essential amino acids.

[0217] A particularly suitable culture medium is the maturation medium used in the examples or the maturation medium disclosed below.

[0218] Any culture medium may further comprise nutrients and / or buffers.Preferred nutrients include carbohydrates, especially monohexoses or monopentoses, such as glucose or fructose.

[0219] Suitable culture conditions for use in the methods of the present invention include, for example:

[0220] (a) culturing at about 36°C-39°C or 36.5°C-37.5°C, preferably about 37°C;

[0221] (b) culturing at about 4% to 6% or 4.5% to 5.5% CO2, preferably about 5% CO2; and / or

[0222] (c) culturing at a humidity of at least about 95%, 96%, 97%, 98% or 99%, preferably at a humidity of about 100%.

[0223] Another exemplary EB medium is (per 50 mL): 40 ml DMEM / F12 (Invitrogen catalog number 11330-032); 10 ml knockout serum replacement (KOSR; Invitrogen catalog number 10828-028); 1.5 ml embryonic stem cell-quality FBS; 0.5 ml GlutaMAX (Invitrogen catalog number 35050-038); 0.5 ml MEM-non-essential amino acids (MEM-NEAA; Sigma catalog number M7145); 100 μl 50 mM 2-ME (e.g., Life Technologies catalog number 31350-010); 4 ng / ml bFGF (e.g., Peprotech catalog number 100-18B) (add immediately before use; 1:2500); and 1:100 Rock inhibitor (Y27632; VWR catalog number 688000-5) (add immediately before use).

[0224] Another exemplary neural induction medium is (per 100 mL): 100 ml DMEM / F12 (Invitrogen catalog number 11330-032); 1 ml N2 supplement (Invitrogen catalog number 17502048); 1 m Glutamax supplement (Invitrogen catalog number 35050-038); 1 ml MEM-NEAA (Sigma catalog number M7145); and 100 ul heparin solution (Sigma catalog number H3149).

[0225] Another exemplary expansion medium is (per 250 mL): 125 ml DMEM / F12 (Invitrogen catalog number 11330-032); 125 ml Neurobasal medium (Invitrogen catalog number 21103049); 1.25 ml N2 supplement (Invitrogen catalog number 17502048); 5 ml B27 supplement (Invitrogen catalog number 12587010 or 17504044); 62.5 ul insulin (Sigma catalog number 19278); 250 ul 50 mM 2-ME solution (e.g., Life Technologies catalog number 31350-010); 2.5 ml Glutamax supplement (Invitrogen catalog number 35050-038); 1.25 ml MEM-NEAA (Sigma catalog number M7145); and 2.5 ml penicillin / streptomycin (Sigma catalog number P0781).

[0226] Another exemplary maturation medium is (per 250 mL): 125 ml DMEM / F12 (Invitrogen catalog number 11330-032); 125 ml Neurobasal medium (Invitrogen catalog number 21103049); 1.25 ml N2 supplement (Invitrogen catalog number 17502048); 5 ml B27 supplement (Invitrogen catalog number 12587010 or 17504044); 62.5 ul insulin (Sigma catalog number 19278); 250 ul 50 mM 2-ME solution (e.g., Life Technologies catalog number 31350-010); 2.5 ml Glutamax supplement (Invitrogen catalog number 35050-038); 1.25 ml MEM-NEAA (Sigma catalog number M7145); and 2.5 ml penicillin / streptomycin (Sigma catalog number P0781).

[0227] Methods for screening and determination

[0228] In one aspect, the present invention provides use of the choroid plexus organoid of the present invention for drug discovery screening or determining drug toxicity.

[0229] In some embodiments, the use is for testing the ability of a candidate agent to cross the blood-cerebrospinal fluid (CSF) barrier.

[0230] In another aspect, the invention provides a method for testing the ability of a candidate agent to cross the blood-cerebrospinal fluid (CSF) barrier, comprising contacting a choroid plexus organoid of the invention with the candidate agent and determining whether the candidate agent crosses the epithelium.

[0231] For example, a candidate agent can be added to a culture medium containing a culture of choroid plexus organoids and incubated. Subsequently, for example, after a suitable period of time, the fluid in one or more cysts of the choroid plexus organoids can be extracted and analyzed for the presence of the candidate agent. Any suitable technique known to the skilled artisan can be used to determine the presence of the candidate agent. For example, the agent can be identified using mass spectrometry.

[0232] The skilled person will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0233] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0234] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of one of ordinary skill in the art and which are explained in the literature. See, e.g., Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodically supplemented) Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference.

[0235] Example

[0236] Materials and Methods

[0237] Stem cell culture

[0238] Human H9 and H1 ES cells were obtained from WiCell and have been approved for use in these studies by the UKSCB Steering Committee. iPS cells (IMR90-4) were obtained from WiCell and all cell lines were confirmed to be pluripotent and mycoplasma-free. Cells were maintained in culture and cultured with StemFlex medium (Gibco A3349401).

[0239] Brain and choroid plexus organoid culture conditions

[0240] As previously described, embryoid bodies (EBs) were prepared from single cell suspensions from hES and iPSCs (1). The brain organoid kit (Catalog Nos. 08570, 08571) reagents from Stem Cell Technologies were used to generate brain and ChP organoids. As previously reported, EBs were generated using fiber microfolding and 18,000 hES or iPS cells, or using fewer cells (2000-4000) to generate smaller EBs without microfolding, both of which were used to increase surface area and improve neural induction efficiency (2). These were plated into 96-well U-bottom low-attachment plates with EB culture medium and 50 μM Y-27632 ROCK inhibitor for 3 days, followed by 2 days of culture in EB culture medium only. On the 5th day, the culture medium was replaced with NI culture medium in the same 96 wells. Two days later, as previously described, EBs were embedded in 20 μl of Matrigel using a recessed paraffin film sheet and incubated at 37°C for 20 min (2). EB is then transferred to a 6-well plate, wherein each well has 3ml amplification medium. For ChP pattern formation, at the 10th day, 3 μM CHIR and 20ng / ml BMP4 in maturation medium are used for processing, thereby maintaining the maximum number of 6 EBs in each 5cm culture dish. The processing is maintained in maturation medium for 7 days. At the 15th day, organoids are moved to an incubator with an oscillator, and maturation medium is fed every 3-4 days. From the 30th day, dissolved matrigel (1:50) is added to maturation medium.

[0241] Immunostaining and immunoblotting

[0242] Organoids were fixed in 4% PFA overnight at 4°C and then washed three times in PBS for 10 min each before being transferred to 30% sucrose buffer at 4°C for at least 24 h. Organoids were then embedded in gelatin and sectioned as previously described (1). After blocking and permeabilization with 0.25% Triton and 1% donkey serum buffer, sections were incubated overnight with the following primary antibodies: sheep anti-TTR (1:500, Abcam, ab9015), mouse anti-ZO1 (1:300, BD Transduction, 610966), rabbit anti-Aqp1 (1:200, Abcam ab15080), mouse anti-MRP1 (1:200, Abcam ab24102), rabbit anti-MRP4 (1:200, Cell Signaling Technology, 12705), rabbit anti-Sox2 (1:300, Abcam, ab97959), rabbit anti-CLIC6 (1:500 for immunostaining, 1:1000 for immunoblotting, Abcam, ab204567), rabbit anti-Foxg1 (1:200 for immunostaining, 1:1000 for immunoblotting, Abcam, ab18259), mouse anti-GAPDH (1:1000, Abcam, ab8245), rabbit anti-IGF2 (1:200 for immunostaining, 1:1000 for immunoblotting, Abcam, ab9574), rat anti-phospho-histone H3 (PH3, 1:300, Abcam ab10543), mouse anti-keratin 18 (Krt18, 1:200, Novusbiologicals NBP2-47985), rabbit anti-serotonin (5HT2C, 1:200, Sigma S5545); rabbit anti-CCDC67 (1:200, Abcam ab102688), mouse anti-Foxj1 (1:100, Thermo Fisher 14-9965-82); rabbit anti-Arl13b (1:200, Proteintech 17711-1-AP), rabbit anti-DLK1 (1:200, Abcam, ab21682), sheep anti-Tbr2 (1:200, R&D Systems, AF6166), mouse anti-HuC / D (1:500, LifeTech, A21271), mouse anti-LAT1 (1:50, SantaCruz, sc-374232), rabbit anti-Pgp / MDR1 (1:200, Cell Signaling Technology, 12683). After three 10 min washes in PBS, secondary antibodies labeled with Alexafluor 488, 568, 647 were applied for 1 h at room temperature. To label cell nuclei, DAPI was added to the secondary antibody incubation.The slides were then washed three times in PBS and mounted with ProlongDiamond mounting medium. For histological analysis, sections were subsequently immersed in hematoxylin and eosin, dehydrated with ethanol and xylene, and finally mounted with xylene-based mounting medium. Images were acquired using a Zeiss LSM 780 confocal microscope (Carl Zeiss) and prepared using Fiji (NIH).

[0243] For immunoblotting, organoids were snap frozen in liquid nitrogen and subsequently homogenized in RIPA buffer with 1:100 Halt protease inhibitor (Thermo Fisher) to produce tissue extracts. To collect iCSF, organoids were washed twice in fresh medium and the fluid was collected by mouth pipetting using a glass capillary. Protein concentration was measured using the Bradford assay. Samples containing approximately 10 μg of protein were then prepared with NuPAGE LDS sample buffer 4x and DTT 1M and then heated at 95°C for 15 min. Protein samples and ladder markers were loaded into polyacrylamide gels and run at 90mV for 2h. The samples were transferred to PVDF membranes (Immobilon) at 4°C for 3h. The membranes were blocked in 5% milk in PBS-T and incubated with primary antibodies overnight at 4°C. After washing 3 times in PBS, secondary antibodies conjugated to Alexafluor were added at room temperature for 1h. The membranes were imaged using the Li-COR Odyssey CLx infrared imaging system.

[0244] Single-cell RNA sequencing and analysis

[0245] Single cell dissociation was performed by first pooling two organoids for each case into 15 ml conical tubes: 55-day telencephalic organoids (sample 1), 53-day choroid plexus (sample 2), 27-day choroid plexus (sample 3) and 46-day choroid plexus (sample 4). Under gentle stirring, the samples were incubated at 37 ° C for 20 min in 1 ml Accumax (Sigma, A7089) with 400 μg DNase I and 15 μM actinomycin D. Every 5 min, flick the sample tube and pipette up and down 10 times with a P1000 pipette tip at the end. Bulk precipitation was made and supernatant was collected, 100 μl FBS was added thereto, and then filtered on a 35 μm filter tube (Corning, 352235). The sample was then spun at 300 x g for 5 min. Dead cells were then removed from the cell pellet using a dead cell removal kit and MACS columns (Miltenyi, 130-090-101) followed by an additional 5 min spin at 300 xg. Cells were resuspended in an appropriate volume of 0.04% BSA in PBS to load 16,000 cells on a 10X Chromium system (10X Genomics).

[0246] Single-cell RNA-seq libraries were prepared using the 10X Genomics Chromium Single Cell 3' Library and Gel Bead Kit v3 (10X Genomics) workflow according to the manufacturer's instructions. The Chromium controller was run according to the protocol for generating single-cell gel beads in an emulsion mixture. Reverse transcriptase reactions and subsequent amplification were performed on a C1000 Touch Thermal Cycler (Biorad), and library quality was tested using a 2100 Bioanalyzer (Agilent). Samples were pooled and sequenced on two lanes of the S1 flow cell of a Novasek sequencer (Illumina).

[0247] The raw sequencing output was converted to standard fastq format by bcl2fastq before running through CellRanger Count (version 3.1.0, 10X Genomics), which also uses STAR to align reads to the GRCh38 human reference genome. The QC output showed that for sample 1, there were 10,327 cells, an average of 39,973 reads per cell, and a median UMI count of 4,660 per cell; for sample 2, there were 8,573 cells, an average of 41,815 reads per cell, and a median UMI count of 11,899 per cell; for sample 3, there were 5,347 cells, an average of 67,938 reads per cell, and a median UMI count of 16,252 per cell; for sample 4, there were 8,603 cells, an average of 45,030 reads per cell, and a median UMI count of 9,927 per cell. The filtered feature barcode matrices for each sample were then further analyzed using the Seurat v3 R package in the following manner: the matrices were first merged, then read depth across cells was normalized, the data were scaled, and variable feature finding was performed using SCTransform. During normalization, both mitochondrial profile percentage and cell cycle were regressed out to eliminate these confounding sources of variation. ElbowPlot was used to guide the choice of dimensionality (4), followed by FindNeighbors, FindClusters, and UMAP dimensionality reduction visualization, and unbiased clustering was performed using principal component analysis (PCA). Clusters were identified based on the top 10 differentially expressed genes in each cluster and analysis of known marker genes.

[0248] To compare with in vivo data, subsets of each individual cluster were first generated. For comparison with scRNA-seq from the developing human brain, expression matrices and metadata from Nowakowski et al. (3) were downloaded from the UCSC Cell Browser and processed with Seurat v3 using cell cycle state regression and PCA cluster analysis (visualized with UMAP) similarly to above to generate 12 clusters based on identification of top differentially expressed genes and known marker genes. Subsets were then generated for each of these clusters. Normalized feature count data from each cluster subset from organoids and humans were averaged across single cells within each cluster, and Pearson correlation coefficients were subsequently calculated across matrices and unbiased hierarchical cluster analysis was performed using the heatmap.2 function with the manhattan method and ward.D2 clustering.

[0249] For comparison with mouse embryonic bulk RNA-seq data, raw feature counts were downloaded from NCBI GEO (GSE66312) (4) and TPMs were calculated, followed by conversion to human gene names and removal of duplicates and genes not present in humans. A cutoff TPM of 1 was set in at least one sample and then averaged across 1) whole choroid plexus samples and 2) sorted epithelial cell samples. Data were then log-transformed and merged with organoid subset clustering (averaged as above). Genes not present in all comparison groups were removed, followed by scaling and principal component analysis.

[0250] Electron microscopy

[0251] At 4 ° C, the organoids were fixed in glass vials with 2.5% glutaraldehyde and 2% paraformaldehyde (EM grade, Agar Scientific) in 0.1M cacodylate buffer, pH 7.4 (CB) overnight. The next day, they were rinsed 6 times with CB over the course of a day and stored at 4 ° C overnight. The samples were then rinsed again and post-fixed in the dark for 1 hour with 1% osmium tetroxide in CB on ice, washed 5 times with distilled water over 1 hour, and dehydrated in an increasing concentration of ethanol series (30%, 50%, 70%, 90%, 100%), and then propylene oxide for 10 min (do so at each step). The dehydrated samples were immersed and embedded in CY212 Araldite epoxy resin. 70nm light gold sections were cut from the embedded samples and contrasted with saturated aqueous uranyl acetate. Electron micrographs were recorded on a FEI Tecnai Spirit TEM at 80kV.

[0252] Mass spectrometry data analysis

[0253] Organoids were washed with PBS or fresh culture medium, and iCSF samples were collected by mouth pipetting with glass microcapillaries. Organoid fluid and culture medium were centrifuged for 5 min at 500 g to remove dead cells and debris, and then analyzed via mass spectrometry. Polyacrylamide gel slices (1-2 mm) containing purified proteins were prepared using Janus liquid handling system (PerkinElmer, UK) for mass spectrometry. In short, the protein gel pieces cut were placed in the wells of 96-well microtiter plates, decolorized with 50% v / v acetonitrile and 50mM ammonium bicarbonate, reduced with 10mM DTT, and alkylated with 55mM iodoacetamide. After alkylation, the protein was digested overnight with 6 ng / μL endoproteinase Asp-N (Promega, UK) at 37 ° C. The resulting peptides were extracted in 2% v / v formic acid, 2% v / v acetonitrile. The digests were analyzed by nanocapillary LC-MS / MS (to provide a flow rate of approximately 300 nL / min) using an Ultimate U3000 HPLC (Thermo Scientific Dionex, San Jose, USA). Pre-capture peptides were separated on a C18 Acclaim PepMap100 5 μm, 100 μm x 20 mm nanoViper (Thermo Scientific Dionex, San Jose, USA). Peptides were eluted with a 60 min acetonitrile gradient (2% to 80%). The analytical column outlet was directly connected to a hybrid quadrupole orbital trap mass spectrometer (Q-Exactive Plus Orbitrap, ThermoScientific, San Jose, USA) via a nanoflow electrospray ionization source. Data-dependent analysis was performed on the entire MS spectrum followed by 10 MS / MS spectra using a resolution of 30,000. MS spectra were collected in the m / z range of 300–2000. MS / MS scans were collected using a threshold energy of 27 for high energy collision dissociation (HCD). The LC-MS / MS data were then searched against a protein database (UniProt KB) using the Mascot search engine program (Matrix Science, UK) (5). The database search parameters were set to a precursor tolerance of 10 ppm and a fragment ion mass tolerance of 0.8 Da. One missing enzyme cleavage was allowed, and variable modifications of oxidized methionine, carbamidomethylcysteine, pyroglutamate, phosphorylated serine, threonine and tyrosine, and methylarginine were included. The Scaffold program (Proteome Software Inc., USA) was used to verify the MS / MS data and calculate emPAI values ​​(6).To compare corresponding proteins in samples from different species, only the highest emPAI value was considered as the peptide count for a given protein in each sample. Thus, for example, only human TTR emPAI values ​​were taken for human samples (organoids and human adult CSF), while bovine TTR emPAI values ​​were taken for fetal bovine CSF, and mouse TTRemPAI values ​​were taken for mouse embryonic CSF. This enabled us to compare corresponding homologous proteins across samples despite their different accession numbers.

[0254] NMR sample preparation and analysis

[0255] For NMR experiments, ChP organoids were incubated in complete IDM+A medium (Gibco, A14132-02) without phenol red and 1:50 Geltrex for 2 h, 12 h, 24 h, 48 h, or 72 h. Organoid culture medium and iCSF were collected and centrifuged at 500 g for 5 min to remove debris. The images were acquired using a Bruker Ultrashield 400 Plus operating at 400.1 MHz. 1 H-NMR spectroscopy. Samples consisting of H2O / D2O mixtures were analyzed using HOD suppression to collect 1 H-NMR data. Chemical shifts (δ) are shown in ppm. 1 The conversion yield was determined by relative integration of the signals in the H-NMR spectra. Data analysis was performed using MestReNova (version 7.0) and GraphPad Prism (version 7.0b).

[0256] Similar to what has been described previously (7), to compare the in vitro and in vivo levels of small molecules in CSF, the ratio of unbound CSF to plasma in vivo, K p,uu,CSF Calculated as the unbound drug in CSF (C u,CSF ) and unbound drug in plasma (C u,p ) ratio (Equation A).

[0257]

[0258] The amount of unbound drug in CSF and plasma, respectively, is the total drug concentration (C p ) or (C CSF ) and the unbound drug fraction (f u,p ) or (f u,CSF ) (Equations B and C).

[0259] C u,p =C p f u,p (B)

[0260] C u,CSF =C CSFf u,CSF (C)

[0261] C p , C CSF and f u,p Extracted from the literature (Table S2). However, due to the difficulty in obtaining large amounts of CSF, most drugs are u,CSF is still unknown. Therefore, f u,p Calculate f u,CSF , where drug-protein dissociation in plasma and CSF is identical (Equation D), by calculating the ratio of albumin CSF to plasma (Q alb ) was taken as 0.005 to correct for the reduced plasma protein content of CSF, as previously reported in humans.

[0262]

[0263] Because NMR measurements of in vitro fluids detect unbound drug, the ratio (K m,uu,iCSF ) is a drug in CSF in vitro (C u,iCSF ) and the drug in the culture medium (C u,m )) is a simple function of the ratio (Equation E).

[0264]

[0265] Example 1

[0266] As previously reported, cerebral organoids are characterized by intrinsic self-organization, with recent protocols primarily generating forebrain features (13, 14), which also include the ChP epithelium (Figure 7A) (9, 13). Therefore, to study the development of the human ChP, we established a cerebral organoid-based protocol to generate the ChP in a reliable and reproducible manner.

[0267] The in vitro derivation of ChP cells using the dorsalizing factor Bmp4 alone (15) and in combination with the Wnt activator molecule CHIR (16) has been reported previously.

[0268] Therefore, in order to promote the ChP fate in cerebral organoids, Bmp4 and CHIR were given as pulses (see methods) (Figures 1A, 7B, 7C) (7, 9, 13) starting from the 10th day of the improved cerebral organoid protocol. On the 14th day, non-directional forebrain organoids developed large circular neuroepithelial lobes, while in the organoids treated with Bmp4 / CHIR, we observed more slender neuroepithelial tissues (Figure 7B). This observation is consistent with the in vivo development of ChP from the dorsal midline neuroepithelium, which gradually becomes more slender and expanded in the ventricular cavity of the brain (1, 17, 18). Compared with the forebrain organoids that mainly develop large neuroepithelial lobes with dorsal cortical characteristics (Figures 1B, 1C), ChP organoids appear to be almost completely enriched in ChP cubic epithelium (Figures 1B, 1C). Comparison of day 40 ChP organoid tissue sections with human embryonic ChP at 15 weeks post-conception and ChP from embryonic mouse brain at E18.5 revealed close similarities in complexity and organization between human embryonic samples and organoid tissue (Figure 1D).

[0269] In vivo, the ChP epithelium initially develops as a pseudostratified epithelium, followed by an intermediate columnar stage, and matures into a highly folded cuboidal epithelium (1, 18, 19). To confirm tissue specificity, we stained the ChP marker TTR, the neural stem cell marker Sox2, and the tight junction marker ZO1 at early and late time points (Figure 1E, Figure 1F). ChP organoids treated with Bmp4 and CHIR developed into pseudostratified neural epithelium with TTR-positive areas by day 28 (Figure 1E). By day 39, the treated organoids developed a more columnar, polarized ChP epithelium that was increasingly enriched in TTR immunostaining, with relatively strong ZO1 expression in cell-cell junctions, and sparse Sox2 immunostaining (Figure 1F). Quantification of TTR-positive areas showed reproducible enrichment in treated organoids compared to untreated controls (Figure 1G). Together, these data demonstrate the reliable and reproducible generation of ChP tissue in vitro from different human pluripotent stem cell lines and using two different seeding methods (Fig. 1C-G).

[0270] ChP expresses the highest levels of the channel protein chloride intracellular channel (CLIC6) in the brain (20). We could detect CLIC6 in the epithelium of ChP organoids both by immunohistochemistry and by immunoblotting compared to control, untreated organoids. In parallel, lower levels of the forebrain marker Foxg1 were detected in treated organoids compared to controls. Cortical-specific Tbr2-positive intermediate progenitors and HuC / D-positive neurons were also greatly reduced in early ChP organoids compared to controls and almost completely absent in mature ChP organoids (Figure 8A).

[0271] ChP contains both epithelial and matrix components: polarized epithelial top faces the ventricular cavity and surrounds the matrix core exposed to fenestrated capillaries. In order to better characterize the different cell populations present in ChP organoids, we performed single-cell RNA sequencing (scRNA-seq), which was sampled from three separate batches of ChP organoids at different maturation stages. Two organoids were sampled, and two untreated telencephalic organoids were used as controls (Fig. 2C, Fig. 2D). Combined analysis identified the expected cell clustering of markers expressing neurons and neuronal progenitors in untreated organoids, while immature and mature ChP features were enriched in ChP organoids (Fig. 2C). Unbiased analysis showed that in treated organoids, the enrichment in ChP colonies was consistent and progressive over time (Fig. 2D). Compared with early organoids and untreated controls, older treated organoids showed larger ChP mature epithelial cell components and enrichment in matrix cell colonies (Fig. 2D). Consistent with our previous staining, we confirmed the expression of CLIC6 and other specific ChP markers such as the serotonin receptor HRT2C (21) (Figure 2E). As expected, the immature ChP / hem cell cluster expressed higher levels of early regulators involved in ChP development, such as the homeobox proteins MSX1, OTX2, R-spondin 3 (RSPO3), and PAX6 (Figure 2E, Figure 2F). The mature ChP cell population expressed higher levels of TTR, carbonic anhydrase 2 and 12 (CA2, CA12), nucleoside diphosphate kinase homolog 5 (NME5), inwardly rectifier potassium channel 13 (KCNJ13), and the epithelial marker keratin 18 (Krt18) (Figure 2F). A maturation gradient of the ChP epithelium in vivo has been previously reported, with more mature Krt18-positive cells located distally to the lateral ventricle (21). Krt18 was enriched in more mature ChP organoids compared to early immature ChP organoids (Figure 2A). To investigate the matrix components of ChP in our system, we visualized the expression of DLK1 (delta-like non-classical notch ligand 1), a gene expressed in the ChP stroma, by immunohistochemistry (21) (Fig. 2B). Expression of DLK1 and other ChP matrix proteins (basement membrane polysaccharide LUM and decorin polysaccharide DCN) was detected in the organoid region surrounding the ChP and within the epithelium (Figs. 2B, 8B). Consistent with these results, the ChP matrix population detected by scRNA-seq expressed high levels of DCN, LUM, and DLK1 (Fig. 2F), as well as the extracellular matrix component collagen alpha-1 chain 1 (COL1A1) (Fig. 2E). These data suggest that in vitro derived ChP organoids develop both epithelial and matrix components.

[0272] We next sought to identify similarities between our in vitro model and in vivo ChP organization. We performed unbiased clustering and principal component analysis (PCA) using published datasets of mouse ChP tissue (1) and developing human brain (2) (Figures 3A-D). First, we performed PCA between our identified organoid clusters and RNAseq data from in vivo E18.5 mouse embryonic whole ChP and isolated epithelial ChP cells (4). We could observe that ChP from in vivo samples and our mature ChP clusters were closest to the other identified clusters (Figure 3A). We then performed correlation analysis between organoid clusters and in vivo human developing brain (22), revealing high correlations between organoid and in vivo ChP clusters and between organoid and in vivo matrix clusters (Figure 3B). Further unbiased hierarchical clustering revealed clustering of organoid mature and immature ChPs together with in vivo ChPs, and clustering of organoid ChP matrix with in vivo matrix features (Figure 3C). To further investigate whether our system can better mimic human ChP specifically, we then compared organoid mature ChP clusters with both humans and mice in vivo (Fig. 3D), revealing a higher correlation with human ChP than with mice (Fig. 3D). Finally, examination of specific regional ChP markers (4, 24) revealed that choroid plexus organoids had lateral ventricle characteristics rather than the third or fourth ventricle (Fig. 3E), consistent with their telencephalic origin. These data generally demonstrate that the ChP epithelium and matrix composition of organoids seem to recapitulate the cellular composition and genetic structure of human ChP in vivo.

[0273] To examine the maturity of the ChP organoids, we looked at previously described markers of ChP maturation. We could observe the presence of ciliary markers, indicating expression during ChP epithelial maturation (3); as well as the presence of a marker for the more mature ChP epithelium, aquaporin 1 (AQP1). We could detect enrichment of ciliary genes such as DYNLRB2 and FOXJ1 in the mature ChP clusters, as well as enrichment of AQP1 (Figure 3F). Staining further revealed the presence of CCDC67 and FOXJ1, both regulators of cilia formation, as well as the presence of the ciliary protein ARL13B, which stained the apical ciliary clusters in the ChP epithelium (Figure 3G). Staining for AQP1 also confirmed the presence of this more mature marker, showing localization on the apical surface of the epithelium, similar to in vivo (Figure 3H). The ChP epithelium is an extremely efficient secretory machinery (23), with numerous microvilli, motile cilia, and a dense network of cytoplasmic cysts on the apical surface that increase its surface area. To further investigate whether ChP organoids recapitulate these basic ultrastructural features, we performed EM imaging. We observed primary cilia, numerous apically located microvilli, intercellular tight junctions, multivesicular bodies, and numerous extracellular vesicles (Fig. 3I; indicated by arrows). In line with their roles in brain lipid metabolism, we also observed enrichment of lipid droplets and lipoproteins (ApoE, clusterin / ApoJ, and phospholipid transfer protein PLTP) (Fig. 3J). These features, along with the absence of actively cycling cells positive for the cell cycle marker phosphorylated histone H3 (PH3) (Fig. 3K), indicated a fairly mature secretory epithelium in ChP organoids.

[0274] We next asked whether ChP organoids could develop a tight barrier by examining the expression of tight junction markers such as claudin and occludin, which seal the intercellular spaces between barrier cells of the ChP epithelium (25, 26). scRNA-seq revealed the presence of several claudin proteins (Fig. 4A) as well as other tight junction components occludin (OCLN), ZO1 (TJP1), ZO2 (TJP2), and the PDZ proteins PATJ (INADL) and MPDZ (Fig. 4B). Staining revealed apical localization of claudin 1, 3, 4, and 5 in the polarized epithelium of ChP organoids and validated their enriched expression (Fig. 4C). Low levels of claudin 2 were also observed in the epithelium of ChP organoids, as well as the presence of OCLN (Fig. 11A). Finally, to directly assess the barrier function of the ChP epithelium, we applied fluorescently labeled dextran of different molecular weights (70, 10, and 3–5 kDa) and examined their incorporation into organoids. After 2 h of incubation, no obvious leakage of 10 kDa Alexa-647 labeled dextran was seen within the organoids ( FIG. 11B ). Fluorescence intensity of the culture medium and internal fluid collected after 2 h of incubation with 70 kDa, 10 kDa, and 3-5 kDa labeled dextran confirmed that these molecules were completely excluded from the organoids ( FIG. 11C ).

[0275] A notable feature of ChP organoids is the subsequent development of large fluid-filled compartments or cysts containing colorless fluid, which are rarely or completely absent in forebrain, untreated organoids (Fig. 4D, Fig. 9A-B). We found that these compartments were completely isolated from the culture medium and could be maintained by continued addition of dissolved Matrigel to the culture medium. We observed identical cuboidal epithelia surrounding these compartments and exhibiting a polarized orientation, with the apical markers Aqp1 and ZO1 on the luminal side oriented toward the fluid (Fig. 4D).

[0276] To investigate the composition of this fluid, we performed mass spectrometry to compare in vitro organoid CSF-like fluid (iCSF) (3 batches of H9, 1 batch of H1, and 1 batch of iPSC IMR-90), with 3 different in vivo samples (adult telencephalic CSF pooled from 3 healthy donors, mouse embryonic CSF at E13.5, and fetal bovine CSF), as well as culture medium. We detected a total of 248 abundant proteins in organoid iCSF, of which 199 were identical to the in vivo CSF ​​samples (Fig. 10A). Gene ontology analysis revealed that the GO:CC categories “extracellular vesicle” and “extracellular space,” as well as the GO:BP categories “exported from the cell” and “secreted from the cell” were enriched in the organoid fluid proteome (Fig. 5A). Insulin-like growth factors, particularly IGF2, are secreted by ChPs and regulate the proliferation of cortical progenitor cells in the developmental CSF (2). Consistent with a role for IGF in developmental CSF, the REAC category “regulation of insulin-like growth factor” was also significantly enriched in organoid iCSF.

[0277] Next, we focused on proteins detected in at least 2 organoid iCSF samples to exclude technical artifacts, and we then examined the most abundant proteins with a reported emPAI value of 1 based on the number of peptides observed. Of these 83 abundant proteins detected in organoid iCSF, 80 were also detected in in vivo samples, further indicating a high degree of similarity (Figures 5B, 10C, 10D). Among these proteins, we examined several biomarkers such as clusterin (CLU), phospholipid transfer protein (PLTP), and apolipoprotein E (APOE) (Figure 5B, Supplementary Table 1). Many of the proteins detected in iCSF, such as insulin-like growth factor binding protein 7 (IGFBP7), Serpin family F member 1 (SERPINF1), Niemann–Pick disease type C2 protein (NPC2), and retinol binding protein 1, were also detected in the scRNA-seq ChP clusters (Figures 5C, 5D), further confirming their generation. A subset of proteins is only present in human organoids and adult CSF samples, indicating potential human-specific secretion (Fig. 5E). We also detected several development-specific CSF proteins, including insulin-like growth factor 2 (IGF2), IGFBP7, and follistatin-like protein 1 (FSTL1), which are not present in adult samples, indicating that iCSF secreted by ChP organoids retains at least some developmental characteristics of human CSF (Fig. 5E). IGF2 levels in organoid fluids were confirmed by immunoblotting (Fig. 5G), and immunohistochemistry revealed that it is present in ChP epithelium (Fig. 5H). Consistent with the unique role of ChP in folic acid and vitamin C transport (both folic acid and vitamin C are essential for brain homeostasis and function (27, 28)), we detected specific transporters involved in the transport of these nutrients: SLC23A2 (vitamin C) and SLC46A2 (folate) are enriched in ChP epithelial clusters (Fig. 5D).

[0278] We next asked which stage iCSF from organoids best matched by further comparing it to a previously published proteomic dataset of human embryonic Carnegie stage 20 CSF (29). We observed a large overlap between organoid iCSF and adult CSF, sharing 121 proteins, compared to 51 proteins shared between iCSF and embryonic CSF (Figure 5E), suggesting that iCSF may be more similar to adult CSF but still retain some developmental features.

[0279] Overall, there was very good consistency between in vitro and in vivo samples; however, in order to fully determine the similarity with in vivo, we further examined proteins with abnormal distribution. First, although the vast majority of proteins detected were not present in the culture medium, indicating the presence of a highly selective barrier, proteomic analysis revealed that 13 of the 14 abundant proteins in the culture medium were also present in organoid iCSF (Figures 10C, 10D). Importantly, most of them were also detected in in vivo samples and included proteins such as albumin (ALB) and transferrin (TF), which are actively transported across the ChP epithelium. Those not detected in vivo were proteins provided by Matrigel, including several laminins. Second, there are several proteins that can be reproducibly detected in in vivo samples, but not in organoid iCSF, which requires further examination because these may be factors that prevent organoids from being produced normally. However, we found that proteins that were only detected in in vivo CSF ​​samples but not in organoid iCSF showed enrichment in GO:CC “blood microparticles”, GO:BP “platelet degranulation”, and KEGG: “complement and coagulation cascades” (Figures 10B, 10E, 10F). This can be explained by the fact that the invasive procedure of lumbar puncture (spinal tap) CSF collection often leads to contamination with peripheral blood and bone marrow. Therefore, one of the advantages of the organoid system is that ChP organoids lack vascular components; therefore, we expect this fluid to be a specialized ChP secretome. In summary, these findings indicate that: 1. ChP organoids develop into self-contained compartments that are distinguished from the surrounding culture medium by a tight epithelial barrier, and 2. ChP organoids actively secrete CSF-like fluid that is rich in human development-specific CSF proteins and free of blood contaminants.

[0280] As part of its selective barrier function, the ChP epithelium is also highly selective for small molecules, including therapeutic agents. Therefore, to test this selective permeability to small molecules, we applied a variety of therapeutically relevant small molecules and determined their permeability by NMR analysis of organoid iCSF. As a proof of principle, we tested the permeability of dopamine (DOPA) and its precursor levodopa (L-DOPA) through the barrier, as they are highly similar molecules with very different permeabilities in vivo and this specificity has not been previously reported in any in vitro BBB model. It is well established that DOPA does not enter the brain in vivo, whereas L-DOPA is actively transported into the CSF via the LAT-1 transporter (30–33) and is therefore used as a therapeutic agent in dopamine replacement therapy in patients with Parkinson's disease. We incubated organoids with DOPA and L-DOPA in the presence of carbidopa, a small molecule that blocks the conversion of L-DOPA to DOPA (34, 35). After 2 h, we could detect the presence of both L-DOPA and DOPA in the culture medium, but only L-DOPA in the iCSF, which is consistent with the in vivo properties of the CNS barrier (Figure 6A, Figure 11E). The ratio of L-DOPA iCSF / culture medium was consistently higher than the ratio of DOPA iCSF / culture medium, indicating that our model exhibits the same selectivity for small molecules as in vivo (Figure 6A).

[0281] These findings suggest that there may be appropriate expression of key transporters in the blood-CNS barrier. In fact, we were able to detect the expression of transporter LAT-1 on the top surface of the ChP organoid epithelium (Fig. 6 D, Fig. 9 C), which explains the correct transport of L-dopa seen in our permeability assay. We also dyed for the well-known efflux pump P-glycoprotein 1 (Pgp or multidrug resistance protein 1, MDR1), which prevents drugs from entering the brain via an ATP-dependent mechanism (36, 37) (Fig. 6 D). We detected the top and near-top expression of Pgp that seems to be located in intracellular vesicles in the ChP epithelium (Fig. 6 D). The positioning of Pgp in ChP has not yet been fully determined (38, 39), however, it is reported that it plays a role in pumping molecules from CSF into the blood (38, 40). Other important efflux transporters in ChP are MRP1 and MRP4, which prevent toxic compounds and unwanted signals from reaching CSF (38, 41). At 40 days in culture, MRP1 was localized to the ChP epithelium both apically and basally (Fig. 6D, Fig. 9C), whereas MRP4 was expressed apically in the ChP epithelium (Fig. 6D). These results indicate that the ChP epithelium generated in vitro endogenously expresses transport and export pumps that are essential for the correct function of B-CSF-B in the human brain.

[0282] We next tested whether the system could quantitatively predict drug permeability. To this end, we performed similar permeability experiments to test bupropion, an antidepressant known to readily cross the BBB (42), and two chemotherapeutic compounds, methotrexate and vincristine, which cross the BBB poorly (43, 44). As predicted, bupropion was readily detectable in iCSF after 2 h, whereas methotrexate and vincristine were completely excluded from iCSF (Figure 6B). Most importantly, we found a very high correlation between the in vitro iCSF / medium ratio and the in vivo CSF / plasma ratio for the same drugs reported in the literature (R 2 =0.9921). Moreover, the actual values ​​also agree very well (as indicated by the slope of 1.004), indicating that this in vitro system can qualitatively and quantitatively recapitulate the drug permeability reported in vivo (Figure 6E).

[0283] These results suggest that the ChP organoid system can be used to model and predict the CNS permeability of unknown drugs. To test this theory, we turned to sephin 1, a novel inhibitor of the regulatory subunit of protein phosphatase 1, PPP1R15A, which has been shown to cross the BBB and have protective CNS effects against protein misfolding in the Charcot-Marie Tooth-1B mouse model (45). No preclinical or clinical data for this molecule in humans are yet available, but a phase I clinical trial is ongoing, so any additional human in vitro data would be informative. Using the same experimental conditions as previously described, sephin 1 was detectable in iCSF after 2 h of incubation, indicating that this compound may also cross the human CNS barrier (Figure 6C). However, it is worth noting that the permeability in the human organoid model is much lower than that described in the mouse model, suggesting that higher doses may be required in humans to achieve the same CNS levels.

[0284] Problems related to the pharmacokinetic profile of drugs are responsible for the failure of most clinical trials (46, 47). An extreme example involves the compound BIA-10-2474, a fatty acid amide hydrolase inhibitor under development for the treatment of several medical conditions such as chronic pain and multiple sclerosis (48, 49). Unfortunately, the Phase I clinical trial was abruptly terminated when six trial participants exhibited severe neurotoxicity, one of which died (49, 50). An investigation conducted by the French Ministry of Health proposed several possible explanations, one of which was accumulation of the toxic drug in the brain, which was not evident in any of the animal models tested (49–51). However, the exact cause remains unknown as further studies were terminated. Given the failure to detect this adverse effect in animal models, we sought to test whether human ChP organoids could more accurately predict human CNS permeability and test the accumulation theory. We compared the pharmacokinetic profile of BIA-10-2474 with that of a safe compound, bupropion, which readily crosses the epithelial barrier but does not exhibit this toxic accumulation. Similar to bupropion, BIA-10-2474 was able to cross the ChP epithelial barrier after 2 h of incubation (Figure 6F). However, in contrast to bupropion, which stabilized at baseline levels after 24 h, BIA-10-2474 continued to accumulate in the iCSF (Figure 6G). These data are consistent with the theory that BIA-10-2474 may accumulate in the brains of trial participants, suggesting a potential explanation for the failure of this drug in clinical trials. In summary, our findings suggest that organoid systems can be used as human in vitro preclinical models to quantitatively predict not only the permeability of unknown compounds, but also their kinetics over time (Figure 6H).

[0285] In conclusion, ChP organoids can be used in an unprecedented and reliable way to study the development and function of the human ChP, a relatively understudied organ that is gaining increasing attention due to its important roles in development and disease, such as neurodegeneration.

[0286] Supplementary Table 1. ChP organoids secrete human disease-associated biomarkers. The table shows the biomarkers detected in iCSF (31 of the 50 most abundant iCSF proteins are shown), the diseases or conditions in which the biomarker levels are altered, and relevant literature. Asterisks indicate proteins that were also detected in the culture medium (8–59).

[0287] Supplementary Table 2. Time point permeability experiments. Table shows cell line, batch number, iCSF collection date (DIV) for the relative drugs tested on ChP organoids (shown in Figure 6).

[0288] Supplementary Table 3. iCSF time points for proteomics. The table shows the cell line, batch generation date, and iCSF collection date (DIV) of samples used for proteomic analysis (shown in Figure 5). Asterisks indicate samples that were pooled together.

[0289] Supplementary Table 4. In vivo CSF ​​time points used for proteomics. Table shows the type and size of in vivo samples used for proteomic analysis (shown in Figure 5). Asterisks indicate samples that were pooled together.

[0290] Supplementary Table 5. In vivo and in vitro data for drug CSF levels. The table shows the measured values ​​and calculated ratios (K) (60–68) of unbound small molecules in CSF and plasma (in vivo) or culture medium (in vitro).

[0291] Supplementary Table 1

[0292]

[0293] Supplementary Table 2

[0294] Time point permeability experiments

[0295]

[0296] Supplementary Table 3

[0297] iCSF time points for proteomics

[0298]

[0299] Supplementary Table 4

[0300] In vivo CSF ​​time points for proteomics

[0301]

[0302] Supplementary Table 5

[0303] Comparison with drug permeability in vivo

[0304]

[0305] Total compound brain to plasma ratio

[0306] Reported unbound concentration, average of two measurements

[0307] In vivo data from mice

[0308] The values ​​marked with ~ are estimated based on graphs in the relevant references.

[0309] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the products, uses and methods disclosed in the present invention will be clear to the technician without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the disclosed mode for implementing the present invention that are obvious to the technician are intended to fall within the scope of the following claims.

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Claims

1. A choroid plexus organoid comprising: (a) an epithelium comprising a tight epithelial barrier; and (b) one or more cysts surrounded by the epithelium; Wherein the epithelium comprises apical microvilli.

2. The choroid plexus organoid of claim 1, wherein the epithelium is thyroxine transporter (TTR)+, multidrug resistance protein 1 (MRP1)+, aquaporin (Aqp1)+, and / or zonula occludens (ZO1)+.

3. The choroid plexus organoid of claim 2, wherein (a) the TTR, Aqp1 or ZO1 is enriched on the apical surface of the epithelium; or (b) The TTR, Aqp1 and ZO1 are enriched on the apical surface of the epithelium.

4. The choroid plexus organoid of claim 2, wherein the MRP1 is enriched on the apical and basal surfaces of the epithelium.

5. The choroid plexus organoid of any one of claims 1-4, wherein the epithelium comprises primary cilia. The choroid plexus organoid according to any one of claims 1 to 4, further comprising (c) stromal cells.

7. The choroid plexus organoid according to any one of claims 1 to 4, which is a lateral ventricular choroid plexus organoid.

8. The choroid plexus organoid of any one of claims 1-4, wherein the one or more cysts are filled with a fluid.

9. The choroid plexus organoid of claim 8, wherein the fluid comprises one or more proteins selected from the group consisting of thyroxine transporter (TTR), clusterin (CLU), apolipoprotein E (APOE), apolipoprotein A4 (APOA4), and lumihane (LUM).

10. A method for producing a choroid plexus organoid according to any one of claims 1 to 9, the method comprising the following steps: (a) generating an embryoid body population by culturing a stem cell population; (b) culturing the embryoid body population under conditions suitable for neural induction; (c) embedding the product of step (b) in a three-dimensional matrix; (d) culturing the product of step (c) in the presence of a wingless gene-related (WNT) pathway activator and a bone morphogenetic protein (BMP) signaling pathway activator, wherein step (d) begins 8-12 days after the start of step (a); and (e) adding ECM to the culture medium, wherein the ECM is dissolved Matrigel, wherein step (e) is initiated 30 days after the initiation of step (a).

11. The method of claim 10, wherein the method comprises (c) embedding the product of step (b) in an extracellular matrix (ECM).

12. The method of claim 10, wherein step (c) begins 7 days after step (a) begins.

13. The method of claim 10, wherein the culturing of step (d) comprises stirring.

14. The method of claim 10 or 11, wherein the WNT pathway activator is selected from CHIR99021, Wnt1, Wnt2, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, Wnt10b, BML-284, 6-bromoindirubin-3'-oxime (BIO), WAY-316606, IQ1, QS11, SB-216763, LY2090314, DCA, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine and lithium.

15. The method of claim 14, wherein the WNT pathway activator is CHIR99021.

16. The method of claim 10 or 11, wherein the BMP signaling pathway activator is selected from the group consisting of Bmp4, Bmp2, Bmp3, Bmp5, Bmp6, Bmp7, Bmp8a, Bmp8b, Bmp10, Bmp11, Bmp15, isoliquiritigenin, 4'-hydroxychalcone, apigenin, diosgenin and ventromorphins.

17. The method of claim 16, wherein the BMP signaling pathway activator is Bmp4.

18. The method of claim 16, wherein the ventromorphins are SJ000291942, SJ000063181 and / or SJ00037178.

19. Use of the choroid plexus organoid according to any one of claims 1 to 9 for drug discovery screening or determining drug toxicity.

20. The use according to claim 19, wherein the use is for testing the ability of a candidate agent to cross the blood-cerebrospinal fluid (CSF) barrier.

21. A method for testing the ability of a candidate agent to cross the blood-cerebrospinal fluid (CSF) barrier, comprising contacting the choroid plexus organoid according to any one of claims 1-9 with the candidate agent and determining whether the candidate agent crosses the epithelium.

Images