A drug testing platform for synucleinopathies using human brain organoids

Abstract

The present invention provides human striatum-like organoids (hSLO), which resemble the human striatum, fusion organoids of hSLO and human midbrain-like organoids (hMLO) with functional connections between hSLO and hMLO, and methods for testing drugs using the organoid culture platform.

Description

[Technical field]

[0001] The present disclosure relates to striatum-like organoid (SLO) and the method of producing SLO.The present disclosure also relates to functionally fused striatum-like organoid (SLO) and mesencephalon-like organoid (MLO).The present disclosure also relates to human striatum-like organoid (hSLO) and the method of producing hSLO.The present disclosure also relates to functionally fused human striatum-like organoid (hSLO) and human mesencephalon-like organoid (hMLO). [Background technology]

[0002] The striatum is a component of the basal ganglia that is centrally located in the human brain and has various functions, including voluntary movement (Hikosaka et al., 2000). The striatum transmits and receives information to and from various brain regions, mainly via the processes of specific neurons (Gerfen, 2006; Ingham et al., 1998; Lovinger, 2010; Macpherson et al., 2014). Thus, the striatum is most often associated with movement and is heavily affected by neurodegeneration in patients suffering from Parkinson's disease (PD).

[0003] Neural activity between striatal-nigral GABAergic neurons in the striatum and nigro-striatal dopaminergic (DA) neurons in the midbrain motivates behavior and movement (Albin et al., 1989). Dysfunction of these neural circuits later contributes to the pathogenesis of PD. However, how these brain regions interconnect and what causes functional defects in neurological diseases is poorly understood. Current cell culture and animal models have been criticized for not accurately mimicking the actual neural circuits in the brain. Therefore, generating in vitro brain models to demonstrate and visualize the human nigro-striatal pathway will provide a platform for future studies of neurological diseases, such as alpha-synuclein (α-syn) pathology. Summary of the Invention

[0004] 3D organoid model systems are engineered to approximate the in vivo organ or tissue from which they were derived. These 3D culture systems can recapitulate the complex morphological features of differentiated epithelia and allow cell-cell and cell-matrix biological interactions. This is in contrast to classical 2D culture models, which often share little physical, molecular, or physiological similarity with the original tissue. Although in vitro modeling holds great promise, current brain organoid systems have certain limitations as they cannot reflect all aspects of human brain diseases, such as PD.

[0005] The generation of human striatal organoids faces several challenges: - there is no stepwise protocol for creating reciprocal nigrostriatal and striatonigral projections between the substantia nigra of the midbrain and the striatum, a specific region of the basal ganglia; - there are no human in vitro neuronal systems to demonstrate synapse formation between DA neurons and medium spiny neurons (MSNs) in the substantia nigra and striatum, respectively; - the absence of a system to model α-syn pathology and study the propagation of pathological proteins between different brain regions; - the absence of drug screening / testing systems based on α-syn pathology as a readout using the brain organoid culture platform; Most drug screening platforms for neurodegenerative diseases are built on 2D neuronal cell culture systems and animal models, which do not reproduce the in vivo human brain environment, increasing the risk of misinterpretation of results.

[0006] In this disclosure, we first describe how to generate human striatum-like organoids, with characterization data including gene expression analysis, immunohistochemical analysis, and calcium imaging. We then generate fusion organoids using hSLO and hMLO, which have been previously established by our laboratory, and demonstrate that the midbrain and striatum are physically and functionally connected, with characterization of reciprocal projections and synapse formation.

[0007] The present disclosure provides, for example, the inventions described below. (1) A method for culturing embryoid bodies (EBs), such as human embryoid bodies, comprising: The method includes culturing EBs in a first culture medium containing a first factor, for example, for 1 to 7 days, 2 to 6 days, or 3 to 5 days, wherein the first factor includes TGF-β and a Wnt inhibitor such as a SMAD2 / 3 signaling pathway inhibitor and / or a GSK inhibitor (e.g., a GSK3 inhibitor or a GSK3β inhibitor), preferably a TGF-β and a SMAD2 / 3 signaling pathway inhibitor and a Wnt inhibitor. (2) culturing the obtained EBs in a second medium containing a third factor for, for example, 7 to 14 days, 8 to 13 days, 9 to 12 days, or 10 to 11 days, optionally under orbital shaking conditions; The method according to (1) above, wherein the third factor comprises a Wnt inhibitor such as a GSK inhibitor (e.g., a GSK3 inhibitor or a GSK3β inhibitor or XAV939), a patterning factor for Sonic Hedgehog pathway activation such as a smoothened agonist and purmorphamine, and an activin such as activin A, in order to obtain LGE neurospheres, and does not comprise the first factor. (3) The method according to (2) above, further comprising culturing the obtained EBs in a third medium containing a third factor, the third factor comprising brain-derived neurotrophic factor (BDNF) and / or ascorbic acid, in order to obtain organoids such as human striatum-like organoids, and not having the first factor and the second factor. (4) The method described in (3) above, wherein the obtained organoid contains one or more markers of mature medium spiny neurons (MSNs). (5) The method according to (4) above, wherein the obtained organoids contain D1 and / or D2 MSNs. (6) The method according to (5) above, wherein the obtained organoid expresses at least one or all of the following markers: interneuron markers such as TH, cholinergic neuron markers such as CHAT and 5-HT, and serotonin neuron markers, and glial cell markers such as MBP and GFAP. (7) The method according to any one of (4) to (6) above, wherein the organoid has a long axis diameter or diameter of 1 mm or more, preferably a long axis diameter or diameter of 1 mm to 2 mm. (8) An isolated organoid containing mature medium spiny neurons (MSNs) that express one or more markers of mature MSNs. (9) The isolated organoid described in (8) above, wherein 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the cells contained in the organoid are DARPP32 positive and GABA positive. (10) The isolated organoid described in (8) or (9) above, comprising D1 and / or D2 GABAergic MSNs. (11) The isolated organoid according to any one of (8) to (10), expressing at least one or all of the following interneuron markers: TH, cholinergic neuron markers such as CHAT and 5-HT, and serotonin neuron markers; MBP, S100β, and GFAP, glial cell markers. (12) The isolated organoid according to any one of (8) to (11), having a long axis diameter or diameter of 1 mm or more, preferably a long axis diameter or diameter of 1 mm to 2 mm. (13) A method for producing an organoid, comprising: Preparing an organoid (first organoid) according to any one of (8) to (12) above, and a second organoid comprising dopaminergic (DA) neurons, for example, A9-like subtype mDA neurons and A10-like subtype mDA neurons, wherein the organoid preferably expresses one or more of DA neuron markers, such as FOXA2, LMX1A, OTX2;TH, DAT, and GIRK2; and A method comprising contacting a first organoid and a second organoid to obtain a fusion organoid of the first organoid and the second organoid, wherein dopaminergic neurons of the second organoid have projections reaching the first organoid and GABAergic MSNs of the first organoid have projections reaching the second organoid. (14) A fusion organoid of the first organoid according to any one of (8) to (12) above, and a second organoid comprising a dopaminergic (DA) neuron, for example, an A9-like subtype mDA neuron and an A10-like subtype mDA neuron, wherein the organoid preferably expresses one or more dopamine neuron markers, such as FOXA2, LMX1A, OTX2;TH, DAT, and GIRK2; A fused organoid, in which the dopaminergic neurons of the second organoid have projections that reach the first organoid and the GABAergic MSNs of the first organoid have projections that reach the second organoid. (15) The fused organoid described above in (14), wherein the cells contained in the fused organoid include DA neurons having α-syn aggregates. (16) A method for testing a candidate drug, comprising the steps of: Contacting a candidate drug with the fused organoid described in (14) or (15) above; To observe α-synuclein aggregation in DA neurons, and selecting candidate drugs that reduce alpha-synuclein aggregation relative to a negative control; The method includes: (17) The method according to (16) above, wherein SNCA is overexpressed in at least DA neurons or all cells of the second organoid entity or organoid. [Brief description of the drawings]

[0008] [Figure 1] Figure 1. Generation of human striatal-like organoids (hSLOs). (A) Schematic illustrating the pathways determining specific human midbrain and striatal development. (B) Schematic showing the strategy to generate hSLOs. (C) DIC images show typical morphological characteristics of cells at days 10, 20, 30, and 60 of differentiation, and in (D) quantification of EB diameter is shown. Scale bar = 500 μm. Error bars represent the mean ± SEM (n = 8). [Diagram 2] Figure 1 shows qRT-PCR results showing expression of pluripotency and early lateral ganglionic eminence (LGE) markers at early times of differentiation. Quantitative RT-PCR analysis of cells dissociated from hSLO for NANOG, OCT4, ASCL1, DLX5, GSX2, and EBF1. Error bars represent mean ± SEM (n=3). [Diagram 3] Immunohistochemical analysis showing differentiation efficiency of GABAergic neurons at day 60. (A) Frozen sections of hSLO at day 60 immunostained for CTIP2 and GABA. Scale bar = 20 μm. (B) Quantification of A. Error bars represent mean ± SEM (n = 3). [Figure 4] Immunohistochemical analysis showing expression and quantification of functional GABAergic markers DARPP32 and GABA. White scale bar = 50 μm. Error bars represent mean ± SEM (n = 3). [Diagram 5] Immunohistochemical analysis showing the presence of subtypes of GABAergic neurons in hSLO. (A) Frozen sections of hSLO immunostained for GABA and substance-P. (B) Frozen sections of hSLO immunostained for GABA and DRD2. Scale bar = 5 μm. [Figure 6]Immunohistochemical analysis showing expression of different cell types in hSLO as well as in human striatum. Frozen sections of hSLO at 60 and 90 days immunostained for (A) MAP2, TH, and DAPI, (B) CHAT, 5'HT, and DAPI, (C) MBP and DAPI, (D) GFAP and DAPI. Scale bar = 50 μm. [Figure 7] Characterization of dissociated neurons in hSLO. (A) Representative image showing dissociated hSLO cells labeled with AAV-mDlx::EGFP reporter. Neurons are immunostained for EGFP and CTIP2. (B) Representative image showing dissociated hSLO cells labeled with AAV-mDlx::EGFP reporter. Enlarged views of panels show (B') growth cones and (B'') dendritic projection spines, with arrows indicating the position of growth cones and dendritic projection spines. White scale bar = 20 μm, yellow scale bar = 5 μm. [Figure 8] Figure 1 shows calcium imaging results of hSLO and trace peaks showing calcium activity. Spikes of nine single neurons in hSLO at day 150 are extracted in the right panel. Scale bar = 200 μm. [Figure 9] Figure 1 shows the results of immunohistochemical analysis to show the neuronal reciprocal projection between hSLO and hMLO. (A) Simplified schematic showing the formation of neuronal projections between the striatum and substantia nigra in the basal ganglia. (B) 3D immunostaining of clearing hSLO organoids expressing AAV-hSyn1::EGFP and hMLO-hSLO fusion organoids with unlabeled hMLO. (C) 3D immunostaining of clearing hMLO organoids with TH-EGFP DA neuron reporter and hMLO-hSLO fusion organoids with unlabeled hSLO. Scale bar = 200 μm. [Figure 10]Figure 1 shows the results of immunohistochemical analysis to show synapse formation of neurons in hMLO-hSLO fusion organoids. (A) Fusion organoids generated from H9 TH-EGFP hMLO and H9 hSLO, immunostained for EGFP, PSD95, and SYN1, show expression of pre- and post-synaptic markers along with projection neurons from hMLO. (B) Fusion organoids generated from H9 TH-EGFP hMLO and H9 hSLO, immunostained for EGFP, VMAT2, and GABA, show expression of VMAT2 and GABA along with projection neurons from hMLO. (C) Fusion organoids generated from H9 hMLO and H9-AAV-hSyn1::EGFP hSLO, immunostained for EGFP, PSD95, and SYN1, show expression of pre- and post-synaptic markers along with projection neurons from hSLO. (B) Fusion organoids generated from H9 hMLO and H9-AAV-hSyn1::EGFP hSLO immunostained for EGFP, VGAT, and GABA, showing expression of VGAT and GABA along with projection neurons from hSLO. Red scale bar = 1 μm. White scale bar = 10 μm. [Figure 11] Figure 1: Results of α-syn pathogenesis modeling in hMLO-hSLO fusion organoids. (A) Whole mount staining images and quantification of W / T and SNCA O / E fusion organoids stained for EGFP showing the projection of TH-EGFP+ neurons from hMLO to hSLO at 21 d.pf (mean ± SEM; *p<0.05, n=4). White scale bar=200 μm, yellow scale bar=50 μm. (B) Lentiviral constructs to generate SNCA-linker-mKO2 overexpressing cell lines. (C) Whole mount images of fusion organoids stained with mKO2 antibody to label SNCA-linker-mKO2 propagation from hSLO to hMLO and vice versa. White scale bar=100 μm, yellow scale bar=50 μm. (D) Quantification of C (mean ± SEM; ****p<0.0001, n=4). [Figure 12] FIG. 1 shows a model for anti-PD drug testing using fused organoids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] As used herein, the term "embryoid body" (EB) refers to a three-dimensional aggregate of pluripotent cells or, preferably, pluripotent stem cells (PSCs). EBs are characterized by the presence of Ca expressed on pluripotent cells. 2+ They are formed by pluripotent cells such as embryonic stem cells (ESCs) through intramolecular binding of the E-cadherin-dependent adhesion molecule. When cultured as single cells in the absence of anti-differentiation factors, PSCs spontaneously aggregate to form EBs. Such spontaneous formation is often achieved in bulk suspension cultures, where the culture dishes are coated with non-adhesive substances such as agar or hydrophilic polymers to promote preferential adhesion between single cells rather than to the culture substrate.

[0010] As used herein, the term "embryonic stem cell" refers to a pluripotent stem cell that can be derived from the inner cell mass of a blastocyst of an animal, e.g., a mammal, such as a rodent, including mouse and rat, or a primate, including human and monkey.

[0011] The term "pluripotent stem cells" as used herein refers to stem cells with pluripotency. Pluripotency is the potential of cells to differentiate into any of the three germ layers, including endoderm, mesoderm, and ectoderm, but not into extraembryonic tissues such as placenta. Pluripotent stem cells can be artificially derived from non-pluripotent cells, such as adult somatic cells, by inducing the forced expression of certain combinations of reprogramming factors. For example, forced expression of Oct4, Sox2, Klf4, and c-Myc can generate induced pluripotent stem cells (iPS cells) from fibroblasts, etc.

[0012] The term "organoid" refers to a cell aggregate that can be cultured in vitro and usually contains one or more types of cells to form a three-dimensional structure. Some organoids have tissue structures similar to those of the original organ in the body. Organoids with functions similar to those of organs can show therapeutic effects against diseases caused by reduced function of organs. Organoids with structures similar to those of organs have been generated to study the development of organs.

[0013] The present disclosure provides a method for culturing embryoid bodies (EBs).EBs are preferably animal EBs, more preferably mammalian EBs, and even more preferably human EBs.EBs can be obtained by culturing pluripotent stem cells, such as ES cells or iPS cells, as described above.

[0014] In one embodiment, the method includes culturing the EBs in a first medium containing a first factor, e.g., for 1-7 days, 2-6 days, or 3-5 days. The first factor may include a TGF-β signaling pathway inhibitor and / or a Wnt inhibitor, preferably a TGF-β signaling pathway inhibitor and a Wnt inhibitor, such as a GSK inhibitor (e.g., a GSK3 inhibitor or a GSK3β inhibitor).

[0015] Examples of TGF-β signaling pathway inhibitors include, but are not limited to, (i) SMAD inhibitors, such as ALK4 inhibitors, ALK5 inhibitors, ALK7 inhibitors, SMAD2 / 3 inhibitors, including SMAD2 / 3 phosphorylation inhibitors, and multiple inhibitors (e.g., dual inhibitors) for two or more selected from the group consisting of ALK4, ALK5, ALK7, SMAD2 / 3, and SMAD2 / 3 phosphorylation, preferably for TGF-β and SMAD2 / 3. (ii) one or more selected from the group consisting of LY-364947, SB-525334, SD-208, and SB-505124; 616452 and 616453; GW788388 and GW6604; LY580276, as disclosed in International Publication WO 2015 / 002724 A, the entirety of which is incorporated herein by reference; or (iii) SB-431542. In one embodiment, examples of TGF-β signaling pathway inhibitors include pan-TGF-beta / Smad inhibitors such as LDN-193189 and K02288; and selective TGF-beta / Smad inhibitors such as SB431542 and galunisertib. Dorsomorphin can also be used as a TGF-β signaling pathway inhibitor.

[0016] Examples of Wnt inhibitors include, but are not limited to, adavint (SM04690), IM-12, lanatoside C, M435-1279, Wnt-C59 (C59), atranorin, Box5, isoquercitrin, AZD2858, CCT251545, PNU-74654, IWP-2, CP21R7 (CP21), IWR-1-endo, ginsenoside Rh4, FIDAS-3, gigantol, AZ6102, IWR-1-exo, stenoparib (E7449), indirubin-3'-oxime, capmatinib (INC B28060), WAY-316606, iCRT3, FH535, IWP-O1, LF3, prodigiosin, KY19382 (A3051), WIKI4, heparan sulfate, fossenvivint (ICG-001), triptonide, XAV-939, IWP-4, LGK-974, Foxy-5, MSAB, raduviglusib (CHIR-99021) HCl, KY-05009, KY1220, IQ-1, KYA1797K, harmine, G244-LM, KY02111, JW55, PH-064, and raduviglusib (CHIR-99021).

[0017] In a preferred embodiment, the first agent comprises SB431542, XAV-939, and dorsomorphin.

[0018] In one embodiment, the method further comprises culturing the obtained EBs in a second medium containing a second factor, for example, for a suitable period of time, for example, 7-14 days, 8-13 days, 9-12 days, or 10-11 days. The second factor may comprise a patterning factor for Sonic Hedgehog pathway activation, such as a smoothened receptor agonist, for example, smoothened agonist (SAG) and purmorphamine. The culturing may be performed in the presence of activin A. In a preferred embodiment, the second factor comprises XAV-939, activin A, SAG, and purmorphamine. The culturing is preferably performed without the first factor. This culturing process may allow the EBs to modulate differentiation into the lateral ganglionic eminence (LGE), thereby giving rise to the striatum during development.

[0019] In one embodiment, the method further comprises culturing the obtained EB in a fourth medium containing a third factor to obtain organoids such as striatum-like organoids (e.g., hSLO).The third factor can contain brain-derived neurotrophic factor (BDNF) and / or ascorbic acid.This culturing is preferably carried out without the first and second factors.

[0020] In one embodiment, striatum-like organoid (for example, hSLO) preferably has a diameter of 500 μm or more, 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, 1000 μm or more, 1100 μm or more, 1200 μm or more, or 1300 μm or more.Striatum-like organoid (for example, hSLO) comprises both D1 subtype and D2 subtype GABAergic medium spiny neuron (MSN), such as D1 GABAergic MSN expressing dopamine receptor D1 (DRD1) and substance-P, and D2 GABAergic MSN expressing DRD2 and enkephalin. In one embodiment, striatal-like organoid (e.g., hSLO) can express one or more markers for lateral ganglionic eminence (LGE) (LGE markers), such as ASCL1, DLX2, GSX2, and EBF1. In one embodiment, striatal-like organoid (e.g., hSLO) can further express one or more early neuroectoderm markers, such as SOX1 and SOX2. In one embodiment, striatal-like organoid (e.g., hSLO) preferably expresses one or more markers of mature medium spiny neuron (MSN), such as DARPP32. In one embodiment, striatal-like organoid (e.g., hSLO) preferably expresses one or more markers selected from the group consisting of interneuron markers, such as TH, cholinergic neuron markers, such as CHAT and 5-HT, and serotonin neuron markers, glial cell markers, such as MBP and GFAP. In a preferred embodiment, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of striatal-like organoid (e.g., hSLO) can be GABAergic neurons. In one embodiment, GABAergic neurons express GABA and COUP-TF interacting protein 2 (CTIP2). In a preferred embodiment, striatal-like organoid (e.g., hSLO) can have one or more axonal collaterals and growth cones at axon terminals, and dendritic projection spines. The present disclosure provides any of these striatal-like organoid (e.g., hSLO).

[0021] The present disclosure provides a method for producing organoids. The method may include providing a first organoid and a second organoid. The first organoid is a striatum-like organoid (e.g., hSLO), and the second organoid comprises dopaminergic neurons (DA), such as midbrain dopaminergic (mDA) neurons. In a preferred embodiment, the mDA neurons are selected from the group consisting of A9-like subtype mDA neurons and A10-like subtype mDA neurons. In a preferred embodiment, the second organoid preferably expresses one or more of dopamine neuron markers, such as FOXA2, LMX1A, OTX2;TH, DAT, and GIRK2. In a preferred embodiment, the second organoid is a midbrain-like organoid (MLO), more preferably a human MLO (hMLO). In a preferred embodiment, the organoid can be obtained by contacting and fusing a striatum-like organoid (SLO) with a midbrain-like organoid (MLO).

[0022] In all of the embodiments, the SLO is preferably hSLO and the MLO is preferably hMLO.

[0023] In one embodiment, organoid or fusion SLO and MLO comprises the projection from SLO to MLO.In one embodiment, organoid or fusion SLO and MLO comprises the projection from MLO to SLO.In a preferred embodiment, organoid or fusion SLO and MLO comprises the mutual projection between SLO and MLO.In a preferred embodiment, projection can form neural circuit, more preferably electrophysiologically functional neural circuit.

[0024] In one embodiment, the neuron of MLO, preferably mDNA neuron, expresses alpha-synuclein (alpha-syn) and preferably shows alpha-syn aggregation. In this embodiment, the neuron, preferably mDNA neuron, may comprise a gene (SNCA) encoding alpha-syn, operably linked to a control sequence, such as a promoter (e.g., PolII promoter). The present disclosure provides organoid or fusion SLO and MLO, where the neuron of MLO expresses alpha-synuclein (alpha-syn), and preferably the organoid or fusion SLO and MLO show detectable alpha-syn aggregation.

[0025] The present disclosure provides a method for testing or screening candidate drugs for treating Parkinson's disease (PD).The method includes providing organoid or fusion SLO and MLO.The method includes contacting candidate drugs with organoid or fusion SLO and MLO, and then observing α-synuclein (particularly the presence or absence of α-synuclein aggregation or the degree of α-synuclein aggregation) in organoid or fusion SLO and MLO (preferably mDA neuron).The method further includes selecting the candidate drug that reduces α-synuclein aggregation compared to negative control such as vehicle treatment group.

[0026] In all of the embodiments, the culture is performed in a suitable medium under suitable conditions. In one embodiment, the medium may be a serum-free medium. In one embodiment, the medium may be a chemically defined composition medium in which all chemicals used are known. Examples of culture media that can be used herein include, but are not limited to, Eagle's Minimum Essential Medium (EMEM), alpha Minimum Essential Medium (aMEM), Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12), Roswell Park Memorial Institute (RPMI or RPMI 1640), Glasgow Minimum Essential Medium (GMEM), Biggers, Gwatkin, and Judah Medium (BGJ), Biggers, Gwatkin, and Judah Medium Fitton-Jackson Modification (BGJb), basal Examples of such a medium include Medium Eagle (BME), Brinstar Oocyte Culture Medium (BMOC-3), Connaught Medical Laboratory Medium (CMRL), Neurobasal Medium, CO2-independent medium, Ham's F-10 Nutrient Mixture, Ham's F-12 Nutrient Mixture, Improved MEM, Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Leibovitz's L-15, McCoy's 5A, MCDB131, Medium 199, mTeSR Medium, Minimum Essential Medium (MEM), Modified Eagle's Medium (MEM), Waymouth's MB752 / 1, Williams' Medium E, or combinations, known alternatives, or modifications thereof. Typically, minimal media include a carbon source, such as glucose; salts; essential elements, such as magnesium, nitrogen, phosphorus, and sulfur; and water. Any cell culture medium may be supplemented with additional components as needed based on the experiment to be performed, the cell type in question, and the cell condition required. Cell culture supplements include, but are not limited to, serum, amino acids (e.g., L-glutamine), chemical compounds, salts, buffer salts or buffers, antibiotics, antimycotics, cytokines, growth factors, hormones, lipids, and derivatives thereof. Culture can typically be performed at 37° C. under 5% CO2 conditions.

[0027] In all of the embodiments, each of the media contains a suitable amount of each of the first, second, and third factors. EXAMPLES

[0028] 1. Protocol Establishment and Characterization of hSLO Similar to the approach we used previously to generate hMLOs ( Jo et al., 2016 ), we applied several small molecules to promote neuroectodermal differentiation into the dorsal striatum ( Figure 1 A). First, hESCs were dissociated to generate single cells, and 10,000 cells were seeded into V-bottom 96-well plates to form embryoid bodies (EBs). On day 1, these EBs were cultured in neural induction medium containing DMEM / F12 (Nacalai):Neurabasal (Gibco) (1:1) supplemented with dual SMAD inhibitors (SB431542, 10 μM, Stemolecule, and Dorsomorphin, 2 μM, Sigma-Aldrich) along with a WNT inhibitor (XAV939, 0.8 μM, StemCell Technologies), 1:100 N2 supplement (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% Minimal Essential Medium Non-Essential Amino Acids (Invitrogen), 0.1% β-mercaptoethanol (Invitrogen). On day 7, patterning factors including SAG (0.5 μM, StemCell Technologies) and purmorphamine (0.5 μM, Stemolecule) were added to the medium to modulate differentiation into the lateral ganglionic eminence (LGE), thereby giving rise to the striatum during development (Figure 1B). Importantly, we include activin A (50 ng / ml, Gibco) to promote differentiation of striatal neurons by LGE patterning, as previously reported (Arber et al., 2015). EBs were transferred to an orbital shaker from day 7 onwards. On day 14, patterning factors were removed and organoids were maintained in neural medium supplemented with BDNF (Peprotech, 10 ng / ml) and ascorbic acid (Sigma-Aldrich, 100 μM) (Figure 1B). Development was tracked across multiple batches, and these organoids grew to a diameter of 1.3 mm by day 60 of differentiation (Figure 1C). Examination of organoid development by qRT-PCR analysis showed decreased expression of pluripotency markers, such as NANOG, OCT4, and robust expression of early LGE markers, such as ASCL1, DLX5, GSX2, and EBF1 (Figure 2).

[0029] It is known that the human striatum is composed of more than 90% GABAergic MSNs. Based on their axonal projection formation ability and neurochemical contents, MSNs can be divided into two semi-subpopulations: dopamine receptor D1 (DRD1) and substance P expressing MSNs and DRD2 and enkephalin expressing MSNs (Graveland and DiFiglia, 1985). Previous studies have shown that D1 MSNs send outputs to the internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr) and form the striatal-nigral pathway directly to the basal ganglia. Meanwhile, the striatum receives dopaminergic input from the substantia nigra pars compacta (SNpc) of the nigro-striatal pathway, and dopaminergic neurons from the SNpc projection form projections to the striatum and release dopamine from their axon terminals to affect GABAergic MSNs located in the striatum (Yager et al., 2015). This neural circuit also plays an important role in locomotion. Dysfunction of the nigro-striatal and striatal-nigral pathways has been published to be the cause of several neurological disorders, including PD ( Goto et al., 1989 ).

[0030] Here, by labeling neurons in our hSLO with CTIP2 and GABA, we conclude that at day 60, 64% of GABAergic neurons are also positive for CTIP2 (Figure 3). Previous studies have shown that the homeobox gene GSX2 is important for LGE fate determination and striatal development (Hsieh-Li et al., 1995; Toresson et al., 2000; Yun et al., 2001). By labeling day 30 hSLO cryosections with GSX2 and the ventral forebrain marker DLX2, we show that GSX2 + We observed that the majority of cells (83%) also expressed DLX2. Further characterization of mature hSLOs at day 60 revealed that the striatal marker COUP-TF interacting protein 2 (CTIP2) was upregulated by GABA +Finally, immunostaining of hSLO cryosections at day 90 confirmed the expression of DARPP32, a marker for mature MSNs, with 90% of cells expressing DARPP32. + / GABA + More interestingly, immunostaining of hSLO neurons with substance P and DRD2 antibodies revealed that GABA + A specific population of neurons was identified that expresses these two markers individually (Figure 5). This feature is of great practical importance since currently there are no 2D or 3D human striatal cultures that show the generation of human striatum with a high percentage of D1 and D2 GABAergic neurons.

[0031] Various studies have concluded that, in addition to MSNs, the human striatum contains a small population of supporting cells to promote neuronal survival and maintain synaptogenesis (Huot et al., 2007). We found that cells in our hSLO expressed TH (a dopaminergic neuron marker), CHAT and 5-HT (cholinergic and serotonin neuron markers), MBP (oligoprojection glial cell marker), and GFAP (astrocyte cell marker) (Figure 6).

[0032] To further characterize the morphological features of MSNs, including dendritic projection spines and growth cones as projection neurons, we labeled the organoids with AAV viruses expressing an EGFP reporter under DLX5 and DLX6 enhancers (AAV-mDlx::EGFP) and reseeded them to promote axonal outgrowth of MSNs. As a result, we found that at day 80, the EGFP-labeled MSNs were labeled with the CTIP2 marker. + We observed a population of cells expressing EGFP by further culturing the neurons for 130–150 days (Figure 7A). +We found that neurons grew into more complex morphological features with numerous axonal branches. More interestingly, we captured the formation of growth cones at the axon terminals as well as dendritic projection spines (Figure 7B), indicating that neurons were mature and active in synapsing with other neurons.

[0033] Calcium imaging allows us to monitor the electrophysiological activity of individual neurons in brain organoids. For this purpose, we performed Fluo-4 acetoxymethyl ester (AM)-based calcium imaging. When hSLO was incubated with Fluo-4 AM, significant spontaneous Ca 2+ This resulted in labeled cells showing transients. Recorded activity was analyzed using ImageJ software, which showed single neuronal spikes in hSLO at day 150 (Figure 8). Because calcium activity in hSLO can be measured using confocal microscopy, measurement of calcium imaging of fused organoids may be a promising assay that allows direct evaluation of the function of fused organoids in PD models.

[0034] All these results suggest that our generated hSLO can generate MSN GABAergic neurons with multiple features similar to those in the human striatum.More importantly, we are analyzing bulk and single-cell RNA-seq from hSLO to further understand the transcriptome characterization and cell type composition of hSLO, and to compare hSLO neurons with neurons derived from previously published protocols.

[0035] 2. Fusion of hSLO and hMLO recapitulates reciprocal projections We expected that the projected neurons of the hMLO-hSLO fusion organoids would be able to form better functional synaptic connections. Therefore, we hypothesized that the more active Ca2+ neurons in the fusion organoids would be able to form better functional synaptic connections. 2+We aim to record the activity of hMLO and hSLO. Reciprocal projections between the substantia nigra of the midbrain and the striatum, a specific region of the basal ganglia, arise during neurogenesis (Figure 9A). To efficiently visualize the formation of neural projections, we constructed reporter systems in hMLO and hSLO. We used CRISPR / Cas9 technology to knock in the EGFP fluorescent protein, which is specifically expressed in DA neurons of hMLO, into the 3'-TH locus. Meanwhile, we used an AAV infection system carrying hSyn1::EGFP (AAV-hSyn1::EGFP) to label GABAergic MSNs in hSLO. To establish such projections in vitro, we placed hSLO and hMLO next to each other in a 24-well plate to promote their direct physical contact and fuse them together. Two days after fusion, the organoids were returned to the orbital shaker and cultured for another two weeks.

[0036] To demonstrate the formation of striatonigral projections, we fused hSyn1::EGFP-infected hSLOs with unlabeled hMLOs. Interestingly, we began to observe projection formation 3 days after fusion. Two weeks after fusion, we observed EGFP-infected hSLOs reaching the opposite side of hMLOs and forming axon bundles. + We found robust outgrowth of processes (Figure 9B), a similar anatomical feature in the human brain (Morello et al., 2015). Similarly, to investigate the ability of hMLO to form projections to hSLO, we fused the TH-EGFP reporter hMLO with unlabeled hSLO. As expected, we observed EGFP outgrowth of the hSLO side 2 weeks after fusion. + We detected EGFP-labeled processes in both fused organoids (Figure 9C). With longer culture, we saw increased intensity of EGFP-labeled projection signals from both fused organoids. Collectively, these results indicated the existence of reciprocal projections between hSLO and hMLO, suggesting that the fused organoids are anatomical replicas of human neural circuits.

[0037] We further carried out a series of assays to investigate the synaptic properties of the fused organoids. First, to determine whether projection neurons can direct axon targeting and synapse formation in the fused organoids, we fused the TH-EGFP reporter hMLO with unlabeled hSLO, and then we labeled projection neurons with presynaptic markers at the projection axon terminals to confirm the establishment of synaptic connections in projection neurons. As expected, we found that TH-EGFP on the hSLO side was strongly correlated with the synaptic properties of the projection neurons. + Together with the projection-forming neurons, we observe expression of the SYN1 presynaptic marker and the PSD95 postsynaptic marker. + We observed expression of vesicular monoamine transporter 2 (VMAT2) in the projection neurons and local GABAergic neurons (Figure ​(Figure10). 10). In addition, we expressed AAV-hSyn1::EGFP in the hMLO side. + Together with the projection-forming neurons, we observe expression of the SYN1 presynaptic marker and the PSD95 postsynaptic marker. + We observed expression of vesicular GABA transporter (VGAT) in projection-forming neurons and local GABAergic neurons, indicating synapse formation between projection-forming DA neurons and GABAergic neurons and local GABAergic neurons.

[0038] We can then use calcium imaging, MEAs, and electrophysiological recordings, including whole-cell patch clamp, to investigate the functional maturation and formation of neural circuits in the fused organoids. We predicted that projection-forming neurons would be able to form better synapses with their counterparts when fused, something that would not happen in each organoid alone.

[0039] To demonstrate that it is possible to model α-syn pathology using our fusion organoid system, we compare hMLO-hSLO fusion organoids between WT and SNCA overexpressing (Figure 11A). +By labeling DA neurons, we showed that elevated α-Syn expression restricted the projection formation of DA neurons from hMLO to hSLO. Interestingly, WT DA neurons formed projections as bundles, whereas DA neurons in SNCA-overexpressing organoids showed fewer random projections. Next, we used a lentiviral system to overexpress the mKO2 reporter (red fluorescent protein) fused with α-syn (Figures 11B and 11C). This reporter system allowed us to visualize the aggregation of α-syn as well as monitor the effect of α-syn propagation in hMLO-hSLO fusion organoids. By performing whole-mount staining using mKO2 antibody, we observe that the propagation level of α-Syn-mKO2 from hSLO to hMLO is significantly higher, but not vice versa, with a nearly four-fold difference (Figures 11C-11D). These data strongly suggest that the SNCA-mKO2 reporter is a viable system to investigate α-syn propagation between the striatum and substantia nigra and the formation of α-syn aggregates using our hMLO-hSLO fusion organoid model.

[0040] 3. Application of fused organoids to drug screening and testing for synucleinopathies The main aim of using the hMLO-hSLO fusion organoid model is to develop a system that will make revolutionary changes to study PD pathogenesis in the reciprocal projection between the midbrain nigra and the striatum, focusing specifically on synuclein pathology. We aim to use the fusion organoid in vitro system to test the effects and impacts of anti-PD drug compounds in clinical trials. Using an isogenic PD hESC line with a TH-EGFP reporter system established in our laboratory (Jo et al., 2021), we generate fusion organoids that display PD phenotypes (Figure 12). First, we will test drugs (e.g., compounds, nucleic acids, or antibodies) or candidate drugs in phase I clinical trials (NPT200-11 and NPT088: inhibitors of α-syn misfolding) and phase II clinical trials (SAR402671: inhibitors of glucosylceramide synthase, and ambroxol: GCase activator) for their rescue effects on α-syn pathology. We will then measure the degree of α-syn aggregation at different time points that represent different degrees of PD phenotype and apply drugs at optimized concentrations to profile the long-term, direct drug response to the fused organoids. + Assessment of amelioration of pathological α-syn levels in DA neurons will be validated by various techniques including multi-omics analysis, imaging, biochemistry, electrical characterization, metabolic assays, and synaptogenic capacity in both physical and functional modes. We anticipate an outcome of reduced α-syn accumulation associated with reduced neurodegeneration following drug treatment.

[0041] Parkinson's disease (PD) is the second most common neurodegenerative disorder and is manifested by the degeneration of dopaminergic (DA) neurons. Dopaminergic (DA) neurons normally form projections from the midbrain to the striatum in the nigro-striatal pathway. Constructing an in vitro system to model neurological diseases is challenging but an achievable goal that numerous research groups are attempting. Herein, we developed a detailed protocol to produce specific human striatum-like organoids (hSLOs) that have characteristics similar to the human striatum, such as the presence of D1 and D2 subtype GABAergic medium spiny neurons (MSNs). By fusing hSLOs with our previously generated midbrain-like organoids (hMLOs), we provide in vitro evidence of connections and communication between the midbrain and the striatum of the basal ganglia. Finally, we provide evidence that our fused organoid system is a suitable drug screening platform for Parkinson's disease, based on alpha-synuclein (α-syn) propagation. This finding represents the first attempt based on observing both structural and functional interactions between hSLO and hMLO that could revolutionize in vitro neurodegenerative disease modeling, particularly synucleinopathies.

[0042] Each of the references and patents and patent applications cited herein is incorporated by reference in its entirety. References Albin, RL, Young, AB, and Penney, JB (1989). The functional anatomy of basal ganglia disorders. Trends Neurosci 12, 366-375. Arber, C., Precious, S.V., Cambray, S., Risner-Janiczek, J.R., Kelly, C., Noakes, Z., Fjodorova, M., Heuer, A., Ungless, M.A., Rodriguez, T.A., et al. (2015). Activin A directs striatal projection neuron differentiation of human pluripotent stem cells. Development 142, 1375-1386. Gerfen, C.R. (2006). Indirect-pathway neurons lose their spines in Parkinson disease. Nat Neurosci 9, 157-158. Goto, S., Hirano, A., and Matsumoto, S. (1989). Subdivisional involvement of nigrostriatal loop in idiopathic Parkinson's disease and striatonigral degeneration. Ann Neurol 26, 766-770. Graveland, G.A., and DiFiglia, M. (1985). The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum. Brain Res 327, 307-311. Hikosaka, O., Takikawa, Y., and Kawagoe, R. (2000). Role of the basal ganglia in the control of purposive saccadic eye movements. Physiol Rev 80, 953-978. Hsieh-Li, H.M., Witte, D.P., Szucsik, J.C., Weinstein, M., Li, H., and Potter, S.S. (1995). Gsh-2, a murine homeobox gene expressed in the developing brain. Mech Dev 50, 177-186. Huot, P., Levesque, M., and Parent, A. (2007). The fate of striatal dopaminergic neurons in Parkinson's disease and Huntington's chorea. Brain 130, 222-232. Ingham, C.A., Hood, S.H., Taggart, P., and Arbuthnott, G.W. (1998). Plasticity of synapses in the rat neostriatum after unilateral lesion of the nigrostriatal dopaminergic pathway. J Neurosci 18, 4732-4743. Jo, J., Xiao, Y., Sun, A.X., Cukuroglu, E., Tran, H.D., Goke, J., Tan, Z.Y., Saw, T.Y., Tan, C.P., Lokman, H., et al. (2016). Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell stem cell 19, 248-257. Jo, J., Yang, L., Tran, H.D., Yu, W., Sun, A.X., Chang, Y.Y., Jung, B.C., Lee, S.J., Saw, T.Y., Xiao, B., et al. (2021). Lewy Body-like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and alpha-Synuclein Mutations. Ann Neurol. Lovinger, D.M. (2010). Neurotransmitter roles in synaptic modulation, plasticity and learning in the dorsal striatum. Neuropharmacology 58, 951-961. Macpherson, T., Morita, M., and Hikida, T. (2014). Striatal direct and indirect pathways control decision-making behavior. Front Psychol 5, 1301. Morello, F., Prasad, A.A., Rehberg, K., Vieira de Sa, R., Anton-Bolanos, N., Leyva-Diaz, E., Adolfs, Y., Tissir, F., Lopez-Bendito, G., and Pasterkamp, R.J. (2015). Frizzled3 Controls Axonal Polarity and Intermediate Target Entry during Striatal Pathway Development. J Neurosci 35, 14205-14219. Toresson, H., Potter, S.S., and Campbell, K. (2000). Genetic control of dorsal-ventral identity in the telencephalon: opposing roles for Pax6 and Gsh2. Development 127, 4361-4371. Yager, L.M., Garcia, A.F., Wunsch, A.M., and Ferguson, S.M. (2015). The ins and outs of the striatum: role in drug addiction. Neuroscience 301, 529-541. Yun, K., Potter, S., and Rubenstein, J.L. (2001). Gsh2 and Pax6 play complementary roles in dorsoventral patterning of the mammalian telencephalon. Development 128, 193-205.

Claims

1. A method for culturing embryoid bodies (EBs), such as human embryoid bodies, The process involves culturing EB in a first medium containing a first factor, wherein the first factor includes a Wnt inhibitor such as a TGF-β signaling pathway inhibitor and / or a GSK inhibitor. method.

2. The method according to claim 1, further comprising culturing the obtained EB in a second medium containing a second factor, wherein the second factor comprises a Wnt inhibitor, a pattern-forming factor for sonic hedgehog pathway activation, and activin, for obtaining LGE neurospheres, and does not contain a first factor.

3. The method according to claim 2, comprising culturing the obtained EB in a third medium containing a third factor, wherein the third factor contains brain-derived neurotrophic factor (BDNF) and / or ascorbic acid for obtaining organoids such as human striatum-like organoids, and does not contain the first and second factors.

4. The method according to claim 3, wherein the obtained organoid contains one or more markers of a mature medium spiny neuron (MSN).

5. The method according to claim 4, wherein the obtained organoid comprises D1 and / or D2 MSN.

6. The method according to claim 5, wherein the obtained organoid expresses at least one or all of the following: interneuron markers such as TH, cholinergic neuron markers such as CHAT and 5-HT, serotonin neuron markers, and glial cell markers such as MBP and GFAP.

7. The method according to claim 4, wherein the organoid has a major axis diameter or diameter of 1 mm or more, preferably a major axis diameter or diameter of 1 mm to 2 mm.

8. Isolated organoids containing mature medium spiny neurons (MSNs) expressing one or more markers of mature MSNs.

9. The isolated organoid according to claim 8, wherein more than 50% of the cells contained in the organoid are DARPP32-positive and GABA-positive.

10. The isolated organoid according to claim 8, comprising D1 and / or D2 MSNs.

11. An isolated organoid according to claim 8, expressing at least one or all of the interneuron markers, cholinergic neuron markers, and serotonin neuron markers.

12. An isolated organoid according to claim 8, having a major axis diameter or diameter of 1 mm or more.

13. A method for producing organoids, To provide the organoid described in claim 8 (first organoid), and a second organoid including a dopaminergic (DA) neuron, and A method comprising bringing the first organoid and the second organoid into contact to obtain a fused organoid of the first organoid and the second organoid, wherein the dopaminergic neurons of the second organoid have projections to the first organoid, and the GABAergic MSNs of the first organoid have projections to the second organoid.

14. A fusion organoid comprising the first organoid described in Claim 8 and a second organoid including dopaminergic (DA) neurons, A fused organoid in which the dopaminergic neurons of the second organoid have projections to the first organoid, and the GABAergic MSNs of the first organoid have projections to the second organoid.

15. The fusion organoid according to claim 14, wherein the cells contained in the fusion organoid include DA neurons having α-synchn aggregation.

16. A method for testing candidate drugs, The candidate drug and the fusion organoid described in claim 14 are brought into contact, Observing α-synuclein aggregation in DA neurons, and Selecting candidate drugs that reduce α-synchn aggregation compared to a negative control. A method that includes this.

17. The method according to claim 16, wherein SNCA is overexpressed in a second organoid entity or in at least DA neurons or all cells of the organoid.

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