Patent Application
20240393320
The disclosure relates to striatal-like organoids (SLO) and a method of producing the SLOs. The disclosure also relates to a functionally fused striatal-like organoid (SLO) and midbrain-like organoid (MLO). The disclosure also relates to human striatal-like organoids (hSLO) and a method of producing the hSLOs. The disclosure also relates to a functionally fused human striatal-like organoid (hSLO) and human midbrain-like organoid (hMLO).
The striatum is a component of basal ganglia, located in the central human brain, that have a variety of functions including voluntary movement (Hikosaka et al., 2000). It receives and sends out information from and to different brain regions mostly through the projection of specific neurons (Gerfen, 2006; Ingham et al., 1998; Lovinger, 2010; Macpherson et al., 2014). Thus, the striatum is most frequently associated with movement, which is heavily affected by neurodegeneration in patients suffering from Parkinson's disease (PD).
Neural activity between striato-nigral GABAergic neurons in striatum and nigro-striatal dopaminergic (DA) neurons in midbrain motivates the behavior and movement (Albin et al., 1989). Dysfunctions in these neural circuits subsequently contribute to pathogenesis of PD. However, it is poorly understood how these brain regions connect to each other and what causes functional defects in neuronal diseases. Current cell culture and animal models are criticized for their failure to accurately mimic the actual neural circuits in brain. Hence, creating an in vitro brain model to demonstrate and visualize human nigro-striatal pathways will provide a platform for future research on studying neuronal diseases, for example alpha-synuclein (α-syn) pathology.
3D organoid model systems are designed to resemble the in vivo organ or tissue from which they were derived. These 3D culture systems can reproduce the complex morphology of differentiated epithelium to enable biologically interaction between cell-cell and cell-matrix. This contrasts with the classical 2D culture models that often share little physical, molecular, or physiological similarity to their tissue of origin. Despite the tremendous promise for in vitro modelling, current brain organoid systems have certain limitations as it cannot reflect every aspect of human brain diseases such as PD.
The generation of human striatal organoids solves several problems:
In the disclosure, we first describe our method to generate human striatum-like organoid together with characterization data including gene expression analysis, immunohistochemistry analysis and calcium imaging. We next generate fusion organoids using hSLOs and hMLOs, which hMLO has been established by our lab previously, to demonstrate the physical and functional connection between midbrain and striatum with characterization of the reciprocal projections and synaptic formation.
The present disclosure provides, for example, an invention as described below.
(1) A method of culturing an embryoid body (EB) such as a human embryoid body, comprising:
(2) The method of (1) above, further comprising:
(3) The method of (2) above, further comprising:
(4) The method of (3) above, wherein the obtained organoid comprising one or more markers of a mature medium spiny neuron (MSN).
(5) The method of (4) above, wherein the obtained organoid comprising D1 and/or D2 MSN.
(6) The method of (5) above, wherein the obtained organoid expresses at least one or all of interneuron makers such as TH, cholinergic neuron and serotonin neuron markers such as CHAT and 5-HT, glial cell markers such as MBP and GFAP.
(7) The method of any one of (4) to (6) above, wherein the organoid has 1 mm or more in major axis diameter or in diameter, preferably 1 mm to 2 mm in major axis diameter or in diameter.
(8) An isolated organoid comprising: a mature medium spiny neuron (MSN) expressing one or more markers of a mature MSN.
(9) The isolated organoid of (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 of (8) or (9) above, comprising D1 and/or D2 GABAergic MSN.
(11) The isolated organoid of any one of (8) to (10) above, wherein the organoid expresses at least one or all of interneuron makers such as TH, cholinergic neuron and serotonin neuron markers such as CHAT and 5-HT, glial cell markers such as MBP, S100β, and GFAP.
(12) The isolated organoid of any one of (8) to (11) above, wherein the organoid has 1 mm or more in major axis diameter or in diameter, preferably 1 mm to 2 mm in major axis diameter or in diameter.
(13) A method of producing an organoid, comprising:
(14) A fusion organoid of the first organoid of any one of (8) to (12) above and the second organoid comprising a dopaminergic (DA) neuron, for example, A9-like subtype mDA neuron and A10-like subtype mDA neuron, wherein the organoid preferably expresses one or more of FOXA2, LMX1A, OTX2, dopamine neuron makers such as TH, DAT, and GIRK2,
(15) The fusion organoid of (14) above, wherein the cells included in the fusion organoid comprises a DA neuron having α-syn aggregation.
(16) A method of testing a candidate drug, comprising:
(17) The method of (16) above, wherein SNCA is overexpressed in at least a DA neuron or all cells in the second organoid entity or the organoid.
The term “embryoid body” (EB) as used herein refers to a three-dimensional aggregate of pluripotent cells or preferably pluripotent stem cells (PSCs). EBs are formed by pluripotent cells such as embryonic stem cells (ESCs) through the intramolecular binding of the Ca2+ dependent adhesion molecule E-cadherin expressed on the pluripotent cells. When cultured as single cells in the absence of antidifferentiation factors, PSCs spontaneously aggregate to form EBs. Such spontaneous formation is often accomplished in bulk suspension cultures whereby the dish is coated with non-adhesive materials, such as agar or hydrophilic polymers, to promote the preferential adhesion between single cells, rather than to the culture substrate.
The term “embryonic stem cells” as used herein refers to a pluripotent stem cell that can be derived from the inner cell mass of a blastocyst from animals, for example, mammals such as rodents including mouse and rat, primates including human and monkeys.
The term “pluripotent stem cell” as used herein refers to a stem cell having a pluripotency. Pluripotency is a potential of cells to differentiate into any of the three germ layers, including endoderm, mesoderm and ectoderm, but not into extraembryonic tissues like the placenta. A pluripotent stem cell can be artificially induced from a non-pluripotent cell such as an adult somatic cell, by inducing a forced expression of a combination of certain reprogramming factors. For example, a forced expression of Oct4, Sox2, Klf4 and c-Myc can generate an induced pluripotent stem cell (iPS cell) from a fibroblast and the like.
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 of organoids have a similar tissue structure to an original organ in a body. Organoids having a similar function to an organ can have a therapeutic effect to a disease caused by a reduced function of the organ. Organoids having a similar structure to an organ were generated to study a development of the organ.
The disclosure provides a method of culturing an embryoid body (EB). The EB is preferably an animal EB, more preferably a mammal EB, still more preferably a human EB. The EB can be obtained by culturing pluripotent stem cells such as ES cells or iPS cells as explained above.
In an embodiment, the method comprises culturing an EB in a first culture medium containing a first factor, for example, for one to seven days, two to six days, or three to five days. The first factor may comprise a TGF-β signaling pathway inhibitor and/or a Wnt inhibitor, preferably a TGF-β signaling pathway inhibitor and a Wnt inhibitor such as GSK inhibitor (e.g., GSK3 inhibitor or GSK3β inhibitor).
Examples of TGF-β signaling pathway inhibitors include, for example, but not limited to, (i) one or more selected from the group consisting of ALK4 inhibitor, ALK5 inhibitor, ALK7 inhibitor, SMAD inhibitor such as SMAD2/3 inhibitor including SMAD2/3 phosphorylation inhibitor, and a multiple inhibitor (e.g., a dual inhibitor) for two or more selected from the group consisting of ALK4, ALK5, ALK7, SMAD such as SMAD2/3 and SMAD2/3 phosphorylation preferably a dual inhibitor for TGF-β and SMAD2/3; (ii) one or more selected from the group consisting of LY364947, SB-525334, SD-208, and SB-505124; 616452 and 616453; GW788388 and GW6604; LY580276, which are disclosed in WO2015/002724A, which is herein incorporated by reference in its entirety; or (iii) SB-431542. In an embodiment, Examples of TGF-β signaling pathway inhibitors includes 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 uses as a TGF-β signaling pathway inhibitor.
Examples of Wnt inhibitors include, for example, but not limited to Adavivint (SM04690), IM-12, Lanatoside C, M435-1279, Wnt-C59 (C59), Atranorin, Box5, Isoquercitrin, AZD2858, CCT251545, PNU-74654, IWP-2, CP21R7 (CP21), IWR1-endo, Ginsenoside Rh4, FIDAS-3, Gigantol, AZ6102, IWR-1-exo, Stenoparib (E7449), Indirubin-3′-oxime, Capmatinib (INCB28060), WAY-316606, iCRT3, FH535, IWP-O1, LF3, Prodigiosin, KY19382 (A3051), WIKI4, Heparan Sulfate, Fosconvivint (ICG-001), Triptonide, XAV-939, IWP-4, LGK-974, Foxy-5, MSAB, Laduviglusib (CHIR-99021) HCl, KY-05009, KY1220, IQ-1, KYA1797K, Harminc, G244-LM, KY02111, JW55, PH-064, and Laduviglusib (CHIR-99021).
In a preferable embodiment, the first factor comprises SB431542, XAV-939, and Dorsomorphin.
In an embodiment, the method further comprises culturing the obtained EB in a second culture medium containing a second factor, for example, for a suitable period, for example, for seven to fourteen days, eight to thirteen days, nine to twelve days, or ten to eleven days. The second factor may comprise a patterning factors for sonic hedgehog pathway activation such as smoothened receptor agonists, for example, smoothened agonist (SAG) and purmorphamine. The culturing can be done in the presence of Activin A. In a preferable embodiment, the second factor comprises XAV939, Activin A, SAG, and purmorphamine. This culturing is preferably performed without the first factors. This culturing process may allow the EB to modulate the differentiation towards lateral ganglionic eminence (LGE), which give rise to the striatum during development.
In an embodiment, the method further comprises culturing the obtained EB in a fourth culture medium containing a third factor to obtain an organoid such as a striatal-like organoid (e.g., hSLO). The third factor may comprise brain-derived neurotrophic factor (BDNF) and/or ascorbic acid. This culturing is preferably performed without the first and second factors.
In an embodiment, the striatal-like organoid (e.g., hSLO) preferably has 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 in diameter. The striatal-like organoid (e.g., hSLO) comprises both of D1 and D2 subtypes GABAergic medium spiny neurons (MSNs) such as D1 GABAergic MSNs expressing dopamine receptor D1 (DRD1) and Substance-P, and D2 GABAergic MSNs expressing DRD2 and enkephalin. In an embodiment, the striatal-like organoid (e.g., hSLO) may express one or more markers for lateral ganglionic eminence (LGE) (LGE markers) such as ASCL1, DLX2, GSX2 and EBF1. In an embodiment, the striatal-like organoid (e.g., hSLO) may further expresses one or more early neuroectodermal markers such as SOX1, and SOX2. In an embodiment, the striatal-like organoid (e.g., hSLO) preferably expresses one or more markers of a mature medium spiny neuron (MSN) such as DARPP32. In an embodiment, the striatal-like organoid (e.g., hSLO) preferably expresses one or more markers selected from the group consisting of interneuron makers such as TH, cholinergic neuron and serotonin neuron markers such as CHAT and 5-HT, glial cell markers such as MBP and GFAP. In a preferable embodiment, at least 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the striatal-like organoid (e.g., hSLO) may be GABAergic neurons. In an embodiment, the GABAergic neurons express GABA and COUP-TF-interacting protein 2 (CTIP2). In a preferable embodiment, the striatal-like organoid (e.g., hSLO) may have one or more axonal branches and growth cones at the axon terminus as well as the formation of dendritic spines. The disclosure provides any of these striatal-like organoid (e.g., hSLO).
The disclosure provides a method of producing an organoid. The method may comprise providing a first organoid and a second organoid. The first organoid is a striatal-like organoid (e.g., hSLO), and the second organoid comprises a dopaminergic neuron (DA), for example, a midbrain dopaminergic (mDA) neuron. In a preferable embodiment, the mDA neuron is selected from the group consisting of A9-like subtype mDA neuron and A10-like subtype mDA neuron. In a preferable embodiment, the second organoid preferably expresses one or more of FOXA2, LMXIA, OTX2, dopamine neuron makers such as TH, DAT, and GIRK2. In a preferable embodiment, the second organoid is midbrain-like organoid (MLO), more preferably human MLO (hMLO). In a preferable embodiment, the organoid can be obtained by contacting and fusing a striatal-like organoid (SLO) with a midbrain-like organoid (MLO).
In all of the embodiments, SLO is preferably hSLO and MLO is preferably hMLO.
In an embodiment, the organoid or fused SLO and MLO comprises projections from SLO to MLO. In an embodiment, the organoid or fused SLO and MLO comprises projections from MLO to SLO. In a preferable embodiment, the organoid or fused SLO and MLO comprises reciprocal projections between SLO and MLO. In a preferable embodiment, the projections may form a neuronal circuit, more preferably an electro-physiologically functional neuronal circuit.
In an embodiment, neurons, preferably a mDNA neurons, in the MLO expresses α-synuclein (α-syn) and preferably has α-syn aggregation. In this embodiment, the neuron, preferably a mDNA neuron, may comprise a gene encoding α-syn (SNCA) operably linked to a control sequence such as a promoter (e.g., PoIII promoter). The disclosure provides the organoid or fused SLO and MLO, wherein the neuron in MLO expresses α-synuclein (α-syn), preferably wherein the organoid or fused SLO and MLO has a detectable α-syn aggregation.
The disclosure provides a method of testing a candidate drug or screening a candidate drug for Parkinson's disease (PD) therapy. The method comprises providing the organoid or fused SLO and MLO. The method comprises contacting the candidate drug with the organoid or fused 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 the organoid or fused SLO and MLO (preferably mDA neurons). The method further comprises selecting the candidate drug that decreases α-synuclein aggregation, compared to a negative control such as a vehicle-treated group.
In all of the embodiments, culturing is performed under suitable conditions in a suitable culture medium. In an embodiment, the culture medium may be a serum-free medium. In an embodiment, the culture medium may be a chemically defined medium, where all of the chemicals used are known. Examples of culture medium that can be used herein include, but not limited to Eagle's minimal essential medium (EMEM), alpha minimum essential medium (aMEM), Dulbecco's modified Eagle's medium (DMEM), Dulbecco's Modified Eagle medium/Nutrient Mixture F-12 (DMEM/F-12) Roswell Park Memorial Institute medium (RPMI or RPMI 1640), Glasgow's Minimal Essential Medium (GMEM), Biggers, Gwatkin, and Judah medium (BGJ), Biggers, Gwatkin, and Judah medium Fitton-Jackson modification (BGJb), Basal Medium Eagle (BME), Brinster's medium for ovum culture (BMOC-3), Connaught Medical Research Laboratories 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, MCDB 131, Media 199, mTeSR media, Minimum Essential Media (MEM), Modified Eagle Medium (MEM), Waymouth's MB 752/1, Williams' Media E, or combinations, known substitutions or modifications thereof. Minimal medium typically contains a carbon source such as glucose; salts; essential elements such as magnesium, nitrogen, phosphorus, and sulphur; and water. Any cell culture media may be supplemented with further components, as and when required based on the experiment to be performed, the cell type in questions, as well as the required status of the cell. Cell culture supplements are, but are not limited to, serum, amino acids (e.g., L-glutamine), chemical compounds, salts, buffering salts or agents, antibiotics, antimycotics, cytokines, growth factors, hormones, lipids, and derivatives thereof. The culture can typically be performed under 5% CO2 conditions at 37° C.
In all of the embodiments, a suitable amount of each of the first, second, and third factors is contained in each of the medium.
Establishment of the Protocol and Characterization of hSLOs
Similar to an approach we previously used to generate hMLOs (Jo et al., 2016), we applied several small molecules to promote neuroectodermal differentiation toward dorsal striatum (
It is known that human striatum consists of over 90% GABAergic MSNs. MSNs can be divided into two half subpopulations based on their axonal projection capacity and neurochemical content: dopamine receptor D1 (DRD1)—and Substance-P-expressing MSNs whereas DRD2—and enkephalin-expressing MSNs (Graveland and DiFiglia, 1985). Previous studies have shown that D1 MSNs send output to the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr), forming direct striato-nigral pathway in basal ganglia. On the other hand, striatum receives dopaminergic input from the substantia nigra pars compacta (SNpc) in nigro-striatal pathway in which dopaminergic neurons from SNpc project to striatum and release dopamine from its axon terminal to influence GABAergic MSNs located in the striatum (Yager et al., 2015). This neural circuit also plays an important role in movement. Dysfunction of the nigro-striatal and striato-nigral pathway has been documented as the cause of multiple neuronal diseases such as PD (Goto et al., 1989).
Here, by labeling our neurons in hSLOs with CTIP2 and GABA, we conclude that there are 64% of GABAergic neurons are also positive with CTIP2 day 60 (
Different studies concluded that besides MSNs, human striatum contains a small population of supporting cells to promote neuronal survival as well as to maintain synaptic formation (Huot et al., 2007). We found that cells in our hSLO express TH (dopaminergic neuron makers), CHAT and 5-HT (Cholinergic neurons and serotonin neuron markers), MBP (oligodendrocyte markers) as well as GFAP (astrocyte markers) (
To further characterize the morphology of MSN including dendritic spines and growth cones as projection neurons, we labeled organoids with an AAV virus expressing EGFP reporter under DLX5 and DLX6 enhancer (AAV-mDlx::EGFP) and replated them to promote axonal outgrowth of MSNs. As a result, we observed a population of EGFP+ cell that are labeled with CTIP2 markers at day 80 (
Calcium imaging allows us to monitor the electrophysiological activity of individual neurons in brain organoids. For that purpose, we performed Fluo-4 acetoxymethyl ester (AM)-based calcium imaging. Incubating hSLOs with Fluo-4 AM resulted in labeled cells with prominent spontaneous Ca2+ transients. The recorded activity was analyzed using ImageJ software showed the spiking of single neurons of day 150 hSLOs (
All these results suggest that the hSLOs we generated can produce MSN GABAergic neurons with multiple features that resemble to the human striatum. More importantly, we are analyzing bulk and single cell RNA sequencing data from hSLOs to further understand the transcriptomic characterization and the composition of cell types in hSLOs, as well as to compare the neurons in hSLOs with those derived from the previous published protocols.
Fusion of hSLOs and hMLOs Recapitulates Reciprocal Projections
We expected that the projected neurons in hMLO-hSLO fusion organoids can form a better functional synaptic connection and hence, we aim to record more active Ca2+ activity in fusion organoids. Reciprocal projections between midbrain substantia nigra and striatum where is a specific region of basal ganglia arise during neural development (
To demonstrate the striato-nigral projection, we fused hSyn1::EGFP infected hSLOs with no-labeled hMLOs. Interestingly, from 3 days post fusion we started to observe the projection. Two weeks after fusion, we found a robust extention of EGFP+ processes from hSLO reaching the opposite side of hMLOs and forming axonal bundles (
We further performed a series of assays to investigate the synaptic properties of fusion organoids. First, to determine whether projected neurons can direct axonal targeting and synaptogenesis in the fusion organoids, we fused TH-EGFP reporter hMLOs with no-labeled hSLO, we then labeled projected neurons together with pre-synaptic markers at the projected axonal terminal to convince the establishment of synaptic connectivity in the projected neuron. As expected, we observe the expression of SYN1 pre-synaptic marker and PSD95 post-synaptic marker along with the THEGFP+ projected neurons on hSLO side. Moreover, we observed the expression of vesicular monoamine transporter 2 (VMAT2) along with the TH-EGFP+ projected neurons and local GABAergic neurons (
Next, electrophysiological recording including calcium imaging, MEA, and whole cell patch clamp can be used to investigate the functional maturation and formation of neuronal circuit in fusion organoids. We expected that projected neurons can form better synapses with their counterparts when fusing, which is missing in each organoid alone.
To demonstrate the possibility of using our fusion organoid system to model the asyn pathologies, we compare hMLO-hSLO fusion organoids between WT and SNCA overexpression (
The main aim of utilizing hMLO-hSLO fusion organoid model is to develop a system that revolutionize to study pathogenesis of PD in reciprocal projections between midbrain substantia nigra and striatum, particularly focusing on synuclein pathology. We aim to test the effect and impact of anti-PD drug compounds in clinical trials using the fusion organoid in vitro system. Utilizing the isogenic PD hESC lines (Jo et al., 2021) with TH-EGFP reporter system that have been established in our lab, we generate fusion organoids demonstrating PD phenotypes (
Parkinson's disease (PD) is the second most common neurodegenerative disorder, demonstrated by the degeneration of dopaminergic (DA) neurons, which normally project from midbrain to striatum in nigro-striatal pathway. Building an in vitro system to model neuronal diseases is challenging but is an achievable goal that many research groups attempt. Here, we developed a detailed protocol to produce specific human striatal-like organoid (hSLOs) with features similar to human striatum, such as the presence of D1 and D2 subtypes GABAergic medium spiny neurons (MSNs). By fusing hSLOs with our previously generated midbrain-like organoids (hMLOs), we provide in vitro evidence of connection and communication between midbrain and striatum in basal ganglia. Finally, we provide evidence that our fusion organoid system is a suitable drug screening platform against Parkinson's disease based on α-synuclein (α-syn) propagation. This finding represents the first attempt that can revolutionize in vitro neurodegenerative disease modeling, especially synucleinopathy, based on looking at both structural and functional interaction between hSLOs and hMLOs.
Each of the references and patents and patent applications cited herein has been incorporated by reference in its entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-152986 | Sep 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/035166 | 9/21/2022 | WO |
