Use of induced pluripotent stem cell-derived midbrain organoids

Abstract

The present invention relates to the use of midbrain organoids in the preparation of a medicament for treating Parkinson's disease, wherein the midbrain organoids are derived from pluripotent stem cells and do not contain serotonergic neurons. The present invention discovers for the first time the therapeutic effect of midbrain organoids on Parkinson's disease, which has advantages such as high compatibility compared to the transplanted cells obtained by two-dimensional culture of iPSCs, and has advantages such as no ethical obstacles, sufficient sources, homogeneity, and no pollution compared to the grafts isolated from fetal ventral midbrain tissue.

Description

Technical Field

[0001] The present invention relates to the fields of neurobiology and stem cells, and particularly relates to the use of induced pluripotent stem cell-derived midbrain organoids. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease, the pathology of which is the degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Current clinical practices for the treatment of PD include the administration of L-DOPA (levodopa) and / or surgical deep brain stimulation. However, these two treatments mainly focus on temporarily relieving clinical symptoms and have no effect on the pathological progression of Parkinson's disease.

[0003] Advances in cell therapy have brought new hope for the treatment of PD. In cell therapy, the loss of dopaminergic neurons and the damaged nigrostriatal pathway can be partially restored by supplementing tissues from the ventral midbrain (fVM) region of allogeneic fetuses (S. Grealish et al., Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease. Cell Stem Cell 15, 653-665, 2014). Animal studies and clinical trials using fetal ventral midbrain tissues have shown that DA neurons within the transplanted tissues can survive, integrate, and release the dopamine neurotransmitters they produce into the host striatum, thereby alleviating the motor symptoms of Parkinson's disease (P. Hagell et al., Cell survival and clinical outcome following intrastriatal transplantation in Parkinson disease. J Neuropathol Exp Neurol 60, 741-752, 2001). However, although the embryonic ventral midbrain tissues can generate new dopaminergic neurons in vivo and integrate with the neural circuits within the host after transplantation, the survival rate is low, with only 3%-5% of the exogenous neurons surviving, far less than the amount required in the brain. In addition, the acquisition of human embryonic ventral midbrain tissues is restricted by ethics and cannot be obtained and utilized in large quantities. These two factors limit the development of embryonic tissue transplantation therapy. Therefore, how to improve the survival rate of exogenous neurons in the graft and obtain sufficient and effective exogenous dopaminergic neurons has become one of the technical problems to be solved (Tao Mengdan et al., The role of human pluripotent stem cells in the treatment and research of Parkinson's syndrome, Acta Academiae Medicinae Sun Yat-seni (Medical Sciences Edition), Vol. 43, No. 2, pp. 173-180, 2022).

[0004] The groundbreaking discovery of induced pluripotent stem cells (iPSCs) has provided a novel and abundant source of grafts for PD treatment, and iPSCs can be used as autologous or allogeneic donor cells. iPSCs, as well as embryonic stem cells (ESCs) and induced neural stem cells (iNSCs), have shown efficacy in improving the motor function of PD animal models after specific differentiation into DA precursors and implantation (X. Tang et al., Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors. Stem Cell Res 16, 236-242, 2016). Two-dimensional culture (2-D culture) is a key approach for directing differentiation and inducing the generation of a canonical DA lineage of cells in vitro by using soluble factors / morphogens involved in normal embryonic development (S. Kriks et al., Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547-551, 2011). However, compared with the three-dimensional embryonic development process, this two-dimensional system lacks the complex and precise temporal and spatial regulation experienced during DA neuron development. Studies have shown that iPSC-derived two-dimensional DA precursor cells seem to be less effective than fVM tissue in improving PD motor behavior (A. Rath et al., Survival and functional restoration of human fetal ventral mesencephalon following transplantation in a rat model of Parkinson's disease. Cell Transplant 22, 1281-1293, 2013). A greater number of surviving DA neurons are required in the animal striatum to achieve a recovery level similar to that of fVM grafts (A. Kirkeby et al., Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep 1, 703-714, 2012).The phenomenon that the symptom improvement is not obvious indicates that the DA progenitor cells obtained by two-dimensional culture are essentially different from the progenitor cells in the normal embryonic midbrain.

[0005] In the past decade, the developmental culture method of induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) has advanced from two-dimensional planar culture to three-dimensional stereoscopic culture, successfully achieving in vitro three-dimensional organ induction culture. This three-dimensional organ is called an "organoid". Currently, research fields around the world have completed the induction and development of organoids such as the colon, liver, retina, and brain. These organoids can, to some extent, simulate normal organ development in a three-dimensional manner and can be used for mechanism research and drug screening. The success rates of in vitro culture of different tissue organoids vary significantly. The success rates of culturing organoids such as the intestine, pancreas, and bile duct are relatively high. However, it wasn't until 2013 that human brain organoids were established. In 2016, Junghyun Jo and his colleagues reported the successful generation of midbrain organoids from human iPSCs (J. Jo et al., Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell Stem Cell 19, 248-257, 2016).

[0006] Currently, human pluripotent stem cells (hPSCs) are widely used to study the therapeutic effects and pathogenesis of Parkinson's disease. For the research on the therapeutic effects of hPSCs, most studies focus on transplanting human pluripotent stem cells (hPSCs) cultured in two dimensions into specific regions such as the striatum after induced differentiation. For the research on the pathogenesis using hPSCs, some research teams have chosen to use the midbrain organoid model as a platform for studying the molecular pathogenesis and drug screening of Parkinson's disease (Tao Mengdan et al., The Role of Human Pluripotent Stem Cells in the Treatment and Research of Parkinson's Syndrome, Acta Scientiarum Naturalium Universitatis Sunyatseni, Vol. 43, No. 2, pp. 173-180, 2022). However, whether iPSC-induced midbrain organoids can be used as donor grafts for transplantation to treat PD has not been reported.

[0007] To date, multiple clinical studies have involved the use of fetal ventral mesencephalic tissue transplantation for the treatment of PD. However, there are unresolved problems with the use of fetal ventral mesencephalon (fVM) tissue transplantation. In an independent double-blind trial, for example, fVM tissue transplantation failed to reach the primary endpoint and was even found to be associated with the emergence of PD dyskinesia, a side effect thought to be closely related to the complex cellular components within the graft and / or allograft-induced immune recognition (D.E. Redmond et al., Influence of cell preparation and target location on the behavioral recovery after striatal transplantation of fetal dopaminergic neurons in a primate model of Parkinson's disease. Neurobiol Dis 29, 103-116, 2008). Existing studies have demonstrated that fVM tissue used in patient transplantation trials inevitably contains several non-dopaminergic neuron populations, including GABAergic neurons, glutamatergic neurons, cholinergic neurons, and serotonergic neurons (S.C. Neto et al., Cell fate analysis of embryonic ventral mesencephalic grafts in the 6-OHDA model of Parkinson's disease. PLoS One 7, e50178, 2012), and the presence of an excessive number of serotonergic neurons in the graft will trigger adverse side effects, including graft-induced dyskinesia (T. Carlsson et al., Serotonin neuron transplants exacerbate L-DOPA-induced dyskinesias in a rat model of Parkinson's disease. J Neurosci 27, 8011-8022, 2007). That is to say, serotonergic neurons are a contaminating cell type in midbrain grafts and can cause adverse side effects, including dyskinesia induced by fVM tissue grafts. Therefore, how to prepare midbrain organoids that are substantially or essentially free of serotonergic neurons has become a technical obstacle to the application of midbrain organoids.

[0008] In addition, although methods for preparing midbrain organoids have been provided, existing methods, as a continuous culture process, still cannot determine which culture stage results in midbrain organoids suitable for achieving the objectives of the present invention.

[0009] Through summarizing existing research, it can be found that existing research results have reflected that fVM grafts containing precursor cells from the normal embryonic midbrain have advantages in PD treatment compared to two-dimensional cultured DA precursor cells. However, fVM grafts are limited by insufficient sources of embryonic tissue and ethical factors. On the other hand, in the existing technology, midbrain organoids induced from hPSCs are only used as a research platform for PD mechanisms, but no research has been conducted on whether midbrain organoids can be used as candidate grafts for treating PD. Therefore, whether iPSC-induced midbrain organoids can be used and which stage of midbrain organoids (if possible) can be used as donor grafts for treating PD have become urgent problems to be solved in the field of PD treatment. In addition, in view of the presence of contaminating cells represented by serotonergic neurons in fVM, how to provide midbrain organoids that are substantially or essentially free of serotonergic neurons is also a problem that needs to be solved simultaneously in this field. Summary of the Invention

[0010] To solve the problems existing in the above-mentioned prior art, the technical solutions of the present invention are provided.

[0011] In the first aspect of the present invention, there is provided the use of midbrain organoids for treating Parkinson's disease, or the use of midbrain organoids in the preparation of a medicament for treating Parkinson's disease.

[0012] In one embodiment, the midbrain organoids are prepared from pluripotent stem cells, and the pluripotent stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or neural stem cells (NSCs). Among them, the neural stem cells can be induced neural stem cells (iNSCs). More preferably, the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0013] In one embodiment, the midbrain organoids do not contain serotonergic neurons.

[0014] In one embodiment, the midbrain organoid has the following characteristics: (1) positive staining for NESTIN, SOX2, TUJ1, KI67, OTX2, FOXA2, EN1, NURR1, and TH, wherein the proportion of cells expressing FOXA2 is 20-40%, the proportion of cells expressing EN1 is 10-30%, the proportion of cells expressing OTX2 is 5%-15%, the proportion of cells expressing NURR1 is 0.01-5%, and the proportion of cells expressing TH is 0.01-5%; (2) the midbrain organoid is negative for staining of DARPP32, GAD67, Vglut1, CHAT, and SEROTONIN; (3) the midbrain organoid does not express FOXG1, NKX2.2, NKX6.1, HOXA2, HOXB, GBX2, and GATA3; and (4) the midbrain organoid exhibits apical-basal polarity. Preferably, the proportion of cells expressing FOXA2 in the midbrain organoid is about 30%, the proportion of cells expressing EN1 is about 18%, the proportion of cells expressing OTX2 is about 10%, the proportion of cells expressing NURR1 is about 2%, and the proportion of cells expressing TH is about 0.8%. More preferably, the proportion of cells expressing FOXA2 in the midbrain organoid is 30%, the proportion of cells expressing EN1 is 18%, the proportion of cells expressing OTX2 is 10%, the proportion of cells expressing NURR1 is 2%, and the proportion of cells expressing TH is 0.8%.

[0015] Those skilled in the art will understand that the positive staining of the midbrain organoid for NESTIN, SOX2, TUJ1, KI67, OTX2, FOXA2, EN1, NURR1, and TH means that the midbrain organoid expresses NESTIN, SOX2, TUJ1, KI67, OTX2, FOXA2, EN1, NURR1, and TH. Accordingly, those skilled in the art will understand that the negative staining of the midbrain organoid for DARPP32, GAD67, Vglut1, CHAT, and SEROTONIN means that the midbrain organoid does not express DARPP32, GAD67, Vglut1, CHAT, and SEROTONIN or the expression levels of DARPP32, GAD67, Vglut1, CHAT, and SEROTONIN are below the detection limit.

[0016] In one embodiment, the midbrain organoid is cut into 0.1-2 mm before treating Parkinson's disease 3The small pieces are resuspended in a transplantation buffer containing 0.1 - 1 μM Rock inhibitor, vitamin A-free B27, and 10 - 30 ng / ml BDNF. Preferably, the transplantation buffer contains 0.5 μM Rock inhibitor, vitamin A-free B27, and 20 ng / ml BDNF.

[0017] In one embodiment, the midbrain organoids are midbrain organoids cultured in vitro for 10 - 25 days. Preferably, the midbrain organoids are midbrain organoids cultured in vitro for 13 - 17 days. Preferably, the midbrain organoids are midbrain organoids cultured in vitro for about 15 days. More preferably, the midbrain organoids are midbrain organoids cultured in vitro for 15 days.

[0018] In one embodiment, the method for preparing the midbrain organoids comprises: (1) Neural induction stage: dissociating induced pluripotent stem cells (preferably hiPSCs) into single cells and forming homogeneous embryoid bodies, culturing the embryoid bodies in medium A containing heparin, SB431542, Noggin, CHIR99021, Y27632, and replacing medium A with medium B containing heparin, SB431542, Noggin, CHIR99021 but without Y27632 after 45 - 50 hours (preferably about 48 hours, more preferably 48 hours) for continued culture to obtain culture a; (2) Midbrain patterning stage: adding SHH-C25II and FGF8 to medium B to form medium C, and continuing to culture culture a for midbrain patterning to obtain culture b, where the concentration of SHH-C25II is 150 - 250 ng / ml (preferably 200 ng / ml); (3) Lamination stage: after culture b extends neural ectodermal buds, completely removing medium C, embedding with low growth factor Matrigel, and growing in medium D containing insulin, laminin, SHH-C25II, FGF8 after the low growth factor Matrigel solidifies to obtain culture c, where the concentration of SHH-C25II is 150 - 250 ng / ml (preferably 200 ng / ml); (4) Final differentiation stage: transferring culture c to medium E containing BDNF, GDNF, ascorbic acid, cAMP for culture to obtain midbrain organoids; wherein, each stage of the preparation method is carried out for 10 - 25 days. Preferably, each stage of the preparation method is carried out for 13 - 17 days. Preferably, each stage of the preparation method is carried out for about 15 days. More preferably, each stage of the preparation method is carried out for 15 days.

[0019] In one embodiment, during the neural induction stage, it is cultured in Medium A for 48 hours and changed to Medium B on the second day and cultured for 2 days; during the midbrain patterning stage, it is cultured in Medium C for 3 days; during the laminarization stage, it is cultured in Medium D for 1 day; during the final differentiation stage, it is cultured in Medium E for 2 - 17 days (i.e., midbrain organoids cultured in vitro for 10 - 25 days). Preferably, during the final differentiation stage, it is cultured in Medium E for 7 days (i.e., midbrain organoids cultured in vitro for 15 days).

[0020] In one embodiment, in the method for preparing the midbrain organoids, the Medium A, Medium B, Medium C, and Medium D all contain a neuronal induction basal medium capable of enabling the growth of induced pluripotent stem cells. The neuronal induction basal medium is DMEM / F12:Neurobasal 0.8 - 1.2:1, 0.5 - 2:100 of N2 additive, 0.5 - 2:50 of vitamin A-free type B27, 0.5 - 2% of minimum essential medium (MEM)-non-essential amino acids, and 0.01 - 1% of β-mercaptoethanol; Medium E contains a medium obtained by removing DMEM / F12 from the neuronal induction basal medium.

[0021] In one embodiment, the neuronal induction basal medium used in the method for preparing the midbrain organoids is DMEM / F12:Neurobasal 1:1, 1:100 of N2 additive, 1:50 of vitamin A-free type B27, 1% of minimum essential medium (MEM)-non-essential amino acids, and 0.1% of β-mercaptoethanol.

[0022] The second aspect of the present invention provides a midbrain organoid, characterized in that: (1) it is positive for the staining of NESTIN, SOX2, TUJ1, KI67, OTX2, FOXA2, EN1, NURR1 and TH, wherein the proportion of cells expressing FOXA2 is 20-40%, the proportion of cells expressing EN1 is 10-30%, the proportion of cells expressing OTX2 is 5%-15%, the proportion of cells expressing NURR1 is 0.01-5%, and the proportion of cells expressing TH is 0.01-5%; (2) the midbrain organoid is negative for the staining of DARPP32, GAD67, Vglut1, CHAT and SEROTONIN; (3) the midbrain organoid does not express FOXG1, NKX2.2, NKX6.1, HOXA2, HOXB, GBX2 and GATA3; and (4) the midbrain organoid exhibits apical-basal polarity. Preferably, the proportion of cells expressing FOXA2 in the midbrain organoid is about 30%, the proportion of cells expressing EN1 is about 18%, the proportion of cells expressing OTX2 is about 10%, the proportion of cells expressing NURR1 is about 2%, and the proportion of cells expressing TH is about 0.8%; wherein, the midbrain organoid is derived from human induced pluripotent stem cells. More preferably, the proportion of cells expressing FOXA2 in the midbrain organoid is 30%, the proportion of cells expressing EN1 is 18%, the proportion of cells expressing OTX2 is 10%, the proportion of cells expressing NURR1 is 2%, and the proportion of cells expressing TH is 0.8%.

[0023] The third aspect of the present invention provides a method for preparing midbrain organoids, comprising: (1) Neural induction stage: dissociating induced pluripotent stem cells (preferably hiPSCs) into single cells and forming homogeneous embryoid bodies, culturing the embryoid bodies in medium A containing heparin, SB431542, Noggin, CHIR99021, Y27632, and replacing medium A with medium B containing heparin, SB431542, Noggin, CHIR99021 but not Y27632 after 45 - 50 hours (preferably about 48 hours, more preferably 48 hours) for continued culture to obtain culture a; (2) Midbrain patterning stage: adding SHH-C25II and FGF8 to medium B to form medium C, and continuing to culture culture a for midbrain patterning to obtain culture b, wherein the concentration of SHH-C25II is 150 - 250 ng / ml (preferably 200 ng / ml); (3) Lamellation stage: after culture b extends neural ectodermal buds, completely removing medium C, embedding with low-growth factor Matrigel, and growing in medium D containing insulin, laminin, SHH-C25II, FGF8 after the low-growth factor Matrigel solidifies to obtain culture c, wherein the concentration of SHH-C25II is 150 - 250 ng / ml (preferably 200 ng / ml); (4) Final differentiation stage: transferring culture c to medium E containing BDNF, GDNF, ascorbic acid, cAMP for culture to obtain midbrain organoids; wherein, each stage of the preparation method is carried out for 10 - 25 days. Preferably, each stage of the preparation method is carried out for 13 - 17 days. Preferably, each stage of the preparation method is carried out for about 15 days. More preferably, each stage of the preparation method is carried out for 15 days.

[0024] In one embodiment, according to the method for preparing midbrain organoids of the present invention, in the neural induction stage, culture in medium A for 48 hours, replace with medium B on the second day and culture for 2 days; in the midbrain patterning stage, culture in medium C for 3 days; in the lamellation stage, culture in medium D for 1 day; in the final differentiation stage, culture in medium E for 2 - 17 days (i.e., midbrain organoids cultured in vitro for 10 - 25 days). Preferably, in the final differentiation stage, culture in medium E for 7 days (i.e., midbrain organoids cultured in vitro for 15 days).

[0025] In one embodiment, in the method for preparing midbrain organoids according to the present invention, the culture media A, B, C, and D in the method for preparing midbrain organoids all contain a neuronal induction basal medium capable of enabling the growth of induced pluripotent stem cells. The neuronal induction basal medium is DMEM / F12:Neurobasal at a ratio of 0.8 - 1.2:1, N2 supplement at a ratio of 0.5 - 2:100, vitamin A-free B27 at a ratio of 0.5 - 2:50, 0.5 - 2% minimum essential medium (MEM)-non-essential amino acids, and 0.01 - 1% β-mercaptoethanol; the culture medium E contains a medium obtained by removing DMEM / F12 from the neuronal induction basal medium. Preferably, in the method for preparing midbrain organoids according to the present invention, the neuronal induction basal medium is DMEM / F12:Neurobasal at a ratio of 1:1, N2 supplement at a ratio of 1:100, vitamin A-free B27 at a ratio of 1:50, 1% minimum essential medium (MEM)-non-essential amino acids, and 0.1% β-mercaptoethanol.

[0026] Compared with existing research, the present invention has good effects, obvious advantages, and progress.

[0027] First, in existing research, differentiated cells obtained from two-dimensional culture of iPSCs are used in the treatment of Parkinson's disease by stem cell therapy. In contrast, the midbrain organoids derived from iPSCs in the present invention are formed under complex spatial and temporal rules, to a certain extent replicating the patient's own embryogenesis. This makes the organoids more compatible with the host in cell transplantation therapy. This dynamic process can provide niche signals at appropriate stages to induce allogeneic populations and tissue-specific cell types with more complete and mature functional activities.

[0028] Second, in existing research, hPSCs have been induced into midbrain organoid models to provide a platform for studying the pathogenesis of Parkinson's disease or drug screening, but there is a lack of research on using midbrain organoids as therapeutic agents for transplantation treatment of Parkinson's disease; the research results of the present invention show that human-induced pluripotent stem cell-derived midbrain organoids can be used as therapeutic products for transplantation treatment of Parkinson's disease, and provide the most suitable growth and culture stage of midbrain organoids as therapeutic grafts.

[0029] Third, there have been successful attempts to treat Parkinson's disease with fetal ventral midbrain (fVM) tissue transplantation in existing research. However, the method of treating PD with fVM has problems in terms of ethical obstacles, scarce sources, and transplantation heterogeneity. However, the midbrain organoids of the present invention show advantages in terms of ethics, source, and transplantation. hMOs derived from iPSCs can well solve the ethical and source problems related to embryo-derived fVM tissue. In terms of the heterogeneity problem, hMOs have the advantage of being applicable to autologous transplantation or homogeneous transplantation.

[0030] Fourth, when using fVM to treat Parkinson's disease in existing research, there are technical difficulties in separating the required ventral midbrain region. Therefore, other contaminated cells are often detected in the fVM grafts. In particular, the serotonergic neurons contained therein can induce serious side effects, including graft-induced dyskinesia, etc. The midbrain organoids at a specific growth and culture stage provided by the present invention do not contain contaminated cells such as serotonergic neurons, thus avoiding serious side reactions such as dyskinesia caused by contaminated cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings are used to provide a further understanding of the technical solutions of the present application and constitute a part of the specification. They are used together with the embodiments of the present application to explain the technical solutions of the present application, but do not constitute a limitation to the technical solutions of the present application.

[0032] Figure 1Figure showing the differentiation of hiPSCs into midbrain organoids: (A) Schematic diagram of the differentiation process from iPSCs to midbrain organoids (hMOs); (B) Immunofluorescence staining used to characterize hiPSC-derived hMOs, scale bars: a, b, c are 250 μm, d is 75 μm, e is 50 μm, f is 100 μm; (C) Evaluation of neuromelanin production in hMOs, a is a representative differential interference contrast (DIC) image of an organoid with neuromelanin granules (within the dashed box), b is Fontana-Masson staining, scale bar: 100 μm; (D) High-performance liquid chromatography (HPLC) analysis of whole organoids. The amounts of DA and metabolite DOPAC were evaluated after high-potassium stimulation at 60 DIV (n = 10). FGF8: fibroblast growth factor 8; SHH: sonic hedgehog; BDNF: brain-derived neurotrophic factor; GDNF: glial cell-derived neurotrophic factor; cAMP: dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt; SOX2: sry-box transcription factor 2; TH: tyrosine hydroxylase; TUJ1: tubulin beta-3 class III; OTX2: orthodenticle homeobox 2; FOXA2: forkhead box protein A2; EN1: engrailed-1; NURR1: nuclear receptor subfamily 4; GIRK2: G-protein-coupled inwardly rectifying potassium; DIV: days in vitro; ap.: apical; bas.: basal; min: minute.

[0033] Figure 2 Figure showing the evaluation of hMO functional maturity: (A) Evaluation of the electrophysiological activity of whole organoids using the MED64 multi-electrode array system, a is a schematic diagram of the electrophysiological signal processing system, b is a micrograph of an organoid on a disk with a 64-electrode array, c is a burst occurring simultaneously in different channels representing network bursts (boxed area), d is one of the representative spike clusters (i.e., the arrow in c); (B) Transplantation of iPSC-EGFP-derived hMOs into the brains of SCID mice to examine electrophysiological properties, a-d are micrographs showing EGFP+ organoids at day 4 and day 7 (a, c are DIC images; b, d are fluorescence micrographs, scale bar: 2 mm), e is a brain section containing EGFP+ organoids 6 weeks after transplantation; (C) Whole-cell current clamp recordings, a is the regular spontaneous AP activity of transplanted GFP-labeled cells, b is APs evoked by step current injection, c is the representative voltage-dependent Na + and K + currents, inward Na +Their respective traces were enlarged. d is the phase-contrast image of the membrane neurons and the corresponding triple staining of EGFP, biocytin, and TH of the recorded neurons. The scale bar is 25 μm. DIV: days in vitro; wks: weeks.

[0034] Figure 3 It is a figure showing the optimal differentiation stage for searching for transplanted hMO: (A) Schematic diagram of the experimental design; (B) Representative RT-PCR results at different differentiation time points, presented as fold change relative to undifferentiated hiPSCs on day 0; (C) Representative immunofluorescence staining results of the dopaminergic progenitor marker FOXA2 and the mature dopaminergic neuron marker TH on hMO at 10, 15, and 25 DIV. Scale bars: a, c are 250 μm, e is 100 μm, b, d are 50 μm, f is 25 μm; (D) Survival and differentiation of hMO after transplantation into the brains of SCID mice (n = 9). a is the transplantation of organoids at day 10, b - d are at day 15, e is at day 25. Most of the organoids at 25 DIV died after transplantation. Scale bars: a is 250 μm, b, c, d are 50 μm, e is 100 μm. D0: day 0; DIV: days in vitro.

[0035] Figure 4 It is a figure showing the transplantation of iPSC-derived midbrain organoids into a PD mouse model: (A) Schematic diagram of the experimental design and schedule of the behavioral test. During the 4-month period, a small number of animals in groups A and C were sacrificed for histological analysis; (B) Immunofluorescence staining confirmed the differentiation and maturation of the transplanted organoids in vivo. Scale bar: 50 μm; (C) a - d are representative confocal microscopy images at three time points after transplantation and the proportion of TH+ cells in the transplanted cells (2.57% at 6 weeks, 14.11% at 12 weeks, 7.33% at 16 weeks); *p < 0.0453, **p < 0.0078, ****p < 0.0001, calculated by two-way ANOVA and Tukey's multiple comparison test. Scale bar: 25 μm, e - g are the results of the behavioral tests, including apomorphine-induced rotation, open field, and rotarod tests. TX: graft; wks: weeks.

[0036] Figure 5Figure showing axonal projections of transplanted neurons: (A) Representative immunofluorescence image showing the distribution of transplanted cells at 6 weeks; (B) Immunofluorescence staining of hNCAM, indicating that graft-derived fibers innervate host brain regions, including the cerebral cortex (a), striatum (b), mfb (c, d), and MB (e). The boxed areas are magnified in I-IV. Scale bar: 250 μm; (C) Immunofluorescence staining of hNCAM, indicating that graft-derived fibers innervate host brain regions at 12 weeks (I corresponds to a, II corresponds to b, III corresponds to c, IV corresponds to d, V corresponds to e). Scale bar: 250 μm. wks: weeks; PFC: prefrontal cortex; fa / CC: corpus callosum; MO: motor cortex; SS: somatosensory cortex; NAc: nucleus accumbens; Strd / Strv: dorsolateral / ventral striatum; GP: globus pallidus; VPL: ventral posterolateral nucleus of the thalamus; mfb: medial forebrain bundle; HY: hypothalamus; TH: thalamus; MB: midbrain; MRN: mesencephalic reticular nucleus; fr: fasciculus retroflexus; SPF: parafascicular nucleus; CLA: claustrum; EP: piriform nucleus; SI: substantia innominata; OT: olfactory bulb; SNr / SNc: reticular / compact parts of the substantia nigra; VTA: ventral tegmental area.

[0037] Figure 6 Figure showing graft-derived dopaminergic neurons innervating host neuronal circuits: (A) TH+ / hNCAM+ fibers innervate the PAL (a) and ipsilateral SNr / SNc (d). The boxed areas in a and d are magnified in b, c and e, f respectively. Scale bar: 250 μm; (B) Transplanted DA neurons receive synaptic (h+m Syn) inputs from host and / or transplanted neurons (the boxed area in a is magnified in b). Transplanted dopaminergic neurons extend axons to adjacent MSNs (the boxed area in c is magnified in d). Scale bars: a, b, c are 50 μm; d is 7.5 μm. MB: midbrain; PAL: globus pallidus; Strd: dorsolateral striatum; SNr: reticular part of the substantia nigra; VTA: ventral tegmental area.

[0038] Figure 7Figure showing the connection between transplanted neurons and host neurons: (A) Transplanted neurons located in the STR project to the PFC and HY (the boxed areas in a and c are magnified in b and d respectively); (B) a is a schematic diagram showing the rabies virus tracing system; b shows that the GFP+ starting neurons infected with G-rabies virus subsequently begin to express mCherry, and the upstream neurons being traced are retrogradely infected by the G rabies virus and also show mCherry labeling; (C) Monosynaptic tracing determines the early host-to-graft connection. a shows that the starting neurons are mainly located in the transplantation site and retrogradely infect adjacent transplanted and / or host cells (the arrows highlight the starting neurons with GFP+ / mCherry+), and b-f show scattered traced neurons located in multiple brain regions (including PFC, fi, Strd, LHA, MB). Scale bars: 250 μm for a, c, d, e, f; 75 μm for b. Puro: Puromycin; fi: Fimbria hippocampi; LHA: Lateral hypothalamic area.

[0039] Figure 8 Figure showing the cell composition within midbrain organoids: (A) The morphology of organoids at different time points; Images a to c were taken by DIC, image d was taken by a stereomicroscope, and image e was taken by a camera. Scale bar: 2 mm; (B) The proportion of cells positive for markers related to individual dopaminergic neurons at day 15 and day 35 (n = 10 organoids); (C) Examination of non-DA cells. A small number of O4+ cells were detected in the organoids at day 35 (a), a small number of OLIG2+ cells were detected in the organoids at day 15 (b), and cells positive for markers related to the forebrain (FOXG1), hindbrain (NKX6.1, NKX2.2, GATA3), and other neuronal subtypes (including CHAT, 5-HT, DARPP32, Vglut1) were not detected by immunofluorescence staining in the organoids at day 15. Scale bar: 250 μm; (D) Real-time PCR results confirmed the absence of serotonin progenitor cells.

[0040] Figure 9 Figure showing that histological analysis reveals the degeneration of unilateral endogenous dopaminergic neurons: (A) IHC analysis shows the degeneration of unilateral endogenous TH-positive midbrain dopaminergic neurons in the SN and striatum of 6-OHDA-lesioned mice; (B) Graft-derived dopaminergic neurons in the lesioned hemisphere. Scale bar: 100 μm. 6-OHDA: 6-Hydroxydopamine; wks: weeks; NAc: Nucleus accumbens; Strd / Strv: Dorsolateral / ventral striatum; HY: Hypothalamus; TH: Thalamus; PAL: Pallidum.

[0041] Figure 10Figures showing tracing using rabies virus: (A) hiPSC, and day 4 and day 7 organoids expressing rabies glycoprotein (RVG) and EGFP, a, c, and e are DIC images, b, d, and f are live cell images observed using a fluorescence microscope; (B) Four weeks after transplantation, mature dopaminergic neurons with GFP expression were found at the implantation site, scale bar: 50 μm; (C) Triple staining of TH, DARPP32, and hSYN confirmed synaptic connections (arrows) between DA and MSN neurons. MSN: medial spiny neuron. Detailed implementation manners

[0042] Various exemplary embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The description of the exemplary embodiments is merely illustrative and does not impose any limitation on the present invention and its application or use. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to make the present invention thorough and complete and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specifically stated, the technical means described in these embodiments should be construed as merely exemplary and not restrictive.

[0043] In the present invention, the term "organoid" means a micro-organ similar to an organ, having the ability of self-renewal and assembly, and highly similar in structure and function to an organ, which is produced by three-dimensional (3D) in vitro culture of stem cells.

[0044] In the present invention, the term "midbrain organoid" (midbrain organoids or midbrain-like organoids, MO) means a micro-organ similar to the midbrain, having the ability of self-renewal and assembly, and highly similar in structure and function to the midbrain, which is produced by three-dimensional in vitro culture of stem cells. Among them, human stem cell-derived midbrain organoids are human midbrain organoids (hMO or hMOs).

[0045] In the present invention, the term "embryoid body" (EB) means a spherical structure formed by embryonic stem cells (ES) or induced pluripotent stem cells (iPS) under certain culture conditions in vitro, having the structures of the inner, middle, and outer germ layers and being highly similar morphologically to the early embryonic development stage of mammals.

[0046] In the present invention, the term "serotonergic neuron" (serotonergic neuron or serotoninergic neuron), also known as "5-hydroxytryptaminergic neuron", means a neuron that uses 5-hydroxytryptamine as a neurotransmitter.

[0047] In the present invention, the singular forms "a / an" and "the" include plural forms unless the context clearly indicates otherwise. As will be apparent to those skilled in the art from the teachings contained herein, when used in the context of numerical values and ranges, the term "about" or "approximately" means approximate or close to the specified value or range of values or ranges such that the embodiments can be carried out as expected. In some embodiments, "about" means ±10% of the numerical quantity.

[0048] In the study of the present invention, the following research materials and methods were used.

[0049] Tissue processing and immunofluorescence staining: Mice were perfused transcardially with 50 mL of 0.9% saline and 100 mL of ice-cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at room temperature (21°C). Brains were removed and stored in 4% PFA for an additional 2 hours, then re-suspended overnight in 20% sucrose (in 0.1 M PB) and dehydrated in 30% sucrose. Twelve series of 40-μm thick coronal slices of mouse brains were collected at -20°C. For IF / IHC, tissues were blocked with 3% donkey serum for 2 hours at room temperature. Table 1 lists the information and working dilutions of the primary antibodies used. A peroxidase-based reaction was used, followed by diaminobenzidine (DAB) precipitation (goat anti-rabbit, ZSGB-BIO, Beijing, China) or appropriate Alexa 488, Alexa 555, or Alexa 647-conjugated secondary antibodies (Jackson ImmunoResearch, Pennsylvania, USA).

[0050] Table 1 Information and working dilutions of primary antibodies

[0051]

[0052]

[0053] Gene expression analysis: Analysis was performed on days 0, 15, 25, and 35 using the RNeasy kit (Qiagen, Valencia, CA). Reverse transcription of total RNA (Quantitech, Qiagen) was used for qRT-PCR analysis. qRT-PCR analysis was performed using an ABI Prism (Applied Biosystems, USA). For each cell culture condition, the expression of the gene of interest was measured in triplicate. GAPDH was used as an internal reference gene.

[0054] High-performance liquid chromatography (HPLC) analysis: The organoid culture medium was replaced with HBSS (Gibco) (3 ml per well), and incubated at 37 °C for 30 minutes. Subsequently, it was replaced with 3 ml of high-potassium HBSS medium (60 mM KCl and 82 mM NaCl) containing the DA uptake blocker Nomifensine (10 μM, Sigma) and the monoamine oxidase inhibitor Pargyline (50 μM, Sigma), and incubated at 37 °C for 45 minutes. Then, 180 μl of the high-potassium medium was transferred from each well to a dark Eppendorf tube containing 20 μl of 1 N perchloric acid (Merck) and antioxidants (0.33 g / L Na2S2O5, 0.083 g / L EDTA) placed on ice and stored at -80 °C until analysis. The neurotransmitter content of 100 μl of the supernatant was measured by performing HPLC analysis.

[0055] MED64 multi-electrode array and whole-cell patch-clamp recording: A multi-electrode array device was used to record the electrophysiological activities of the midbrain organoids on day 60. The area of each planar microelectrode at the center of the MED64 Probe was 50 μm × 50 μm, and the spacing was 150 μm (model P515A). Whole-cell patch-clamp recording was used to analyze the electrophysiological activities of the transplanted EGFP+ organoids. Whole-cell patch-clamp recording of acute brain slices was performed using ACSF bath solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4, 1 mM MgSO4, 25 mM glucose, and 26 mM NaHCO3). The internal solution consisted of 143 mM KCl, 8 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 2% Biocytin, 2 mM Na-ATP, and 0.4 mM Na-GTP. EGFP+ cells were observed using a fluorescence microscope.

[0056] Microscopy and quantification: All bright-field images were taken using a Leica microscope, and a Leica DMI6000 confocal microscope was used to acquire fluorescence images. To evaluate the number of EN1-, FOXA2-, OTX2-, NURR1-, and TH-expressing cells among the total DAPI-labeled cells in vitro, at least 10 sampling regions in three organoids were calculated using ImageJ software. The grafts on the brain slices were delineated, and images were captured using a confocal microscope to show TH+ and hNA+ signals. Single-labeled or double-labeled cells were manually counted using Image J. The data were represented as the ratio of TH+ to total hNA+ cells.

[0057] Statistical analysis: All data are presented as mean ± SEM and were analyzed statistically using GraphPad Prism 9. Two-way ANOVA and Tukey's multiple comparison test were used to analyze the proportion of hNA+ cells co-expressing TH. For all behavioral test results, two-way ANOVA and Bonferroni multiple comparisons were used. Significance was set at a p-value ≤ 0.05.

[0058] Example 1 Cell culture and generation of midbrain organoids

[0059] The method for generating midbrain organoids (hMO) provided by the present invention can be divided into four stages: neural induction, midbrain patterning, laminarization, and final differentiation, as Figure 1 shown in A. The cell source for generating midbrain organoids is pluripotent stem cells, such as human induced pluripotent stem cells (hiPSC). Before generating midbrain organoids, hiPSC can be cultured, for example, on feeder-free Matrigel in E8-mTESR medium (STEMCELL TM , catalog number 05990). The preparation method comprising the following four stages was used to induce the generation of human midbrain organoids.

[0060] Neural induction stage: Use TrypLE TMExpress (Gibco, 12563029) dissociates hiPSCs into single cells from intact colonies and plates them at 10,000 cells per well on a low cell adhesion 96-well culture plate (Corning) with V-bottom conical wells to form uniform embryoid bodies (EBs); the wells contain neuronal induction basal medium, which contains DMEM / F12 (Gibco, 10565-018): Neurobasal (Gibco, 21103049) 1:1, 1:100 N2 supplement (Gibco, 17502048), 1:50 vitamin A-free B27 (Gibco, 12587010), 1% minimum essential medium (MEM)-non-essential amino acids (Gibco, 11130051), and 0.1% β-mercaptoethanol (Invitrogen, 31350010), and 1 μg / ml heparin (Sigma-Aldrich), 10 μM SB431542 (Stemgent), 200 ng / ml Noggin (Peprotech), 0.8 μM CHIR99021 (Reagents Direct), and 10 μM ROCK inhibitor Y27632 (Calbiochem) are added to the above neuronal induction basal medium. The ROCK inhibitor is added in the first 48 hours, and the medium is changed on the second day.

[0061] Midbrain patterning stage: On the 4th day, 200 ng / ml of SHH-C25II (Peprotech) and 100 ng / ml of FGF8 are added to the neuronal induction basal medium of hMLO for midbrain patterning.

[0062] Lamination stage: After 3 days, hMLO begins to extend neuroectodermal buds. At this time, the medium is completely removed, and immediately 30 μl of low growth factor Matrigel (BD Biosciences, 356230) is added to each well using a pipette equipped with a pre-cooled 200 μl pipette tip. The Matrigel-embedded midbrain organoids are placed in a 37 °C incubator for 30 minutes to solidify the Matrigel and grown in neuronal induction basal medium supplemented with 2.5 μg / ml insulin (Sigma-Aldrich), 200 ng / ml laminin (Sigma-Aldrich), 200 ng / ml SHH-C25II, and 100 ng / ml FGF8 for 24 hours.

[0063] Final differentiation stage: Once the hMLOs are embedded in Matrigel, more expanded neuroepithelia start to form in the organoids. To promote growth and differentiation, the hMLOs are transferred to ultra-low attachment 6-well plates (Costar) by pipetting using a cut 1000 μl pipette tip. These plates contain the final differentiation medium, which is obtained by adding 10 ng / ml BDNF (Peprotech), 10 ng / ml GDNF (Peprotech), 100 μM ascorbic acid (Sigma-Aldrich), and 125 μM db-cAMP (Sigma-Aldrich) to the neuronal induction basal medium and removing DMEM / F12. The hMLOs are cultured using an orbital shaker (set at 70 rpm) to enhance nutrient and oxygen exchange. The medium is changed every 2 - 3 days.

[0064] In multiple attempts to prepare midbrain organoids, the inventors found that when the concentration of SHH-C25II in the above-mentioned "midbrain patterning stage" and "lamination stage" is less than 150 ng / ml, for example, 100 ng / ml, the reproducibility of forming midbrain organoids is poor, that is, the desired midbrain organoids cannot be stably formed.

[0065] Example 2 Characterization of in vitro iPSC-derived midbrain organoids

[0066] hMOs obtained at different time points are analyzed by using immunofluorescence staining ( Figure 1 B) and qPCR ( Figure 3 C). At 15 days in vitro (DIV), the hMOs are positive for staining of the neural stem / progenitor cell markers NESTIN and SOX2, the pan-neuronal and proliferation markers TUJ1 and KI67, the embryonic midbrain brain progenitor cell marker OTX2, and the floor-plate-derived dopaminergic progenitor cell markers FOXA2 and EN1, indicating that the organoids are mainly composed of midbrain DA lineage cells in the early stage of development ( Figure 1 B, a to d). At 25 days in vitro, the hMOs are positive for staining of the midbrain post-mitotic DA cell markers Nurr1 and tyrosine hydroxylase (TH) (Figure 1B, e). At 45 days in vitro, the expression of the midbrain A9 region-specific marker GIRK2 is confirmed ( Figure 1 B, f). In addition, the neuroepithelium within the organoids starts to show apical-basal polarity from 15 days in vitro, which can be illustrated by the expression of KI67 (a proliferation marker mainly located on the apical side of the organoids) and TUJ1+ (a marker located on the basal side) ( Figure 1 B, b).

[0067] After characterizing the marker expression profiles, we evaluated the function of hMO. Neuromelanin-like structures began to appear at 45 days in vitro, and Fontana-Masson staining was performed to show neuromelanin, which are black / brown granular pigments usually produced by dopaminergic neurons located in the SNpc region. As Figure 1 shown in C, the positively stained deposits were mainly located within the neuronal cytosol. To confirm the release of the neurotransmitter dopamine, we performed high-performance liquid chromatography (HPLC) analysis on the culture supernatants stimulated with high potassium, and a large amount of DOPAC (3.806 ng / ml, n = 10) and its metabolite DOPA (6.149 ng / ml, n = 10) were detected at 60 days in vitro ( Figure 1 D, a, b). The secretion of the neurotransmitter serotonin was not detected in the cultures using HPLC (data not shown).

[0068] To further understand the functional activity of the neuronal networks formed in hMO, we used the MED64 multi-electrode array system, which is a non-invasive method for recording extracellular field potentials generated by the entire organoid. Several synchronous network-wide bursts of cellular potentials were found in the organoids at 60 days in vitro, indicating that the neurons in the midbrain organoids had developed proper network connectivity and electrophysiological activity ( Figure 2 A).

[0069] In addition, to examine the electrophysiological properties of single dopaminergic neurons, midbrain organoids were transplanted into the brains of immunodeficient mice, and whole-cell patch-clamp recordings were performed on acute brain slices.

[0070] First, a reporter hiPSC line with lentiviral expression of enhanced green fluorescent protein (EGFP) was generated. EGFP was constitutively expressed during the differentiation of hiPSC into hMO ( Figure 2 B, a-d). EGFP+ organoids were transplanted into the striatum of SCID mice. Six weeks after transplantation, EGFP+ grafts were detected in all transplanted animals, and the electrophysiological properties of the transplanted cells were examined by whole-cell patch-clamp recordings ( Figure 2 B, e). Whole-cell current-clamp recordings showed regular spontaneous action potentials in the transplanted EGFP+ cells with a longer single action potential (AP) activity duration (exceeding 10 ms, Figure 2C, a). In current-clamp mode, APs can be induced by injecting currents from -200 pA to 500 pA in 20-pA incremental steps, and a typical afterhyperpolarization (AHP) "sag" is presented at the start of the response and a delayed inward rectification is presented at the end of the response ( Figure 2 C, b). In voltage-clamp mode, voltage-gated potassium (K + ) channels and sodium (Na + ) channels can be induced at a holding potential from -60 mV to +60 mV in 10-mV steps ( Figure 2 C, c). Patch cells were identified as positive for TH, EGFP, and biocytin ( Figure 2 C, d).

[0071] Example 3 determines the optimal stage of transplanted DA differentiation

[0072] An ideal differentiation stage would ensure good survival of the graft after transplantation and successful differentiation and maturation of its DA neurons. To find the optimal differentiation time window, first, the mRNA expression of genes related to midbrain DA neuron development in the organoids during differentiation was measured. These related genes include FOXA2, EN1, OTX2, NURR1, TH, and TUJ1. The transcription factors FOXA2, EN1, and OTX2 related to early DA lineage development peaked on day 7 and then gradually decreased. On the other hand, transcription factors such as NURR1 and TH expressed by post-mitotic DA cells were detected starting on day 15 and peaked on day 25 and day 30, respectively ( Figure 3 B). The level of the pan-neuronal marker TUJ1 gradually increased during the observation period (day 0 - day 35) ( Figure 3 B). The results of immunofluorescence staining showed a similar trend. FOXA2 staining increased from day 10 to day 15 and gradually decreased on day 25, while TH staining was detected starting on day 15 and increased on day 25 (Figure 3C). The organoids showed high expression of the progenitor cell marker FOXA2 on day 10 but did not express the mature DA neuron marker TH ( Figure 3 C, a and b). Therefore, in the organoids on day 25, the upregulation of TH+ signal was correlated with the downregulation of FOXA2+ signal ( Figure 3C, e, and f). Previous studies using iNSC-derived DA cells for transplantation have shown that DA cells in the late maturation stage cannot survive in transplantation (Y. Yuan et al., Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model. Theranostics 8, 4679-4694, 2018). Therefore, we narrowed down the differentiation stage to days 10 to 25 for further transplantation testing. We transplanted the organoids on days 10, 15, and 25 into the striatum of SCID mice (Figures 3A and 3D). Six weeks later, the mice were sacrificed for analysis. Transplanted cells were identified by using antibodies specific for human nuclei (hNA) or human cytoplasm (STEM121). Figure 3 D). One month after transplantation, surviving cells were detected in the brains of mice receiving day 10 and day 15 hMOs. Figure 3 D, a to d). The vast majority of day 25 organoid grafts died within 1 month after transplantation. Figure 3 D, e). In contrast, day 10 hMOs showed good survival rates but produced only a small number of TH+ cells. Figure 3 D, a). Compared with day 10 and day 25 hMOs, day 15 hMOs achieved a balance between survival rate and maturation into DA neurons. Figure 3 D, b to d). Based on the above results, day 15 hMOs were selected for further study.

[0073] Before in vivo potency testing, the marker expression and cell type composition of day 15 hMOs were characterized. Immunofluorescence staining results showed that day 15 hMOs contained approximately 30.65% FOXA2-positive cells, 17.54% EN1-positive cells, and 10.04% OTX2-positive cells, while only a small fraction of post-mitotic DA markers NURR1 (2.06%) and TH (0.79%) were positive in the cells. Figure 8 At 35 days in vitro, the proportions of NURR1- and TH-positive cells increased to 50.44% and 32.22%, respectively. Figure 8 B, b).

[0074] Previous studies have shown that the fetal ventral mesencephalic tissue used in patient transplantation trials contains several non-dopaminergic neuronal populations, including GABAergic neurons, glutamatergic neurons, cholinergic neurons, and serotonergic neurons (S. C. Neto et al., Cell fate analysis of embryonic ventral mesencephalic grafts in the 6-OHDA model of Parkinson's disease. PLoS One 7, e50178, 2012). Excessive serotonergic neurons in the grafts result in adverse side effects, including graft-induced dyskinesias (T. Carlsson et al., Serotonin neuron transplants exacerbate L-DOPA-induced dyskinesias in a rat model of Parkinson's disease. J Neurosci 27, 8011-8022, 2007). Given the above problems, the inventors evaluated the composition of various cell types within hMO by staining cell lineage markers, including DARPP32 for medium spiny neurons (MSN), GAD67 for GABAergic, Vglut1 for glutamatergic, CHAT for cholinergic, and SEROTONIN for serotonergic neurons. None of these markers were detected in hMO (Figure 8C). Therefore, in addition to immunofluorescence staining, we also detected the expression of NKX2.2, NKX6.1, HOXA2, HOXB, GBX2, and GATA3 at the transcriptional level, which are important transcription factors for determining serotonergic neurons (M. P. Smidt et al., Subset specification of central serotonergic neurons. Front Cell Neurosci 7, 200, 2013), and no evidence was found to support the expression of these genes ( Figure 8 D). Regarding the oligodendrocyte lineage, a small number of Olig2+ cells (<1%) were observed in the organoids at day 15 by immunofluorescence staining, and a small number of O4+ cells were identified in the organoids at day 35. No MBP+ mature oligodendrocytes were observed in the organoids at day 15 and day 35 ( Figure 8 C, a, b, d).

[0075] Example 4 Establishment of PD Model and hMO Transplantation

[0076] All animal experiments were conducted in accordance with the 3R principles (Reduction, Refinement, Replacement) and approved by the Ethics Committee of Xuanwu Hospital, Capital Medical University. This study included 86 SCID mice (8 - 12 weeks old, Charles River, Beijing, China), among which 76 were used to establish a PD model according to the previously reported method (Y. Yuan et al., Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model. Theranostics 8, 4679 - 4694, 2018). Briefly, 2 μl of 6-OHDA (5 mg / ml, in saline containing 0.2% ascorbic acid) was directly injected into the right striatum (anteroposterior [AP] = +0.5 mm, lateral [L] = -2.1 mm, vertical [V] = -3.2 mm). Four weeks after 6-OHDA lesion, 26 animals that showed more than 100 rotations within 30 minutes after apomorphine induction were selected for transplantation experiments.

[0077] Ten of the 26 6-OHDA-lesioned mice were assigned to the vehicle control group that received only buffer injection into the striatum. The remaining 16 PD mice were assigned to the hMO transplantation group. hMOs on the 15th day were transplanted into the striatum of unilaterally 6-OHDA-lesioned SCID PD mice ( Figure 4 A). Three time points after hMO transplantation were selected, namely 6, 12, and 16 weeks after transplantation, and the mice were sacrificed for histological analysis. The number of mice at each time point was as follows: 6 weeks (n = 4, group A), 12 weeks (n = 8, group B), and 16 weeks (n = 4, group C). During the study, one of the 16 mice died accidentally. The remaining 15 transplanted PD mice all showed graft survival, and the number of mice at each time point was as follows: 6 weeks (n = 4), 12 weeks (n = 8), and 16 weeks (n = 3). Organoids corresponding to 2×10 5 cells were cut into small pieces (about 0.2 mm per piece) by physical means 3 ) and resuspended in 4 μl of transplantation buffer containing Rock inhibitor (0.5 μM), vitamin A-free B27, and BDNF (20 ng / ml), and then injected into the right striatum ([AP] = +0.5 mm, lateral [L] = -2.1 mm, vertical [V] = -3.0 mm). The injection rate was 0.5 μl / min, and the needle was placed in situ for another 5 minutes and then slowly retracted.

[0078] At 6, 12, and 16 weeks after transplantation, the mice were sacrificed for histological analysis. The transplanted cells were identified by staining for hNA and human neural cell adhesion molecule (hNCAM). Differentiation and maturation of the transplanted organoids in vivo were confirmed by co-labeling mDA-specific markers, including FOXA2, NURR1, TH, and the A9 region-specific DA neuron marker GIRK2( Figure 4 B, a to c). At 6 weeks after transplantation, the transplanted cells were negative for OCT4 and KI67, indicating a low risk of tumorigenesis( Figure 4 B, d).

[0079] At 6 weeks after transplantation, approximately 2.57% of the surviving hNA+ cells were co-labeled with TH( Figure 4 C, d); at 12 and 16 weeks after transplantation, the proportions of transplanted cells (hNA+) co-expressing TH were 14.11% and 7.33%, respectively (Figure 4C, d). Compared with 6 weeks, the TH+ grafts at 12 and 16 weeks after transplantation showed a more mature morphology, with larger somata and more extensive dendritic structures( Figure 4 C, a to c), indicating that the grafts gradually matured over time.

[0080] Example 5 Transplantation of iPSC-derived midbrain organoids improves motor function in PD mice

[0081] To examine the efficacy of the grafts in restoring motor function in PD mice, behavioral tests, including apomorphine (APO)-induced rotation, open field test, and rotarod test, were performed. The specific test methods are as follows.

[0082] APO-induced rotation test: Apomorphine (APO)-induced rotation was studied at different time points before and after transplantation of human midbrain organoids (hMO). Rotation was recorded 5 minutes after intradermal injection of APO (10 mg / ml in saline, 0.5 mg / kg). Analysis was performed using the total net rotation count (contralateral minus ipsilateral) within 30 minutes.

[0083] Open field test: Spontaneous locomotion was evaluated by measuring the average speed and total distance moved using an automated open field system. Mice were acclimated to the open field arena (25.4 cm × 25.4 cm) for 10 minutes before the test, followed by a 30-minute observation period. Each animal was tested 1 week before organoid transplantation and retested at 6 and 12 weeks after transplantation. The open field measurements of the total distance moved were in meters (m).

[0084] Rotarod test: The passive motor coordination function was tested using a Harvard Instruments rotarod. To obtain stable performance, all animals were pre-trained for two days. On day 1, mice were trained on a rotarod rotating at 2 to 20 revolutions per minute (rpm) for 300 seconds three times. On day 2, mice were trained on a rod that accelerated twice from 3 rpm to 30 rpm and once from 4 rpm to 40 rpm within 300 seconds. The test started on the third day, using a rotarod that accelerated from 4 to 40 rpm within 300 seconds. The duration of staying on the rod was measured. For data analysis, the average length of three repeated tests for each animal was used. Statistical analysis was performed using GraphPad. For multiple comparisons, two-way ANOVA and Bonferroni's post hoc test were used.

[0085] Results showed that PD mice receiving hMO grafts showed statistically significant improvement in all three tests, which was not observed in the vehicle control group ( Figure 4 C, e to g). The results indicate that transplantation of hMO can restore motor function impaired by 6-OHDA lesion.

[0086] Example 6 Transplanted neurons gradually send out axonal projections to innervate the target brain regions

[0087] For the functional recovery of brain-lesioned mice, specific axonal projections and integration into the host neuronal circuitry are crucial (K.S. Ziemba et al., Targeting axon growth from neuronal transplants along preformed guidance pathways in the adult CNS. J Neurosci 28, 340 - 348, 2008). To analyze the projections from the grafts, we used a human cell-specific hNCAM antibody to label the transplanted cells and their projections. During the examined time period, the graft mass mostly remained in the same position; however, these processes (dendrites and axons) extended to positions away from the transplant core ( Figure 5 A). Brains collected at 6 and 12 weeks after transplantation were coronally sectioned and analyzed along the anterior-posterior (A-P) axis.

[0088] At 6 weeks, some hNCAM+ fibers extended from the striatal transplant area along the corpus callosum (fa / CC) to the cerebral cortex ( Figure 5B, a), is mainly located in the primary somatomotor cortex (MO) of the prefrontal cortex (PFC), which is a natural target area for A9 or A10 neurons. No hNCAM+ fibers were observed to enter the primary somatosensory (SS) area. On the other hand, a portion of hNCAM+ fibers extended posteriorly and entered the globus pallidus (GP), the ventral posterolateral nucleus of the thalamus (VPL), and the medial forebrain bundle (MFB)( Figure 5 B, c and d). Some hNACM+ fibers continued to grow through the midbrain (MB) motor-related nuclei, such as the mesencephalic reticular nucleus (MRN) and the retrorubral flexor (fr), and entered the parafascicular nucleus (SPF) and the posterior complex of the thalamus (TH)( Figure 5 B, e; the white boxes in B-e correspond to I, II, and III). Some hNCAM+ fibers eventually extended to the posterior cerebral peduncle (cpd)( Figure 5 B, e; the white box in B-e corresponds to IV).

[0089] By 12 weeks, obvious hNCAM+ projections were visible covering the claustrum (CLA) and the piriform nucleus (EP), and a few fibers reached the rostral olfactory bulb (OT) and the substantia innominata (SI)( Figure 5 C, a and I). Analysis of more rostral structures, including the dorsolateral / ventral striatum (Strd / Strv), the nucleus accumbens (NAc / ACB), showed that the Strv and NAc regions (which are mainly A10 targets) lacked obvious graft-derived innervation. The graft was placed in the dorsal striatum( Figure 5 C, b, c, and III), which has been confirmed to be closely related to the improvement of motor function in cell therapy research. Careful observation of the direction of axon extension revealed that several hNCAM fibers entered the contralateral cortex along the corpus callosum (CC)( Figure 5 C, II). Compared with the mice at 6 weeks, a significant increase in the hNCAM+ terminal network in Strd was seen, closer to the substantia nigra (SN)( Figure 5 C, d, IV). A small portion of fibers extended to the reticular / compact parts of the substantia nigra (SNr / SNc) and the ventral tegmental area( Figure 5 C, e and V; Figure 6 A, d to f).

[0090] Example 7 Graft-derived dopaminergic neurons innervate the host circuit

[0091] Histological analysis showed the degeneration of unilateral endogenous TH-positive midbrain dopaminergic neurons in the SN and Str of the vehicle control group mice, indicating a complete Str lesion( Figure 9A), which is consistent with the rotation asymmetry results induced by APO. To examine the growth of DA fibers, we double-stained TH and hNCAM sections (Figure 6A). At 6 weeks post-transplantation, some TH+ / hNCAM+ fibers extended posteriorly and entered the globus pallidus (PAL) composed of the internal and external globus pallidus ( Figure 6 A, a to c). At 12 weeks post-transplantation, a small portion of TH+ / hNCAM+ fibers continued to extend and entered the ipsilateral substantia nigra reticulata / compacta (SNr / SNc) and ventral tegmental area ( Figure 6 A, d to f).

[0092] The functional recovery in the graft area depends not only on the survival of DA neurons and the increase in dopamine release, but also on the integration of the graft with the host neuronal circuitry to achieve long-term remission of PD-related motor symptoms (A.F. Adler et al., hESC-Derived Dopaminergic Transplants Integrate into Basal Ganglia Circuitry in a Preclinical Model of Parkinson's Disease. Cell Rep 28, 3462-3473.e3465, 2019). In this example, synaptophysin antibodies that can recognize human and mouse species were used to identify the formed synapses. The staining showed that graft-derived TH+ neurons had received synaptic inputs from host (mouse) and / or transplanted (human) neurons ( Figure 6 B, a and b). In addition, midbrain DA neurons naturally project to medial spiny neurons (MSN) in the striatum. To examine whether the transplanted neurons had formed synapses, DARPP32 (a marker of MSN), TH, and synaptophysin were stained ( Figure 6 B, c and d; Figure 10 C). The results showed that the transplanted DA neurons were able to form synapses with endogenous MSN.

[0093] Example 8 Retrograde Tracing Reveals Axonal Growth Patterns

[0094] Cholera toxin B subunit (CTB) is a retrograde axonal tracer that was microinjected into the rostral prefrontal cortex (PFC) or posterior region hypothalamus (HY) of different groups of mice. CTB deposits within the expected target sites were observed by CTB-specific antibodies. To confirm that the CTB deposits were clearly separated from the graft itself, CTB and hNA were double-stained, and the results showed that there were no transplanted cells at the CTB injection site (data not shown). Two weeks later, brain sections were double-stained with CTB and hNA or hNCAM, and CTB-labeled grafts were detected in the striatum ( Figure 7A), indicating that the transplanted cells had projected into the PFC and HY four weeks after transplantation.

[0095] The selective retrograde spread of rabies virus through individual synapses allows for the precise identification of host presynaptic inputs onto transplanted cells. To achieve monosynaptic retrograde transmission, hiPSC-derived midbrain organoids expressing a lentiviral vector (rabies virus helper construct) were transplanted into the midbrain ( Figure 7 B, a; Figure 10 A), and this lentiviral vector could encode TVA (avian sarcoma leukosis virus subgroup A), RVG (rabies virus glycoprotein), and GFP. The tracing vector and the control vector were purchased from AddGene (IDs 30195 and 30456 respectively, Watertown, MA). Human iPSCs (passages 30 - 35) were transduced with the lentiviral expression tracing vector or the control vector, and the successfully transduced cells were confirmed by puromycin selection and GFP expression. The titer of the control virus was 4×10 8 TU / mL; the tracer virus was 2×10 8 TU / mL, and RV-ENVA-G-mCherry G-rabies virus (BrainVTA, Wuhan, China) was 2×10 8 TU / ml. In vivo injections were performed using a working dilution of 0.1 - 0.5% (about 1 μl per mouse). Histone-tagged GFP can be used for starting cell confirmation; TVA receptor and RVG can promote the monosynaptic retrograde spread of rabies virus after the initial infection with EnvA-pseudotyped rabies virus ( Figure 7 B, b). The inventors employed a control vector that included GFP and TVA but lacked RVG simultaneously to control for non-specific labeling. Four weeks after transplantation, differentiated dopaminergic neurons co-labeled with GFP and TH were observed at the implantation site ( Figure 10 B). To label the synaptic inputs onto the transplanted cells (here called starting neurons), EnvA-pseudotyped G-rabies virus was injected into the same site as the hMO graft ( Figure 7 C, a).

[0096] GFP expression was confirmed in the starting cells ( Figure 10A). After infection with a G-rabies virus vector expressing the same mCherry, the starting neurons expressing the TVA receptor are infected by the G-rabies virus and are easily recognized by the co-expression of GFP and mCherry. The G-rabies virus can assemble into infectious particles in the starting cells because these neurons express RVG. Since rabies virus prefers to spread retrogradely through active synapses, each neuron forming a presynaptic connection with the starting neuron, called a traced neuron, will be transduced with mCherry (G. Ugolini et al., Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups. J Comp Neurol 356, 457-480, 1995). Therefore, only red fluorescence (mCherry, but not GFP expression) can be recognized in the traced neurons connected to the starting neurons. Mice were microinjected with pseudotyped rabies virus and sacrificed after one week for histological analysis. Transplanted neurons successfully infected with pseudotyped rabies virus were detected, and traced neurons located inside the grafts as well as several brain regions (including the PFC, fimbria (fi), Strd, lateral hypothalamic area (LHA), and MB) were identified ( Figure 7 C, c to f).

[0097] Based on the results of Examples 1-8 above, the hiPSC-derived midbrain organoids of the present invention were transplanted into an immunodeficient mouse model of Parkinson's disease. After implantation, the midbrain organoids matured into FOXA2 / NURR1 / TH / GIRK2 immunoreactive DA neurons, exhibited electrophysiological activity, and improved the motor function of PD mice. The transplanted midbrain organoids showed strong neurite / axon extension and bidirectional synaptic connections with host neurons.

[0098] Compared with the ethical issues, scarce sources, and transplantation heterogeneity shown in the existing research on using fVM to treat PD, the midbrain organoids of the present invention show advantages in terms of ethics, sources, and transplantation. hMOs derived from iPSCs can well solve the ethical and source problems related to embryo-derived fVM tissues. In terms of the heterogeneity problem, hMOs are also advantageous and can be applied to autologous transplantation or isologous transplantation.

[0099] In terms of cell composition, in the fVM tissue, neurons account for only about 5.6% of the total cells, and most cells are astrocytes, microglia, endothelial cells, etc. (F.A. Azevedo et al., Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol 513, 532 - 541, 2009). Among neurons, approximately 70% are DA lineage cells, 30% are GABAergic cells, and 2 - 3% are glutamatergic cells (M.A. Ungless et al., Are you or aren't you? Challenges associated with physiologically identifying dopamine neurons. Trends Neurosci 35, 422 - 430, 2012). Given the technical difficulties in simply isolating the required ventral midbrain region in small embryos at 7 - 9 weeks after conception, other contaminating cells are often detected in the grafts, such as serotonergic neurons, which are thought to be associated with severe side effects, such as dyskinesia. The 15-day hMOs derived from iPSCs provided by the present invention mainly consist of neuronal lineage cells, and no astrocytes or oligodendrocytes are detected ( Figure 8 C); in the 15-day hMOs, more than 30% of these neuronal cells are DA lineage cells (30.64% of FOXA2+, 17.54% of EN1+, 10.04% of OTX2+, 2.10% of NURR1+, and 0.80% of TH+; Figure 8 B, a) and no GABAergic cells, glutamatergic cells, or serotonergic cells are found ( Figure 8 C, f). For serotonergic lineage cells that may cause severe side effects such as dyskinesia, we also examined genes related to serotonergic cell specification during development at the transcriptional level, and none reached detectable levels ( Figure 8 C, e and Figure 8 D).

[0100] iPSC-derived DA precursors in two-dimensional culture are also able to address some of the above-mentioned problems associated with fVM tissue. However, the biggest concern regarding iPSC derivatives is the tumorigenic risk associated with incomplete differentiation of pluripotent stem cells. Compared with single-cell culture, the development of organoids also depends on internal structural contact / support and intrinsic signals that coordinate / synchronize the development of all relevant cells at the organ / tissue level. This process is more similar to embryonic development, and the resulting organoids have a relatively low tumorigenic risk comparable to fVM tissue. As revealed in the examples of the present invention (especially Example 4), four months after transplantation into the brains of immunodeficient mice, no tumor formation or graft overgrowth was observed, and the grafts were negative for KI67 and OCT4 ( Figure 4 B, d), confirming that the tumorigenic risk of the midbrain organoids of the present invention is at a low level.

[0101] Previous studies have shown that iPSC-derived DA cells cultured in a two-dimensional manner are different from DA cells in fVM tissue. Compared with fVM transplantation, a larger number of iPSC-derived DA cells cultured in a two-dimensional manner are required to achieve a similar level of motor function recovery. Approximately 500–700 surviving DA neurons from fVM are sufficient to reverse drug-induced rotation (A. Rath et al., Survival and functional restoration of human fetal ventral mesencephalon following transplantation in a rat model of Parkinson's disease. Cell Transplant 22, 1281-1293, 2013); while it seems that thousands of iPSC-derived two-dimensional cultured DA neurons are required to completely reverse drug-induced rotation (G. Hargus et al., Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc Natl Acad Sci U S A 107, 15921-15926, 2010). The higher efficiency of DA cells in fVM is related to their greater arborization ability: viable DA neurons from fVM can extend their processes up to 6 mm and arborize almost the entire striatum (A. Rath et al., Survival and functional restoration of human fetal ventral mesencephalon following transplantation in a rat model of Parkinson's disease. Cell Transplant 22, 1281-1293, 2013); in contrast, iPSC-derived two-dimensional cultured DA neurons extend their processes to 2-3 mm and only about 10% of the striatum is dendritic (P. Brundin et al., Behavioural effects of human fetal dopamine neurons grafted in a rat model of Parkinson's disease. Exp Brain Res 65, 235-240, 1986).In the current study, hMO generated approximately 200 - 800 viable DA neurons and could reverse apomorphine - induced rotation at 6 weeks post - transplantation (Figure 4C, e). In addition, hMO - derived DA neurons not only arborized throughout the striatum but also sent projections to the midbrain, thalamus, and hypothalamic regions ( Figure 5 B and C), and received afferent projections from the prefrontal cortex, midbrain, and hypothalamic regions ( Figure 7 C). hMO has greater similarity to native SN A9 DA neural progenitor / precursor cells, and this intrinsic proximity and a more homogeneous population with fewer contaminating cells endow hMO with a stronger ability to establish bidirectional synaptic connections with natural projection targets in the brain.

Claims

1. Use of midbrain organoids in the preparation of a medicament for treating Parkinson's disease, wherein the midbrain organoids are prepared from pluripotent stem cells, the pluripotent stem cells are induced pluripotent stem cells, and the midbrain organoids do not contain serotonergic neurons, wherein the pluripotent stem cells are human induced pluripotent stem cells, wherein the midbrain organoids are prepared by the following method: (1) Neural induction stage: Dissociate the induced pluripotent stem cells into single cells and form uniform embryoid bodies, and culture the embryoid bodies in medium A containing heparin, SB431542, Noggin, CHIR99021, Y27632. After 45 - 50 hours, replace medium A with medium B containing heparin, SB431542, Noggin, CHIR99021 but not containing Y27632 and continue culturing to obtain culture a; (2) Midbrain patterning stage: Add SHH - C25II and FGF8 to medium B to form medium C, and continue culturing culture a for midbrain patterning to obtain culture b, wherein the concentration of SHH - C25II is 150 - 250 ng / ml; (3) Laminarization stage: After culture b extends neural ectodermal buds, completely remove medium C, add low - growth - factor Matrigel for embedding, and grow in medium D containing insulin, laminin, SHH - C25II, FGF8 after the low - growth - factor Matrigel solidifies to obtain culture c, wherein the concentration of SHH - C25II is 150 - 250 ng / ml; (4) Final differentiation stage: Transfer culture c to medium E containing BDNF, GDNF, ascorbic acid, cAMP for culturing to obtain midbrain organoids; Among them, The total duration of each stage of the preparation method is 10 - 25 days.

2. The use according to claim 1, wherein the proportion of cells expressing FOXA2 in the midbrain organoids is 30%, the proportion of cells expressing EN1 is 18%, the proportion of cells expressing OTX2 is 10%, the proportion of cells expressing NURR1 is 2%, and the proportion of cells expressing TH is 0.8%.

3. The use according to claim 1, wherein the midbrain organoids are cut into small pieces of 0.1-2 mm before treating Parkinson's disease and resuspended in a transplantation buffer containing 0.1-1 μM Rock inhibitor, vitamin A-free B27, and 10-30 ng / ml BDNF. 3 ​ 4. The use according to claim 1, wherein the midbrain organoids are midbrain organoids cultured in vitro for 10 - 25 days.

5. The use according to claim 4, wherein the midbrain organoids are midbrain organoids cultured in vitro for 15 days.

6. The use according to claim 1, wherein the midbrain organoids have the following characteristics: (1) Positive staining for NESTIN, SOX2, TUJ1, KI67, OTX2, FOXA2, EN1, NURR1 and TH, wherein the proportion of cells expressing FOXA2 is 20 - 40%, the proportion of cells expressing EN1 is 10 - 30%, the proportion of cells expressing OTX2 is 5% - 15%, the proportion of cells expressing NURR1 is 0.01 - 5%, and the proportion of cells expressing TH is 0.01 - 5%; (2) The midbrain organoids are negative for staining of DARPP32, GAD67, Vglut1, CHAT, and SEROTONIN; (3) The midbrain organoids do not express FOXG1, NKX2.2, NKX6.1, HOXA2, HOXB, GBX2, and GATA3; (4) The midbrain organoids exhibit apical-basal polarity.

7. The use according to claim 6, wherein each stage of the preparation method is carried out for 15 days.

8. A midbrain organoid, characterized in that: The midbrain organoid is prepared by the following method: (1) Neural induction stage: Dissociate induced pluripotent stem cells into single cells and form homogeneous embryoid bodies, and culture the embryoid bodies in medium A containing heparin, SB431542, Noggin, CHIR99021, and Y27632. After 45 - 50 hours, replace medium A with medium B containing heparin, SB431542, Noggin, CHIR99021 but without Y27632 and continue culturing to obtain culture a; (2) Midbrain patterning stage: Add SHH-C25II and FGF8 to medium B to form medium C, and continue culturing culture a for midbrain patterning to obtain culture b, wherein the concentration of SHH-C25II is 150 - 250 ng / ml; (3) Lamination stage: After culture b extends neural ectodermal buds, completely remove medium C, add low-growth factor Matrigel for embedding, and grow in medium D containing insulin, laminin, SHH-C25II, and FGF8 after the low-growth factor Matrigel solidifies to obtain culture c, wherein the concentration of SHH-C25II is 150 - 250 ng / ml; (4) Final differentiation stage: Transfer culture c to medium E containing BDNF, GDNF, ascorbic acid, and cAMP for culturing to obtain midbrain organoids; Wherein, each stage of the preparation method is carried out for 10 - 25 days; Wherein, the midbrain organoid is derived from human induced pluripotent stem cells.

9. The midbrain organoid according to claim 8, wherein the proportion of cells expressing FOXA2 in the midbrain organoid is 30%, the proportion of cells expressing EN 1 is 18%, the proportion of cells expressing OTX2 is 10%, the proportion of cells expressing NURR1 is 2%, and the proportion of cells expressing TH is 0.8%.

10. The midbrain organoid according to claim 9, wherein each stage of the preparation method is carried out for 15 days.

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