A method for differentiating pluripotent stem cells into midbrain nigral dopaminergic neural cells

Abstract

A specific method for differentiating pluripotent stem cells into midbrain substantia nigra dopaminergic (A9 mDA) neural cells is provided. The differentiation forms mature A9 mDA neurons that express surface molecular markers of midbrain substantia nigra dopaminergic neurons, including TH, FOXA2, EN1, LMX1A, NURR1, and GIRK2, while expressing little of the marker CB for ventral tegmental area dopaminergic neurons. The A9 mDA neural cells are transplanted into the substantia nigra, and their axons specifically project to the target brain region dorsal striatum innervated by endogenous substantia nigra dopaminergic neurons; the transplanted A9 mDA neurons themselves exhibit the classic electrophysiological properties of endogenous substantia nigra dopaminergic neurons, including low frequency of spontaneous firing and sag induced by hyperpolarizing current stimulation. Transplanting A9 mDA neural cells into the substantia nigra or striatum of a subject with a neurodegenerative disease can improve motor dysfunction.

Description

Technical Field

[0001] This invention belongs to the field of neural stem cell science, and more specifically, this invention relates to a method for differentiating pluripotent stem cells into dopaminergic neurons in the substantia nigra of the midbrain. Background Technology

[0002] Parkinson's disease (PD) is a long-term degenerative disease of the central nervous system, with an average age of onset of 60 years and a prevalence of 1.7% among people over 65. International epidemiological surveys show that in 2005, China had approximately 2 million PD patients, accounting for half of the global total. It is projected that by 2030, China will have nearly 5 million PD patients. Patients with Parkinson's disease often exhibit motor disorders, such as resting tremor, increased muscle tone, bradykinesia, and postural instability. The pathological basis of Parkinson's disease is the degeneration and loss of dopaminergic neurons in the substantia nigra of the midbrain, resulting in a significant decrease in striatal dopaminergic levels, thus leading to motor dysfunction.

[0003] Dopaminergic neurons, named for their ability to secrete the catecholamine neurotransmitter dopamine, are widely distributed throughout the brain. Dopaminergic neurons are characterized by tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, which catalyzes the conversion of tyrosine to L-DOPA. In the midbrain, dopaminergic neurons are primarily located in three nuclei: the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA), and the retrorubral field (RrF). SNc dopaminergic neurons are also known as A9 mDA neurons, VTA neurons as A10 mDA neurons, and RrF neurons as A8 mDA neurons; the A8-10 designations are based on anatomical location. A9 mDA neurons and A10 mDA neurons are two important subtypes of midbrain dopaminergic neurons (mDA). Besides their different locations, these two types of neurons also differ in their projection circuits. A9 mDA neurons primarily project to the dorsolateral striatum, forming the mesostriatal pathway, which mainly regulates motor function and is a subgroup of dopaminergic neurons specifically lost in Parkinson's disease. A10 mDA neurons primarily project to the nucleus accumbens, cortex, olfactory cortex, amygdala, etc., forming the mesolimbic cortex pathway, and are involved in regulating reward and emotion functions.

[0004] A9 mDA neurons and A10 mDA neurons exhibit different susceptibility in Parkinson's disease. In the brains of Parkinson's patients, A9 mDA neurons in the SNc (subsynaptic fossa) degenerate preferentially, with cells on the ventral side of the SNc being more sensitive than those on the dorsal side, while A10 mDA neurons in the adjacent VTA (ventral synaptic plexus) are relatively unaffected. The intracellular calcium-binding protein calbindin (CALB) is used as a marker for A10 mDA neurons. Calbindin is used to distinguish antagonistic neurons in dopaminergic neurons in the brains of PD patients and PD model animals, most of which are A10 mDA neurons. Another protein, the G-protein-gated inward rectifier potassium channel (GIRK), regulates the activity of dopaminergic neurons by activating D2 or GABA receptors to generate a slow inhibitory postsynaptic potential. GIRK2 is specifically expressed in susceptible dopaminergic neurons (mostly A9 mDA neurons).

[0005] There is currently no effective cure for Parkinson's disease. Current treatments can only improve symptoms but cannot stop the progression of the disease. Drug therapy is the primary treatment for Parkinson's disease, aiming to achieve therapeutic effects by supplementing dopamine or enhancing dopamine receptor function. Levodopa (L-DOPA) remains the most effective drug; however, it is only effective in the early stages of PD. As dopaminergic neurons are further lost, the drug gradually becomes ineffective and causes significant side effects. Deep brain stimulation (DBS) has also been used to treat Parkinson's patients. However, like drug therapy, surgery can only improve symptoms and cannot cure the disease. Moreover, due to the side effects of DBS, this treatment is only suitable for some patients. Exogenous transplantation of dopaminergic neurons to replace the function of lost dopaminergic neurons in the brain (stem cell therapy) is one of the promising treatment methods. Clinical trials have shown that transplanting cells from the ventral midbrain of aborted fetuses (the brain region containing midbrain dopaminergic progenitor cells) into the striatum of patients can lead to long-term recovery of motor function in some patients, requiring little or no medication. Transplanted neural progenitor cells can differentiate into dopaminergic neurons and release dopaminergic receptors (DA) [16,17]. These clinical studies demonstrate the great potential of stem cell therapy in the treatment of Parkinson's disease (PD). However, the source of brain tissue from aborted fetuses is limited, and ethical issues exist. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to differentiate into all types of cells throughout the body, making them an ideal cell source for obtaining various functional cells for treatment. Following the principles of in vivo development, human pluripotent stem cells can be induced to differentiate into midbrain dopaminergic neurons. These cells, when transplanted into the striatum of PD model mice, can survive and salvage the behavioral performance of Parkinson's disease, bringing new hope to the treatment of Parkinson's disease.

[0006] However, none of these studies clearly distinguished the subtypes (A9 / A10) of midbrain dopaminergic neurons in the transplanted cells. As mentioned above, different endogenous dopaminergic neuron subtypes have completely different electrophysiological characteristics, innervated brain regions, and physiological functions. Therefore, there is an urgent need to develop differentiation methods targeting different subtypes of midbrain dopaminergic neurons, especially for cell therapy of Parkinson's disease, requiring the development of efficient differentiation methods for enriching midbrain substantia nigra dopaminergic neurons. Furthermore, the subtype specificity of the differentiated dopaminergic neurons needs to be verified at multiple levels, including the expression of biomarker molecules, electrophysiological characteristics, and axonal projection characteristics. Summary of the Invention

[0007] The purpose of this invention is to provide a specific method for differentiating pluripotent stem cells into dopaminergic neurons in the substantia nigra of the midbrain, as well as the cells or cell preparations obtained by this method.

[0008] In a first aspect of the invention, a method for preparing dopaminergic neurons in the substantia nigra of the midbrain is provided, comprising: (1) placing stem cells in a culture medium containing a neuroinducing agent and inducing them in multiple stages with added components; and (2) obtaining the stem cell-derived dopaminergic neurons in the substantia nigra of the midbrain from the culture.

[0009] In a preferred embodiment, (1) induction is performed in multiple stages with different added components: first stage: addition of SB431542, DMH-1, SHH and CHIR99021; second stage: addition of SAG, SHH and CHIR99021; third stage: addition of SHH, SAG and FGF8b; fourth stage: addition of SHH and FGF8b.

[0010] In another preferred embodiment, in the first stage, 1–15 μM (e.g., 2, 5, 8, 12, 14 μM) of SB431542, 1–5 μM (e.g., 0.4, 0.6, 1, 3, 5, 10, 15, 18 μM) of DMH-1, 200–1000 ng / mL (e.g., 300, 400, 600, 700, 800, 900 ng / mL) of SHH, and 0.1–1 μM (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 μM) of CHIR99021 are added.

[0011] In a more preferred embodiment, in the first stage, 10±5 μM of SB431542, 2±1 μM of DMH-1, 500±200 ng / ml of SHH, and 0.4±0.2 μM of CHIR99021 are added. More preferably, 10±2 μM of SB431542, 2±0.5 μM of DMH-1, 500±100 ng / ml of SHH, and 0.4±0.1 μM of CHIR99021 are added.

[0012] In another preferred embodiment, in the second stage, 0.1–5 μM (e.g., 0.2, 0.5, 0.8, 1, 2, 3, 4 μM) of SAG, 50–300 ng / ml (e.g., 80, 100, 150, 200, 250 ng / ml) of SHH, and 0.1–1 μM (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 μM) of CHIR99021 are added.

[0013] In a more preferred embodiment, in the second stage, 2±1 μM of SAG, 100±50 ng / ml of SHH, and 0.4±0.2 μM of CHIR99021 are added. More preferably, 2±0.5 μM of SAG, 100±20 ng / ml of SHH, and 0.4±0.1 μM of CHIR99021 are added.

[0014] In another preferred embodiment, in the third stage, 5–100 ng / ml (e.g., 10, 15, 20, 30, 40, 50, 60, 70, 80 ng / ml); preferably 5–50 ng / ml) of SHH, 0.1–5 μM (e.g., 0.2, 0.5, 0.8, 1, 2, 3, 4 μM) of SAG, and 5–200 ng / ml (e.g., 10, 15, 20, 40, 60, 80, 100, 150, 200, 250 ng / ml) of FGF8b are added.

[0015] In a more preferred embodiment, in the third stage, 20±10 ng / ml of SHH and 0.5±0.2 μM of SAG, and 100±50 ng / ml of FGF8b are added. More preferably, 20±5 ng / ml of SHH and 0.5±0.1 μM of SAG, and 100±20 ng / ml of FGF8b are added.

[0016] In another preferred embodiment, in the fourth stage, 5–100 ng / ml (e.g., 10, 15, 20, 30, 40, 50, 60, 70, 80 ng / ml) of SHH and 5–80 ng / ml (e.g., 10, 15, 20, 30, 40, 50, 60, 70 ng / ml) of FGF8b are added.

[0017] In a more preferred embodiment, in the fourth stage, 20±10 ng / ml of SHH and 20±10 ng / ml of FGF8b are added. More preferably, 20±3 ng / ml of SHH and 20±3 ng / ml of FGF8b are added.

[0018] In another preferred embodiment, the stages are as follows: Stage 1: from the start of cultivation to 6-8 days; preferably 7 ± 0.5 days; Stage 2: from 6-8 days to 11-13 days; preferably 12 ± 0.5 days; Stage 3: from 11-13 days to 18-20 days; preferably 19 ± 0.5 days; Stage 4: from 18-20 days to 31-33 days; preferably 32 ± 0.5 days.

[0019] In another preferred embodiment, the stem cells include: embryonic stem cells or induced pluripotent stem cells; preferably, the stem cells are human stem cells, and the embryonic stem cells or induced pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0020] In another aspect of the invention, a midbrain substantia nigra dopaminergic neuron is provided, which is prepared by any of the methods described above.

[0021] In another aspect of the invention, a midbrain substantia nigra dopaminergic neuron is provided, wherein the midbrain substantia nigra dopaminergic neuron expresses surface molecular markers of midbrain substantia nigra dopaminergic neurons, including tyrosine hydroxylase (TH), FOXA2, EN1, LMX1A, NURR1 and / or GIRK2, after 5 to 10 days (e.g., 6, 7, 8, 9 days) after differentiation, while rarely expressing the ventral tegmental dopaminergic neuron marker CALB (CB).

[0022] In another preferred embodiment, the differentiation is in vitro differentiation, which includes: in vitro attachment and maturation, differentiating into mature midbrain substantia nigra dopaminergic neurons.

[0023] In another preferred embodiment, the differentiation is in vivo differentiation, including in vivo maturation.

[0024] In another preferred embodiment, the low expression of the ventral tegmental dopaminergic neuron marker CALB includes: less than 20%, more preferably less than 15%, more preferably less than 12%, 10%, and 8% CALB expression.

[0025] In another preferred embodiment, after the midbrain substantia nigra dopaminergic neurons are transplanted into the substantia nigra region of the brain, the axons of the A9 midbrain dopaminergic (mDA) neurons differentiated after transplantation specifically project to the target brain region innervated by endogenous substantia nigra dopaminergic neurons—the dorsal striatum.

[0026] In another preferred embodiment, the A9 midbrain dopaminergic neurons differentiated after transplantation exhibit the classic electrophysiological characteristics of endogenous substantia nigra dopaminergic neurons, including low-frequency spontaneous firing and the ability of hyperpolarizing current stimulation to induce sag.

[0027] In another preferred embodiment, the A9 midbrain dopaminergic neurons differentiated after transplantation into the substantia nigra or striatum of the brain can improve motor dysfunction.

[0028] In another preferred embodiment, the midbrain substantia nigra dopaminergic neurons are A9 midbrain dopaminergic cells; preferably, after 5 to 10 days (e.g., 6, 7, 8, 9 days) of differentiation, more than 80% express the A9 mDA neuronal marker GIRK2; more preferably, more than 85% express the A9 mDA neuronal marker GIRK2.

[0029] In another preferred embodiment, after 5 to 10 days of differentiation, more than 40% (e.g., 45%, 50%, 55%, 60%, 70%) of the total cells or more than 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%) of the TUJ1+ neurons have this characteristic; more preferably, more than 50% (e.g., 55%, 60%, 65%, 70%, 75%) of the total cells or more than 60% (e.g., 65%, 70%, 75%) of the TUJ1+ neurons have this characteristic.

[0030] In another aspect of the invention, a midbrain substantia nigra dopaminergic neuron is provided, which is derived from the differentiation of any of the aforementioned midbrain substantia nigra dopaminergic neurons; preferably, it expresses surface molecular markers of midbrain substantia nigra dopaminergic neurons, including TH, FOXA2, EN1, LMX1A, NURR1 and / or GIRK2, while rarely expressing the ventral tegmental dopaminergic neuron marker CALB (CB).

[0031] In another aspect of the invention, the use of any of the aforementioned midbrain substantia nigra dopaminergic neurons is provided in the preparation of formulations (including cell cultures or isolates) for the treatment of neurodegenerative diseases.

[0032] In another aspect of the invention, an agent for treating neurodegenerative diseases is provided, comprising: dopaminergic neurons in the substantia nigra of the midbrain as described above; and a pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, the formulation is also used as a graft (drug) for transplantation of the substantia nigra or striatum of the brain.

[0034] In another preferred embodiment, the formulation is also used to prevent and treat motor dysfunction.

[0035] In another preferred embodiment, the neurodegenerative disease includes (but is not limited to): Parkinson's disease, Alzheimer's disease, Lewy body dementia, Huntington's disease, amyotrophic lateral sclerosis, and nerve damage.

[0036] In another aspect of the present invention, a method is provided for screening substances (including potential substances) that improve neurodegenerative diseases, the method comprising: (1) treating a model system with a candidate substance, the model system being a model system of damaged brain neural circuits or impaired neural function, and containing midbrain dopaminergic neurons (cells); and, (2) testing the model system, if the candidate substance statistically promotes (significantly promotes, such as promoting more than 10%, more than 20%, more than 50%, more than 80%, etc.) dopaminergic neurons for damaged neural circuits in the brain or promotes their remodeling of neural function, then the candidate substance is a substance useful for repairing damaged neural circuits in the brain or remodeling neural function.

[0037] In another preferred embodiment, the model system described in step (1) is an animal model system, a tissue model system, an organ model system, or a cell (culture) model system.

[0038] In another preferred embodiment, step (2) includes: observing the effect of the candidate substance on midbrain dopaminergic neurons; if it can promote (significantly promote, such as promoting more than 10%, more than 20%, more than 50%, more than 80%, etc.) the repair of the substantia nigra-striatal pathway by midbrain dopaminergic neurons, then it is a substance useful for repairing damaged neural circuits or reshaping neural function in the brain.

[0039] In another preferred embodiment, step (2) includes: observing the effect of the candidate substance on midbrain dopaminergic neurons; if it can promote (significantly promote, such as promoting more than 10%, more than 20%, more than 50%, more than 80%, etc.) presynaptic and postsynaptic integration of midbrain dopaminergic neurons, then it is a substance useful for repairing damaged neural circuits or remodeling neural function in the brain.

[0040] In another preferred embodiment, step (2) includes: observing the effect of the candidate substance on midbrain dopaminergic neurons; if it can promote (significantly promote, such as promoting more than 10%, more than 20%, more than 50%, more than 80%, etc.) the projection of midbrain dopaminergic neuron axons to the dorsal side (Caudate putamen, CCu), then it is a substance useful for repairing damaged neural circuits or reshaping neural function in the brain.

[0041] In another preferred embodiment, step (2) includes: observing the effect of the candidate substance on midbrain dopaminergic neurons. If it can promote (significantly promote, such as promoting more than 10%, more than 20%, more than 50%, more than 80%, etc.) the growth of nerve fibers from midbrain dopaminergic neurons, specifically growing and extending to their endogenous target region - the striatum, forming neural connections with striatal neurons, and projecting to the striatum, then it is a substance useful for repairing damaged neural circuits or reshaping neural function in the brain.

[0042] In another preferred embodiment, step (2) includes: the model system is an animal system, the animal has motor dysfunction, and also includes: observing the animal's motor ability, if the candidate substance further improves (significantly improves, such as by more than 10%, more than 20%, more than 50%, more than 80%, etc.) its motor dysfunction, then it is a substance useful for repairing damaged neural circuits in the brain or reshaping neural function.

[0043] In another preferred embodiment, step (2) includes: step (1) includes: treating a model system with the candidate substance in the test group; and / or, step (2) includes: detecting the effect of dopaminergic neurons in the system on damaged neural circuits in the brain or their remodeling neural function, or detecting the repair effect of midbrain dopaminergic neurons on the substantia nigra-striatal pathway, or detecting the presynaptic and postsynaptic integration of midbrain dopaminergic neurons, or observing the motor dysfunction of animals; and comparing with a control group, wherein the control group is an expression system without the addition of the candidate substance; if the candidate substance statistically promotes the effect of dopaminergic neurons on damaged neural circuits in the brain or their remodeling neural function, or promotes the repair effect of midbrain dopaminergic neurons on the substantia nigra-striatal pathway, or promotes the presynaptic and postsynaptic integration of midbrain dopaminergic neurons, or improves the motor dysfunction of animals, then the candidate substance is a substance useful for repairing damaged neural circuits in the brain or remodeling neural function.

[0044] In another preferred embodiment, the screening method does not include methods with the direct purpose of treating the disease.

[0045] In another preferred embodiment, the candidate substances include (but are not limited to): compounds, interacting molecules, biomacromolecules, etc.

[0046] In another preferred embodiment, the method further includes conducting further cell experiments, animal (e.g., rats, non-human primates) experiments, or human clinical trials on the obtained or potential substances to further select and identify substances from the candidate substances that are useful for repairing damaged neural circuits in the brain or remodeling neural function.

[0047] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0048] Figure 1 Axonal projections of human neurons transplanted into the substantia nigra

[0049] (A and B) Sagittal sections of mouse brain slices after substantia nigra transplantation, hNCAM immunohistochemical staining of mDA(A) and Glu(B) neurons. Scale bar, 250µm. The black-boxed area is magnified in the upper right. The white-boxed area is magnified in the lower right. Scale bar, magnified image is 25µm.

[0050] (C) Schematic diagram of three representative sagittal sections of the anatomical structure.

[0051] (D) TH immunostaining of wild-type mice (left) or PD mice transplanted with mDA neurons (middle) or Glu neurons (right) on the corresponding sagittal plane.

[0052] (E) Quantification of the distribution of hNCAM+ fibers in different regions. mDA group n=8, Glu group n=6.

[0053] (F) Relative distribution of hNCAM+ fibers in the dorsal (CPu) and ventral striatum (Acb) of mDA-transplanted mice. Images in (A), (B), and (D) were automatically stitched together from multiple high-magnification images. See also Figure 8 .

[0054] Figure 2 Axonal projection pathways of transplanted neurons in the substantia nigra

[0055] (A) Schematic diagram of approximate mid-lateral plane and corresponding continuous sagittal plane hNCAM immunostaining in mouse brains transplanted with mDA neurons.

[0056] (B) Draw hNCAM+ axon projections in different sagittal planes. The boxed areas are enlarged in (C)-(F).

[0057] (CF) High magnification shows the projection path and region of the axon. Scale bar, 250 μm. In (C), the red arrow indicates hNCAM+ axons in the MFB. In (D), the red arrow indicates ascending hNCAM+ axons. (F) shows the distribution of hNCAM+ fibers in the lateral striatum. The blue arrows in (D) and (F) indicate hNCAM+ fibers in the cortex.

[0058] (G) Morphological distribution of hNCAM+ fibers in mDA or Glu neuronal fibers transplanted to different host brain regions and their co-labeling with human STEM121 and TH. Scale bar, 50 mm (top) and 25 mm (bottom).

[0059] (H) Quantitative percentage of human STEM121 pixels co-labeled with TH in CPU of mDA or Glu neuron transplanted mice. Data are expressed as mean ± SEM. t-test. ***p<0.001.

[0060] The images in (A) and (CF) were automatically stitched together from multiple highly magnified images. See also Figure 9 .

[0061] Figure 3 Axonal projections and electrophysiological characteristics of genetically marked human mDA neurons

[0062] (A) Visualization and electrophysiological recording strategies for transplanted human mDA and non-mDA neurons.

[0063] (B) Strategy for constructing TH-tdTomato / ChR2-EYFP dual-site knock-in hESC cell lines (TH-tdTomato / AAVS1-ChR2-EYFP hESCs).

[0064] (C) Immunostaining of the above hESCs cell lines on day 42 of differentiation showed that tdTomato and EYFP were co-expressed in TH+ neurons (white arrows), while tdTomato was absent in TH- neurons (white arrows). Scale bar, 20 μm.

[0065] (D) Immunohistochemical images show that the transplanted block of transgenic human mDA neurons in the substantia nigra contains tdTomato+ / EYFP+ mDA neurons (white arrows) and tdTomato- / EYFP+ non-mDA neurons (white arrows).

[0066] (E) Immunostaining of serial coronal sections of brain slices from PD mice with substantia nigra transplantation. Scale bar, 1 mm. The area within the box is magnified as shown in the figure. Scale bar, 0.1 mm. The red arrows indicate ascending axons from the ventral striatum.

[0067] (F) Co-labeling of human STEM121 and tdTomato. Scale bar, 50 μm.

[0068] (GI) Coronal sections of the transplant site, immunostaining of tdTomato and EYFP on striatum (G and H) or substantia nigra (I) transplant blocks from the brains of PD mice. The boxed areas in (G) are magnified below. The image in (I) is a composite of two separate images of the top and bottom of the same transplant block. Scale bars: 1 mm, top (G); 100 mm, bottom (G); 250 μm (H) and (I).

[0069] (J) Typical spontaneous action potentials (sAPs) were observed 3 months after transplantation of endogenous SNc mDA neurons or striatal or black transplanted human mDA neurons in mDA neuron reporter mice (DAT-cre / Ai9).

[0070] (KM) Typical voltage dimming characteristics were detected 6 months post-transplantation of endogenous intermediate VTA or SNc (K) from mDA neuron reporter mice or human non-mDA neurons or mDA neurons (L) transplanted into the striatum. The numbers in parentheses indicate the number of neurons showing dimming in the recorded cells. The dimming amplitude is expressed as (M), and the sample size is shown in the bars. Data are expressed as mean ± SEM. t-test, ##p<0.01, ##p<0.001.

[0071] The images in (E), (G), and (H) are automatically stitched together from multiple high-magnification images. The image in (I) is stitched together from two separate images of the upper and lower halves of the same cross-section. See also Figure 10 and 11 .

[0072] Figure 4 Tracking of rabies-mediated gene markers in human mDA neuronal input

[0073] (A) A strategy for tracking inputs to gene-labeled human mDA neurons in the SN or striatum of PD mice. (B) A schematic diagram of the construction of the TH-iCre hESC cell line.

[0074] (C) Neurons expressing EGFP and tdTomato at the transplantation site. Scale bar, 1 mm. (D) Immunohistochemical images showing the expression of tdTomato, EGFP, and TH in neurons at the SN transplantation site. White arrows indicate co-expression of EGFP and tdTomato in TH+ neurons. Scale bar, 100 mm.

[0075] (E) Serial coronal sections show the distribution of human mDA neurons transplanted into the substantia nigra or striatum by the traced host neurons (EGFP+ / tdTomato-). Only the ipsilateral side of the graft is shown. Scale bar, 1 mm.

[0076] (F) Quantitative statistical labeling of ipsilateral labeled inputs to human mDA neurons transplanted into the substantia nigra or striatum, expressed as a percentage of all ipsilateral inputs. Data are presented as mean ± SEM. Only brain regions with substantia nigra or striatum transplants showing a mean input percentage greater than 1%. For striatum transplants, n = 3; for substantia nigra transplants, n = 5. ND, not detected. Student's t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

[0077] (G) Magnified image of labeled neurons input to transplanted mDA neurons in different host brain regions. Scale bar, 200 mm.

[0078] (H) The coronal section shows the neuronal inputs marked in Acb to the transplanted mDA neurons. The boxed area is magnified below (H1). An example distribution of input neurons from another animal is shown (H2). White arrows indicate that the marked input neurons are distributed in patches. Scale bars are 1 mm (top) and 0.5 mm (bottom).

[0079] The images in (CE) and (H) were automatically stitched together from multiple high-magnification images. The image in (G) was enlarged from a tiled image. See also Figure 12 .

[0080] Figure 5 Electrophysiological characteristics of human mDA or non-mDA neuronal inputs

[0081] Human mDA or non-mDA neurons transplanted into the striatum (A) or substantia nigra (B) within 3 or 6 months post-transplantation exhibit typical sEPSC and sIPSC.

[0082] (C and D) Frequency and amplitude of sIPSC(C) and sEPSC(D). Data are expressed as mean ± SEM. One-way ANOVA followed by Holm-Sidak post-hoc test. *p<0.05, **p<0.01, ***p<0.001; comparison of mDA or nonDA neurons transplanted at 3 months and 6 months. ##p<0.01, ###p<0.001; comparison between mDA and non-mDA neurons.

[0083] (E) The ratio of sIPSC / sEPSC in endogenous striatal or SNc neurons of wild-type SCID mice to non-mDA or mDA neurons in the striatum or substantia nigra 6 months post-transplantation. Data are presented as mean ± SEM. One-way ANOVA was performed, followed by Holm-Sidak post-hoc tests. ##p<0.01, ###p<0.001. Sample numbers used for statistical analysis are shown in this column.

[0084] Figure 6 Behavioral consequences of transplanted animals

[0085] (A) The experimental process of establishing animal models, transplantation, and behavioral analysis. These animals underwent monthly behavioral tests, including amphetamine-induced rotation, wheeling, and cylinder tests.

[0086] (B) Amphetamine-induced rotational behavior changed within 6 months post-transplantation.

[0087] (C) The rotating tripod test showed the change in drop time before and after transplantation.

[0088] (D) The cylinder test shows changes in ipsilateral preference before and after transplantation.

[0089] In all three behavioral tests, the substantia nigra mDA group (n=11), substantia nigra Glu group (n=8), black ACSF group (n=8), and striatum mDA group (n=8). Data are presented as mean ± SEM. Two-step ANOVA was performed, followed by the Holm-Sidak test. ***p<0.001.

[0090] Figure 7 Bidirectional control of PD mice after transplantation

[0091] (A) Schematic diagram of human mDA neurons transplanted in PD mice under bidirectional regulation.

[0092] (B) Schematic diagram of the production of mCherry and Bi-DREADD hESC strains.

[0093] (C) Immunostaining showed that on day 42 of Bi-DREADD hESC differentiation, TH, hM3Dq-mCherry, and hemagglutinin (HA)-labeled KORD were co-expressed in the differentiated mDA neurons. Scale bar, 50 mm.

[0094] (D) Immunohistochemical images show that mDA neurons differentiated from Bi-DREADD hESCs co-expressed human nuclei (hNs), mCherry, and TH after transplantation. Scale bar, 50 mm.

[0095] (E) Experimental procedures, transplantation, and behavioral analysis of animal models. S-rotation, spontaneous rotation.

[0096] (F and G) Amphetamine-induced rotation and cylinder tests showed changes in rotational behavior (F) or ipsilateral tactile preference (G).

[0097] (H) The cylinder test showed that treatment with mediators, CNO or SALB altered ipsilateral preferential tactile sensation in PD mice.

[0098] (I and J) Spontaneous rotation tests show the changes in ipsilateral net rotation (I) and ipsilateral preferred rotation (J) caused by CNO or SALB.

[0099] Figure 8 In vitro differentiation of mDA and Glu neurons and in vivo distribution of TH fibers

[0100] (AB) Immunostaining from day 32 cultures of hESCs shows labeled mDA progenitor cells (A) and forebrain glutamate progenitor cells (B). Ho, Hoechst, scale bar = 25 μm. (C) Quantification of cell differentiation as presented in (A) and (B). (DE) Immunostaining from day 42 cultures of hESCs shows labeled mDA neurons (D) and forebrain glutamate neurons (E). Scale bar = 25 μm. (F) Quantification of the cell differentiation process described in (D) and (E). (G) Regional quantification of the distribution of TH+ fibers in wild-type mice, as shown in... Figure 1 As shown in D. n = 5. TH+ positive cell bodies in the cortex are also included in our calculations.

[0101] Figure 9 Survival, axonal projection, and synapse formation of mDA and Glu neurons transplanted into the substantia nigra

[0102] (A) Immunohistochemical image of hNCAM from the brain of a PD mouse. Transplanted mDA neurons show the distribution and branching of hNCAM+ fibers in the CPU. Red arrows indicate the beaded structure of hNCAM+ fibers. Scale bar = 50 μm. (B) Immunohistochemical image of the damaged brain of a mouse with transplanted nDA. Pixels show the morphology of hNCAM+ fibers in different brain regions. Scale bar = 125 μm.

[0103] (C) Immunohistochemical image of hNCAM from the brain of PD mice. Transplanted Glu neurons show the distribution and branching of hNCAM+ fibers in the mouse cortex and OB (olfactory bulb). Scale bar = 125 μm.

[0104] (D) Immunohistochemical images show transplanted cells in the substantia nigra expressing both human nuclei (hN) and GIRK2TH positivity. The boxed areas are magnified below. Scale bar = 100 μm for large images and 25 μm for magnified images. Arrows indicate double-positive cells.

[0105] (E) Immunohistochemical images show that human nuclear (hN) positive cells transplanted in the substantia nigra co-express FOXA2 and LMX1A. Scale bar = 100 μm.

[0106] Quantification of cell identity in (F)(D and E).

[0107] (G) Human-specific staining of synaptophysin and TH (top panel) or GABA (bottom panel) in the host CCu of mouse brains with human-derived mDA neurons was performed. The boxed areas are enlarged on the right. White arrows indicate human-specific synaptophysin colocalization along TH fibers with TH. White arrows indicate human-specific synaptophysin colocalization with the cell body of GABA around GABA neurons.

[0108] (H) Staining of mouse brains with transplanted Glu neurons and human-specific synaptophysin and GABA in host CCu. The boxed area is enlarged on the right. White arrows indicate the colocalization of human-specific synaptophysin around GABA neurons.

[0109] Figure 10 Establishment and characterization of the hESC TH-tdTomato / AAVS1-ChR2-EYFP cell line.

[0110] (A) Schematic diagram of the cell line strategy for hESC TH-tdTomato / AAVS1-ChR2-EYFP. PCR primers showing TH site insertion or homozygosity are indicated by red and black arrows, respectively. PCR primer insertion or homozygosity at the AAVS1 locus is indicated by green and blue arrows.

[0111] (B) PCR genotyping of hESC TH-tdTomato / AAVS1-ChR2-EYFP. The expected PCR products correctly targeting the TH or AAVS1 site are respectively... (red arrow) or (Green arrows). These are used to identify heterozygosity at the TH locus or the AAVS1 locus, respectively, through... (Black arrow) or (Blue arrow) PCR products. Those without or The PCR products were homozygous. The maternal cell line H9 ESCs served as a control. The TH-tdTomato / AAVS1-ChR2-EYFP hESC cell line was homozygous at both the TH and AAVS1 loci.

[0112] (C) DIC and fluorescence images show the expression of tdTomato and EYFP in hESCs and ESCs during mDA neuron differentiation. tdTomato is expressed in the intermediate (D15) and terminal (D48) stages, but not in the early (D9) stage of ES or mDA neuron differentiation. Scale bar = 100 μm;

[0113] (D) Immunostaining from day 42 cultures of the above hESCs shows co-expression of TH+ neurons and tdTomato. The boxed areas are magnified below. White arrows indicate neurons with high tdTomato and TH expression. White arrows indicate neurons with low tdTomato and TH expression. Scale bar = 20 μm;

[0114] (E) Immunohistochemical images showing the expression of tdTomato in TH+ neurons of the substantia nigra mDA graft. The boxed areas are magnified regions. White arrows and white barbs indicate neurons with high and low expression of tdTomato and TH, respectively. Scale bar = 20 μm;

[0115] (F) Immunohistochemical images showing 5-HT-positive serotonin neurons in a substantia nigra mDA graft. The box indicates a magnified area. White arrows indicate tdTomato- and 5-HT+ neurons. Scale bar = 20 μm;

[0116] (G) Coronal section of the transplantation site after immunostaining with tdTomato and EYFP. mDA neurons derived from TH-tdTomato / AAVS1-ChR2-EYFP were transplanted into the striatum of the brain of PD mice, showing specific projections of the mDA neurons;

[0117] Up to the CPU, but excluding adjacent brain regions. Scale bar = 1 mm. The area within the box is magnified below. Scale bar = 100 μm. Images are automatically stitched from high-magnification images from multiple locations.

[0118] (H) DIC and fluorescence images of mDA neuron slices derived from TH-tdTomato / AAVS1-ChR2-EYFP transplanted into the brains of PD mice. White arrows indicate tdTomato+mDA neurons, and white arrows indicate tdTomato-non-mDA neurons in EYFP+ grafts. Scale bar = 50 μm.

[0119] Figure 11 Electrophysiological examination of functionally matured human mDA or non-mDA neurons transplanted into the substantia nigra or striatum.

[0120] (AD) Typical whole-cell patch-clamp recordings of blue light-induced action potentials (A and B) or current-induced action potentials (C and D) in the striatum (A and C) or substantia nigra (B and D) of transplanted human mDA or non-mDA neurons 3 months post-transplantation.

[0121] (E and F) Typical whole-cell patch-clamp recordings of spontaneous action potentials (sAPs) following 3 months of striatal (E) or substantia nigra (F) transplantation of human non-mDA neurons. Dashed lines indicate threshold potentials.

[0122] (G and H) Potential frequencies of spontaneous action potentials (sAP) (G) and subthreshold oscillations (H) from endogenous SNc mDA neurons from mDA neuron reporter mice (DAT-Cre / Ai9), or after transplantation of human mDA neurons in the striatum or substantia nigra at 3 months. Data are presented as mean ± SEM. Sample statistics are indicated in columns. One-way ANOVA, p > 0.05.

[0123] (I and J) Typical whole-cell patch-clamp recordings of spontaneous action potentials (sAPs) in the striatum (I) or substantia nigra (J) 6 months after transplantation of human mDA neurons. Dashed lines indicate threshold potentials.

[0124] (K and L) Tau plots of input resistance (Rm), membrane capacitance (Cm), and striatum (K) or substantia nigra (L) of human non-mDA and mDA neurons 6 months post-transplantation. Data are presented as mean ± SEM. The sample size for the statistics is indicated in the column. Student's t-test, #p < 0.05.

[0125] Figure 12 Constructing the TH-icre hESC cell line and using rabies virus tracer gene markers to identify human and mouse endogenous mDA neurons.

[0126] (A) Schematic diagram of the genotyping strategy for the TH-iCre hESC cell line. PCR primers used for TH site insertion or homozygosity are indicated by red and black arrows, respectively. Green arrows indicate PCR primers used to remove PGK-Pur.

[0127] (B) PCR genotyping analysis of the TH-iCre hESC line. The PCR product correctly targeting the TH site is approximately 1000 bp (red arrow). Homozygous clones were identified by a PCR product of approximately 1000 bp (black arrow), and clones without a PCR product were homozygous. The PCR product used to remove PGK is approximately 750 bp (green arrow). The mother cell line (H9 ESCs) served as a control. Heterozygous clones with TH site removed by PGK-pur (red asterisk) were selected for the experiment.

[0128] (C) Illustration of lentivirus encoding cre-dependent mCherry expression driven by ubiquitin promoter (Lenti-Ubi-DIO-mCherry).

[0129] (D) mDA neurons differentiated from TH-icre are infected with the lentivirus Lenti-Ubi-DIO-mCherry at TH +mCherry is expressed in mDA neurons. The boxed area is enlarged to the right. White arrows indicate co-expression of mCherry and TH in mDA neurons. White arrows indicate mCherry-expressing neurons with low TH expression. Scale bar = 20µm.

[0130] (E) Immunohistochemical images show that mDA neurons derived from TH-icre hESC cells transplanted into the striatum were infected with AAV-DIO-TVA-2A-NLS-tdTomato virus 6 months after transplantation. tdTomato and TH were co-expressed in the transplanted mDA neurons. The boxed area is magnified to the right. White arrows indicate co-expressing neurons. Scale bar = 20 μm.

[0131] (F) Immunohistochemical images show that cortical CTIP2+ or SATB2+ neurons, CCu zone GABA+ or DARPP32+ neurons, and DR zone 5-HT+ neurons are all connected to human mDA neurons transplanted into the substantia nigra. White arrows indicate co-expressed neurons. Scale bar = 100 μm.

[0132] (G) Transsynaptic tracing of rabies-mediated endogenous mDA neurons. Confocal images show EGFP and tdTomato expressing neurons in the SNc of DAT-Cre mice. Scale bar = 0.5 mm.

[0133] (H) A series of coronal sections show the distribution of tracking neurons (EGFP+ / tdTomato-) in the endogenous mDA neurons of date-cre mice. Only the ipsilateral side of the transplant site is shown. Scale bar = 1 mm.

[0134] (I) Magnified view of the area within the frame. (I) The magnified image shows that the labeled neurons afferent to endogenous mDA neurons are distributed in a patchy pattern. Scale bar = 0.5 mm.

[0135] (J) High-magnification images of mDA neurons transplanted to the striatum from different brain regions of the host, labeled. Scale bar 200 μm.

[0136] The images in (G) and (H) were automatically stitched together from multiple high-magnification images.

[0137] The images in (I) and (J) are magnified from the tiled images.

[0138] Figure 13 Functional afferent input from endogenous neurons to transplanted neurons and the dynamics of sIPSCs and sEPSCs of transplanted neurons.

[0139] (A and B) Typical illustrations of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) of non-mDA or mDA neurons on brain slices of the striatum (A) or substantia nigra (B) at 3 or 6 months post-transplantation.

[0140] (CF) Separate quantitative analyses of sEPSCs and sIPSCs in A and B. Data are expressed as mean ± SEM. Sample numbers for statistical data are shown in columns. Holm-Sidak post-hoc test was performed after one-way ANOVA. p>0.05. (G and H) Endogenous lateral SNc mDA in brain slices from wild-type SCID mice.

[0141] Whole-cell patch-clamp recordings of typical sEPSCs and sIPSCs in (G) or striatal neurons (H).

[0142] Figure 14 Construction and characterization of hESC cell lines expressing mCherry- and Bi-DREADD

[0143] (A) PCR genotyping of hESC clones expressing mCherry or Bi-DREADD. The expected PCR product correctly targeting the AAVS1 locus is approximately 2000 bp (red arrow). Homozygous clones do not have this characteristic. Homozygosity testing in clones of PCR products (black arrows) The clones of the PCR product are heterozygous. Select homozygous clones (marked with a red asterisk) for the experiment.

[0144] (B) Immunostaining showed the expression of mCherry, hM3Dq-mcherry, or HA-labeled KORD in mCherry or Bi-DREADD hESC cell lines. Scale bar = 50 μm.

[0145] (C) Immunostaining of progenitor cells on day 16. Ho, Hoechst. Scale bar = 50 μm.

[0146] (D) Immunostaining of mDA neurons cultured from mCherry or Bi-DREADD hESC cell lines on day 42. Scale bar = 50 μm.

[0147] (E) Immunostaining on day 42 of differentiation showed co-expression of TH and mCherry, but not in HA-labeled KORD cell lines. Scale bar = 50 μm.

[0148] Immunohistochemical images of mDA neurons transplanted from the substantia nigra of mCherry hESC(F) or Bi-DREADD hESC(G) (F and G) show co-expression of transgenic mCherry or hM3Dq-mCherry and human STEM121. Scale bar = 20 μm. Detailed Implementation

[0149] Through in-depth research, the inventors have revealed a specific method for differentiating pluripotent stem cells into dopaminergic neurons in the substantia nigra of the midbrain. The differentiated mature A9 mDA neurons express surface molecular markers of dopaminergic neurons in the substantia nigra, including TH, FOXA2, EN1, LMX1A, NURR1, and GIRK2, but rarely express CALB, a marker of dopaminergic neurons in the ventral tegmental area. The A9mDA neurons transplanted into the substantia nigra can have their axons specifically project to the dorsal striatum, the target brain region innervated by endogenous substantia nigra dopaminergic neurons. The transplanted A9 mDA neurons in the substantia nigra receive more inhibitory input and less excitatory input, a regulatory pattern similar to that of endogenous substantia nigra dopaminergic neurons. The transplanted A9 mDA neurons themselves exhibit the classic electrophysiological characteristics of endogenous substantia nigra dopaminergic neurons, including a low-frequency spontaneous firing rate, and hyperpolarization current stimulation can induce sag. Transplanting A9 mDA neurons into the substantia nigra or striatum of individuals with neurodegenerative diseases can improve motor dysfunction.

[0150] the term

[0151] As used in this invention, the term “treatment” here includes preventive (e.g., prophylactic), curative, or palliative treatment of a mammal (particularly a human); and includes (1) preventing, treating, or alleviating an individual’s morbidity for a disease (e.g., cancer), wherein the individual is in a high-risk group for the disease or has the disease but has not yet been diagnosed; (2) suppressing a disease (e.g., inhibiting its occurrence); or (3) alleviating a disease (e.g., alleviating symptoms associated with the disease).

[0152] As used in this invention, the terms "midbrain substantia nigra dopaminergic neurons", "stem cell-derived midbrain dopaminergic neurons" and "A9 mDA neurons" are interchangeable / can be used interchangeably.

[0153] As used in this invention, the term "cell" includes both "cell population" and "cell culture".

[0154] As used in this invention, “individual,” “organism,” “subject,” or “test subject” refers to an animal (such as a rodent or primate) that includes a human being, who is eligible to receive treatment with the cells (midbrain substantia nigra dopaminergic neurons) or cell preparations of this invention.

[0155] As used in this invention, "prevention and treatment" includes "prevention", "alleviation" and "treatment".

[0156] As used in this invention, a "neural circuit" is a connection of neurons of different properties and functions in the brain through various forms; in particular, this invention focuses on the substantia nigra-striatal neural circuit.

[0157] As used in this invention, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not themselves essential active ingredients and do not exhibit excessive toxicity after administration.

[0158] As used in this invention, "effective amount" refers to an amount of a drug (in this invention, a cell or cell preparation) sufficient to produce the desired therapeutic response. Effective amount also includes situations where the therapeutic benefit of the drug outweighs its toxic or harmful effects. An effective amount of a drug may not necessarily cure a disease or condition, but may delay, inhibit, or prevent its occurrence, or may alleviate symptoms associated with the disease or condition. The therapeutically effective amount may be divided into one, two, or more doses and administered once, twice, or more times within a specified period in an appropriate dosage form.

[0159] Stem cell-derived neurons and their preparation

[0160] This invention provides a midbrain substantia nigra dopaminergic neuron (or cell group), which is mainly A9 midbrain dopaminergic neuron.

[0161] As a preferred embodiment of the present invention, more than 80% of the stem cell-derived neurons obtained by the present invention express the A9mDA neuronal marker GIRK2; more preferably more than 85% express the A9mDA neuronal marker GIRK2.

[0162] In a preferred embodiment of the present invention, the midbrain substantia nigra dopaminergic neurons express the markers FOXA2, LMX1A, and EN1. If further differentiated for approximately one week, they also express tyrosine hydroxylase (TH) as well as EN1, FOXA2, LMX1A, and NURR1 (preferably, more than 40% of the total cells or more than 50% of the TUJ1+ neurons possess this characteristic; more preferably, more than 50% of the total cells or more than 60% of the TUJ1+ neurons possess this characteristic).

[0163] The present invention also provides a method for preparing midbrain substantia nigra dopaminergic cells in vitro, comprising: (1) placing stem cells in a culture medium containing a neuroinducing agent; and (2) obtaining the midbrain substantia nigra dopaminergic cells from the culture.

[0164] In a preferred embodiment of the present invention, (1) a neural inducer is added to the culture medium; and induction is performed in multiple stages with different added components: first stage: SB431542, DMH-1, SHH and CHIR99021 are added; second stage: SAG, SHH and CHIR99021 are added; third stage: SHH and CHIR99021 are added; fourth stage: SHH and FGF8b are added.

[0165] In a more preferred embodiment of the present invention, the inventors have further optimized the timing of the addition of each component. Preferably, in each stage: the first stage: from the start of cultivation to 6-8 days; more preferably 7 ± 0.5 days; the second stage: from 6-8 days to 11-13 days; more preferably 12 ± 0.5 days; the third stage: from 11-13 days to 18-20 days; more preferably 19 ± 0.5 days; the fourth stage: from 18-20 days to 31-33 days; more preferably 32 ± 0.5 days.

[0166] In a more preferred embodiment of the invention, the inventors further optimized the amount of each additive component. Optimizing the amount of additive facilitates the acquisition / enrichment of specific midbrain substantia nigra dopaminergic cells as described in this invention.

[0167] The midbrain substantia nigra dopaminergic neurons obtained by the optimized method of this invention have good effects, including repairing damaged neural circuits or remodeling neural function in the brain; more specifically, they can be repaired by forming nerve fibers in the substantia nigra region and projecting them to the striatum; or by forming synaptic connections with target cells in the brain; or by projecting axons to the dorsal side (Caudate putamen, CCu); or by growing nerve fibers, specifically growing along the endogenous substantia nigra-striatal neural connection path, extending to its endogenous target region—the striatum, forming neural connections with striatal neurons, and projecting them to the striatum.

[0168] Repair function of stem cell-derived nerve cells

[0169] Neurons are the basic functional units of the brain. An individual's brain contains thousands of different types of neurons, forming complex and precise networks (neural circuits) that are the foundation for an individual's perception of the world, thinking, and behavior. Many neurological diseases, including stroke, traumatic brain injury, and neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), lead to the loss of neurons and the disruption of neural connections, resulting in severe neurological dysfunctions such as hemiplegia, bradykinesia, muscle rigidity, and impaired learning and memory. However, the regenerative capacity of the brain in adult mammals, including humans, is very limited. For these diseases caused by neuronal loss leading to disrupted neural connections and impaired neurological function, there are currently no effective clinical treatments. The key to stem cell therapy for neurological diseases is the repair and functional reconstruction of damaged neural circuits. However, the precise network connections between neurons in an individual's brain are gradually formed during development, involving complex mechanisms guiding nerve fiber growth. In the adult disease brain environment, it remains unclear whether transplanted nerve cells can grow nerve fibers, bridge the "lost" upstream and downstream brain regions, and thus repair damaged neural circuits. More importantly, is this repair a result of random integration of transplanted cells or a specific repair process? What are the underlying mechanisms and principles? These are key questions that urgently need to be addressed in the field of stem cell therapy for nervous system diseases.

[0170] To address the above issues, the inventors used Parkinson's disease as a disease model to investigate the feasibility and mechanism of repairing damaged neural circuits with stem cell-derived nerve cells transplanted into the adult brain. Parkinson's disease, characterized by resting tremor, rigidity, and bradykinesia, is the second most prevalent neurodegenerative disease worldwide. Its primary cause is the progressive loss of dopaminergic neurons in the substantia nigra region of the brain, leading to the disruption of neural connections between the substantia nigra and the striatum, resulting in insufficient dopamine secretion in the striatum and ultimately causing motor dysfunction in patients. The inventors have been dedicated to developing human stem cell neural differentiation techniques targeting different types of neurons, and based on this, have established an efficient method for differentiating human stem cells into midbrain substantia nigra dopaminergic neurons. Furthermore, genetic markers were applied to human stem cells using gene editing technology, enabling the specific tracing of stem cell-derived human dopaminergic neurons and their nerve fibers. The inventors transplanted genetically marked human dopaminergic neurons into the damaged substantia nigra region of a Parkinson's disease model mouse brain. The results showed that the transplanted human dopaminergic neurons in the substantia nigra region grew a large number of nerve fibers, specifically growing and extending along the endogenous substantia nigra-striatal neural connection pathway to their endogenous target region—the striatum—forming neural connections with striatal neurons, and the vast majority of these nerve fibers projected to the striatum. The inventors further used genetic techniques and rabies virus-mediated tracing technology to track the upstream neural innervation received by the transplanted human dopaminergic neurons in the substantia nigra, finding that the transplanted human dopaminergic neurons received similar neural innervation to the endogenous substantia nigra dopaminergic neurons. Studies of the neuronal electrophysiological function revealed that the transplanted human dopaminergic neurons exhibited similar electrophysiological characteristics to the endogenous animal substantia nigra dopaminergic neurons, receiving similar neurotransmitter regulation. These results indicate that the human dopaminergic neurons transplanted into the brains of Parkinson's disease model animals specifically repaired and reconstructed the damaged substantia nigra-striatal neural connection, and their structure and function were highly consistent with the endogenous neural connection. Finally, through behavioral testing, the inventors found that motor dysfunction in the cell transplantation group gradually improved with prolonged transplantation time. However, inhibiting the activity of transplanted nerve cells using chemogenetic techniques caused the improvement in motor function to disappear, suggesting that the reconstructed neural connections of the transplanted cells mediated the recovery of behavior in the model animals. Interestingly, when the inventors transplanted another type of nerve cell—human cortical glutamatergic neurons—into the substantia nigra of Parkinson's disease model animals, the nerve fibers of these neurons primarily projected to the cortex and olfactory bulb region, with almost no projection to the striatum. This failed to repair the damaged substantia nigra-striatal neural circuit, and the motor dysfunction in the model animals did not improve, indicating that only specific cell types can repair specific neural functional circuits.

[0171] This invention suggests that damaged neural connections in the adult brain can be structurally and functionally repaired and remodeled through the transplantation of stem cell-derived nerve cells. Furthermore, this invention reveals that different types of nerve cells have varying effects on circuit repair, suggesting that targeted transplantation of specific nerve cells is necessary for circuit repair and treatment in neurological diseases caused by different types of neuronal loss. These findings provide new ideas and theoretical foundations for the treatment of brain injury and neurodegenerative diseases. Currently, the main types of nerve cells in the human brain can be efficiently obtained in vitro using stem cell neural differentiation technology. The development of stem cell technology will bring new hope for the treatment of many neurological diseases.

[0172] Therefore, based on the inventor's new discovery, the technical solution of the present invention has the following characteristics: (1) Dopaminergic neurons derived from stem cells (e.g., human embryonic stem cells) have the characteristics of midbrain dopaminergic neurons in the substantia nigra; (2) Functional input depends on the type of transplanted nerve cells rather than the location of transplantation; (3) Midbrain dopaminergic neurons can precisely repair the substantia nigra-striatal pathway; (4) Functionally repaired substantia nigra-striatal pathway restores motor function in models of neurodegenerative diseases (e.g., Parkinson's disease).

[0173] Drug screening

[0174] The inventors transplanted midbrain dopamine (mDA) or glutamate (Glu) cortical neurons derived from human embryonic stem cells (hESCs) into the substantia nigra or striatum of animal PD models and found extensive integration of the transplanted cells with host circuits. Axonal pathways toward the dorsal striatum were determined by the type of transplanted neurons, presynaptic input largely depended on the transplantation site, and inhibitory and excitatory inputs were determined by the type of transplanted neurons. hESC-derived mDA neurons exhibited characteristics of A9 neurons and restored the function of the reconstructed substantia nigra circuits to modulate improved motor function. These results demonstrate the cell-type-specific presynaptic and postsynaptic integration similarities between the transplanted reconstructed circuits and endogenous neural networks, highlighting the ability of hESC-derived neuronal subtypes to perform specific circuit repair and functional restoration in the adult brain.

[0175] Based on the inventors' new discoveries, substances that can be screened to repair damaged neural circuits or reshape neural function in the brain can be identified. Drugs useful for brain injury, neurodegenerative diseases, and other conditions may be found among these substances.

[0176] Therefore, the present invention provides a method for screening substances (including potential substances) that repair damaged neural circuits or remodel neural function in the brain, the method comprising: (1) treating a model system with a candidate substance, the model system being a model system of damaged neural circuits or impaired neural function in the brain, and containing midbrain dopaminergic neurons (midbrain dopaminergic cells); and, (2) testing the model system, if the candidate substance statistically promotes the dopaminergic neurons in repairing damaged neural circuits in the brain or promoting their remodeling neural function, then the candidate substance is a substance useful for repairing damaged neural circuits in the brain or remodeling neural function.

[0177] Based on the research results of this invention, the effectiveness of the potential substance (candidate substance or candidate drug) can be determined by analyzing the repair of the substantia nigra-striatal pathway by midbrain dopaminergic neurons, the presynaptic and postsynaptic integration of midbrain dopaminergic neurons, and the ability of midbrain dopaminergic neuron axons to project to the dorsal side and / or the utilization ability of animal models after the action of the candidate substance.

[0178] Based on the research results of this invention, the effectiveness of the potential substance (candidate substance or candidate drug) can be determined by systematically analyzing the changes in the ability of midbrain dopaminergic neurons to grow nerve fibers, specifically grow and extend to their endogenous target region—the striatum—along with striatal neurons, and project to the striatum after the candidate substance has been acted upon.

[0179] In a preferred embodiment of the present invention, during screening, a control group can be set up to make it easier to observe the changes of the candidate substance before and after treatment. The control group can be a model system without the addition of the candidate substance (such as a blank control or a placebo control).

[0180] On the other hand, the present invention also provides potential substances obtained using the aforementioned screening method. These initially screened substances can form a screening library, from which substances that are truly useful for repairing damaged neural circuits in the brain or reshaping neural function can ultimately be selected.

[0181] pharmaceutical preparations

[0182] The present invention also provides a pharmaceutical composition (formulation) containing an effective amount (e.g., 0.000001-50 wt%; preferably 0.00001-20 wt%; more preferably 0.0001-10 wt%) of midbrain substantia nigra dopaminergic cells prepared by the method of the present invention, and a pharmaceutically acceptable carrier.

[0183] The term "pharmaceutically acceptable carrier" refers to a carrier used for therapeutic administration, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0184] The effective amount of dopaminergic cells in the substantia nigra of the midbrain described in this invention can vary depending on the administration method and the severity of the disease to be treated. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0185] The specific therapeutically effective dose depends on a variety of factors, such as the specific condition to be treated, the individual's physiological condition (e.g., body weight, age, or sex), the type of individual receiving treatment, the duration of treatment, the nature of any concurrent treatments, and the structure of the specific formulation and compound or its derivative used. For example, the therapeutically effective dose can be expressed as the total weight of the active ingredient, for example in grams, milligrams, or micrograms; or as the ratio of the weight of the active ingredient to body weight, for example in milligrams per kilogram of body weight (mg / kg). Alternatively, the effective dose can be expressed as the concentration of the active ingredient (such as the cells or cell preparations of the present invention), for example, molar concentration, weight concentration, volumetric concentration, weight-molar concentration, molar fraction, weight fraction, and mixing ratio. Those skilled in the art can calculate the human equivalent dose (HED) of the agent (such as the cells or cell preparations of the present invention) based on animal model dosages. For example, those skilled in the art can estimate the maximum safe dose for human use based on the "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the U.S. Food and Drug Administration (FDA).

[0186] In specific embodiments of the present invention, some dosing regimens for animals such as mice are provided. Converting animal dosages (e.g., mouse) to human dosages is readily achievable by those skilled in the art, for example, using the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. Where A is the body surface area, expressed in meters. 2 Calculations are as follows: W represents body weight in grams; K is a constant that varies depending on the animal species, generally 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions can vary depending on the drug and clinical circumstances, and are based on the assessment of an experienced pharmacist.

[0187] The present invention also provides a medicine box containing the aforementioned pharmaceutical composition or directly containing the aforementioned dopaminergic cells in the substantia nigra of the midbrain. Furthermore, the medicine box may also include instructions on how to use the medicine contained therein.

[0188] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0189] Materials and methods

[0190] Cell culture

[0191] The H9 human embryonic stem cell line and the H9 human embryonic stem cell reporter line were cultured in stem cell culture medium seeded with irradiated mouse embryonic fibroblasts (MEF). The culture medium consisted of DMEM / F-12, KOSR, 1x NEAA, 0.5x Glutamax, 0.1 mM 2-mercaptoethanol, and 4 ng / ml FGF-2. The medium was changed daily and passaged weekly with Dispase II.

[0192] The generation of midbrain dopaminergic neurons and forebrain glutamatergic neurons

[0193] The induction method for midbrain dopaminergic (mDA) neuronal precursor cells involved culturing human embryonic stem cells (1 day post-passage) on MEF feeder layers or vetronection in neural induction medium (NIM) (DMEM / F-12, 1xNEAA, 1xN2 supplement) supplemented with SB431542 (10 μM) and DMH-1 (2 μM). To induce the differentiation of cells into midbrain basal plate precursor cells, SHH (C25II, 500 ng / ml) and CHIR99021 (0.4 μM) were added to the culture from day 1 to day 7. On day 7, neuroepithelial cell colonies were gently pipetted off and reattached to mouse embryonic fibroblast feeder layers, and then cultured for another 6 days (D7-12) in NIM supplemented with SAG (2 μM), SHH (100 ng / ml), and CHIR99021 (0.4 μM). On day 12, CHIR99021 was removed, the SHH concentration was reduced to 20 ng / ml, and SAG (0.5 μM) and FGF8b (100 ng / ml) were added to the culture medium to allow the precursor cells to expand in suspension until day 19. On day 32, the cells were cultured in neural induction medium containing 20 ng / ml SHH and 20 ng / ml FGF8b until transplantation. For in vitro differentiation, on day 32, the neurospheres were dissociated by incubation with Accutase at 37°C for 3–5 minutes. Then, it was placed on a glass coverslip coated with Matrigel and cultured in neurobasal medium (1xN2 supplement, 1xB27) supplemented with brain-derived neurotrophic factor (BDNF, 10 ng / ml), glial cell line-derived neurotrophic factor (GDNF, 10 ng / ml), ascorbic acid (AA, 200 μM), cAMP (1 μM), transforming growth factor β3 (TGFβ3, 1 ng / ml) and compound E (Compound E, 0.1 μM).

[0194] Method for inducing forebrain glutamate (Glu) neurons from human embryonic stem cells: H9 human embryonic stem cell clones were cultured for 1 week, with the medium changed daily. Then, hESC clones were isolated from the feeder layer and grown in ES medium for 4 days to aid in cell aggregate formation. For neural induction, the cell aggregates were cultured for 3 days in NIM supplemented with 2 μM SB431542 and 2 μM MMH-1. The ESC aggregates were then adhered to 6-well plates coated with fibronectin and cultured in NIM until approximately day 16 when neural tube-like rosette structures formed. The rosette structures were gently blown off with a 1 mL pipette and resuspended in the same medium for another 10 days. On day 26, the precursor cells were digested with Accutase for 4 minutes to form small spheres. After culturing in culture flasks containing NIM for another day, collect the microspheres and transplant them into animal models, or culture them for one week in NDM supplemented with BDNF (10 ng / ml), GDNF (10 ng / ml), AA (200 μM), cAMP (1 μM), and IGF1 (10 ng / ml) for attachment and maturation, and then use them for immunofluorescence staining.

[0195] PD models and cell transplantation

[0196] The surgical procedure for establishing a PD model in SCID mice includes: anesthetizing adult SCID mice (8-12 weeks old) with a mixture of 1-2% isoflurane and oxygen. 1 μL of 6-OHDA (3 mg / ml, dissolved in saline containing 1% ascorbic acid) is injected directly into the left substantia nigra of the brain (anteroposterior [AP] = -2.9 mm, lateral [L] = -1.1 mm, vertical [V] = 4.5 mm, vertical depth calculated from the skull). Four weeks after the 6-OHDA injury surgery, cells are transplanted into animals that can rotate at more than 6 revolutions / minute within 1.5 hours after amphetamine induction. Animals are randomly assigned to groups and transplanted with glutaminergic neuronal precursor cells, dopaminergic neuronal precursor cells, or artificial cerebrospinal fluid (ACSF) (control). 50,000 cells were resuspended in 1 μL of ACSF containing Rock inhibitor (0.5 μM), B27, and 20 ng / ml BDNF, and injected into the left substantia nigra (anteroposterior [AP] = -2.9 mm, lateral [L] = 1.1 mm, vertical [V] = 4.4 mm, vertical depth calculated from the skull) or the left striatum (AP = +0.6 mm, L = 1.8 mm, V = 3.2 mm, vertical depth calculated from the dura mater).

[0197] Donor plasmid construction

[0198] The TALEN tool, human codon-optimized wild-type Streptococcus pyogenes Cas9 (Cas9-2A-GFP), Cas9 nickase (Cas9D10A-2A-GFP), and pCAG-Flpo (plasmids #52342, #52341, #44719, #44720, and #60662) were obtained from Addgene. A PL652 donor plasmid vector containing a PGK-Puro expression cassette flanked by FRT sequences was constructed by replacing the loxP sequence (#68407) in PL552 with the FRT sequence.

[0199] To generate the TH-iCre donor plasmid, DNA fragments with left and right homologous arms were amplified from sequences immediately upstream or downstream of the STOP codon of the TH gene in the genomic DNA. Using primers containing the P2A sequence, the iCre gene DNA fragment fused with the P2A sequence was amplified from pDIRE (Addgene plasmid #80945). These three fragments were then cloned into the multiple cloning site of plasmid PL652.

[0200] To generate the TH-tdTomato donor plasmid, a DNA fragment fused with the P2A sequence was amplified from pAAV-FLEX-ArchT-tdTomato (Addgene plasmid #28305) using primers containing the P2A sequence. A DNA fragment containing the hGH polyA signal sequence was amplified from AAVS1-pur-CAG-EGFP plasmid (Addgene plasmid #80945). Then, DNA fragments from the left and right homologous arms of the TH-iCre donor plasmid, the DNA fragment containing the P2A-tdTomato sequence, and the DNA fragment containing the hGH polyA signal sequence were cloned into the multiple cloning site of plasmid PL552.

[0201] To construct the AAVS1-neo-CAG-ChR2-EYFP donor plasmid, the inventors amplified the ChR2-EYFP gene from pAAV-hSyn-hChR2(H134R)-EYFP (Addgene plasmid #26973) and replaced the FlpeERT2 gene in the AAVS1-Neo-CAG-Flpe-ERT2 plasmid (Addgene plasmid #68460).

[0202] To construct the AAVS1-pur-CAG-Bi-DREADD donor plasmid (AAVS1-pur-CAG-hM3Dq-mcherry-P2A-HA-KORD) and the AAVS1-pur-CAG-mCherry donor plasmid, the inventors amplified mCherry or hM3Dq-mCherry from AAVS1-pur-CAG-hM3Dq-mCherry (Addgene plasmid #80948), and amplified HA- from pAAV-hSyn-dF-HA-KORD-IRES-mCitrine (Addgene plasmid #65417). KORD. Two DREADD genes, hM3Dq-mcherry and HA-KORD, are linked via P2A (hM3Dq-mcherry-P2A-HA-KORD) to ensure simultaneous expression of these two genes in the same cell. Finally, the EGFP in AAVS1-pur-CAG-EGFP is replaced with either hM3Dq-mcherry-P2A-HA-KORD or the mCherry gene. SA-Neo consists of a T2A self-cleaving peptide sequence and a neomycin resistance gene linked sequentially after the splice acceptor sequence. CAG is a synthetically produced CAGGS promoter containing an actin enhancer and an early cytomegalovirus promoter.

[0203] Electroporation and Construction of Human Embryonic Stem Cell Reporter Lines

[0204] H9 hESCs were pretreated with a Rho kinase (ROCK) inhibitor for 6–8 hours (0.5 mM). Then they were treated with TrypLE. TM Express Enzyme digestion resulted in single-celled cells. Appropriate plasmid ratios were added to 500 mL of electroporation buffer (5 mM KCl, 5 mM MgCl2, 15 mM HEPES, 102.94 mM Na2HPO4, and 47.06 mM NaH2PO4, pH 7.2). Electroporation was performed in a 0.4 cm electroporation cuvette (Phenix Research Products) using a Gene Pulser Xcell (Bio-Rad) at 250 V and 500 mF. Cells were then seeded into MEF feeder layers in 6-well plates and cultured in medium containing a ROCK inhibitor. The medium was changed daily with MEF-fed ESC (CM). After 72 hours, puromycin (0.5 μg / ml) or G418 (50-100 μg / ml) was added to the CM, and selection was performed for two weeks. After selection, cells were pretreated with a ROCK inhibitor for 6-8 hours, and then single colonies were picked. Genomic PCR is used to identify whether a foreign gene has been integrated.

[0205] To construct the TH-iCre knock-in hESC strain, a codon-optimized Cre recombinase (iCre) gene containing a P2A linker and the STOP codon were knocked into the endogenous TH gene of H9 ESCs immediately upstream of the STOP codon using CRISPR, followed by a PGK-Puro sequence flanked by FRT (PGK promoter-driven Puro). The PGK-Puro sequences flanking FRT were then removed by transient expression of Flpo.

[0206] To construct the TH-tdTomato / AAVS1-ChR2-EYFP hESC line, a codon-optimized tdTomato gene with a P2A linker and a STOP codon were knocked into the endogenous TH gene of H9ESCs immediately upstream of the STOP codon using CRISPR, followed by a polyA sequence and a PGK-Pur sequence. The ChR2 expression cassette was then knocked into the AAVS1 gene locus via TALEN. To construct Bi-DREADD or mCherry hESC cell lines, the hM3Dq-mCherry-P2A-HA-KORD or mCherry expression cassette was inserted into the AAVS1 gene locus of H9 hESCs via TALEN.

[0207] Whole-cell patch-clamp and brain slice recording

[0208] Three and six months post-transplantation, 350 μm thick coronal brain slices at the forebrain or midbrain level were prepared using a Leica VT1200S vibratory microtome in pre-cooled sectioning solution (composition of sectioning solution: 100 mM glucose, 75 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 2 mM MgCl2-6H2O, 1.25 mM NaH2PO4-6H2O, and 0.7 mM CaCl2). The brain slices were then transferred to ACSF (124 mM NaCl, 4.4 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 25 mM NaHCO3, 1 mM NaH2PO4, and 10 mM glucose) and incubated for 1 hour before recording. The electrical signals were recorded using an Axon 700B amplifier (Axon). Both the external recording solution and the incubation solution for brain slices were ACSF. The recording electrodes (3-5 MΩ) contained an internal electrode solution (composed of: 112 mM Cs-Gluconate, 5 mM TEA-Cl, 3.7 mM NaCl, 0.2 mM EGTA, 10 mM HEPES, 2 mM MgATP, 0.3 mM Na3GTP, and 5 mM... QX-314 (adjusted to pH 7.2 with CsOH) was used for recording spontaneous excitatory postsynaptic currents (sEPSC) and spontaneous inhibitory postsynaptic currents (sIPSC). For sEPSC or sIPSC recording, cells were clamped to 60 mV and 0 mV, respectively. Membrane resistance Rm was maintained between 15 and 30 MΩ throughout the experiment. Cells with Rm changes greater than 15% during recording were discarded. Data acquisition was filtered at 1 kHz and sampled at 10 kHz. Action potentials (APs) induced by blue light stimulation (473 nm, 5 Hz, 10 mM / mm²) or depolarization current (0–100 pA, 10 pA steps, 2 s duration) were recorded under current-clamped conditions. In current-clamped mode, 90 pA or 120 pA current was injected into transplanted or endogenous mDA neurons for Sag detection. The tdTomato fluorescence signal in EYFP-positive transplanted cells was used to distinguish between mDA and non-mDA neurons.

[0209] Virus injection and rabies virus tracking experiment

[0210] For rabies virus tracking experiments, 200 nL of AAV virus expressing Cre-induced TVA and tdTomato (AAV2 / 9-Ef1a-DIO-TVA-2A-NLS-tdTomato, titer 1.29*10^12 gene copies (gc) / ml), or 200 nL of AAV expressing Cre-induced rabies glycoprotein (AAV2 / 9-Ef1a-DIO-G, titer 1.29*10^12 gc / ml) was co-injected into the transplant site (substantia nigra: AP = 2.9 mm, L = 1.1 mm, V = 4.4 mm, vertical depth calculated from the skull; striatum: AP = +0.6 mm, L = 1.8 mm, V = 3.2 mm, vertical depth calculated from the dura mater) in PD mice 5 months post-transplantation. Three weeks later, EnVA pseudotyped, rabies protein G-deficient, EGFP-expressing rabies virus (RVdG-EGFP, 400 nl, titer 2*10^8 pfu / ml) was injected into the same site for antisynaptic labeling. One week later, mice were sacrificed for histological analysis. For endogenous mDA neurons, the virus was injected into the SNc of DAT-Cre / Ai9 mice (AP = 2.9 mm, L = 1.1 mm, V = 4.5 mm, vertical depth calculated from the skull). After fixation, sections were prepared using a cryostat (30 mm thick). All unstained coronal sections (1:4 series) were imaged using a fluorescence microscope (Olympus VS120) through a 20x objective. Tiled images were automatically stitched together using VS-ASW (Olympus) software with a 10% overlap. The locations of labeled neurons and the outlines of brain regions were manually marked using Photoshop, following Paxinos and Franklin (2007). Some sections underwent immunostaining to clarify cell identity.

[0211] Tissue preparation and immunohistochemistry

[0212] Animals were euthanized with an excessive amount of pentobarbital (250 mg / kg, intraperitoneally) and perfused first with saline, then with 4% ice-cold phosphate-buffered paraformaldehyde (PFA). The brain was removed and successively immersed in 20% and 30% sucrose until submerged. Continuous sagittal (0.12 to 3.12 mm from medial to lateral) or coronal (1.42 to 0.10 mm from Bregma) sections were cut to a thickness of 30 mm using a cryostat (Leica SM2010R) and preserved in cryoprotectant solution at 20°C. The floating brain slices were incubated with primary antibody at 4°C for 1–2 nights, then unbound primary antibody was removed. For DAB staining, the sections were incubated with the corresponding biotinylated secondary antibody for 1 h, then with anti-biotin-biotin peroxidase for 1 h at room temperature. Immunoreactivity was observed using a DAB staining kit. The sections were then dehydrated with ethanol, permeabilized in xylene, and fixed in neutral resin. For fluorescent immunolabeling, the sections were incubated with the appropriate fluorescent secondary antibody at room temperature for 1 hour. They were then mounted using Fluoromount-G.

[0213] Lentiviral packaging

[0214] Lentiviral cells were generated in 293T cells by transfecting the packaging plasmid and backbone plasmid using a calcium phosphate / DNA coprecipitation method. The 293T cells were cultured in Dulbecco MEM (DMEM) containing 10% FBS. 72 hours after transfection, the supernatant containing viral particles was collected and concentrated by ultracentrifugation at 27,000 rpm for 2 hours at 4°C. The viral particles were then resuspended in DPBS.

[0215] Imaging and cellular quantification

[0216] To quantify the number or proportion of TH cells expressing EN1, FOXA2, LMX1A, NURR1, GIRK2, and TUJ-1, counting was performed on at least five randomly selected images from coverslips using ImageJ software. Data were repeated three times and expressed as mean ± SEM. To measure the density of human fibrils in brain slices, tiled images were captured using a Nikon TE600 or Olympus VS120 microscope. Optical density of human brain in different regions of the mouse brain was measured using an image processing and analysis system (Image Pro Plus 5.1 software). Data are shown as optical density in different regions. For TH, GIRK2, LMX1A, human nuclei (hN), and FOXA2 staining, grafts were delineated and captured using a Nikon A1R-Si laser scanning confocal microscope (Nikon) or a fluorescence microscope (Olympus VS120) with a 60x objective. Single-stained or double-stained cells were manually counted using ImageJ. Data are expressed as the ratio of TH-, LMX1A-, FOXA2- to total hN, or the ratio of GIRK2 / TH / hN to TH / hN cells. All data are expressed as mean ± SEM.

[0217] Behavioral test: Rotation test

[0218] Amphetamine-induced rotation was tested before transplantation and monthly to 6 months post-transplantation. Amphetamine (2 mg / ml in normal saline, 5 mg / kg) was administered intraperitoneally 5–10 minutes later, and recording was performed for 1.5 hours via camera. Data are expressed as the mean net rotations per minute over 90 minutes.

[0219] For the spontaneous rotation test, the animal was recorded for 60 minutes after injection of CNO (1.2 mg / kg) 20 minutes later, SALB (5 mg / kg) 5 minutes later, or saline 20 minutes later.

[0220] Behavioral Testing: Cylinder Experiment

[0221] Individual animals were placed in glass cylinders and recorded with a camera for 3 minutes. The number of times the paws on the ipsilateral and contralateral sides of the mouse brain injury came into contact with the cylinder wall was counted. Data are expressed as the percentage of ipsilateral touches out of the total touches. For drug treatment, animals were treated with CNO (1.2 mg / kg) for 20 minutes, SALB (5 mg / kg) for 5 minutes, or saline for 20 minutes before the cylinder experiment.

[0222] Behavioral testing: Rotating rod experiment

[0223] Motor coordination was tested using a roulette wheel (Med Associates Instruments). All animals underwent two days of pre-training to achieve stable performance. On Day 1, mice were trained on a roulette wheel that accelerated from 2 rpm to 20 rpm over 300 seconds, repeated three times. On Day 2, mice were trained on the roulette wheel twice, accelerating from 3 rpm to 30 rpm, and once, accelerating from 4 rpm to 40 rpm, over 300 seconds. Starting on Day 3, testing was conducted on the roulette wheel, accelerating from 4 rpm to 40 rpm over 300 seconds. The time the mice remained on the wheel was monitored. The average duration of the three repeated tests for each animal was used for data analysis.

[0224] Quantitative and statistical analysis

[0225] Statistical analysis was performed using SPSS software. In all studies, data were analyzed using the Student-T test, paired t-test, two-way ANOVA, Holm-Sidak test, two-way RM ANOVA and Tukey's post hoc test, or one-way ANOVA and Holm-Sidak test. Statistical significance was determined by p < 0.05.

[0226] The English annotations for the various abbreviations in this invention are shown in Table 1.

[0227] Table 1

[0228]

[0229] Example 1: Transplanted human dopaminergic and glutamatergic neurons project to different brain regions.

[0230] During development, the directional projection of axons typically depends on the intrinsic properties of the cell. To understand whether these intrinsic properties still determine the cell's projection target in the adult brain, the inventors transplanted mDA or forebrain glutamatergic neuronal precursor cells differentiated from hESCs into the midbrain of PD model mice. The inventors' method can differentiate hESCs into mDA or Glutamate neuronal precursor cells. On day 32 of mDA neuron differentiation (the day of transplantation), most precursor cells expressed the basal plate and midbrain markers CORIN, FOXA2, LMX1A, and EN1 (…). Figure 8 A and 8C). By day 42, 69% of total cells or 84% of TUJ1+ neurons expressed tyrosine hydroxylase (TH) as well as EN1, FOXA2, LMX1A, and NURR1 ( Figure 8D and 8F) indicate the characteristics of mDA neurons. Furthermore, most TH+ neurons co-express the A9 mDA neuronal marker GIRK2 (>85%), but less frequently express the A10 mDA neuronal marker Calbindin (CALB) (15%). Figure 8 D and 8F). Therefore, most TH+ neurons possess A9 mDA neuron characteristics. On day 32 of Glu neuron differentiation, most cells expressed the dorsal forebrain marker PAX6 and the forebrain marker FOXG1, indicating that they are dorsal forebrain neural progenitor cells (D and 8F). Figure 8 B and 8C). By day 42, 82% of the cells were vGLUT1 positive, and most of these neurons expressed CTIP2 (>80%) or TBR1 (>85%), indicating that the differentiation yielded Glu neurons from layers 5 / 6 of the forebrain cortex. Figure 8 E and 8F).

[0231] Subsequently, the inventors will use mDA( Figure 1 A) or Glu precursor cells ( Figure 1 B) Transplanted into the SN brain region of PD mice. Six months post-transplantation, transplanted cells were present in all transplanted animals. Serial sagittal sections of the mDA neuron transplanted brain showed that most hNCAM+ nerve fibers were distributed in the caudate putamen (CPu). Figure 1 (A and 1D), this region is mainly innervated by A9 mDA neurons (Bjorklund and Dunnett, 2007). Quantitative analysis of hNCAM+ fiber density was performed from three representative sagittal planes (A and 1D). Figure 1 C) indicates that in the CCu brain region, 72% of the total hNCAM+ fibers are present in plane L2.16, 62% in plane L1.44, and 45% in plane L0.72. Figure 1 E). Fewer hNCAM+ fibers were detected in the olfactory tubercle (Tu) (17%–20%) and the nucleus accumbens (Acb; 9%–16%), brain regions that primarily receive A10 mDA neuronal projections. The remaining regions, including the amygdala and cortex, accounted for less than 15% of the total fibers. Figure 1 E). Within the striatum, 72% and 87% of human-derived fibers project to the dorsal striatum (CPu) at planes L0.72 and L1.44, respectively. Figure 1 F). The distribution pattern of hNCAM+ fibers, especially in CPU, is similar to the distribution pattern of endogenous TH+ fibers in normal animals. Figure 1 D and 8G).

[0232] Conversely, axonal growth in Glu neurons is localized, projecting throughout the midbrain ( Figure 1B). They also send axons to distant sites, but primarily to the amygdala, olfactory bulb (OB), asymptomatic substance (SI), and cerebral cortex, while only a small fraction (2%–8%) of hNCAM+ fibers are detected in CPU. Figure 1 B, 1D and 1E).

[0233] In summary, these results indicate that transplanted human neural progenitor cells differentiate into their respective neuronal types, while axons project to different brain regions.

[0234] Example 2: Transplanted human mDA neurons project via a homologous pathway

[0235] From continuous sagittal sections ( Figure 2 A) shows that the transplanted mDA neurons extend along the well-defined medial forebrain tract (MFB) axons in the medulla oblongata. Figure 2 B, L1.08 and the red arrow are at Figure 2 (C in the middle). Viewed from the side, they extend through SI ( Figure 2 B, L1.44) and amygdala ( Figure 2 B, L2.04). Viewed from the snout side, hNCAM+ fibers pass through Acb ( Figure 2 B-2E), in which most hNCAM+ fibers enter CCu along the boundary between the cortex and striatum. Figure 2 A and Figure 2 (Red arrow in D). A small number of hNCAM+ fibers were detected in the cortex. Figure 2 (Blue arrows in D and 2F). These results indicate that most axonal projections of the transplanted mDA neurons follow the intrinsic nigrostriatal pathway.

[0236] In the dorsal / lateral striatum, dense hNCAM+ fibers are branched, forming a dense branched network. Figure 2 D–2F and Figure 9 A). In Tu ( Figure 2 Axonal branching was also observed in C and 2D, but not in Acb, amygdala, and MFB. Figure 9 No axonal branching was observed in B), indicating projection-specific axonal branching. Most fibers stained with STEM121 were TH-positive ( Figure 2 G and 2H) indicate that these nerve fibers are dopaminergic. Branches of hNCAM+ fibers from transplanted Glu neurons were observed in the OB and cortex. Figure 9 C).

[0237] Immunohistochemical analysis of mDA grafts showed that 68% of the transplanted cells co-expressed TH and human nuclei (hNs), and most TH+ cells also expressed GIRK2, as well as FOXA2 and LMX1A. Figure 9 D– Figure 9 F) suggests A9 mDA characteristics. Human-specific synaptophysin (hSyn) sites are distributed along TH+ fibers in CPU, with some located on GABA+ cell bodies. Figure 9 In contrast, almost no hSyn+ synapses were observed in the forebrain neuron transplantation group, and almost none were localized on the GABA+ cell body. Figure 9 These results indicate that transplanted human mDA neurons can form synaptic connections with target cells in the host brain.

[0238] Example 3: Genetic markers reveal specific axonal neural distribution in human mDA neurons

[0239] To elucidate the specific axonal neural distribution induced by transplanted mDA neurons, the inventors established an hESC line carrying the TH reporter gene with tdTomato expression, summarizing the expression of endogenous TH genes (method details). The inventors further knocked the ChR2-EYFP fusion protein expression cassette into the AAVS1 gene locus to achieve specific labeling and manipulation of transplanted human cells (…). Figure 3 A, 3B, 10A, and 10B). The final hESC, called TH-tdTomato / AAVS1-ChR2-EYFPhESC, structurally expresses ChR2-EYFP throughout mDA neuron differentiation, while tdTomato is only expressed in the later stages of mDA neuron differentiation. Figure 10 C), and is expressed only in TH+mDA neurons. Figure 3 C and 10D) highlight the specific expression of TH+ cells.

[0240] Then, the inventors transplanted mDA precursor cells derived from TH-tdTomato / AAVS1-ChR2-EYFP hESC into the SN or striatum of PD model mice. Figure 3 A). Six months after transplantation, EYFP+ transplanted cells appeared in all transplanted animals. Figure 3 D), while tdTomato is expressed only in TH+ mDA neurons, and not in non-mDA neurons; for example, 5-HT+ neurons ( Figure 10 E and 10F). Serial coronal sections of the brain from SN cell transplantation show that in most individuals, mDA neurons project into the CCu, where fibers form a dense and branching network. Figure 3E, b, and c). Only a small fraction of mDA neuron projections exist in Acb ( Figure 3 E, sections 1-3). In these coronal sections ( Figure 3 In sections E (slices 1 and 2, a and b, red dashed arrows), the medullary projection pathway of mDA neuron axons was determined. tdTomato+ projection showed STEM121 positivity. Figure 3 F) confirmed that it was of human cell origin. These results validated the specific axonal pathway and targeting function of transplanted human mDA neurons shown by hNCAM staining. Figure 1 and 2 In striatal transplantation, tdTomato+mDA neuronal fibers occupy the entire CPU, and most fibers are confined within the CPU. Figure 3 G, 3H, and 10G). In substantia nigra transplantation, tdTomato+ fibers showed extensive dendritic growth limited to the surrounding graft (G, 3H, and 10G). Figure 3 I) indicates cell and target-specific branching of human mDA dendrites and axons. Furthermore, hESC-derived dopamine (DA) neurons are distributed around the striatum and substantia nigra grafts (I). Figure 3 This phenomenon (H and 3I) is very similar to human fetal brain transplantation in PD patients.

[0241] Cell characteristics ( Figure 8 D, 9D and 9F) and specific projections ( Figure 1 , 2 (3) indicates that the inventors' mDA neurons are similar to neurons in SN pars compacta (SNc). Using a genetic reporter gene, the inventors performed whole-cell patch-clamp recordings on mDA neurons (EYFP+ / tdTomato+) and non-mDA neurons (EYFP+ / tdTomato+). Figure 10 H). The inventors have discovered that human mDA neurons and non-mDA neurons in striatal or substantia nigra grafts exhibit current- or blue light-induced action potentials (APs) and spontaneous APs (sAPs) within 3 months post-transplantation. Figure 3 J and 11A–11F) indicate functional maturation of the transplanted human neurons. Importantly, the sAPs of human mDA neurons transplanted into the striatum and substantia nigra showed regular firing patterns with low firing frequencies (0.87±0.20 Hz in the striatum and 0.83±0.15 Hz in the substantia nigra) and slow subthreshold oscillatory potentials (0.29±0.06 Hz in the striatum and 0.26±0.13 Hz in the substantia nigra). Figure 3 J, 11G, and 11H). Six months after transplantation, significant hyperpolarization (AHP) was observed in the sAP of human mDA neurons. Figure 11I and 11J). These physiological characteristics are consistent with those of endogenous SNc(A9)mDA neurons (I and 11J). Figure 3 J; Guzman et al., 2009; Lammel et al., 2008; Nedergaard et al., 1993). Furthermore, compared to non-mDA neurons, transplanted mDA neurons showed a trend towards higher membrane capacitance (Cm) and lower neuronal input resistance (Rm) in striatal or substantia nigra grafts at 6 months post-transplantation. Figure 11 K and 11L). Furthermore, endogenous SNc mDA neurons are characterized by a concave amplitude in response to hyperpolarization current injection (Evans et al., 2017; Lammel et al., 2008; Neuhoff et al., 2002). Using mDA neuron reporter mice (DAT-Cre / Ai9), the inventors found that all recorded SNc neurons exhibited typical concave amplitudes in the sub-threshold range (37.2 ± 3.8 mV, n = 15 / 15). Figure 3 K and 3M). However, among the 11 endogenous VTA mDA neurons, only 5 showed a voltage dip, and the amplitude was much smaller (23.0 ± 1.6 mV, n = 5 / 11). Figure 3 K and 3M). Interestingly, the human mDA neurons transplanted into the striatum showed a significant voltage dip (32.5 ± 3.3 mV, n = 13 / 16). In contrast, only 11 of the 29 transplanted non-mDA neurons showed a much smaller voltage dip (14.9 ± 2.9 mV, n = 11 / 29). Figure 3 L and 3M).

[0242] These results indicate that transplanted human mDA neurons possess the functional characteristics of A9 mDA neurons.

[0243] Example 4: Structurally, the synaptic input of human neurons is related to the transplantation site.

[0244] Rabies virus-mediated tracing has been used to track anatomical inputs to transplanted cells in PD models. To reveal the presynaptic signal inputs of transplanted human mDA neurons, the inventors combined the Cre-loxP gene expression system with rabies virus-mediated synaptic tracing. Figure 4 A). The inventors established the TH-iCre hESC line, enabling Cre recombinase to be directly expressed in TH-expressing cells without disrupting the expression of endogenous TH. Figure 4B, 12A, and 12B). The specificity of this system was demonstrated by the unique mCherry expression in TH+mDA neurons after infection of TH-iCre hESC-derived neuronal cultures with a lentivirus expressing Cre-dependent mCherry. Figure 12 Five months after transplanting TH-iCre mDA precursor cells into the substantia nigra or striatum of PD mice, Cre-dependent TVA and NLS-tdTomato expressing AAV (AAV-DIO-TVA-2A-NLS-tdTomato) and Cre-dependent rabies glycoprotein (G) expressing Cre (AAV-DIO-G) were co-injected into the transplantation site. Figure 4 A). One month later, EnvA pseudotyped and G-deficient rabies virus expressing EGFP (RVdG-EGFP) was injected into the transplant site ( Figure 4 A). Because only transplanted human mDA neurons express Cre recombinase, the expression of TVA, tdTomato, and G is limited to human mDA neurons, not just mDA neurons. In fact, tdTomato is expressed only in TH+ human mDA neurons. Figure 12 E). Because RVdG-EGFP only infects (tdTomato+) human mDA neurons expressing TVA, the transplanted initiating human mDA neurons co-express EGFP and tdTomato. Co-expression of G in transplanted human mDA neurons allows RVdG-EGFP to synaptactically propagate to their presynaptic partners (Wickersham et al., 2007); therefore, the host presynaptic neurons express only EGFP ( Figure 4 A).

[0245] The initiating neuron (EGFP+ / tdTomato+) was found only in human transplanted cells and was TH+ ( Figure 4 (C and 4D). Synaptically labeled host neurons (EGFP+ / tdTomato+) are readily detectable in brain regions far from the transplant site. Figure 4 E). In the transplanted brain, the most abundant host neurons with labeling were found in the striatum, including CCu and Acb ( Figure 4 E-4G). Host neurons labeled in CPU express GABA and DARPP32, suggesting they are striatal spinous neurons (E-4G). Figure 12 F). In Acb, labeled host neurons form plaques, a phenomenon observed in different samples. Figure 4 H). In the cortical region, CTIP2+ and SATB2+ cortical neurons were found to project to human substantia nigra mDA neurons (H). Figure 4E-4G and 12F). In the hypothalamus, the peduncle portion of the lateral hypothalamus (PLH) and the paraventricular nucleus (Pa) strongly project to human mDA neurons in the SN. Densely labeled host neurons were also found in the terminal striatum (ST) and the bed nucleus of the central amygdala (Ce), but not in other amygdala regions. Figure 4 E-4G). Furthermore, scattered neurons were observed in the globus pallidus (GP), ventral globus pallidus (VP), and extended amygdala (EA). More in the caudal region, dorsal suture (DR), and periaqueductal gray (PAG). The pontine reticular nucleus (Pn) contains a large number of labeled neurons. Figure 4 E-4G). The inventors discovered 5-HT+ neurons in DR that project to human mDA neurons in SN (E-4G). Figure 12 F). The presynaptic input distribution of transplanted human mDA neurons was strikingly similar to that of endogenous SNc mDA neurons in DAT-Cre mice, which express Cre in DA neurons. Figure 12 G and 12H), and endogenous mDA neurons with sheet-like input distribution were also observed in Acb.

[0246] In striatal transplanted brains, dense presynaptic input was found in the CCu, but not in the Acb. More input was observed in the GP and cortical regions compared to human mDA neurons transplanted into the substantia nigra. The parafascicular nucleus (PF) and ventromedial nucleus (MD) tended to project onto striatal human DA neurons. Labeled host neurons were also found in the Ce and SN reticular formation (SNR) in striatal transplanted brains. In areas with more coccygeal regions, such as the PAG, DR, parabrachial nucleus (PB), and Pn, few labeled neurons were observed. Figure 4 E, 4F and 12J).

[0247] Therefore, the input received by human mDA neurons transplanted into the striatum and substantia nigra comes from different brain regions, indicating location-dependent presynaptic input. Like endogenous mDA neurons, human mDA neurons transplanted into the substantia nigra also receive a significant amount of input from similar brain regions.

[0248] Example 5: The type of transplanted neuron determines its functional input characteristics.

[0249] Electrophysiological recordings showed that 3 months post-transplantation, sEPSCs and sIPSCs (spontaneous excitatory and inhibitory postsynaptic currents, respectively) were almost undetectable in human mDA and non-mDA neurons in the striatum or substantia nigra transplanted blocks. Figure 5 A-5D). Surprisingly, 6 months post-transplantation, the mean frequency, rather than the amplitude, of sIPSCs and sEPSCs from mDA and non-mDA neurons in the striatal or substantia nigra graft significantly increased. Figure 5 A-5D). There was no difference in the rise or decay time of sEPSC or sIPSC between transplanted non-mDA neurons and mDA neurons. Figure 13 These results indicate that functional input is established within 3 to 6 months, regardless of transplantation location or neuron type.

[0250] Interestingly, in the substantia nigra transplant, the sIPSC frequency in mDA neurons was higher than that in non-mDA neurons. Figure 5 C). In contrast, the frequency of sEPSCs in mDA neurons was significantly lower than that in non-mDA neurons in striatal and substantia nigra grafts. Figure 5 D). By calculating the sIPSC / sEPSC ratio, the inventors found that transplanted human mDA neurons received more inhibitory input, with an sIPSC / sEPSC ratio of 3.28, similar to the pattern of endogenous mDA neurons, while human non-mDA neurons received less inhibitory / excitatory input, with an sIPSC / sEPSC ratio of 0.95. Figure 5 E and 13G). The trend of higher sIPSC / sEPSC ratios (2.61) in human mDA neurons compared to non-mDA neurons was also observed in striatal graft blocks. Figure 5 E), while endogenous striatal neurons receive more excitatory input, with a sIPSC / sEPSC ratio of 0.86 (E). Figure 5 E and 13H).

[0251] These results indicate that the type of transplanted neuron, rather than the transplantation site, determines the inhibitory and excitatory input characteristics of the transplanted neuron.

[0252] Example 6: Transplantation of mDA rather than Glu neurons can correct motor dysfunction in PD mice.

[0253] The functional effects of the transplanted substantia nigra cells were assessed before and every 4 weeks after transplantation using amphetamine-induced rotation, rotation test, and cylinder test. Figure 6 A). In striatal transplanted PD mice, amphetamine-induced rotation began to recover at 3 months and was fully recovered at 4 months post-transplantation. Over time, the mice gradually showed overcompensation by rotating to the contralateral side. Figure 6 B). Functional recovery was also observed in PD mice with mDA transplantation into the substantia nigra 4-5 months later; however, overcompensation disappeared by 6 months (p<0.001). Figure 6 B). Conversely, mice whose substantia nigra received either glutamate neurons or artificial cerebrospinal fluid (ACSF; control) showed no signs of recovery in amphetamine-induced rotation (p>0.05). Figure 6 B).

[0254] In rotational tests used to assess motor coordination and balance without relying on dopaminergic system pharmacological stimulation, the duration of falls increased significantly over time in PD mice that received human mDA neuron transplants into the substantia nigra or striatum (p<0.001), but not in mice that received substantia nigra Glu neurons or ACSF (p>0.05). Figure 6 C).

[0255] In the cylinder test, a method for measuring forelimb motor ability, all groups showed preferential ipsilateral limb touch after 6-OHDA injury. Four months after transplantation of human mDA into the substantia nigra or striatum, ipsilateral contact preference was significantly reduced in mice (nearly 50%) (p<0.001), but this was not changed in mice with Glu neurons or ACSF transplanted into the substantia nigra (p>0.05). Figure 6 D).

[0256] Example 7: Motor recovery depends on the functional reconstruction of the nigrostriatal neural circuit.

[0257] To determine whether behavioral recovery in PD model animals depends on the reconstructed nigrostriatal circuit, the inventors configured transplanted mDA neurons derived from hESCs with a "bidirectional switch" ( Figure 7 A). The inventors have established an hESC cell line called Bi-DREADD-hESCs by inserting two DREADD (drug-specifically activated receptors) into the AAVS1 locus: an excitatory hM3Dq receptor activated by CNO (Alexander et al., 2009) and a KORD inhibitory receptor activated by Salvinorin B (SALB) (Vardy et al., 2015). Figure 7 B, 14A, and 14B). hESCs (mCherry-hESCs) with mCherry expression knocked into the AAVS1 locus were used as controls. Figure 7 B, 14A, and 14B). The expression of the transgenes was readily detectable in neurons derived from Bi-DREADD-hESC or mCherry-hESC during culture or after transplantation. Figure 7 C, 7D, and 14C-14G). Six months post-transplantation, motor recovery was observed in both the Bi-DREADD group and the mCherry (control group), as evidenced by amphetamine-induced reduction in rotation and ipsilateral contact preference (p<0.05). Figure 7 E-7G).

[0258] Using cylinder tests and spontaneous rotation tests that do not require amphetamine stimulation for DA release, the inventors found that CNO (1 mg / kg) treatment can further reduce ipsilateral preferential contact (p<0.05). Figure 7 H), while SALB (5 mg / kg) treatment increased ipsilateral tactile sensation in the same animals transplanted with Bi-DREADD (p<0.01); Figure 7 H). CNO or SALB treatment had no effect on ipsilateral contact in mice receiving mCherry expression (p>0.05); Figure 7 H). In the spontaneous rotation test, control mice showed no significant changes in motor balance. However, in mice transplanted with Bi-DREADD cells, CNO treatment significantly induced more contralateral rotation than ipsilateral rotation (p<0.01). Figure 7 I) The ipsilateral rotation ratio decreased from 48.49±8.10 to 34.83±6.23 (p<0.05); Figure 7 Conversely, SALB significantly increased ipsilateral net rotation (p<0.05), with the ipsilateral rotation ratio increasing from 48.49±8.10 to 58.42±9.91 (p<0.01). Figure 7 J). No significant changes were observed in mCherry mice regardless of CNO or SALB treatment (p>0.05). Figure 7 I and 7J).

[0259] These results indicate that recovery from forelimb motor dysfunction and asymmetric rotation depends on the activity of transplanted cells.

[0260] discuss

[0261] In this invention, a genetic marker strategy was developed to precisely map the projections and synaptic inputs of transplanted human mDA neurons in a PD mouse model. The inventors discovered that human mDA neurons transplanted into the substantia nigra specifically project to the dorsal striatum. Rabies virus-mediated tracking revealed that transplanted mDA neurons receive synaptic input in a manner remarkably similar to endogenous mDA neurons. Electrophysiological recordings showed that the primarily inhibitory input to transplanted mDA neurons appeared to depend on cell type rather than transplantation site. Through presynaptic and postsynaptic integration, orthotopically transplanted human mDA neurons could rescue motor dysfunction in PD mice based on the activity of the transplanted cells. These findings reveal cell type-dependent functional circuit integration in transplanted neurons, highlighting the potential for repairing neural circuits using specific neuronal types from stem cells to treat neurological diseases.

[0262] What determines the targeted projection of transplanted neurons in the mature brain remains unknown. By transplanting two types of projection neurons, mDA and Glu neurons, into the tunica albuginea of ​​PD mice, the inventors found that both neuron types can project axons over long pathways, but they project axons to different targets via different pathways, with the majority of transplanted mDA neuron axons projecting to CCu. This was further confirmed by using the TH reporter cell line, which showed almost exclusive projection to the dorsal (CPu) rather than the ventral striatum (Acb). Since CCu is the primary target of SNc(A9) mDA neurons (Bjorklund and Dunnett, 2007; Joel and Weiner, 2000), the inventors' findings suggest that human mDA neurons are predominantly A9 mDA neurons. Indeed, the inventors' cellular signatures, particularly electrophysiological recordings, confirmed the A9 morphology of human mDA neurons. This interpretation suggests that many axonal projections to other brain regions observed in previous studies may originate from non-mDA neurons. In summary, these results strongly suggest that the projection pathway and target brain region depend to a large extent on the type of transplanted neurons.

[0263] Properly delivering synaptic input to transplanted neurons is also crucial for restoring lost function. In this invention, the TH-iCre system specifically identifies monosynaptic inputs to transplanted human mDA neurons, revealing an interesting pattern. Generally, anatomical synaptic input in grafts appears to be related to transplantation location, even though mDA neurons transplanted into the substantia nigra and striatum receive input from overlapping regions (such as the dorsal striatum), similar to the observations of Adler et al. However, the striking similarity in input between transplanted human and endogenous mDA neurons suggests that cell type plays a role in determining synaptic input. This is even clearer at the functional level. Compared to non-mDA neurons, transplanted mDA neurons receive more inhibitory but less excitatory input, regardless of whether the cells are transplanted into the striatum or substantia nigra, indicating that the type of transplanted neuron determines functional synaptic input.

[0264] By observing the presynaptic and synaptic integration of transplanted mDA neurons, it is natural to assume that the reconstructed substantia nigra-striatal circuit contributes to motor recovery in PD mice. It has been demonstrated that human mDA neurons transplanted into the striatum are functionally connected to striatal neurons, and optogenetic and chemogenetic tools have been used to promote behavioral recovery in animals. In the current study, the Bi-DREADD strategy demonstrated activation and inhibition on the same transplanted cells, clearly indicating that the reconstructed substantia nigra-striatal circuit is functional and fundamental to behavioral recovery in PD mice.

[0265] By combining precise genetic markers and functional testing, the inventors discovered that the restoration of neural circuits (including pathway finding, target specificity, and functional input establishment) by transplanted cells in the mature brain largely depends on the intrinsic characteristics of the transplanted neurons. Therefore, it is crucial to transplant highly enriched, fate-determined neural progenitor cells to reconstruct specific circuits for therapeutic effects. Thus, cell-based therapies for treating neurological diseases are a realistic possibility.

[0266] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing A9 midbrain substantia nigra dopaminergic neurons, comprising: (1) Stem cells were placed in a culture medium containing a neural inducer and induced in multiple stages with added components, including: Phase 1: Add 1–15 μM DMH-1, 200–1000 ng / mL SHH, and 0.1–1 μM CHIR99021; Second stage: Add 0.1-5 μM SAG, 50-300 ng / ml SHH, and 0.1-1 μM CHIR99021. The second stage is: attach the cells cultured in the first stage to a fibroblast feeder layer for culture. The third stage involves adding 5–100 ng / ml of SHH, 0.1–5 μM of SAG, and 5–200 ng / ml of FGF8b, and the third stage is a suspension culture. Fourth stage: Add 5–100 ng / ml of SHH and 5–80 ng / ml of FGF8b, and the fourth stage is a suspension culture; and (2) Obtain midbrain substantia nigra dopaminergic neurons derived from the aforementioned stem cells from the culture. The nerve cells mentioned are dopaminergic precursor cells of the A9 brain.

2. The method as described in claim 1, characterized in that, When induction is performed in multiple stages with different added components: Phase 1: Add 10±5 μM of SB431542, 2±1 μM of DMH-1, 500±200 ng / ml of SHH, and 0.4±0.2 μM of CHIR99021; Second stage: Add 2±1μM SAG, 100±50 ng / ml SHH, and 0.4±0.2μM CHIR99021; Phase 3: Add 20±10 ng / ml of SHH and 0.5±0.2 μM of SAG, and 100±50 ng / ml of FGF8b; Phase 4: Add 20±10 ng / ml of SHH and 20±10 ng / ml of FGF8b.

3. The method as described in claim 1, characterized in that, In each stage, Phase 1: From the start of culture to 6-8 days of culture; Second stage: Culture for 6-8 days to 11-13 days; Phase 3: Culture for 11-13 days to 18-20 days; Phase 4: Culture for 18-20 days to 31-33 days.

4. The method as described in claim 1, characterized in that, In each stage, Phase 1: From the start of culture to 7±0.5 days; Second stage: Culture for 6-8 days to 12 ± 0.5 days; Phase 3: Cultured for 11–13 days to 19 ± 0.5 days; Phase 4: Culture for 18-20 days to 32±0.5 days.

5. The method as described in claim 1, characterized in that, The stem cells mentioned include embryonic stem cells or induced pluripotent stem cells.

6. The method as described in claim 5, characterized in that, The embryonic stem cells or induced pluripotent stem cells mentioned are human embryonic stem cells or human induced pluripotent stem cells.

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