Use of forebrain neural progenitor cells in the treatment of diseases associated with death and / or dysfunction of forebrain neurons

Abstract

The present invention provides use of a neural cell population for treating a neuronal injury disease, a neurodegenerative disease, a neurodevelopmental disease, or a pathology associated with the death and / or dysfunction of forebrain neurons, wherein the neural cell population comprises forebrain neural progenitor cells (FNPCs) with specific developmental characteristics. Preferably, the forebrain neural progenitor cells with specific developmental characteristics highly express markers including FOXG1, PAX6, and NESTIN. Preferably, the neural cell population is obtained by culturing human pluripotent stem cells (hPSCs).

Description

[Technical Field]

[0001] [Cross reference] This application claims priority to a Chinese patent application for invention bearing application number 202211466612.0, filed on November 22, 2022, and entitled "Application of forebrain neural progenitor cells in treating diseases associated with the death and / or dysfunction of forebrain neural cells," the entire contents of which are incorporated herein by reference.

[0002] [Technical field] The present invention relates to the field of cell therapy in biotechnology, and in particular to the application of neuronal cell populations to treat neurological diseases, disorders, or conditions associated with neuronal injury, neurodegenerative, or neurodevelopmental disorders, or the death and / or dysfunction of forebrain neurons. The neural cell population contains forebrain neural progenitor cells that have specific developmental characteristics. [Background technology]

[0003] During early development, the forebrain differentiates into the telencephalon and diencephalon. The telencephalon then develops into the cerebral cortex and subcortical brain tissues, such as the basal ganglia, olfactory bulb, and hippocampus, while the diencephalon develops into the thalamus, inferior thalamus, and superior thalamus. Each part of the forebrain has a complex structure and function, and many diseases and pathologies are associated with damage or dysfunction of forebrain neurons. These diseases and pathologies include neurodegenerative disorders such as stroke, post-stroke syndrome, traumatic brain injury (TBI), and post-brain injury syndrome; neurodegenerative disorders such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS); and neurodevelopmental disorders such as epilepsy, autism, and cerebral palsy.

[0004] Stroke is one of the leading causes of cranial nerve damage and loss of function worldwide. According to statistics from the World Health Organization (WHO), approximately 15 million people suffer from stroke each year worldwide, of which approximately one-third die and another one-third suffer permanent serious disabilities, with high incidence, mortality, and disability rates.

[0005] Currently, there are approximately 20 million surviving stroke patients in China, with the number increasing by approximately 2.7 million each year. As the aging of society progresses, the number of stroke patients will continue to grow. The prognosis and quality of life of stroke patients is poor, causing great inconvenience to patients and their families and creating a serious social and economic burden.

[0006] Strokes are classified into hemorrhagic strokes and ischemic strokes, with the latter accounting for approximately 80% of cases. The brain is extremely sensitive to ischemia. When ischemia occurs, the brain immediately suffers from a lack of energy supply, causing functional impairment. If the situation is not improved, the functional impairment will worsen and lead to cerebral infarction.

[0007] Currently, the primary treatment for ischemic stroke is rapid thrombolytic therapy or endovascular thrombectomy within 3 to 6 hours of the onset of the stroke to restore cerebral blood flow and reduce brain tissue damage. This treatment is typically performed using drugs such as t-PA, which was the first thrombolytic drug approved by the US FDA in 1996 for the treatment of ischemic stroke. A drawback of this type of treatment is the narrow window of application, which often results in patients being unable to receive treatment. Another drawback is that this type of treatment does not reverse neurological damage, and because certain neurological damage caused by ischemia still exists, there is a risk of residual aftereffects such as functional impairment even after treatment.

[0008] In addition, neuroprotective agents such as calcium channel blockers (e.g., nimodipine), free radical scavengers (e.g., edaravone, tocopherol, SOD), neurotrophic factors (e.g., nerve growth factor), NMDA antagonists, gangliosides, excitatory glycine receptor antagonists, and Enbip are also used in stroke treatment. These drugs are targeted at acute stroke patients (within 14 days after the onset of stroke) and aim to reduce the impact on neuronal function by reducing neuronal damage, inflammation, and cell death. However, most of these drugs have low clinical efficacy or harmful side effects, and are unable to alleviate or treat the aftereffects of hemiplegia caused by stroke.

[0009] Cell therapy is a new therapeutic approach currently available. Several clinical trials using mesenchymal cells to treat acute stroke patients have shown that these cells have anti-inflammatory, tissue repair, and vascular regeneration effects. However, in controlled clinical trials or clinical studies with expanded populations, mesenchymal cells have not demonstrated clear therapeutic effects.

[0010] Rehabilitation is currently the standard treatment for functional disorders caused by neurological damage such as stroke, but there are limits to how much it can promote functional recovery, and it is difficult to bring about improvement again in the stable period of hemiplegia (six months or more after onset).

[0011] These treatments can reduce the damage caused by stroke to a certain extent or achieve a certain degree of functional recovery, but they cannot reorganize the function of damaged tissues or improve the aftereffects of hemiplegia.

[0012] Because most stroke patients suffer from sequelae due to brain damage, even physical disabilities, it is extremely important to reverse or compensate for the loss of neurons caused by stroke. Currently, no FDA-approved neuroprotective drugs can reverse or restore these neuronal losses, and clinically, no drugs have been shown to improve the function of patients in the stable hemiplegic stage.

[0013] Research has shown that certain areas of the adult cerebral cortex (such as the dentate gyrus and subventricular zone) have regenerative capabilities and can induce compensatory neurogenesis after a stroke. However, the number of newly generated neurons and the repair effect of such endogenous neurogenesis-promoting therapies are very limited, making it difficult to overcome the functional impairment caused by brain tissue damage after a stroke.

[0014] With advances in stem cell technology, research is underway to promote tissue repair and replacement of lost neurons by transplanting tissue-derived neural stem cells into the brains of patients with ischemic stroke.

[0015] ReNeuron's patent application WO2011137117A1 discloses a method for treating stroke using the immortalized human fetal neural stem cell line CTX0E03. A key feature of this method is that neural stem cells can be delivered intravenously or intraarterially without the need for invasive craniotomy. This application was validated in a rat model. However, this application requires treatment within seven days, preferably two days, after the onset of stroke.

[0016] NeuralStem's patent application WO2006055685A2 discloses a method for transplanting human neural cells for the treatment of neurodegenerative diseases. The method involves isolating neural stem cells from tissue regions corresponding to the neurons in need of replacement, culturing and expanding them in vitro, and then transplanting them into the corresponding neural tissue to replenish cells in the neural circuit that produce specific neurotransmitters. This application does not address stroke and the resulting neuronal damage, and calls for the transplantation of tissue-derived primary neural stem / progenitor cells.

[0017] The cells used in the above method are all derived from neural stem cells obtained from aborted fetal tissue, which poses ethical restrictions for clinical commercialization. Furthermore, due to the late stage of obtaining so-called "neural stem cells," cells obtained from aborted fetal brain or spinal cord tissue are primarily glial progenitor cells. Therefore, after intracranial implantation of CTX0E03 products into animals, only 2% differentiated into FOXA3 neurons and approximately 20% into astrocytes. There are no reports of the transplanted cells possessing electrophysiological function or forming synaptic connections with the original animal neurons. This indicates that the transplanted cells did not develop into neurons capable of forming new neural circuits (EJ Smith et al., Stem Cell 2012, 30:785-796).

[0018] On the other hand, immortalized neural stem cell products (those that can be continuously proliferated by introducing tumor genes or fluorescent reporter genes) can be expanded in vitro and produced in batches, but clinically there is a certain risk of tumor formation.

[0019] Similar to neuronal damage caused by stroke, many other diseases characterized by the death and / or dysfunction of forebrain neurons, such as the aforementioned neuronal injury, neurodegenerative, and neurodevelopmental disorders, are expected to be ameliorated by providing patients with effective cell replacement therapies. Therefore, there is an urgent need in the art for cell products that are not dependent on fetal tissue, and that can differentiate into forebrain neurons with desired functions, enabling compensatory cell therapy for neurodegenerative diseases or neuronal damage. Summary of the Invention

[0020] To solve the above problems, the inventors developed forebrain neural progenitor cells (FNPCs) obtained by directional differentiation of pluripotent stem cells, and applied them to a stroke model animal of a primate (cynomolgus monkey) with ischemic nerve damage to study the therapeutic potential of these cells for cranial nerve damage and neurodegenerative diseases.

[0021] The inventors discovered that after administering the forebrain neural progenitor cells to a primate stroke model animal, they survived for a long time, differentiated into neurons at a high rate, and significantly improved the clinical symptoms of the diseased animals, demonstrating excellent therapeutic effects in terms of improving motor activity, reducing the lesion area, and increasing the animals' weight, etc. These results suggest that the forebrain neural progenitor cells are promising for the treatment of related diseases in the human body, such as repairing nerve injury diseases, treating neurodegenerative diseases, or treating other similar pathologies.

[0022] Furthermore, the inventors discovered that the forebrain neural progenitor cells of the present invention are suitable for administration after the acute phase of stroke and can provide sustained improvement. For example, when administered at high doses, cynomolgus monkeys with a permanent middle cerebral artery occlusion (pMCAO) disease model recovered their ladder walking speed to the level of normal healthy monkeys 5 months after transplantation of forebrain neural progenitor cells on the 15th day after onset. Meanwhile, the walking time of the model animals in the saline-injected control group was approximately twice that of normal healthy monkeys. This indicates that the therapeutic window for stroke, as previously thought, has been unexpectedly significantly expanded. When using the forebrain neural progenitor cells or cell preparations of the present invention to treat indications such as stroke and associated neuronal damage, the therapeutic window is not limited to the acute phase, but can also be used after the acute phase. Based on the above findings, the present invention was completed.

[0023] Thus, in a first aspect, the present invention provides use of a cell population for the manufacture of a medicament for treating a neuronal injury disease, a neurodegenerative disease, a neurodevelopmental disease, or a neurological disease associated with the death and / or dysfunction of forebrain neurons, the cell population comprising forebrain neural progenitor cells (FNPCs) with specific developmental characteristics. Preferably, the forebrain neural progenitor cells with specific developmental characteristics highly express markers including FOXG1, PAX6, and NESTIN. Preferably, the cell population is composed of a specific type of cell, the highest proportion of which are neural progenitor cells, preferably forebrain neural progenitor cells. Preferably, the cell population is obtained by culturing human pluripotent stem cells (hPSCs) using a specific method.

[0024] In a second aspect, the present invention provides a method of treating a subject having a neuronal injury disease, a neurodegenerative disease, a neurodevelopmental disease, or a neurological disease associated with death and / or dysfunction of forebrain neurons, said method comprising administering to said subject a therapeutically effective amount of a cell population comprising forebrain neural progenitor cells having particular developmental characteristics.

[0025] In preferred embodiments of the first and second aspects, the cell population is obtained by culturing human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0026] In a preferred embodiment of the first and second aspects, at least about 70% or more, preferably at least 80% or more, and more preferably at least 90% of the cells in the cell population express one or more markers selected from NESTIN, FOXG1, PAX6, and SOX2. Preferably, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells in the cell population express NESTIN, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express FOXG1, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express PAX6, and at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express SOX2.

[0027] In preferred embodiments of the first and second aspects, at least about 70% or more, preferably at least 80%, and more preferably at least 90% of the cells in the cell population express one or more markers selected from EFNB2, WNT7B, RSPO2, and FEZF2. Preferably, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells in the cell population express EFNB2, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express WNT7B, at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express RSPO2, and at least about 70% or more, preferably at least about 80% or more, more preferably at least 85%, and even more preferably at least 90% of the cells express FEZF2.

[0028] In preferred embodiments of the first and second aspects, the proportion of neural progenitor cells in the cell population as a percentage of all cell types is ≥ 60%, more preferably ≥ 70%, even more preferably ≥ 80%. Preferably, the proportion of pluripotent stem cells in the cell population as a percentage of all cell types is ≤ 3%, preferably ≤ 2%, more preferably ≤ 1%.

[0029] In preferred embodiments of the first and second aspects, the cell population can be further differentiated into neuronal cells in vitro and in vivo. Preferably, the neuronal cells generated by the differentiation of the cell population have neuroelectrophysiological functions. Preferably, the neuronal cells generated by the differentiation of the cell population express MAP2 and / or TUJ1. Preferably, on day 35 after differentiation of the cell population, neuronal cells in the cell population account for approximately 80% or more of the total cells. Preferably, the cell population also differentiates to generate astrocytes. Preferably, the astrocytes generated by differentiation of the cell population express GFAP and / or S100β. Preferably, on day 35 after differentiation of the cell population, astrocytes in the cell population account for approximately 20% or less of the total cells.

[0030] In specific embodiments of the first and second aspects, the neurological injury disorder is a stroke or a stroke-related disease, condition, or symptom, such as a sequela or complication, and in these embodiments, the cell population is administered preferably at least 14 days after the stroke.

[0031] In specific embodiments of the first and second aspects, the neurological injury disorder is cerebral palsy or a disease, condition, or symptom associated with cerebral palsy, such as a sequela or complication.

[0032] In specific embodiments of the first and second aspects, the cell population is about 1 x 10 4 ~1×10 10 is administered at a dose of about 1 x 10 cells, preferably about 1 x 10 5 ~6×10 7 For example, if the subject is a human, the cell population may be administered at a dose of 2 x 10 cells. 5 ~2×10 9 is administered at a dose of approximately 2 x 10 cells, preferably 6 ~1.2×10 9 The cells are administered in a dose of 100 cells.

[0033] In specific embodiments of the first and second aspects, the cell population is administered to the subject in one or more doses.

[0034] In specific embodiments of the first and second aspects, the administration is by injection. Preferably, the injection is a local injection.

[0035] Advantages of the present invention include, but are not limited to: (1) Regarding the restoration of damaged neural function, treatment with the forebrain neural progenitor cells of the present invention demonstrated significantly faster recovery in cerebral infarction model animals. Specifically, after high-dose cell transplantation, animals in the treatment group gained weight more rapidly compared to control animals. Within six weeks, cage-free behavioral changes in treated animals improved their scores (overall neural function score) approximately eight weeks earlier, and they gained weight more rapidly. Seven months after cell administration, animals in the high-dose group reached the ladder walking speed of normal healthy monkeys. Furthermore, cerebral palsy model animals treated with high-dose forebrain neural progenitor cells of the present invention showed significant recovery of motor and balance functions compared to control animals, with a maximum recovery of motor function of approximately 50%. This remarkable effect may be due to the fact that the forebrain neural progenitor cells of the present invention exhibit differentiation trends similar to those of normal human brain development and have the ability to develop and form functional neural networks. (2) Regarding the treatment window, the treatment time window of the application and method of the present invention is not limited to the acute phase (e.g., 1-3 days after the onset of stroke) where current common treatments and drugs for related diseases are applied, but can be used even after 14 days after the onset of stroke. Therefore, it can provide an option for patients who miss acute treatment or hemiplegic patients who remain with nerve damage even after treatment, and fill the gap in current treatment. (3) In terms of source, compared to methods using neural stem cells derived from fetal tissue, the neural progenitor cells of the present invention are derived from pluripotent stem cells, such as iPSCs or ESCs, and are obtained using a specific induced differentiation method. The advantages of this method include the ability to stably prepare multiple batches of consistent cell products, the ability to obtain cell numbers sufficient for hundreds of individuals per batch, strict quality control, and the ability to overcome ethical issues associated with using fetal tissue. (4) Compared with neural stem cells, neural progenitor cells are highly differentiated cells and can rapidly and efficiently differentiate into functional neurons in specific brain regions at a high rate, thereby reducing the risk of excessive cell division in the body. At the same time, compared with terminally differentiated neuronal cells, neural progenitor cells have a higher survival rate after cryopreservation and transplantation, making them more suitable for therapeutic applications.

[0036] In summary, the method of the present invention is expected to realize the indication treatment of stroke in the non-acute phase and fill the gap in the treatment of hemiplegic sequelae of stroke. At the same time, forebrain neural progenitor cells with specific developmental characteristics can form cell and tissue replacements in the body, thereby more effectively improving functional impairment caused by brain injury. [Brief explanation of the drawings]

[0037] [Figure 1] Immunofluorescence images at days 7 and 14 of induced differentiation are shown, showing the expression of DAPI, MAP2, and FOXG1 with different color fluorescence. [Figure 2] Immunofluorescence analysis of cortical neuronal cell markers on day 35 of induced differentiation is shown. [Figure 3] Immunofluorescence analysis of excitatory and inhibitory neuronal cell markers on day 35 of induced differentiation is shown. [Figure 4] Immunofluorescence analysis of presynaptic and postsynaptic membrane protein markers on day 35 of induced differentiation is shown. [Figure 5] Immunofluorescence analysis of astrocyte and neuronal markers on day 35 of induced differentiation is shown. [Figure 6] Immunofluorescence analysis of cortical neuronal cell markers on day 70 of induced differentiation is shown. [Figure 7] Immunofluorescence analysis of excitatory and inhibitory neuronal cell markers on day 70 of induced differentiation is shown. [Figure 8] Immunofluorescence analysis of astrocyte and neuronal markers on day 70 of induced differentiation is shown. [Figure 9]Immunofluorescence analysis of oligodendrocyte markers on day 70 of directed differentiation is shown. [Figure 10] Electrophysiological diagrams of induced differentiated cortical neurons are shown. [Figure 11] An experimental design diagram is shown in which a cerebral infarction model animal is treated with hNPC01 and tested. [Figure 12] The weight gain rate of cerebral infarction model animals before and after hNPC01 treatment (0 to 28 weeks) is shown, with the X-axis representing the number of days. [Figure 13] This shows the change curves (0-28 weeks) in the neurofunctional behavioral scores of the affected side of permanent cerebral infarction model animals treated with different doses (high, medium, and low doses) of hNPC01. The behavioral scores at each observation time point are expressed as a percentage change relative to the score on day 2 after treatment (i.e., score 0). Day 0 on the horizontal axis is day 2 after treatment, i.e., day 17 after stroke. [Figure 14] Individual data (from the previous day to 4 weeks) on the neurofunctional behavioral scores of the affected side of cerebral infarction model animals treated with hNPC01 are shown. [Figure 15] The graph shows the average time (seconds) for a single ladder walk in cerebral infarction model animals after hNPC01 treatment (5 to 7 months). [Figure 16] This shows MRI images of the brain of a cerebral infarction model animal after hNPC01 treatment (D183), with each small image showing a different cross section of the same animal. [Figure 17] The brain damage volume (%) of cerebral infarction model animals after hNPC01 treatment is shown. [Figure 18] Representative brain tissues and bright field images of brain sections from cerebral infarction model animals after hNPC01 treatment (7 months) are shown. [Figure 19] Immunofluorescence staining of the brain of a cerebral infarction model animal after hNPC01 treatment (7 months) is shown. [Figure 20] 1 shows the results of a rotorod test in cerebral palsy model animals after hNPC01 treatment. [Figure 21A] FIG. 21 shows the results of a balance beam test in cerebral palsy model animals at a certain time point after hNPC01 treatment, with FIG. 21A showing the crossing time. [Figure 21B]The results of the balance beam test in cerebral palsy model animals at a certain time point after hNPC01 treatment are shown in Figure 21B, where Figure 21B shows the number of slips. [Figure 22] 1 shows the results of TTC staining of cerebral palsy model animals after treatment with hNPC01. [Figure 23] The results of a comparative analysis of the cellular composition of hNPC01 and fetal forebrain samples are shown. DETAILED DESCRIPTION OF THE INVENTION

[0038] Unless otherwise specified in the present invention, the meanings of all technical terms used herein are to be interpreted as commonly understood by those skilled in the art of the present invention.

[0039] In this specification, unless stated otherwise, "or" means the same as "and / or."

[0040] Unless otherwise specified in the context of the present invention, "comprises," "comprises," and "containing" should be understood to mean the inclusion of the stated elements, e.g., compositions, properties, steps, or groups thereof, but not the exclusion of other elements, such as other compositions, properties, and steps. As used herein, "comprises" or any variation thereof can be replaced with "included," "containing," or equivalent variations. In some embodiments, "comprising" also includes "consisting of."

[0041] Cellular Therapy One of the features of the present invention is the use of specialized cells, i.e., forebrain neural progenitor cells (FNPCs), or cell populations primarily containing these cells, as a cell therapy. Unless otherwise specified, all therapeutic cells used in the present invention are of human origin.

[0042] In a preferred embodiment, the forebrain neural progenitor cells are obtained by inducing directed differentiation using human pluripotent stem cells, such as ESCs or iPSCs, as the starting material for directed differentiation.

[0043] In a preferred embodiment, the cell population of the present invention is obtained by a specific differentiation method. To understand this differentiation method, the cells at each stage involved in differentiation are defined below.

[0044] The term "cell population" refers to a collection of cells, and can include one or several types of cells.

[0045] The term "pluripotent stem cells" or its abbreviation "PSC" refers to a type of cell present in animals and humans that has the potential for multidirectional differentiation and the ability to self-renew.

[0046] The term "embryonic stem cells" or "ESCs" refers to pluripotent stem cells that are derived from the inner cell mass of a blastocyst resulting from the development of a fertilized egg and have the potential to differentiate into all human germ layers and are capable of self-renewal. Note that this does not refer to fetal stem cells.

[0047] The term "induced pluripotent stem cells" or "iPSCs" refers to pluripotent stem cells generated by reprogramming from human cells, capable of self-renewal, and capable of differentiating into all human germ layers.

[0048] The term "neuron" refers to all relevant cells that make up the human nervous system.

[0049] The term "neural progenitor cells (NPCs)" as used herein refers to a type of cell with a certain self-renewal capacity that can differentiate into specific brain regions or neural lineages (e.g., neurons and glial cells).

[0050] The term "forebrain neural progenitor cells (FNPCs)," as used herein, refers to neural progenitor cells that can differentiate into neurons in specific brain regions, i.e., neurons in the forebrain (e.g., primarily the cerebral cortex). The difference between NPCs that can differentiate into neurons in specific brain regions and general NPCs is that the former express markers for specific brain regions (e.g., the forebrain), whereas general NPCs do not. For example, forebrain NPCs primarily express markers such as FOXG1 and PAX6, while midbrain NPCs primarily express markers such as FOXA2 and LMX1A. Furthermore, upon differentiation, forebrain NPCs primarily differentiate into neurons and glial cells in layer 6 of the cerebral cortex, while midbrain NPCs primarily differentiate into midbrain dopamine neurons.

[0051] The term "embryoid bodies" or "EBs" as used herein refers to cell aggregates formed by three-dimensional culture of ESCs and iPSCs.

[0052] The terms "rosette-like neural stem cell-derived neural aggregates," "rosette-like neural aggregates," or "RONA" are used interchangeably herein to refer to neural aggregates of neural stem cells derived from ESCs or iPSCs, which are highly compact three-dimensional neural aggregates that spontaneously organize to form neural stem cells.

[0053] The term "neurosphere" or "neurosphere," as used herein, refers to a spherical aggregate formed by culture of neural cells in suspension. Neurospheres are heterogeneous populations containing neural stem cells, neural progenitor cells, and some differentiated neural cells.

[0054] "ECM" is an abbreviation for extracellular matrix, which is composed primarily of proteins such as collagen, elastin, and / or laminin.

[0055] A "cell preparation" refers to a preparation containing cells as the primary active ingredient, to which a certain amount of pharmaceutically acceptable additives may be added. For example, a cell preparation is a cell injection, specifically a sodium chloride injection of cells. In some cases, the terms "cell preparation" and "cell population" have the same meaning. For example, a cell population produced by a particular process can be used directly for therapy. A cell population can be a cell culture cultivated by the methods of the present invention, or a derived cell product obtained by further processing, e.g., purifying and culturing, the culture.

[0056] In a preferred embodiment, in the cell population of the present invention, at least 70% or more of the cells are forebrain neural progenitor cells, preferably at least 75% or more, more preferably at least 80% or more, and even more preferably at least 85% or more, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0057] The identity of the forebrain neural progenitor cells of the present invention can be determined by one or more of the following methods: 1) morphological observation, 2) detection of cell-specific biomarkers, and 3) in vitro differentiation and identification of specific neural cell types (e.g., cortical neurons) with specific biomarkers and electrophysiological activity.

[0058] For example, the forebrain neural progenitor cells of the present invention can exhibit the morphology of typical neural progenitor cells, exhibiting a uniform, transparent, spindle-shaped morphology, with some cells aggregated to form rosette-shaped arrangements.

[0059] For example, neural stem cell markers and forebrain neural progenitor cell markers can be used to jointly confirm the identity of cells in the cell populations of the present invention. The markers are selected from NESTIN, SOX2, DCX, FOXG1, and PAX6. In a preferred embodiment, the majority of cells in the cell populations or cell preparations of the present invention express FOXG1, PAX6, and NESTIN. In a preferred embodiment, the majority of cells in the cell populations or cell preparations of the present invention express FOXG1, PAX6, NESTIN, and SOX2. In a preferred embodiment, in the cell population of the present invention, at least about 70% or more, preferably about 80% or more, more preferably about 85% or more, and even more preferably about 90% or more of the cells express NESTIN, at least about 70% or more, preferably about 80% or more, more preferably about 85% or more, and even more preferably about 90% or more of the cells express FOXG1, at least about 70% or more, preferably about 80% or more, more preferably about 85% or more, and even more preferably about 90% or more of the cells express PAX6, and / or at least about 70% or more, preferably about 80% or more, more preferably about 85% or more, and even more preferably about 90% or more of the cells express SOX2. In a more preferred embodiment, in the cell population of the present invention, at least about 70% or more, preferably about 80% or more of the cells simultaneously express NESTIN, FOXG1, and PAX6. In a more preferred embodiment, at least 70% or more, preferably 80% or more, of the cells in the cell population of the present invention simultaneously express NESTIN, FOXG1, PAX6, and SOX2. The expression of the above markers can be measured by methods commonly known in the art, such as flow cytometry.

[0060] The abbreviation "FOXG1" or "Foxg1," as used herein, stands for "Forkhead box G1," which is one of the earliest expressed transcription factors during human brain development, directing the development of the telencephalon into several important structures, including the cerebral cortex.

[0061] The abbreviation "PAX6," as used herein, stands for "Paired Box 6," and is a key transcription factor in human neuroectodermal cell fate determination and forebrain development.

[0062] The abbreviation "NESTIN" as used herein stands for "Nestin protein," which is a type of class VI IF protein and a neural stem cell marker.

[0063] The abbreviation "SOX2," as used herein, stands for "Sex determining region Y-box 2," and is a key transcription factor that maintains pluripotency and self-renewal, as well as a neural stem cell marker.

[0064] The abbreviation "DCX," as used herein, stands for "Doublecortin," a neural stem and progenitor cell marker.

[0065] In a preferred embodiment, the cell populations or cell preparations of the present invention express one or more, preferably all, of the following characteristic markers: EFNB2, WNT7B, RSPO2, and FEZF2. A "characteristic marker" is a gene expression signature in which the co-expression of these markers distinguishes the cell populations of the present invention from other products of different developmental stages, cell types, and / or compositions. In a preferred embodiment, the cell populations or cell preparations of the present invention simultaneously express EFNB2, WNT7B, RSPO2, and FEZF2. In a specific embodiment, the expression of the above markers can be measured by methods commonly known in the art, such as single-cell transcriptome analysis (scRNA-seq).

[0066] The abbreviation "EFNB2," as used herein, stands for Ephrin B2, a cell surface transmembrane receptor and key regulator of angiogenesis.

[0067] The abbreviation "WNT7B" as used herein stands for "Wnt Family Member 7B."

[0068] The abbreviation "RSPO2" as used herein stands for "R-spondin 2."

[0069] The abbreviation "FEZF2" as used herein stands for "FEZ Family Zinc Finger 2."

[0070] In some embodiments, the cell population of the present invention comprises primarily neural progenitor cells, particularly forebrain neural progenitor cells, as well as a plurality of specific cell types. The cells are selected from one or more, preferably all, of immature neurons, GABAergic neurons, glutamatergic neurons, ependymal cells, and pericytes. Preferably, the proportion of the above cells is 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less of the total number of cells.

[0071] In some embodiments, the cell population of the present invention is derived from human pluripotent stem cells, and the proportion of pluripotent stem cells in the cell population is 3% or less of the total number of cells, preferably 2% or less, and more preferably 1% or less.

[0072] For example, the forebrain neural progenitor cells or cell populations containing forebrain neural progenitor cells of the present invention can further differentiate into neuronal cells in vitro and in vivo, preferably into a cell population mainly composed of neuronal cells. When the differentiation is performed in vitro or in vivo, particularly when performed in vivo, the differentiation is spontaneous and does not require the addition of additional reagents (e.g., inducers). Furthermore, the forebrain neural progenitor cells or cell populations containing the forebrain neural progenitor cells used as cell therapeutic agents of the present invention exhibit differentiation trends similar to the normal human brain development process in the differentiation process and differentiation results.

[0073] For example, the cell populations used as cell therapeutic agents of the present invention can express neuron-specific markers after differentiation. For example, the neuronal cells generated by differentiation of the cell populations express MAP2 and / or TUJ1. MAP2 and TUJ1 are markers for mature neurons. Preferably, as determined by the expression of MAP2 and / or TUJ1, neuronal cells in the cell populations of the present invention account for more than half of the total cells by around day 35 of differentiation, e.g., about 50% or more, 60% or more, 70% or more, or 80% or more of the total cells by around day 35 of differentiation.

[0074] For example, the cell population used as the cell therapy agent of the present invention contains cells that express different cortical-specific neuronal markers after differentiation. For example, MAP2 produced by differentiation of the cell population of the present invention + Neuronal cells include neuronal cells that express BRN2, a marker for cerebral cortex layers II-IV. For example, MAP2, which is produced by differentiation of the cell population of the present invention, + Neuronal cells include those expressing CTIP, a marker for cerebral cortex layers V-VI. These cortex-specific neuronal cells can appear around day 35 after differentiation. This means that the therapeutic cells or cell populations of the present invention can differentiate into different neural cell types that resemble the normal structure of the human brain.

[0075] More importantly, the neuronal cells generated by differentiation of the cells or cell populations used as cell therapeutic agents of the present invention have neuroelectrophysiological functions. For example, when electrophysiological function tests are performed around 25 days after in vitro differentiation, the cell populations exhibit one or more of the following characteristics after differentiation: number of active electrodes ≥ 5, weighted average discharge frequency (Hz) ≥ 0.5, and / or cluster discharge frequency (Hz) ≥ 0.05.

[0076] Preferably, around day 35 after in vitro differentiation, the cell population can differentiate to generate cells that co-express synapsin and PSD95. Co-expression of synapsin and PSD95 indicates that synaptic structures have been successfully formed between neurons, establishing a basis for signal transduction between neurons.

[0077] Preferably, around day 35 after in vitro differentiation, the cell population contains neurons expressing VGLUT (an excitatory neuron marker) and VGAT (an inhibitory neuron marker), with the number of VGLUT-expressing neurons significantly higher than the number of VGAT-expressing neurons. This distribution of numbers resembles the phenomenon in the normal cerebrum, where VGLUT-positive neurons are absolutely dominant in the cerebral cortex.

[0078] During cerebral development, early neuronal cells are absolutely dominant, but as neurons mature, they cease to divide. Meanwhile, astrocytes are continuously generated, gradually increasing their proportion. Therefore, the cell population used as the cell therapeutic agent of the present invention preferably also generates astrocytes through differentiation. Preferably, the astrocytes generated by differentiation of the cell population express GFAP and / or S100β. Both GFAP and S100β are astrocyte-specific markers. Preferably, the astrocytes generated by differentiation of the cell population account for approximately 20% or less of the cells generated around day 35 of differentiation.

[0079] In a specific embodiment, the cell population used as the cell therapeutic agent of the present invention differentiates ex vivo around day 35 into a cell population mainly composed of neurons (80% or more) that highly express MAP2 and / or TUJ1 and a minority of astrocytes (20% or less) that highly express GFAP / S100β, and has the corresponding neuroelectrophysiological functions. Once differentiation is complete in vivo, the cell population containing the forebrain neural progenitor cells of the present invention can differentiate into a cell population mainly composed of neurons in about 30 to 90 days.

[0080] In a preferred embodiment, the cell population of the present invention can be administered as a cell therapeutic agent to a subject at the site of nerve damage and its surrounding tissue region, and then reorganize neural circuits in the administration region.

[0081] In a preferred embodiment, the cell population of the present invention can secrete cytokines and microRNAs after transplantation, which exert neuroprotective and pro-angiogenic effects, contributing to the realization of neurorepair effects.

[0082] 1. Preparation of Cells The following provides exemplary methods for preparing the forebrain neural progenitor cells and cell populations comprising the same of the present invention. It should be understood by those skilled in the art that specific steps of the above methods can be adjusted or modified, and that the method can begin at any intermediate step. The present invention is not limited to completing each step of the following methods, as long as cell products with equivalent properties and composition are obtained.

[0083] In a specific embodiment, the forebrain neural progenitor cells of the present invention or a cell population comprising said forebrain neural progenitor cells are generated by directed differentiation from human pluripotent stem cells, such as ESCs or iPSCs, said method comprising the steps of: (1) Cultivate and grow ESCs or iPSCs in human pluripotent stem cell medium (hPSC medium); (2) Digesting the ESCs or iPSCs from step (1) and culturing them in suspension in EB medium to form embryoid bodies (EBs); (3) The EBs obtained in step (2) are cultured in a neural induction medium (RONA) to form rosette-like neural aggregates (RONAs). (4) culturing the RONAs formed in step (3) and forming neurospheres in suspension culture, wherein the neurospheres contain forebrain neural progenitor cells (FNPCs); (5) optionally, further culturing and subculturing the neurospheres formed in step (4) using NPC medium; and (6) Optionally, recovering the FNPCs formed in step (5).

[0084] Optionally, prior to step (1), the ESCs or iPSCs are maintained in culture and grown to 80% to 90% confluence. Preferably, they are cultured on a laminin-coated culture surface. The culture surface can be a culture plate.

[0085] In step (1), ESCs or iPSCs can be dispersed into single cells or cell aggregates, for example, by digestive enzymes or mechanical means, and then inoculated into hPSC medium to induce the formation of EBs. The ESCs or iPSCs can be obtained commercially or prepared by any known method. Those skilled in the art will recognize that the ESCs are ESCs formed from the inner layer cells of blastocysts within 5 to 7 days of fertilized egg development, and have not undergone in vivo development.

[0086] In step (2), one or more types of EB medium of the present invention, for example, two, three, or more types, can be used.

[0087] In step (3), one or more RONA of the present invention can be used, for example, two, three, or more types.

[0088] Step (4) can use the same medium as the previous or next step. Step (4) can be performed using RONA or NPC medium. In one embodiment, the medium used in step (4) is the same as that used in step (3), specifically RONA.

[0089] Preferably, the culture in steps (3) and (5) above is an adherent culture, which can be performed, for example, on a culture surface coated with extracellular matrix (ECM). In step (5), the neurospheres can be dispersed using digestive enzymes or mechanical means.

[0090] In the above preparation methods, the EB medium, RONA medium, and NPC medium may contain a basal medium and additives. Each medium may be independently clinical grade, preferably cGMP grade or CTS grade. TM It is a grade.

[0091] Exemplary media used in each step are described in detail below.

[0092] A) hPSC medium hPSC media include NutriStem® hPSC XF Medium, Essential 8 Medium, StemFit® Basic03 Medium, and StemMACS TM iPS-Brew medium, Stem-Partner® ACF medium, TeSR TM - AOF medium, or TeSR2 medium, preferably NutriStem® hPSC XF medium.

[0093] Optionally, a ROCK inhibitor can be added to the hPSC culture medium. In a preferred embodiment, the ROCK inhibitor is Y-27632. Preferably, the ROCK inhibitor Y-27632 is added at a concentration of about 10 μM.

[0094] B) EB medium It is important that the EB medium contains a factor that promotes differentiation into neuroectodermal cells (eg, neurons), thereby generating a cell population that is primarily composed of forebrain neural progenitor cells.

[0095] EB medium may contain: (i) a basal medium selected from any of a) to c): a) Single KnockOut TM DMEM / F12 medium, b) DMEM / F12 medium and Neurobasal TM Media combinations, c) KnockOut TM DMEM / F12 medium and Neurobasal TMMedia combinations, and, (ii) an additive comprising or consisting of d) or e): d) N-2 Additive and GlutaMAX TM -I additive, e) N-2 additive, GlutaMAX TM -I additives and Vitamin A-free B-27 TM Additives (B-27 TM Supplement,minus vitamin A).

[0096] Optionally, the EB medium comprises (iii) an inhibitor, which comprises a BMP inhibitor, an AMPK inhibitor, and an ALK inhibitor, or consists of a BMP inhibitor, an AMPK inhibitor, and an ALK inhibitor. Preferably, the EB medium comprises one or more of Noggin, SB431542, LDN-193189, DMH-1, and Dorsomorphin, or consists of one or more of Noggin, SB431542, LDN-193189, DMH-1, and Dorsomorphin. More preferably, the EB medium comprises a combination of SB431542 and any one or more of Noggin, LDN-193189, DMH-1, and Dorsomorphin, for example, a combination of SB431542 and one or two of Noggin, LDN-193189, DMH-1, and Dorsomorphin, or consists of them.

[0097] In a specific embodiment, the EB medium comprises: (i) a basal medium, TM DMEM / F12 medium and Neurobasal TM It is a combination of media; (ii) additives, including N-2 additive and GlutaMAX TM -I containing a combination of additives; and (iii) SB431542, Noggin, and Dorsomorphin.

[0098] Preferably, when two types of basal media are used, the two media are combined in a 1:1 volume ratio.

[0099] Preferably, the additive is added at a 1x concentration.

[0100] For example, Noggin is added at a concentration of 25 to 100 ng / mL, preferably 40 to 60 ng / mL, and more preferably about 50 ng / mL. For example, dorsomorphin is added at a concentration of 0.5 to 2 μM, preferably 0.75 to 1.5 μM, and more preferably 1 μM. For example, SB431542 is added at a concentration of 5 to 15 μM, preferably 7 to 13 μM, more preferably 8 to 12 μM, and most preferably 10 μM.

[0101] C) RONA medium RONA medium contains factors that promote differentiation into forebrain neurons, making it possible to generate a cell population primarily composed of forebrain neural progenitor cells.

[0102] RONA medium may contain: (i) a basal medium selected from any of a) to c): a) Single KnockOut TM DMEM / F12 medium, b) DMEM / F12 medium and Neurobasal TM Media combinations, c) KnockOut TM DMEM / F12 medium and Neurobasal TM A combination of culture media, and (ii) an additive comprising or consisting of d) or e): d) N-2 Additive and GlutaMAX TM -I additive, e) N-2 additive, GlutaMAX TM -I additives and Vitamin A-free B-27 TM Additives.

[0103] In a specific embodiment, RONA medium is used, which contains the following components: (i) a basal medium, TM DMEM / F12 medium and Neurobasal TM a combination of culture media; and (ii) additives, including N-2 additive and GlutaMAX TM -I Contains a combination of additives.

[0104] In a specific embodiment, RONA medium is used, which contains the following components: (i) a basal medium, TM DMEM / F12 medium and Neurobasal TM a combination of culture media; and (ii) Additives, such as N-2 Additive, B-27 TM Additives (B-27 TM Supplement, XenoFree, minus vitamin A), and GlutaMAX TM -I Contains a combination of additives.

[0105] In a specific embodiment, multiple RONA media are used. For example, in the first stage of RONA differentiation, a first RONA medium is used, which includes the following components: (i) a basal medium, KnockOut TM DMEM / F12 medium and Neurobasal TM (ii) a combination of media, and (iii) an additive, N-2 additive and GlutaMAX TM In the second stage of RONA differentiation, a second RONA medium is used that contains the following components: (i) a basal medium, which is a KnockOut-I additive combination; TM DMEM / F12 medium and Neurobasal TM and (ii) an additive, which is a combination of a medium, N-2 additive, B-27 TM Additives (B-27 TM Supplement, XenoFree, minus vitamin A), and GlutaMAX TMPreferably, the first and second stages of RONA differentiation each last for about 5 to 14 days, preferably about 7 days.

[0106] Preferably, when two types of basal media are used, the two media are combined in a 1:1 volume ratio.

[0107] Preferably, the additive is added at a 1× concentration.

[0108] D) NPC medium The basal medium for NPC culture is Neurobasal TM The NPC medium is a medium, and the additives of the medium are: (a) GlutaMAX TM -I additive, and (b) Vitamin A-free B-27 TM It is an additive.

[0109] Optionally, the NPC medium further comprises brain-derived neurotrophic factor (BDNF), and / or glial cell line-derived neurotrophic factor (GDNF), and / or L-ascorbic acid, and / or N 6 ,O 2’ -Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (DB-cAMP).

[0110] The BDNF is animal-free recombinant BDNF or GMP-grade recombinant BDNF, and / or the GDNF is animal-free recombinant GDNF or GMP-grade recombinant GDNF.

[0111] In a specific embodiment, an NPC medium is used that contains the following components: (i) a basal medium, Neurobasal TM The medium is (ii) an additive, B-27 TM Additives (B-27 TM Supplement, XenoFree, minus vitamin A) and GlutaMAX TM -I containing a combination of additives, and (iii) BDNF, GDNF, L-ascorbic acid, and N 6 ,O 2’ -Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (DB-cAMP).

[0112] Preferably, when two types of basal media are used, the two media are combined in a 1:1 volume ratio.

[0113] Preferably, the additive is added at a 1× concentration.

[0114] For example, BDNF is added at a concentration of about 5 to 50 ng / mL, preferably 10 to 30 ng / mL, and more preferably about 20 ng / mL.

[0115] For example, GDNF is added at a concentration of about 5 to 50 ng / mL, preferably 10 to 30 ng / mL, and more preferably about 20 ng / mL.

[0116] For example, L-ascorbic acid is added at a concentration of 0.02 to 2 mM, preferably 0.05 to 0.5 mM, more preferably about 0.1 to 0.3 mM, and most preferably about 0.2 mM.

[0117] For example, DB-cAMP is added at a concentration of 0.1 to 5 mM, preferably 0.2 to 2.5 mM, more preferably about 0.3 to 1 mM, and most preferably about 0.5 mM.

[0118] In the above-described preferred production method, the use of specific neuroectodermal induction factors in the EB culture process and specific factors promoting forebrain neural cell differentiation in the RONA formation process ensures the reliable production of a cell population consisting primarily of forebrain neural progenitor cells. In fact, in the above-described preferred production method, the formed neurospheres already contain forebrain neural progenitor cells expressing specific markers. Therefore, it is understood that after neurosphere formation, other methods can be used to maintain and proliferate forebrain neural progenitor cells and / or promote their differentiation into neurons.

[0119] Treatment method In the present invention, the term "subject" refers to an animal receiving the administration, preferably a vertebrate, more preferably a mammal (e.g., a rodent, e.g., a mouse, a rat), most preferably a primate (e.g., a cynomolgus monkey, a rhesus monkey), and specifically a human.

[0120] The term "effective amount" or "therapeutically effective amount" refers to the amount of a pharmaceutical composition (e.g., a pharmaceutical composition (cell preparation or cell population) containing the human forebrain neural progenitor cells of the present invention), which is sufficient to exert a desired function when administered to a subject in need thereof. The desired function can include at least one of delaying the onset of symptoms of a disease, preventing or delaying the progression of the disease, or alleviating the disease.

[0121] The term "treatment" refers to the improvement, alleviation, or elimination (i.e., cure) of symptoms of a disease. In some cases, treatment includes prophylactic treatment.

[0122] When used for repairing nerve damage or treating neurological disorders such as neurodegenerative diseases, the forebrain neural progenitor cells or cell preparation of the present invention can be transplanted 14 days or more after the subject has suffered nerve damage. For example, if the subject is a stroke patient, the cell population of the present invention can be administered at least 14 days after the stroke.

[0123] The forebrain neural progenitor cells of the present invention or a cell population comprising the forebrain neural progenitor cells can be administered via a transcranial route (e.g., intraparenchymal injection), a transventricular route (e.g., intrathecal injection, intraventricular catheter, intraventricular injection), or the like. In a preferred embodiment, the neural progenitor cells of the present invention or a cell population comprising the neural cells can be administered by local injection (e.g., local injection into an infarcted or damaged brain region). For example, they can be injected into the motor cortex and / or basal ganglia region surrounding the infarct. For example, local injection can be performed with the aid of an imaging device (e.g., a 3D Lab navigation system).

[0124] The administration or transplantation of the cells or cell population (e.g., administration or transplantation by injection) can be performed using an injection device such as a syringe. The injection device can include a needle (e.g., a puncture needle) or any suitable needle known to those skilled in the art. The needle should be configured to be sufficiently smooth and rigid to penetrate the corresponding tissue and long enough to reach the desired location. The needle length can be any length from about 1 to 25 cm. The size and thickness of the needle can be selected based on several factors, such as the number of cells, the volume and formulation of the cell preparation, and the site of needle insertion.

[0125] For injection, the cells to be transplanted can be provided as a suspension. For example, the cells of the present invention can be suspended in a buffer solution having an appropriate osmotic pressure (e.g., physiological saline, i.e., 0.9% sodium chloride solution) and used for injection.

[0126] When the total volume of the cell suspension is large (e.g., for intraparenchymal injection), multiple needle tracts can be used to inject at multiple points at different depths, e.g., 2, 3, 4, 5, or more needle tracts can be used, with each tract containing a portion of the total volume of the cell suspension.

[0127] The forebrain neural progenitor cells or cell populations containing the forebrain neural progenitor cells of the present invention can be administered by single or multiple injections. Multiple injections can be, for example, two, three, or more times. The number of injections can be determined depending on the ameliorative effect. The interval between multiple injections can be one or several days, one or several weeks, one or several months, or longer.

[0128] In a preferred embodiment, the forebrain neural progenitor cells of the present invention or a cell population containing the forebrain neural progenitor cells are injected into a single or multiple points within the skull. For example, injections can be made using one to three needle tracts with different needle angles within a single cranial foramen. For example, cells can be injected into one to ten different injection sites at different depths using each needle tract. This can be achieved by inserting the injection device and then stopping it at different depths during the retraction process. Alternatively, injections can be made slowly and continuously during the retraction process, leaving cells in the path of the needle as it is retracted.

[0129] The single dose of the cell population containing forebrain neural progenitor cells of the present invention is about 1 × 10 4 ~1×10 10 For example, 1 x 10 cells 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces, 1×10 10 In a preferred embodiment, a single dose is about 1 x 10 cells. 5 ~1×10 7 For example, about 1 x 10 cells 5 pieces, 2×10 5 pieces, 3×10 5 pieces, 4×10 5 pieces, 5×10 5 pieces, 6×10 5 pieces, 7×10 5 pieces, 8×10 5 pieces, 9×10 5 pieces, 1×10 6 pieces, 2×10 6 pieces, 3×10 6 pieces, 4×10 6 pieces, 5×10 6 pieces, 6×10 6 pieces, 7×10 6 pieces, 8×10 6 pieces, 9×10 6 In some embodiments, when the subject is a human, the cell population is 2 x 10 5 ~2×10 9cells, preferably about 2 x 10 6 ~1.2×10 9 In a single administration, the amount of the forebrain neural progenitor cells of the present invention should be at least 70%, preferably at least 80%, of the total number of cells. That is, at least 70%, preferably at least 80%, of the total number of cells in a single administration should express FOXG1, PAX6, and NESTIN.

[0130] Indications / Effectiveness evaluation index After administering the forebrain neural progenitor cells or cell populations containing forebrain neural progenitor cells of the present invention, several indicators can be used to evaluate the therapeutic effect.

[0131] For example, within one month to one year after transplantation or administration of the cell population or cell preparation of the present invention, improvement is observed in one or more of the following aspects: neurological function in stroke sequelae, scores for assessing motor function (e.g., Modified Rankin score, FMMS score, or NIHSS score), language function, cognitive function, and specific symptoms.

[0132] Preferably, the improvement is significant compared to before transplantation or administration. For example, the improvement is manifested as an increase of at least 10%, preferably at least 20%, in the quantified score, and / or reaching or approaching the corresponding score of a healthy individual. For example, the improvement in specific symptoms is manifested in one or more of the following aspects: improvement from clenching a fist due to hypertonic muscles in one hand to being able to normally control hand activities (e.g., writing, using tableware, etc.), improvement from weakness or disability in one leg to being able to walk and / or run freely, improvement of a bedridden cognitively impaired patient to being able to recognize and / or communicate with family members, and / or improvement of aphasic patient to being able to speak and / or communicate.

[0133] For example, the age range of patients eligible for treatment may be 20 to 85. For example, the indicator range for cerebral infarction is cortical / subcortical ischemic infarction due to unilateral cerebral artery / lenticulostriate artery infarction, an ischemic focus of 3 cm or greater, and an FMMS score of less than 55 on two assessments within 3 weeks before surgery.

[0134] Purpose The forebrain neural progenitor cells of the present invention or cell populations comprising said forebrain neural progenitor cells can be used alone or in combination with other drugs to treat neuronal injury diseases, neurodegenerative diseases, neurodevelopmental diseases, or other neurological diseases, illnesses, or conditions associated with the death and / or dysfunction of forebrain neurons.

[0135] The term "brain injury," as used herein, refers to damage to brain tissue and includes brain damage caused by internal or external forces. Brain injury includes contusion of brain tissue, which is included in "intracranial injury," and also includes damage caused by ischemia, hypoxia, and reperfusion. Brain injury can result in various forms of cognitive impairment and symptoms, such as impaired attention, memory, or movement.

[0136] The term "neuropathic disease" as used herein specifically refers to neuropathic damage to the brain, including brain damage, including neuropathic damage caused by cerebral ischemia, cerebral hemorrhage, or physical injury.

[0137] The term "neurodegenerative disease" refers to a disease characterized by the gradual loss of function and death of specific nerve cells over time. In specific embodiments, the neurodegenerative disease includes amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), etc.

[0138] The term "neurodevelopmental disorder" as used herein refers to a disorder caused by abnormal development of brain tissue and cells, particularly forebrain tissue and cells.

[0139] The term "other neurological disorders associated with death and / or dysfunction of forebrain neurons" as used herein refers to other neurological disorders caused by a decrease in the number and / or function of forebrain neurons.

[0140] The term "stroke" in the present invention should be interpreted broadly and includes ischemic stroke and hemorrhagic stroke. Ischemic stroke, also known as cerebral infarction or cerebral infarction, occurs when the blood supply to a local brain tissue region is impaired due to various causes, resulting in acute interruption of blood flow, ischemia, hypoxia, softening, and necrosis of brain tissue, resulting in the rapid appearance of clinical neurological function deficits such as hemiplegia and aphasia. Hemorrhagic stroke is also known as cerebral hemorrhage.

[0141] In the present invention, the term "cerebral palsy" refers to non-progressive brain damage that occurs during the perinatal period (before birth and in the neonatal period), and is characterized primarily by motor disorders, intellectual retardation, epileptic seizures, and the like.

[0142] In a specific embodiment, the forebrain neural progenitor cells of the present invention or a cell population comprising the forebrain neural progenitor cells can be used to treat a nerve injury disease selected from the group consisting of diseases associated with ischemic brain injury, diseases associated with hemorrhagic brain injury, and diseases associated with traumatic brain injury.

[0143] Those skilled in the art generally believe that treatment during the acute phase of nerve injury provides more pronounced benefits. However, even if a patient receives appropriate treatment during the acute phase, nerve injury may remain and cause permanent sequelae. The cell population of the present invention can be used not only for subjects in the acute phase of a nerve injury disease, but also for subjects in the stable phase after the acute phase. Without being limited by theory, the cell population of the present invention exerts a therapeutic effect on subjects in the acute phase through the release of cellular factors, and on subjects in the stable phase through functions such as neural circuit reorganization due to cell replacement.

[0144] In a specific embodiment, the ischemic brain injury is ischemic stroke, also called cerebral infarction.For example, the subject has or has experienced total anterior circulation infarction, partial anterior circulation infarction, posterior circulation infarction, and lacunar infarction, or the patient has or has experienced cerebral ischemia due to large artery atherosclerosis, cardiogenic embolism, and small artery occlusion.The disease associated with ischemic brain injury can be stroke or stroke-related disease (e.g., complications, sequelae).

[0145] In a specific embodiment, the hemorrhagic brain injury is a hemorrhagic stroke, also called cerebral apoplexy. The disease associated with hemorrhagic brain injury may be a stroke or a condition associated with a stroke (e.g., a complication or sequela).

[0146] Symptoms associated with stroke include, but are not limited to, hemiplegia, hemiplegia, numbness of the limbs, facial muscle weakness, numbness, aphasia, slurred speech, and hemianopia.

[0147] Traumatic brain injury is, for example, intracranial injury caused by a collision injury, a puncture injury, or the like, or symptoms associated therewith (for example, complications, sequelae).

[0148] In a preferred embodiment, the forebrain neural progenitor cells of the present invention are particularly suitable for treating ischemic brain damage, particularly ischemic stroke, or related conditions (e.g., complications and sequelae). In this case, the forebrain neural progenitor cells of the present invention are preferably administered after the acute phase of stroke, for example, 14 days or later after the onset of stroke.

[0149] In a specific embodiment, the forebrain neural progenitor cells of the present invention or a cell population containing the forebrain neural progenitor cells can be used to treat cerebral palsy. The cell therapy of the present invention can improve the motor and / or cognitive abilities of subjects with cerebral palsy.

[0150] In a specific embodiment, the forebrain neural progenitor cells of the present invention or cell populations comprising the forebrain neural progenitor cells can be used to treat neurodegenerative diseases. Neurodegenerative diseases are characterized by the gradual loss of function of nerve cells, particularly neurons. Neurodegenerative diseases suitable for treatment with the cells or cell populations of the present invention include diseases associated with the loss of function of forebrain nerve cells, particularly forebrain neurons, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD).

[0151] In a specific embodiment, the forebrain neural progenitor cells of the present invention or a cell population comprising said forebrain neural progenitor cells can be used to treat neurodevelopmental diseases. Neurodevelopmental diseases are also called neurodevelopmental disorders. Neurodevelopmental diseases suitable for treatment with the cells or cell populations of the present invention include diseases associated with loss of function of forebrain neurons, particularly forebrain neurons, such as epilepsy, autism, and cerebral palsy.

[0152] The forebrain neural progenitor cells of the present invention or cell populations containing the forebrain neural progenitor cells can be used for research purposes. Such research includes clinical and non-clinical research. For example, the research can be a study evaluating an animal model of the above-mentioned disease. For example, the research can be a study evaluating the role of forebrain neural progenitor cells in the therapeutic mechanism (e.g., injury repair mechanism) of the above-mentioned disease. [Example]

[0153] The following is an illustrative description of the embodiments of the present invention. Those skilled in the art should not understand that the present invention should be implemented only by the following specific methods. The scope of protection of the present invention is defined by the claims.

[0154] Example 1. Cell preparation In this example, a cell preparation, hNPC01, mainly composed of forebrain neural progenitor cells was prepared from human-derived iPSCs (induced pluripotent stem cells) through directed differentiation (iPSC → EB → RONA → neurosphere → forebrain neural progenitor cells (FNPC)).

[0155] It should be understood that one need not start with iPSCs to practice the present invention.

[0156] 1. iPSC Culture Approximately 2×10 5 The cell suspension was seeded onto MX521 substrate-coated 6-well cell culture plates at a density of viable cells / well, supplemented with NutriStem hPSC XF Xeno-free Medium (containing 10 μM Y-27632), and cultured in a 37°C, 5% CO2 incubator until the cells reached 90% confluence.

[0157] 2. Pseudoembryonic (EB) differentiation culture A pseudo-embryonic differentiation medium EB-M was prepared according to the following table. TIFF2025537905000001.tif60152The procedure for EB differentiation is as follows.

[0158] (1) Inoculation of pseudoembryonic bodies (EBs) Remove the old medium and rehydrate with DPBS (Ca 2+ , Mg 2+ Cells were washed with 1 mL of Cell Therapy Systems TrypLE in each well. TM Select Enzyme digestion enzyme was added and digestion continued until cells were extensively detached. Digestion was stopped by adding 3 mL of medium to each well, and the cell suspension was transferred to a centrifuge tube.

[0159] After centrifugation at 200 g for 3 minutes, the supernatant was removed and the cells were resuspended in NutriStem hPSC XF Xeno-free Medium containing 10 μM Y-27632 and counted.

[0160] Approximately 0.3×10 5 The cell suspension was seeded into a low-adhesion 96-well cell culture plate at a density of viable cells / mL and cultured at 37°C in a CO2 incubator for 48 hours.

[0161] (2) Medium exchange for pseudoembryonic bodies (EBs) On day 2 of EB differentiation, the EBs in the 96-well low-adhesion cell culture plate were transferred to a 6-well low-adhesion cell culture plate. 32 EBs were transferred to each well of the 6-well cell culture plate. On days 2-6 of EB differentiation, the medium in each well was removed and 2 mL of fresh EB-M medium was added to each well. After medium replacement, the cells were cultured in a 37°C, 5% CO2 incubator for a total of 7 days.

[0162] 3.RONA differentiation culture RONA differentiation stage 1 (Day 1 to Day 7) medium RONA-M1 was prepared according to the table below. TIFF2025537905000002.tif41152 RONA differentiation stage 2 (Day 8 to Day 14) medium RONA-M2 was prepared according to the table below. TIFF2025537905000003.tif47152

[0163] The procedure for RONA differentiation is as follows. A 6-well cell culture plate coated with MX521 substrate was prepared, and 3 mL of RONA-M1 medium was added to each well. EBs in the center of the low-adhesion 6-well cell culture plate were transferred to the plate. RONA was cultured in a 37°C, 5% CO2 incubator.

[0164] RONA-M1 medium was used on days 1 to 7 of RONA culture, and RONA-M2 medium was used on days 8 to 17.

[0165] 4. Neurosphere Culture A basal medium for forebrain neural progenitor cells, NPC-M, was prepared according to the table below. TIFF2025537905000004.tif57163

[0166] Neurospheres were cultured as follows. After the RONA differentiation step was completed, the RONA central cell cluster was transferred to a 6-well low-adhesion culture plate containing 2.5 mL of NPC-M. The cells were cultured at 37°C in a CO2 incubator to form neurospheres.

[0167] After culturing the neurospheres for 12–72 h, the neurospheres are enriched for FNPCs and are ready for seeding.

[0168] 5. Forebrain Neural Progenitor Cell Expansion Culture The neurospheres were collected in a 50 mL centrifuge tube and centrifuged at 300 rpm for 3 minutes. After removing the supernatant, 10 mL of Accutase was added to the tube. The tube was digested at 37°C in a CO2 incubator for 15 minutes. The old medium was added to stop the digestion, and the tube was centrifuged at 300 g for 3 minutes. After removing the supernatant, 3 mL of NPCM was added and pipetted to prepare a single-cell suspension. Cell counting was performed. The final total number of viable cells was 5.67 × 10 8 The number of viable cells was 98%, and the cell viability was 98%.

[0169] Using NPCM, the cell concentration was increased to 1.52 x 10 6 The concentration was adjusted to 100 viable cells / mL. The cells were seeded into a T175 cell culture flask coated with 8 μg / mL of the substrate coating agent MX521. The cells were cultured at 37°C in a 5% CO2 incubator. The medium was replaced the next day (46-50 hours after seeding), and the cells were subcultured on the fourth day. After four passages, the cells were cryopreserved.

[0170] 6. Preparation of Cell Preparations The cell stock solution was thawed and a cell suspension was prepared using 0.9% sodium chloride injection. The cell concentration was 5.0 × 10 4 The cells were then diluted to 1000 cells / μL and dispensed into a transport bottle. This is the cell preparation hNPC01.

[0171] Furthermore, cell preparations can also be prepared using FNPC cells undergoing passage.

[0172] 7. Identification and Characterization of hNPC01 Cell Preparations Flow cytometry was used to detect the cell markers FOXG1, PAX6, NESTIN, and SOX2. The hNPC01 cell preparation showed a positive rate of 93% for FOXG1, 97% for PAX6, over 98% for NESTIN, and 94% for SOX2. These results indicate that most cells in the cell preparation expressed the expected FNPC cell markers.

[0173] Example 2. In vitro differentiation experiment To verify the biological activity of the cell preparation prepared in Example 1, the cell preparation was induced to undergo differentiation. At different time points during differentiation, the differentiation potential of the cell preparation was evaluated by detecting the expression of multiple markers using immunofluorescence staining and monitoring neural electrophysiological activity.

[0174] 2.1 Differentiation of hNPC01 hNPC01 was used for neuronal differentiation. The NPCM described in Example 1 was used as the neural differentiation medium. 2.5 × 10 cells were cultured. 4 The cells were seeded at a viable cell density of 1000 cells / well onto a 24-well culture plate coated with MX521 (BioLamina), and 500 μL of NPCM was added to each well. The next day, hNPCs were observed to have adhered and grown. Half of the medium was replaced with fresh medium every 3–7 days.

[0175] 2.2 Immunofluorescence staining experiment Table 1 below shows the markers detected in the immunofluorescence staining experiments and the results at different time points. [Table 1]

[0176] As shown in the table above, neural progenitor cell markers (FOXG1, 88.00%) and neuronal cell markers (MAP2) (Figure 1) were detected on day 7 of in vitro induced differentiation, indicating that the majority of cells were still in the neural progenitor state.

[0177] On day 14 of in vitro induced differentiation, neural progenitor cell markers (FOXG1, 41.91%) and neuronal cell markers (MAP2) (Figure 1) were detected. The proportion of cells expressing neural progenitor cell markers was decreasing, confirming that cell differentiation was progressing.

[0178] At day 35 of in vitro differentiation, the differentiated cells contained >90% neuronal cells and expressed cerebral neuron markers. Detected cerebral neuron markers included layer 6 cortical neuron markers (CTIP2: 37.55%, BRN2: 20.18%, TBR1: 33.36%, SATB2: 6.90%) (Figure 2), excitatory glutamatergic neuron marker (VGLUT1, approximately 70-80%), inhibitory neuron-specific vesicular γ-aminobutyric acid transporter marker (VGAT, approximately 20-30%), inhibitory neuron neurotransmitter γ-aminobutyric acid marker (GABA, approximately 10-20%) (Figure 3), presynaptic and postsynaptic membrane protein markers (Synapsin, PSD95, >99%) (Figure 4), astrocyte marker (GFAP, approximately 2%), and neuronal marker (MAP2, >90%) (Figure 5).

[0179] On day 70 of in vitro induced differentiation, the differentiated cells were detected to contain more than 65% neurons, approximately 30% astrocytes, and 1.37% oligodendrocyte precursor cells. The detected cerebral neuron cell markers included layer 6 cerebral cortical neuron cell markers (CTIP2: 75.89%, BRN2: 8.49%, TBR1: 18.43%, SATB2: 13.15%) (Figure 6), excitatory glutamatergic neuron marker (VGLUT1, approximately 70-80%), excitatory neuron AMPA receptor GLUR2 subunit marker (GLUR2, approximately 70-80%), inhibitory neuron-specific vesicular γ-aminobutyric acid transporter marker (VGAT, approximately 20-30%), inhibitory neuron neurotransmitter γ-aminobutyric acid marker (GABA, approximately 20-25%) (Figure 7), astrocyte marker (GFAP, approximately 30%), neuron marker (MAP2, approximately 65%) (Figure 8), and oligodendrocyte marker (OLIG2, approximately 1.37%) (Figure 9).

[0180] These results demonstrate that hNPC01 can differentiate into mature human cortical functional neurons and glial cells, the composition of which is highly similar to that of the human cerebrum.

[0181] 2.3 Electrophysiological monitoring On days 8, 17, 20, and 25 after the initiation of hNPC01 differentiation, the electrophysiological activity of the cells was monitored using a multi-electrode array (MEA).

[0182] As shown in Figure 10, spontaneous electrophysiological activity and relatively mature features such as synchronized discharges were detected approximately 3 weeks after differentiation (day 20). On day 25 after differentiation, the number of active electrodes was 16, the weighted average discharge frequency (Hz) was 3.2, and the cluster discharge frequency (Hz) was 0.16.

[0183] The above results indicate that the cortical neurons obtained by induced differentiation of the FNPCs of the present invention were observed to form complex neural networks in approximately 3 weeks, and that the complexity of the neural network increased with the passage of differentiation time.

[0184] Example 3. Cell therapy for a cynomolgus monkey permanent ischemic stroke model In this example, the inventors used cynomolgus monkeys as experimental subjects to perform cell therapy in a primate stroke model. The brain volume and functional areas of cynomolgus monkeys are similar to those of humans, and the process of pathological changes after stroke is also similar to that of humans.

[0185] The cynomolgus monkey permanent ischemic stroke model in the examples was created by permanently ligating the M1 segment of the left middle cerebral artery in cynomolgus monkeys using surgical sutures, i.e., a pMCAO model. MRI and functional impairment confirmed that the level of damage in this model was comparable to that of severe stroke patients in humans, and that some neurological and motor dysfunction persisted even 6-7 months after cerebral infarction. In contrast, reperfusion ischemia models in mice and rats only detect functional impairment for 14 days to 1 month. This model overcomes the problem of rapid spontaneous recovery in animals and can better mimic long-term functional impairment after stroke (cerebral infarction) in human subjects and evaluate the effectiveness of treatment after the acute phase.

[0186] See Figure 11 for the specific experimental design.

[0187] 3.1 Creation of a permanent ischemic stroke model Experimental animals: Twenty-four cynomolgus monkeys, aged 4-8 years, were tested and confirmed negative for the pathogenic microorganisms Mycobacterium tuberculosis (TB), simian retrovirus (SRV), simian T-cell leukemia virus (STLV), and simian immunodeficiency virus (SIV).

[0188] Grouping: The animals were randomly divided into four groups, each consisting of six animals, half male and half female. These groups were designated the model control group, the low-dose hNPC01 group, the medium-dose hNPC01 group, and the high-dose hNPC01 group. Hereinafter, the model control group will be abbreviated as the control group, and the latter three groups will sometimes be collectively referred to as the experimental groups.

[0189] Model creation: On day 0, the left middle cerebral artery M1 segment of cynomolgus monkeys in the control and experimental groups was ligated using surgical sutures to create a cynomolgus monkey permanent ischemic stroke model.

[0190] 3.2 Cell injection (transplant) On day 13 (D13) after the ischemic stroke model was created, brain MRI images were obtained for each group, and the location of the infarct was identified based on the images to serve as a guide for the injection site.

[0191] Starting 4 days before cell injection (transplantation), each group was given cyclosporine A orally at a dose of 15 mg / kg / day for immunosuppression, and this was administered for 28 consecutive days.

[0192] On day 15 (D15) after ischemic stroke model creation, each group of cynomolgus monkeys received a single injection of the neural progenitor cell preparation hNPC01 into the motor cortex and basal ganglia region surrounding the infarct. Specifically, intracranial multipoint injections (three needle tracts, three different injection depths per needle tract) were performed. The doses administered to the model control group, low-dose hNPC01 group, medium-dose hNPC01 group, and high-dose hNPC01 group were 0 (0.9% sodium chloride injection), 4.5 × 10 cells, and 10 cells, respectively. 5 cells / mouse, 9.0×10 5 cells / animal, and 18 x 10 5 cells / animal.

[0193] 3.3 Pharmacodynamic evaluation plan After cell injection, each group of cynomolgus monkeys was continuously observed for 28 weeks, and pharmacological and toxicological evaluations were performed. Specific observation and measurement methods and indicators used are summarized in Table 2. [Table 2]

[0194] 3.3.1 Physiological Monitoring All model animals were clinically observed daily after middle cerebral artery occlusion surgery. During the test period, the animals showed clinical symptoms of stroke, but their body temperature, blood pressure, and other physical indicators were normal, their moods were stable, and no other abnormal signs were found. No animal deaths occurred during the test period.

[0195] The animals' body weights were measured one day before model creation, three days after model creation, one day before cell transplantation, and two days (D2), seven days (D7), 14 days (D14), and 28 days (D28) after transplantation, and then once a week thereafter.

[0196] The weight gain rate of experimental animals of different sexes after cell therapy was calculated according to the following formula, and the results are shown in Figure 12, which shows the weight changes after treatment.

[0197] Weight gain rate = (weight on the day before model creation - weight on the day before model creation) / weight on the day before model creation x 100% As shown in Figure 12, the weight gain rates of the medium-dose and high-dose cell transplantation groups were higher than those of the vehicle control group, and the high-dose group's weight gain rate was higher than that of the medium-dose group. Two-way ANOVA statistical analysis revealed significant differences (p<0.001) between the high-dose group, the vehicle control group, and the low-dose and medium-dose groups. This result indirectly indicates that the animals in the high-dose cell transplantation group recovered their appetite and feeding ability more quickly after model creation, and therefore regained their weight more quickly.

[0198] 3.3.2 Behavioral observations Para-cage behavioral assessment and upper limb feeding ability test were performed 1 day before model creation, 3 days after model creation, 1 day before cell transplantation, 2 days, 7 days, and 14 days after transplantation, and every 2 weeks thereafter. From 176 days after cell transplantation, a ladder walking test was performed once a week.

[0199] 3.3.2.1 Paracage behavioral studies (neurologic function scores) Before surgical model creation, cage-parallel behavioral assessments were performed on cynomolgus monkeys that had completed behavioral training and were grouped. Behavioral scores were used to assess neurological function based on the animal's state of consciousness, sensory system (contralateral to the ischemic side), motor system, and coordination of skeletal muscle movements. Scores were assessed on a scale of 0 to 100, with lower scores indicating closer to normal neurological function and higher scores indicating more severe neurological dysfunction.

[0200] As a result, before the model was created, the cynomolgus monkeys in each group showed normal behavior in terms of consciousness, sensory system, motor system, and skeletal muscle coordination, and were able to complete the movements according to the instructions of the keepers, indicating that the behavioral adaptation training was successful.

[0201] The cage-based behavioral assessment scores of the animals were significantly decreased on days 3 and 14 after the ischemic surgery model (12 and 1 days before cell transplantation). On day 14, the animals showed significant behavioral impairments in consciousness, sensory system, motor system, and skeletal muscle coordination, indicating that the extent of brain damage was as expected and that the model was successfully created.

[0202] After treatment, considering the influence of the cell transplantation process on animal behavior and to eliminate behavioral errors due to the process, the second day after cell transplantation (day 3, D17) was set as the reference point (i.e., point 0), and the degree of change in cage para-ethological behavior for each group over 28 weeks after cell transplantation was statistically analyzed. Specifically, cage para-ethological evaluations were performed on days 2 (D17), 7 (D22), and 14 (D29) after treatment, and then every two weeks thereafter. The results are shown in Figures 13 and 14.

[0203] Statistical analysis of cage-parallel behavioral data for each group 28 weeks after cell transplantation showed that animals in the low-, medium-, and high-dose cell-treated groups showed a rapid behavioral recovery compared to the vehicle control group, with a significant difference of p<0.0001 determined by two-way ANOVA (Figure 13). Furthermore, the experimental results also showed that, except for two animals with mild modeling, the initial behavioral scores (before treatment) of the affected side of the treated experimental groups were 30-50% higher than those of the vehicle control group, indicating a faster recovery rate despite more severe neurological dysfunction (Figure 14).

[0204] 3.3.2.2 Ladder towing walking test To intuitively explore the recovery status of each function of the animals after cell therapy and the differences between the groups, a ladder walking test was added from 23 weeks after the treatment (D176).

[0205] Using a short-gap ladder (10cm bar spacing), the animals were pulled and asked to walk back and forth three times. The single walking time and upper limb usage were recorded and statistically analyzed. The results of this test indicate the recovery status of the animals' motor behavior, physical coordination, sensory system, muscle tone, etc.

[0206] In this experiment, normal, unmodeled cynomolgus monkeys (n=6) were used for synchronization training and compared with the control and experimental animals as a normal animal group. The results are shown in Figure 15.

[0207] Figure 15 shows the statistical results of the mean single walking time for the ladder walking test measured at 5 to 7 months (23 to 28 weeks). Two-way ANOVA statistical analysis revealed a significant difference between the groups (p<0.001). By 5 months, the high-dose group had almost returned to the level of the untreated control group, while the vehicle control group still required approximately twice the walking time.

[0208] Five months after non-acute injection treatment with the hNPC01 cell preparation of the present invention, the motor function and coordination of the upper and lower limbs of the cerebral ischemia model animals recovered to the level of healthy animals, approximately two months earlier than the control group.

[0209] 3.3.3 Magnetic Resonance Imaging (MRI) of Animal Brain Tissue On the 13th day after model creation (2 days before transplantation), MRI scans were performed on the animals in each group to analyze the extent of brain damage and evaluate whether the model had been created successfully.

[0210] In addition, brain MRI images were obtained on days 3 (D18), 28 (D43), 56 (D71), 84 (D99), and 168 (D183) after cell transplantation, and the changes in brain lesion volume were analyzed to evaluate the therapeutic effect.

[0211] Evaluation of the model animals' condition two weeks after surgery showed that the animals showed symptoms of ischemic stroke in terms of cage behavior, feeding ability, and MRI imaging, indicating that the model was successfully created.

[0212] Regarding the MRI detection results at various time points after transplantation, typical magnetic resonance images of the animal brain after cell transplantation at the end of the test are shown in FIG.

[0213] Furthermore, based on the MRI images, a statistical analysis of the brain damage volume ratio was performed using a 3D slicer, and the results are shown in Figure 17.

[0214] The results in Figure 17 show that the extent of brain damage in all animals in each group reached its maximum before cell transplantation. Analysis based on pre-treatment (D14) revealed that within 4 weeks of treatment, the high-dose hNPC01 group showed the greatest reduction in brain lesion volume. This means that the high-dose group had the strongest brain lesion repair effect, followed by the medium-dose group, and the low-dose group had the weakest. These results suggest that this cell preparation can be used to improve brain injury and infarction after the acute phase of ischemic stroke and can achieve consistent tissue repair. Furthermore, the cell preparation administered at a higher dose may have a stronger ameliorative effect.

[0215] 3.4 Immunohistochemical staining At the end of the study, all remaining animals were euthanized, and brain tissue was collected (Figure 18). Pathological examination was performed on the brain tissue of some animals, and the brain tissue on the infarcted side was fixed, cut into coronal sections, and scanned for images. HE staining was performed on the brain tissue from the lesion area to observe the state of inflammatory cell infiltration in the brain tissue of the infarcted area. The results of immunofluorescence staining from animals in the medium dose group are shown in Figure 19. Immunofluorescence staining was performed using anti-human nuclear marker STEM101 and mature neuron marker NEUN.

[0216] As shown in Figure 19, in brain sections from animals injected with hNPC01 cells, over 80% of human-derived cells (confirmed by staining with the anti-human nuclear marker STEM101) expressed the mature neuron marker NEUN, indicating that the human neural progenitor cells differentiated into a population primarily composed of mature neuronal cells 7 months after transplantation.

[0217] In summary, all of these results demonstrate that after transplantation of the hNPC01 cell preparation, containing differentiated forebrain neural progenitor cells derived from iPSCs, the experimental group of animals receiving cell therapy showed significant improvements in neurological and gait function, weight loss, and recovery of brain tissue damage compared to the control group. The administered FNPC cells were able to differentiate into neurons. Furthermore, some experiments demonstrated that the effects of FNPC treatment were dose-dependent, suggesting that a specific high dose may be preferable.

[0218] Example 4. Cell therapy for ischemic-hypoxic cerebral palsy rat model In this example, the inventors used an animal model to test the therapeutic effect of the cells of the present invention on ischemic-hypoxic pediatric cerebral palsy, and demonstrated the therapeutic efficacy of the cell product of the present invention on different types of brain damage.

[0219] 4.1 Modeling and Dosing Regimen Using the classical RICE model, unilateral common carotid arteries were permanently ligated in postnatal day 7 (P7) SFP-class SD rats. After 1 hour of postoperative recovery, the rats were subjected to 2.5 hours of hypoxia using a mixture of 8% oxygen and 92% nitrogen. After model creation, the rats' limb movement and unilateral eye opening were monitored to confirm the success of the model creation. On day 7 after model creation, 10 μL of hNPC01 cells at different cell densities were stereotaxically injected into the lateral ventricle using a brain stereotaxic device. Control and non-control mice were treated with saline in the same manner. The specific experimental groupings are shown in Table 3 below. [Table 3]

[0220] 4.2 Pharmacodynamic evaluation After cell injection, rats in each group were continuously observed for 10 weeks and the efficacy of the drugs was evaluated. Specific experiments included rotorod experiments, balance beam experiments, and 2,3,5-triphenyltetrazolium chloride (TTC) staining experiments.

[0221] 4.2.1 Rotating rod experiment Rotating rod experiments will be performed before administration and 2, 4, and 6 weeks after administration to examine the effect of hNPC01 administration on the motor abilities of rats.

[0222] Before the experiment, the animals were moved from the breeding room to the experimental room and allowed to acclimate for 60 minutes. The rats were placed on a 90mm diameter rotor rod. After 2 minutes of acclimation, the rotor rod was set to an initial speed of 4 rpm and accelerated to 40 rpm within 300 seconds, allowing the rats to move independently along the rotor rod. The time spent on the rotor rod was recorded. After the experiment, the rats were returned to the breeding room and the experimental apparatus was cleaned with 70% ethanol. The temperature, humidity, and lighting intensity of the experimental room must be kept constant during all experiments.

[0223] The results of the rotorod experiment are shown in Figure 20. The high-dose group showed longer motor time on the rotorod compared to the model saline group at 2, 4, and 6 weeks after administration (*, P<0.05; **, P<0.01; **, P<0.01). The high-dose group showed stable performance and no tendency for deterioration was observed. The medium-dose group showed longer motor time on the rotorod compared to the model saline group at 6 weeks after administration (#, P<0.05). No significant differences were observed between the low-dose group and the model saline group at any time point.

[0224] 4.2.2 Balance beam experiment Balance beam tests were performed before and after administration (2, 4, and 6 weeks) to detect the effects of hNPC01 administration on the motor abilities of rats.

[0225] During the training phase, the animals were moved from the breeding room to the experimental room and allowed to acclimate for 60 minutes before undergoing training using a 25mm diameter square pillar. The animals were placed at the beginning of the horizontal pillar, and the time it took to cross a 100cm distance before entering the dark box (i.e., latency) was measured with a stopwatch. The rats were tested for latency on the horizontal pillar and the number of slips of their left and right hind paws. After the test, they were returned to the breeding room, and the experimental apparatus was cleaned with 70% ethanol. All tests were performed in a laboratory with constant temperature, humidity, and lighting intensity.

[0226] The results of the balance beam experiment are shown in Figures 21A and 21B. The high-dose group had shorter balance beam times than the model saline group at 2, 4, and 6 weeks after administration (****, P<0.0001; **, P<0.01). The high-dose group showed stable performance and no deterioration was observed. The medium-dose and low-dose groups showed no significant differences from the model saline group in detection at each time point, and no deterioration was observed.

[0227] 4.2.3 TTC staining TTC staining was performed at the end of the experimental observation. Dehydrogenases in viable cells of normal brain tissue can reduce TTC to the stable, insoluble, red triphenylformazan (TTF). If brain tissue cells are dead or have reduced vitality, they will not stain or will stain weakly. Therefore, the vitality of brain tissue can be determined based on the staining site and intensity of the staining.

[0228] The results of the TTC staining experiment are shown in Figure 22. No significant residual damage was observed in the unprepared saline group (G1), the medium-dose group (G4), and the high-dose group (G5). Significant brain tissue damage was observed in the low-dose group and the model saline group.

[0229] Example 5. Cellular composition study of hNPC01 In this example, we describe the cellular composition of hNPC01 using single-cell transcriptome sequencing (scRNA-seq). Furthermore, we compared hNPC01 with NPCs from the fetal forebrain. Data from the fetal forebrain NPCs were obtained from a published paper (Zhong, S. et al., A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex. Nature 2018, 555(7697), 524-528). Data comparison confirmed that the transcriptome data and molecular markers from hNPC01 are consistent with naturally occurring human forebrain neural progenitors.

[0230] Based on the results of scRNA-seq, we annotated the cell types in the hNPC01 sample and confirmed the phenotype of the cells in the sample through gene expression profiles. The results are shown in Figure 23. As shown in Figure 23, the predominant cell type in the hNPC01 sample was neural progenitor cells, which phenotypically matched the predominant cell type in the fetal forebrain sample, accounting for over 70% of the hNPC01 sample. Furthermore, the hNPC01 sample contained immature neurons, GABAergic neurons, glutamatergic neurons, and small amounts (≤10%) of ependymal cells and pericytes, which are widely distributed throughout the brain. The neural progenitor cells in hNPC01 can further differentiate in vivo and in vitro to form cortical neurons with the correct subtype composition and mature electrophysiological functions (including excitatory and inhibitory neurons, astrocytes, and oligodendrocyte progenitor cells). At the same time, these transplanted neural progenitor cells secrete multiple cytokines and microRNAs that promote neuroprotection and angiogenesis, helping to enhance endogenous repair processes.

[0231] Further analysis of the sequencing data from the fetal samples and the hNPC01 samples showed that the two samples shared a high degree of concordance in the expression profiles of neural progenitor and neuronal markers.

[0232] From the above scRNA-seq comparison data, we found that the forebrain NPC cell product hNPC01 of the present invention, produced by iPSC-induced differentiation, exhibited neural progenitor cell characteristic genes consistent with those found in naturally occurring fetal neural progenitor cells, but the expression levels of pluripotent stem cell marker genes were lower than those of fetal cells, indicating that the cell product of the present invention contained fewer or less highly divisive cells. These results indicate that the cell product of the present invention contained no residual iPSC phenotype cells, nor any mesodermal or endodermal phenotype cells, demonstrating that the cell product of the present invention contained forebrain neural progenitor cells of higher purity and was therefore safer.

[0233] Furthermore, differential comparison analysis of single-cell sequencing data from human embryonic cerebral development and human mesoderm- and endoderm-derived organs confirmed that the cell product of the present invention specifically and highly expresses molecular markers of forebrain neural progenitor cells (EFNB2, WNT7B, RSPO2, FEZF2, etc.), supporting the conclusion that the cell product of the present invention contains highly purified forebrain neural progenitor cells.

[0234] Example 6. In vivo neural circuit reorganization study In addition to the survival and differentiation of transplanted cells into the desired type, the ability of differentiated cells to form neural circuits is an important indicator of transplantation efficacy. This study demonstrated that after transplanting hNPC01 of the present invention into the ischemic-damaged brain region of immunodeficient experimental mice, these hNPCs formed by in vitro induced differentiation not only further differentiated to form mature neurons in vivo, but also integrated into the recipient's existing central nervous system, forming functional neural connections and reorganizing neural circuits. This is crucial for functional recovery after brain injury.

[0235] After transplanting 2 x 10E5 hNPCs into the perinfarct region of the somatosensory cortex of each rat, the animals were injected with the recombinant viral vector scAAV2 / 1-hSyn-Cre into the ventral thalamus. This serotype of AAV virus has the ability to label downstream synapses and deliver a certain amount of Cre recombinase to neurons at the next level of synaptic signaling. At the same time, the same animals were injected with another recombinant viral vector, AAV2 / 8-EF1α-DIO-mCherry, into the somatosensory cortex. When infected neurons encounter the Cre recombinase delivered upstream, the loxp sequence is inverted, resulting in expression of the mCherry fluorescent reporter gene. A red mCherry signal was observed in the AAV2 / 8-EF1α-DIO-mCherry-injected tissue, indicating that neurons in that region established direct synaptic connections with the ventral thalamus. During the experimental process, red mCherry signaling was first observed in intact somatosensory cortex, confirming the effectiveness of this strategy.

[0236] Furthermore, we confirmed the expression of red mCherry signals in progeny neurons differentiated from GFP-positive human forebrain NPCs, and these red signals overlapped with anti-human nuclei antibody HNA staining, further confirming that the progeny neurons of transplanted hNPCs could establish direct synaptic connections with the host ventral thalamus, indicating successful establishment of neural circuits.

Claims

1. Use of a cell population comprising human forebrain neural progenitor cells for the treatment of a neurological disease, pathology or condition associated with the death and / or dysfunction of forebrain neurons in a subject, or for the manufacture of a medicament for treating a neurological disease, pathology or condition associated with the death and / or dysfunction of forebrain neurons in a subject, said cell population comprising human forebrain neural progenitor cells.

2. The use of claim 1, wherein in the cell population, at least about 70% of the cells express NESTIN, at least about 70% of the cells express FOXG1, at least about 70% of the cells express PAX6 and / or at least about 70% of the cells express SOX2.

3. The use of claim 2, wherein in the cell population, at least about 70% of the cells express NESTIN, at least about 70% of the cells express FOXG1, and at least about 70% of the cells express PAX6.

4. The use of claim 3, wherein in the cell population, at least about 70% of the cells express NESTIN, at least about 70% of the cells express FOXG1, at least about 70% of the cells express PAX6, and at least about 70% of the cells express SOX2.

5. The use of claim 4, wherein in the cell population, at least about 80% of the cells express NESTIN, at least about 80% of the cells express FOXG1, at least about 80% of the cells express PAX6, and at least about 80% of the cells express SOX2.

6. The use according to any one of claims 1 to 5, wherein the cell population expresses one or more genes selected from EFNB2, WNT7B, RSPO2, and FEZF2.

7. The use according to claim 6, wherein the cell population simultaneously expresses EFNB2, WNT7B, RSPO2, and FEZF2.

8. 8. The use according to any one of claims 1 to 7, characterized in that in said cell population, neural progenitor cells, preferably forebrain neural progenitor cells, represent ≧60%, more preferably ≧70%, even more preferably ≧80% of all cell types.

9. The use according to any one of claims 1 to 8, characterized in that the cell population further comprises one or more types selected from immature neurons, GABAergic neurons, and glutamatergic neurons.

10. The use according to any one of claims 1 to 9, characterized in that the cell population further comprises ependymal cells and / or pericytes.

11. The use according to claim 10, characterized in that in the cell population, the total proportion of the immature neurons, GABAergic neurons, glutamatergic neurons, ependymal cells, and pericytes is 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less of the total number of cells.

12. The use according to any one of claims 1 to 11, characterized in that the forebrain neural progenitor cells are obtained by induced differentiation from human pluripotent stem cells (hPSCs).

13. The above-mentioned induced differentiation method is as follows: (1) Cultivating and expanding ESCs or iPSCs in human pluripotent stem cell medium (hPSC medium); (2) digesting the ESCs or iPSCs from step (1) and culturing them in suspension in EB medium to form embryoid bodies (EBs); (3) Adherently culturing the EBs obtained in step (2) using a neural induction medium (RONA) to form rosette-like neural aggregates (ROSette Neural Aggregates, RONAs); (4) collecting neuroectodermal cells in the RONAs formed in step (3) and culture them in suspension to form neurospheres; The use according to claim 12, characterized in that it comprises the steps of:

14. The above-mentioned induced differentiation method further comprises the steps of: (5) culturing and subculturing the neurospheres formed in step (4) using NPC medium; 14. The use according to claim 13, characterized in that it comprises the steps of:

15. The above-mentioned induced differentiation method further comprises the steps of: (6) recovering the FNPCs formed in step (5); 15. The use according to claim 14, characterized in that it comprises the steps of:

16. The hPSC culture medium includes NutriStem® hPSC XF medium, Essential 8 medium, StemFit® Basic 03 medium, and StemMACS TM iPS-Brew medium, Stem-Partner (registered trademark) ACF medium, TeSR TM The use according to any one of claims 13 to 15, characterized in that the medium is an AOF medium or a TeSR2 medium.

17. The use according to any one of claims 13 to 16, wherein a ROCK inhibitor is added to the hPSC culture medium.

18. The use according to claim 17, wherein the ROCK inhibitor is Y-27632.

19. The use according to claim 18, wherein the Y-27632 is added at a concentration of about 10 μM.

20. The EB medium contains the following: (i) a basal medium selected from a) to c); a) KnockOut alone TM DMEM / F12 medium, b) DMEM / F12 medium and Neurobasal TM Media combinations, c) Knock Out TM DMEM / F12 medium and Neurobasal TM Media combinations, and, (ii) an additive comprising or consisting of d) and e); d) N-2 Additive and GlutaMAX TM -I additives, e) N-2 Additive, GlutaMAX TM -I additives and vitamin A-free B-27 TM Additive (B-27 TM Supplement, minus vitamin A), Use according to any one of claims 13 to 19, characterized in that it comprises

21. The use described in claim 20, characterized in that the EB culture medium further contains inhibitors, which include a BMP inhibitor, an AMPK inhibitor, and an ALK inhibitor, or consist of a BMP inhibitor, an AMPK inhibitor, and an ALK inhibitor.

22. The use according to claim 21, wherein the inhibitor comprises one or more selected from Noggin, SB431542, LDN-193189, DMH-1, and Dorsomorphin, or consists of one or more of Noggin, SB431542, LDN-193189, DMH-1, and Dorsomorphin.

23. The use of claim 22, wherein the inhibitor comprises or consists of a combination of SB431542 and one or more selected from Noggin, LDN-193189, DMH-1, and Dorsomorphin.

24. The use according to claim 23, wherein the inhibitor comprises or consists of a combination of SB431542 and one or two selected from Noggin, LDN-193189, DMH-1, and Dorsomorphin.

25. The EB medium contains the following: (i) a basal medium comprising: TM DMEM / F12 medium and Neurobasal TM It is a combination of media; (ii) Additives, including N-2 Additive and GlutaMAX TM -I is a combination of additives; and, (iii) SB431542, Noggin, and Dorsomorphin; Use according to any one of claims 13 to 24, characterized in that it comprises

26. In the above step (3), (i) a basal medium comprising: TM DMEM / F12 medium and Neurobasal TM It is a combination of media; and, (ii) Additives, including N-2 Additive and GlutaMAX TM -I is a combination of additives; Use according to any one of claims 13 to 25, characterized in that a RONA medium containing

27. In the above step (3), (i) a basal medium comprising: TM DMEM / F12 medium and Neurobasal TM It is a combination of media; and, (ii) Additives, such as N-2 Additive and B-27 TM Additive (B-27 TM Supplement, XenoFree, minus vitamin A), and GlutaMAX TM -I is a combination of additives; Use according to any one of claims 13 to 26, characterized in that a RONA medium containing

28. The basal medium for the NPC culture medium is Neurobasal TM The NPC medium is a medium, and the additives of the NPC medium are (a) GlutaMAX TM -I additive, and (b) Vitamin A-free B-27 TM The use according to any one of claims 13 to 27, characterized in that it is an additive.

29. The use according to claim 28, characterized in that the NPC culture medium further contains brain-derived neurotrophic factor (BDNF), and / or glial cell line-derived neurotrophic factor (GDNF), and / or L-ascorbic acid, and / or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (DB-cAMP).

30. In the above step (5), (i) a basal medium comprising: TM It is a medium; (ii) Additives, such as B-27 TM Additive (B-27 TM Supplement, XenoFree, minus vitamin A) and GlutaMAX TM -I additive combinations; and, (iii) BDNF, GDNF, L-ascorbic acid, and DB-cAMP; 30. The use according to any one of claims 12 to 29, characterized in that an NPC medium comprising:

31. The use according to any one of claims 13 to 30, characterized in that in step (4), the RONA medium or the NPC medium is used.

32. 32. The use according to any one of claims 1 to 31, characterized in that in said cell population, pluripotent stem cells account for ≦3% of all cell types, preferably ≦2% or less, more preferably ≦1%.

33. The use according to any one of claims 1 to 32, characterized in that the human forebrain neural progenitor cells have the ability to differentiate into neuronal cells.

34. The use according to any one of claims 1 to 33, characterized in that the human forebrain neural progenitor cells have the ability to differentiate into astrocytes.

35. 34. The use according to claim 33, characterized in that the neuronal cells express MAP2.

36. 35. The use according to claim 34, wherein the astrocytes express GFAP and / or S100β.

37. The use according to any one of claims 1 to 36, wherein the cell population has the ability to construct a new neural circuit in a subject.

38. The use according to any one of claims 1 to 37, wherein the disease is a neurological injury disease selected from diseases associated with ischemic brain injury, diseases associated with hemorrhagic brain injury, and diseases associated with traumatic brain injury.

39. The use according to claim 38, wherein the disease associated with ischemic brain damage is ischemic stroke or a condition associated with ischemic stroke, such as a complication or sequela.

40. The use according to claim 38, wherein the disease associated with hemorrhagic brain injury is hemorrhagic stroke or a pathology associated with hemorrhagic stroke, such as a complication or sequela.

41. The disease is a neurodegenerative disease selected from Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). The use according to any one of claims 1 to 37.

42. Use according to any one of claims 1 to 37, characterized in that the disease is a neurodevelopmental disease and is selected from autism, epilepsy and cerebral palsy.

43. 41. The use according to claim 39 or 40, wherein the administration of the cell population is carried out after the acute phase of the stroke or at least 14 days after the onset of the stroke.

44. The use according to any one of claims 1 to 43, characterized in that the cell population is administered by intracranial injection.

45. 45. The use according to claim 44, wherein the cell population is injected into the infarcted or damaged brain region, or into the motor cortex and / or basal ganglia region surrounding the infarcted or damaged brain region.

46. 46. ​​Use according to claim 44 or 45, characterized in that the injection is a single-site injection or a multi-site injection.

47. Use according to any one of claims 44 to 46, characterized in that the injection is a single injection or multiple injections.

48. 48. The use according to claim 47, wherein when multiple injections are administered, the interval between two adjacent injections is 30 days or more.

49. The use according to any one of claims 1 to 48, wherein the cell population is prepared as a suspension.

50. 50. The use according to claim 49, wherein the suspension is prepared using 0.9% sodium chloride injection.

51. The cell concentration in the suspension was 1.0 x 10 4 ~5.0 x 10 5 51. Use according to claim 49 or 50, characterized in that the concentration of viable cells / μL.

52. The injection dose was 1.0 x 10 5 ~1 x 10 10 cells / time, preferably 1.5 x 10 5 cells / time ~ 6 x 10 7 The use according to any one of claims 44 to 48, characterized in that the number of cells per treatment is 100.

53. The subject is a human, and the injected dose is 2 x 10 5 ~2 x 10 9 cells, preferably 2 x 10 6 ~1.2 × 10 9 The use according to any one of claims 44 to 48, characterized in that the cells are cells.

54. The use according to any one of claims 1 to 53, characterized in that said cell population or said cell preparation is used alone or in combination with other drugs or treatments.

55. 1. A method of treating a neuronal injury disease, neurodegenerative disease, neurodevelopmental disease, or neurological disease, condition, or state associated with forebrain neuronal cell death and / or dysfunction in a subject, comprising: A therapeutic method comprising administering to the subject the cell population of the present invention, wherein the cell population contains human forebrain neural progenitor cells.

56. The method according to claim 55, wherein the cell population is a cell population according to any one of claims 2 to 37.

57. The method according to claim 55 or 56, characterized in that the disease is a disease according to any one of claims 31 to 35.

58. 58. The method according to any one of claims 55 to 57, characterized in that the cell population is administered by the method according to any one of claims 36 to 46.

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