Use of forebrain nerve precursor cells in treatment of diseases associated with death and / or dysfunction of forebrain nerve cells

Abstract

Provided is the use of a population of nerve cells comprising forebrain nerve precursor cells (FNPCs) having specific developmental characteristics in the treatment of a nerve injury disease, a neurodegenerative disease, a neurodevelopmental disease, or other conditions associated with death and / or insufficiency of forebrain nerve cells, in particular in the treatment of a disease associated with death and / or insufficiency of the nerve cells of the nerve cells of the nerve cells of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell of the nerve cell. Preferably, the forebrain nerve precursor cells with specific developmental characteristics highly express markers including FOXG1, PAX6 and NESTIN. Preferably, the population of nerve cells is obtained by culturing human pluripotent stem cells (hPSCs).

Description

Use of forebrain neural progenitor cells in treating diseases associated with forebrain neural cell death and / or dysfunction

[0001] Cross-references

[0002] This application claims priority to Chinese invention patent application No. 202211466612.0, filed on November 22, 2022, entitled “Application of forebrain neural precursor cells in treating diseases associated with forebrain neural cell death and / or dysfunction.” The contents of that application are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of cell therapy in biotechnology, and more particularly to the use of a neural cell population in treating neural injury diseases, neurodegenerative diseases, neurodevelopmental diseases, or other neural diseases, disorders, or conditions associated with the death and / or dysfunction of forebrain neural cells, wherein the neural cell population comprises forebrain neural progenitor cells with specific developmental characteristics. Background Art

[0004] The forebrain differentiates into the telencephalon and diencephalon during early development. The telencephalon then develops into the cerebral cortex and subcortical brain tissue such as the basal ganglia, olfactory bulb, and hippocampus, while the diencephalon develops into the thalamus, hypothalamus, and epithalamus. Due to the complex structure and function of the various parts of the forebrain, a variety of diseases and conditions are associated with damage or dysfunction of forebrain nerve cells. These diseases or conditions include neurological injury diseases such as stroke, stroke sequelae, traumatic brain injury (TBI), and sequelae of brain injury; neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS); and neurodevelopmental diseases such as epilepsy, autism, cerebral palsy, and so on.

[0005] Stroke is a leading cause of forebrain nerve damage and functional loss worldwide. According to the World Health Organization (WHO), approximately 15 million people suffer from stroke each year, with approximately one-third of these patients dying and another third experiencing severe permanent disability. The disease results in high morbidity, mortality, and disability rates.

[0006] Currently, approximately 20 million people in China are surviving stroke sequelae, with approximately 2.7 million new cases each year. With the increasing aging of society, the number of people suffering from stroke sequelae is expected to grow even larger. Post-stroke patients experience a poor quality of life, causing significant inconvenience to patients and their families, and placing a heavy socioeconomic burden.

[0007] Strokes are categorized as hemorrhagic and ischemic, with the latter accounting for approximately 80% of all strokes. The brain is extremely sensitive to ischemia. Once ischemic, insufficient energy supply immediately leads to functional impairment. If this condition persists, the impairment worsens and may eventually lead to cerebral infarction.

[0008] Currently, the main treatment for ischemic stroke involves rapid thrombolysis or intravascular clot removal within 3-6 hours of stroke to restore cerebral blood flow and reduce brain tissue damage. This type of treatment includes the use of drugs represented by t-PA. t-PA was the first thrombolytic drug approved by the US FDA in 1996 for the treatment of ischemic stroke. A disadvantage of this type of treatment is that the time window for application is too narrow, resulting in patients often not receiving treatment in time. Another disadvantage is that this type of treatment cannot reverse neurological damage, resulting in patients still having sequelae such as functional impairment after treatment because the ischemia has already caused a certain degree of neurological damage.

[0009] In addition, neuroprotective drugs are also used in stroke treatment, including calcium channel blockers (such as nimodipine), free radical scavengers (such as edaravone, tocopherol, SOD), neurotrophic factors (such as nerve growth factor), NMDA antagonists, gangliosides and excitatory glycine receptor antagonists, Enbipu, etc. These drugs are all targeted at patients with acute stroke (within 14 days after stroke) and reduce the impact on neurological function by reducing nerve damage, inflammation, and cell death. However, the clinical efficacy of most of these drugs is unsatisfactory, or they have adverse side effects, and they cannot alleviate or treat the sequelae of hemiplegia caused by stroke.

[0010] Cell therapy is a new treatment option currently available. Clinical trials using mesenchymal cells to treat acute stroke patients have shown that these cells have certain anti-inflammatory, tissue repair, and angiogenesis effects. However, in clinical trials or studies with larger populations and controlled controls, mesenchymal cells have yet to demonstrate clear therapeutic efficacy.

[0011] Rehabilitation is currently the standard treatment for functional impairment caused by neurological damage such as stroke, but it can only promote functional recovery to a certain extent and is unlikely to bring about further improvement during the stable period of hemiplegia (6 months after stroke).

[0012] Although the above treatments can reduce the damage caused by stroke to a certain extent or achieve a certain degree of functional recovery, they cannot rebuild the function of damaged tissues or improve the sequelae of hemiplegia.

[0013] Because most stroke patients suffer sequelae or even disability due to brain damage, reversing or compensating for the loss of nerve cells caused by stroke is extremely important. Currently, there are no FDA-approved neuroprotective drugs that can reverse or restore these lost nerve cells, and no clinically available drugs can achieve functional improvement in patients with stable hemiplegia.

[0014] Studies have found that certain areas of the adult brain, such as the dentate nucleus of the hippocampus and the subventricular zone, possess regenerative capacity, enabling compensatory neurogenesis after stroke. However, therapeutic approaches that attempt to promote this endogenous neurogenesis have been limited in terms of both the number of newly generated neurons and the effectiveness of repair, making it difficult to overcome the functional impairments caused by brain tissue damage after stroke.

[0015] With the continuous development of stem cell technology, some studies have attempted to promote tissue repair and replenish the loss of nerve cells by transplanting tissue-derived neural stem cells into the brains of patients with ischemic stroke.

[0016] ReNeuron's patent application WO2011137117A1 discloses a method for treating stroke using an immortalized human fetal neural stem cell line, CTX0E03. This method allows for intravenous or intra-arterial delivery of neural stem cells, eliminating the need for invasive craniotomy. The application was validated in a rat model. However, the application requires treatment to be administered within seven days, preferably within two days, of the stroke.

[0017] NeuralStem's patent application WO2006055685A2 discloses a method for transplanting human neural cells to treat neurodegenerative diseases. This method involves isolating neural stem cells from the tissue region in need of neuronal recruitment, culturing and expanding them in vitro, and then transplanting them into the corresponding neural tissue, thereby replenishing cells that produce specific neurotransmitters within neural circuits. This application does not address stroke and the resulting neural damage, but rather calls for the transplantation of primary neural stem cells / progenitor cells derived from tissue.

[0018] The cells used in the above methods are all derived from neural stem cells obtained from aborted fetal tissue, which is subject to ethical restrictions in clinical commercial applications. In addition, due to the late period of obtaining the so-called "neural stem cells", the cells obtained from the fetal brain or spinal cord tissue at the aborted stage are mainly glial precursor cells. Therefore, after the CTX0E03 product was transplanted into the animal's skull, only 2% of the cells were observed to differentiate into FOXA3 neurons and about 20% into astrocytes. There are no reports that the transplanted cells have electrophysiological functions or can form synaptic connections with the original animal neurons, indicating that the transplanted cells failed to develop into neurons capable of forming new neural circuits (EJ Smith, et al., Stem Cell 2012; 30:785-796).

[0019] On the other hand, although immortalized neural stem cell products (introduced with tumor genes, fluorescent reporter genes, etc., and capable of continuous proliferation) can be expanded in vitro and produced in batches, they have a certain risk of tumor formation in clinical practice.

[0020] Similar to the neurological damage caused by stroke, there are many other diseases characterized by the death and / or dysfunction of forebrain nerve cells, including the aforementioned neurological damage diseases, neurodegenerative diseases, and neurodevelopmental diseases. These diseases are expected to be improved through effective cell replacement therapy. Therefore, there is an urgent need in the art for a cell product that is not derived from fetal tissue and can differentiate into forebrain nerve cells with the desired function, thereby providing compensatory cell therapy for neurodegenerative diseases or neural damage.

[0021] Summary of the Invention

[0022] To address the above issues, the inventors developed forebrain neural progenitor cells (FNPCs) obtained by directed differentiation of pluripotent stem cells, and used them to treat primate (crab-eating macaque) stroke model animals with neural damage caused by ischemia, in order to study the ability of the cells to treat brain neural damage and neurodegenerative diseases.

[0023] The inventors discovered that the forebrain neural progenitor cells, when administered to primate stroke models, were able to survive for extended periods and differentiate into neurons at a high rate, significantly improving the animals' clinical manifestations and providing excellent therapeutic benefits in terms of improved motor behavior, reduced lesion size, and increased animal weight. These results suggest that the forebrain neural progenitor cells hold promising promise for treating related human diseases, such as repairing nerve damage, treating neurodegenerative diseases, and other similar conditions.

[0024] In addition, the inventors have also found that the forebrain neural precursor cells of the present invention are suitable for administration after the acute phase of stroke and can provide a sustained improvement effect. For example, when administered in high doses, the walking speed of the crab-eating macaque monkey, a disease model of permanent middle cerebral artery occlusion (pMCAO), on the 15th day after onset of the disease, after receiving the forebrain neural precursor cell transplantation for 5 months, can be restored to the level of normal healthy monkeys on the ladder, while the walking time of the control group model animals injected with normal saline at this time point is about 2 times that of normal healthy monkeys. This means that the window period for treating stroke that was previously thought to be effective has been greatly expanded unexpectedly. When using the forebrain neural precursor cells or cell preparations of the present invention to treat indications such as stroke and the nerve damage caused by it, the treatment window period is no longer limited to the acute phase, and treatment can also be carried out after the acute phase. Based on the above findings, the present invention has been completed.

[0025] Thus, in the first aspect, the present invention provides a use of a cell colony in the preparation of a medicine, the medicine is used to treat a neurological injury disease, a neurodegenerative disease, a neurodevelopmental disease or other neurological disease associated with the death and / or dysfunction of forebrain nerve cells in a subject, and the cell colony comprises forebrain neural precursor cells (FNPC) with specific developmental characteristics. Preferably, the forebrain neural precursor cells with specific developmental characteristics highly express markers including FOXG1, PAX6 and NESTIN. Preferably, the cell colony is composed of a specific type of cell, and the highest proportion of which is neural precursor cells, preferably forebrain neural precursor cells. Preferably, the cell colony is obtained by culturing human pluripotent stem cells (hPSC) using a specific method.

[0026] In a second aspect, the present invention provides a method for treating a neurological injury disease, a neurodegenerative disease, a neurodevelopmental disease or other neurological disease associated with the death and / or dysfunction of forebrain neural cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a cell population comprising forebrain neural progenitor cells having specific developmental characteristics.

[0027] 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).

[0028] In preferred embodiments of the first and second aspects, in the cell population, at least about 70% or more, preferably 80% or more, more preferably 90% or more of the cells express one or more markers selected from the group consisting of NESTIN, FOXG1, PAX6, and SOX2. Preferably, in the cell population, at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express NESTIN; at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express FOXG1; at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express PAX6; and at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express SOX2.

[0029] In a preferred embodiment of the first and second aspects, in the cell population, at least about 70% or more, preferably 80% or more, more preferably 90% or more of the cells express one or more markers selected from the group consisting of EFNB2, WNT7B, RSPO2 and FEZF2. Preferably, in the cell population, at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express EFNB2; at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express WNT7B; at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express RSPO2; and at least about 70% or more, preferably at least about 80% or more, more preferably 85% or more, and even more preferably 90% or more of the cells express FEZF2.

[0030] In preferred embodiments of the first and second aspects, the proportion of neural precursor cells in the cell population as a whole is ≥ 60%, preferably ≥ 70%, and more preferably ≥ 80%. Preferably, the proportion of pluripotent stem cells in the cell population as a whole is ≤ 3%, preferably ≤ 2%, and more preferably ≤ 1%.

[0031] In a preferred embodiment of the first and second aspects, the cell population can further differentiate into neuronal cells both in vitro and in vivo. Preferably, the neuronal cells produced by differentiation of the cell population have neuroelectrophysiological functions. Preferably, the neuronal cells produced by differentiation of the cell population express MAP2 and / or TUJ1. Preferably, when the cell population is differentiated for 35 days, the neuronal cells in the cell population account for about 80% or more of the total cells. Preferably, the cell population also produces astrocytes by differentiation. Preferably, the astrocytes produced by differentiation of the cell population express GFAP and / or S100β. Preferably, when the cell population is differentiated for 35 days, the astrocytes in the cell population account for about 20% or less of the total cells.

[0032] In specific embodiments of the first and second aspects, the neurological injury disease is a stroke or a disease, condition or symptom associated with a stroke, such as a sequelae or complication. In these embodiments, the cell colony is preferably administered at least 14 days after the onset of a stroke.

[0033] In specific embodiments of the first and second aspects, the neurological injury disease is cerebral palsy or a disease, disorder or symptom associated with cerebral palsy, such as a sequelae or complication.

[0034] In specific embodiments of the first and second aspects, the cell population is in the range of about 1×10 4 to 1×10 10 cells, preferably in the range of about 1×10 5 to 6×10 7 For example, when the subject is a human, the cell population is administered in a dose ranging from 2×10 5 to 2×10 9 cells, preferably in the range of about 2×10 6 to 1.2×10 9 The dose of cells was administered.

[0035] In specific embodiments of the first and second aspects, the cell population is administered to the subject single or multiple times.

[0036] In a specific embodiment of the first and second aspects, said administration is performed by injection. Preferably, said injection is a local injection.

[0037] The advantages of the present invention are at least as follows:

[0038] (1) In terms of repairing damaged neural functions, the use of the forebrain neural progenitor cells of the present invention resulted in significantly faster recovery of cerebral infarction model animals. Specifically, after high-dose cell transplantation, the weight of the treated animals increased faster than that of the control group. Cage-side behavioral changes within 6 weeks showed that the scores of the treated animals (comprehensive neurological function scores) improved faster than those of the treated animals, and their weight increased faster. Seven months after cell administration, the high-dose group of animals reached the ladder walking speed of normal healthy monkeys. In addition, the movement and balance functions of cerebral palsy model animals treated with high-dose forebrain neural progenitor cells of the present invention were significantly restored compared to the model control group animals, with the highest recovery of movement function reaching about 50%. This excellent effect may be due to the fact that the forebrain neural progenitor cells of the present invention can show a differentiation trend similar to the normal human brain development process and can develop and form functional neural networks.

[0039] (2) In terms of 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 a stroke) for which common treatments and drugs are currently applicable to related diseases. Treatment can even be performed 14 days after a stroke. Therefore, it can provide an option for hemiplegic patients who have missed the acute phase treatment or who still have nerve damage after treatment, thus filling the gap in current treatment.

[0040] (3) In terms of source, compared with methods that use neural stem cells 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 advantage of this method is that it can stably produce consistent cell products in multiple batches, obtaining hundreds of human cells in a single batch, and can strictly control quality, which also overcomes the ethical issues of using fetal tissue.

[0041] (4) Compared with neural stem cells, neural progenitor cells are more differentiated cells and can differentiate into functional neural cells in specific brain regions more quickly, efficiently and at a higher ratio, reducing the risk of large-scale 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, thawing and transplantation, making them more suitable as therapeutic products.

[0042] In summary, the method of the present invention enables the treatment of stroke indications in the non-acute phase, potentially filling a gap in the treatment of hemiplegic sequelae of stroke. Furthermore, by using forebrain neural progenitor cells with specific developmental characteristics to form cell and tissue replacements in vivo, it can better ameliorate the functional impairment caused by brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 shows immunofluorescence identification images on days 7 and 14 of differentiation induction, showing the expression of DAPI, MAP2, and FOXG1 with different colors of fluorescence.

[0044] FIG2 shows immunofluorescence identification of cortical neuron cell markers on day 35 of induction of differentiation.

[0045] FIG3 shows immunofluorescence identification of excitatory and inhibitory neuronal cell markers on day 35 of induced differentiation.

[0046] FIG4 shows immunofluorescence identification of presynaptic membrane protein and postsynaptic membrane protein markers on day 35 of differentiation induction.

[0047] FIG5 shows immunofluorescence identification of astrocyte markers and neuronal markers on day 35 of differentiation induction.

[0048] FIG6 shows immunofluorescence identification of cortical neuron cell markers on day 70 of induction of differentiation.

[0049] FIG7 shows immunofluorescence identification of excitatory and inhibitory neuronal cell markers on day 70 of induction of differentiation.

[0050] FIG8 shows immunofluorescence identification of astrocyte markers and neuronal markers on day 70 of differentiation induction.

[0051] FIG9 shows immunofluorescence identification of oligodendrocyte markers on day 70 of differentiation induction.

[0052] FIG10 shows the electrophysiological images of induced differentiated cortical neurons.

[0053] FIG11 is a diagram showing the experimental design of hNPC01 treatment and testing of cerebral infarction model animals.

[0054] FIG12 shows the weight gain rate of cerebral infarction model animals before and after hNPC01 treatment (weeks 0 to 28), where the X-axis represents days.

[0055] Figure 13 shows the change in behavioral scores of the ipsilateral side of the permanent cerebral infarction model animals treated with different doses (high, medium, and low) of hNPC01 (weeks 0 to 28). The percentage change in behavioral scores at different observation times relative to the score on day 2 after treatment (i.e., time 0) is shown on the abscissa, representing day 2 after treatment, or day 17 after the stroke.

[0056] FIG14 shows individual data of neurological functional behavioral scores on the affected side of cerebral infarction model animals treated with hNPC01 (from the previous day to 4 weeks).

[0057] FIG15 shows the average time (s) of a single ladder walking in cerebral infarction model animals after hNPC01 treatment (5-7 months).

[0058] FIG16 shows brain MRI images of cerebral infarction model animals after hNPC01 treatment (D183), with each small image showing a different cross-section of the same animal.

[0059] FIG17 shows the brain injury volume (%) of cerebral infarction model animals after hNPC01 treatment.

[0060] FIG18 shows bright field images of representative brain tissues and brain slices of cerebral infarction model animals after hNPC01 treatment (7 months).

[0061] FIG19 shows immunofluorescence staining of the brain of cerebral infarction model animals after hNPC01 treatment (7 months).

[0062] FIG20 shows the results of the rotarod test in cerebral palsy model animals after hNPC01 treatment.

[0063] FIG21A and FIG21B show the results of a balance beam test on cerebral palsy model animals over a period of time after hNPC01 treatment, wherein FIG21A shows the passing time, and FIG21B shows the number of falls.

[0064] FIG22 shows the TTC staining results of cerebral palsy model animals after hNPC01 treatment.

[0065] FIG23 shows the results of comparative analysis of cellular composition between hNPC01 and fetal forebrain samples.

[0066] Detailed Description of the Invention

[0067] Unless otherwise specified herein, the meanings of all technical and scientific terms used herein are to be interpreted according to the common understanding by one of ordinary skill in the art to which this invention belongs.

[0068] “Or” has the same meaning as “and / or” herein unless expressly stated otherwise.

[0069] In the context of the present invention, unless otherwise specified, "comprising", "including" and "containing" should be understood to mean including the listed elements, such as a component, a feature, a step or a group thereof, but not excluding any other elements, such as other components, properties and steps. When used herein, the term "comprising" or any variation thereof can be replaced with "containing", "including" or "having" or synonymous variations. In certain embodiments, "comprising" also includes the case of "consisting of".

[0070] Cell therapy agents

[0071] One of the characteristics of the present invention is that it uses special cells, namely forebrain neural precursor cells (FNPCs), or a cell population mainly comprising these cells as a cell therapy agent. Unless otherwise specified, the therapeutic cells used in the present invention are all human cells.

[0072] In a preferred embodiment, the forebrain neural precursor cells can be obtained by using human pluripotent stem cells such as ESCs or iPSCs as the starting material for directed differentiation and inducing directed differentiation.

[0073] In a preferred embodiment, the cell population of the present invention is obtained by a specific differentiation method. To help understand the differentiation method, the cells at various stages involved in differentiation are defined below.

[0074] The term "cell population" refers to a collection of cells, which may comprise one or more cells.

[0075] The term "pluripotent stem cells" or its abbreviation "PSC" refers to a type of cell existing in animals and humans that has multidirectional differentiation potential and self-renewal ability.

[0076] The term "embryonic stem cells" or "ESCs" refers to self-renewing pluripotent stem cells derived from the inner cell mass of the blastocyst that develops from a fertilized egg and has the potential to develop into all germ layer tissues of the human body. It should be noted that embryonic stem cells do not refer to fetal stem cells.

[0077] The term "induced pluripotent stem cells" or "iPSCs" refers to self-renewing pluripotent stem cells derived from human somatic cells that have the potential to develop into tissues of all germ layers of the human body.

[0078] The term "neuronal cells" refers to all related cells that make up the human nervous system.

[0079] The term "neural precursor cells (NPCs)" as used herein refers to a type of cells that can differentiate into cells of certain brain regions or neural lineages (including neurons and glial cells, etc.) and have a certain self-renewal ability.

[0080] The term "forebrain neural precursor cells (FNPC)" as used herein refers to neural precursor cells that can differentiate into neural cells in a specific brain region, i.e., neural cells in the forebrain (mainly including the cerebral cortex, etc.). The difference between NPCs that can differentiate into neural cells in a specific brain region and general NPCs is that the former express markers of a specific brain region, while general NPCs do not express markers of specific brain regions such as the forebrain. For example, forebrain NPCs mainly express markers such as FOXG1 and PAX6, and midbrain NPCs mainly express markers such as FOXA2 and LMX1A. In addition, after continuing to differentiate downward, forebrain NPCs will mainly differentiate into neurons and glial cells in the 6 layers of the cerebral cortex, and midbrain NPCs will mainly differentiate into midbrain dopamine neurons.

[0081] The term "embryoid bodies" or "EBs" as used herein refers to cell aggregates formed by three-dimensional culture of ESCs and iPSCs.

[0082] The terms "rosette-like neural stem cell-derived neural aggregates," "rosette-like neural aggregates," or "RONA" are used interchangeably herein to refer to a special type of aggregate of neural stem cells derived from ESCs or iPSCs that spontaneously organizes into highly compact three-dimensional neural aggregates.

[0083] The term "neurosphere" or "neurosphere" as used herein refers to a spherical aggregate of neural cells formed by suspension culture. Neurospheres are a heterogeneous population composed of different types of neural cells, including neural stem cells, neural progenitor cells, and some differentiated neural cells.

[0084] "ECM" is short for extracellular matrix and is mainly composed of proteins such as collagen, elastin and / or laminin.

[0085] "Cell preparation" refers to a preparation with cells as the main active ingredient, which allows the addition of a certain amount of pharmaceutically acceptable excipients. For example, the cell preparation can be a cell injection solution, specifically a sodium chloride injection solution for cells. In some cases, "cell preparation" and "cell population" have the same meaning. For example, a cell population produced by a specific process can be used directly for treatment. The cell population can be a cell culture obtained by culturing the method of the present invention, or a derivative cell product obtained by further processing the culture, such as purification or culturing.

[0086] In a preferred embodiment, at least 70% or more of the cells in the cell population of the present invention are forebrain neural precursor cells, preferably at least 75% or more, more preferably at least 80% or more, even more preferably at least 85% or more, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0087] 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 into specific neural cell types (such as cortical neurons, etc.) with specific biomarkers and electrophysiological activities.

[0088] For example, the forebrain neural progenitor cells of the present invention can exhibit typical neural progenitor cell morphology, including uniform morphology, a translucent spindle shape, and some cells can be seen aggregated to form a rosette-shaped arrangement.

[0089] For example, the identity of cells in the cell population of the present invention can be determined by neural stem cell markers and forebrain neural precursor cell markers. The markers can be selected from the group consisting of NESTIN, SOX2, DCX, FOXG1, and PAX6. In a preferred embodiment, a majority of cells in the cell population or cell preparation of the present invention express FOXG1, PAX6, and NESTIN. In a preferred embodiment, a majority of cells in the cell population or cell preparation of the present invention express FOXG1, PAX6, NESTIN, and SOX2. In preferred embodiments, 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 in the cell population of the present invention 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, at least about 70% or more, preferably about 80% or more of the cells in the cell population of the present invention simultaneously express NESTIN, FOXG1, PAX6, and SOX2. In a more preferred embodiment, at least 70% or more, preferably about 80% or more of the cells in the cell population of the present invention simultaneously express NESTIN, FOXG1, PAX6, and SOX2. The expression of these markers can be determined by conventional methods in the art, such as flow cytometry.

[0090] The abbreviation "FOXG1" or "Foxg1" as used herein stands for "forkhead box G1", which is one of the earliest transcription factors expressed during human brain development, inducing the development of the telencephalon into several key structures, including the cerebral cortex.

[0091] The abbreviation "PAX6" as used herein stands for "Paired Box 6," a transcription factor important for determining human neuroectodermal cell fate and forebrain development.

[0092] The abbreviation "NESTIN" as used herein stands for "Nestin protein", which is a class VI IF protein and a marker for neural stem cells.

[0093] The abbreviation "SOX2" as used herein stands for "Sex determining region Y-box 2", which is an important transcription factor for maintaining pluripotency and self-renewal, and is also a neural stem cell marker.

[0094] The abbreviation "DCX" as used herein stands for "Doublecortin," a marker for neural stem cells and neural precursor cells.

[0095] In a preferred embodiment, the cell population or cell preparation of the present invention expresses one or more, preferably all, characteristic markers selected from the group consisting of EFNB2, WNT7B, RSPO2, and FEZF2. "Characteristic markers" means that the co-expression of these markers is a gene expression feature that distinguishes the cell population of the present invention from other products of different developmental stages, cell types, and / or compositions. In a preferred embodiment, the cell population or cell preparation of the present invention simultaneously expresses EFNB2, WNT7B, RSPO2, and FEZF2. In a specific embodiment, the expression of the markers can be determined by conventional methods in the art, such as single-cell transcriptome sequencing such as scRNA-seq.

[0096] The abbreviation "EFNB2" as used herein stands for Ephrin B2, a transmembrane receptor on the cell surface that is an important regulator of angiogenesis.

[0097] The abbreviation "WNT7B" as used herein stands for "Wnt Family Member 7B."

[0098] The abbreviation "RSPO2" as used herein stands for "R-spondin 2."

[0099] The abbreviation "FEZF2" as used herein stands for "FEZ Family Zinc Finger 2."

[0100] In some embodiments, the cell population of the present invention, in addition to primarily comprising neural progenitor cells, particularly forebrain neural progenitor cells, further comprises a plurality of cells of a specific type, including one or more, preferably all, selected from the group consisting of immature neurons, GABAergic neurons, glutamatergic neurons, ependymal cells, and pericytes. Preferably, the proportion of such cells does not exceed 30% of the total cell number, preferably does not exceed 20%, more preferably does not exceed 10%, and even more preferably does not exceed 5%.

[0101] In some embodiments, the cell population of the present invention is derived from human pluripotent stem cells, but the proportion of pluripotent stem cells in the cell population does not exceed 3% of the total cell number, preferably does not exceed 2%, and more preferably does not exceed 1%.

[0102] For example, the forebrain neural precursor cells of the present invention or the cell colony comprising the forebrain neural precursor cells can further differentiate into neuronal cells both in vitro and in vivo, preferably differentiate into a cell colony mainly composed of neuronal cells. When the differentiation is carried out in vitro or in vivo, particularly in vivo, the differentiation is spontaneous differentiation, i.e., no additional reagents such as inducers need to be added. In addition, the forebrain neural precursor cells or the cell colony comprising the forebrain neural precursor cells used as cell therapy agents of the present invention exhibit a differentiation trend similar to that of normal human brain development in terms of differentiation process and differentiation results.

[0103] For example, the cell population used as a cell therapy agent of the present invention can express neuron-specific markers after differentiation. For example, the neuronal cells generated by differentiation of the cell population express MAP2 and / or TUJ1. Both MAP2 and TUJ1 are markers of mature neurons. Preferably, as determined by the expression of MAP2 and / or TUJ1, the cell population of the present invention comprises more than half of the total cells in the cell population at approximately 35 days of differentiation. For example, at approximately 35 days of differentiation, the neuronal cells in the cell population comprise more than about 50%, more than 60%, more than 70%, or more than 80% of the total cells.

[0104] For example, the cell population used as a cell therapy agent of the present invention comprises cells expressing different cortical-specific neuronal markers after differentiation. + Neuronal cells include those expressing BRN2, a marker of cerebral cortex layers II-IV. For example, MAP2 is produced by differentiation in the cell population of the present invention. +Among the neuronal cells, there are those expressing the cerebral cortex layer V-VI marker CTIP. These cortex-specific neuronal cells can appear around 35 days after differentiation. This means that the therapeutic cells or cell colonies of the present invention are capable of differentiating into different neural cell types similar to the normal structure of the human brain.

[0105] More importantly, the neurons generated by differentiation of the cells or cell populations used as cell therapeutic agents of the present invention exhibit neuroelectrophysiological functions. For example, when electrophysiological function testing is performed around day 25 after in vitro differentiation, the differentiated cell population exhibits one or more characteristics selected from the group consisting of: number of active electrodes ≥ 5, weighted average discharge frequency (Hz) ≥ 0.5, and / or clustered discharge frequency (Hz) ≥ 0.05.

[0106] Preferably, around day 35 after in vitro differentiation, the cell population is differentiated to produce cells that co-express synapsin and PSD95. The co-expression of synapsin and PSD95 indicates that synaptic structures have been successfully established between neurons, forming the basis for information exchange between neurons.

[0107] Preferably, on the 35th day after in vitro differentiation, the cell population comprises neurons expressing VGLUT (excitatory neuron marker) and VGAT (inhibitory neuron marker), wherein the number of neurons expressing VGLUT is significantly higher than the number of neurons expressing VGAT. This distribution of numbers is similar to the phenomenon in the normal brain, i.e., VGLUT is expressed in the normal brain. + Neurons are overwhelmingly dominant in the cerebral cortex.

[0108] During brain development, early neuronal cells will have an absolute advantage, but neurons will no longer divide after maturation. On the other hand, astrocytes will continue to be produced, so that the proportion of astrocytes gradually increases. Therefore, preferably, the cell population used as a cell therapy agent in the present invention also produces astrocytes through differentiation. Preferably, the astrocytes produced by differentiation of the cell population express GFAP and / or S100β. GFAP and S100β are both astrocyte-specific markers. Preferably, the astrocytes produced by differentiation of the cell population account for less than about 20% of the cells produced after about 35 days of differentiation.

[0109] In specific embodiments, the cell population used as a cell therapy agent of the present invention can differentiate in vitro within approximately 35 days into a cell population composed primarily of neurons (over 80%) that highly express MAP2 and / or TUJ1, with a minority of astrocytes (less than 20%) that highly express GFAP / S100β, and exhibiting corresponding neuroelectrophysiological functions. Upon completion of differentiation in vivo, the cell population comprising forebrain neural progenitor cells of the present invention can differentiate into a cell population primarily composed of neurons within approximately 30 to 90 days.

[0110] In a preferred embodiment, the cell population of the present invention is capable of forming neural circuit reconstruction in the administered area after being administered as a cell therapeutic agent to a subject's nerve injury and adjacent tissue areas.

[0111] In a preferred embodiment, the cell population of the present invention can secrete cytokines and microRNA after transplantation. The cytokines can play a role in neuroprotection and promoting angiogenesis, which helps to achieve the effect of nerve repair.

[0112] Cell preparation

[0113] The following provides exemplary methods for preparing forebrain neural precursor cells and cell populations comprising them. Those skilled in the art will appreciate that this does not necessarily mean that the specific steps of the methods cannot be adjusted or modified, and that any intermediate step can be initiated. The present invention is not limited to requiring completion of each step of the methods listed below, as long as a cell product with comparable properties and composition can be obtained.

[0114] In a specific embodiment, the forebrain neural progenitor cells of the present invention or a cell population comprising the forebrain neural progenitor cells are generated from human pluripotent stem cells, such as ESCs or iPSCs, via directed differentiation, and the method comprises the following steps:

[0115] (1) Culturing and expanding ESCs or iPSCs in human pluripotent stem cell culture medium (hPSC culture medium);

[0116] (2) digesting the ESCs or iPSCs in step (1) and culturing them in suspension in EB medium to form embryoid bodies (EBs);

[0117] (3) culturing the EBs obtained in step (2) with neural induction medium (RONA) to form rosette neural aggregates (RONAs);

[0118] (4) culturing the RONAs formed in step (3) and culturing them in suspension to form neurospheres, wherein the neurospheres contain forebrain neural precursor cells (FNPCs);

[0119] (5) optionally, further culturing the neurospheres formed in step (4) using an NPC medium for passage expansion; and

[0120] (6) Optionally, harvest the FNPCs formed in step (5).

[0121] Optionally, before step (1), the ESCs or iPSCs are maintained and expanded to 80% to 90% confluence, preferably on a culture surface coated with laminin, which may be a culture plate.

[0122] In the above step (1), ESCs or iPSCs can be dispersed into single cells or cell aggregates, for example, by digestive enzymes or by mechanical means, and then inoculated into hPSC culture medium to induce EBs formation. The ESCs or iPSCs can be obtained from commercial sources or prepared by any known method. It will be understood by those skilled in the art that the ESCs are ESCs formed from the inner layer cells of the blastocyst within 5-7 days of development of the fertilized egg without in vivo development.

[0123] More than one, for example, two, three or more EB culture media of the present invention may be used in step (2).

[0124] More than one, for example two, three or more RONAs of the present invention may be used in step (3).

[0125] Step (4) can use the same culture medium as the previous step or the next step. Step (4) can be performed using RONA or NPC culture medium. In one embodiment, the culture medium used in step (4) is the same as that used in step (3), specifically RONA.

[0126] Preferably, the culture in steps (3) and (5) is adherent culture, for example, it can be performed on a culture surface coated with an extracellular matrix (ECM). In step (5), the neurospheres can be dispersed using digestive enzymes or mechanical means.

[0127] In the above preparation method, EB culture medium, RONA culture medium and NPC culture medium can all contain basal culture medium and additives. Each culture medium can be independently clinical grade, preferably cGMP grade or CTS grade. TM level.

[0128] Exemplary culture media used in each step are described in detail below.

[0129] A) hPSC culture medium

[0130] hPSC culture medium can be selected from hPSC XF Medium, Essential 8 Medium, Basic03 culture medium, StemMACS TM iPS-Brew medium, ACF medium, TeSR TM -AOF medium or TeSR2 medium, preferably hPSC XF Medium.

[0131] Optionally, a ROCK inhibitor can be added to the hPSC culture medium. In a preferred embodiment, the ROCK inhibitor is Y-27632. Preferably, a ROCK inhibitor such as Y-27632 is added at a concentration of about 10 μM.

[0132] B) EB culture medium

[0133] EB culture medium contains factors that promote the differentiation of cells into neuroectodermal cells such as neurons, which is critical for generating a cell population dominated by precranial neural progenitor cells.

[0134] EB culture medium may contain:

[0135] (i) a basal medium selected from a) to c):

[0136] a) Single KnockOut TM DMEM / F12 medium,

[0137] b) DMEM / F12 medium and Neurobasal TM Combination of culture medium,

[0138] c)KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and

[0139] (ii) an additive comprising or consisting of d) or e):

[0140] d) N-2 Supplement and GlutaMAX TM -I additives;

[0141] e) N-2 additives, GlutaMAX TM -I Supplement and B-27 without Vitamin A TM Additive (B-27 TM Supplement,minus vitamin A).

[0142] Optionally, the EB culture medium comprises (iii) an inhibitor. The inhibitor 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 culture 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 culture 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 it.

[0143] In a specific embodiment, the EB culture medium comprises:

[0144] (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combination of culture media;

[0145] (ii) Supplements comprising N-2 Supplements and GlutaMAX TM - a combination of additives; and

[0146] (iii) SB431542, Noggin and Dorsomorphin.

[0147] Preferably, when two basal culture media are used, the two culture media are combined in a volume ratio of 1:1.

[0148] Preferably, the additive is added at a 1x concentration.

[0149] For example, Noggin is added at a concentration of 25-100 ng / mL, preferably 40-60 ng / mL, more preferably about 50 ng / mL.

[0150] For example, Dorsomorphin is added at a concentration of 0.5-2 μM, preferably 0.75-1.5 μM, more preferably 1 μM.

[0151] For example, SB431542 is added at a concentration of 5-15 μM, preferably 7-13 μM, more preferably 8-12 μM, and most preferably 10 μM.

[0152] C) RONA medium

[0153] RONA culture medium contains factors that promote the differentiation of cells into prefrontal neural cells, which is critical for generating a cell population dominated by prefrontal neural progenitor cells.

[0154] RONA medium may contain:

[0155] (i) a basal medium selected from a) to c):

[0156] a) Single KnockOut TM DMEM / F12 medium,

[0157] b) DMEM / F12 medium and Neurobasal TM Combination of culture medium,

[0158] c)KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and

[0159] (ii) an additive comprising or consisting of d) or e):

[0160] d) N-2 Supplement and GlutaMAX TM -I additives;

[0161] e) N-2 additives, GlutaMAX TM -I Supplement and B-27 without Vitamin A TM additive.

[0162] In a specific embodiment, RONA medium comprising the following components is used:

[0163] (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and

[0164] (ii) Supplements comprising N-2 Supplements and GlutaMAX TM -I Combination of additives.

[0165] In a specific embodiment, RONA medium comprising the following components is used:

[0166] (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and

[0167] (ii) additives comprising N-2 additive, B-27 TM Additive (B-27 TM Supplement,XenoFree,minus vitamin A) and GlutaMAX TM -I Combination of additives.

[0168] In a specific embodiment, more than one RONA culture medium is used. For example, in the first stage of RONA differentiation, a first RONA culture medium is used that comprises the following components: (i) a basal medium that is a KnockOut TM DMEM / F12 medium and Neurobasal TM A combination of a culture medium; and (ii) a supplement comprising N-2 supplement and GlutaMAX TM -I supplement combination; and in the second stage of RONA differentiation using a second RONA medium comprising the following components: (i) basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM and (ii) an additive comprising N-2 additive, B-27 TM Additive (B-27 TM Supplement,XenoFree,minus vitamin A) and GlutaMAX TM Preferably, the first and second stages of RONA differentiation last for about 5-14 days, preferably about 7 days, respectively.

[0169] Preferably, when two basal culture media are used, the two culture media are combined in a volume ratio of 1:1.

[0170] Preferably, the additive is added at a 1× concentration.

[0171] D) NPC culture medium

[0172] The basal medium of NPC culture medium can be Neurobasal TM culture medium, and the additive of the NPC culture medium is (a) GlutaMAX TM -I supplement, and (b) B-27 without vitamin A TM additive.

[0173] Optionally, the NPC culture 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 ,O2’ -Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (DB-cAMP).

[0174] The BDNF may be animal-free recombinant BDNF or GMP-grade recombinant BDNF, and / or the GDNF may be animal-free recombinant GDNF or GMP-grade recombinant GDNF.

[0175] In a specific embodiment, an NPC culture medium comprising the following components is used:

[0176] (i) Basic culture medium, which is Neurobasal TM culture medium;

[0177] (ii) an additive comprising B-27 TM Additive (B-27 TM Supplement,XenoFree,minus vitamin A) and GlutaMAX TM - a combination of additives; and

[0178] (iii) BDNF, GDNF, L-ascorbic acid and N 6 ,O 2’ -Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (DB-cAMP).

[0179] Preferably, when two basal culture media are used, the two culture media are combined in a volume ratio of 1:1.

[0180] Preferably, the additive is added at a 1x concentration.

[0181] For example, BDNF is added at a concentration of about 5-50 ng / mL, preferably 10-30 ng / mL, more preferably about 20 ng / mL.

[0182] For example, GDNF is added at a concentration of about 5-50 ng / mL, preferably 10-30 ng / mL, more preferably about 20 ng / mL.

[0183] For example, L-ascorbic acid is added at a concentration of 0.02-2 mM, preferably 0.05-0.5 mM, more preferably about 0.1-0.3 mM, and most preferably about 0.2 mM.

[0184] For example, DB-cAMP is added at a concentration of 0.1-5 mM, preferably 0.2-2.5 mM, more preferably about 0.3-1 mM, and most preferably about 0.5 mM.

[0185] In the preferred preparation method described above, by using specific neural ectoderm induction factors during EB culture and specific factors that promote forebrain neural cell differentiation during RONA formation, a cell population dominated by forebrain neural progenitor cells is obtained. In fact, in the preferred preparation method described above, the neurospheres formed already contain forebrain neural progenitor cells that express specific markers. Therefore, it is understood that after neurosphere formation, other methods can also be used to passage the forebrain neural progenitor cells to maintain and amplify them and / or promote their differentiation into neurons.

[0186] Treatment

[0187] The term "subject" in the present invention refers to an animal to be administered, preferably a vertebrate, more preferably a mammal, such as a rodent, for example, a mouse, a rat; most preferably a primate, such as a monkey, including a cynomolgus monkey, a rhesus monkey; specifically a human.

[0188] The term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical composition, such as a pharmaceutical composition (such as a cell preparation or cell population) comprising human forebrain neural precursor cells of the present invention, that is sufficient to produce a desired function when delivered to a subject in need thereof. The desired function may include delaying the manifestation of a condition, preventing or delaying the progression of a condition, or alleviating at least one effect of a condition.

[0189] The term "treating" refers to the improvement, alleviation or elimination of disease symptoms (ie, cure). In some cases, treatment includes prophylactic treatment.

[0190] When used to repair nerve damage or treat neurodegenerative diseases such as neurodegeneration, the forebrain neural progenitor cells or cell preparations of the present invention can be transplanted 14 days after the subject suffers nerve damage. For example, when the subject is a stroke patient, the cell population of the present invention can be administered at least 14 days after the stroke.

[0191] The forebrain neural precursor cells of the present invention or the cell colony comprising the forebrain neural precursor cells can be administered via a transcranial route (e.g., intraparenchymal injection), a transventricular route (e.g., by intrathecal injection, intraventricular catheterization, intraventricular injection, etc.). In a preferred embodiment, the neural precursor cells of the present invention or the cell colony comprising the neural cells can be locally injected, e.g., locally injected into an infarcted or damaged brain region. For example, injection is performed in the motor cortex and / or basal ganglia region surrounding the infarct. For example, local injection can be performed with the assistance of an imaging device, e.g., injected under a 3D Lab navigation system.

[0192] The administration or transplantation of the cells or cell colonies, for example, by the administration or transplantation of injection, can be carried out by an injection device such as a syringe. The injection device can include a needle, for example a puncture needle, or any suitable needle known 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 position. The length of the needle can be any length between about 1-25 cm. The size thickness of the needle etc. can be selected according to various factors, including the volume and formula of cell number, cell preparation and the position into which the needle is inserted.

[0193] 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 having an appropriate osmotic pressure for injection. The buffer can be physiological saline, i.e., 0.9% sodium chloride solution.

[0194] When the total volume of the cell suspension is large, such as during brain parenchymal injection, injections can be performed at multiple points at different depths through multiple needle channels, such as 2, 3, 4, 5 or more needle channels, each containing a portion of the total volume of the cell suspension.

[0195] The forebrain neural progenitor cells of the present invention or a cell population comprising the forebrain neural progenitor cells can be injected once or multiple times. Multiple injections can be performed, for example, two, three, or more times. The number of injections can be determined based on the improvement effect. The intervals between multiple injections can be one or more days, one or more weeks, one or more months, or even longer.

[0196] In a preferred embodiment, the forebrain neural precursor cells of the present invention or a cell population comprising the forebrain neural precursor cells are injected through a single point or multiple points in the skull. For example, injections can be performed through 1 to 3 needle tracks with different needle insertion angles in a single cranial hole. For example, during injection, cells can be injected into 1 to 10 injection sites of different depths for each needle track. This can be accomplished by inserting the injection device and then stopping at positions of different depths during the withdrawal process and injecting. Alternatively, the injection device can be inserted and then slowly and continuously injected during the withdrawal process, so that the cells are left on a path where the needle is withdrawn.

[0197] The single administration dose of the cell population comprising the forebrain neural precursor cells of the present invention can be about 1×10 4 to 1×10 10 cells, for example, 1×10 4 cells, 1×10 5 cells, 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, 1×1010 In a preferred embodiment, a single dose can be about 1×10 5 cells to approximately 1×10 7 cells, for example, approximately 1×10 5 cells, 2×10 5 cells, 3×10 5 cells, 4×10 5 cells, 5×10 5 cells, 6×10 5 cells, 7×10 5 cells, 8×10 5 cells, 9×10 5 cells, 1×10 6 cells, 2×10 6 cells, 3×10 6 cells, 4×10 6 cells, 5×10 6 cells, 6×10 6 cells, 7×10 6 cells, 8×10 6 cells, 9×10 6 In some embodiments, when the subject is a human, the cell population is in the range of 2×10 5 to 2×10 9 cells, preferably in the range of about 2×10 6 to 1.2×10 9 In a single administration, the amount of the forebrain neural precursor cells of the present invention should be at least 70%, preferably at least 80% of the total cell amount, that is, at least 70%, preferably at least 80% of the total cells in a single administration express FOXG1, PAX6 and NESTIN.

[0198] Applicable diseases / efficacy evaluation indicators

[0199] After treatment with the forebrain neural progenitor cells or a cell population comprising forebrain neural progenitor cells of the present invention, the effect of the treatment can be evaluated using various indicators.

[0200] For example, after transplantation or administration of the cell population or cell preparation of the present invention, within 1 month to 1 year, there may be improvement in one or more of the following aspects: neurological function and motor function scores used to evaluate stroke sequelae, such as the Modified Rankin score, FMMS score or NIHSS score; language function; cognitive function; specific symptoms.

[0201] Preferably, the improvement is significant relative to that before transplantation or administration. For example, the improvement can be reflected in a quantitative score of more than 10%, preferably at least 20%, and / or reaching or approaching the corresponding score of a healthy individual. For example, the improvement of the specific symptoms can be manifested in one or more of the following aspects: improvement from a state of unilateral hand muscle tension and clenching a fist to normal control of hand activities, such as writing, using tableware, etc.; patients with weakness or inflexibility on one side of the leg are improved to be able to walk and / or run freely; bedridden patients with cognitive impairment are improved to be able to recognize family members and / or communicate, etc.; and / or patients with aphasia are improved to be able to speak and communicate, etc.

[0202] For example, the age range of patients eligible for treatment could be 20 to 85 years old. For example, the range of cerebral infarction indicators could include cortical / subcortical ischemic infarction caused by unilateral middle cerebral artery / lenticulostriate artery infarction, an ischemic lesion no less than 3 cm, and a FMMS score less than 55 on two preoperative assessments three weeks apart.

[0203] use

[0204] The forebrain neural progenitor cells of the present invention or a cell population comprising the forebrain neural progenitor cells can be used alone or in combination with other drugs to treat neural damage diseases, neurodegenerative diseases, neurodevelopmental diseases or other neurological diseases, disorders or conditions associated with the death and / or dysfunction of forebrain neural cells.

[0205] The term "brain injury" in this article refers to damage to brain tissue, whether caused by internal or external forces. This includes injuries to brain tissue, such as contusions, as defined under "intracranial injury," as well as damage caused by ischemia, hypoxia, and reperfusion. Brain injury can lead to various cognitive impairments and symptoms, such as impairments in attention, memory, or movement.

[0206] The term "neurovascular disease" herein specifically refers to brain neurological damage, including brain damage caused by cerebral ischemia, cerebral hemorrhage or physical injury.

[0207] The term "neurodegenerative disease" refers to a disease characterized by the gradual loss of function and death of certain neurons over the course of the disease. In specific embodiments, the neurodegenerative disease includes amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Alzheimer's disease (AD), etc.

[0208] The term "neurodevelopmental disease" as used herein refers to diseases resulting from abnormal development of brain tissue and cells, particularly forebrain tissue and cells.

[0209] The term "other neurological diseases associated with death and / or dysfunction of forebrain nerve cells" herein refers to other neurological diseases caused by damage to the number and / or function of forebrain nerve cells.

[0210] The term "stroke" in the context of this invention should be understood broadly to include both ischemic and hemorrhagic strokes. Ischemic stroke, also known as cerebral infarction or cerebral infarction, is caused by various factors that disrupt the blood supply to a localized area of ​​brain tissue, leading to acute blood flow interruption, ischemia, hypoxia, softening, and necrosis of brain tissue, which rapidly manifests as clinically relevant neurological deficits such as hemiplegia and aphasia. Hemorrhagic stroke, also known as cerebral hemorrhage, is caused by a variety of factors.

[0211] The term "cerebral palsy" in the context of the present invention refers to non-progressive brain damage occurring in the perinatal period (before birth and neonatal period), which is mainly manifested by movement disorders, mental retardation, epileptic seizures, etc.

[0212] 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 neurological injury disease. The neurological injury disease can be selected from the group consisting of a disease associated with ischemic brain injury, a disease associated with hemorrhagic brain injury, and a disease associated with brain injury caused by trauma.

[0213] It is generally believed in the art that treatment during the acute phase of nerve damage can produce significantly better results. However, even if the patient receives timely treatment in the acute phase, he or she may still be disabled due to nerve damage, leaving a permanent disability. The cell population of the present invention can be used not only for subjects in the acute phase of nerve damage diseases, but also for subjects after the acute phase of nerve damage diseases, such as those in the stable phase. Without wishing to be limited by theory, the cell population of the present invention can achieve a therapeutic effect for subjects in the acute phase through cytokine release, and for subjects in the stable phase, a therapeutic effect can be achieved through functions such as neural circuit reconstruction achieved by cell replacement.

[0214] In specific embodiments, the ischemic brain injury is ischemic stroke, also known as cerebral infarction. For example, the subject suffers from or has experienced total anterior circulation infarction, partial anterior circulation infarction, posterior circulation infarction, or lacunar infarction; or the patient suffers from or has experienced cerebral ischemia caused by large artery atherosclerosis, cardioembolism, and arteriolar occlusion. The disease associated with ischemic brain injury can be stroke or a condition related to stroke, such as complications or sequelae.

[0215] In a specific embodiment, the hemorrhagic brain injury is hemorrhagic stroke, which may also be called cerebral hemorrhage. The disease associated with hemorrhagic brain injury may be cerebral stroke or a condition associated with cerebral stroke, such as complications and sequelae.

[0216] The symptoms associated with stroke may include, but are not limited to, hemiplegia, hemilimb disorder, limb numbness, facial muscle weakness, numbness, aphasia, slurred speech, and hemianopsia.

[0217] Brain damage caused by trauma, such as craniocerebral injury caused by impact injury, puncture injury, etc., or related symptoms such as complications and sequelae.

[0218] In a preferred embodiment, the forebrain neural progenitor cells of the present invention are particularly suitable for treating ischemic brain injury, particularly ischemic stroke or its related conditions, such as complications and sequelae. In this case, the forebrain neural progenitor cells of the present invention are suitable for administration after the acute phase of stroke, for example, 14 days after the onset of stroke.

[0219] 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 cerebral palsy. The cell therapy of the present invention can improve the motor ability and / or cognitive ability of a subject suffering from cerebral palsy.

[0220] In a specific embodiment, the forebrain neural precursor cells of the present invention or a cell population comprising the forebrain neural precursor cells can be used to treat neurodegenerative diseases. Neurodegenerative diseases are diseases 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 are those involving 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).

[0221] In a specific embodiment, the forebrain neural precursor cells of the present invention or a cell colony comprising the forebrain neural precursor cells can be used to treat neurodevelopmental diseases. Neurodevelopmental diseases may also be referred to as neurodevelopmental disorders. Neurodevelopmental diseases suitable for treatment with the cells or cell colonies of the present invention are those involving forebrain neural cells, particularly forebrain neuronal function loss, such as epilepsy, autism, cerebral palsy.

[0222] The forebrain neural progenitor cells of the present invention or cell populations comprising the forebrain neural progenitor cells can be used for research purposes, including clinical and non-clinical studies. For example, the research can be conducted to evaluate animal models of the above-mentioned diseases. For example, the research can be conducted to investigate the therapeutic mechanisms of forebrain neural progenitor cells in the above-mentioned diseases, such as injury repair mechanisms. Example

[0223] The following examples illustrate the embodiments of the present invention. Those skilled in the art should understand that this does not mean that the present invention must be implemented by the following specific methods. The scope of protection of the present invention shall be based on the claims.

[0224] Example 1. Preparation of cells

[0225] In this example, a cell preparation hNPC01 consisting mainly of forebrain neural progenitor cells was prepared from human iPSCs (induced pluripotent stem cells) through directed differentiation (iPSC→EB→RONA→neurosphere→forebrain neural progenitor cells (FNPC)).

[0226] It should be understood that this does not mean that one must start with iPSCs to practice the present invention.

[0227] 1. iPSC Culture

[0228] About 2×10 5 The cell suspension was seeded into 6-well cell culture plates coated with MX521 Matrigel in NutriStem hPSC XF Xeno-free Medium (containing 10 μM Y-27632) at a density of 10 viable cells / well. The cells were cultured in a 37°C, 5% CO2 incubator until 90% confluence was achieved.

[0229] 2. Embryoid Body (EB) Differentiation Culture

[0230] Prepare embryoid body differentiation medium EB-M according to the table below.

[0231] The operational procedure for EB differentiation was performed as follows.

[0232] (1) Inoculation of embryoid bodies (EBs)

[0233] Remove the old medium and replace with DPBS (without Ca 2+ , Mg 2+ ) Wash the cells. Add 1 mL of Cell Therapy Systems TrypLE TM Digest with Select Enzyme until cells are largely detached. Add 3 mL of culture medium to each well to terminate digestion and transfer the cell suspension to a centrifuge tube.

[0234] Centrifuge at 200 g for 3 minutes, discard the supernatant, resuspend the cells in NutriStem hPSC XF Xeno-free medium containing 10 μM Y-27632, and count the cells.

[0235] About 0.3×10 5 The cell suspension was seeded into a low-attachment 96-well cell culture plate at a density of 10 viable cells / mL and cultured in a 37°C, CO2 incubator for 48 hours.

[0236] (2) Embryoid body (EB) medium change

[0237] On day 2 of EB differentiation, transfer EBs from the low-attachment 96-well cell culture plate to a low-attachment 6-well cell culture plate, transferring 32 EBs to each well of the 6-well cell culture plate. On days 2-6 of EB differentiation, discard the culture medium in each well and replace each well with 2 mL of fresh EB-M culture medium. After the culture medium change, transfer the cells to a 37°C, 5% CO2 incubator and culture for a total of 7 days.

[0238] 3. RONA Differentiation Culture

[0239] Prepare RONA-M1 culture medium for the first stage of RONA differentiation (Day 1-Day 7) according to the table below.

[0240] Prepare RONA differentiation second stage (Day 8-Day 14) culture medium RONA-M2 according to the table below.

[0241] The protocol for RONA differentiation was as follows.

[0242] Prepare a 6-well cell culture plate coated with MX521 Matrigel and add 3 mL of RONA-M1 medium to each well. Transfer EBs from the center of the well of the low-attachment 6-well cell culture plate to the plate. Incubate the plate in a 37°C, 5% CO2 incubator to culture the RONA cells.

[0243] During the RONA culture stage, RONA-M1 medium was used from day 1 to day 7, and RONA-M2 medium was used from day 8 to day 17.

[0244] 4. Neurosphere Culture

[0245] Prepare NPC-M, a basal medium for forebrain neural progenitor cells, according to the table below.

[0246] Neurosphere culture was performed as follows. After the aforementioned RONA differentiation stage was completed, the cell cluster in the middle of the RONA was transferred to a 6-well low-attachment culture plate containing 2.5 mL of NPCM. The plates were cultured in a 37°C, CO2 incubator to allow neurosphere formation.

[0247] After 12 to 72 hours of neurosphere culture, FNPCs are enriched in the neurospheres and can be plated.

[0248] 5. Expansion and Culture of Forebrain Neural Progenitor Cells

[0249] The neurospheres were collected into 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 placed in a 37°C, CO2 incubator for 15 minutes. Digestion was terminated by adding old culture medium and centrifuged at 300 g for 3 minutes. After discarding the supernatant, 3 mL of NPCM was added and pipetted to form a single-cell suspension for cell counting. The final total number of viable cells was 5.67 × 10 8 The cell viability was 98%.

[0250] The cell concentration was adjusted to 1.52 × 10 6 Viable cells / mL were plated onto T175 cell culture flasks coated with 8 μg / mL Matrigel MX521. Culture was performed in a 37°C, 5% CO2 incubator. The medium was changed the next day (46-50 hours after plating) and passaged on the fourth day. After the fourth passage, cells were cryopreserved.

[0251] 6. Preparation of Cell Preparations

[0252] After thawing, the cell suspension was prepared using 0.9% sodium chloride injection. The cell concentration was 5.0×10 4 viable cells / μL and dispensed into transport bottles to obtain the cell preparation hNPC01.

[0253] Cell preparations can also be prepared using FNPC cells in passage.

[0254] 7. Identification and Characterization of hNPC01 Cell Preparations

[0255] Flow cytometry was used to detect the cell markers FOXG1, PAX6, NESTIN, and SOX2. The hNPC01 cell preparation showed a 93% FOXG1 positivity rate, a 97% PAX6 positivity rate, over 98% NESTIN positivity rate, and a 94% SOX2 positivity rate. These results indicate that the majority of cells in the obtained cell preparation expressed the desired FNPC cell markers.

[0256] Example 2. In vitro differentiation experiment

[0257] To test the biological activity of the cell preparation prepared in Example 1, the cell preparation was induced to differentiate. The expression of various markers was detected by immunofluorescence assay at different time points of differentiation, and neuroelectrophysiological activity was monitored to test the differentiation ability of the cell preparation.

[0258] 2.1 Differentiation of hNPC01

[0259] hNPC01 was used for neuronal differentiation. NPCM described in Example 1 was used as neural differentiation medium. Cells were cultured at 2.5×10 4 Live cells were plated at a density of 100 μL in MX521 (BioLamina)-coated 24-well plates, with 500 μL of NPCM per well. The next day, hNPCs were observed to grow in an adherent manner. The medium was replaced with 1 / 2 of fresh medium every 3-7 days.

[0260] 2.2 Immunofluorescence staining experiment

[0261] Table 1 below shows the markers detected by immunofluorescence staining experiments and the results at different time points.

[0262] Table 1. Summary of cell marker detection results Note: "+" indicates that the corresponding cell marker was detected at this time point and the detection result was positive; the number containing % indicates that the corresponding cell marker was detected at this time point, and the results were displayed as the percentage of positive cells; " / " indicates that the cell marker was not detected at this time point and there is no corresponding result.

[0263] From the results in the table above, we can see that neural progenitor cell markers (FOXG1, 88.00%) and neuronal cell markers (MAP2) can be detected after 7 days of in vitro differentiation (Figure 1), indicating that most cells are still in the neural progenitor cell state.

[0264] After 14 days of in vitro differentiation, neural progenitor cell markers (FOXG1, 41.91%) and neuronal cell markers (MAP2) were detected (Figure 1). The proportion of cells expressing neural progenitor cell markers decreased, indicating cell differentiation.

[0265] After induction of differentiation in vitro to day 35, the induced differentiated cells were detected to contain >90% neuronal cells and express brain neuronal markers. Detectable brain neuronal cell markers include: markers for neuronal cells in the six layers of the cerebral cortex (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 marker γ-aminobutyric acid (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).

[0266] After induction of differentiation in vitro to day 70, the induced differentiated cells were detected to contain >65% neurons, approximately 30% astrocytes, and 1.37% oligodendrocyte precursor cells. Corresponding brain neuronal cell markers were detected, including: 6-layer cerebral cortical neuronal 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%), neuronal marker (MAP2, approximately 65%) (Figure 8), and oligodendrocyte marker (OLIG2, approximately 1.37%) (Figure 9).

[0267] The above results show that hNPC01 can differentiate and obtain mature human cerebral cortical functional neurons and glial cells, whose composition is highly similar to that of the human brain.

[0268] 2.3 Electrophysiological monitoring

[0269] The electrophysiological activity of hNPC01 cells was monitored using a multi-electrode array (MEA) on days 8, 17, 20, and 25 after the initiation of hNPC01 differentiation.

[0270] The results in Figure 10 show that relatively mature characteristics such as spontaneous electrical physiological activity and synchronous discharge can be detected around 3 weeks of differentiation (day 20); on day 25 after differentiation, the results showed that 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.

[0271] The above results indicate that the formation of a complex neural network can be observed in about 3 weeks of cortical neurons obtained by induced differentiation of FNPCs of the present invention, and the complexity of the neural network increases with the increase of differentiation time.

[0272] Example 3. Cell therapy for permanent ischemic stroke model in cynomolgus monkeys

[0273] In this example, the inventors used cynomolgus monkeys as experimental subjects and conducted cell therapy on a primate stroke model. The brain volume and functional areas of cynomolgus monkeys are more similar to those of humans, and the pathological changes after stroke are more similar to those of humans.

[0274] The permanent ischemic stroke model of the cynomolgus monkey in the embodiment is established by permanently ligating the M1 segment of the left middle cerebral artery of the cynomolgus monkey using surgical sutures, i.e., the pMCAO model. MRI and functional damage confirm that the degree of damage of the model is equivalent to that of severe stroke patients in humans, and there is still certain neurological and motor function damage 6-7 months after cerebral infarction. In contrast, the reperfusion cerebral ischemia model of mice and rats can only detect functional damage from 14 days to 1 month. The model solves the problem of faster autologous recovery of animals, and can better simulate the long-term functional damage after stroke (stroke) in human subjects and evaluate the therapeutic effect after the acute phase.

[0275] The detailed design of the experiment is shown in Figure 11.

[0276] 3.1 Establishment of a permanent ischemic stroke model

[0277] Experimental animals: 24 cynomolgus macaques, aged 4-8 years, tested negative for Mycobacterium tuberculosis (TB), simian retrovirus (SRV), simian T-cell leukemia virus (STLV), and simian immunodeficiency virus (SIV).

[0278] Grouping: Rats were randomly divided into four groups, each consisting of six rats, half male and half female: 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 sometimes be referred to as the control group, and the remaining three groups will be collectively referred to as the experimental groups.

[0279] Model establishment: On day 0, the M1 segment of the left middle cerebral artery of the control and experimental groups of cynomolgus monkeys was ligated with surgical sutures to establish a permanent ischemic stroke model in cynomolgus monkeys.

[0280] 3.2 Cell injection (transplantation)

[0281] On the 13th day (D13) after the establishment of the ischemic stroke model, brain MRI images of each group were collected, and the location of the infarct was determined based on the images for injection positioning guidance.

[0282] Starting from 4 days before cell injection (transplantation), each group was orally administered cyclosporine A at a dose of 15 mg / kg / day for immunosuppression for 28 consecutive days.

[0283] On day 15 of the ischemic stroke model, hNPC01, a single injection of the neural progenitor cell product, was administered to the motor cortex and basal ganglia surrounding the infarct in each group of cynomolgus monkeys. Specifically, multiple intracranial injections were performed (three needle tracks, with three injection sites at different depths in each track). The doses administered to the model control group, hNPC01 low-dose group, hNPC01 medium-dose group, and hNPC01 high-dose group were: 0 (0.9% sodium chloride injection), 4.5×10 5 cells / cell, 9.0×10 5 cells / cell and 18×10 5 Cells / cell.

[0284] 3.3 Pharmacodynamic evaluation plan

[0285] After cell injection, the cynomolgus monkeys in each group were observed for 28 weeks, and pharmacodynamic and toxicological evaluations were performed. The specific observation and measurement methods and indicators are summarized in Table 2.

[0286] Table 2. Observation and measurement methods and indicators

[0287] 3.3.1 Physiological monitoring

[0288] All model animals underwent daily clinical observation after the middle cerebral artery occlusion surgery. Throughout the experiment, despite exhibiting clinical symptoms of stroke, all other physical indicators, including body temperature and blood pressure, remained normal, and the animals were emotionally stable. No other abnormalities were observed, and no animals died during the experiment.

[0289] Animal body weight was measured 1 day before modeling, 3 days after modeling, 1 day before cell transplantation, 2 days (D2), 7 days (D7), 14 days (D14), 28 days (D28) after transplantation, and once a week thereafter.

[0290] The weight growth rate of experimental animals of different sexes after cell therapy was calculated according to the following formula, and the results are shown in FIG12 to reflect the weight changes after treatment.

[0291] Weight gain rate = (weight on the day of modeling - weight on the day before modeling) / weight on the day before modeling × 100%

[0292] As shown in Figure 12, the weight gain rates of both the medium-dose and high-dose cell transplant groups were higher than those of the vehicle control group, and the growth rate of the high-dose group was higher than that of the medium-dose group. Two-way ANOVA statistical analysis showed that the differences between the high-dose group and the vehicle control group, as well as the low- and medium-dose groups, were significant at p < 0.001. This result indirectly reflects that the animals in the high-dose cell transplant group recovered their appetite and feeding ability more quickly after modeling, resulting in a faster weight recovery.

[0293] 3.3.2 Behavioral Observation

[0294] Cage-side behavioral evaluation and upper limb feeding skill test were performed 1 day before modeling, 3 days after modeling, 1 day before cell transplantation, 2 days, 7 days, 14 days after transplantation, and every 2 weeks thereafter; ladder traction walking test was performed once a week after 176 days after cell transplantation.

[0295] 3.3.2.1 Cage-based behavioral assessment (neurological function score)

[0296] Prior to surgical modeling, cage-based behavioral assessments were performed on all enrolled cynomolgus monkeys that had completed behavioral training. Behavioral scoring assessed neurological function based on the animal's state of consciousness, sensory system (contralateral to the ischemic side), motor system, and skeletal muscle coordination. Scoring was based on a 0-100 scale, with lower scores indicating closer-to-normal neurological function and higher scores indicating more severe neurological impairment.

[0297] The results showed that before modeling, all groups of crab-eating macaques performed normally in terms of consciousness, sensory system, motor system, skeletal muscle coordination, etc., and could complete the instructed movements guided by the breeders, indicating that the behavioral adaptation training was successful.

[0298] Three and 14 days after the cerebral ischemia model was established through ligation (corresponding to 12 days and 1 day before cell transplantation), the animals' cage-side behavioral assessment scores decreased significantly. On day 14, the animals showed significant behavioral impairments in consciousness, sensory systems, motor systems, and skeletal muscle coordination. The degree of brain damage reached the expected level, indicating that the model was successful.

[0299] After treatment, to account for the impact of the cell transplantation procedure on animal behavior and to eliminate behavioral errors caused by this procedure, cage-side behavioral changes in each group were assessed over the 28 weeks following cell transplantation, with Day 3 (D17) serving as the baseline (i.e., time zero). Specifically, cage-side behavioral assessments were performed 2 days (D17), 7 days (D22), and 14 days (D29) after injection (treatment), and then every two weeks thereafter. The results are shown in Figures 13-14.

[0300] Statistical analysis of cage-side behavioral data for each group within 28 weeks after cell transplantation showed that animals in the low, medium, and high dose cell treatment groups all showed faster behavioral improvement than the vehicle control group, and the two-way ANOVA statistical analysis reached a significant difference of p<0.0001 (Figure 13). At the same time, the experimental results also showed that, with the exception of two animals with milder modeling, the initial behavioral scores (before treatment) of the affected side of almost all treated experimental groups were 30-50% higher than those in the vehicle control group, indicating that although they had more severe neurological damage, they still showed a faster recovery rate (Figure 14).

[0301] 3.3.2.2 Ladder traction walking test

[0302] In order to intuitively explore the recovery of various functions of animals after cell therapy and the differences between groups, a ladder traction walking test was added starting from 23 weeks after treatment (D176).

[0303] Using a closely spaced ladder (10 cm between ladder bars), the animal is led up the ladder for three round trips. The duration of each walk and upper limb use are recorded and statistically analyzed. The results of this test reflect the recovery of the animal's motor behavior, body coordination, sensory system, and muscle tone.

[0304] At the same time, in this experiment, normal non-modeled cynomolgus monkeys (n=6) were supplemented with synchronous training as a normal animal group and compared with the control group and experimental group animals. The results are shown in Figure 15.

[0305] Figure 15 shows the statistical results of the average single walking time of ladder traction walking measured at 5-7 months (23rd to 28th week). Two-way ANOVA statistical analysis showed that the overall inter-group differences reached a significant difference of p < 0.001. At 5 months, the high-dose group basically recovered to the level of the normal animal group without modeling, while the animals in the solvent control group still needed nearly twice as much walking time.

[0306] Five months after the non-acute injection treatment of 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, about 2 months earlier than the control group.

[0307] 3.3.3 Magnetic resonance imaging (MRI) of animal brain tissue

[0308] MRI scans were performed on the animals in each group 13 days after model establishment (2 days before transplantation) to analyze the degree of brain damage and evaluate whether the model was successfully established.

[0309] In addition, brain MRI images were collected on the 3rd day (D18), 28th day (D43), 56th day (D71), 84th day (D99), and 168th day (D183) after cell transplantation to analyze changes in brain lesion volume and evaluate the therapeutic effect.

[0310] Two weeks after surgery, the evaluation results of the modeling animals showed that the animals showed ischemic stroke symptoms in terms of cage-side behavior, feeding ability and MRI imaging, indicating that the modeling was successful.

[0311] Regarding the MRI examination results at various time points after transplantation, a typical magnetic resonance image of the animal brain after cell transplantation at the end point of the experiment is shown in FIG16 .

[0312] Furthermore, statistical analysis of brain injury volume ratios was performed using a 3D slicer based on the MRI images, and the results are shown in FIG17 .

[0313] As shown in Figure 17, 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 four weeks of treatment, the high-dose hNPC01 group experienced the greatest reduction in brain lesion volume. This indicates that the high-dose group demonstrated the greatest repair effect on brain damage, followed by the medium-dose group, and the low-dose group the weakest. These results suggest that this cell preparation can be used to improve brain damage and infarction following acute ischemic stroke, achieving a certain degree of tissue repair. Furthermore, the improvement may be even greater with high-dose administration of the cell preparation.

[0314] 3.4 Immunohistochemical staining

[0315] At the end of the experiment, all remaining animals were euthanized and their brain tissues were collected (Figure 18). Pathological examination was performed on the brain tissues of some animals. After fixing the brain tissues on the infarcted side, coronal sections were cut and scanned to collect images. HE staining was performed on the brain tissues in the lesion area to observe the inflammatory cell infiltration of the brain tissues in the infarcted area. The results of immunofluorescence staining from animals in the medium dose group are shown in Figure 19, where immunofluorescence staining was performed using anti-human nuclear marker STEM101 and mature neuron marker NEUN.

[0316] As shown in Figure 19, in brain slices from animals injected with hNPC01 cell preparations, >80% of human-derived cells (determined by staining with the anti-human nuclear marker STEM101) expressed the mature neuronal marker NEUN. This indicates that human neural progenitor cells successfully differentiated into a population primarily composed of mature neurons 7 months after transplantation.

[0317] Taken together, these results suggest that when hNPC01, a cell preparation containing forebrain neural progenitor cells differentiated from iPSCs, is transplanted into animal models, the treated animals exhibited significant improvements in neurological function and walking ability, as well as weight loss and recovery of brain tissue damage compared to the control group. The administered FNPC cells were capable of differentiating into neurons. Furthermore, in some experiments, the effects of FNPC treatment were dose-dependent, suggesting that specific high doses may be more effective.

[0318] Example 4. Cell therapy for rats with hypoxic-ischemic cerebral palsy model

[0319] In this example, the inventors tested the therapeutic effect of the cells of the present invention on hypoxic-ischemic cerebral palsy in children in an animal model to demonstrate the therapeutic effectiveness of the cell products of the present invention on different types of brain injuries.

[0320] 4.1 Modeling and Dosage Regimen

[0321] Using the classic RICE model, permanent unilateral common carotid artery ligation was performed on SFP-grade Sprague-Dawley rats on postnatal day seven (P7). After a 1-hour postoperative recovery period, the rats were subjected to hypoxia for 2.5 hours using a mixture of 8% oxygen and 92% nitrogen. Successful modeling was confirmed by observing the movement of the rats' limbs and the opening of one eye. On the seventh day after modeling, 10 μL of hNPC01 at varying cell densities was injected into the lateral ventricle using a stereotaxic device. Both untreated and treated mice were treated with saline in the same manner as controls. The specific experimental groups are shown in Table 3 below.

[0322] Table 5. Treatment methods of the experimental and control groups

[0323] 4.2 Pharmacodynamic evaluation

[0324] After cell injection, rats in each group were observed for 10 weeks, and pharmacodynamic evaluation was performed. The specific experiments included: rotarod test, balance beam test and 2,3,5-triphenyltetrazolium chloride (TTC) staining test.

[0325] 4.2.1 Rotarod test

[0326] Rotarod test was performed before administration and 2 weeks, 4 weeks and 6 weeks after administration to detect the effect of hNPC01 administration on the motor ability of rats.

[0327] Before the experiment, animals were moved from the housing room to the experimental room and allowed to acclimate for 60 minutes. Rats were placed on a 90 mm diameter rotarod. After acclimation for 2 minutes, the rotarod was set to an initial speed of 4 rpm and accelerated to 40 rpm over 300 seconds. The rats were allowed to move voluntarily with the rotarod, and the time spent on the rotarod was recorded. After the test, the rats were returned to the housing room and the experimental apparatus was cleaned with 70% ethanol. The laboratory temperature, humidity, and light intensity were maintained consistent throughout all testing.

[0328] The results of the rotarod test are shown in Figure 20. The high-dose group had longer rotarod time than 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, with no deterioration observed. The medium-dose group had longer rotarod time than 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.

[0329] 4.2.2 Balance beam experiment

[0330] Balance beam tests were performed before and after administration (2 weeks, 4 weeks, and 6 weeks) to detect the effect of hNPC01 administration on the motor ability of rats.

[0331] During the training phase, animals were moved from the housing to the experimental room and acclimated for 60 minutes. Training was performed using a 25 mm diameter square rod. The animals were placed at the starting end of the rod, and a stopwatch was used to count the time it took them to cross the 100 cm distance between the rods and enter a dark box, which was the latency period. The latency period and the number of hind paw slips on the rod were measured. After testing, the rats were returned to the housing room and the test apparatus was cleaned with 70% ethanol. The laboratory temperature, humidity, and light intensity were maintained consistent throughout all testing.

[0332] The results of the balance beam test are shown in Figures 21A and 21B. The high-dose group showed shorter beam crossing times compared to the saline group at 2, 4, and 6 weeks after administration (****, P < 0.0001; **, P < 0.01). The high-dose group also showed stable performance, with no observed deterioration. No significant differences were observed between the medium-dose and low-dose groups and the saline group at any time point, nor was a deterioration observed.

[0333] 4.2.3 TTC staining

[0334] TTC staining is performed at the end of the experimental observation. Dehydrogenases in living brain tissue cells reduce TTC to insoluble, red, stable triphenylformamide (TTF). If brain cells are dead or their vitality has declined, they will not stain or will stain only slightly. Therefore, brain tissue vitality can be assessed based on the location and intensity of the staining.

[0335] The results of the TTC staining experiment are shown in Figure 22. No significant residual damage was observed in the non-modeled saline group (G1), the medium-dose administration group (G4), and the high-dose administration group (G5). Significant brain tissue damage was observed in both the low-dose administration group and the modeled saline group.

[0336] Example 5. Study on the Cellular Composition of hNPC01

[0337] This example describes an investigation of the cellular composition of hNPC01 using single-cell RNA-seq (scRNA-seq) technology. hNPC01 was also compared with fetal forebrain NPCs, where fetal forebrain NPC data were obtained from a published article (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). This data comparison revealed that the transcriptome data and molecular markers of hNPC01 were consistent with those of naturally occurring human forebrain neural progenitor cells.

[0338] Based on the scRNA-seq results, cell types in the hNPC01 sample were annotated, and gene expression profiles were used to confirm the phenotypes of the cells in the sample. The results are shown in Figure 23. As shown in Figure 23, the predominant cell type in the hNPC01 sample is neural progenitor cells, which are phenotypically identical to those in fetal forebrain samples, accounting for ≥70% of the hNPC01 sample. In addition, the hNPC01 sample also contains some immature neurons, GABAergic neurons, glutamatergic neurons, and a small number (≤10%) of ependymal cells and pericytes, which are widely present in the brain. The hNPC01 neural progenitor cells are capable of further differentiation in vitro and in vivo into cerebral cortical neural cells (including various excitatory and inhibitory neurons, astrocytes, and oligodendrocyte progenitor cells) with the correct subtype composition, mature electrophysiological functions, and long-term survival in vitro and in vivo. At the same time, these transplanted neural progenitor cells can secrete a variety of neuroprotective and angiogenic cytokines and microRNAs, which help enhance the endogenous repair process.

[0339] Further analysis of the sequencing data of fetal samples and hNPC01 samples revealed that the expression profiles of neural progenitor cell and neuronal markers in the two samples were highly consistent.

[0340] The scRNA-seq comparison data above revealed that the hNPC01 forebrain NPC cell product of the present invention, produced by iPSC-induced differentiation, expressed the same types of neural precursor-specific genes as naturally occurring fetal neural progenitor cells. However, the expression levels of pluripotent stem cell marker genes were lower than those in fetal cells, indicating that there were fewer cells with strong division capacity or a lower division capacity. This result indicates that the cell product of the present invention contains no residual iPSC phenotype cells, nor cells with mesodermal or endodermal phenotypes, indicating that the cell product of the present invention has a higher purity of forebrain neural progenitor cells and is safer.

[0341] Further comparative analysis of single-cell sequencing data from human embryonic brain development and single-cell sequencing data from organs derived from mesoderm and endoderm development revealed that the cell product of the present invention specifically and highly expressed molecular markers for forebrain neural progenitor cells, including EFNB2, WNT7B, RSPO2, and FEZF2. This result also supports the conclusion that the cell product of the present invention contains highly purified forebrain neural progenitor cells.

[0342] Example 6. In vivo neural circuit reconstruction study

[0343] In addition to whether the transplanted cells can survive and differentiate into the desired type, whether the differentiated cells can form neural circuits is an important indicator of transplantation efficacy. This study found that after hNPC01 of the present invention was transplanted into the ischemic brain region of immunodeficient experimental mice, these hNPCs induced to differentiate in vitro not only further differentiated into mature neurons in vivo but also integrated into the recipient's pre-existing central nervous system, forming functional neural connections and thus reconstructing neural circuits, which is crucial for functional recovery after brain injury.

[0344] After transplanting 2 x 105 hNPCs per rat into the peri-infarct area of ​​the somatosensory cortex, the recombinant viral vector scAAV2 / 1-hSyn-Cre was injected into the ventral thalamus. This AAV serotype has the ability to label downstream synapses and induces a certain amount of Cre recombinase expression in neurons at the next level. Simultaneously, another recombinant viral vector, AAV2 / 8-EF1α-DIO-mCherry, was injected into the somatosensory cortex of the same animal. When neurons infected with this virus encounter Cre recombinase delivered upstream, the loxP sequence is reversed, leading to expression of the mCherry fluorescent reporter gene. Subsequently, red mCherry signals were observed in the AAV2 / 8-EF1α-DIO-mCherry-injected tissue, indicating that neurons in this area have established direct synaptic connections with the ventral thalamus. During the experiment, red mCherry signal expression was first observed in normal somatosensory cortex, confirming the effectiveness of this strategy.

[0345] In addition, the expression of red mCherry signals was successfully observed on the daughter neurons differentiated from GFP-positive human forebrain NPCs, and these red signals overlapped with anti-human nuclear antibody HNA staining, further confirming that the transplanted hNPCs daughter neurons can establish direct synaptic connections with the host ventral thalamus, indicating the successful establishment of neural circuits.

Claims

1. A cell population for use in treating a neurological injury disease, a neurodegenerative disease, a neurodevelopmental disease, or other neurological disease, disorder, or condition associated with the death and / or dysfunction of forebrain neural cells in a subject, or for use in preparing a medicament for treating a neurological injury disease, a neurodegenerative disease, a neurodevelopmental disease, or other neurological disease, disorder, or condition associated with the death and / or dysfunction of forebrain neural cells, wherein the cell population comprises human forebrain neural progenitor cells.

2. The use of claim 1, wherein at least about 70% of the cells in the cell population 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 at least about 70% of the cells in the cell population 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 at least about 70% of the cells in the cell population 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 at least about 80% of the cells in the cell population 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 of any one of claims 1-5, wherein the cell population expresses one or more genes selected from the group consisting of EFNB2, WNT7B, RSPO2 and FEZF2.

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

8. The use according to any one of claims 1 to 7, wherein in the cell population, neural progenitor cells, preferably forebrain neural progenitor cells, account for ≥60% of all cell types, more preferably ≥70%, even more preferably ≥80%.

9. The use according to any one of claims 1 to 8, wherein the cell population further comprises one or more selected from the group consisting of immature neurons, GABAergic neurons, and glutamatergic neurons.

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

11. The method of claim 10, wherein the total proportion of immature neurons, GABAergic neurons, glutamatergic neurons, ependymal cells, and pericytes in the cell colony is no more than 30% of the total cell number, preferably no more than 20%, more preferably no more than 10%, and even more preferably no more than 5%.

12. The use according to any one of claims 1 to 11, wherein the forebrain neural precursor cells are obtained from human pluripotent stem cells (hPSCs) by an induced differentiation method.

13. The use according to claim 12, wherein the differentiation induction method comprises the following steps: (1) Culturing and expanding ESCs or iPSCs in human pluripotent stem cell culture medium (hPSC culture medium); (2) digesting the ESCs or iPSCs in step (1) and culturing them in suspension in EB medium to form embryoid bodies (EBs); (3) culturing the EBs obtained in step (2) with neural induction medium (RONA) to form rosette neural aggregates (RONAs); (4) The neuroectodermal cells in the RONAs formed in step (3) are picked and cultured in suspension to form neurospheres.

14. The use according to claim 13, wherein the differentiation induction method further comprises, after step (4): (5) The neurospheres formed in step (4) were cultured using NPC medium for passage expansion.

15. The use according to claim 14, wherein the differentiation induction method further comprises, after step (5): (6) Harvesting the FNPCs formed in step (5).

16. The use according to any one of claims 13 to 15, wherein the hPSC culture medium is hPSC XF Medium, Essential 8 Medium, Basic03 culture medium, StemMACS TM iPS-Brew medium, Stem- ACF medium, TeSR TM -AOF medium or TeSR2 medium.

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

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 use according to any one of claims 13 to 19, wherein the EB culture medium comprises: (i) a basal medium selected from a) to c): a) Single KnockOut TM DMEM / F12 medium, b) DMEM / F12 medium and Neurobasal TM Combination of culture medium, c)KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and (ii) an additive comprising or consisting of d) or e): d) N-2 Supplement and GlutaMAX TM -I additives; e) N-2 additives, GlutaMAX TM -I Supplement and B-27 without Vitamin A TM Additive (B-27 TM Supplement,minus vitamin A). The use according to claim 20 , wherein the EB culture medium further comprises an inhibitor, and the inhibitor comprises or consists of a BMP inhibitor, an AMPK inhibitor and an ALK inhibitor.

22. The use according to claim 21, wherein the EB culture medium further comprises an inhibitor comprising or consisting of one or more of Noggin, SB431542, LDN-193189, DMH-1 and Dorsomorphin.

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

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

25. The use according to any one of claims 13 to 24, wherein the EB culture medium comprises: (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combination of culture media; (ii) Supplements comprising N-2 Supplement and GlutaMAX TM - a combination of additives; and (iii) SB431542, Noggin and Dorsomorphin.

26. The use according to any one of claims 13 to 25, wherein the step (3) uses a RONA medium comprising the following components: (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and (ii) Supplements comprising N-2 Supplement and GlutaMAX TM -I Combination of additives.

27. The use according to any one of claims 13 to 26, wherein the step (3) uses a RONA medium comprising the following components: (i) Basal medium, which is KnockOut TM DMEM / F12 medium and Neurobasal TM Combinations of culture media; and (ii) additives comprising N-2 additive, B-27 TM Additive (B-27 TM Supplement,XenoFree,minus vitamin A) and GlutaMAX TM -I Combination of additives.

28. The use according to any one of claims 13 to 27, wherein the basal medium of the NPC culture medium is Neurobasal TM culture medium, and the additive of the NPC culture medium is (a) GlutaMAX TM -I supplement, and (b) B-27 without vitamin A TM additive.

29. The method of claim 28, wherein the NPC culture 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).

30. The use according to any one of claims 12 to 29, wherein the step (5) uses an NPC culture medium comprising the following components: (i) Basic culture medium, which is Neurobasal TM culture medium; (ii) an additive comprising B-27 TM Additive (B-27 TM Supplement,XenoFree,minus vitamin A) and GlutaMAX TM - a combination of additives; and (iii) BDNF, GDNF, L-ascorbic acid, and DB-cAMP.

31. The use according to any one of claims 13 to 30, wherein the step (4) is performed using the RONA medium or the NPC medium.

32. The use according to any one of claims 1 to 31, wherein in the cell population, the proportion of pluripotent stem cells in all cell types is ≤3%, preferably ≤2%, more preferably ≤1%.

33. The use according to any one of claims 1 to 32, wherein the human forebrain neural precursor cells have the ability to differentiate into neuronal cells.

34. The use of any one of claims 1 to 33, wherein the human forebrain neural precursor cells have the ability to differentiate into astrocytes.

35. The use of claim 33, wherein the neuronal cells express MAP2.

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

37. The use of any one of claims 1-36, wherein the cell population is capable of establishing new neural circuits in a subject.

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

39. The use according to claim 38, wherein the disease associated with ischemic brain injury is ischemic stroke or a condition associated with ischemic stroke, such as complications and sequelae.

40. The use according to claim 38, wherein the disease associated with hemorrhagic brain injury is hemorrhagic stroke or a condition associated with hemorrhagic stroke, such as complications and sequelae.

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

42. The use of any one of claims 1-37, wherein the disease is a neurodevelopmental disease and is selected from autism, epilepsy, and cerebral palsy.

43. The use of claim 39 or 40, wherein the cell population is administered after the acute phase of the stroke, or at least 14 days after the stroke occurs.

44. The use of any one of claims 1-43, wherein the cell population is administered by intracranial injection.

45. The use of claim 44, wherein the cell population is injected into the infarct or damaged brain area, or into the motor cortex and / or basal ganglia area surrounding the infarct or damaged brain area.

46. ​​The use according to claim 44 or 45, wherein the injection is a single-point injection or a multi-point injection.

47. The use of any one of claims 44-46, wherein the injection is a single injection or multiple injections.

48. The method of claim 47, wherein when multiple injections are performed, the interval between two adjacent injections is 30 days or more.

49. The use of any one of claims 1-50, wherein the cell population is prepared as a suspension.

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

51. The use according to claim 49 or 50, wherein the cell concentration in the suspension is 1.0×10 4 ~5.0×10 5 viable cells / μL.

52. The use according to any one of claims 44 to 48, wherein the injection dose is 1.0×10 5 ~1×10 10 cells / time, preferably 1.5×10 5 cells / time~6×10 7 Cells / times.

53. The use of any one of claims 44-48, wherein the subject is a human and the injection dose is 2×10 5 to 2×10 9 cells, preferably 2×10 6 to 1.2×10 9 cells.

54. The use of any one of claims 1-53, wherein the cell population or the cell preparation is used alone or in combination with other drugs or treatments.

55. A method of treating a neurological injury disease, a neurodegenerative disease, a neurodevelopmental disease, or other neurological disease, disorder, or condition associated with death and / or dysfunction of forebrain neural cells in a subject, comprising administering to the subject a cell population of the invention, wherein the cell population comprises human forebrain neural precursor cells.

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

57. The method of claim 55 or 56, wherein the disease is the disease of any one of claims 31-35.

58. The method of any one of claims 55-57, wherein the cell population is administered as described in any one of claims 36-46.

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