Methods for the production of a rodent model of neurodegenerative disease and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to a preparation method of a rodent model of neurodegenerative disease, a rodent model prepared by the method, the use of the model as an animal model for studying neurodegenerative disease, the use of the model for screening or identifying candidate agents that can alleviate or treat neurodegenerative disease, and a method for screening or identifying candidate agents that can alleviate or treat neurodegenerative disease.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing a rodent model of neurodegenerative diseases and its uses. Background Technology

[0002] Neurodegenerative diseases affect millions of people worldwide and pose a major threat to human health. The incidence of neurodegenerative diseases increases with age. These include diseases such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), prefrontal dementia, and spinocerebellar ataxia. The pathologies of these diseases vary—some cause memory and cognitive decline, while others affect motor, language, and respiratory abilities.

[0003] Neurodegenerative diseases occur due to the gradual loss of function or eventual death of nerve cells in the central or peripheral nervous system. The central nervous system is composed of billions of nerve cells, and we depend on these cells to function every minute of every day. Due to the complexity of the brain, abnormal brain function often originates from tiny errors in communication between cells. Many neurodegenerative diseases originate in different brain regions. Parkinson's disease, whose main symptoms are related to movement, is caused by the death of dopamine neurons in the substantia nigra. Huntington's disease affects the basal nucleus, causing neuronal death due to abnormal protein aggregation, leading to motor incoordination. In neurodegenerative diseases, the death of any neuron can cause brain atrophy. This further affects the patient's memory and thinking abilities. Therefore, memory and cognitive impairment are common pathologies in neurodegenerative diseases.

[0004] Current research models for neurodegenerative diseases include transgenic animals, in vitro stem cell models, pathological samples, non-invasive human imaging, and chimeric models. Transgenic animals can be used for long-term behavioral experiments. Transgenic animals are artificially injected with disease-related genes to rapidly develop the disease. However, transgenic animals cannot fully reflect the true pathological condition because existing research has confirmed that human nerve cells, especially glial cells, differ significantly from glial cells in other animals.

[0005] In vitro stem cell models can directly obtain human nerve cells. However, in vitro culture often presents cells in an early progenitor state. External application of growth factors or nutrients can promote relative cell maturation. Due to the overly simple stimulation conditions, these cells are far less mature than cells in vivo. Many neurological diseases are related to adult cell pathology, and the results obtained from in vitro induced cell culture cannot truly reflect the long-term responses among various cell types. Current stem cell spheroids can achieve co-culture of multiple cell types, but due to the lack of vascular structures, the health status of these cells cannot be guaranteed. Artificial cell bodies also cannot simulate brain structure, thus many disease-related brain regions cannot be fully simulated. Recent research shows that artificial cell bodies contain multiple cell types, but cannot reproduce different subtypes of the same cell type or the maturation process of similar progenitor cells. Although the molecular characteristics of neurons can be reproduced in cell bodies, spatial structural information cannot be obtained. Cell spheroids exhibit activation of cellular stress pathways, and stress can impair cell type specialization.

[0006] The advancements in gene sequencing technology in recent years have greatly advanced our understanding of neurodegenerative diseases at the molecular level. Many research institutions use brain samples donated after the death of patients for research on neurodegenerative diseases. Studies on Alzheimer's disease (AD) have revealed significant differences in the response of various brain cells to pathology, and that gender differences in disease pathology can be reflected at the cellular level. Although we can directly study patients' cells without using any models, directly using human samples also has serious limitations. First, we can only obtain a fragment of the disease pathology, unable to understand the disease progression, and therefore unable to select effective treatment windows and methods. Second, we cannot scientifically evaluate the results obtained; each patient has significant individual pathological characteristics, and we lack a scientifically rigorous reference group as a control. Moreover, most patients have undergone long-term and complex drug treatments, making it difficult to distinguish between primary pathological features and secondary reactions induced by drug treatment. Human pathological samples can serve as a reference for our research on neurodegenerative diseases, but they cannot provide us with comprehensive information in terms of time and space dimensions. We can only describe pathological features, but cannot mediate or interfere with the scientific study of the disease process, and therefore cannot serve as an effective disease model.

[0007] Current in vivo imaging technology allows for non-invasive detection of the human brain's health. To monitor brain health, we must first understand the differences between the brains of healthy young people and the elderly. The aging brain exhibits significant changes in brain volume, basic cognition, blood vessels, and neurovascular coupling. Changes in brain volume are the most pronounced. With age, after age 40, brain volume decreases by 5% every 10 years. This change accelerates significantly after age 70. Additionally, there is specific atrophy and myelin degeneration in the white matter. Other significant changes are in blood vessels and blood pressure; vascular size and health are associated with the incidence of Alzheimer's disease.

[0008] Functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and diffuse optical tomography (DOT) are the main imaging techniques for studying brain function and metabolism. Through differential comparison, all these imaging techniques can provide macroscopic information about the brain's functional state under resting and stress conditions. fMRI is sensitive to blood oxygen-dependent signals and can detect neuronal activity through neurovascular coupling. Because this technique is non-invasive, it is currently the most popular tool for studying cognitive impairment. It shows a decrease in cerebral hemodynamics in a normally aging brain upon receiving visual stimuli. PET relies on the enrichment of radiolabeled substances to monitor brain metabolic status and other pathophysiological indicators. Although it has lower resolution and requires radiolabeling, PET is a powerful tool for studying normal brain aging and changes in neurodegenerative diseases due to its high sensitivity and specificity. Compared to fMRI and PET, DOT is a relatively new functional brain imaging method. DOT works similarly to fMRI by detecting changes in cerebral blood oxygenation; increased blood flow and elevated blood oxygen content are its primary measurements. Mainly limited by the projection capabilities of near-infrared spectroscopy, DOT primarily detects functional changes in the cerebral cortex. Compared to fMRI and PET, DOT is simpler and allows for rapid, real-time monitoring of brain function. However, current imaging technologies primarily target humans, and individual differences and the complexity of disease progression make it difficult to use the results scientifically and rationally. Furthermore, current imaging technologies have low spatial and temporal resolution, failing to achieve the resolution of microscopic studies at the cellular and molecular levels. Optical imaging has poor penetration, making it difficult to detect brain regions below the cortex, thus hindering the study of differences in brain damage across different brain regions.

[0009] Chimeric mouse models utilize induced cellular stem cells to competitively occupy the brains of immunodeficient mice, gradually replacing glial cells. Glial cells are non-neuronal cells of the nervous system, including oligodendrocytes, astrocytes, and microglia. Oligodendrocytes typically form myelin sheaths in white matter and peripheral cell bodies in gray matter. They are large and have few branches surrounding neurons. Astrocytes typically form connections between blood vessels and neurons. They are smaller than oligodendrocytes and have extensive branching. Microglia play an immune role in the nervous system, specializing in the elimination of macromolecules, cytophagy of apoptotic or necrotic cells, recognition and elimination of pathogens, and regulation of immune responses. Once activated or stimulated, microglia can engulf debris. They are very small cells but enlarge upon activation or stimulation.

[0010] Existing experiments have shown that transplanting macroglia can replace corresponding mouse cells with human oligodendrocytes and astrocytes. These "macroglia" chimeric mice can be used to study schizophrenia and Huntington's disease. However, current research primarily focuses on using macroglia for cell transplantation to treat demyelinating diseases. Macroglia chimeric mice lack microglia. Therefore, we cannot obtain information on the function and potential therapeutic effects of microglia in diseases.

[0011] Microglia, as glial cells of a unique origin, have also been used to establish chimeric models. Recent studies have reported that hematopoietic stem cell transplantation can achieve the occupation of human microglia in mouse brain regions, with significant regional differences in microglia distribution, and mouse microglia also present in the brains of chimeric mice. Due to the immune characteristics of microglia, the functions of mouse and human microglia in chimeric mice cannot currently be distinguished. Therefore, there are currently no mature microglia chimeric models available for disease simulation or treatment.

[0012] Existing chimeric mouse models are still immature, with uneven integration and distribution of human cells. New animal models need to overcome the shortcomings of current models. We must directly study changes in human cells to eliminate the pathological differences between transgenic mice and humans. Furthermore, long-term monitoring of human cell responses during pathological development is necessary to compensate for the limitations of in vitro stem cell models. Simultaneously, human cells must exhibit adult cell characteristics, as young progenitor cells may respond completely differently from adult cells. Summary of the Invention

[0013] This invention relates to a complete solution encompassing brain tissue sample collection, cell nucleus extraction, chromatin immunoprecipitation, library construction, high-throughput sequencing, data analysis, core algorithm development, and the design and development of derivative products based on the obtained results. The specific technical solution is as follows:

[0014] In a first aspect, the present invention provides a method for preparing a rodent model of neurodegenerative diseases, comprising the following steps:

[0015] 1) Inducing human stem cells to differentiate into macroglial precursor cells, microglial precursor cells, or a combination thereof, wherein the human stem cells are induced pluripotent stem cells;

[0016] 2) Cell transplantation is performed using newborn or intrauterine rodents as hosts to create chimeric animals with human-derived cells;

[0017] 3) Accelerate the directed differentiation, expansion, and maturation of macroglia, microglia, or combinations thereof transplanted into chimeric animals into human glial cells.

[0018] In one specific implementation, step 2) involves cell transplantation of intrauterine rodents using macroglia, microglia, or a combination thereof.

[0019] In another specific implementation, step 2) involves cell transplantation of newborn rodents using macroglia, microglia, or a combination thereof.

[0020] In another specific implementation, in step 2), cell transplantation is first performed on fetal rodents using microglia precursor cells, and then on newborn rodents using microglia precursor cells after delivery.

[0021] In another specific implementation, step 3) is achieved by removing the host's own glial cells from the brain using pharmaceutical or genetic methods.

[0022] In another specific embodiment, the drug specifically removes glial cells from the host's own brain, such as copper hydrazone, colony-stimulating factor 1 receptor inhibitors like PLX3397, and glutamate homolog L-α-aminoglycolic acid.

[0023] In another specific embodiment, the rodent is a transgenic animal lacking glial cells, such as lacking colony-stimulating factor 1 receptor or its enhancer or myelin protein MBP.

[0024] In another specific embodiment, the rodent is a rat or a mouse.

[0025] In another specific embodiment, the rodent is a newborn mouse 0-4 days old, preferably 0-2 days old, or a fetal mouse with embryonic development of 9-14 days.

[0026] In another specific embodiment, the mouse is an immunodeficient mouse, preferably expressing human MCSF-1 and IL-34. Human MCSF-1 and IL-34 are required for microglia transplantation; for macroglia chimeras, MCSF-1 and IL-34 are not essential.

[0027] In another specific embodiment, the mice carry specific neurodegenerative disease genes, thereby exhibiting the pathology of neurodegenerative diseases, preferably early-stage neurodegenerative diseases. However, this does not mean that chimeric mice without degenerative pathology are without value. In fact, chimeric mice without degenerative pathology will be very important normal controls in the study of degenerative diseases. Furthermore, we can also use these glial chimeric mice to study neurological diseases unrelated to gene mutations, such as brain trauma, neurological diseases caused by viral infections, and aging.

[0028] In another specific implementation, the mouse is a 5xFAD transgenic mouse.

[0029] In another specific embodiment, the mouse is a Tau P301L transgenic mouse.

[0030] In another specific embodiment, the mouse is a novel AD mouse model produced by crossing 5xFAD transgenic mice with immunodeficient mice containing human MCSF-1 and IL-34.

[0031] In another specific embodiment, the mouse is a novel AD mouse model produced by crossing Tau P301L transgenic mice with immunodeficient mice containing human MCSF-1 and IL-34.

[0032] In another specific implementation, the induced pluripotent stem cells are derived from patients with neurodegenerative diseases.

[0033] In another specific implementation, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0034] Secondly, the present invention provides a rodent model prepared by the method described above.

[0035] In one specific implementation, the rodent model is selected from oligodendrocyte-dominated chimeric mice, oligodendrocyte precursor cell and astrocyte chimeras, microglia chimeras, and macroglia and microglia chimeras. This invention can establish either a single glial chimera or multiple glial cell chimeras. When using multiple glial cell chimeras, a single glial chimera is required as an appropriate control group.

[0036] Thirdly, the present invention provides the use of rodent models prepared by the method described above as animal models for studying neurodegenerative diseases.

[0037] In one specific implementation, the neurodegenerative disease is in its early stages.

[0038] Fourthly, the present invention provides the use of rodent models prepared by the method described above for screening or identifying candidate agents that can alleviate or treat neurodegenerative diseases.

[0039] Fifthly, the present invention provides a method for screening or identifying candidate agents that can alleviate or treat neurodegenerative diseases, comprising the following steps:

[0040] 1) Apply the candidate reagent to the rodent model prepared by the method described above;

[0041] 2) Determine whether the candidate reagent has a relieving or therapeutic effect on one or more signs, symptoms, and / or conditions associated with neurodegenerative diseases; and

[0042] 3) Identify candidate reagents that have a relieving or therapeutic effect on one or more signs, symptoms and / or conditions associated with neurodegenerative diseases.

[0043] The glial cell chimeric animal model of this invention will replace conventional transgenic animals, and the indicators to be detected will focus on changes in human glial cells. These improvements will greatly increase the success rate of clinical trials for screened drugs. Attached Figure Description

[0044] Figure 1 A schematic diagram illustrating the induced directed differentiation of iPS cells into macroglial precursor cells and microglial precursor cells.

[0045] Figure 2 This is a schematic diagram of a cell transplantation surgery.

[0046] Figure 3 This is a schematic diagram illustrating the role of glial cells in the early pathological development of Alzheimer's disease (AD).

[0047] Figure 4 This is an immunohistochemical staining image showing the distribution of human glial cells in the brains of glial chimera mice. Three months after cell transplantation, human astrocytes were labeled with the human cell-specific marker hN and the glial cell-specific marker hGFAP; human oligodendrocytes were labeled with the human cell-specific marker hN and the oligodendrocyte-specific marker PDGFRa; and human microglia were labeled with the human cell-specific marker hN and the microglia-specific marker P2Y12R. Detailed Implementation

[0048] This invention is not limited to the specific methods, schemes, reagents, etc., described herein, as these can vary. The terminology used herein is for the purpose of describing particular embodiments only and not for limiting the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0049] I. Scheme Description

[0050] Glial cell pathology includes microglial activation, astrocyte activation, and demyelination due to oligodendrocyte loss, all of which are prevalent in various neurodegenerative diseases. However, the relationship between glial cell pathology and the development of neurodegenerative diseases remains unclear. This invention proposes using a novel glial cell chimera model to simulate neurodegenerative diseases, predicting these diseases by detecting the responses of human glial cells, for early diagnosis and drug screening.

[0051] In the central nervous system, glial cells are mainly divided into two categories: macroglia and microglia. Macroglia account for 30%-80% of the human brain, while microglia account for 5-10%. Macroglia include oligodendrocytes and astrocytes. Oligodendrocytes include NG2-positive oligodendrocyte precursor cells and mature oligodendrocytes. In vivo two-photon imaging shows that the responses of NG2 cells to injury include cell migration, proliferation, and swelling, exhibiting diverse characteristics. Moreover, NG2 cells proliferate extensively and aggregate at the site of injury to form glial scars. Besides these studies, research on the function of NG2 cells in pathological and physiological states is still limited. The main function of oligodendrocytes is to encapsulate axons to form myelin sheaths, enabling neurons to conduct signals in a jump manner. After myelination, oligodendrocytes establish close connections with neurons and provide nutrition. Therefore, oligodendrocytes not only possess important functions but are also highly sensitive to nerve injury. Demyelination is widespread in neurodegenerative diseases. Studies have reported that oligodendrocytes also participate in the formation of glial scars after nerve injury.

[0052] Astrocytes are the most abundant type of keratinocyte in the brain. Their functions are highly complex, including maintaining extracellular potassium levels and supplying glutamate to neurons. Astrocytes can encapsulate neuronal synaptic structures to form triposomes. Astrocyte processes can also encapsulate blood vessels, forming lymphovascular structures. These glial lymphovascular structures are crucial for clearing waste products from the intercellular spaces of the brain and are thought to be the structural basis for how sleep helps the brain clear waste. Astrocytes have also been reported to participate in intrinsic immune responses in the brain. One group of astrocytes, called A1 astrocytes, produces pro-inflammatory factors that drive damaging inflammatory responses. Another group, called A2 astrocytes, produces anti-inflammatory factors and trophic factors that protect neurons. These two types of astrocytes have primarily been reported in mouse models but have not yet been confirmed in human astrocytes.

[0053] Oligodendrocytes and astrocytes both originate from the ectoderm. Current research allows for the acquisition of precursor cells for these two cell types through induced embryonic stem cell (ES) or induced pluripotent stem cell (IPS) methods. This invention utilizes stem cell-induced macroglia precursor cells for cell transplantation to obtain macroglia chimeric mice.

[0054] The primary function of microglia is to monitor the brain's internal environment and respond rapidly to cellular damage. During development, microglia play a crucial role by clearing excess neurons and pruning synaptic structures. Recent research has found that microglia can directly contact neuronal cell bodies and protect neurons during ischemic injury. The most significant function of microglia is their ability to engulf cellular debris and metabolic waste, thereby maintaining a healthy internal brain environment. Similar to astrocytes, microglia are also important members of the brain's immune response. Microglia also divide into two types when responding to injury: M1 type promotes a damaging immune response by releasing inflammatory factors; M2 type protects neurons by releasing inflammatory suppressor factors and trophic factors.

[0055] Microglia differ from macroglia in origin. Current research suggests that microglia primarily originate from the endoderm, developing from macrophages that initially derive from hematopoietic stem cells and then differentiate into nascent hematopoietic stem cells. Microglia appear in the brain earlier than macroglia. Existing methods obtain microglia by inducing embryonic stem cells (ES) or induced pluripotent stem cells (IPS) to differentiate into nascent hematopoietic stem cells. This invention uses stem cell-induced microglia precursor cells for cell transplantation to obtain "microglia" chimeric mice.

[0056] In the development and pathology of the central nervous system, macroglia and microglia interact and support each other to maintain neuronal function. Therefore, it is essential to develop novel glial chimeric models to study neurodegenerative diseases. This new chimeric mouse contains human oligodendrocytes, astrocytes, and microglia. Since macroglia and microglia precursor cells can be obtained through in vitro induced stem cell transplantation, this invention will perform mixed cell transplantation to obtain novel human glial cell chimeric mice. During normal development, microglia are formed by the invasion and specialization of macrophages derived from naïve hematopoietic stem cells into the brain.

[0057] To mimic the normal developmental process, this invention involves the transplantation of microglia precursor cells into fetal mice at embryonic stage E9-14. Larger glial precursor cells are then injected into the brain within the first four days after birth, preferably the first two days. By the time the fetal mice mature, all three types of human glial cells will have expanded and matured in the mouse brain. Because microglia appear earlier than large glial cells during development, this approach more closely resembles the normal developmental process and is more important for research related to developmental neurodegenerative diseases.

[0058] Previous studies have shown that human glial precursor cells can competitively occupy brain regions in mice and gradually replace their glial cells. This competitive advantage is particularly pronounced in mice with brain damage. Existing research has developed pharmacological and genetic methods to remove glial cells from the mouse brain. As a pharmacological approach, systemic administration of colony-stimulating factor 1 receptor (CSF-1R) inhibitors such as PLX3397 effectively eliminates microglia. The glutamate homolog L-α-aminoglycolic acid (L-AAA) specifically eliminates astrocytes in the brains of healthy adult mice without toxicity to neurons or other glial cells. Copper hydrazones, copper chelators, can specifically kill oligodendrocytes in mice. Experiments have shown that long-term feeding of mice with copper hydrazone-containing diets reduces oligodendrocytes in the brain, thereby stimulating the proliferation of human oligodendrocyte precursor cells.

[0059] When macroglia progenitor cells are transplanted into immunodeficient mice lacking the myelin protein MBP, they proliferate and differentiate faster than when transplanted into immunodeficient mice with normal myelination. This indicates that the growth of macroglia progenitor cells is influenced by the brain environment. Therefore, it is necessary to select different immunodeficient mice as hosts to obtain human glial chimeric models. Studies have reported that macroglia progenitor cells can differentiate into oligodendrocytes and astrocytes. Since the differentiation of macroglia progenitor cells can be regulated by the brain environment, chimeric mice dominated by different cell types can be obtained by using different immunodeficient mice. When macroglia progenitor cells are directly transplanted into the brains of immunodeficient mice, chimeric mice with both oligodendrocytes and astrocytes present in equal numbers can be obtained. Oligodendrocyte-dominated chimeric mice can be obtained by using transgenic mice with demyelinating disease as hosts. This invention uses copper hydrazone to stimulate the proliferation and differentiation of human oligodendrocytes.

[0060] Human microglia depend on specific human cytokines; therefore, immunodeficient mice expressing hMCSF and hIL-34 are required when establishing microglia precursor cell chimeras. Since microglia invade the brain before the blood-brain barrier forms during development, this invention involves transplantation in fetal mice at embryonic days 9-14. Mouse microglia reject exogenous microglia, thus it is necessary to help the transplanted human microglia survive in the early stages of transplantation. Experiments have reported that CSF1R inhibitors can effectively kill mouse microglia. This invention administers CSF1R inhibitors to pregnant mice before transplantation, thereby creating space for human microglia survival. In addition, this invention also uses CSF1R-deficient transgenic mice as hosts to obtain microglia chimeras.

[0061] Single glial cell chimeras (oligodendrocyte-astrocytocyte chimeras, oligodendrocyte-lineage chimeras, and microglia chimeras) can be used to study the role of single cells in neurodegenerative diseases. However, degenerative diseases often present with multiple cellular pathologies; therefore, novel chimeras containing multiple glial cell types are needed. This means that human oligodendrocytes, astrocytes, and microglia will be present in the mouse central nervous system. Under pathological conditions, these glial cells not only respond to damage but may also be pathogenic.

[0062] To differentiate the roles of various cells in the pathological development of degenerative diseases, this invention utilizes a series of chimeric mice to study the pathology of specific degenerative diseases. When chimeric mice carry specific degenerative disease genes, these chimeric mice progressively exhibit the pathological characteristics of degenerative diseases. The technical solution of this invention allows for long-term detection of specific responses of human glial cells under different disease pathological states, such as determining the morphological characteristics of glial cells at different lesions through immunostaining, and further obtaining the transcriptomic characteristics of glial cells in different brain regions through emerging single-cell sequencing technology.

[0063] These methods allow us to identify disease progression through cell morphology and transcriptional characteristics, providing new targets and directions for early disease diagnosis. Furthermore, they can help identify the "double-edged sword" effect of glial cells in pathological development. Based on the location of glial cells in the lesion and their transcriptomic characteristics, we can obtain the properties of cells with different characteristics and regulate their behavior as needed. This can achieve the goal of disease treatment by inhibiting the proliferation of pathogenic cell subsets and promoting the expansion of protective cell subsets.

[0064] II. Analysis of Innovation Points

[0065] Glial cells play an important role in the physiological and pathological processes of the central nervous system.

[0066] NG2-positive oligodendrocyte precursor cells are not only precursor cells capable of differentiating into mature oligodendrocytes, but their unique characteristics also classify them as a separate type of glial cell. Although extensive research has been conducted, the function of NG2 cells, at least in the adult brain, remains largely unknown. Reported functions include: as an oligodendrocyte lineage, NG2 cells produce myelinating oligodendrocytes throughout their lives. In the adult mouse brain, NG2 cells form a tight homeostatic network, maintaining cell number under physiological conditions. When cell number decreases due to differentiation or cell death, this homeostatic network is activated, and nearby oligodendrocytes rapidly proliferate and migrate to maintain cell number balance. NG2 cells are rapidly proliferating cells in the brain parenchyma, giving them stem cell-like characteristics. Most interestingly, NG2 cells can form synaptic connections with neurons, initially found only in the hippocampus, but now widely reported in other brain regions. However, the function of these synaptic connections remains unclear. What is known is the directionality of this synaptic connection; NG2 cells can only receive signals from neurons but cannot generate action potentials or transmit electrical signals themselves.

[0067] The function of mature oligodendrocytes is relatively clear: they are myelin-producing cells that wrap around neuronal axons, aiding in the transduction of neuronal electrical signals and providing nutrients to the axons. Unmyelinated oligodendrocytes also exist in the brain, distributed in sparsely myelinated brain regions such as the gray matter of the cerebral cortex; their function remains unstudied. Because NG2 cells continuously differentiate into mature oligodendrocytes, these two seemingly independent cell types are closely linked. By knocking out myrf in oligodendrocyte precursor cells, the production of mature oligodendrocytes is completely blocked. Experiments have shown that continuous differentiation of mature oligodendrocytes is crucial for brain physiological function, especially in the early stages of learning complex motor functions.

[0068] Under physiological conditions, astrocytes control water transport and distribution and balance extracellular potassium ion concentration. They regulate the release of neurotrophic factors and the concentration of neurotransmitters near synaptic structures. An important function of astrocytes is to neutralize reactive oxygen species (ROS) produced near other cells. Astrocytes participate in the regulation of neurovascular coupling and can affect cerebral hemodynamics. They play a vital metabolic support role for surrounding neurons by absorbing glucose from blood vessels, converting it into lactose, and supplying it to neurons. Astrocyte function is impaired under pathological conditions. Stressed astrocytes' inability to control water transport leads to cerebral edema. Dysfunctional astrocytes also release harmful peroxides such as hydroxyl radicals, nitric oxide, and peroxynitrite.

[0069] Astrocytes also participate in synapse formation, synaptic maturation, neurotransmitter homeostasis, cerebral microcirculation, brain metabolism, and control and maintenance of the blood-brain barrier. Conversely, pathological or stressed astrocytes can reverse the direction of certain neurotransmitter transporters, thereby releasing glutamate and calcium ions, which can cause cytotoxicity. Increased intracellular calcium ions can activate calcium-dependent cysteine ​​proteases, leading to neuronal death. If the concentration of potassium ions in vitro cannot be balanced, it can lead to neuronal death due to overactivation. Astrocytes can also communicate with surrounding cells through exocytosis and endocytosis via cell membrane channels, transporters, and receptors.

[0070] Microglia are key members of the brain's defense system, resisting various physiological challenges and injuries. Morphologically, microglia resemble chameleons, exhibiting numerous protrusions in a resting state that transforms into a macrophage-like (amoebic) state in response to various stimuli. In damaged or diseased states, while astrocytes release various chemical factors, microglia are rapidly recruited to the damaged site. They can engulf various cellular debris and remove dead and non-essential cells. Besides responding to injury and infection, they can also release neurotoxic proteins and cytokines, causing pathological inflammation. New evidence supports the idea that microglia influence the formation of the nervous system and neural connections by regulating neuronal cell generation and synaptic pruning.

[0071] Glial cells play a crucial role in the physiology and pathology of the central nervous system. Neuron-dominated research can only reveal parts of the physiological and pathological processes. This invention's model will focus on the role of glial cells in the nervous system. This will not only enrich our existing understanding of the nervous system but also provide new ideas and directions for understanding the pathological development of diseases.

[0072] A novel glial cell chimeric mouse model containing human glial cells

[0073] While rabbits, weasels, guinea pigs, and rhesus macaques are also used as model animals in current research, the novel gelatinous chimeric mouse used in this invention is based on existing transgenic mice. Because mice and humans share 99% of their genes, and because mice and rats are relatively inexpensive to breed and raise, and reproduce rapidly, researchers can simultaneously study the function of specific genes in several generations of offspring over a suitable period. Furthermore, their physiology and genetics have been extensively studied, and they can be easily compared to humans. Techniques used to study genetics and specific gene functions, such as transgenic methods, have been developed for decades. Many mouse models of human diseases have also been established to facilitate research on pathogenesis and to evaluate the efficacy and toxicity of various candidate drugs. For decades, transgenic mice have been a commonly used model for studying neurodegenerative diseases due to their ease of operation.

[0074] Most neurodegenerative diseases are sporadic, with the risk typically increasing with age. However, some patients also exhibit familial inheritance, often linked to single-gene mutations. This familial nature of single-gene mutations allows us to establish animal models to study underlying pathogenesis. For example, up to 40% of frontotemporal dementia (FTD) cases and 10% of ALS cases are caused by a single pathogenic gene mutation. Up to 5% of AD is autosomal dominant, usually caused by a mutation in one of the APP gene or either presenilin 1 (PS1) or presenilin 2 (PS2). Approximately 15% of PD occurs in individuals with a family history, and about 2% of PD is caused by a single-gene (usually dominant) mutation. The pathogenic alleles also vary considerably among different populations. Given the increasing sophistication of gene-editing tools available for genetic engineering, such as CRISPR-CAS9, more mature methods can be employed to create mouse models to study the mechanisms of specific diseases, without necessarily requiring perfect simulations of human diseases.

[0075] Lifespan is another important factor to consider when designing mouse models of neurodegenerative diseases in middle-aged or elderly individuals. Transgenic mouse models expressing multiple copies of a mutant gene may exhibit accelerated mid-to-late-stage disease (if the number of mutant proteins is proportional to the incidence of disease), which is essential for studying the later stages of neurodegenerative diseases. However, the disease phenotype may be due to overexpression of the gene itself, rather than mutations in the transgenic DNA. The practicality of transgenic mouse models is exemplified in the field of prion disease research. Transgenic mice overexpressing human prion proteins help mimic bovine spongiform encephalopathy (BSE), also known as "mad cow disease," which spreads among mammals.

[0076] Another advantage of using mouse models to study neurodegenerative diseases is the ability to crossbreed independent mouse models and use the resulting double-mutant offspring to study disease networks, such as those involved in demyelinating and axonal neuropathies. Similarly, this classic genetic hybridization can reveal novel genetic interactions, such as the interaction between mutant SOD1 and the cytoplasmic dynein heavy chain in ALS models. Clearly, a single mouse model of any particular disease cannot provide all the results needed to study the molecular mechanisms of early and late stages of disease, as well as to develop treatments and biomarkers. A range of different animal models is required to address diverse questions regarding disease pathogenesis and treatment.

[0077] However, few transgenic mouse models perfectly mimic the realities of human diseases or prove effective in treating neurodegenerative diseases. Much of the uncertainty surrounding mouse models stems from the numerous differences between mice and humans, ranging from their genome and neuroanatomy to their immune system and behavior.

[0078] First, from a genomic perspective, approximately 90% of the 46 human chromosomes and 40 mouse chromosomes are conserved. Both species have a similar number of protein-coding genes, but about 1% of mouse genes appear to lack human homologs, and vice versa. These genetic differences may explain why mouse models cannot perfectly mimic human diseases. Second, humans and mice have different gene copy numbers. Regarding gene expression, the ENCODE (Encyclopedia of DNA Elements) project has revealed differences in many cis-regulatory structures between mice and humans, including the deoxyribonuclease I hypersensitive site (DHS). Additionally, mice and humans have different spliceosomes; humans produce more spliceosomes (an average of 3.4 subtypes per gene) compared to the average of 2.4 spliceosomes per gene in mice. Mouse non-coding RNAs differ significantly in sequence from those in humans. Mutations in non-protein-coding gene regions can lead to neuropathic disorders. By 2017, more than 3,000 different genome-wide association studies (GWAS) had reported more than 30,000 single nucleotide polymorphisms (SNPs) associated with diseases, the vast majority of which were located in non-coding regions, demonstrating the importance of gene regulation in diseases including neurodegeneration.

[0079] Secondly, subtle differences in amino acid sequences between humans and mice can have a profound impact on phenotype. While cellular and metabolic pathways may be similar in both species, their importance to each species can differ. Humans and mice may have evolved different primary biochemical, metabolic, and physiological pathways to meet different environmental needs. Therefore, fundamental differences in the mouse and human immune systems must be considered when explaining the link between immunology and neurodegenerative diseases. Genes may be more critical to one species than another. For example, while most shared genes would be lethal if deleted, the SOD1 gene is essential in humans but not in mice. This is important in mimicking SOD1-ALS, as most mutations leading to a loss of SOD1 activity may have more severe consequences in human motor neurons than in mice. Morphological differences are also important for establishing models of human diseases. Regarding the gut and microbiota, the cecum is important for microbial fermentation of food in mice, resulting in a longer relative length from small intestine to colon compared to humans. Although there are fewer differences in the peripheral nervous system between mice and humans, there are significant neuroanatomical differences in the brain and spinal cord.

[0080] Therefore, in the study of neurological diseases, it is necessary to target human cells. The novel glial cell chimeric mouse model of this invention contains human oligodendrocytes, astrocytes, and microglia. While transgenic mice can mimic the pathology and progression of human neurological diseases, they cannot reflect the responses of human cells. By transplanting human glial cells to replace mouse glial cells, it is possible to detect the progressive responses of human glial cells to specific neuropathologies. This approach leverages the advantages of existing transgenic animals while allowing for the specific study of human cellular responses.

[0081] A novel glial cell chimeric mouse model can obtain adult human glial cells.

[0082] Some researchers have questioned the practicality and effectiveness of mouse models in finding treatments, and have therefore turned to other cell models, such as those created using induced pluripotent stem cells (iPSCs). Neurons derived from iPSCs have been used to model various neurodegenerative diseases, most notably Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis (ALS). These in vitro models can deepen our understanding of the pathology of these diseases and provide insights into new treatment options. However, two-dimensional in vitro models cannot effectively reproduce the complexity of the nervous system, let alone simulate the neural structures and network connections of the brain.

[0083] In recent years, the establishment of three-dimensional neural cell culture systems or brain spheroids has helped us study human brain development. The first reported brain spheroid structure was created using optimized serum-free cultured mouse embryonic stem cell-derived brain progenitor cells. The authors demonstrated that this spheroid structure can spontaneously assemble neuroepithelial structures and form secondary brain regions under extracellular induction signals. The brain spheroid can reproduce human cortical development, including the regional composition of progenitor cells, neurogenesis, gene expression, and the human-specific extracellular glial cell layer. Several research institutions have reported cortical spheroids, midbrain spheroids, cerebellar spheroids, and neural tubes. However, whether these neurosomes can truly be used to study neural development or neurological diseases remains unknown.

[0084] Human iPSCs provide cell models and robust in vitro assays for high-throughput analysis, such as drug screening, which is not feasible in mouse models. However, the application of this model still faces many limitations. First, the variability between cell lines needs further optimization. Significant differences exist among different pluripotent stem cells, even among pluripotent stem cell lines established within the same research institution. Second, identifying the association between abnormalities observed in neurons induced by patient-derived pluripotent stem cells and the actual neuropathology in the patient's brain remains a challenge. Third, aging is a problem that cannot be addressed by using induced pluripotent stem cells to simulate disease. The vast majority of neurodegenerative diseases are age-related. During reprogramming, most age-related genes are turned off, and neurons or glial cells induced under in vitro conditions cannot truly mature and are younger than the patient's cells. Neurons at different developmental stages cannot accurately simulate the actual pathological stages of the disease. Researchers have used various stimuli to accelerate mitochondrial stress, nutrient deprivation, or oxidative stress to accelerate disease pathology. However, whether these stimuli reflect the actual disease pathology is unknown. In contrast, transplantation into the mouse brain into an intensive environment and allowing for prolonged differentiation allows cells to more closely resemble mature cells.

[0085] Brain spheroids mimic brain structure, exhibiting developmental stages similar to those of endogenous neurons, and generating various brain tissue types and regions. Brain spheroids can survive for extended periods under culture conditions, containing more mature and functional neurons, and may exhibit a relatively "aged" state. They may serve as an alternative method for studying aging. Since living human brain tissue is unavailable, brain spheroids are currently used to study brain structure. Brain spheroids cultured from pluripotent stem cells induced from patients with familial Alzheimer's disease or Down syndrome can spontaneously exhibit AD-related pathologies, including amyloid deposits and neurofibrillary tubercles.

[0086] However, mouse models remain essential because they allow us to explore the whole animal lifecycle, enabling us to understand the interactions between systems within the body, cell types, tissues, and the entire animal system. Mouse models also provide a means of studying the development, metabolic, and behavioral outcomes of diseases in their natural course. In particular, many symptoms in human patients undergoing treatment are behavioral (e.g., memory impairment and cognitive deficits), making it crucial to understand how molecular, cellular, and circuit changes lead to functional changes in the organism's behavior. Mouse models can also simulate environmental effects and individual responses throughout the lifecycle, which is not possible with cell line models.

[0087] Despite the existence of hundreds of mouse models of human neurodegenerative diseases, there remains no effective treatment for any form of neurodegenerative disease, and treatment options are very limited for the few diseases that are currently being explored. Many published articles focus on research into the mechanisms of disease in mice and the translation of therapies from mouse diseases into human clinical trials. The differences between mice and humans may not only be a significant factor contributing to current translational failures but may also be a source of research into pathogenesis.

[0088] III. Effect Evaluation

[0089] 1. Distribution of human glial cells in a novel glial cell chimeric mouse model.

[0090] In macroglia chimeras, human glial cells continuously proliferate in the mouse brain, while the number of mouse glial cells steadily decreases. By tracking the proportion of Ki67-positive glial cells over one year, we found that the proliferation rate of human glial cells was 20% in the early stages (within 3 months), but this rate decreased significantly with age, reaching approximately 2% at one year. Mouse glial cell proliferation showed a similar pattern, but the initial proliferation rate was lower than that of human glial cells, at only 5%, and dropped sharply to 1% after 2 months. Therefore, as mice age, the proportion of human glial cells in the mouse brain continuously increases, eventually replacing the vast majority of mouse glial cells. After one year of transplanting 200,000 cells, there were 10,000 human cells per cubic millimeter in the macroglia, based on the average mouse brain volume of 508.91 ± 23.42 mm². 3 The total number of human cells in each chimeric mouse was calculated to be 5 million. In the corpus callosum white matter region, the proportion of mature human oligodendrocytes was 10%, human oligodendrocyte precursor cells were 60%, and human astrocytes were 10%. If macroglia chimeric mice were fed a copper hydrazone diet from 2 months of age until 7 months of age, and the number of human glial cells was measured at 1 year of age, we found a significant increase in human glial cells, with a total number of human cells of approximately 50 million in each chimeric mouse. In the corpus callosum white matter region, the proportion of mature human oligodendrocytes was 50%, human oligodendrocyte precursor cells were 95%, and human astrocytes were 50%.

[0091] In microglia chimeras, human microglia exhibit similar characteristics, continuously proliferating in the mouse brain. By tracking the proportion of Ki67-positive glial cells, we found that the proliferation rate of human glial cells was 15% in the early stages (within one month). As the mice aged, the proliferation rate of human microglia decreased significantly, reaching approximately 2% at six months. After transplanting 200,000 cells for six months, there were 9,000 human cells per cubic millimeter in the large chimeric mice, based on the average brain volume of 508.91 ± 23.42 mm². 3 The total number of human cells in each chimeric mouse was calculated to be 4.5 million. Feeding two-month-old microglia chimeric mice a diet containing CSF1R inhibitors further stimulated microglia proliferation, increasing the total number of human microglia.

[0092] In dual-chimeric mice (macroglia and microglia), human glial cells continuously proliferate in the mouse brain, competitively rejecting and occupying the spaces of mouse glial cells. The proliferation rate of glial cells in monoglia is approximately 15%-20%, gradually decreasing to a level of 2%. Due to the specific and close interactions between human glial cells, the actual proliferation rate of glial cells is predicted to be even higher in monoglia mice.

[0093] 2. Application of a novel glial cell chimeric mouse model.

[0094] Using specific transgenic mice to study specific pathological features, focusing on early pathological prediction and early diagnostic markers.

[0095] When using transgenic mice to study neurodegenerative diseases, the mouse model should be selected based on specific needs. Transgenic mouse models are not suitable for clinical trials developing treatments for advanced stages of human diseases. Mouse models are generally better suited for understanding the function of specific genes or proteins or specific disease processes; some animals with slow-progressing diseases and relatively mild phenotypes are best suited for understanding the pathogenesis in the early stages of disease.

[0096] All neurodegenerative diseases raise several key questions for discussion. While significant progress has been made in understanding the key pathologies of Alzheimer's disease (AD), there are currently no effective drugs to treat or delay its progression. Numerous phase 3 clinical trials have failed due to lack of therapeutic efficacy. Many drug screening platforms face enormous challenges, and some pharmaceutical companies have even abandoned their AD drug development divisions. These studies demonstrate that the vast majority of degenerative diseases become untreatable in their late stages. Intervention must be initiated before irreversible pathological progression. Therefore, rapid and accurate diagnosis of disease progression is crucial for the treatment of neurodegenerative diseases.

[0097] This invention helps answer the above questions by constructing precise mouse models. Existing mouse models include gene knock-in mouse models, humanized mouse models, transgenic mouse models, chemically induced mutant mouse models, conditionally induced mutant mouse models, point mutation mouse models, chromosomal mutant mouse models, and trans chromosomal mutant mouse models. Each model provides relevant information for studying the process of neurodegenerative diseases. For early disease diagnosis, mouse models that mimic the early pathology of the disease are selected when choosing mouse models for neurodegenerative diseases.

[0098] In biomedical research, studies are typically limited to a specific aspect of a disease to minimize variability and maximize the chances of identifying significant differences between experimental and control conditions. This is achieved by breeding transgenic mice with identical genetic backgrounds and maintaining them under constant environmental conditions. Consequently, any significant findings in animal research, especially those related to treatment, may only be relevant to a subset of the clinical population.

[0099] To better understand the differences between mouse models and human pathology, new informatics methods, including machine learning, can help us find new phenotypes from the vast amounts of data generated by analysis. Developing human-mouse phenotypes, combined with new imaging and computational methods to facilitate cross-referencing between mouse and human datasets, will greatly enrich our understanding of human (and mouse) biology and will help elucidate neurodegenerative processes.

[0100] A comprehensive analysis of the role of glial cells through a series of chimeras.

[0101] Glial cell pathology, including microglia activation, astrocyte activation, and demyelination due to oligodendrocyte loss, is widely present in various neurodegenerative diseases. These cells are often the first to respond to brain injury. However, the relationship between glial cell pathology and the development of neurodegenerative diseases remains unclear. There are close interactions among glial cells; while focusing on a single cell type can provide insights into its role in neurodegenerative diseases, it does not allow for a comprehensive understanding of the ultimate consequences of these interactions. This invention uses a novel glial cell chimera model to simulate neurodegenerative diseases for early diagnosis.

[0102] The glial cell chimeric mouse of this invention comprises: an oligodendrocyte-dominated chimeric mouse, an oligodendrocyte-astrocytic chimera, a microglia chimera, and a macroglia-microglia chimera. The oligodendrocyte-dominated chimeric mouse can help us study the role of human oligodendrocytes and mature oligodendrocytes in neurodegenerative diseases. Since oligodendrocytes are the most actively proliferating cells in the adult brain, we can also study oligodendrocyte transplantation for the treatment of demyelinating diseases.

[0103] Oligodendrocyte precursor cell (ORC) and astrocyte chimeras can help us study the roles of both OCCs and astrocytes in neurodegenerative diseases. Current research in mouse models shows that these two cell types can interact to participate in the formation of glial scars in the brain. Existing studies have shown that this chimeric mouse can support the transmission of human-specific polyomavirus (JCV). By specifically regulating glial cell proliferation, we demonstrated the interaction between JCV and host cells.

[0104] Microglia chimeras can help us understand the role of human microglia in the central nervous system. In this invention's microglia model, human microglia completely replace mouse microglia. Since microglia and brain macrophages share the same origin during development, microglia will also contain some macrophages. This model will greatly advance our understanding of the contribution of microglia to intrinsic neuroimmunity. Because microglia in mice can rapidly proliferate and recover to normal cell numbers after being cleared by toxic drugs, microglia can be used for cell transplantation therapy or as drug delivery carriers.

[0105] The chimera of macroglia and microglia helps in studying the role of major human glial cells in neurodegenerative diseases. Oligodendrocyte precursor cells continuously differentiate into mature oligodendrocytes, and the dynamic balance between them affects normal brain function. Oligodendrocyte precursor cells can interact with astrocytes, playing a role in glial scar formation. The interaction between microglia and astrocytes is highly complex, as they jointly participate in intrinsic neuroimmune responses. On one hand, microglia can transmit signals to astrocytes after sensing external stimuli. Depending on the degree of damage, astrocytes further amplify the signal, activating the microglia's response. This forms an immune-inflammatory cycle that may drive the onset or progression of neurodegenerative diseases. Within microglia and astrocytes, individual cell responses to stimuli or damage can be completely different. They can be broadly categorized into two types: those that promote inflammatory responses and those that suppress inflammation and provide protection. Currently, there are no methods to study these changes in these two cell types, and it is unknown whether this difference in response can be controlled or transformed. The macroglia and microglia models of this invention will serve as a powerful tool to help us explore and regulate the neurointrinsic immune system.

[0106] Improve the ability to translate research findings into clinical practice

[0107] The slogan for clinical translation, "from the workbench to the bedside," encompasses two separate processes: first, the discovery of disease pathogenesis; and second, the development of new therapies and their clinical trials. Therefore, most attempts to translate findings from animal models into heterogeneous clinical populations (differing in age, sex, genetic background, environment, and life history) fail. The core issue remains the inherent variability in preclinical mouse studies and the significant variability in human clinical patient populations. Therefore, in mouse studies, variability should be accepted rather than rejected, and understanding its sources and underlying mechanisms may be helpful for successful translation into clinical patients.

[0108] Traditional mouse models are highly useful for exploring biological processes, but their applicability to effective treatments in humans has not yet been proven, with very few exceptions, including the successful use of antisense oligonucleotides to treat spinal muscular atrophy (SMA). Therefore, the current utility of mouse models is limited to discovering the pathogenesis of diseases. Their potential utility as drug screening tools may only become apparent after a better understanding of the pathological mechanisms and the reasons for current clinical translational failures. The novel glial cell chimeric mouse of this invention will enhance the translational capabilities of existing transgenic mouse research.

[0109] The primary purpose of animal models is to identify key pathological processes that can serve as drug targets. To date, in clinical trials for AD, ALS, FTD, and PD, drugs targeting animal models have largely failed, especially in treating cases or delaying disease progression. Reasons for this failure include inadequate preclinical design, unsuitable animal models, overly optimistic assessments of preclinical trials, lack of direct targets in humans, and inability to effectively evaluate clinical trial outcomes. There is a lack of correspondence between preclinical and clinical trials; therefore, it is crucial to pay attention to whether the treatment time window changes under different experimental conditions when designing and interpreting clinical trials. Drugs that can influence the early onset of disease may fail to improve disease progression in already affected individuals.

[0110] Therefore, the practicality (or other) questions regarding mouse models associated with human neurodegenerative diseases seem to only be answered after addressing the central issue of variability. Although treatments for human neurodegenerative diseases are currently scarce and our understanding of the mechanisms of neuronal death is relatively limited, we are rapidly entering a new era of personalized precision medicine. Using appropriate mouse models to address specific problems and examining the variability of these models can help in studying the different mechanisms of neurological dysfunction and neuronal death. Accepting and understanding variability can be of great help to translational medicine. On the other hand, novel chimeric models can be created using patient-specific induced pluripotent stem cells to understand individualized differences in neurodegenerative diseases, providing a diagnostic tool for further screening of specific therapeutic agents.

[0111] The gel chimeric mouse of the present invention can:

[0112] 1) Preclinical drug screening for the development of drugs to treat neurodegenerative diseases.

[0113] 2) For the development of personalized medicine, personalized glial chimeric mice are established by using glial progenitor cells differentiated from induced pluripotent stem cells of patients for personalized drug screening and evaluation.

[0114] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be understood that the embodiments and figures should not be construed as limiting. Those skilled in the art will be able to clearly conceive of further modifications to the principles set forth herein.

[0115] Example:

[0116] Example 1: Induction of iPS cells to differentiate into glial progenitor cells

[0117] Referring to published methods for directed differentiation of glial cells, and improving upon them, the cell culture process was used to induce the preparation of glial precursor cells. Macroglia and microglia precursor cells were induced separately, and then glial chimeric cells were prepared through cell transplantation. The specific experimental procedure is as follows: Figure 1 .

[0118] Induction steps for macroglial precursor cells:

[0119] iPS cells were expanded and cultured in suspension to form embryoid bodies (EBs). After 5 days of EB culture, RA or Purmorphomine was added to induce neuroectoderm formation. After 9 days, the neuroectoderm cells were cultured in adherent form. After 10 days, further expansion of the neuroectoderm was induced by adding bFGF, and the neuroectoderm bodies were cultured in suspension. The culture medium was replaced with glial cell induction medium containing PDGFaa, NT3, and IGF-1, and the suspension of large glial precursor cell spheroids was cultured for up to 90 days. The large glial precursor cell spheroids were mechanically cut into small cell clusters and cultured in adherent form for 1-2 months. The purity of the cultured large glial precursor cells was determined by flow cytometry analysis of the proportion of PDGFaa and olig2 positivity.

[0120] Microglial precursor cell induction steps:

[0121] iPS cells were seeded into Matrigel-coated cell plates. The density of the germinal cells was adjusted, and cell culture medium containing BMP4 and Activin was added to induce endoderm development. Four days later, VEGF was added to induce and expand the proliferation and transformation of vascular endothelial cells. After 10 days, a large number of CD43-positive primitive hematopoietic stem cells were obtained. Further differentiation and maturation of hematopoietic stem cells were induced by the addition of IL-34, MCSF-1, and TGF-β to obtain microglial precursor cells. The purity of the cultured microglial precursor cells was identified by flow cytometry analysis of the proportion of P2Y12R and SALL1 positivity.

[0122] The induced glial progenitor cells can be cryopreserved in batches, and detailed information on cell purity and induction cycle will be archived for later experiments. A portion of the glial progenitor cells will be co-cultured for post-transplantation cell viability testing and quality control.

[0123] Example 2: Cell transplantation surgery

[0124] Freshly induced glial progenitor cells or cryopreserved and revived glial progenitor cells can be used for cell transplantation experiments. To study the amyloid-related pathology of Alzheimer's disease, we used a novel AD mouse model created by crossing 5xFAD transgenic mice with immunodeficient mice containing human MCSF-1 and IL-34 for cell transplantation research. 5xFAD transgenic mice accurately mimic early AD pathology, including the production of toxic Aβ, with pathogenic Aβ first appearing in the Papez circuit. In the late stages of AD pathology, 5xFAD mice showed significant Aβ deposition, neuronal death, and cognitive decline. Another important pathological feature of AD is neurofibrillary tangles; we used a novel AD mouse model created by crossing Tau P301L transgenic mice with immunodeficient mice containing human MCSF-1 and IL-34 for cell transplantation research.

[0125] Based on experimental requirements, single glial progenitor cells can be transplanted using P0-2 newborn mice or E9-14 fetal mice from the aforementioned novel AD mouse model. When a chimeric model with multiple glial cell types is needed, a mixture of macroglia and microglia progenitor cells can be transplanted simultaneously. See details... Figure 2 .

[0126] Cells intended for transplantation were resuspended in calcium- and magnesium-free HBSS to 10⁻⁶. 5 Cells / µL. For experiments involving the simultaneous transplantation of a mixture of macroglia and microglia, the cell density will be adjusted to 10-1 cells per cell type. 5 Cells / µL, total cell concentration 2 x 10⁻⁶ 5 Cells per microliter. Unless otherwise specified, the cells used for transplantation below may be macroglial precursor cells, microglial precursor cells, or a mixture of macroglial precursor cells and microglial precursor cells.

[0127] Transplantation was performed using fetal mice. E9-14 fetal mice were transplanted under inhalation anesthesia. The abdomen of the pregnant mice was disinfected with alcohol and iodine, and the hair was removed. A 1.5 cm incision was made in the abdominal skin, 1 cm above the urethra. The muscle layer was then cut open to expose the fetal mice. Cells were injected using a pulled glass needle. Injections were made one by one along the Y-shaped uterus of the mouse, adjusting the position so that the brain was directly attached to the uterine wall. Then, 3 μL of cells were injected into the ventricle of the mouse by piercing the uterine wall with a glass needle. After all injections were completed, all fetal mice were returned to the abdominal cavity of the mouse, and 0.9% saline solution was added. The muscle and skin layers of the mouse were sutured in two layers. The pregnant mice were kept separate after regaining consciousness post-surgery. After birth, the fetal mice were fed with fostermom until weaning.

[0128] Cell transplantation was performed using newborn mice. Newborn mice, 1-2 days old, were anesthetized using a rapid freezing method. After disinfection with alcohol and iodine, the mice were placed in a homemade mold, and cells were injected into the brain via direct puncture of the skin and skull. Four injection sites were chosen: AP ± 1.0 mm, ML ± 1.0 mm, and ventral 1.2 mm. 50,000 cells were injected at each site, for a total of 200,000 cells per mouse. After injection, the newborn mice were disinfected with alcohol and iodine and placed on a heating plate to recover. Once recovered from anesthesia, the mice were returned to their mothers. The transplanted mice were weaned and separated into different cages after 21 days.

[0129] Example 3: Accelerating the expansion and maturation of human glial cells in chimeric mice

[0130] The distribution of human glial cells in mouse brains was examined at 1 and 3 months post-transplantation. Human astrocytes were labeled with the human cell-specific marker hN and the glial cell-specific marker hGFAP, human oligodendrocytes with the human cell-specific marker hN and the oligodendrocyte-specific marker PDGFRa, and human microglia with the human cell-specific marker hN and the microglia-specific marker P2Y12R. The total number of glial cells was calculated by counting the number of cells per square centimeter of brain region. Ki67 was used to track cell proliferation.

[0131] Human cells have a much longer proliferation cycle than mouse cells. When human cells enter a mouse brain, they slowly compete with and eventually replace the mouse's glial cells. This process naturally takes 1-2 years. This invention accelerates this process by externally inducing apoptosis in mouse glial cells.

[0132] Chemical inhibitors were used to stimulate the proliferation of human glial cells. To increase the expansion of human macroglia, mice that had undergone macroglia progenitor cell transplantation were given a diet containing 0.2% copper hydrazone after weaning. To increase the expansion of human microglia, mice that had undergone microglia transplantation were given a diet containing PLX3397 (290 mg / kg) after weaning. Another method is to use cell transplantation experiments using existing transgenic mice lacking microglia. CSF1R-deficient mice cannot develop macrophages and osteoclasts normally, thus lacking microglia in the central nervous system and macrophages and osteoclasts in the peripheral nervous system. Transplantation of human hematopoietic stem cells can simultaneously compensate for the cell losses in the central and peripheral nervous systems of these mice, thereby enabling them to have normal lifespan and fully humanized microglia.

[0133] Glial cells cultured in vitro often remain in a progenitor state and cannot truly mature due to the lack of abundant brain environment nutrients. RNA-seq and ATAC-seq sequencing were used to identify the maturity level of human glial cells in chimeric mice.

[0134] Example 4: Collection of chimeric mouse brain samples

[0135] Animal samples were collected at corresponding time points in the pathological development of the animal model of the simulated disease. Mice were anesthetized and fixed by cardiac perfusion with PBS and 4% PFA. The brain or spinal cord, the organs to be studied, were isolated from the mice according to experimental requirements. The tissues were fixed in 4% PFA for 2 hours, then transferred to PBS overnight. Finally, the brain tissue was transferred sequentially to 10% and 30% sucrose solutions for dehydration and cryopreservation. The tissues were then embedded and stored at -80°C or in liquid nitrogen.

[0136] Example 5: Brain Sample Detection

[0137] Brain samples were analyzed using a strategy combining immunohistochemistry and gene sequencing. Frozen sections 14 micrometers thick were prepared. Following the traditional immunohistochemical staining procedure, a staining chamber was first created by drawing circles along the brain tissue with a water-resistant pen. Next, PBS was added to allow the brain tissue to fully absorb water; a buffer containing perforated material was added to increase the permeability of the brain tissue; and a buffer containing serum was added to block non-specific antibody binding sites. The brain tissue was then incubated at 4°C for 24 hours with a buffer containing antibodies. Finally, the brain tissue was rinsed with PBS and stained with the appropriate fluorescent secondary antibody. After staining, the slides were mounted with mounting medium. The stained brain tissue can be used for microscopic imaging (see [link to article]). Figure 4 And store it for a long time.

[0138] Slide sequencing was used to obtain spatial-level single-cell transcriptome information. Freshly frozen brain tissue slices, 5 micrometer thick, were placed directly onto a slide rich in magnetic beads containing T-enriched primers encoding spatial UMI. Brain tissue was then lysed and reverse transcription was performed in situ. The reverse transcription products were collected and amplified to construct sequencing libraries for gene sequencing. Gene analysis was then used to obtain cellular transcriptome information at the single-cell level in specific anatomical brain regions.

[0139] By combining immunohistochemical staining with slide sequencing, we can link morphological changes in specific cells with transcriptomic alterations. This will provide important evidence for our understanding of cell function and potential transcriptomic changes.

[0140] Example 6: Validation using patient samples (development of early diagnostic markers, detection of disease progression milestones)

[0141] Cellular phenotypes or molecular targets obtained using the human glial chimera model will be validated using patient samples. First, suitable human brain samples are selected based on pathological characteristics. Second, for cell phenotype detection, immunohistochemical staining is used to display the morphological characteristics of glial cells, and a database of human glial cell images is obtained through cell imaging. Machine learning is used to classify cell morphology types and compare them with the image database obtained through the glial chimera model. For the detection of cellular molecular targets, gene sequencing technology is used to obtain the transcriptomic characteristics of glial cells in specific brain regions at the single-cell level. Validated molecular targets will be used to develop specific PET probes. Ultimately, clinically applicable diagnostic markers will be obtained.

Claims

1. A method for preparing a rodent model of neurodegenerative disease, comprising the following steps: 1) Inducing human stem cells to differentiate into macroglial precursor cells and microglial precursor cells, wherein the human stem cells are induced pluripotent stem cells; 2) Cell transplantation is performed using newborn or intrauterine rodents as hosts, transplanting macroglia and microglia from step 1) to form chimeric animals with human cells. 3) Accelerate the directed differentiation, expansion, and maturation of macroglia and microglia progenitor cells transplanted into chimeric animals into human glial cells; Step 3) is achieved by removing the host's own glial cells from the brain using pharmaceutical or genetic methods; The drug specifically removes glial cells from the host's own brain and is selected from copper hydrazone, colony-stimulating factor 1 receptor inhibitor PLX3397, and glutamate homolog L-α-aminoglycolic acid. The rodents are novel AD mice produced by crossing 5xFAD transgenic mice with immunodeficient mice containing human MCSF-1 and IL-34.

2. The method of claim 1, wherein the rodent is a transgenic animal lacking glial cells, and the transgenic animal lacks colony-stimulating factor 1 receptor or myelin protein MBP.

3. The method of claim 1, wherein the rodent is a newborn mouse of 0-4 days old, or a fetal mouse of 9-14 days of embryonic development.

4. The method of claim 3, wherein the rodent is a newborn mouse of 0-2 days old, or a fetal mouse of 9-14 days of embryonic development.

5. The method according to any one of claims 1-4, wherein the induced pluripotent stem cells are derived from patients with neurodegenerative diseases.

6. The method of any one of claims 1-4, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

7. The rodent model prepared by the method of any one of claims 1-6 is used for the study of neurodegenerative diseases.

8. The use as claimed in claim 7, wherein the neurodegenerative disease is in the early stage of the disease.

9. The use of the rodent model prepared by the method of any one of claims 1-6 for screening or identifying candidate agents that can alleviate or treat neurodegenerative diseases.

10. A method for screening or identifying candidate agents that can alleviate or treat neurodegenerative diseases, comprising the following steps: 1) Apply the candidate reagent to the rodent model prepared by the method according to any one of claims 1-6; 2) Determine whether the candidate reagent has a relieving or therapeutic effect on one or more signs, symptoms and / or conditions associated with neurodegenerative diseases; and 3) Identify candidate reagents that have a relieving or therapeutic effect on one or more signs, symptoms and / or conditions associated with neurodegenerative diseases.

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