CN116478923B - Preparation method of astrocyte - Google Patents

Abstract

The present application provides a method for preparing astrocytes, comprising culturing neural stem cells or neural precursor cells in a chemically defined medium by adherence. The invention also relates to functional astrocytes prepared by said method and to the use thereof. In particular, the astrocyte preparation method of the present invention may be adapted for preparing astrocytes suitable for clinical use.

Description

Preparation method of astrocyte

Technical Field

The present application relates to a cell culture method, and in particular, to a culture method for preparing astrocytes having a desired function using neural stem cells and/or neural precursor cells.

Background

Astrocytes, one of the large number of glial cells that make up neural tissue, play a vital role in maintaining an extracellular environment suitable for neurons. Astrocytes do not conduct electrical impulses and have been considered support cells. Astrocytes and other glial cells have not been well studied to a large extent compared to neurons.

It is now known that altered astrocyte function plays a key role in Amyotrophic Lateral Sclerosis (ALS) and other neurodegenerative diseases. Astrocytes have also been reported to be responsible for controlling the strength of neuronal connections, mediating the elimination of synapses by phagocytizing the "synaptic pruning" mechanism of old synapses, thus playing an important role in synaptic remodeling (Won-Suk Chung, et al 2013, nature. Dec 19;504 (7480): 394-400). Astrocytes are therefore considered to be useful in the treatment of neurodegenerative disorders and central nervous system injury, playing a role in the repair of Brain injury and for therapeutic purposes (barbeitto l.stem Cell Res ter.2018; 9 (1): 241;Weber RZ,et al., brain pathl.2021; 31 (5): e 12999). Human astrocytes are large in volume and complex in structure, propagate calcium waves at a higher speed than rodent astrocytes, and are suitable for repairing injury of human brain.

As a method for producing astrocytes, a classical differentiation method is to obtain astrocytes having a high purity by natural differentiation after culturing neurospheres (neurospheres) in suspension for 180 days (Krencik R, et al, nat Biotechnol.2011;29 (6): 528-534). The cell functionality and maturity obtained by such methods are high, but limited by low yield and long cycle times.

Later, a method was developed that uses less time. The method uses adherent Neural Precursor Cells (NPC) cultured for 28-90 days and 1-10% Fetal Bovine Serum (FBS) added to expand astrocytes (Tcw J et al, stem Cell reports.2017;9 (2): 600-614). The purity of the cells obtained by such a method is low, sorting and purification are required, and FBS is required. In view of the fact that FBS is a serum and not a chemically defined reagent, the use of FBS can cause product lot variations, introduce contamination with animal-derived components, and therefore it is desirable to minimize the use of FBS in the process of obtaining astrocytes.

Recent approaches have been to promote differentiation by allowing cells to express high levels of genes associated with astrocyte development, mainly by means of viral infection or gene editing (Canals I et al, nat methods.2018Sep;15 (9): 693-696.). Such a method can differentiate to obtain mature astrocytes of higher purity after about 21-60 days. However, there is a potential safety hazard due to the need to genetically engineer cells.

Current astrocyte differentiation methods based on human induced pluripotent stem cells (hipscs) generally rely on Neural Precursor Cells (NPCs) or oligodendrocyte progenitor cell intermediates. While it has been widely demonstrated that hiPSCs can differentiate into functional astrocytes for use in cell-based models of in vitro neuropsychiatric diseases or in vivo implantation to treat neurological diseases, the current methods are slow (up to 6 months) or require sorting to improve purity.

In addition, from the standpoint of using cells for clinical treatment, it is difficult to simply convert the above methods into production methods that are suitable for clinical-grade use. In order to meet the clinical level, it is impossible to use a preparation containing animal components, and it is also necessary to avoid the introduction of genetic modification or the like with unknown safety as much as possible.

Thus, there remains a need in the art for a relatively short time-consuming and reproducible process for the preparation of human astrocytes, and the astrocytes thus prepared can be adapted for clinical use. Therefore, in order to mass-produce therapeutic astrocytes in a short period of time and to study the functions of astrocytes, a new method for producing human astrocytes is required.

Disclosure of Invention

The inventors developed a method of adherent culture that obtains an astrocyte population having a desired function in a relatively short time by a simple operation using cells having the differentiation potential of astrocytes as a starting cell material, thereby completing the present invention.

The method of the invention is simple and easy to implement, and can use various cell types as initial cells, including neural stem cells, neural precursor cells or cultures of neural stem cells and neural precursor cells which undergo certain differentiation. The method of the present invention does not involve gene editing and does not use reagents of animal origin such as serum, and thus has high safety. On the basis, the culture medium and the reagent used in the method can be replaced by corresponding substances meeting the production standard of clinical products, so that the method and the products obtained by the method can meet the clinical use standard and specification.

Accordingly, in a first aspect, the present application provides a method of preparing astrocytes comprising a rapid and convenient method of obtaining functional astrocytes from a cell population having astrocyte differentiation potential (e.g., neural Stem Cells (NSCs), neural Precursor Cells (NPCs), NSCs-or NPC-derived cell products), comprising subjecting said cell population to adherent culture. The proportion of GFAP positive cells in astrocytes obtained by the method is not less than 60%. In a preferred embodiment, the astrocytes obtained by the method are astrocytes suitable for clinical use.

In a second aspect, the present application provides astrocytes or a population of cells containing astrocytes obtained by the method of the first aspect.

In a third aspect, the present application provides a method for producing an astrocyte comprising the astrocyte or population of cells of the second aspect, wherein the astrocyte is further comprised of astrocyte precursor cells, said astrocyte precursor cells being s100deg.P positive.

In a fourth aspect, the present application provides the use of the astrocytes of the second aspect or the cell population of the third aspect for the preparation of a medicament. For example, the medicament is useful for treating diseases associated with the Central Nervous System (CNS). For example, the medicament is for the treatment of neurodegenerative diseases.

In a fifth aspect, the present application provides a method of preparing a neuron comprising co-culturing an astrocyte or cell population of the second aspect with a cell having the potential to differentiate into a neuron, such as a neural stem cell or neural precursor cell.

In a sixth aspect, the invention relates to a reagent and medium combination for use in the method of the first aspect, or a kit comprising them.

The methods of the present application allow for the obtaining of astrocytes differentiated into different brain region attributes by using populations of astrocyte differentiation potential cells of different brain region origin, all of which are included within the scope of the present application.

The advantages of the present application are at least the following.

The method for differentiating cells is simple and does not need to carry out separation purification.

The methods of the present application do not involve genetic engineering of cells, do not involve the use of vectors such as viruses to introduce genes into cells, do not require artificial increases in the expression of specific genes or secretion of specific factors, or allow cell surface expression of tags or expressions for sorting. The method of the invention thus saves costs and shortens the time required to obtain cells. More importantly, the safety of the cells obtained by the method of the invention when used in cell therapy is increased because the introduction of exogenous genes is not involved.

The astrocytes obtained by the method of the present invention have a desired function, and thus are capable of performing important functions of astrocytes in vitro, including neuronal support, glutamate uptake and phagocytosis.

The method has high yield, and the obtained mature astrocytes have high purity and high maturity. Not only suitable for research use, but also can be used for clinical use.

The method of the present application can obtain cells suitable for clinical applications, such as clinical grade cells, due to the production and preparation of cells using clinical grade reagents. Therefore, the method of the invention has great economic value.

Meanwhile, since the astrocytes prepared by the method of the present application have high purity and high yield and meet clinical grade standards, the astrocytes of the present invention are more suitable for clinical applications, such as clinical therapeutic applications, for example, for treating central nerve injury, neurodegenerative diseases such as parkinson's disease, huntington's disease or alzheimer's disease, etc., than the astrocytes obtained by other methods.

Drawings

The following detailed description and accompanying drawings are provided to facilitate a better understanding of the features and advantages of the present application. However, those skilled in the art will appreciate that these are for the purpose of illustrating the present application only and are not limiting of the present application. The scope of the application is defined by the claims.

FIG. 1A is a flow chart of a method of preparing astrocytes according to the present application.

FIG. 1B is a schematic flow chart of the preparation of CNS neural cells starting from somatic cells or embryonic stem cells.

Fig. 2A-B are fluorescence micrographs of the cells of example 1, each plot showing a different color channel, corresponding to a different marker. (A) Fluorescent photographs of glial precursor cells obtained by serial passage in example 1, blue channel on the top left, show DAPI staining; the upper right is red channel, showing s100deg.S expression; the lower left is a mixing channel; (B) Fluorescent photographs of cells (astrocytes-based) at day 24 of differentiation, first column blue channel, showing DAPI staining; the second column is the red channel, showing GFAP expression; the third column is purple channel, showing the expression of s100deg.P; the fourth column is the mixing channel. DAPI:4', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole) shows a dye of nuclei by binding to DNA; GFAP: a glial cell marker; s100deg.P: glial precursor cell marker.

FIG. 3 is a histogram showing the results of the glutamate uptake assay in example 1 comparing glutamate uptake levels at 0 and 3 hours in the control group and the EAAT2/4/5 inhibitor L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC) treated group. EAAT2/4/5: excitatory amino acid transporter2/4/5 (Excitatory amino acid transporter 2/4/5), which is an astrocyte surface glutamate transporter.

FIG. 4 is a photograph showing the results of the mechanical scratch test of the cells obtained in example 1, showing the diffusion of calcium waves (green fluorescence) at various time points.

FIG. 5 is a photograph showing the results of the cells obtained in example 1 in an ATP (adenosine triphosphate) stimulus test, showing the diffusion of calcium waves (green fluorescence) at various time points.

FIGS. 6A-B are histograms showing the results of the cells obtained in example 1 in a neuron support experiment, comparing the levels of (A) the number of Segments/number of neurons (Segments/Number of neurons) and (B) the Length/number of neurons (Length/Number of neurons) of the (A) newly-generated neurons on days 1, 2, 3, 4, and 7 of NPC co-culture differentiated group with astrocytes and NPC alone differentiated group. AS-neurons: neurons co-cultured with astrocytes; neurons: neurons cultured alone.

FIGS. 7A-E are bright field photographs of cells in example 2, (A) representative bright field photographs of NPC cells in example 2 that initiate differentiation; (B) Representative bright field photographs of cells serially passaged 1-2 passages in example 2; (C) Representative bright field photographs of cells serially passaged 3-5 passages in example 2; (D) Representative bright field photographs of astrocytes precursor cells in example 2; (E) Representative bright field photographs of astrocytes in example 2.

FIG. 8 is a fluorescence micrograph of astrocytes differentiated on day 22 in example 2. Blue channel on the top left, showing DAPI staining; the upper right is the green channel, showing s100deg.S expression; the lower left is the red channel, showing GFAP expression; the lower right is the mixing channel.

FIG. 9 is a histogram of the results obtained in the glutamic acid uptake experiment for the cells obtained in example 2, comparing the glutamic acid uptake levels at 0 hours (0 h) and 1 hour (1 h) for the control group and the EAAT2/4/5 inhibitor PDC treated group.

FIG. 10 is a photograph of the results of the mechanical scratch test of the cells obtained in example 2, showing the diffusion of calcium waves (green fluorescence) at various time points.

FIG. 11 is a photograph of the results of the cells obtained in example 2 in an ATP (adenosine triphosphate) stimulus test, showing the diffusion of calcium waves (green fluorescence) at various time points.

FIGS. 12A-B are flowcharts of different culturing methods in the examples. (A) the cultivation method of example 1; (B) the cultivation method of example 2.

Detailed Description

Definition of the definition

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Unless specifically defined elsewhere in this disclosure, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

In this document, including in the following claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

The term "or" means and is used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

In the context of the present disclosure, unless otherwise indicated, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated element, e.g. component, attribute, step or group thereof, but not the exclusion of any other element, e.g. component, attribute and step. When the terms "comprise," "include," or any variation thereof, are used herein, they may be substituted with the terms "comprise," "include," or sometimes "have" or equivalents thereof. In certain embodiments, the term "comprising" also includes "consisting of.

Cell types at different stages

The present invention is directed to the preparation of high quality astrocytes, a central nervous system neural cell. For a better understanding of the flow of preparing neural cells of the central nervous system, this section describes the various cell types and their relationships, and their roles in the methods of the present application, with reference to the flow chart in FIG. 1B. The various cell types referred to in FIG. 1B are described substantially in the order from downstream to upstream.

The term "central nervous system" or "CNS" refers to the nervous system consisting of the brain and spinal cord. Cell types in the central nervous system include neuronal cell types (neuronal cell types), glial cells (glial cell types) and some other non-neural cells, such as immune system cells. In the context of the present invention, cells belonging to the neural lineage and located within the central nervous system are collectively referred to as "central nervous system neural cells".

The term "astrocyte" or "astrocyte" is a central nervous system nerve cell. Astrocytes are the most abundant glial cells in the brain. Because of the many protrusions, they are morphologically similar to "stars" and are named. Astrocytes possess a variety of functions including neuronal support, glutamate uptake and phagocytosis. Astrocytes can be identified by expression of GFAP and s100deg.P.

"GFAP" refers to glial fibrillary acidic protein (glial fibrillary acidic protein). GFAP is expressed in many cell types. In the central nervous system, GFAP is expressed in astrocytes. GFAP can be used as a marker for astrocytes.

"S100deg.P" refers to S100 calbindin B, belonging to the S100 calbindin family. s100deg.S is expressed mainly in astrocytes. During astrocyte differentiation, s100deg.P is often used as a marker for astrocyte precursor cells, and s100deg.P is expressed earlier than GFAP.

The term "astrocyte precursor cells" is understood in a broad sense as stem cells or precursor cells capable of forming mature astrocytes. In the context of the present invention, "astrocyte precursor cells" more specifically refer to precursor cells derived from NPC and capable of forming astrocytes. Astrocytes also belong to the central nervous system nerve cells. Astrocytes may have a high level of s100deg.beta.expression compared to astrocytes, whereas GFAP is not expressed or expressed in a lower amount.

As the starting cellular material preferably used in the adherent culture step of the invention, "neural precursor cells (neural progenitor cell)" or "NPC" as used herein refers to a cell type capable of differentiating into cells in the neural lineage (neurol lineage) including, but not limited to, neurons, glial precursor cells and glial cells, such as astrocytes, oligodendrocytes and oligodendrocytes. The precursor cell is a stem cell-like pluripotent cell, but has a lower replication and proliferation capacity than the stem cell. However, precursor cells still have the ability to replicate and can differentiate further into different cell types compared to fully differentiated cells. Whether NPC successfully differentiates to a specific cell type can be determined by morphological observation, cell specific molecular marker detection, and in vitro differentiation to a specific neural cell type with specific molecular markers and electrophysiological activity. NPCs used in the methods of this invention can exhibit typical neural precursor cell morphology, including relatively homogeneous columnar cell and rosette-like radial cell arrangements.

NPC can express a variety of characteristic markers selected from Nestin, SOX2, PAX6, FOXG1, FOXA2, LMX1A, OLIG2, HOXA3. In general, NPC that can differentiate into cells in different brain regions will express both Nestin and SOX2. In addition, NPC expresses markers for specific brain regions. For example, PAX6 and FOXG1 can be markers for forebrain neural precursor cells; FOXA2 and LMX1A can be markers for the midbrain neural precursor cells; OLIG2 and HOXA3 can be used as markers for spinal cord nerve precursor cells.

"NESTIN" is an intermediate fibrin that is expressed at an early stage of central nervous system development. Nestin is a common marker of NPC.

"SOX2" refers to sex-determining region Y-related HMB box protein 2 (sex-determining region Y-related HMG box 2). SOX2 is a key regulator protein in embryonic stem cells and neural precursor cells and is thus used as a marker of NPC.

"PAX6" refers to p-box 6 (paired box 6). PAX6 is involved in the proliferation of neural precursor cells and in the determination of neural fate, one of the key regulatory proteins and markers in neural precursor cells.

"FOXG1" or "FOXG1" as used herein represents "fork G1", which is one of the transcription factors expressed earliest in the human brain development process, inducing development of the terminal brain into several critical structures, including the cerebral cortex. The cortex is derived from an organ primordium that expresses forebrain fork box G1 (FOXG 1). FOXG1 can therefore serve as a marker of precursor cells that eventually differentiate into forebrain cells.

In a preferred embodiment, the NPC used in the present invention expresses one or more, preferably all, NESTIN, SOX2, PAX6 and FOXG 1. In another embodiment, at least a majority of NPCs, e.g., at least 60% NPCs, at least 70% NPCs, at least 80% NPCs, at least 90% NPCs, in a population of cells comprising NPCs for use in the invention express one or more, preferably all, of nettin, SOX2, PAX6 and FOXG 1.

NPC can be obtained from Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs) by in vitro methods. In a specific embodiment, the NPCs of the invention are obtained from ipscs by an in vitro method. In a specific embodiment, the NPC of the invention is obtained from an ESC by an in vitro method. For example, the embryonic stem cells are commercially available embryonic stem cells. For example, the embryonic stem cells are derived from human embryos (specifically, blastocyst inner cell mass) within 14 days of fertilization without in vivo development.

Methods for obtaining NPC from pluripotent stem cells are well known in the art. In a preferred embodiment, the NPC of this invention are obtained using the RONA method. The method provides for the differentiation of iPSC or ESC to provide RONA, from which neurospheres are then formed, and finally NPCs are formed. For a specific description of the RONA method, reference is made to the method described in the JC.xu article (Xu et al 2016,Science Translational Medicine,Apr 6;8 (333): 333ra 48). The method for obtaining clinical grade NPC using the RONA method can be found in the method described in CN 113604434A.

The term "neurosphere" refers to spherical neural fate cell aggregates isolated from RONA formed from suspension cultures. Neurospheres are heterogeneous populations of different types of neural cells, including neural stem cells, neural progenitor cells, and some differentiated neural cells. In some embodiments, the neurosphere consists essentially of NPC. The neurospheres can be dispersed or digested into single cells and then inoculated onto the culture surface coated with matrigel, such as in a cell culture plate, to form a high purity monolayer of NPC.

The terms "neural aggregates derived from rosette-like neural stem cells", "rosette-like neural aggregates" or "RONA" are used interchangeably herein to refer to aggregates of neural stem cells derived from ESCs or iPSCs that spontaneously organize from neural stem cells to form highly compact three-dimensional columnar neural aggregates. Preferably, the RONA is positive for FOXG1 and nettin in immunostaining.

The term "stem cell" refers to a cell that has the potential to differentiate into a variety of different cell types. Stem cells are able to form more stem cells that are identical in nature by dividing.

The term "embryonic stem cells (embryonic stem cell)" or its abbreviation "ESCs" refers to pluripotent (pluripotent) stem cells derived from the blastocyst stage of a mammalian early embryo. As a stem cell, embryonic stem cells have a strong self-renewal capacity and are capable of differentiating into various embryonic cell types. In a specific embodiment, the embryonic stem cells used in the invention are human embryonic stem cells, preferably embryonic stem cells (specifically, blastocyst cell mass) from human embryos within 14 days of fertilization that have not undergone in vivo development.

The term "induced pluripotent stem cells (induced pluripotent stem cell)" or its abbreviation "iPSC" refers to pluripotent stem cells produced by reprogramming from differentiated somatic cells. The most commonly used somatic cells include fibroblasts, blood cells, epithelial cells, and the like.

In a specific embodiment, the iPSC may be obtained using a reprogramming method as disclosed in WO 2021/018296. The method uses a combination of two transcription factors and three small chemical molecules. The two transcription factors are Oct4 and Nanog. Three chemical inducers are tgfβ receptor inhibitors, cyclic AMP agonists and glycogen synthase kinase inhibitors, specifically 616452, forskolin and TD114-2. However, it will be understood by those skilled in the art that in embodiments in which the NPC is derived from an iPSC, the specific method of obtaining the iPSC is not intended to be limiting.

Another cell that may be used as a starting material is a neural stem cell. The term "neural stem cells" exists in the nervous system and has the potential to differentiate into neurons, astrocytes and oligodendrocytes, producing a large number of brain cells and self-renewing. In the context of the present invention, neural stem cells may be considered as a class of intermediate stage cells between iPSC/ESC and NPC. Neural stem cells differentiate to a lesser extent than neural precursor cells, particularly those that can differentiate into multiple brain regions, whereas neural precursor cells are generally considered to have committed to a single brain region stage, differentiating into only a specific brain region or a certain type of neural cell. ESCs/iPSCs are cells that differentiate to a lesser degree than neural stem cells. ESCs/iPSCs are theoretically capable of differentiating into all cell types except trophoblasts, whereas neural stem cells as adult stem cells can only differentiate into neural cells. Neural stem cells may be obtained by extraction from tissue, or may be obtained by differentiation of ESCs/iPSCs into neural stem cells by the "dual-SMAD" method.

It will be appreciated that in the process flow of the present invention as shown in FIG. 1A, the cell type in each box is the most predominant and critical cell type in this stage, but it is not meant to be limiting to the cell type alone in the culture of this stage. The culture at each stage may be a mixture comprising a plurality of cell types, the cells of which may have different degrees of differentiation, and even different differentiation types. The key to the protection intended in this application is the method of transitioning the population of NPC-based cells as a whole to the population of astrocyte-based cells.

Steps of the method

In this section, the key steps of the method of the present invention are described in connection with fig. 1A.

As can be seen from fig. 1B, NPCs may further form cells in a variety of neural lineages, including, but not limited to, neurons, glial precursor cells, and glial cells, such as astrocytes, oligodendrocytes, and oligodendrocytes. To achieve the objects of the present application, namely to obtain a population of cells that is predominantly mature astrocytes, the present invention employs multiple steps to promote astrocyte formation while reducing the fraction of other cell types in the final obtained population of cells.

The term "enriching" refers to increasing the fraction of cells of a particular class in a population of cells, or concentrating a large number of cells of a particular class in a given region such that the region has a high proportion of cells of that class.

The term "purifying" refers to reducing the proportion of cells outside a particular class in a population of cells by removing cells outside that class.

The term "adherent culture" refers to culture in which cells adhere to a solid or semi-solid phase surface, and cells may be grown in a planar manner, and is not limited to a culture in which cells are in direct contact with the surface of a culture vessel. In contrast, the "suspension culture" refers to a culture in which cells are not adhered to a solid or semi-solid surface, and the cells are suspended in a culture medium and may be in the form of cell aggregates or spheroids.

The present invention provides a method for preparing astrocytes, the method comprising:

(1) Providing a neural stem cell or neural precursor cell as a 0 th generation cell (P0) in the method;

(2) Seeding the neural stem cells or neural precursor cells onto a plane of a culture vessel coated with a cell adhesion medium, or seeding the neural stem cells or neural precursor cells together with a cell adhesion medium into a culture vessel, wherein the neural stem cells or neural precursor cells after seeding are first generation cells (P1);

(3) Performing an adherent culture of the neural stem cells or neural precursor cells (P1) under conditions suitable for differentiation of the neural stem cells or neural precursor cells into astrocytes to obtain astrocytes;

(4) Inoculating the astrocyte precursor cells obtained in step (3) onto the plane of a culture vessel coated with a cell adhesion medium; and

(5) The astrocytes are subjected to an adherent culture under conditions suitable for the astrocytes to differentiate into astrocytes, to obtain astrocytes.

In step (1), the neural stem cells or neural precursor cells may be from any source as long as they are suitable for the application of the present invention. In the context of the present invention related to the above-described method, for convenience of description, if not specified, cells in the method as an astrocyte differentiation starting material are defined as the 0 th generation (P0) cells. It is to be understood that this does not mean that the P0 generation cells in the methods of the invention must be the 0 th generation cells of neural stem cells or neural precursor cells.

In an optional embodiment, step (1) comprises culturing the neural stem cells or neural precursor cells for later use in the step. The culture in step (1) may use a maintenance-like medium or a medium that promotes differentiation of stem cells or precursor cells. For example, neural stem cells can be cultured using a neural stem cell medium. For example, neural precursor cells can be cultured using a neural precursor cell culture medium. For example, a differentiation medium may be used to culture neural stem cells or neural precursor cells, preferably to the point where astrocyte-like cells (cells similar in morphology to astrocytes) appear for subsequent steps. For example, neural stem cells or neural precursor cells can be cultured using a neural differentiation medium. The incubation may last for different periods of time, for example up to 2 months. The culture conditions can be 37 ℃ and 5% CO ₂ Half a week, 2 changes.

The cells prepared in step (1) are referred to as the 0 th generation (P0) cells. If not specified, P0 cells in the context of the present invention are cells for which astrocyte differentiation is initiated.

In step (2), the prepared (optionally cultured) neural stem cells or neural precursor cells are seeded into a culture vessel, which is previously coated with a cell adhesion medium. It should be noted that although the cell adhesion medium is preferably Matrigel, the culture of step (2) is different from the 3D organoid culture performed in Matrigel, because the type and concentration of Matrigel (e.g., laminin such as MX521,6 μg/mL) are different from those in 3D organoid culture (e.g., matrigel, stock solution) in the present invention. The culturing of step (2) attempts to attach cells to a planar surface and form a monolayer adherent culture by the use of a cell adhesion medium, rather than forming spheroids, which such cells typically tend to form. It is possible to avoid death due to insufficient oxygen and culture medium available to the cells inside the sphere.

In some embodiments, in step (2) at 1.8X10 ⁵ To 4.5X10 ⁵ Individual cells/cm ² Inoculating the cells, preferably in an amount of 2.0X10 ⁵ To 4.0X10 ⁵ Individual cells/cm ² More preferably 2.6X10) cells are inoculated ⁵ To 3.1X10 ⁵ Individual cells/cm ² Inoculating the cells in an amount. For example, when a 6-well plate is used, the cell plating density in step (2) is 2.5 to 3X 10 ⁶ Individual cells/wells.

In a preferred embodiment, the cells are treated with digestive enzymes to disperse the cells prior to seeding the cells in step (2).

The cells inoculated in the step (2) are referred to as the 1 st generation cells (P1).

In step (3), the cells (P1) inoculated in step (2) are cultured. Step (3) uses the astrocyte precursor cell expansion medium of the present invention. Culture conditions were 37℃and 5% CO ₂ 。

In a preferred embodiment, medium half-volume medium exchange is performed every 2-3 days, i.e. half the volume of medium is replaced with fresh medium.

In a preferred embodiment, the passage is made every 5-7 days. Starting from passage 2 (P2), the cells may be seeded at a lower seeding density than in step (2) for passaging. For example, 1.2X10 can be used ⁵ Up to 2.5X10 ⁵ Individual cells/cm ² Is inoculated.

In a preferred embodiment, the cells are treated with digestive enzymes to disperse the cells prior to each subculture. After 3 passages, the cells treated with digestive enzymes may be in a single cell state.

In step (4), the astrocyte precursor cells obtained by the culture in step (3) are inoculated into a culture vessel previously coated with a cell adhesion medium. In step (4), the ratio of 7.0X10 ⁴ Up to 2X 10 ⁵ Individual cells/cm ² Inoculating the cells, preferably at 7.0X10 ⁴ Up to 1.5X10 ⁵ Individual cells/cm ² More preferably 7.0X10 cells ⁴ Up to 1.2X10 ⁵ Individual cells/cm ² Inoculating the cells in an amount of 8.0X10 are particularly preferred ⁴ Up to 1.0X10 ⁵ /cm ² Inoculating the cells in an amount.

In step (5), the astrocyte precursor cells inoculated in step (4) are subjected to an adherent culture. Step (5) uses the astrocyte maturation medium of the present invention. Culture conditions were 37℃and 5% CO ₂ 。

Step (5) typically lasts for a period of about 1 to 3 weeks, i.e., mature astrocytes are typically observed at around 7 days. During step (5), the astrocytes gradually mature into astrocytes, which can be manifested by a change in the morphology of the cells and a change in the marker protein expressed by the cells: the cell morphology is slowly transformed into star shape, and the expression level of GFAP reaches more than 90%. After the culture is completed, the cells may be subjected to staining identification or functional experiments.

In the culture method of the present invention, the culture vessel may be a culture plate such as a porous culture plate, a culture dish, a culture flask, a cell factory, a microcarrier, a hollow fiber tube reactor, a fixed bed reactor, or the like, or may be a culture surface such as a glass slide.

Among the cells obtained by the above-described method of the present invention, GFAP positive cells account for 60% or more, more preferably 80% or more, and still more preferably 90% or more of the total number of cells.

For the method of the present application, the total length of time (40-60 days) for obtaining astrocytes by the adherent culture method is shorter than that by the suspension culture method. Starting from stem cells, the astrocytes finally obtained by the method of the present invention have a higher expansion factor (100-150 times) than the suspension culture method (less than 5 times).

By utilizing the principle that the neuron cells gradually die in the continuous passage process, the method can obtain the astrocytes (90%) with higher purity. In addition, FBS is not needed in the culture process, so that the expansion of non-astrocytes is avoided.

The method has the advantages of good repeatability, convenient operation and rapidness (40-60 days). The astrocyte GFAP expression level obtained by the method is more than 90%, and the astrocyte GFAP has the capabilities of taking in glutamic acid, supporting neuron growth and forming calcium waves.

Method for identifying astrocyte function

Astrocytes produced by the method of the present invention have the function of mature astrocytes, said function comprising one or more of the following:

(1) Neuron support ability;

(2) Glutamate uptake ability; and

(3) Intracellular calcium (Ca) ²⁺ ) Signal transduction capability.

Conventional assay means may be used to verify the above-described functions and properties of the obtained astrocytes.

For example, the support capacity of astrocytes for neurons can be verified by co-culturing the obtained astrocytes with cells having the potential to develop into neurons, such as neural stem cells or neural precursor cells, under conditions suitable for forming neurons, and examining the growth of neuronal processes.

For example, the glutamate uptake ability of astrocytes can be detected by a glutamate uptake inhibition assay.

For example, intracellular calcium (Ca ²⁺ ) Signal transduction capability.

Culture medium

The term "medium" refers to a mixture that contains various nutrients necessary for the growth of a certain type of cell. The medium may be prepared by adding additives to the basal medium. Additives in the broad sense refer to additional components required for cell culture but not contained in the basal medium, including proteins, lipids, amino acids, vitamins, hormones, cytokines, growth factors, etc.

Preferably, the various media used in the methods of the invention are chemically defined media. By "chemically defined medium" is meant that all components contained in the medium are known and thus are distinguished from media comprising components of animal origin, in particular serum. Thus, the chemically-defined medium does not contain any serum preparations as a source of nutrients. The advantage of using a chemically defined medium is that the potential risk due to uncertainty in composition and unknown safety is reduced. Another advantage resides in facilitating adapting the overall method to the production criteria of clinically used cells.

In one embodiment of the present invention, any basal medium and additives used in order to obtain astrocytes suitable for clinical use are clinical grade, preferably GMP grade, cGMP grade or CTS grade ^TM A stage.

For convenience of description, in the context of the present invention, proteins capable of stimulating proliferation and cell differentiation of cells (particularly neural cells, more particularly astrocytes and their precursors) are referred to as "growth factors". Accordingly, growth factors (growth factors) herein include cytokines (cytokines) in addition to those proteins referred to as "growth factors", such as Fibroblast Growth Factor (FGF), epidermal Growth Factor (EGF). In a preferred embodiment, the growth factors used in the culture medium of the invention are animal component free protein preparations produced by recombinant techniques.

In the method of the present invention, the two most critical media are the astrocyte precursor cell expansion medium (or "astrocyte precursor expansion medium") and the astrocyte maturation medium (or "astrocyte maturation medium"), which are used in the adherent culture step to produce astrocyte precursor cells and mature astrocytes, respectively.

In one embodiment, the astrocyte precursor cell expansion medium comprises components that maintain cell growth and promote differentiation of neural stem cells or neural precursor cells into astrocyte precursor cells.

In a preferred embodiment, the astrocyte precursor cell expansion medium comprises growth factors in addition to the basal medium and the components necessary to maintain cell growth, and the growth factors are EGF and FGF. In one embodiment, the astrocyte precursor cell expansion medium contains only EGF and FGF as growth factors, and no other growth factors.

In one embodiment, the astrocyte precursor cell expansion medium comprises or consists of DMEM/F-12, N2 supplements, EGF, FGF and MEM nonessential amino acids (MEM Non-Essential Amino Acids Solution; MEM-NEAA). "DMEM/F-12" refers to Dulbecco's Modified Eagle Medium/F-12, a commonly used serum-free medium. An "N-2 supplement" is a chemically defined, serum-free supplement. "MEM nonessential amino acids" refers to those nonessential amino acids contained in "standard minimal essential Medium (minimum essential medium)". In one embodiment, the astrocyte precursor cell expansion medium comprises EGF at a concentration of 1-100ng/mL, preferably 5-50ng/mL EGF, more preferably about 10ng/mL EGF. In one embodiment, the astrocyte precursor cell expansion medium comprises FGF at a concentration of 1-100ng/mL, preferably 5-50ng/mL, more preferably about 10ng/mL FGF. In a preferred embodiment, the FGF is FGF2.

In a specific embodiment, the astrolThe composition of the precursor cell expansion medium is as follows: 1X KnockOut ^TM DMEM/F-12CTS ^TM 1 XN 2 supplement-A CTS ^TM Grade, 10ng/mL animal component free recombinant human FGF-basic form (FGF-basic), 10ng/mL animal component free recombinant human EGF, and 1 XMEM nonessential amino acids.

In a specific embodiment, the astrocyte precursor cell expansion medium of the present invention does not comprise a B-27 supplement. The B-27 supplement is a medium supplement commonly used to support the growth of nerve cells, particularly neurons.

In one embodiment, the astrocyte maturation medium comprises components that maintain cell growth and promote the maturation of astrocyte precursors into astrocytes.

In a preferred embodiment, the astrocyte maturation medium comprises, in addition to the basal medium and the components necessary to maintain cell growth, growth factors, and the growth factors are BMP-4 and CNTF. "BMP-4" refers to bone morphogenic protein 4 (bone morphogenetic protein 4). "CNTF" refers to ciliary neurotrophic factor (ciliary neurotrophic factor), a cytokine of the interleukin-6 family. In one embodiment, the astrocyte maturation medium contains only BMP-4 and CNTF as growth factors, and no other growth factors.

In one embodiment, the astrocyte maturation medium comprises DMEM/F-12, N2 supplements, BMP-4, CNTF, MEM nonessential amino acids, and L-glutamine supplements. As the L-glutamine supplement, glutamax is preferably used ^TM -I supplement. In one embodiment, the astrocyte precursor cell expansion medium comprises BMP-4 at a concentration of 1-100ng/mL, preferably 5-50ng/mL, more preferably about 10ng/mL BMP-4. In one embodiment, the astrocyte precursor cell expansion medium comprises CNTF at a concentration of 1-100ng/mL, preferably 5-50ng/mL, more preferably about 10 ng/mL.

In a specific embodiment, the composition of the astrocyte maturation medium is as follows: 1X KnockOut ^TM DMEM/F-12CTS ^TM 1 XN 2 supplement-A CTS ^TM Grade, 10ng/mL animal component free recombinant human BMP-4, 10ng/mL animal component free recombinant human CNTF, 1 XMEM nonessential amino acid solution and 1 XCTS ^TM GlutaMAX ^TM -I supplement.

In a specific embodiment, the astrocyte maturation medium of the present invention does not comprise a B-27 supplement.

In addition to the two media used for the adherent culture, neural stem cells and/or neural precursor cells may also be cultured prior to the adherent culture. In such pre-cultured embodiments, the methods of the invention may further comprise the use of a neural stem cell culture medium and a neural precursor cell culture medium for culturing neural stem cells and neural precursor cells, respectively, which cells are subsequently subjected to adherent culture.

The "neural stem cell medium" may use any medium suitable for maintenance culture of neural stem cells. For example, the neural stem cell medium may comprise Neurobasal medium, B-27 supplement, and L-glutamine. In one embodiment, the growth factors in the neural stem cell medium may comprise or consist of: BDNF and GDNF. "BDNF" refers to brain-derived neurotrophic factor (brain-derived neurotrophic factor). "GDNF" refers to glial cell line-derived neurotrophic factor. In a specific embodiment, the neural stem cell culture medium comprises or consists of: CTS (clear to send) ^TM Neurobasal ^TM A culture medium; CTS (clear to send) ^TM B-27 ^TM Supplements, xenofole, desvitamin a; and CTS (clear to send) ^TM GlutaMAX ^TM -I supplement.

The "neural precursor cell medium" may use any medium suitable for maintenance culture of neural precursor cells.

In a specific embodiment, the neural precursor cell culture medium comprises Neurobasal medium, B27 supplement, BDNF, GDNF, and L-glutamine supplement. In one embodiment, the neural precursor cell culture medium comprises BDNF at a concentration of 1-100ng/mL, preferably 5-50ng/mL, more preferably about 20ng/mL BDNF. In one embodiment, the pre-nerve The somatic cell culture medium contains GDNF at a concentration of 1-100ng/mL, preferably 5-50ng/mL, more preferably about 20 ng/mL. As the L-glutamine supplement, glutamax is preferably used ^TM -I supplement.

In a specific embodiment, the composition of the neural precursor cell culture medium is as follows: 1X CTS ^TM Neurobasal ^TM Medium, 1 XB 27 supplement CTS ^TM Grade, 20ng/mL animal component free recombinant human BDNF, 20ng/mL animal component free recombinant human GDNF and 1 XCTS ^TM GlutaMAX ^TM -I supplement.

In addition to the two media used for the adherent culture, in embodiments in which neural stem cells and/or neural precursor cells are subjected to the differentiation culture in advance, the method of the present invention may further comprise using a differentiation medium for culturing neural stem cells and/or neural precursor cells, which cells are subjected to the adherent culture of the present invention after being cultured by the differentiation medium.

The "differentiation medium" may comprise a medium that promotes differentiation of neural stem cells and/or neural precursor cells into neurons.

In exemplary embodiments, the differentiation medium may comprise 1 XCTS ^TM Neurobasal ^TM Medium, 1 XB 27 supplement CTS ^TM A stage, and one or more of the following: 1X CTS ^TM GlutaMAX ^TM -medium of supplement I, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), L-ascorbic acid, N6, O2' -di Ding Xianxian glycoside 3',5' cyclic monophosphate sodium salt (DB-cAMP).

In a specific embodiment, the composition of the differentiation medium is as follows: 1X CTS ^TM Neurobasal ^TM Medium, 1 XB 27 supplement CTS ^TM Stage, 1×CTS ^TM GlutaMAX ^TM -I supplement, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), N ⁶ O2' -bis Ding Xianxian glycoside 3',5' cyclic monophosphate sodium salt (DB-cAMP) and L-ascorbic acid.

In a specific embodiment, the differentiation medium may be a neural differentiation medium as described in CN108359638B, differentiation medium a and/or differentiation medium B as described in example 1.

Any particular product, such as a medium, supplement, inhibitor, neurotrophic factor, or other agent, as exemplified by the present disclosure, may be replaced by the same type of product but under a different trademark or product name, or may be replaced by an equivalent function. Those skilled in the art will appreciate that substantially identical products having different brands or product names, and functional equivalents, are also encompassed by the present disclosure.

Enzyme for desorption

In the adherent culture process of the present invention, cells are preferably desorbed and dispersed by using an enzyme at the time of passage. Digestive enzymes are commonly used for this purpose. For example, enzymes useful in the methods of the invention for cell detachment and/or dispersion include, but are not limited to Accutase, trypLE, versene, CTS ^TM TrypLE、CTS ^TM Versene, preferably CTS ^TM TrypLE。

Preferably, the enzyme or enzyme preparation suitable for use in the methods of the present application is clinical grade, GMP grade, cGMP grade or CTS ^TM A stage. The digestive enzymes may be used in amounts recommended by the manufacturer thereof.

In a preferred embodiment, the digestive enzyme is CTS ^TM TrypLE. In a preferred embodiment, the digestion time is 10-15min and the temperature is 37 ℃.

In one embodiment, the digestive enzyme used to desorb cells is CTS ^TM TrypLE。

Cell adhesion medium

The term "cell adhesion medium" refers to a medium capable of facilitating the adherent culture of cells that are not prone to adherent growth, such a medium should be non-toxic to the cells and may be a medium commonly used in cell culture, such as matrigel-like media.

The term "matrigel" is used in the same sense as "extracellular matrix (ECM)". For astrocytes or astrocyte precursor cells, an extracellular matrix is used to support the adherent growth of the cells. ECM is an extracellular cell surface matrix, consisting mainly of proteins such as collagen, elastin, and laminin. ECM is widely used in mammalian cell culture and is known to those skilled in the art.

Non-limiting examples of ECMs that can be used to coat a solid support or culture surface (e.g., a culture plate) include: matrigel ^TM Laminin, polylysine, polyornithine (PO)/Fibronectin (FN)/Laminin (Laminin) combinations, fibronectin (FN), vitronectin (Vitronectin), and the like.

In one embodiment, matrigel may be of the type commonly used in cell culture, e.g., matrigel, vitronectin (e.g., VTN-N), laminin (e.g., LN521, MX521, CT 521) may be used. In a preferred embodiment, the culture surface is coated, for example with laminin, such as MX 521. The coating concentration of matrigel such as MX521 may be 5. Mu.g/mL to 10. Mu.g/mL, preferably 6. Mu.g/mL to 8. Mu.g/mL.

The thickness and amount of the cell adhesion medium in the cell culture plate or dish depends on the specific culture conditions, such as the type of cells being cultured. In general, the cell adhesion medium is used in an amount that enables (most) cells to grow in an adherent state and that does not undergo cell shedding or float in the medium under observation under a mirror. Matrigel was used as a cell adhesion medium in much lower amounts than when used for 3D culture.

For example, the culture plane may be coated with a cell adhesion medium. Specifically, after the cell adhesion medium is added to the culture vessel, it is left for a while and then discarded, thereby forming a thin layer. It should be appreciated that the thickness of the cell adhesion medium may be non-uniform, facilitating cell attachment. The time left after the addition of matrigel may be 1 to 72 hours. For example, the residence time of the matrigel may be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours.

For example, the cell adhesion medium may be mixed with the cells to be seeded and the mixture used for seeding.

Use of the same

Studies have shown that astrocytes from different brain regions exhibit different physiological functions, e.g., have different transcriptome properties (Xinzhu Yu et al Nature Reviews Neuroscience,21.3 (2020): 121-138). The methods of the present application can be used to prepare astrocytes from different brain regions. Specifically, neural precursor cells of different brain regions can be used as a starting material, and astrocytes corresponding to brain regions can be obtained by the adherent culture step of the present invention. For example, the neural precursor cell used in the present invention may be a neural precursor cell of the cerebral cortex, the metabrain, the midbrain, the cerebellum, or the spinal cord. Accordingly, the astrocytes or astrocytes produced may be astrocytes of the cerebral cortex, the metabrain, the midbrain, the cerebellum or the spinal cord.

The methods of the present application are suitable for producing high purity astrocytes suitable for clinical and preclinical use. For example, astrocytes produced by the present method can be used for drug development, disease modeling, pre-clinical and clinical research, as well as for existing therapies and new therapies being developed.

Astrocytes prepared by the methods of the present application are particularly useful, for example, in the clinical treatment of diseases associated with the central nervous system, such as diseases associated with damage to the central nervous system, including neurodegenerative diseases such as parkinson's disease, huntington's disease, or alzheimer's disease, among others.

In the methods of the present application, the term "clinical grade" with respect to materials is used to produce astrocytes, particularly materials comprising one or more media, and matrices and enzymes in the present application, without limiting the use of other known or unknown reagents, media, and sources of cell-derived materials, means that such reagents, media, or cell-derived materials are suitable for clinical use per se, and/or allow for the directed differentiation of NPCs and derived products to produce safe and stable cells, particularly astrocytes suitable for clinical use.

More preferably, the agent, medium or cell-derived material used to produce the astrocytes of the present application, in particular the one or more media constituting the present application, is GMP (pharmaceutical manufacturing practice) grade or cGMP (dynamic pharmaceutical manufacturing practice) grade, meaning that the agent, medium or cell-derived material has been approved for GMP or cGMP quality and is produced according to the GMP or cGMP standard defined by the authorities, e.g. provided by WHO, MOH (chinese health), the U.S. food and drug administration or the european pharmaceutical administration. "GMP" or "cGMP" refers to the (current) standard that manufacturers should follow to ensure that the manufacturing process of their products is properly monitored and controlled and to provide consistent high security to the end user.

The term "CTS" or "CTS ^TM "stands for cell therapy system. This is a term used by the manufacturer, sameiser, to indicate that a certain product for cell therapy manufacture is of high quality and meets cGMP standards.

In the context of the present application, the term "clinically used" or "suitable for clinical use" in reference to astrocytes means that the astrocytes meet at least the criteria required by certain regulations both clinically and clinically. Preclinical practices such as Good Manufacturing Practice (GMP). In particular, the astrocytes enriched and purified by the method of the present application have properties suitable for clinical use.

Detailed Description

The present application is illustrated in detail by the following examples, but the present application is not limited to the embodiments shown in these examples.

Experimental materials

1. Culture medium

1.1 astrocyte precursor cell expansion Medium

Astrocyte precursor cell expansion medium is used to culture neural progenitor cells to form a cell population containing astrocyte precursor cells. In the embodiment using CTS ^TM The medium of grade DMEM/F-12 and N2 supplements were supplemented with FGF and EGF formulations free of animal-derived components, as well as non-essential amino acid solutions.

1.2 astrocyte maturation Medium

The astrocyte maturation medium is used to promote the formation of mature astrocytes from astrocyte precursors. In the embodiment using CTS ^TM Grade DMEM/F-12 medium, N2 supplement and Glutamax for glutamate supplementation ^TM -I supplement supplemented with BMP-4 and CNTF formulations free of animal derived components, and non-essential amino acid solutions.

1.3 neural differentiation Medium

2. Cellular material

NPC used in the examples of this application were prepared from iPSC. Specifically, the reprogramming method as disclosed in WO2021/018296 was used to obtain iPSC, and the NPC was obtained by using the method as described in CN 113604434A.

FOXG1 positive Neural Precursor Cells (NPCs) accounting for 90% were differentiated from ipscs.

Example 1 preparation of mature astrocytes from differentiated neural cells

In this example, a population comprising astrocytes was prepared using the differentiated neural cells, and a cell population of mainly astrocytes was finally obtained. The method flowchart of this embodiment is shown in fig. 12A.

1. From human embryonic stem cells to neural cells

Human embryonic stem cell (hESC) cell line H1 was purchased from Shanghai Ezetoshima Biotech Inc. (product catalog number AC-2001002H 1). NPC used in the examples of the present application were prepared from hESC. Specifically, NPC was obtained using the method described in CN113604434a, and differentiated neural cells were obtained using the method described in CN 108359638B.

2. From nerve cells to astrocytes (serial passage)

First, differentiated neural cells (P0) obtained from hESCs according to the above method were used with 0.1mL/cm ² 1X digestive enzyme CTS of (C) ^TM TrypLE ^TM Select Enzyme (thermo#a 1285901) was digested for 15min and blown into single cells. The culture dish was previously coated with MX521 matrigel at a concentration of 6. Mu.g/mL, and then with 15-26 ten thousand/cm ² Neurons of the P0 generation were inoculated into matrigel coated dishes to obtain P1 generation cells.

Culturing P1 generation cells by using astrocyte precursor cell expansion medium, wherein the amount of the medium is 0.3mL/cm ² . Half-volume medium was replaced every 3 days with fresh medium and passaged every 5-7 days (cells were passaged at confluence of about 100%). And obtaining the P7 generation cell, namely the astrocyte precursor cell.

Nuclei were stained with DAPI by immunofluorescent staining and expression of s100deg.P and GFAP was detected with a luminescent labeled antibody. The results of observation under the fluorescence microscope are shown in fig. 2A. As shown, the obtained astrocyte precursor cells express s100deg.P.

3. From astrocyte precursor cells to mature astrocytes

A circular slide of 10mm diameter was placed in the well of a 48-well plate and then the slide was coated with MX521 (at a concentration of 6. Mu.g/mL). The P7 cells obtained above were cultured in a culture medium of 1.3 to 2.0X10 ⁵ Wells were inoculated into 48-well plate slides and cultured with 0.5mL astrocyte maturation medium added to each well. Half liquid change is carried out every 1-3 days. After inoculation ofAnd identifying the expression conditions of the S100.beta.and the GFAP by immunofluorescence staining in 7-21 days. When the GFAP expression level reaches 90%, that is, 90% of the cells express GFAP, astrocytes are considered to be differentiated and mature.

The astrocytes obtained are mature astrocytes. After immunofluorescence staining, the cell bodies are visible to be star-shaped under a fluorescence microscope, and the periphery of the cell membrane is smooth and the boundary is clear; the cell nucleus is elliptical and deviates to one side of the cell; the cell processes are long and contact each other. FIG. 2B shows a fluorescence photograph of astrocytes. As shown in fig. 2B, the obtained astrocytes expressed GFAP and s100deg.S.

Example 2 preparation of mature astrocytes from neural precursor cells

In this example, a population comprising astrocytes was prepared using FOXG1 positive neural precursor cells, and a cell population of mainly astrocytes was finally obtained. The method flowchart of this embodiment is shown in fig. 12B.

1. Neural precursor cells to astrocyte precursor cells (serial passage)

First, neural precursor cells (P0) from human induced pluripotent stem cells (hiPSCs) were used with 0.1mL/cm ² 1X digestive enzyme CTS of (C) ^TM TrypLE ^TM Select Enzyme (thermo#a 1285901) was digested for 15min and blown into single cells. The dishes were previously coated with matrigel MX521 at a concentration of 6. Mu.g/mL. Then 15-26 ten thousand/cm ² The neural precursor cells were inoculated into a matrigel-coated culture dish to obtain P1 generation cells.

The P1 generation cells were cultured using an astrocyte precursor cell expansion medium in an amount of 0.3mL/cm ² . Half-volume medium was replaced every 3 days with fresh medium and passaged every 5-7 days (cells were passaged at confluence of about 100%). Obtaining P6-7 generation cells, namely astrocyte precursor cells. The results of observation of cells passaged 7 times under a bright field microscope are shown in fig. 7D.

2. From astrocyte precursor cells to mature astrocytesGlial cells

A circular slide of 10mm diameter was placed in the well of a 48-well plate and then the slide was coated with MX521 (at a concentration of 6. Mu.g/mL). The P7 cells obtained above were cultured in a culture medium of 1.3 to 2.0X10 ⁵ Wells were inoculated into 48-well plate slides and cultured with 0.5mL astrocyte maturation medium added to each well. Half liquid change is carried out every 1-3 days. The expression of s100deg.P and GFAP was identified by immunofluorescent staining on days 7-21 post-inoculation. When the GFAP expression level reached 90%, astrocytes were considered to differentiate and mature.

The astrocytes obtained are mature astrocytes. After immunofluorescence staining, the cell bodies are visible to be star-shaped under a fluorescence microscope, and the periphery of the cell membrane is smooth and the boundary is clear; the cell nucleus is elliptical and deviates to one side of the cell; the cell processes are long and contact each other. Fig. 7E shows a bright field photograph of astrocytes; FIG. 8 shows a photograph of fluorescent staining of astrocytes, which obtained astrocytes express GFAP and S100deg.S.

Example 3 functional detection of functional astrocytes

In this example, the functions of astrocytes obtained in examples 1-2 were examined.

1. Neuron support experiments

This experiment tested its neuronal support function by co-culturing astrocytes obtained by the method of the present invention with NPC cells. The following experiment was performed using the mature astrocytes obtained in example 1.

NPC cells with stably transformed GFP fluorescent protein were seeded alone or on the above astrocytes and subjected to differentiation culture for 7 days. In the differentiation culture, the medium used was a neural differentiation medium, and the proportion of cells in the co-culture group was astrocytes: npc=80: 1.

On days 1-7 of co-cultivation, cell photographs were taken daily by high content microscope imaging. Since the added cells are capable of expressing GFP, no antibody staining is required. The degree of maturity of the differentiated neurons was determined by directly analyzing the number of Segments (Segments) and Length (Length) of green fluorescent cell neurites (neurites) in culture using analysis software equipped with a high content microscope imaging system. The greater the number of segments and the greater the length of the neuronal processes, the better the maturity of neuronal differentiation.

The results of FIG. 6 show that the astrocytes obtained in example 1 show significant differences in Segments/Number of neurons and Length/Number of neurons values from the coculture group of astrocytes and NPC cells, compared to NPC differentiation alone, indicating that astrocytes obtained by the method of the present application are capable of promoting NPC differentiation maturation when co-cultured with NPC, and have a neuronal support function.

2. Glutamic acid uptake assay

Glutamate is the most prominent excitatory neurotransmitter in mammals. To maintain sensitivity to synaptic transmission, glutamate must be cleared rapidly and in time. Astrocyte uptake is the most predominant route to maintain stable extracellular glutamate concentrations. Although both neurons and astrocytes express glutamate transporters, astrocytes have a much higher capacity to uptake glutamate than neurons.

PDC (L-trans-pyrrolidine-2, 4-dicarboxylic acid, L-trans-pyrrosidine-2, 4-dicarboxylic acid) is a glutamate transporter uptake inhibitor. Treatment of astrocytes with PDC inhibits the transport function of EAAT2/4/5 on the surface of astrocytes and inhibits the uptake of glutamic acid.

Astrocytes obtained in examples 1 and 2 were divided into two groups, PDC-treated and no drug-treated as a control.

PDC treatment group: cells were incubated with PDC and 100. Mu. M L-monosodium glutamate (sigma # 49621) at 1mM concentration for 1 hr (cells of example 2) or 3 hr (cells of example 1).

Drug-free treatment group: cells were incubated with 100 μ M L-monosodium glutamate (sigma # 49621) for 1 hour (cells of example 2) or 3 hours (cells of example 1).

Culture supernatants of both groups of astrocytes were collected before the start (0 h) and after the end (3 h or 1 h), respectively. The concentration of glutamic acid in the culture supernatant was read by an enzyme-labeled instrument, and the concentration difference was calculated, and the statistics of the results are shown in FIGS. 3 and 9.

The results show that the concentration of the glutamic acid is obviously reduced after the group without the drug is incubated for 3 hours or 1 hour, which indicates that the astrocytes prepared by the method have the function of taking in the glutamic acid. Meanwhile, after astrocytes were treated with PDC as an EAAT2/4/5 inhibitor, glutamate uptake function was significantly inhibited in both the cells obtained in example 1 and the cells obtained in example 2. Indicating that astrocytes were inhibited from glutamate uptake by PDC treatment.

3. Scratch test

Astrocyte scratch experiments are scratch models that simulate mechanical damage. It was found that scratch stimulation can cause astrocytes to produce intracellular calcium signals that are transmitted as calcium waves from scratched cells to the non-scratched side.

In this example, cells were transfected with lentivirus to express the calcium ion response protein Gcamp6s. GCaMPs are the products of fusion of a single Green Fluorescent Protein (GFP), calmodulin (CaM) and smooth muscle cell myosin light chain kinase fragment M13. GFP in GCaMPs is a circular sequence altered enhanced green fluorescent protein (cpEGFP) and the N-and C-termini of EGFP are linked together using the hexapeptide GGTGGS, and at the same time the peptide bond at a specific position on the EGFP amino acid sequence is cleaved, resulting in new N-and C-termini. The C-terminus was fused to CaM and the N-terminus was fused to the M13 fragment as a target sequence for CaM binding. Inter astrocyte chemical signaling-Ca ²⁺ Concentration rise (calcium oscillation wave) for information transfer and signal amplification. In one aspect, the calcium waves are transmitted from glial cells to glial cells through a gap junction. On the other hand, calcium waves can also be transmitted via extracellular signal substances such as glutamic acid and ATP. Ca (Ca) ²⁺ Elevated concentrations may trigger the release of glutamate by astrocytes. This information can be transmitted to other astrocytes, and can also be fed back to the neuronal cells, affecting the synaptic function of the neuronal cells. Mechanical damage causes an increase in calcium ion concentration, causing M13 to bind to CaM, thereby altering the conformation of cpEGFP, from absentThe state of fluorescence changes to a green fluorescence state. Therefore, the signaling ability of astrocytes can be reflected by observing a change in fluorescence.

The change in fluorescence emitted by Gcamp6s was recorded under a fluorescence microscope. The specific test procedure is shown below.

The population of astrocyte-containing precursor cells thus obtained is 1.6 to 3.6X10 ⁵ Wells were inoculated onto 24-well plates with 10mm slides coated with MX521 and infected with lentiviruses Lenti-EF1A-Gcamp6s expressing the Gcamp6s sequence in an amount MOI=5-10.

After 2-3 days of stable cell status after inoculation of the slide, lentiviruses of the required volume converted according to virus titer, number of inoculated cells and MOI value were added to the 24-well plate and infected overnight. The next day, the culture medium is replaced by an astrocyte maturation culture medium, the liquid is half-changed every 1 to 3 days, and the astrocytes are obtained after 7 to 21 days of differentiation.

The cell layer on the surface of the glass slide is scored by a needle, and mechanical damage is generated. The scratch process was recorded in real time by fluorescence microscopy 1min before the scratch, resulting in a change in fluorescence emitted by Gcamp6s. The results are shown in fig. 4 and 10, and the shear head in fig. 4 points to a mechanical scratch area, and calcium waves (green fluorescence) are diffused from the center to the periphery.

The results showed that the scratched edge portions of the cells were peeled off from the slide glass after the scratching, and the cells were damaged. After scoring, a large number of green fluorescent signals were observed to propagate from the cells at the score along the cell body to the surrounding cell network. The calcium wave transmission of astrocytes after injury was demonstrated, and the obtained astrocytes were confirmed to be functional astrocytes.

ATP stimulation assay

Astrocyte ATP stimulation experiments are models that mimic ATP transfer in vivo. It was found that glial cells, when excited, release ATP. ATP, when bound to receptors on adjacent glial cells, can cause calcium influx and increase intracellular calcium concentration. The increased concentration of calcium ions in turn can promote the release of ATP, thereby allowing information to be transferred and amplified.

The pre-treatment of this example was performed in the same scratch assay, and cells were transfected with lentivirus to express the calcium ion response protein Gcamp6s. After overnight transfection with lentivirus, the medium was changed to astrocyte maturation medium the next day, half-changed every 1-3 days, and mature astrocytes were obtained after 7-21 days of differentiation.

To the cell layer side of the slide surface, 100. Mu.M ATP solution was slowly added in an equal volume to the medium. The change in fluorescence emitted by Gcamp6s was obtained by fluorescence microscopy recording from 1min before addition, and the addition was recorded in real time. The results are shown in fig. 5 and 11.

The results show that after ATP addition, a large number of green fluorescent signals were observed to propagate from the cells where ATP was added along the cell body to the surrounding cell network; some cells have an elevated intracellular calcium ion concentration (yellow arrow); through tight junctions (gap junctions) between astrocytes, the intracellular calcium ion concentration of the surrounding astrocytes is also rapidly increased in linkage (white arrows), and obvious calcium waves are formed, which are one of important markers for the functional maturation of astrocytes; the results demonstrate that calcium wave transmission of astrocytes after ATP stimulation occurs, confirming that the astrocytes obtained are functional astrocytes.

Claims (17)

1. A method of preparing astrocytes, the method comprising the steps of:

(1) Providing neural stem cells or neural precursor cells from human embryonic stem cells or human induced pluripotent stem cells as the 0 th generation cells (P0) in the method;

(2) Seeding the neural stem cells or neural precursor cells onto a plane of a culture vessel coated with a cell adhesion medium, or seeding the neural stem cells or neural precursor cells together with a cell adhesion medium into a culture vessel, wherein the neural stem cells or neural precursor cells after seeding are first generation cells (P1);

(3) Performing an adherent culture on the neural stem cells or neural precursor cells (P1) using an astrocyte precursor cell expansion medium, which is a medium containing growth factors in addition to a basal medium and essential components for maintaining cell growth, under conditions suitable for differentiation of the neural stem cells or the neural precursor cells into astrocyte precursor cells, to obtain astrocyte precursor cells, and the growth factors are FGF and EGF;

(4) Seeding the astrocyte precursor cells obtained in step (3) onto the surface of a culture vessel coated with a cell adhesion medium, or seeding the astrocyte precursor cells together with the cell adhesion medium into a culture vessel; and

(5) Performing adherent culture on the astrocytes using an astrocyte maturation medium, which is a medium containing growth factors in addition to a basal medium and essential components for maintaining cell growth, and which are BMP-4 and CNTF, under conditions suitable for the astrocyte precursor cells to differentiate into astrocytes, to obtain astrocytes,

The human embryonic stem cells are derived from human embryos within 14 days of fertilization that have not undergone in vivo development,

the medium used in step (3) and step (5) is serum-free.

2. The method of claim 1, wherein step (1) comprises culturing neural stem cells or neural precursor cells.

3. The method of claim 2, wherein the culturing is performed by one of:

(a) Culturing in a neural stem cell culture medium;

(b) Culturing in a neural precursor cell culture medium; or (b)

(c) Culturing in differentiation medium.

4. The method of claim 3, wherein neural stem cells or neural precursor cells are cultured in a differentiation medium and the culturing is continued until astrocyte-like cells appear.

5. The method of claim 1, wherein the cell adhesion medium in step (2) or step (4) is matrigel, vitronectin (Vitronectin) or Laminin (Laminin).

6. The method of claim 5, wherein the cell adhesion mediator is laminin.

7. The method of claim 6, wherein the laminin concentration is 5 μg/mL to 10 μg/mL.

8. The method of claim 7, wherein the laminin concentration is 6 μg/mL to 8 μg/mL.

9. The method of claim 1, wherein during the adherent culture of step (3), the cells are passaged every 5-7 days and to passage 7 (P7), 8 (P8) or 9 (P9).

10. The method of claim 9, wherein the cells are passaged every 5-7 days and at about 100% confluency during the adherent culture of step (3).

11. The method of claim 1, wherein step (5) ends the culture when the GFAP expressing positive cells are 90% or more.

12. The method of claim 1, wherein the entire culture medium is a chemically defined medium.

13. The method of claim 12, wherein the astrocytes obtained are suitable for clinical use.

14. An astrocyte culture obtainable by the method of any one of claims 1-13.

15. Use of the astrocyte culture according to claim 14 for the preparation of a medicament for the treatment of neurodegenerative diseases.

16. A method of preparing a neuron comprising co-culturing the astrocyte culture of claim 15 with a neural stem cell or a neural precursor cell.

17. A kit comprising an astrocyte precursor cell expansion medium and an astrocyte maturation medium,

The astrocyte precursor cell expansion medium comprises DMEM/F-12, N2 supplements and nonessential amino acids contained in a standard minimal essential medium, wherein the growth factors are EGF and FGF; and is also provided with

The astrocyte maturation medium comprises DMEM/F-12, N2 supplements, nonessential amino acids and glutamine contained in a standard minimal essential medium, wherein the growth factors are BMP-4 and CNTF,

the astrocyte precursor cell expansion medium and the astrocyte maturation medium are serum-free.