Method for efficiently and rapidly differentiating microglial cells

Abstract

The invention discloses a method for efficiently and rapidly differentiating microglial cells. According to the invention, the iPSC is creatively differentiated into the neural stem cells, and then the neural stem cells are induced and differentiated to obtain the microglial cells. The microglial cells obtained by applying the scheme show excellent homogeneity and relatively high yield; the differentiation scheme is simple and convenient, and microglial cells with relatively high purity can be obtained without suspension culture or a low-oxygen environment; meanwhile, the method disclosed by the invention is short in time consumption, the microglial cells with relatively high purity can be obtained only in 14 days, and the differentiation time is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell induction. Specifically, the present invention relates to a method for differentiating induced pluripotent stem cells into microglia. Background Art

[0002] As a key innate immune cell in the central nervous system, microglia play important roles in neurogenesis, synaptic remodeling, neuron function maintenance, tissue repair, blood-brain barrier integrity, angiogenesis, inflammation regulation, and myelin regeneration. In the field of in vitro research on microglia, directed differentiation of pluripotent stem cells such as iPSCs to obtain microglia is an important technical means for studying their differentiation pathways and functional mechanisms. Currently, the existing technical solutions for obtaining microglia mainly include the following several types:

[0003] (1) The first prior art solution is based on the understanding that only when iPSCs are first differentiated into hematopoietic progenitor cells and then induced to differentiate into microglia can the true entity of microglia be obtained. By inducing iPSCs to differentiate into hematopoietic progenitor cells, then differentiating hematopoietic progenitor cells into primitive macrophage progenitor cells, and finally adding specific inducing factors to the differentiation system to obtain microglia-like cells. However, this differentiation process has strict dynamic regulation requirements for oxygen concentration. Since most laboratories lack corresponding hypoxic culture and dynamic oxygen concentration control equipment, this technical solution is difficult to effectively repeat, limiting its wide application in the scientific research and clinical fields.

[0004] (2) The second prior art solution adopts a three-step differentiation path of "iPSC→embryoid bodies→primitive hematopoietic progenitor cells→microglia-like cells". Specifically, first, iPSCs are induced to form embryoid bodies (EBs), then EBs are differentiated into primitive hematopoietic progenitor cells, and finally, inducing factors are added to induce differentiation into microglia-like cells. This solution has a complex and cumbersome operation process, and the final yield of microglia is extremely low, making it difficult to meet the actual application requirements.

[0005] (3) The third prior art solution is to obtain microglia-like cells by jointly inducing and stimulating monocytes derived from peripheral blood with IL-34 and GM-CSF. This solution has a fundamental defect that the obtained cells cannot truly reflect the generation process of microglia entities, and the cell population has high heterogeneity, seriously affecting the stability and reliability of experimental results.

[0006] (4) The prior art solution four uses Extended Pluripotent Stem Cells (EPSC) as the starting cells, which need to go through four stages, and the differentiation process takes more than 28 days. During the implementation of this solution, not only are multiple small molecule compounds required to be added, but suspension culture operations are also required. The entire process is long and complex, with low differentiation efficiency, which is not conducive to large-scale popularization and application.

[0007] (5) Other solutions, such as first differentiating iPSC into hematopoietic progenitor cells in the differentiation stage, and then co-culturing with astrocytes to obtain microglia-like cells. Compared with the aforementioned methods, this solution additionally adds steps for the purification and extraction of microglia, resulting in cumbersome subsequent operations and a reduced cell yield.

[0008] Therefore, it is of great significance to provide a method for efficiently and rapidly differentiating microglia. Summary of the Invention

[0009] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a method for rapidly, simply, and efficiently differentiating highly homogeneous microglia from iPSC.

[0010] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention provides a method for preparing microglia.

[0012] Further, the method includes the following steps:

[0013] 1) Provide neural stem cells or a culture containing neural stem cells;

[0014] 2) Culture the neural stem cells or the culture containing neural stem cells in a microglia induction medium to obtain microglia.

[0015] Further, the microglia induction medium is Advanced DMEM / F12 medium supplemented with Activin A, CHIR 99021, M-CSF, GM-CSF, and TGF-β1.

[0016] Further, in the microglia induction medium, the concentration of Activin A is 100 ng / mL - 200 ng / mL, the concentration of CHIR 99021 is 3 μM - 6 μM, the concentration of M-CSF is 50 ng / mL, the concentration of GM-CSF is 20 ng / mL, and the concentration of TGF-β1 is 5 ng / mL.

[0017] Preferably, the concentration of Activin A in the microglia induction medium is 150 ng / mL, and the concentration of CHIR99021 is 4.5 μM.

[0018] Further, microglia are obtained after culturing in the microglia induction medium for 7 days in step 2).

[0019] Further, the microglia induction medium is replaced every 24 h.

[0020] Further, the neural stem cells or the culture containing neural stem cells are obtained by culturing pluripotent stem cells in a neural stem cell induction medium.

[0021] Further, the neural stem cell induction medium comprises a basal medium, a nutrient additive, and an induction factor; the basal medium is Advanced DMEM / F12 and Neurobasal TM Medium; the nutrient additive comprises B-27 (without vitamin A) additive and N-2 additive; the induction factor comprises SB431542, LDN193189, and CHIR 99021.

[0022] Preferably, the dosage ratio of Advanced DMEM / F12:Neurobasal TM Medium:B-27 (without vitamin A) additive:N-2 additive in the neural stem cell induction medium is 48:48:2:1.

[0023] Preferably, the concentration of SB431542 in the neural stem cell induction medium is 10 μM, the concentration of LDN193189 is 100 nM, and the concentration of CHIR 99021 is 3 μM.

[0024] Further, the pluripotent stem cells are cultured in the neural stem cell induction medium for 6 days.

[0025] Further, the neural stem cell induction medium is replaced every 24 h.

[0026] Preferably, the pluripotent stem cells are induced pluripotent stem cells.

[0027] Further, the method further comprises the steps of digesting and inoculating the product obtained by culturing the pluripotent stem cells in the neural stem cell induction medium for 6 days.

[0028] Further, the digestion includes: adding a digestive solution to the product after culturing for 6 days and incubating.

[0029] Furthermore, the digestion solution includes EDTA digestion solution, trypsin digestion solution, trypsin-EDTA digestion solution, collagenase digestion solution, and accutase digestion solution.

[0030] Preferably, the digestion solution is EDTA digestion solution.

[0031] Preferably, the concentration of the EDTA digestion solution is 0.5 mM.

[0032] Preferably, the incubation conditions are 37°C and 5 min.

[0033] Furthermore, the inoculation includes: collecting the cell suspension after digestion, resuspending the cells with culture medium, and inoculating them in a culture plate for continued cultivation.

[0034] Preferably, the culture medium is Advanced DMEM / F12 medium supplemented with Y-27632.

[0035] Preferably, the concentration of Y-27632 in Advanced DMEM / F12 medium is 10 μM.

[0036] Preferably, the continued culture time is 24 hours.

[0037] The present invention pioneered the differentiation of iPSC into neural stem cells (NSC) for 6 days, and then differentiated the neural stem cells into microglia (MG) for 7 days. In both differentiation stages, only induction factors need to be added to the basal culture medium to quickly, simply and efficiently obtain highly homogeneous microglia, which can significantly shorten the induction differentiation time and has a high microglia yield.

[0038] In the present invention, "induced pluripotent stem cells (iPSCs)" refer to stem cells with totipotency or pluripotency obtained by artificially inducing the expression of certain genes from certain adult cells (such as fibroblasts). In some methods known in the art, iPSCs can be obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. In some methods, the transfected genes may include transcription factors Oct4, Sox2, Klf4, and c-Myc, although co-transfection of other genes may potentially improve the induction efficiency. In other methods, somatic cells can be transformed using the Oct4, Sox2, Nanog, and Lin28 genes with a lentiviral system. Genes whose expression is induced in iPSCs include but are not limited to Oct-3 / 4; certain members of the Sox gene family (such as Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (such as Klf1, Klf2, Klf4, and Klf5); certain members of the Myc family (such as C-myc, L-myc, and N-myc); Nanog, Lin28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-Catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof. Currently, various reagents for preparing iPSCs, such as reprogramming vectors, expression cassettes, culture media, etc., and even commercialized iPSCs, are available on the market. hiPSCs refer to iPSCs induced from human cells.

[0039] In some embodiments, it can be determined whether induced pluripotent stem cells are successfully induced to differentiate into neural stem cells by observing the morphological changes of cells at different stages and detecting the expression of neural stem cell markers NESTIN / SOX2. Among them, detecting the expression of the marker refers to measuring the relative levels of the RNA transcripts of the gene (marker) or its expression products. Methods for detecting gene expression / transcription, namely gene expression / transcription profiling, include methods based on polynucleotide hybridization analysis, methods based on polynucleotide sequencing, immunohistochemical methods, and proteomics-based methods.

[0040] In some embodiments, it can be determined whether neural stem cells are successfully induced to differentiate into microglia by detecting the morphological changes of cells and detecting the expression of microglia-specific genes such as IBA1 and TMEM119.

[0041] In some embodiments, the present invention does not particularly limit the specific contents of the components in the microglia induction medium and the neural stem cell induction medium, as long as the microglia induction medium and the neural stem cell induction medium composed of the components with specific contents can exert or basically exert the expected effects (for example: can effectively or basically effectively induce the differentiation of extended pluripotent stem cells into neural stem cells), they are all within the protection scope of the present invention.

[0042] In some embodiments, the components referred to in the present invention refer to any compound or other material that can be used in cell culture media to maintain and / or promote cell growth and / or differentiation, regardless of its source being chemical or biological. The terms "component" and "ingredient" can be used interchangeably. Conventional ingredients for cell culture media may include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other ingredients for promoting and / or maintaining in vitro or ex vivo cell cultures can be selected by those of ordinary skill in the art according to the needs of the desired effects.

[0043] The second aspect of the present invention provides a culture system for preparing microglia.

[0044] Furthermore, the culture system includes the microglia induction medium described in the first aspect of the present invention.

[0045] Preferably, the culture system further includes the neural stem cell induction medium and the inoculated medium described in the first aspect of the present invention.

[0046] The third aspect of the present invention provides a kit for preparing microglia.

[0047] Furthermore, the kit includes the microglia induction medium described in the first aspect of the present invention.

[0048] Preferably, the kit further includes the neural stem cell induction medium and the inoculated medium described in the first aspect of the present invention.

[0049] The fourth aspect of the present invention provides a microglia or cell population derived from pluripotent stem cells.

[0050] Furthermore, the microglia or cell population is induced and differentiated by the method described in the first aspect of the present invention.

[0051] The fifth aspect of the present invention provides a pharmaceutical composition.

[0052] Furthermore, the pharmaceutical composition contains an effective amount of the microglia or cell population described in the fourth aspect of the present invention.

[0053] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutically acceptable carrier and / or excipient include, but are not limited to: diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants. These substances are used as needed to help the stability of the formulation or to enhance the activity or its bioavailability. The formulations that can be used in such drugs can be in the form of the original compound itself, or optionally in the form of its pharmaceutically acceptable salts. The pharmaceutical composition thus formulated can be administered in any suitable manner known to those skilled in the art as needed.

[0054] In some embodiments, the suitable dosage of the pharmaceutical composition of the present invention can be prescribed in various ways according to factors such as the mode of administration, the age, weight, gender, morbidity, diet, administration time, administration route, excretion rate, and responsiveness of the patient. Skilled doctors can usually easily determine the prescription and the dosage of the prescription effective for the desired treatment.

[0055] The sixth aspect of the present invention provides an application in any of the following aspects, and the application includes:

[0056] 1) The application of the culture system described in the second aspect of the present invention in the preparation of microglia;

[0057] 2) The application of the kit described in the third aspect of the present invention in the preparation of microglia;

[0058] 3) The application of the microglia or cell population described in the fourth aspect of the present invention in the preparation of a product for treating neurodegenerative diseases;

[0059] 4) The application of the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a product for treating neurodegenerative diseases.

[0060] In addition, the present invention also provides a method for treating and / or preventing neurodegenerative diseases, and the method includes the following steps: administering to a subject in need a therapeutically and / or prophylactically effective amount of the microglia or cell population or pharmaceutical composition provided by the present invention.

[0061] In the present invention, the subject refers to any animal, and also refers to humans and non-human animals. Non-human animals include all vertebrates, for example, mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and any domestic animals or pets; and non-mammals such as chickens, amphibians, reptiles, etc. In a specific embodiment of the present invention, the subject is preferably a human.

[0062] In the present invention, the treatment and / or prevention refers to preventing and reducing the occurrence or development of a disease, inhibiting, containing, alleviating, improving, slowing down, stopping, delaying or reversing the development or aggravation of the disease course, and various indicators of the disease, disorder or pathological state during the described maintenance and / or medication use include alleviating or reducing the symptoms or complications of a specific disease, or curing or eliminating the disease, disorder or condition. Therefore, the treatment and / or prevention described in the present invention includes preventing, alleviating and / or treating the disease (e.g., neurodegenerative diseases).

[0063] In the present invention, the neurodegenerative disease or disorder includes at least one of the following: Parkinson's disease, Alzheimer's disease, prion disease, motor neuron diseases (MND) such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Lewy body disease, epilepsy, encephalitis, hydrocephalus, stroke, chronic traumatic encephalopathy (CTE); synucleinopathy; tauopathy, spongiform encephalopathy; familial amyloidotic polyneuropathy; Dutch hereditary cerebral hemorrhage with amyloidosis; congophilic angiopathy; corticobasal degeneration; Pick's disease; progressive supranuclear palsy; Creutzfeldt-Jakob disease; Gerstmann syndrome; fatal familial insomnia; kuru disease; bovine spongiform encephalopathy; scrapie; chronic wasting disease; Lewy body variant of Alzheimer's disease; diffuse Lewy body disease; Lewy body dementia; multiple system atrophy; type I neuronal degeneration with brain iron accumulation; diffuse Lewy body disease; frontotemporal degeneration; hereditary dentatorubral-pallidoluysian atrophy; Kennedy disease; Alexander disease; Cockayne syndrome; and Icelandic type hereditary amyloidotic cerebral hemorrhage.

[0064] Advantages and beneficial effects of the present invention:

[0065] 1) High homogeneity: The present invention pioneered the differentiation of microglia from iPSCs via the neural stem cell pathway, and the obtained microglia have extremely high homogeneity and yield.

[0066] 2) Simple and convenient differentiation protocol: The present invention only requires a two-step method, and highly pure microglia can be harvested without suspension culture or hypoxic environment.

[0067] 3) Short time-consuming: The present invention only requires 14 days to harvest highly pure microglia, greatly shortening the differentiation time. Brief Description of the Drawings

[0068] Figure 1 Schematic diagram of the iMG induction differentiation protocol;

[0069] Figure 2 Cell morphology diagrams at different time points;

[0070] Figure 3 It is the immunofluorescence staining result diagram of genes related to iPSC stemness;

[0071] Figure 4 It is the immunofluorescence staining result diagram of cells on the 6th day of differentiation expressing neural stem cell marker genes;

[0072] Figure 5 It is the immunofluorescence staining result diagram of neural stem cell marker genes after iNSC digestion and re - adherent;

[0073] Figure 6 It is the cell morphology diagram under different small molecule concentrations;

[0074] Figure 7 It is the cell differentiation efficiency result diagram under different small molecule concentrations;

[0075] Figure 8 It is the identification result diagram of iMG marker genes;

[0076] Figure 9 It is the result diagram of detecting cell senescence status by β - galactosidase staining;

[0077] Figure 10 It is the morphological change diagram of microglia before and after PBS washing. Detailed implementation manners

[0078] The following combines specific embodiments to further elaborate the present invention. The specific embodiments are only used to explain the present invention and cannot be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and purposes of the present invention, various changes, modifications, substitutions, and variations can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents.

[0079] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Without special instructions, they can all be obtained from commercial channels. The experimental methods without specific conditions noted in the present invention are usually implemented according to conventional conditions or the conditions recommended by the manufacturers. In particular, the following embodiments are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0080] Example 1 iMG (microglia derived from iPSC) differentiation protocol and cell morphology record at key time points

[0081] 1. Experimental materials

[0082] iPSC cell line, B8 medium, Advanced DMEM / F12 medium, Neurobasal medium, B-27™ Supplement (50X) minus vitamin A, N-2 Supplement (100X), SB431542, LDN193189, CHIR99021, Activin A, M-CSF, GM-CSF, TGF-β1. Their specific sources are shown in Table 1.

[0083] Table 1 Sources of experimental materials

[0084] Reagent Manufacturer Article number B8 Medium Bioinnova BIB-ALMA025 Advanced DMEM / F-12 Gibco 12634-010 Neurobasal™ Medium Life technologies 21103049 B-27™ Supplement (50X), minus vitamin A Gibco 12587-010 N-2 Supplement (100X) Thermo 17502048 SB431542 Abcam ab120163 LDN193189 Sigma SML0559 CHIR99021 StemCell Technologies 72054 Human Recombinant Activin A StemCell Technologies 78001.3 Human GM-CSF PeproTech 300-03 Recombinant Human M-CSF (carrier-free) Biolegend 574808 TGF-β1 Saibei Biotechnology CA32002

[0085] 2. Experimental methods

[0086] ① Prepare a well plate coated with Matrigel matrix gel 2 hours in advance (Matrigel matrix gel: DMEM / F12 = 1:240);

[0087] ② Prepare neural stem cell induction medium (24 mL of Advanced DMEM / F12, 24 mL of Neurobasal TM Medium, 1 mL of B-27™ Supplement (50X) minus vitamin A, 0.5 mL of N-2 Supplement (100X), 10 μM of SB431542, 100 nM of LDN193189, 3 μM of CHIR99021);

[0088] ③ Culture iPSC cells with B8 medium. When their growth density reaches 80%-90%, randomly select well plates for the stemness identification of iPSCs. Randomly select other well plates and inoculate them into new well plates coated with Matrigel matrix gel at an inoculation ratio of 1:10, and culture them with neural stem cell induction medium for 6 days. Change the medium every day and take pictures to record the cell morphology. On the 6th day, obvious neural rosette structures appear ( Figure 2 ), and at the same time, samples are reserved for immunofluorescence verification;

[0089] ④ Prepare microglia induction medium (50 mL of Advanced DMEM / F12, 150 ng / mL of Activin A, 4.5 μM of CHIR 99021, 50 ng / mL of M-CSF, 20 ng / mL of GM-CSF, 5 ng / mL of TGF-β1);

[0090] ⑤ Digest the neuro-rosette cells in step ③ with 0.5 mM EDTA at 37°C for 5 min, inoculate them on a 6-well plate coated with Matrigel matrix gel at a passage density of 1:6, and culture them in Advanced DMEM / F12 (+10 μM Y-27632) basal medium for 1 day. From the 8th day, change to the microglia induction medium, change the medium every day and take pictures to record the cell morphology until obvious branched cell morphology appears on the 14th day ( Figure 2 ), and at the same time, keep samples for immunofluorescence and flow cytometry detection.

[0091] Generally, in the present invention, iPSCs are first differentiated into neural stem cells (NSCs) for 6 days, and the NSCs are differentiated into microglia 1 day after digestion and re-plating, with a differentiation time of 7 days. In both of these two differentiation stages, only by adding induction factors to the basal medium can homogeneous microglia be obtained quickly, simply and efficiently, which can significantly shorten the induction and differentiation time and the yield of microglia is high ( Figure 1 ).

[0092] At different time points of differentiation, take pictures of the cells in the culture state and record the morphology. On the 6th day of differentiation, obvious neuro-rosette morphology can be observed. On the 7th day of differentiation, significant neural stem cell morphology can be observed after the NSC cells are digested and re-plated and adhere to the wall. At the 14th day of the second-stage differentiation, significant microglia morphology can be observed ( Figure 2 ).

[0093] Example 2 Identification of iPSCs

[0094] At the beginning of differentiation (day0), identify the pluripotency-related genes SOX2, OCT4, NANOG, SSEA4, and TRA-1-60 for the iPSCs. The results show that these five genes related to pluripotency are highly expressed in this strain of iPSCs, indicating that its pluripotency is very strong and it can undergo the next step of differentiation ( Figure 3 ).

[0095] Example 3 Identification of NSCs Induced from iPSCs

[0096] On the 6th day of differentiation, fix the cells with obvious neuro-rosettes with 4% PFA and perform immunofluorescence staining. The results show that the cells highly express the neural stem cell-related genes NESTIN and SOX2, and obvious neuro-rosette structures appear, indicating that the iPSCs have been successfully differentiated into the NSC stage ( Figure 4 ).

[0097] After NSC digestion and re - plating, the cells were fixed with 4% PFA and subjected to immunofluorescence staining. The results showed that the cells highly expressed the neural stem cell - related genes NESTIN and SOX2, indicating that iPSC had been successfully differentiated into the NSC stage ( Figure 5 ).

[0098] Example 4 Identification of iMG

[0099] To explore the effect of different small molecule concentrations on microglial differentiation, we set up concentration gradients to verify the differentiation efficiency. Low concentration (Small concentration, SC) (Activin A 100 ng / mL, CHIR 99021 3 μM, M - CSF 50 ng / mL, GM - CSF 20 ng / mL, TGF - β1 5 ng / mL), medium concentration (Middle concentration, MC) (Activin A 150 ng / mL, CHIR 99021 4.5 μM, M - CSF 50 ng / mL, GM - CSF 20 ng / mL, TGF - β1 5 ng / mL), high concentration (High concentration, HC) (Activin A 200 ng / mL, CHIR 99021 6 μM, M - CSF 50 ng / mL, GM - CSF 20 ng / mL, TGF - β1 5 ng / mL). It was observed that obvious microglial - like cells could be seen at the end - point of differentiation in all three groups. However, compared with the low - concentration and medium - concentration groups, the high - concentration small - molecule group had more floating cells and sparser cells, indicating that higher - concentration small molecules might affect cell survival ( Figure 6 ).

[0100] Furthermore, to explore the differentiation efficiency of cells under different concentrations of small molecules, on the 14th day of differentiation, we detected the proportion of Iba1 - positive cells by flow cytometry. The results showed that under the medium - concentration differentiation protocol, the differentiation efficiency of microglia was the highest, reaching 96.70%. Therefore, we selected the medium concentration as the optimal differentiation protocol ( Figure 7 ).

[0101] On the 14th day of differentiation, the cells were fixed with 4% PFA and subjected to immunofluorescence staining. The results showed that the cells highly expressed the microglia - common gene IBA1 and simultaneously highly expressed the microglia - specific gene TMEM119, indicating that iPSC had been successfully differentiated into microglia ( Figure 8 ).

[0102] On the 14th day of differentiation, the cells were stained with β-galactosidase. The results showed that the differentiated microglial cells did not show senescence ( Figure 9 ).

[0103] On the 14th day of differentiation, when changing the culture medium of the cells, PBS washing was performed. The results showed that the morphology of microglial cells would shrink significantly before and after PBS washing, indicating that the cells would have an obvious reaction even in the face of a buffer such as PBS, which is in line with the property of strong sensitivity of microglial cells ( Figure 10 ).

[0104] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing microglia, characterized in that, The method includes the following steps: 1) Providing neural stem cells or a culture comprising neural stem cells; 2) Culturing the neural stem cells or the culture comprising neural stem cells in a microglia induction medium to obtain microglia; The microglia induction medium is Advanced DMEM / F12 medium supplemented with Activin A, CHIR 99021, M-CSF, GM-CSF, and TGF-β1.

2. The method according to claim 1, characterized in that, In the microglia induction medium, the concentration of Activin A is 100 ng / mL to 200 ng / mL, the concentration of CHIR 99021 is 3 μM to 6 μM, the concentration of M-CSF is 50 ng / mL, the concentration of GM-CSF is 20 ng / mL, and the concentration of TGF-β1 is 5 ng / mL; Preferably, in the microglia induction medium, the concentration of Activin A is 150 ng / mL and the concentration of CHIR 99021 is 4.5 μM.

3. The method according to claim 1, characterized in that, In step 2), microglia are obtained after culturing in the microglia induction medium for 7 days; Preferably, the microglia induction medium is replaced every 24 h.

4. The method according to claim 1, characterized in that, The neural stem cells or the culture comprising neural stem cells are obtained by culturing pluripotent stem cells in a neural stem cell induction medium; Preferably, the neural stem cell induction medium comprises a basal medium, a nutrient additive, and an induction factor; the basal medium is Advanced DMEM / F12 and Neurobasal TM Medium; the nutrient additive comprises a B-27 (without vitamin A) additive and an N-2 additive; the induction factor comprises SB431542, LDN193189, and CHIR 99021; Preferably, in the neural stem cell induction medium, the ratio of Advanced DMEM / F12: Neurobasal TM Medium: B-27 (without vitamin A) additive: N-2 additive is 48:48:2:1; Preferably, in the neural stem cell induction medium, the concentration of SB431542 is 10 μM, the concentration of LDN193189 is 100 nM, and the concentration of CHIR 99021 is 3 μM; Preferably, the pluripotent stem cells are cultured in the neural stem cell induction medium for 6 days; Preferably, the neural stem cell induction medium is replaced every 24 h; Preferably, the pluripotent stem cells are induced pluripotent stem cells.

5. The method according to claim 4, characterized in that, The method further includes the steps of digesting and inoculating the product obtained after culturing the pluripotent stem cells in the neural stem cell induction medium for 6 days; Preferably, the digestion includes: adding a digestive solution to the product after culturing for 6 days and incubating; Preferably, the digestive solution includes EDTA digestive solution, trypsin digestive solution, trypsin-EDTA digestive solution, collagenase digestive solution, accutase digestive solution; Preferably, the digestive solution is EDTA digestive solution; Preferably, the concentration of the EDTA digestive solution is 0.5 mM; Preferably, the conditions for the incubation are 37°C and 5 min; Preferably, the inoculation includes: collecting the digested cell suspension, resuspending the cells with a medium, and inoculating them into a culture plate for further culture; Preferably, the medium is Advanced DMEM / F12 medium supplemented with Y-27632; Preferably, the concentration of Y-27632 in the Advanced DMEM / F12 medium is 10 μM; Preferably, the time for the further culture is 24 h.

6. A culture system for preparing microglia, characterized in that, The culture system includes the microglia induction medium described in claim 1 or 2; Preferably, the culture system further comprises the neural stem cell induction medium described in claim 3 and the medium described in claim 4.

7. A kit for preparing microglia, characterized in that, The kit comprises the microglia induction medium described in claim 1 or 2; Preferably, the kit further comprises the neural stem cell induction medium described in claim 3 and the medium described in claim 4.

8. A microglia or cell population derived from pluripotent stem cells, characterized in that, The microglia or cell population is induced and differentiated by the method described in any one of claims 1-4.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the microglia or cell population described in claim 7.

10. Application in any of the following aspects, the application comprising: 1) Application of the culture system described in claim 6 in the preparation of microglia; 2) Application of the kit described in claim 7 in the preparation of microglia; 3) Application of the microglia or cell population described in claim 8 in the preparation of a product for treating neurodegenerative diseases; 4) Application of the pharmaceutical composition described in claim 9 in the preparation of a product for treating neurodegenerative diseases.

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