CN117778313B - Differentiation method and application of mesenchymal stem cells obtained from brain organoids - Google Patents
Differentiation method and application of mesenchymal stem cells obtained from brain organoids Download PDFInfo
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Abstract
The invention relates to the technical field of stem cells, in particular to a method for obtaining mesenchymal stem cells from brain organoids and application thereof. The method for obtaining the mesenchymal stem cells by the brain organoids comprises the following steps in sequence: the pluripotent stem cells are cultured by a pluripotent stem cell culture medium containing Y27632 to form cell spheres; culturing the cell ball by using an NIM differentiation medium to obtain a neurosphere; culturing neurospheres by using an NDM differentiation medium to obtain a rose ring structure brain organoid; culturing the brain organoids with the rose ring structure by using an NMM differentiation medium to obtain the brain organoids; the brain organoid is digested into single cells, and mesenchymal stem cells are obtained after screening culture and subculture. The technical scheme can solve the technical problem that the mesenchymal stem cells have an unsatisfactory effect of treating the sicca syndrome. The mesenchymal stem cells of the invention do not use matrigel in the preparation process, have no animal-derived components, are simple and convenient to operate, and have ideal application prospect.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a method for obtaining mesenchymal stem cells from brain organoids and application thereof.
Background
Sjogren's Syndrome (SS) is a chronic inflammatory autoimmune disease that primarily involves exocrine glands, also known as autoimmune exocrine gland epithelial cell inflammation or autoimmune exocrine disease. In clinical practice, there are symptoms of multiple system damage caused by the involvement of other exocrine glands and organs outside the glands besides dry mouth and dry eyes caused by the impaired function of salivary glands and lacrimal glands. There are various autoantibodies and hyperimmune globulinemia in the serum.
Mesenchymal Stem Cells (MSCs) are widely available and can be isolated from a variety of adult tissues including bone marrow, umbilical cord blood, adipose tissue, dental tissue, skin and placenta. MSCs, with their powerful immunomodulatory functions, are very productive in the treatment of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis and type I diabetes. Cell therapy using allogeneic or autologous MSCs has become a promising emerging therapeutic strategy for the treatment of sjogren's syndrome. Ideally, however, mesenchymal stem cell therapy requires a convenient and relatively uniform source of cells and the ability to produce cells with stable phenotype and function. At present, the effect of mesenchymal stem cells on treating sicca syndrome still needs to be further improved.
Disclosure of Invention
The invention aims to provide a method for obtaining mesenchymal stem cells by brain organoids so as to solve the technical problem that the mesenchymal stem cells have an unsatisfactory effect of treating sicca syndrome.
In order to achieve the above purpose, the invention adopts the following technical scheme:
The method for obtaining the mesenchymal stem cells by the brain organoids comprises the following steps in sequence:
S1: the pluripotent stem cells are cultured by a pluripotent stem cell culture medium containing Y27632 to form cell spheres;
S2: culturing the cell ball by using an NIM differentiation medium to obtain a neurosphere; NIM differentiation medium contains dorsomorphin and SB-431542;
S3: culturing neurospheres by using an NDM differentiation medium to obtain a rose ring structure brain organoid; the NDM differentiation medium contains EGF and bFGF;
S4: culturing the brain organoids with the rose ring structure by using an NMM differentiation medium to obtain the brain organoids; the NMM differentiation medium contains BDNF and NT-3;
S5: the brain organoid is digested into single cells, and mesenchymal stem cells are obtained after screening culture and subculture.
The technical scheme also provides the neural mesenchymal stem cells obtained by the method for obtaining the mesenchymal stem cells by the brain organoids.
The technical scheme also provides application of the neural mesenchymal stem cells obtained by the method for obtaining the mesenchymal stem cells by the brain organoids in preparing medicaments for treating sicca syndrome.
Further, in S2, the basal medium of the NIM differentiation medium is DMEM/F12; the addition components of NIM differentiation medium consist of KOSR, NEAA, glutaMAX, 2-mercaptoethanol, dorsomorphin and SB-431542.
Further, in S3, the basal medium of the NDM differentiation medium is a Neurobasal-A medium; the added components of the NDM differentiation medium consist of B27, glutamax, EGF and bFGF.
Further, in S4, the basal medium of the NMM differentiation medium is Neurobasal-a medium; the added components of the NDM differentiation medium consisted of B27, glutaMAX, BDNF and NT-3.
Further, the time required for S1-S4 is: 1 day, 6 days, 20 days, 10 days.
Further, in S5, the screening culture is performed in a culture vessel coated with VTN protein.
To sum up, the principle and the beneficial effects of the technical scheme are as follows:
In the technical scheme, the pluripotent stem cells are used in U-shaped holes to form brain organoids with stable structure and uniform phenotype in the presence of Rock inhibitors. Induced differentiation is largely divided into four stages. In the first stage, double SMAD inhibitors dorsomorphin and SB-431542 are used for suspension culture to promote nerve differentiation; in the second stage, two cytokines EGF and bFGF are used for continuous suspension culture, so that the generation and proliferation of a cerebral organoid cortex layer are promoted; in the third stage, brain organoids are further differentiated to maturity by exposure to neurotrophic factors BDNF and NT 3. And in the fourth stage, the digestive juice is used for decomposing the brain organoid into single cells, then the single cells are subjected to adherence culture, and the non-adherence mixed cells are removed in the subculture process, so that nMSC (mesenchymal stem cells of nerve origin) with uniform phenotype can be obtained. The technical scheme uses the mesenchymal stem cells from the nerve source, and has stronger T cell proliferation inhibition capability and more ideal effect of inhibiting the secretion of T cell inflammatory factors compared with the mesenchymal stem cells similar to umbilical cord sources. Serum SSA/SSB antibodies showed significantly lower than control after three weeks of continuous treatment in mice with the sjogren syndrome model, and submaxillary HE staining showed a significant decrease in the number and proportion of focal points after treatment. The upper prompt nMSC can have more excellent treatment effect on the Sjogren syndrome.
Compared with the prior art, the technical scheme has the advantages that:
(1) The multipotent stem cells are used for forming brain organoids, nMSC is obtained from the brain organoids, and the pain points with unstable MSC material taking and large batch-to-batch difference in clinic are solved.
(2) The used equipment is conventional cell culture equipment, and expensive instruments and equipment such as a fermentation tank, a bioreactor and the like are not required to be purchased separately.
(3) Compared with other methods such as a 6-hole plate, a stirrer, a bioreactor and the like, the method for forming the brain organoids by using the 96-hole plate can accurately control the initial brain organoids, ensure the homogeneity of the brain organoids in the same batch, and reduce quality control points in the cell preparation process.
(4) The whole induced differentiation process does not use matrigel, does not use animal-derived components, and is more suitable for clinical application of products. nMSC obtained in this scheme can be used directly for therapeutic purposes without considering adverse effects (e.g., antigenicity, safety, etc.) caused by residues of animal-derived components.
(5) Small molecules and recombinant proteins in the induced differentiation process are few in variety, the differentiation is directionally regulated and controlled, the path is simple, and the cost is saved.
(6) The SMAD inhibitor was changed to SB431542 and dorsomorphin, and the neurosphere structure formed in the first stage was more stable, with higher phenotypic uniformity and less batch-to-batch variation than the a83-01 and dorsomorphin used in patent CN113025569 a.
Drawings
FIG. 1 is a nMSC flow chart (A: mesenchymal stem cell positive marker flow chart; B: mesenchymal stem cell negative marker flow chart) of the preparation of example 1.
FIG. 2 shows the morphology map nMSC (A: nMSC clear field map under an optical microscope; B: nMSC alizarin red staining map after osteogenic differentiation; C: nMSC oily red O staining map after adipogenic differentiation; D: nMSC Paraffin section Allinn blue staining map after chondrogenic differentiation) of the preparation of example 1.
FIG. 3 is a graph comparing the inhibition of T cell proliferation by nMSC and ucMSC of example 2.
FIG. 4 is a graph comparing the inhibition of T cell inflammatory factor secretion by nMSC and ucMSC of example 3.
FIG. 5 is a graph showing the relative amounts of IDO and HLA-G mRNA secreted by nMSC and ucMSC after IFN-. Gamma.stimulation in example 4 (data format: cell IDO/HLA-G mRNA relative amount.+ -. Standard deviation, n= 3;A is a statistical chart showing the results of measuring IDO mRNA relative amount, and B is a statistical chart showing the results of measuring HLA-G mRNA relative amount).
FIG. 6 is a diagram of the serum antibody detection of nMSC mice for treatment of Sjogren syndrome of example 5 (A is a statistical diagram of the serum SSA antibody detection results, data form: mean.+ -. Standard deviation of SSA antibody content per ml, n=10; B is a HE staining diagram; C is a statistical diagram of the serum SSB antibody detection results, data form: mean.+ -. Standard deviation of SSB antibody content per ml, n=10; D is a statistical diagram of the foci area ratio, data form: foci area ratio.+ -. Standard deviation, n= 9;F is a statistical diagram of foci number, data form: foci number.+ -. Standard deviation in a single field, n=9).
Fig. 7 is a graph showing brain organoid phenotypes of EB spheres of example 1 and comparative example 1 under different SMAD inhibitors.
FIG. 8 shows the removal of Matrigel and the use of nMSC streams containing Matrigel to measure CD90 expression in the third stage of comparative example 2 (A is a stream cytometry image of the Matrigel-removed experimental set; B is a stream cytometry image of the Matrigel-containing experimental set).
FIG. 9 is a graph showing morphology of brain organoids and nMSC at various times in the EB ball of example 1.
FIG. 10 is a graph of morphology of brain organoids and nMSC at various times with EB pellets of comparative example 3 (CN 113025569A).
FIG. 11 is a statistical plot of nMSC yields (data format: number of adherent cells on day 6 of culture P0.+ -. Standard deviation, n=2) for example 1 and comparative example 3 (CN 113025569A).
FIG. 12 shows the results of the sequencing of the transcriptome mRNA of nMSC and ucMSC (A: ucMSC and nMSC differential gene expression heat maps; B: nMSC differential gene volcanic maps compared to ucMSC).
FIG. 13 is a bar graph showing the GO enrichment of ucMSC and nMSC genes.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1: method for inducing formation of brain organoids by pluripotent stem cells and obtaining mesenchymal stem cells
(1) Balling step (day 0): human pluripotent stem cells (hpscs) with a confluence of about 85% were used, digested into single cells with cell digests Accutase (a cell digests containing proteolytic and collagenase activities, commercially available for digesting stem cells), centrifuged, resuspended in hPSC medium (stemcell, 100-0276) with 10 μ M Y27632 (ROCK inhibitor) added, and the single cell suspension was added to an ultra-low adsorption U bottom 96 well plate at 4000 cells/well, centrifuged at 300g for 5min, and left standing in a cell incubator to pellet. Wherein, the hPSC used in the step is from the middlemost traceable biotechnology Co., ltd, and the serial number is 20211011-LLY-C1-P6. Y27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor with CAS number 146986-50-7.
(2) Transfer wells (day 1), select cell pellets between about 260-300 μm in diameter, transfer to a 6-well plate without TC treatment, place the 6-well plate on a shaker, and incubate in an incubator. The swing of the shaker was 24mm, the rotational speed was set at 60rpm, the carbon dioxide concentration in the incubator was set at 5% and the temperature was set at 37 ℃.
(3) The first phase is the neuroinduction phase (day 1-day 6): on day 1, the medium was changed to NIM differentiation medium, after which the new medium was changed every other day, and the shaking table rotation speed was 60rpm. NIM differentiation medium configuration: the basal medium was DMEM/F12 supplemented with 20% Knockout ™ SR (serum replacement, KOSR), 1% NEAA (nonessential amino acids, a prior art conventional cell culture supplement, ready-made), 1% GlutaMAX (L-alanyl-L-glutamine), 0.1mM 2-mercaptoethanol (mercaptoethanol), 5. Mu.M dorsomorphin (Compound C or BML-275, CAS number: 866405-64-3) and 10. Mu.M SB-431542 (CAS number: 866405-64-3). The brain organoids gradually develop into neurospheres with darker middle colors and transparent edge colors from round spheres with similar sizes and uniform internal tissues.
(4) The second stage is the neural differentiation stage (day 7-day 26): on day 7, the culture medium was replaced with NDM differentiation medium, and thereafter replaced with fresh medium every other day, and the rotation speed of the shaking table was 80rpm. NDM differentiation medium configuration: the basal medium was Neurobasal-A medium (Neurobasal ™ -A medium), supplemented with 2% B27 (B27 cell culture supplement, a prior art conventional cell culture supplement, ready-made), 1% GlutaMAX, 20ng/mL EGF (epidermal growth factor) and 20ng/mL bFGF (basic fibroblast growth factor). The brain organoids gradually increase at this stage, the rosette-like structure begins to appear in the interior on day 9, the rosette-like structure gradually becomes clear and increases on day 9-21, and the central tissue of the spheroids on day 21-26 is darkened and radiated to the periphery.
(5) Third stage neural maturation stage (day 27-day 36): the NMM differentiation medium was changed at day 27, after which the new medium was changed every other day, and the shaking table was rotated at 100rpm. Along with the extension of the induced differentiation time, the rotation speed is increased, the mutual adhesion of the organoids is avoided, and the exchange of the organoids of the brain with small molecules in a culture medium is better promoted. NMM differentiation medium configuration: the basal medium was a Neurobasal-A medium supplemented with 2% B27, 1% GlutaMAX, 20ng/mL BDNF (brain-derived neurotrophic factor ) and 20ng/mL NT-3 (neurotrophic factor-3). The brain organoids are further enlarged at this stage, the color is deepened by spreading from the center to the periphery of the sphere, the rose ring structure disappears, and the peripheral part of the nerve-like tissue lines are distributed.
(6) Fourth stage nMSC screening stage: brain organoids on day 36 were digested with Accutase for 10-15min, after termination of digestion, single cell suspensions were formed by filtration using a 100 μm cell screen, the cells were inoculated in VTN (vitronectin) -coated flasks/dishes at 5×10 4cells/cm2, counted as P0 passages, cultured with mesenchymal stem cell medium (medium tracing, RP 02010), and passaged when the degree of confluence was around 90%. The P1 seed density was 2X 10 4cells/cm2, the P2 seed density was 1X 10 4cells/cm2, and thereafter the seed density was 8X 10 3cells/cm2. nMSC expansion passaging mesenchymal stem cell medium (friend, NC 106) was used.
(7) Serial subculture nMSC, collecting P5 cells (5 th generation cells) and performing flow detection, wherein the detection results are shown in figure 1, and the positive index (CD 73/CD90/CD 105) and the negative index (CD 14/CD19/CD34/CD 45/HLA-DR) meet the mesenchymal stem cell standard.
(8) After P5 cells are subjected to adherent culture, the cells are replaced by adipogenic, osteogenic and chondrogenic differentiation media, and after induction is finished, oil red O staining, alizarin red staining and paraffin section alisxin blue staining are respectively used for photographing, and the results are shown in figure 2, so that the obtained cells are proved to have the three-lineage differentiation capability.
Example 2: mesenchymal stem cells inhibit T cell proliferation
NMSC (prepared in example 1, neural-derived mesenchymal stem cells) and ucMSC were plated and wall-mounted on the first day at a density of 1×10 5, wherein ucMSC is umbilical cord-derived mesenchymal stem cells (commercially available as conventional cells in the prior art). The following day donor Peripheral Blood Mononuclear Cells (PBMCs) were isolated using lymphocyte separation fluid and PBMCs were collected for CytoTell Blue staining. After the completion of the staining, co-culture was performed with mesenchymal stem cells according to 1×10 6, and simultaneously stimulation was performed by adding PHA, and no stimulator was added to the unstimulated control group. After 5 days of culture, the supernatant was stained for Zombie and CD3, and flow-through detection was performed. The results are shown in fig. 3, nMSC has a greater capacity to inhibit T cell proliferation (68.25%) than ucMSC has to inhibit T cell proliferation (58.32%).
Example 3: mesenchymal stem cells inhibit T cells from secreting inflammatory factors
NMSC and ucMSC were plated for adherent culture at a density of 2X 10 5 a day in advance. Donor PBMCs were isolated using lymphoid isolation, as mesenchymal stem cells: t cells = 1:10 ratio PBMC cells were inoculated, incubated for 43h followed by stimulation with PMA, lonomycin and BFA, and unstimulated groups were set without addition of the stimulator. After 5h of stimulation, the supernatant was collected and stained for cell surface markers CD3 and intracellular factor IFN-gamma, TNF-alpha for flow detection. As shown in FIG. 4, ucMSC has an IFN-gamma inhibition of 86.99% and TNF-alpha inhibition of 50.71%; nMSC has an IFN-gamma inhibition of 97.09% and a TNF-alpha inhibition of 92.58%. nMSC prepared by the technical scheme has more ideal effect of inhibiting the secretion of T cell inflammatory factors than other types of mesenchymal stem cells.
Example 4: mesenchymal stem cells induce IDO and HLA-G secretion
Inoculating nMSC and ucMSC at density of 2×10 5, adding 10ng/ml IFN-gamma into the culture system, culturing for 24 hr, collecting cells, extracting RNA, reverse transcribing into cDNA, performing RT-qPCR as template, and calculating relative expression of IDO (indoleamine 2, 3-dioxygenase) and HLA-G (human leukocyte antigen-G). The results are shown in FIG. 5, where nMSC and ucMSC secrete more IDO and HLA-G under IFN-gamma stimulation, demonstrating similar immunomodulatory functions of nMSC and ucMSC.
From the experimental data of examples 2-4, nMSC has better effect than ucMSC, has more ideal function in immune regulation and has more ideal potential for alleviating the sicca syndrome. Therefore, a subsequent test of the effect of treatment on the Sjogren's syndrome was performed on nMSC.
Example 5: in vivo experiments
Control mice were used as CD-1 (ICR) mice IGS SPF grade (CD-1. RTM. ICR mice, beijing Vitolihua laboratory animal Co., ltd.) and model mice were used as NOD/ShiLtJGpt mice (Jiangsu Jiugai Kangsu Biotech Co., ltd., N000235). Mice were divided into three groups: mice in the control group, naCl treated group, nMSC treated group, nMSC treated group were treated three times continuously with a week interval at 2X 10 6 nMSC/tail vein injection only. The control group and the NaCl-treated group were injected with an equal amount of physiological saline each time. Two weeks after treatment, mice were sacrificed to collect serum for SSA/SSB antibody detection and submaxillary glands were collected for HE staining. The specific experimental results are shown in fig. 6. The SSA/SSB antibody assays in fig. 6A and C showed a significant increase in the serum antibody content of mice in the NaCl treated group compared to the control group, and a significant decrease in the serum antibody content of mice after nMSC treatment, demonstrating that nMSC significantly reduced the serum SSA/SSB antibody content of mice with sjogren's syndrome. HE staining showed the same result (fig. 6B), and the number of foci and the area ratio of foci decreased significantly after mice were continuously treated with nMSC (fig. 6D and 6F). The results prove that the mice with the Sjogren syndrome model can effectively relieve symptoms after nMSC treatment.
Comparative example 1
This comparative example is basically the same as example 1, except that: smad inhibitor in NIM differentiation medium during "(3) first phase was the neuro-induction phase (day 1-day 6)": 5. Mu.M dorsomorphin and 10. Mu.M SB-431542 were replaced with: 5. Mu.M dorsomorphin and 10. Mu M A83-01. Other procedures and parameters were the same as in example 1. The morphological observations of the brain organoids (day 5, day 11) of example 1 (below fig. 7) and of this comparative example 1 (above fig. 7) are detailed in fig. 7. From the images, the rosette structure was more pronounced in brain organoids and the phenotype was more uniform for each brain organoid when the SMAD inhibitors were SB431542 and dorsomorphin (example 1). The inventors analyzed the reason for: SB-431542 and A83-01 are potent inhibitors of TGF-beta type I receptor ALK5 kinase, ALK4 kinase and ALK7 kinase. A83-01 additionally weakly inhibits transcription induced by constitutively active ALK-6, ALK-2, ALK-3 and ALK-1. Compared with A83-01, the SB-431542 inhibitor has more specific acting molecules, reduces the interference caused by other non-targeted inhibition, and forms more uniform brain organoids. The first discovery of the inventors is that the inhibitor effect is concentrated on TGF-beta type I receptors ALK5 kinase, ALK4 kinase and ALK7 kinase, and can effectively promote the formation of brain organoids with more uniform quality. The phenotype of each brain organoid obtained by the technical scheme is more uniform, namely the quality of nMSC obtained is more uniform, and the difference of wholesale pieces can be reduced and the consistency of products can be improved.
Comparative example 2
HPSC is induced to form brain organoids by directly adopting an induction scheme of CN113025569A, matrigel components in a 3-stage culture medium of the brain organoids are omitted, and finally nMSC flow detection is unqualified (CD 90 is less than 95%). The third stage of the method of CN113025569a, which was performed by removing Matrigel and using nMSC flow assay with Matrigel, was seen in fig. 8, and it was found that the proportion of CD90 positive cells was reduced from 97% to less than 20% without Matrigel. The experimental results demonstrate that the method of the prior patent (CN 113025569 a) is highly dependent on Matrigel components and it is difficult to avoid the use of such animal derived components. In fact, matrigel is a necessary component in the brain organoid induction method of the prior art. The lack of Matrigel results in poor brain organogenesis, and mesenchymal stem cells obtained from such brain organoids are also difficult to meet nMSC requirements. The technical proposal aims to obtain the neurogenic mesenchymal stem cells with qualified quality from brain organoids, and simultaneously avoid the use of animal-derived component Matrigel. However, with the prior art, the lack of Matrigel directly brings nMSC unacceptable results, which the inventors did not expect before the experiment. The preparation nMSC by the traditional method brings the result that the nMSC qualification rate does not reach the standard, so that the preparation method of brain organoids and the preparation scheme of nMSC in the prior art need to be improved, and the quality of nMSC is ensured while Matrigel is avoided.
Comparative example 3
Using the procedure of example 1 and CN113025569a (referred to as the procedure of comparative example 3, see example 1 of this patent for specific procedures), hpscs were induced to form brain organoids, nMSC was obtained after adherent culture, and details of the results of brain organoid phenotypes and nMSC production are shown in fig. 9, 10 and 11.
In patent CN113025569a, the induction factor and the scheme adopted in the process of inducing and obtaining brain organoids are different, and although both brain organoids and nMSC can be obtained, the dependence of the whole method on matrigel is different, and the obtaining efficiency of qualified nMSC is different. By adopting the technical scheme, a large number of brain organoids with compact structures and uniform batch-to-batch phenotypes can be obtained through induction under the condition that Matrigel is not used at all, and the yield of nMSC is ideal, so that the unexpected technical effect of omitting Matrigel is obtained relative to the prior patent scheme. The number of qualified nMSC obtained by the scheme is approximately 4 times that of qualified nMSC obtained by the method of the patent CN113025569A, so that the Matrigel is not applied in the culture process, nMSC is ensured, and the quality of nMSC is ensured.
Comparative example 4
NMSC and ucMSC, which were serially cultured to P5, were collected and co-fed to Shanghai Biotechnology Co., ltd for transcriptome mRNA sequencing. nMSC and ucMSC transcriptome mRNA sequencing results are shown in FIGS. 12 and 13, and numbering in FIG. 13 is shown in Table 1. The related channels of the differential genes are mainly enriched in the functions of synaptic structure and development, embryonic bone development, receptor protein composition and the like, and the mesenchymal stem cells from different sources have different phenotypes and functions. Experimental results show that nMSC obtained by the technical scheme is greatly different from common ucMSC, more than 50% of up-regulated genes in nMSC are related to neural development and synaptic structure, and compared with ucMSC, nMSCs have better immune regulation function and neurogenic function.
Table 1: ucMSC and nMSC differential Gene GO enrichment pathway summary
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (3)
1. The differentiation method for obtaining the mesenchymal stem cells by the brain organoids is characterized by comprising the following steps:
The method comprises the following steps of:
S1: the pluripotent stem cells are cultured by a pluripotent stem cell culture medium containing 10 mu M of Y27632 to form cell spheres;
S2: culturing the cell ball by using an NIM differentiation medium to obtain a neurosphere; the basic culture medium of the NIM differentiation culture medium is DMEM/F12; the addition components of NIM differentiation medium consisted of 20% KOSR, 1% NEAA, 1% GlutaMAX, 0.1mM 2-mercaptoethanol, 5. Mu.M dorsomorphin and 10. Mu.M SB-431542;
S3: culturing neurospheres by using an NDM differentiation medium to obtain a rose ring structure brain organoid; the basic culture medium of the NDM differentiation culture medium is a Neurobasal-A culture medium; the added components of the NDM differentiation medium consist of 2% of B27, 1% of GlutaMAX, 20ng/mL of EGF and 20ng/mL of bFGF;
s4: culturing the brain organoids with the rose ring structure by using an NMM differentiation medium to obtain the brain organoids; the basic culture medium of the NMM differentiation culture medium is a Neurobasal-A culture medium; the added components of the NMM differentiation medium consist of 2% of B27, 1% of GlutaMAX, 20ng/mL of BDNF and 20ng/mL of NT-3;
S5: the brain organoid is digested into single cells, and mesenchymal stem cells are obtained after screening culture and subculture.
2. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 1, wherein: the required time for S1-S4 is respectively as follows: 1 day, 6 days, 20 days, 10 days.
3. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 2, wherein: in S5, the screening culture is performed in a culture vessel coated with VTN protein.
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