CN117778313A - 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 additional components of NIM differentiation medium consisted 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 the nMSCs (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. In combination, the nMSCs are suggested to have more excellent treatment effect on the Sjogren's syndrome.
Compared with the prior art, the technical scheme has the advantages that:
(1) The pluripotent stem cells are used for forming brain organoids, and nMSCs are obtained from the brain organoids, so that the pain points with unstable MSC material acquisition 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. The nMSCs obtained in the scheme can be directly used for treatment purposes without considering negative effects (such as antigenicity, safety and other problems) caused by the 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 phase was more stable, with higher phenotypic uniformity and less batch-to-batch variability than the a83-01 and dorsomorphin used in patent CN113025569 a.
Drawings
FIG. 1 is an nMSC flow assay chart (A: mesenchymal stem cell positive marker flow chart; B: mesenchymal stem cell negative marker flow chart) prepared in example 1.
FIG. 2 shows the morphology of nMSCs prepared in example 1 (A: clear field of nMSCs under an optical microscope; B: alizarin red staining pattern after osteogenic differentiation of nMSCs; C: oil red O staining pattern after lipid differentiation of nMSCs; D: paraffin section Aristolochian blue staining pattern after chondrogenic differentiation of nMSCs).
FIG. 3 is a graph showing the comparison of the inhibition of T cell proliferation by nMSCs and ucMSCs of example 2.
FIG. 4 is a graph showing the comparison of the inhibition of T cell inflammatory factor secretion by nMSCs and ucMSCs of example 3.
FIG. 5 shows the relative expression levels of IDO and HLA-G mRNA secreted by nMSCs and ucMSCs after IFN-. Gamma.stimulation in example 4 (data format: cell IDO/HLA-G mRNA relative expression level.+ -. Standard deviation, n= 3;A is a statistical chart of the results of measuring the relative expression level of IDO mRNA, and B is a statistical chart of the results of measuring the relative expression level of HLA-G mRNA).
FIG. 6 is a graph showing the results of the detection of serum antibodies in mice with the nMSC treatment Sjogren syndrome of example 5 (A is a statistical graph showing the results of the detection of serum SSA antibodies, data form: mean.+ -. Standard deviation of SSA antibodies per ml, n=10; B is a graph showing the results of the staining HE, C is a statistical graph showing the results of the detection of serum SSB antibodies, data form: mean.+ -. Standard deviation of SSB antibodies per ml, n=10; D is a statistical graph showing the percentage of the area of foci, data form: the percentage of the area of foci.+ -. Standard deviation, n= 9;F is a statistical graph showing the number of foci, data form: the number of foci.+ -. Standard deviation in a single field of view, 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 is a flow cytometry image of the comparative example 2 in which Matrigel was removed and CD90 expression was measured using an nMSC flow obtained with Matrigel (A is a flow cytometry image of the Matrigel-removed experimental group; B is a flow cytometry image of the Matrigel-containing experimental group).
Fig. 9 is a graph of brain organoids and nMSC morphology at various times for the EB spheres of example 1.
FIG. 10 is a graph of morphology of brain organoids and nMSCs 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 result of mRNA sequencing of the transcriptome of nMSCs and ucMSCs (A: thermal map of the differential gene expression of ucMSCs and nMSCs; B: volcanic map of the differential gene expression of nMSCs compared to ucMSCs).
FIG. 13 is a graph showing the GO enrichment of the ucMSC and nMSC differential 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 (steecel, 100-0276) supplemented with 10 μ M Y27632 (ROCK inhibitor), and centrifuged at 300g for 5min in a cell culture incubator with single cell suspensions added to ultra low adsorption U bottom 96 well plates at 4000 cells/well. Wherein, the hPSC used in the step is from the midwifery biosciences, inc., and the 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, 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 phase nMSC screening phase: digesting brain organoids on day 36 with Ackutase for 10-15min, stopping digestion, and filtering with 100 μm cell filter to form single cell suspension at 5×10 4 cells/cm 2 Inoculating cells into VTN (vitronectin) coated culture flask/dish, counting as P0 generation, and culturing with mesenchymal stem cell culture medium (medium tracing source, RP02010 Culturing, and performing passage treatment when the concentration is about 90%. P1 inoculation density of 2X 10 4 cells/cm 2 P2 inoculation density of 1X 10 4 cells/cm 2 Thereafter the inoculation density was 8X 10 3 cells/cm 2 . The nMSC expansion passaging used mesenchymal stem cell medium (friend kang, NC 106).
(7) The nMSCs were serially subcultured, and P5 cells (5 th generation cells) were collected and subjected to flow assay, and the results of the flow assay are shown in FIG. 1, wherein both 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
The first day is 1×10 5 Density nMSCs (prepared in example 1, neural-derived mesenchymal stem cells) and ucMSCs were plated for adherent culture, wherein ucMSCs were umbilical cord-derived mesenchymal stem cells (conventional cells of the prior art commercially available). The following day donor Peripheral Blood Mononuclear Cells (PBMCs) were isolated using lymphocyte separation fluid and PBMCs were collected for cytotoll Blue staining. After dyeing is completed, the dyeing is finished according to the ratio of 1 multiplied by 10 6 Co-culturing with mesenchymal stem cells, adding PHA for stimulation, and setting non-stimulated control group without adding stimulator. 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, which shows that nMSCs have a greater capacity to inhibit T cell proliferation (68.25%) than ucMSCs have to inhibit T cell proliferation (58.32%).
Example 3: mesenchymal stem cells inhibit T cells from secreting inflammatory factors
One day ahead by 2X 10 5 Density nMSCs and ucMSCs were plated for adherent culture. Donor PBMCs were isolated using lymphoid isolation, as mesenchymal stem cells: t cell = 1:10 ratio of seeded PBMC cellsAfter 43h of co-culture, PMA, lonomycin and BFA were added for stimulation, and the unstimulated group was set without addition of a 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, the inhibition rate of ucMSC to IFN-gamma is 86.99% and the inhibition rate to TNF-alpha is 50.71%; the inhibition rate of nMSC to IFN-gamma is 97.09%, and the inhibition rate to TNF-alpha is 92.58%. Compared with other mesenchymal stem cells, the nMSCs prepared by the technical scheme have more ideal effect of inhibiting the secretion of T cell inflammatory factors.
Example 4: mesenchymal stem cells induce IDO and HLA-G secretion
According to 2X 10 5 Density the nMC and ucMSC were inoculated, 10ng/ml IFN-gamma was added to the culture system, after normal culture for 24h, the cells were collected to extract RNA, reverse transcribed into cDNA and then used as template for RT-qPCR, the relative expression amounts of IDO (indoleamine 2, 3-dioxygenase) and HLA-G (human leukocyte antigen-G) were calculated. The results are shown in FIG. 5, in which both nMSCs and ucMSCs can secrete more IDO and HLA-G under IFN-gamma stimulation, demonstrating that nMSCs and ucMSCs have similar immunomodulatory functions.
From the experimental data of examples 2-4, the effect of nMSC is better than ucMSC, and the nMSC has more ideal function in immune regulation and has more ideal potential for alleviating sicca syndrome. Thus, an experiment of the effect of treatment on nMSC was subsequently performed.
Example 5: in vivo experiments
Control mice were used as CD-1 (ICR) mice IGS SPF grade (CD-1 Xie ICR mice, beijing Vitolihua laboratory animal Co., ltd.), model mice were used as NOD/ShiLtd JGpt mice (Jiangsu Jiuyaokang Biotech Co., ltd., N000235). Mice were divided into three groups: control group, naCl treatment group, nMSC treatment group, mice of nMSC treatment group were treated at a rate of 2X 10 6 nMSC/tail vein injection only, three consecutive treatments, one week apart. 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. SSA/S in FIGS. 6A and CThe SB antibody detection shows that the content of the mouse serum antibody in the NaCl treatment group is obviously increased compared with that in the control group, and the mouse serum antibody is obviously reduced after nMSC treatment, so that the nMSC can obviously reduce the content of the mouse serum SSA/SSB antibody of the Sjogren syndrome. HE staining showed the same result (fig. 6B), and the number of foci and the area ratio of foci decreased significantly after the mice underwent nMSC continuous treatment (fig. 6D and 6F). The results prove that the mice with the Sjogren syndrome model can effectively relieve symptoms after being treated by the nMSCs.
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 the obtained nMSCs is more consistent, and the difference of wholesale parts 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, the obtained nMSCs are unqualified in flow detection (CD 90 is less than 95%). The third stage of the method of CN113025569a, in which Matrigel was removed and CD90 expression was detected using nMSC flow from Matrigel-containing, was seen in fig. 8, 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 the use of such animal-derived components is difficult to avoid. 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, once Matrigel is missing, the result of nMSC failure is directly felt, which was unexpected by the inventors prior to the experiment. The conventional method for preparing the nMSCs brings the result that the qualification rate of the nMSCs does not reach the standard, so that the preparation method of brain organoids and the preparation scheme of the nMSCs in the prior art need to be improved, and the quality of the nMSCs is ensured while Matrigel is avoided.
Comparative example 3
Using the procedure of example 1 and patent CN113025569A (referred to as the procedure of comparative example 3, see example 1 of the patent for specific procedures), hPSC was induced to form brain organoids, and nMSCs were obtained after adherent culture, and details of the brain organoid phenotype and nMSC production results are shown in FIGS. 9, 10 and 11.
In patent CN113025569a, the induction factor used in the process of obtaining brain organoids and the present solution are different, but the overall method has different dependence on matrigel and the obtaining efficiency of qualified nMSC is different, although both brain organoids and nMSC can be obtained. 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, the yield of nMSCs is ideal, and the unexpected technical effect of omitting Matrigel is obtained relative to the prior patent scheme. The number of qualified nMSCs obtained by the scheme is approximately 4 times that of the qualified nMSCs obtained by the method of the patent CN113025569A, so that not only is the fact that Matrigel is not applied in the culture process ensured, but also nMSCs are ensured, and the quality of the nMSCs is also ensured.
Comparative example 4
The nMSCs serially cultured to P5 were collected and co-delivered to Shanghai-Katsuwon Biotechnology Co., ltd for transcriptome mRNA sequencing. The results of the mRNA sequencing of the nMSCs and ucMSC transcriptomes are shown in FIGS. 12 and 13, and the numbering meanings in FIG. 13 are 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 the nMSCs obtained by the technical scheme have great differences from the common ucMSCs, more than 50% of up-regulated genes in the nMSCs are related to neural development and synaptic structure, and compared with the ucMSCs, the 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 invention, 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 invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be 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 (7)
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 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.
2. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 1, wherein: in S2, the basic culture medium of the NIM differentiation culture medium is DMEM/F12; the additional components of NIM differentiation medium consisted of KOSR, NEAA, glutaMAX, 2-mercaptoethanol, dorsomorphin and SB-431542.
3. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 2, wherein: 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.
4. A method of differentiating mesenchymal stem cells obtained from brain organoids according to claim 3, wherein: in S4, the basal medium of the NMM differentiation medium is a Neurobasal-A medium; the added components of the NDM differentiation medium consisted of B27, glutaMAX, BDNF and NT-3.
5. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 4, wherein: the required time for S1-S4 is respectively as follows: 1 day, 6 days, 20 days, 10 days.
6. The method for differentiating mesenchymal stem cells obtained from brain organoids according to claim 5, wherein: in S5, the screening culture is performed in a culture vessel coated with VTN protein.
7. Use of the neural mesenchymal stem cells obtained by the differentiation method of brain organoids to obtain mesenchymal stem cells according to any one of claims 1-6 for the preparation of a medicament for treating sjogren's syndrome.
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