CN113025569B

CN113025569B - Mesenchymal stem cells derived from human pluripotent stem cells, and preparation method and application thereof

Info

Publication number: CN113025569B
Application number: CN202110324254.9A
Authority: CN
Inventors: 马峰; 张勇刚; 赖默温
Original assignee: Chengdu Yunce Medical Biotechnology Co ltd
Current assignee: Chengdu Yunce Medical Biotechnology Co ltd
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2023-05-05
Anticipated expiration: 2041-03-26
Also published as: CN113025569A

Abstract

The invention belongs to the field of cells, and particularly relates to a mesenchymal stem cell derived from a human pluripotent stem cell, and a preparation method and application thereof. The preparation method of the intermediate mesenchymal stem cells comprises the following steps: performing suspension culture on the human pluripotent stem cells by using a brain organoid 1-stage culture medium; sequentially changing the culture medium into brain organoids culture medium at different stages; transferring the cells into a micro organoid bioreactor, culturing for 3-4 days, inoculating into a gelatin coated culture container, and performing wall-attached culture by adopting an N-MSCs first-stage culture medium to obtain mesenchymal stem cells. The invention successfully separates the mesenchymal stem cells from the brain organoids, and the early-stage pluripotent stem cells adopt a suspension culture method, so that the damage to cells is small, the yield is high, and polyvinyl alcohol is creatively added into a later-stage cell culture medium, so that the whole system stably and efficiently generates organoids; the obtained mesenchymal stem cells have application in preparing medicaments in the field of nerve repair, and particularly have wide application prospects in preparing medicaments for treating Alzheimer's disease.

Description

Mesenchymal stem cells derived from human pluripotent stem cells, and preparation method and application thereof

Technical Field

The invention belongs to the field of cells, and particularly relates to a mesenchymal stem cell derived from brain organoids of human pluripotent stem cells, a preparation method thereof and application in the field of nerve repair.

Technical Field

Organoids (Organoids) are micro-organs with three-dimensional structures that are cultured by induced differentiation of human pluripotent stem cells or adult stem cells in an in vitro environment, and possess cell types of most of the true organs. Organoids can be self-aligned to form complex structures of tissues and organs, and can partially mimic the developmental processes and physiological functions of the tissue or organ from which they are derived. By means of organoids, researchers can deeply observe the development process and pathophysiological changes of human organs, understand the development process better, and can be used for regenerative medicine and curative effect screening of medicines. Because the organoid differentiated cells are close to the natural development state, the organoid can isolate and culture the cells with possible therapeutic value in the development process of the human embryo, and are not influenced by ethical and source instability. Therefore, organoid studies have broad prospects for development. Under specific culture conditions, human brain organoids can be formed by culturing human pluripotent stem cells, the human brain organoids have a 5-layer structure of human brain cortex, have mature functional cells of human brain such as neurons, glial cells and neural stem cells, and can detect neural activity of the brain organoids similar to human brain waves.

Mesenchymal stem cells are a research branch of stem cells, are a type of cells with self-replication and multidirectional differentiation capacity, can be continuously self-updated, have immunoregulation and tissue repair capacities, and are widely applied to clinical researches. The common sources of mesenchymal stem cells are mainly human umbilical cord, bone marrow, fat, dental pulp. However, these sources of mesenchymal stem cells have heterogeneity, and there may be a large difference between batches produced on a large scale as a result of drug development, thereby affecting the cell therapeutic effect.

The human pluripotent stem cell-derived organoid functions are prepared in vitro in large quantities, and the differentiation and development process is close to that of a natural human body, so that the organoid functions are a good way for obtaining therapeutic cells, however, how to effectively extract various cells with therapeutic value from brain organoids is a difficult problem which is needed to be overcome by those skilled in the art.

Disclosure of Invention

In the process of extracting nerve cells from the end brain organoids, the inventor finds that a group of mesenchymal stem cells exist in the end brain organoids, and successfully prepares the mesenchymal stem cells (N-MSCs) by optimizing various culture conditions.

Specifically, the technical scheme of the invention is as follows:

the first aspect of the invention discloses a preparation method of mesenchymal stem cells, which comprises the following steps:

s1: culturing human pluripotent stem cells (human embryonic stem cells H1, induced pluripotent stem cells iPS);

s2: digesting the human pluripotent stem cells (hPSCs) obtained in the step S1, marking the digested cells as D0, and performing suspension culture by using a brain organoid 1-stage culture medium;

s3: sequentially replacing the culture medium with a brain organoid 2 stage culture medium, a brain organoid 3 stage culture medium and a brain organoid 4 stage culture medium in the steps of D4-D6, D7-D14 and D15-D21;

s4: transferring the cells into a micro organoid bioreactor, culturing for 3-4 days, inoculating into a gelatin coated culture container, and performing wall-attached culture by adopting an N-MSCs first-stage culture medium to obtain mesenchymal stem cells.

In some embodiments of the invention, in S1, hPSCs are cultured in conventional manner in 35mm dishes, the fluid is changed daily, the time of changing fluid is gradually advanced, and the medium is gradually increased from the initial 2ml to 3ml. When the culture was carried out at a density of 90%, the obtained human pluripotent stem cells were digested and designated day 0.

In some embodiments of the invention, the hPSCs enzyme-free digest is 0.5mM EDTA-DPBS.

Preferably, in S2, Y-27632 is added to the brain organoid 1 stage medium.

More preferably, the concentration of Y-27632 is 8-12. Mu.M and the rotational speed of the micro-organoid bioreactor is 60RPM.

Preferably, the brain organoid 1 stage medium comprises:

GMEM basal medium, NEAA (non-essential amino acids) 1% -2%, glamax (glutamine) 1% -2%, mercaptoethanol (ethanol bisulfate) 80-120nM, KOSR (serum replacement) 12-18%, dorsomorphin (dihydrodeoxymorphine) 0.8-1.5 mu M, A83-01 (ALK-4, 5,7 inhibitor) 0.8-1.2 μm and polyvinyl alcohol 0.5-1.5%.

Dorsominorphin and A83-01 are small molecule compounds.

Preferably, the brain organoid 2 phase medium comprises: GMEM basal medium, NEAA1% -2%, glumax1% -2%, mercap alcohol 80-120nM, N2 1% -2%, SB-431542 (small molecule inhibitor) 0.8-1.2 mu M, CHIR-99021 0.8-1.2 mu M, polyvinyl alcohol 0.8-1.5%.

SB 431542 is a potent small molecule inhibitor, selectively inhibiting transforming growth factor β (TGF- β) type I receptor activin receptor-like kinase ALK5 (ic50=94 nM), ALK4 (ic50=140 nM) and ALK7. There is no inhibitory activity on other ALK family branching members such as ALK2, ALK3 or ALK6 that bind BMP proteins. SB 431542 inhibits TGF- β induced proliferation of osteosarcoma cells and promotes proliferation, differentiation and lamellar formation of endothelial cells derived from Embryonic Stem Cells (ESCs). By specifically blocking ALK/Smad signaling pathways, SB 431542 is able to maintain not only self-renewal and embryoid body formation in human embryonic stem cells, but also pluripotent stem cells. SB 431542 induces DC cell maturation in vitro while exhibiting anti-tumor activity in vivo. Patients with immune tolerance associated with tgfβ activity may activate an anti-tumor immune response using SB 431542.

CHIR-99021 (CT 99021) is a potent and selective GSK-3 alpha/beta inhibitor with IC50 of 10nM and 6.7nM. CHIR-99021 has selectivity to GSK-3 over CDC2, ERK2 and other protein kinases by more than 500 times. CHIR-99021 is also a potent Wnt/β -catenin signaling pathway activator. CHIR-99021 enhances self-renewal of mouse and human embryonic stem cells. CHIR-99021 can induce autophagy (autophagy).

Preferably, the brain organoid 3-phase medium comprises: MEM basal medium, NEAA1% -2%, glumax1% -2%, mercap alcohol 80-120nM, N2 1% -2%, SB-431542 0.8-1.2 μ M, CHIR-99021 0.8-1.2 μ M, martigel 0.8-1.5%, polyvinyl alcohol 0.8-1.5%.

Preferably, the brain organoid 4 stage medium comprises: GMEM basal medium, NEAA1% -2%, glamax 1% -2%, mercaptoethanol80-120nM, 1% -2%, B27% -3%, insulin 2 μg/mL.

Preferably, the first stage medium of N-MSCs comprises:

GMEM basal medium, NEAA1-2%, glumax 1-2%, mercap ethanol100nM, N2 1-2%, B27-3%, B-FGF 5-6ng/ml.

Preferably, the method further comprises step S5: and when the confluence rate of the mesenchymal stem cells in the S4 is 70-80%, adopting a serum-free MSCs culture medium for subculturing. In some embodiments of the invention, in S5, the digestive juice is Ackutase or type I collagenase (1 mg/ml).

The subsequent passaging digestive enzyme of mesenchymal stem cells was recombinant pancreatin (0.05% pancreatin in DHanks).

In some embodiments of the invention, in S5, subculturing is performed at a confluence of 70% cells to be adherent, using commercial serum-free MSCs medium, at which time the cultured N-MSCs are designated as P1 generation. At the P3 generation, cells were collected for MSCs-related surface marker identification and multipotent differentiation potential identification.

In some embodiments of the invention, the serum-free MSCs medium is LONZA 12-725FultraCULTURE serum-free medium or a ncTarget medium from well-traced company, cat# RP 01020.

The invention discloses a mesenchymal stem cell obtained by the method in a second aspect.

In a third aspect, the invention discloses a medicament comprising mesenchymal stem cells; preferably, the medicament is a medicament for treating Alzheimer's disease.

The fourth aspect of the invention discloses application of the mesenchymal stem cells in the field of nerve repair; preferably, the mesenchymal stem cells are used for preparing a medicament for treating Alzheimer's disease.

Compared with the prior art, the invention has the following beneficial effects:

the invention successfully separates the mesenchymal stem cells from the brain organoid, the early-stage pluripotent stem cells adopt a suspension culture method, the damage to cells is small, the yield is high, polyvinyl alcohol is added in the later-stage cell culture medium creatively, the shearing force of the culture is changed, the early-stage induced differentiation condition is adapted to the suspension culture shearing force, and the use of induction factors can be reduced. The obtained mesenchymal stem cells have application in preparing medicaments in the field of nerve repair, and particularly have wide application prospects in preparing medicaments for treating Alzheimer's disease.

Drawings

Fig. 1 is a microscopic schematic diagram of a prepared mesenchymal stem cell (A: a morphological diagram of the mesenchymal stem cell under a microscope, B: a schematic diagram of mesenchymal stem cell adipogenic differentiation, C: a schematic diagram of mesenchymal stem cell osteogenic differentiation, D: a schematic diagram of mesenchymal stem cell chondrogenic differentiation).

FIG. 2 is a graph showing the flow positive results of the prepared mesenchymal stem cells;

FIG. 3 is a graph showing the flow negative results of the prepared mesenchymal stem cells;

FIG. 4 is a graph of the results of a mouse behavioural experiment-open field experiment-of mesenchymal stem cells implanted-Centimeter: cm; total Distance: total Distance.

FIG. 5 is a graph of the results of a mouse behavioural experiment-open field experiment with mesenchymal stem cells implanted (Entries in central zone: number of central zone entries; time in central zone: time in central zone; distance in central zone: distance in central zone; injection amountof: depression).

FIG. 6 is a diagram of a new Object recognition of a mouse behavioural experiment in which mesenchymal stem cells were implanted.

FIG. 7 is a graph of the behavioural experiments of mice implanted with mesenchymal stem cells-conditioned fear (continuous fear: hippocampal dependency; feed fear: amygdala dependency).

FIG. 8 is a diagram of the experimental behavioural of mice implanted with mesenchymal stem cells-Barns maze-one (escape latency).

FIG. 9 is a mouse behavioural experiment-Barns maze pattern two (Latency 1st Entrance to Target: delay first entry target) of mesenchymal stem cells.

FIG. 10 is a diagram of the mouse behavioural experiment in which mesenchymal stem cells were implanted-Barns maze pattern three (Latency 1st Entrance to Target: delay first entry into the target.

FIG. 11 is a graph showing the results of sequencing differential gene expression in different mesenchymal stem cells.

FIG. 12 is a flow chart of a length calculation of a nascent neuron;

FIG. 13 is a schematic of neurons under a confocal microscope;

FIG. 14 is a graph of Neuron Length (Neuron Length) for different groups of mesenchymal stem cells.

FIG. 15 is a graph of mouse brain slice-neuron complexity (Intersection Number: segment number).

Fig. 16 is a schematic diagram of a confocal microscope of brain sections of mice.

FIG. 17 is a graph of mouse brain slice-spin morphometric (Thin: spindle: stubby: dumbbell, mushroom: mushroom).

FIG. 18 is a graph of brain section-number of neural stem/progenitor cells in mice (SOX2+: SOX2 single positive; SOX2+GFAP+: SOX2 and GFAP co-positive).

FIG. 19 is a graph of mouse brain slice-microglial morphometric analysis (Iba-1positive cell:Iba-1 positive cells; intersection Number: segment number).

FIG. 20 is a graph of mouse brain slice-microglial morphometric analysis two (Average soma size: average body size; branch length; branch: branching).

FIG. 21 is a graph comparing the absence of added polyvinyl alcohol and the addition of 1% polyvinyl alcohol during brain organoids.

Detailed Description

The present application is further illustrated by the following detailed examples, it being understood that the following examples are for purposes of illustration only and are not limiting of the invention.

The instruments, devices, reagents used in the examples may be obtained from a variety of sources, such as purchased, or may be prepared.

Example 1

The embodiment discloses a method for preparing mesenchymal stem cells, which specifically comprises the following steps:

1. hPSCs were cultured in 35mm dishes according to conventional culture, with daily changing of the liquid, with the changing time gradually advancing, and the medium gradually increasing from the initial 2ml to 3ml. When the culture was carried out until the confluency rate reached 90%, the culture was designated as day 0. Wherein hPSCs are human embryonic stem cells H1 or are prepared by inducing human pluripotent stem cells with the reagent of Erythroid Progenitor Reprogramming Kit of STEMCELL company under the trade designation 05924.

2. DPBS was used to wash the cells twice, 1 ml/dish of hPSCs enzyme-free digest was added, and the cells were placed in a 37℃incubator for 8 minutes. 3ml of ice-cold brain organoid 1 phase medium was prepared and Y-27632 was added to a final concentration of 10. Mu.M.

3. Carefully aspirate hPSCs without enzyme digest, add 1ml of ice cold brain organoid 1 phase medium containing Y-27632, shake the dish quickly left and right to shed hPSCs, transfer to one well of a common 6-well plate with a Pasteur pipette, and add Y-27632 of ice cold brain organoid 1 phase medium to 3ml.

4. The 6-well plate was placed in an incubator horizontal shaker (amplitude 24 mm), and incubated overnight at 60RPM.

5. day1, microscopic cell spheres, approximately 250 μm to 350 μm in diameter. The number is about 600. Carefully transferring the culture to a 15ml centrifuge tube by using a Pasteur pipette, naturally settling for 3 minutes, taking about 2.5ml of clear solution into a new 15ml centrifuge tube, centrifuging at 3000RPM for 5 minutes, removing single cells and cell debris, adding the single cells and cell debris into settled cell pellets, adding the brain organoid 1-stage culture medium to 9ml, carefully mixing, and sub-packaging into 3 6-hole plate holes, wherein each hole contains about 200 cell pellets and 3ml of culture medium. The culture was continued in a incubator horizontal shaker (amplitude 24 mm) at 60RPM.

6. day2-day3 half-changed, brain organoid 1 stage medium, incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

7. day4-day6, half a day, and medium in 2 stages of brain organoids. Incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

8. day7-day14, half-changed every day, changed into brain organoid 3 stage medium, incubator horizontal shaker (amplitude 24 mm), 60RPM culture.

9. day15-day21, half-changing liquid every day, changing into brain organoid 4 stage culture medium, transferring into micro organoid bioreactor (patent application number: 2017212099229;2017212027930;2017212119025; 2017108559648), culturing for 21 days, and adjusting the number of each hole to 20.

10. Cultures were removed at day22-day25, settled and digested with accutase for 15min before being blown into single cells, and half-changed every 3 days in flasks coated with gelatin, the first stage medium of N-MSCs. In the second half-change, the suspension cells are removed.

11. When the confluency rate of the adherent cells is 70%, subculturing is carried out, and a commercial serum-free MSCs culture medium is used, wherein the cultured N-MSCs are marked as P1 generation. The serum-free MSCs culture medium is LONZA 12-725FultraCULTURE serum-free culture medium or ncTarget culture medium of the stock name RP01020 of the company of the Ministry of Biotechnology of Anhui.

12. At the P3 generation, the collected cells are subjected to MSCs related surface marker identification and multi-directional differentiation potential identification, and a schematic diagram under a cell microscope is shown in figure 1.

The mesenchymal stem cells of the generation P3 were then subjected to flow assay, and the obtained cells were further verified to be mesenchymal stem cells, and the results are shown in FIGS. 2 to 3.

The formulation of the brain organoid 1 stage medium in this example is as follows:

GMEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, KOSR 15%, dorsomophin 1 μ M, A-01 μM and polyvinyl alcohol 1%.

The brain organoid 2 phase medium includes: GMEM basal medium, NEAA1%, glumax1%, mercapethanol 100nM, N2 1%, SB-431542 1. Mu. M, CHIR-99021 1. Mu.M, polyvinyl alcohol 1%.

The brain organoid 3-stage medium comprises: MEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, SB-431542 1 μ M, CHIR-99021 1 μ M, martigel1%, polyvinyl alcohol 1%.

The brain organoid 4 stage medium comprises: GMEM basal medium, NEAA1%, glumax1%, mercap ethanol100nM, N2 1%, B27%, insulin 2 μg/mL.

Example 2

This example investigated the effect of mesenchymal stem cells on the nerve repair function of mice. This example uses AD model (alzheimer's disease) mice (double transgenes, human presenilins and aβ) for treatment efficacy evaluation, and the experiments are divided into four groups:

PBS group: a control group;

MSC group: umbilical cord derived MSCs;

TC group: another subject, genetically modified MSCs, over-express TIMP 2N-MSCs in human umbilical MSCs using AAV; N-MSC group: the brain-derived MSCs, i.e., the mesenchymal stem cells N-MSCs prepared in example 1.

The experimental design is as follows:

experiment design: and randomly grouping 9 mice in each group, wherein three mice are selected to be used as GFP retrovirus markers of DG proliferation-stage neural stem cells, and the method is mainly used for evaluating the development of new-born neurons and the integration capacity of neural networks after MSCs are analyzed in the future.

Treatment: the treatment period was 8 weeks, the tail vein injection was carried out once a week, and the cell group dose was 10 ⁵ Individual cells/mice. After treatment, carrying out a mining field experiment and a new object identification experiment at the 9 th week; performing a Barns maze experiment on the 10 th week to the 12 th week; conditioned fear experiments were performed at week 13.

After the mice are sacrificed, the whole viscera are checked, and no obvious tumor is generated; the experimental mice were fixed by 6 perfusion and 3 direct liquid nitrogen cryopreservation and hippocampal tissue was taken for sequencing.

The specific test results are as follows:

1. the mine experiments are carried out on four groups of mice, the experimental results are shown in figures 4-5, and the four groups of mice have similar activity degrees, and the mice in the N-MSC treatment group are more active and more general.

2. New object recognition experiments were performed on four groups of mice, scoring% = new object explored time/new object explored time + old object explored time. The results are shown in FIG. 6, and it can be seen from the graph that the treatment group has higher score for new object recognition and the N-MSC treated group mice have better short-term memory.

3. The four groups of mice were subjected to a conditional fear test, and the contextual fear of the N-MSC treated group was found to be significantly improved, while the fear test with clue cues was not significantly different, and the test results are shown in FIG. 7.

4. The barnes maze test was performed on four groups of mice, and a significant difference was found in the first training time after the adaptation period, indicating that the N-MSC treated group of mice had a greater learning ability, and the results are shown in fig. 8.

The fourth day training results showed that all mice could find the target hole within 30 seconds, indicating that training was successful and available for testing, and the results are shown in fig. 9.

The test results on the fifth day show no obvious difference, which indicates that the short-time memories of the four groups of mice are similar; the results on day 12 show that the long term memory of the mice in the N-MSC treated group is improved and the results are shown in FIG. 10.

6. Differential gene expression sequencing was performed on the genes of four groups of mice, and the results are shown in fig. 11.

7. The flow chart of the brain slice-neuron length analysis of the four groups of mice is shown in fig. 12, and the result schematic diagrams are shown in fig. 13-14.

Further studies on neuronal complexity, the results are shown in FIG. 15, where the N-MSC treated group of mice had longer neurons and more branched differences were statistically significant.

8. Four groups of mice were studied for neuron terminal axons (spines), and confocal microscopy was used to photograph the terminal 5 μm of the newly generated neurons, and the spines were counted using a Neurolucida classification, as shown in FIGS. 16-17, in which Thin refers to spindle type, stubby refers to dumbbell type, and Mushroom refers to Mushroom type. As can be seen from the figure, the total number of spines in the mice of the N-MSC treated group is the greatest, and the neuron axons are more mature.

9. The number of neural stem/progenitor cells in brain sections of four groups of mice was studied and the results are shown in fig. 18. The number of the treated mouse neural stem cells (common yang) is more, which indicates that MSC has stronger effect of maintaining dryness.

10. The results of analysis of microglial cell morphology in brain sections of four groups of mice are shown in fig. 19, and after treatment, the number of microglial cells in mice DG and CA1 regions Iba-1 is reduced without obvious difference, and the difference is statistically significant.

Further studies, as shown in figure 20, found that the N-MSC treated group mice had smaller microglial cell bodies, fewer branches and shorter branch lengths. FIG. 21 is a graph comparing the absence of added polyvinyl alcohol and the addition of 1% polyvinyl alcohol during brain organoids.

From the above, it can be seen that the method of the invention utilizes human pluripotent stem cells to differentiate human brain organoids, and obtains a mesenchymal stem cell with better nerve repair function from human brain organoids, and the cell can be used as a cell medicine for treating Alzheimer's disease, and the method also has the characteristics of stable and controllable preparation process, and is suitable for large-scale standardized preparation.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method for preparing mesenchymal stem cells, comprising:

s1: culturing human pluripotent stem cells;

s2: digesting the human pluripotent stem cells obtained in the step S1, marking the cells as Day0, and performing suspension culture by using a brain organoid 1-stage culture medium;

s3: sequentially replacing the culture medium with a brain organoid 2 stage culture medium, a brain organoid 3 stage culture medium and a brain organoid 4 stage culture medium in the stages of Day4-Day6, day7-Day14 and Day15-Day 21;

s4: transferring the cells into a micro organoid bioreactor, culturing for 3-4 days, inoculating into a gelatin-coated culture container, and performing wall-attached culture by adopting an N-MSCs first-stage culture medium to obtain mesenchymal stem cells;

the brain organoid 1 stage medium comprises: GMEM basal medium, NEAA1% -2%, glumax1% -2%, mercap alcohol 80-120nM, KOSR12-18%, dorsosporphin 0.8-1.5 mu M, A-010.8-1.2 mu M and polyvinyl alcohol 0.5-1.5%;

the brain organoid 2 stage medium comprises: GMEM basal medium, NEAA1% -2%, glumax1% -2%, mercap alcohol 80-120nM, N2 1% -2%, SB-431542 0.8-1.2 mu M, CHIR-99021 0.8-1.2 mu M, polyvinyl alcohol 0.8-1.5%;

the brain organoid 3-stage medium comprises: MEM basal medium, NEAA1% -2%, glumax1% -2%, mercap alcohol 80-120nM, N2 1% -2%, SB-431542 0.8-1.2 μ M, CHIR-99021 0.8-1.2 μ M, martigel 0.8-1.5%, polyvinyl alcohol 0.8-1.5%;

the brain organoid 4 stage medium comprises: GMEM basal medium, NEAA1% -2%, glamax 1% -2%, mercaptoethanol80-120nM, N2 1% -2%, B272% -3%, insulin 2 μg/mL;

the first stage medium of the N-MSCs comprises:

2. The method of claim 1, wherein the brain organoid stage 1 medium comprises Y-27632, the concentration of Y-27632 is 8-12 μΜ, and the rotational speed of the micro organoid bioreactor is 60RPM.

3. The method according to claim 1, further comprising step S5: and when the confluence rate of the mesenchymal stem cells in the S4 is 70-80%, adopting a serum-free MSCs culture medium for subculturing.

4. A mesenchymal stem cell obtained by the method of any one of claims 1-3.

5. A medicament comprising mesenchymal stem cells obtained according to the method of any one of claims 1 to 3.

6. The use of the mesenchymal stem cells of claim 4 in the preparation of a medicament for treating alzheimer's disease.