Method for preparing mesenchymal stem cell from neural crest cell line and use of mesenchymal stem cell as osteoarthritis drug

Abstract

The present invention belongs to the technical field of biomedicines. Provided are a method for preparing mesenchymal stem cells from a neural crest cell line and the use of the obtained mesenchymal stem cells as osteoarthritis drugs. The method for preparing the mesenchymal stem cells from the neural crest cell line comprises: S1: inducing human pluripotent stem cells to form neural crest cells; and adding a specific neural crest cell induction and differentiation medium to the human pluripotent stem cells for culture, and adding a YAP activator to the specific neural crest cell induction and differentiation medium; S2: performing expansion culture on the neural crest cells; and S3: differentiating the neural crest cells after the expansion culture to mesenchymal stem cells. The mesenchymal stem cells obtained by the method described above can be used for preparing drugs for the treatment of osteoarthritis.

Description

Method for preparing mesenchymal stem cells through neural crest cell line and application as osteoarthritis medicine Technical Field

[0001] The present invention belongs to the field of stem cell biology and relates to the lineage-specific differentiation of human induced pluripotent stem cells, and specifically to a method for preparing mesenchymal stem cells through a neural crest cell line and its pharmaceutical application. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by articular cartilage degeneration, synovitis, subchondral bone sclerosis, and osteophyte formation. Worldwide, 15% of the population suffers from OA. With increasing aging and obesity, 80% of people over 65 will develop OA. Because articular cartilage lacks blood vessels, nerves, and lymph nodes, OA is difficult to heal on its own once it develops, often resulting in severe pain and mobility limitations, which in turn impacts patients' quality of life and increases their financial burden. Clinical treatments for OA primarily focus on early prevention with moderate exercise, topical nonsteroidal anti-inflammatory drugs, oral acetaminophen, and intra-articular injections of corticosteroids or hyaluronic acid. These methods have limited effectiveness in preventing late-stage OA progression. For advanced treatment, in addition to traditional methods such as bone marrow stimulation and osteochondral transplantation, cell-based therapies (including chondrocytes and mesenchymal stem cells (MSCs)) have also gained clinical application. MSCs, due to their unique immunomodulatory properties and potential for chondrogenic differentiation, have recently attracted significant attention from researchers and biopharmaceutical companies.

[0003] MSCs are multipotent stem cells with the ability to self-renew and differentiate into adult tissue cells, including bone, cartilage, and fat. MSCs were first discovered in bone marrow and subsequently found in human and animal tissues, including amniotic fluid, adipose tissue, umbilical cord, placenta, and dental pulp. Their unique anti-inflammatory and immunomodulatory properties, as well as their ability to differentiate into adult cells, hold broad application prospects in cell therapy and tissue regeneration. However, the use of primary MSCs from either autologous or allogeneic sources presents several challenges: 1) sourcing is difficult and the number of cells extracted is limited; 2) MSCs have limited expansion and differentiation potential; and 3) the MSC production process is challenging to control (there are significant and unpredictable variability between individuals and tissues). These shortcomings hinder the standardized and scalable production of MSCs, significantly limiting the stability and applicability of MSC-based therapies.

[0004] Therefore, in order to realize the clinical application of MSCs, it is imperative to address the source of MSCs. In addition to extracting (adult) MSCs in vivo, induced differentiation of pluripotent stem cells (PSCs) to obtain i-MSCs is also an important source of MSCs. In vivo, MSCs primarily develop from early mesodermal and ectodermal neural crest cells (NCCs). To this end, extensive research has focused on developing biomimetic MSC development pathways, where PSCs are progressively induced to differentiate in vitro through mesodermal or neural crest cell lines to obtain MSCs. Derivation of MSCs from neural crest cell lines is particularly advantageous due to their self-renewal capacity and multipotency (phenotypically stable). Furthermore, the resulting MSCs exhibit strong chondrogenic differentiation potential, making it easier to obtain chondrocytes with a hyaline cartilage phenotype, making them more suitable for the treatment of osteoarthritis and repair of cartilage damage.

[0005] Currently, research on the process of inducing neural crest cell lineage-based PSC differentiation to MSCs primarily involves a two-step induction culture process: inducing PSCs to differentiate into NCCs and then NCCs to differentiate into MSCs. The process of inducing PSCs to differentiate into NCCs primarily involves the regulation of the SMAD signaling pathway (inhibition) and the WNT signaling pathway (activation). NCCs obtained using current regulatory strategies are often of low purity and require flow cytometry sorting. Technical issues

[0006] In summary, although many researchers have developed a number of methods for differentiating hPSCs into MSCs in vitro, the existing methods are still not mature enough. For example, non-specific and random components such as serum are needed to induce PSCs into MSCs, and flow cytometry and other screening techniques are needed to obtain high-purity cells. As a result, the differentiation effect is unstable, the efficiency is low, and the time is long. In addition, MSCs obtained through the mesodermal cell line differentiation pathway have a low final cell yield because the mesodermal cell line is not easy to store. However, MSCs obtained through the neural crest cell line pathway can obtain a large number of secondary cells for storage during the entire cell production process due to the amplification and storability of the neural crest cells themselves. Technical Solutions

[0007] The present invention provides a method for differentiating human pluripotent stem cells (hPSCs) into MSCs and its application in osteoarthritis.

[0008] Specifically, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for preparing mesenchymal stem cells from a neural crest cell line, comprising the following steps:

[0010] S1: Inducing human pluripotent stem cells to form neural crest cells: adding a specific neural crest cell induction differentiation medium to human pluripotent stem cells for culturing, and adding a YAP activator to the specific neural crest cell induction differentiation medium; wherein the human pluripotent stem cells are commercial human embryonic stem cell lines or human induced pluripotent stem cells;

[0011] S2: Expand and culture the neural crest cells;

[0012] S3: The neural crest cells after expansion and culture differentiate into the mesenchymal stem cells.

[0013] The method for preparing mesenchymal stem cells by using a neural crest cell line as described above, wherein the mesenchymal stem cells express CD90, CD73 and CD105, and do not express CD14, CD34, CD45 and HLA-DR.

[0014] The method for preparing mesenchymal stem cells by using a neural crest cell line as described above is obtained by inducing differentiation of neural crest cells that express P75 and SOX10 but do not express PAX6.

[0015] The method for preparing mesenchymal stem cells from a neural crest cell line as described above, further comprises S4 after S3: expanding and culturing the mesenchymal stem cells.

[0016] The method for preparing mesenchymal stem cells from a neural crest cell line as described above, further comprises, after S4, S5: identifying the mesenchymal stem cells obtained by expansion and culture.

[0017] The method for preparing mesenchymal stem cells from a neural crest cell line as described above further includes step S0 before step S1: culturing human pluripotent stem cells.

[0018] The method for preparing mesenchymal stem cells by using a neural crest cell line as described above, wherein the step S1 comprises: removing the supernatant of the human pluripotent stem cells, adding the specific neural crest cell induction differentiation medium and culturing for 10 days, and sequentially adding the following signal pathway regulators to the specific neural crest cell induction differentiation medium: a BMP inhibitor at a concentration of 100 nM-1 μM for 0-2 days, one of the three inhibitors of Activin, Nodal, and TGFb at a concentration of 2 μM-20 μM for 0-3 days, a GSK-3 inhibitor at a concentration of 1 μM-5 μM for 3-6 days, and a YAP activator at a concentration of 2 μM-20 μM for 6-10 days;

[0019] The specific neural crest cell induction differentiation medium is composed of the following components: DMEM / F12 medium, L-ascorbic acid at a concentration of 64 mg / L, sodium selenite at a concentration of 14 μg / L, transferrin at a concentration of 10.7 mg / L, sodium bicarbonate at a concentration of 543 mg / L, insulin at a concentration of 19.4 mg / L, fibroblast growth factor 2 at a concentration of 100 μg / L, and transcription factor β-3 at a concentration of 2 μg / L.

[0020] In the method for preparing mesenchymal stem cells from a neural crest cell line as described above, step S2 is:

[0021] The specific neural crest cell differentiation induction medium is removed, and a neural crest cell expansion medium is added for expansion culture for 7 days, and bFGF at a concentration of 2-20 ng / mL, EGF at a concentration of 5-50 ng / mL, and one of the following three inhibitors at a concentration of 2 μM-20 μM: Activin, Nodal, and TGFb are added to the expansion medium; during this period, the neural crest cells are cryopreserved using a freezing solution;

[0022] The neural crest cell expansion medium is composed of the following components: DMEM / F12 medium, L-ascorbic acid at a concentration of 64 mg / L, sodium selenite at a concentration of 14 μg / L, transferrin at a concentration of 10.7 mg / L, sodium bicarbonate at a concentration of 543 mg / L, insulin at a concentration of 19.4 mg / L, fibroblast growth factor 2 at a concentration of 100 μg / L, and transcription factor β-3 at a concentration of 2 μg / L; the freezing solution contains dimethyl sulfoxide and serum albumin.

[0023] In the method for preparing mesenchymal stem cells from a neural crest cell line as described above, step S3 is:

[0024] The neural crest cell expansion medium was removed, and the cells were resuspended in a specific mesenchymal stem cell differentiation medium and cultured for 7 days to induce differentiation into mesenchymal stem cells.

[0025] The specific mesenchymal stem cell differentiation medium consists of the following components: minimum essential culture medium, 5% serum replacement, 5 ng / mL basic fibroblast growth factor and 1% antibiotics.

[0026] Another aspect of the present invention provides use of mesenchymal stem cells obtained by the method for preparing mesenchymal stem cells from neural crest cell lines as described above as a drug for osteoarthritis. Beneficial effects

[0027] Compared with the existing technology, the beneficial effects of the present invention are as follows: although many researchers have developed a number of methods for differentiating hPSCs into MSCs in vitro, the existing methods are still not mature enough. For example, non-specific and random components such as serum are required to induce PSCs into mesenchymal stem cells, and high-purity cells need to be screened using flow cytometry and other technologies. As a result, the differentiation effect is unstable, the efficiency is low, and the time is long. In addition, MSCs obtained through the mesodermal cell line differentiation pathway have a low final cell yield because the mesodermal cell line is not easy to store. The method for preparing cells of the present invention has a clear differentiation pathway, high differentiation efficiency, and stable differentiation effect. The preparation method of the present invention does not use a serum-containing culture system and trophoblast cells, and the obtained cells are high in purity and quantity, and the cells can be stored in a multi-stage manner, so they are suitable for the subsequent production and application of clinical-grade cell preparations. The present invention solves the following problems:

[0028] (1) The MSCs obtained by traditional differentiation pathways have unstable effects, low purity and low yield;

[0029] (2) The problem of difficulty in storing intermediate cell lines obtained through traditional differentiation pathways;

[0030] (3) The problem of insufficient chondrogenic differentiation ability of MSCs obtained by traditional differentiation pathways;

[0031] (4) By adding activators that regulate the Hippo / YAP mechanical transduction signaling pathway in the differentiation pathway through the neural crest cell lineage pathway, the MSCs obtained can obtain a large amount of secondary cell storage during the entire cell production process due to the amplification and storability of the neural crest cells themselves. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 shows a technical solution flow chart of an implementation case of the present invention.

[0033] Figure 2 shows the verification of the pluripotency of hiPSCs.

[0034] (A) Expression of pluripotency proteins in hiPSCs (scale bar 200 μm);

[0035] (B) The positive expression percentage of hiPSCs pluripotency markers after flow cytometry detection;

[0036] (C) Immunofluorescence staining images of hiPSCs after induction into endoderm, mesoderm, and ectoderm in vitro (bar, 200 μm).

[0037] Figure 3 shows the expression of neural crest cell marker genes P75, SOX10, and PAX6 on days 6 and 10 by RT-PCR (***p < 0.001).

[0038] Figure 4 shows the effect of BMP inhibitors on the induction of differentiation into the neural crest cell marker SOX10 (***p < 0.001).

[0039] Figure 5 shows the effect of TGFb inhibitor on the induction of differentiation and formation of the neural crest cell marker SOX10 (***p < 0.001).

[0040] Figure 6 shows the effect of GSK-3 inhibitors on the induction of differentiation into the neural crest cell marker SOX10 (***p < 0.001).

[0041] Figure 7 shows the effect of YAP activators on the induction of differentiation into the neural crest cell marker SOX10 (***p < 0.001).

[0042] Figure 8 Flow cytometry was used to detect CD90 in i-MSCs + 、CD73 + 、CD105 + 、CD14 - 、CD34 - 、CD45 - 、CD79a - and HLA-DR - The proportion of phenotypes.

[0043] FIG9 shows the ability of i-MSCs to differentiate into osteoblasts, chondrocytes, and adipocytes (bar: 200 μm).

[0044] FIG10 shows the inhibitory ability of i-MSCs and umbilical cord mesenchymal stem cells (UC-MSCs) on T cell proliferation, respectively.

[0045] FIG11 shows the ratio of the regenerative and repair efficacy of i-MSCs and UC-MSCs on rat osteoarthritis.

[0046] (A) Synovial inflammation score 1 week after injection (**p < 0.05);

[0047] (B) Glycosaminoglycan synthesis 4 weeks after injection (**p < 0.05). Best Mode for Carrying Out the Invention

[0048] Example 1:

[0049] A method for preparing mesenchymal stem cells from a neural crest cell line comprises the following steps:

[0050] S0: culture of human pluripotent stem cells;

[0051] S1: Induction of human pluripotent stem cells into neural crest cells;

[0052] S2: Expansion, culture and storage of neural crest cells;

[0053] S3: Neural crest cells differentiate into mesenchymal stem cells after expansion and culture.

[0054] Specifically, the flow chart is shown in Figure 1, and the steps are as follows:

[0055] (1) Step S0: Human pluripotent stem cell culture (D-3 to D0), Day -3 to Day 0:

[0056] The hPSCs used were rigorously validated for pluripotency: they expressed pluripotency markers and formed teratomas containing endoderm, mesoderm, and ectoderm in immunodeficient mice (see Figure 2 for specific data). The hPSCs were cultured normally in pluripotent stem cell maintenance medium, such as E8, TeSR, or other similar media.

[0057] When human pluripotent stem cells are cultured to a confluence of 70-80%, use Accutase or TrypLE to digest them into complete single cells, resuspend them in an appropriate volume of pluripotent stem cell maintenance medium at a certain density, and add Rock inhibitor to the medium. The cell suspension is plated on iMatrix or Vitronectin-coated well plates and cultured in an incubator at 37 °C, 5% CO2 concentration, and saturated humidity for 3 days, with the medium changed every day. The Rock inhibitor can be Y-27632, and the concentration can be 10 μM; the cell density can be 0.1-10×10 4 cells / cm 2 .

[0058] (2) Step S1: Induction of human pluripotent stem cells into neural crest cells (D0-D10), Day 0 to Day 10:

[0059] When the undifferentiated human induced pluripotent stem cells in good condition are cultured to a confluence of 70-80%, the maintenance medium is aspirated, and a neural crest cell induction medium is added. BMP inhibitors, Activin, Nodal, one of the three TGFb inhibitors, GSK-3 inhibitors and YAP activators are added to the culture medium in sequence according to the pre-designed time points. Continue to culture in an incubator at 37 ° C, 5% CO2 concentration, and saturated humidity for 10 days. During these 10 days, BMP inhibitors are added to the specific neural crest cell induction medium on days 0-2, one of the three inhibitors of Activin, Nodal, and TGFb is added on days 0-3, GSK-3 inhibitors are added on days 3-6, and YAP activators are added on days 6-10. Fresh culture medium is replaced every day, and the medium usage volume is 0.2-0.4mL / cm 2The BMP inhibitor was LDN193189, one of the three inhibitors of Activin, Nodal, and TGFb was SB431542, the GSK-3 inhibitor was CHIR99021, and the YAP activator was PY-60. Fresh culture medium was replaced daily at a volume of 0.2-0.4 mL / cm 2 .

[0060] The resulting neural crest cells were tested on days 6 and 10: RT-PCR was used to assess neural crest cell markers to confirm the differentiation of the cells, confirming their identity. Specific data are shown in Figure 3. P75 and SOX10 levels were significantly higher on day 10 than on day 6, while PAX6 levels were significantly lower on day 10 than on day 6. RNA was extracted using the RNAprep Pure Cultured Cell / Bacterial Total RNA Extraction Kit (TIANGEN, catalog number DP430); reverse transcription was performed using the HiScriptÒ Reverse Transcriptase kit (Vazymebiotech, catalog number R101-01 / 02); and RT-PCR was performed using the TransStart Top Green qPCR SuperMix kit (Transgen, catalog number AQ131).

[0061] (3) Step S2: Expansion and culture of neural crest cells (D10-D17), Day 10 to Day 17:

[0062] The D10 culture medium was aspirated and fresh neural crest cell expansion medium was added, along with optimized concentrations of Activin / Nodal / TGFb inhibitors, EGF, and FGF. The cells were then cultured in an incubator at 37°C, 5% CO2, and saturated humidity for 7 days. During the 7-day culture, Nodal signaling pathway inhibitors, EGF, and bFGF were added to the neural crest cell expansion medium. The Nodal signaling pathway inhibitor was SB431542. Fresh culture medium was replaced daily, and the volume of culture medium used was 0.2-0.4 mL / cm 2 .

[0063] (4) Step S3: Differentiation of neural crest cells into mesenchymal stem cells (D17-D24), Day 17 to Day 24:

[0064] Remove the D17 culture medium and add fresh mesenchymal stem cell differentiation medium. Continue to culture in an incubator at 37°C, 5% CO2 concentration, and saturated humidity for 7 days. Replace the culture medium daily at a rate of 0.2-0.4 mL / cm 2When the cell confluence reaches 70-80%, trypsinize the cells into single cells to obtain i-MSCs at passage 0. The obtained cells are trypsinized into single cells, counted, and expanded for further passage and expansion.

[0065] (5) Step S4: MSCs expansion culture

[0066] Remove the D24 culture medium and add fresh mesenchymal stem cell maintenance medium. Continue to culture in an incubator at 37°C, 5% CO2 concentration, and saturated humidity. Replace with fresh medium every other day. The medium usage is 0.2-0.4 mL / cm 2 MSCs were subcultured to P3. Modes for Carrying Out the Invention

[0067] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0068] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and implementation examples, but the present invention is not limited to the scope of the implementation examples.

[0069] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0070] The flowchart of the method for preparing mesenchymal stem cells (i-MSCs) suitable for use in osteoarthritis in the present invention is shown in FIG1 . Specifically, the operation is as follows:

[0071] 1. D-3 – D0: Culture of human pluripotent stem cells

[0072] Undifferentiated human pluripotent stem cells with a polymerization degree of 70-80% were digested into complete single cells using cell digestion solution and cultured at a concentration of 0.1-10×10 4 cells / cm 2 Resuspend in 3 mL of pluripotent stem cell maintenance medium, seed on a protein-coated plate, and culture in an incubator at 37 °C, 5% CO2 concentration, and saturated humidity.

[0073] Cell digestion solution can use Accutase (Thermo Fisher) or TrypLE (Thermo Fisher) or other.

[0074] iMatrix (Thermo Fisher) or Vitronectin (Thermo Fisher) can be used as the protein.

[0075] D-3 – D0 experimental operation details and optimization:

[0076] The human pluripotent stem cells used in this experiment underwent rigorous pluripotency verification (expressing various pluripotency markers and capable of in vitro differentiation into endoderm, mesoderm, and ectoderm). The stem cells were cultured in Essential 8 medium (Gibco), TeSR medium (Stem Cell), or other similar medium, consisting of the following components: DMEM / F12 medium (DMEM / F12), L-ascorbic acid (64 mg / L), sodium selenium (14 μg / L), transferrin (10.7 mg / L), sodium bicarbonate (543 mg / L), insulin (19.4 mg / L), fibroblast growth factor 2 (FGF2, 100 μg / L), and transcriptional growth factor beta-3 (TGF-b3, 2 μg / L).

[0077] When human pluripotent stem cells are cultured to 70-80% polymerization according to the above method, they are digested into complete single cells using Accutase or TrypLE, resuspended in an appropriate volume of pluripotent stem cell maintenance medium at a certain density, and Rock inhibitor is added to the medium. The cell suspension is plated on iMatrix or Vitronectin-coated well plates and cultured in an incubator at 37 °C, 5% CO2 concentration, and saturated humidity. The medium is changed every day. The Rock inhibitor can be Y-27632 (R&D) at a concentration of 5-20 μM; the cell density can be 0.1-10×10 4 cells / cm 2 .

[0078] 2. D0-10: Differentiation of human pluripotent stem cells into neural crest cells

[0079] When healthy, undifferentiated human pluripotent stem cells reach 70-80% confluence, the maintenance medium is aspirated and replaced with neural crest cell induction medium. At pre-specified time points, a BMP inhibitor (days 0-2), one of the three inhibitors of Activin, Nodal, or TGFb (days 0-3), a GSK-3 inhibitor (days 3-6), and a YAP activator (days 6-10) are added to the culture medium. Continue culturing in a 37°C, 5% CO2, and saturated humidity incubator.

[0080] D0-D10 experimental operation details and optimization:

[0081] The specific operation is as follows: when the human induced pluripotent stem cells are cultured to a confluence of 70-80%, the human pluripotent stem cell maintenance medium is aspirated and replaced with 1× PBS (w / o Ca 2+ / Mg 2+ ) Wash once, add Accutase or TrypLE digestion solution, and incubate in a 37°C, 5% CO2, saturated humidity incubator for 3-8 minutes. Gently shake to detach the cells from the bottom of the culture plate. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Aspirate the supernatant and resuspend the cells in 1mL of fresh neural crest cell induction medium. Gently pipette 1-2 times. Count the cells and seed them into Fibronectin (Gibco) / iMatrix-coated well plates at a seeding density of 0.1-1×10 4 cells / cm 2 Add fresh neural crest cell induction medium. During the 10 days of culture, add BMP inhibitors to the specific neural crest cell induction medium on days 0-2, add one of the three inhibitors of Activin, Nodal, and TGFb on days 0-3, add GSK-3 inhibitors on days 3-6, and add YAP activator on days 6-10. Replace fresh medium every day, and the medium usage volume is 0.2-0.4mL / cm 2 The neural crest cell induction medium consists of the following components: DMEM / F12 medium (DMEM / F12), L-ascorbic acid (64 mg / L), sodium selenium (14 μg / L), transferrin (10.7 mg / L), sodium bicarbonate (543 mg / L), insulin (19.4 mg / L), fibroblast growth factor 2 (FGF2, 100 μg / L), and transcriptional growth factor β-3 (TGF-b3, 2 μg / L).

[0082] BMP inhibitors: Substances that inhibit the BMP signaling pathway. Examples include Noggin, LDN193189, and Dorsomorphin. The preferred BMP inhibitor used in the present invention is LDN193189. The concentration of LDN193189 (R&D) in the culture medium is not particularly limited, and may include, but is not limited to, 200 nM, 300 nM, 500 nM, 0.1 μM, 0.2 μM, 0.5 μM, and 1 μM. The optimal concentration is 500 nM.

[0083] One of the three inhibitors of Activin, Nodal, or TGFb: Substances that inhibit the Activin, Nodal, or TGFb signaling pathways can be selected from SB431542, A83-01, LT580276, and the like. The preferred TGFb inhibitor used in the present invention is SB431542 (R&D). The concentration of SB431542 in the culture medium is not particularly limited, and examples include, but are not limited to, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM. The optimal concentration is 10 μM.

[0084] GSK-3 inhibitors: Substances that inhibit the GSK-3 signaling pathway. Examples include BIO, TWS119, and CHIR99021. The preferred GSK-3 inhibitor used in the present invention is CHIR99021 (R&D). The concentration of CHIR99021 in the culture medium is not particularly limited, and may include, but is not limited to, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 3 μM, 5 μM, and 10 μM. The optimal concentration is 3 μM.

[0085] YAP activator: A substance that activates the Hippo-YAP signaling pathway. Options include PY-60, GA-107, and TT-10. The YAP activator used in the present invention is PY-60 (Sigma). The concentration of PY-60 in the culture medium is not particularly limited, and may include, but is not limited to, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM. The optimal concentration is 10 μM.

[0086] The obtained neural crest cells were detected on D6 and D10:

[0087] Method 1: RT-PCR was used to detect neural crest cell markers to determine the type of differentiated cells, proving that the obtained cells were neural crest cells. P75 and SOX10 levels were significantly higher on D10 than on D6, while PAX6 levels were significantly lower on D10 than on D6.

[0088] Method 2: Immunofluorescence staining was used to detect neural crest cell markers to determine the type of differentiated cells, proving that the obtained cells were neural crest cells. P75 and SOX10 were significantly higher on D10 than on D6, while PAX6 was significantly lower on D10 than on D6.

[0089] III. D10-D17: Expansion and culture of neural crest cells

[0090] The D10 culture medium was removed and fresh neural crest cell expansion medium was added, along with optimized concentrations of Activin, Nodal, one of the three TGFb inhibitors, EGF, and FGF. Culture was continued in an incubator at 37°C, 5% CO2, and saturated humidity.

[0091] D10-D17 experimental operation details and optimization:

[0092] The specific operation is as follows: remove the culture plate from the incubator, remove the supernatant, add fresh neural crest cell expansion medium, and add Activin, Nodal or TGFb signaling pathway inhibitor, EGF and bFGF. Replace the culture medium with fresh medium every day, and the medium usage volume is 0.2-0.4mL / cm 2 .

[0093] Activin, Nodal, or TGFb inhibitors: Substances that inhibit the Activin, Nodal, or TGFb signaling pathways can include SB431542, A83-01, and LT580276. SB431542 is preferred as the Activin, Nodal, or TGFb inhibitor used in the present invention. The concentration of SB431542 in the culture medium is not particularly limited and may include, but is not limited to, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM. The optimal concentration is 10 μM.

[0094] bFGF: The concentration of bFGF in the present invention is not particularly limited, and examples include, but are not limited to, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 15 ng / mL, and 20 ng / mL. The optimal concentration is 8 ng / mL.

[0095] EGF: The concentration of EGF in the present invention is not particularly limited, and may be, for example, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 50 ng / mL, or 100 ng / mL, but is not limited thereto. The optimal concentration is 20 ng / mL.

[0096] D17-D24: Differentiation of Neural Crest Cells into Mesenchymal Stem Cells

[0097] The D17 culture medium was removed, and fresh mesenchymal stem cell differentiation medium was added, and the cells were cultured in an incubator at 37°C, 5% CO2 concentration, and saturated humidity.

[0098] D17-D24 experimental operation details and optimization:

[0099] The specific operation is: remove the culture plate from the incubator, remove the supernatant, and add fresh mesenchymal stem cell differentiation medium. Replace the fresh medium every day, and the amount of medium used is 0.2-0.4mL / cm 2 The mesenchymal stem cell culture medium consists of the following components: minimal essential medium (alpha-MEM, aMEM), 5% serum replacement, 5 ng / mL basic fibroblast growth factor (bFGF), and 1% antibiotic antimycotic.

[0100] 5. D24-D28: MSC expansion culture

[0101] The D24 culture medium was removed, and fresh mesenchymal stem cell maintenance medium was added, and the cells were cultured in an incubator at 37°C, 5% CO2 concentration, and saturated humidity.

[0102] D24-D28 experimental operation details and optimization

[0103] The specific operation is: remove the culture plate from the incubator, remove the supernatant, and add fresh mesenchymal stem cell maintenance medium. Replace the fresh medium every other day, and the amount of medium used is 0.2-0.4mL / cm 2 MSCs were subcultured to P3.

[0104] The obtained P3 cells were tested:

[0105] Method 1: Flow cytometry was used to detect cell phenotypes, and the phenotype of the obtained cells was confirmed to be CD90 + 、CD73 + 、CD105 + 、CD14 - 、CD34 - 、CD45 - 、CD79a - and HLA-DR - ; Among them, three positive surface factors (CD90 + 、CD73 + 、CD105 + ) expression rate reached more than 96%, five negative surface factors (CD14 - 、CD34 - 、CD45 - 、CD79a - and HLA-DR -) expression rate was less than 2%, indicating that the purity of differentiated MSCs was very high.

[0106] Method 2: MSCs were induced to differentiate using osteogenic, chondrogenic and adipogenic differentiation medium, and the obtained cells were proved to have the ability to differentiate into osteogenic, chondrogenic and adipogenic types.

[0107] Method 3: Co-culture with T cells to detect whether the obtained cells have immunosuppressive ability.

[0108] In addition, the present invention also includes the step of freezing the neural crest cells: the neural crest cells can be frozen using a neural crest cell freezing solution.

[0109] Specifically, the culture medium was aspirated and replaced with 1× DPBS (w / o Ca 2+ / Mg 2+ ) Wash once, add Accutase or TrypLE digestion solution, and incubate in a 37°C, 5% CO2, saturated humidity incubator for 5-10 minutes, gently shaking to completely detach the cells from the bottom of the culture dish. Transfer the cell suspension to a 15mL offline tube, centrifuge at 1000 rpm for 3 minutes, aspirate the supernatant, add 1mL of neural crest cell freezing solution, and gently pipette 1-2 times to disperse the cells into single cells as much as possible and count them. The recommended cell number is 1-4×10 6 cells / tubes.

[0110] The main components of the neural crest cell freezing solution of the invention are DMSO and HAS.

[0111] The DMSO concentration in the cryopreservation solution is not particularly limited as long as it can maintain the viability and differentiation efficiency of neural crest cells at low temperatures. Examples include, but are not limited to, 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, and 30%. A preferred concentration is 10%.

[0112] The HSA concentration in the cryopreservation solution is not particularly limited as long as it can maintain the viability and differentiation efficiency of motor neuron precursor cells at low temperatures. Examples include, but are not limited to, 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, and 30%. A preferred concentration is 10%.

[0113] The above optimization conditions can be arbitrarily combined in accordance with common knowledge in the art without exceeding the concept and protection scope of the present invention.

[0114] The key points and points to be protected of the present invention include at least the following two points:

[0115] 1) The key innovation of this invention lies in the type, concentration, and addition time of the small molecule compounds used. Differentiation is used to more precisely control the differentiation direction of human pluripotent stem cells. The resulting cells are highly pure, eliminating the need for flow cytometry to screen the cells.

[0116] 2) Methods for differentiating pluripotent stem cells into mesenchymal stem cells and components of differentiation / expansion media.

[0117] The cell differentiation pathway of the present invention is to induce differentiation of PSCs through the neural crest cell line pathway to obtain i-MSCs, and add an activator that regulates the Hippo / YAP mechanical transduction signaling pathway to the differentiation pathway to optimize PSCs to obtain high-quality i-NCCs, and then obtain high-quality i-MSCs, which are applied to the treatment of osteoarthritis. Neural crest cells have excellent proliferation ability and multipotency, can be used as secondary cells, and the final yield of MSCs is greater. Adding an activator that regulates the Hippo / YAP mechanical transduction signaling pathway to the differentiation pathway makes the differentiation efficiency higher, the obtained NCCs are of higher purity, and ultimately, higher-purity MSCs can be obtained.

[0118] Example 1:

[0119] A method for preparing mesenchymal stem cells from a neural crest cell line comprises the following steps:

[0120] S0: culture of human pluripotent stem cells;

[0121] S1: Induction of human pluripotent stem cells into neural crest cells;

[0122] S2: Expansion, culture and storage of neural crest cells;

[0123] S3: Neural crest cells differentiate into mesenchymal stem cells after expansion and culture.

[0124] Specifically, the flow chart is shown in Figure 1, and the steps are as follows:

[0125] (1) Step S0: Human pluripotent stem cell culture (D-3 to D0), Day -3 to Day 0:

[0126] The hPSCs used were rigorously validated for pluripotency: they expressed pluripotency markers and formed teratomas containing endoderm, mesoderm, and ectoderm in immunodeficient mice (see Figure 2 for specific data). The hPSCs were cultured normally in pluripotent stem cell maintenance medium, such as E8, TeSR, or other similar media.

[0127] When human pluripotent stem cells are cultured to a confluence of 70-80%, use Accutase or TrypLE to digest them into complete single cells, resuspend them in an appropriate volume of pluripotent stem cell maintenance medium at a certain density, and add Rock inhibitor to the medium. The cell suspension is plated on iMatrix or Vitronectin-coated well plates and cultured in an incubator at 37 °C, 5% CO2 concentration, and saturated humidity for 3 days, with the medium changed every day. The Rock inhibitor can be Y-27632, and the concentration can be 10 μM; the cell density can be 0.1-10×10 4 cells / cm 2 .

[0128] (2) Step S1: Induction of human pluripotent stem cells into neural crest cells (D0-D10), Day 0 to Day 10:

[0129] When the undifferentiated human induced pluripotent stem cells in good condition are cultured to a confluence of 70-80%, the maintenance medium is aspirated, and a neural crest cell induction medium is added. BMP inhibitors, Activin, Nodal, one of the three TGFb inhibitors, GSK-3 inhibitors and YAP activators are added to the culture medium in sequence according to the pre-designed time points. Continue to culture in an incubator at 37 ° C, 5% CO2 concentration, and saturated humidity for 10 days. During these 10 days, BMP inhibitors are added to the specific neural crest cell induction medium on days 0-2, one of the three inhibitors of Activin, Nodal, and TGFb is added on days 0-3, GSK-3 inhibitors are added on days 3-6, and YAP activators are added on days 6-10. Fresh culture medium is replaced every day, and the medium usage volume is 0.2-0.4mL / cm 2 The BMP inhibitor was LDN193189, one of the three inhibitors of Activin, Nodal, and TGFb was SB431542, the GSK-3 inhibitor was CHIR99021, and the YAP activator was PY-60. Fresh culture medium was replaced daily at a volume of 0.2-0.4 mL / cm 2 .

[0130] The resulting neural crest cells were tested on days 6 and 10: RT-PCR was used to assess neural crest cell markers to confirm the differentiation of the cells, confirming their identity. Specific data are shown in Figure 3. P75 and SOX10 levels were significantly higher on day 10 than on day 6, while PAX6 levels were significantly lower on day 10 than on day 6. RNA was extracted using the RNAprep Pure Cultured Cell / Bacterial Total RNA Extraction Kit (TIANGEN, catalog number DP430); reverse transcription was performed using the HiScriptÒ Reverse Transcriptase kit (Vazymebiotech, catalog number R101-01 / 02); and RT-PCR was performed using the TransStart Top Green qPCR SuperMix kit (Transgen, catalog number AQ131).

[0131] (3) Step S2: Expansion and culture of neural crest cells (D10-D17), Day 10 to Day 17:

[0132] The D10 culture medium was aspirated and fresh neural crest cell expansion medium was added, along with optimized concentrations of Activin / Nodal / TGFb inhibitors, EGF, and FGF. The cells were then cultured in an incubator at 37°C, 5% CO2, and saturated humidity for 7 days. During the 7-day culture, Nodal signaling pathway inhibitors, EGF, and bFGF were added to the neural crest cell expansion medium. The Nodal signaling pathway inhibitor was SB431542. Fresh culture medium was replaced daily, and the volume of culture medium used was 0.2-0.4 mL / cm 2 .

[0133] (4) Step S3: Differentiation of neural crest cells into mesenchymal stem cells (D17-D24), Day 17 to Day 24:

[0134] Remove the D17 culture medium and add fresh mesenchymal stem cell differentiation medium. Continue to culture in an incubator at 37°C, 5% CO2 concentration, and saturated humidity for 7 days. Replace the culture medium daily at a rate of 0.2-0.4 mL / cm 2 When the cell confluence reaches 70-80%, trypsinize the cells into single cells to obtain i-MSCs at passage 0. The obtained cells are trypsinized into single cells, counted, and expanded for further passage and expansion.

[0135] (5) Step S4: MSCs expansion culture

[0136] Remove the D24 culture medium and add fresh mesenchymal stem cell maintenance medium. Continue to culture in an incubator at 37°C, 5% CO2 concentration, and saturated humidity. Replace with fresh medium every other day. The medium usage is 0.2-0.4 mL / cm 2 MSCs were subcultured to P3.

[0137] Example 2:

[0138] This example investigates the effects of different concentrations of the BMP inhibitor LDN193189 on experimental results. LDN193189 concentrations were set at 200 nM, 500 nM, and 1 μM. As shown in Figure 4, when the LDN193189 concentration was 500 nM, SOX10 expression was highest in neural crest cells induced from pluripotent stem cells.

[0139] Therefore, LDN193189 plays a significant role in the formation of neural crest cells during the 0-2 days of induction differentiation of our human pluripotent stem cells into neural crest cells.

[0140] Example 3:

[0141] This example investigated the effects of varying concentrations of SB431542, one of three inhibitors of Activin, Nodal, and TGFb, on experimental results. SB431542 concentrations were set at 1 μM, 5 μM, 10 μM, and 20 μM, respectively. As shown in Figure 5, when the SB431542 concentration was set at 10 μM, SOX10 expression was highest in neural crest cells induced by differentiation from pluripotent stem cells.

[0142] Therefore, SB431542 plays a significant role in the formation of neural crest cells during the 0-3 days of induction differentiation of our human pluripotent stem cells into neural crest cells.

[0143] Example 4:

[0144] This example investigates the effects of different concentrations of the GSK-3 inhibitor CHIR99021 on experimental results. CHIR99021 concentrations were set at 1 μM, 2 μM, 3 μM, and 5 μM. As shown in Figure 6, when the concentration of CHIR99021 was set at 3 μM, neural crest cells differentiated from pluripotent stem cells expressed the highest levels of SOX10.

[0145] Therefore, SB431542 plays a significant role in the formation of neural crest cells during the 3-6 days of induction differentiation of our human pluripotent stem cells into neural crest cells.

[0146] Example 5:

[0147] This example investigates the effects of different concentrations of the YAP activator PY-60 on experimental results. The concentrations of PY-60 were set at 0 μM, 2 μM, 5 μM, 10 μM, and 20 μM, respectively. As shown in Figure 7, when a certain amount of PY-60 was added, neural crest cells induced and differentiated from pluripotent stem cells all expressed high levels of SOX10. Specifically, when the concentration of PY-60 was set at 10 μM, neural crest cells induced and differentiated from pluripotent stem cells expressed the highest levels of SOX10.

[0148] Therefore, during the 6-10 days of induction differentiation of our human pluripotent stem cells into neural crest cells, PY-60 plays a significant role in the formation of neural crest cells.

[0149] Example 6:

[0150] The P3 generation i-MSCs in Example 1 were tested:

[0151] Method 1: Flow cytometry was used to detect cell phenotypes, and the phenotype of the obtained cells was confirmed to be CD90 + 、CD73 + 、CD105 + 、CD14 - 、CD34 - 、CD45 - 、CD79a - and HLA-DR - ; Among them, three positive surface factors (CD90 + 、CD73 + 、CD105 + ) expression rate reached more than 96%, five negative surface factors (CD14 - 、CD34 - 、CD45 - 、CD79a - and HLA-DR - ) expression rate was less than 2%. Specific data are shown in Figure 8. This indicates that the purity of the differentiated MSCs is very high.

[0152] The specific steps are as follows:

[0153] (1) Each time the cells were passaged, 1×10 5 cells / tube, a total of nine tubes (8 tubes for each sample test, plus one tube for blank control) were added to 1.5 mL of flow cytometry wash buffer, centrifuged at 200 g for 3 min, and the supernatant was discarded.

[0154] (2) Add 100 μL of flow cytometry buffer and gently suspend the cells.

[0155] (3) Label the cell numbers and the following antibody types and tube bodies respectively: CD73 / CD90 / CD105 / CD45 / HLA-DR / CD34 / CD14. No reagent is added to the blank tube. Add appropriate volumes of antibodies to the remaining tubes according to the reagent usage requirements and gently tap the tube wall to mix.

[0156] (4) Incubate at 4°C in the dark for 20-30 minutes.

[0157] (5) After the dark-proof incubation, add 1 mL of flow cytometry washing buffer, centrifuge at 200 g for 3 minutes, discard the supernatant, add 500 μL of FACS buffer, mix well, and prepare for loading. Figure 8 shows that the three cell surface factors (CD73 / CD90 / CD105) of the i-MSCs obtained by the present invention are all positive, and the five surface factors (CD CD79a / CD45 / HLA-DR / CD34 / CD14) are all negative, indicating that the i-MSCs obtained in this example have the characteristics of mesenchymal stem cells.

[0158] Among them, the flow cytometry antibody information is as follows: CD90-FITC, Abcam, ab25672; CD73-APC, Abcam, ab155378; CD105, Abcam, ab2529; CD14, Abcam, ab133503; CD34, Abcam, ab81289; CD45, Abcam, ab40763; CD79a, Abcam, ab187269; HLA-DR, Abcam, ab136320.

[0159] Example 7:

[0160] The P3 mesenchymal stem cells obtained in Example 1 were tested:

[0161] Method 2: Cells were differentiated using osteogenic, chondrogenic, and adipogenic differentiation media to demonstrate that the resulting cells had the ability to differentiate into bone, cartilage, and adipogenesis. The results are shown in FIG9 .

[0162] The specific experimental steps are as follows:

[0163] (1) Identification of osteogenic differentiation

[0164] a. Seed the obtained i-MSCs onto a cell culture plate at an appropriate seeding density, add an appropriate amount of preheated fresh MSCs culture medium, and culture in an incubator at 37 °C, 5% CO2 concentration, and saturated medium.

[0165] The bi-MSCs spread and grew evenly. After reaching approximately 80% confluence, the culture medium on the plate was aspirated and replaced with osteogenic differentiation medium. Specifically, the osteogenic differentiation culture method involved adding ascorbic acid (L-ascorbic acid), b-glycerophosphate (sodium b-glycerophosphate), and dexamethasone to a-MEM medium containing 10% FBS and 1% GlutaMAX Supplement. The cells were cultured for 21 days and observed under a light microscope. During normal differentiation, the cells gradually became elongated.

[0166] c. After 21 days, wash the osteogenic i-MSCs with pure water and add an appropriate volume of Alizarin red staining solution. Incubate in the dark for 30 minutes. Remove excess staining solution and add an appropriate volume of saline or DPBS to each well for infiltration. Observe under a microscope and take photos.

[0167] (2) Identification of chondrogenic differentiation

[0168] a. Seed the obtained i-MSCs onto a cell culture plate at an appropriate seeding density and add an appropriate amount of preheated fresh MSCs culture medium; culture in an incubator at 37°C, 5% CO2 concentration, and a saturated medium.

[0169] b. i-MSCs were allowed to spread and grow evenly. After the confluence reached approximately 90%, the culture medium on the culture plate was aspirated and replaced with chondrogenic differentiation medium. The specific osteogenic differentiation culture method was as follows: Ascorbic Acid (L-ascorbic acid), Dexamethasone (dexamethasone), ITS+Primix tissue culture supplements (ITS+Premix tissue culture supplements), TGF-β1, etc. were added to DMEM high-glucose medium containing 10% FBS for culturing, and the cell density was adjusted to 1×10 6 / mL;

[0170] c. Take a labeled 15 mL centrifuge tube and fill each tube with 500 μL of cell suspension (the final cell suspension in each tube is 0.5×10 6 After loosening the tube cap, place the tube in a cell culture incubator and change the medium every three days for 28 days.

[0171] d. After 28 days, the cell spheres were sectioned, stained with Alcian blue, and photographed.

[0172] (3) Identification of adipogenic differentiation

[0173] a. Seed the obtained i-MSCs onto a cell culture plate at an appropriate seeding density, add an appropriate amount of preheated fresh MSCs culture medium, and culture in an incubator at 37°C, 5% CO2 concentration, and a saturated medium.

[0174] The bi-MSCs spread and grew evenly. After reaching approximately 90% confluence, the culture medium on the plate was aspirated and replaced with adipogenic differentiation medium. Specifically, the adipogenic differentiation culture method involved adding IBMX, dexamethasone, and indomethacin to DMEM high-glucose medium containing 10% FBS for 21 days and observing under a light microscope. During normal differentiation, cells will gradually become wider and shorter.

[0175] c. After 21 days, wash the adipogenically differentiated i-MSCs with pure water and add an appropriate volume of Oil Red O staining solution. Incubate in the dark for 30 minutes. Remove excess staining solution and add an appropriate volume of saline or DPBS to each well for infiltration. Observe under a microscope and take photos.

[0176] Implementation Case 8:

[0177] The P3 mesenchymal stem cells obtained in Example 1 were studied and found to have immunosuppressive capacity when co-cultured with T cells. The T cells and umbilical cord mesenchymal stem cells (UC-MSCs) in this example are commercial T cell lines and UC-MSCs cell lines.

[0178] The specific steps are as follows:

[0179] (1) UC-MSCs and i-MSCs obtained in Example 1 were cultured until the confluence reached more than 90%, and 5 μg / mL mitomycin C was directly added to the old culture medium and incubated at 37 °C for 2 h.

[0180] (2) After 2 hours, discard the old culture medium, wash twice with DPBS, digest the cells normally (digest with Accutase for 3-5 minutes), and collect the cell pellet by centrifugation at 200g for 3 minutes.

[0181] (3) After removing the supernatant, add an appropriate amount of DPBS to the cell pellet, blow off the cell pellet, and take 500 μL of cell suspension to count on a vi-cell cell counter.

[0182] (4) Inoculate the two groups of cells counted in step 3 with TPA (T Cell Proliferation Assay) medium (RPMI+10% FBS+GlutaMAX) to a concentration of 5×10 5Cells / well were plated into six-well plates. After the cells adhered to the wall the next day, the next step of the experiment was continued.

[0183] (5) PBMCs activated 4 days after activation (activated with T cell culture medium containing CD3 / CD28 antibodies and 100 IU / mL IL-2) were transferred to a 15 mL centrifuge tube and centrifuged at 200 g for 3 min to remove the culture medium containing protein components. 5 mL of DPBS was added to wash the tube once. At the same time, the cell suspension was diluted 1:10 and 500 μL was taken to count on a vi-cell cell counter. The cell pellet was collected by centrifugation.

[0184] (6) Resuspend PBMCs in DPBS to 1-3×10 6 A sufficient number of PBMCs were reserved as negative control, and the rest were added with CFSE at a final concentration of 5 μM, mixed and incubated at 37 °C for 10 min.

[0185] (7) Add an equal volume of TPA medium to terminate the CFSE reaction (PBMC pellet should be bright yellow after successful CFSE staining), centrifuge at 200 g for 3 min, discard the supernatant, add 10 mL of DPBS to resuspend the cells, take the cell suspension and count, and collect the cell pellet by centrifugation.

[0186] (8) Resuspend PBMC in TPA medium to 2.5×10 5 / tube of cells for later use, each group of cells after staining was 5×10 5 The number of cells / well was the same as that of the MSCs in the six-well plate seeded in step 4) and co-cultured with MSCs. The final volume per well was adjusted to 4 mL using TPA medium.

[0187] (9) After 4 days of co-culture, cells were photographed using a fluorescence microscope, and the number of T cells in the photographs was recorded. Figure 10 shows that T cells cultured alone expanded normally, but T cells co-cultured with UC-MSCs and i-MSCs obtained by the present invention did not expand normally, demonstrating that these two cell types have immunosuppressive effects, and that i-MSCs have similar immunosuppressive effects as UC-MSCs.

[0188] Example 9:

[0189] This example studies the effect of UC-MSCs and i-MSCs obtained in Example 1 on the repair of osteoarthritis in rats. The specific steps include:

[0190] To evaluate the repair function of i-MSCs obtained in Example 1 for osteoarthritis, a comparative experiment on repair function was conducted with UC-MSCs. Each rat was ovariectomized bilaterally to simulate an osteoarthritis animal model caused by estrogen deficiency. UC-MSCs (1×10 8 / ) and i-MSCs obtained in Example 1 (1×10 8 UC-MSCs (i-MSCs) were injected into the joint cavity of rats (per rat) using an intra-articular injection method. The rats were dissected 1 and 4 weeks later to assess inflammation and proteoglycan (GAG) content within the joint cavity. Figure 11 shows that both UC-MSCs and i-MSCs obtained in Example 1 exhibit anti-inflammatory effects and promote proteoglycan synthesis, demonstrating that i-MSCs have therapeutic efficacy in treating osteoarthritis, with comparable potency to UC-MSCs.

[0191] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the essence and principles of the present invention should be considered as equivalent replacement methods and included in the scope of protection of the present invention.

[0192] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates. Industrial Applicability

[0193] It is easy for those skilled in the art to know from the above description that the technical solution of the present invention is suitable for industrial manufacturing and use in production and life, so the present invention has industrial applicability.

Claims

1. A method for preparing mesenchymal stem cells by using a neural crest cell line, characterized in that , including the following steps: S1: Inducing human pluripotent stem cells to form neural crest cells: adding a specific neural crest cell induction differentiation medium to human pluripotent stem cells for culturing, and adding a YAP activator to the specific neural crest cell induction differentiation medium; wherein the human pluripotent stem cells are commercial human embryonic stem cell lines or human induced pluripotent stem cells; S2: Expand and culture the neural crest cells; S3: The neural crest cells differentiate into mesenchymal stem cells after expansion culture.

2. The method for preparing mesenchymal stem cells by using a neural crest cell line according to claim 1, characterized in that: The mesenchymal stem cells express CD90, CD73 and CD105, but do not express CD14, CD34, CD45 and HLA-DR.

3. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 1, characterized in that: The mesenchymal stem cells are obtained by inducing differentiation of neural crest cells that express P75 and SOX10 but do not express PAX6.

4. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 1, characterized in that: The S3 further includes S4: expanding and culturing the mesenchymal stem cells.

5. The method for preparing mesenchymal stem cells by using a neural crest cell line according to claim 4, characterized in that: The step S4 also includes S5: identifying the mesenchymal stem cells obtained by expansion and culture.

6. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 1, characterized in that: The method also includes step S0 before S1: culturing human pluripotent stem cells.

7. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 1, characterized in that: The S1 step is: removing the supernatant of human pluripotent stem cells, adding the specific neural crest cell induction differentiation medium and culturing for 10 days, and sequentially adding the following signal pathway regulators to the specific neural crest cell induction differentiation medium: a BMP inhibitor with a concentration of 100nM-1μM for 0-2 days, one of the three inhibitors of Activin, Nodal, and TGFb with a concentration of 2μM-20μM for 0-3 days, a GSK-3 inhibitor with a concentration of 1μM-5μM for 3-6 days, and a YAP activator with a concentration of 2μM-20μM for 6-10 days; The specific neural crest cell induction differentiation medium is composed of the following components: DMEM / F12 medium, L-ascorbic acid with a concentration of 64 mg / L, sodium selenite with a concentration of 14 μg / L, transferrin with a concentration of 10.7 mg / L, sodium bicarbonate with a concentration of 543 mg / L, insulin with a concentration of 19.4 mg / L, fibroblast growth factor 2 with a concentration of 100 μg / L, and transcription growth factor β-3 with a concentration of 2 μg / L.

8. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 2, characterized in that: The step S2 is: The specific neural crest cell induction differentiation medium is removed, and a neural crest cell expansion medium is added for expansion culture for 7 days, and bFGF at a concentration of 2-20 ng / mL, EGF at a concentration of 5-50 ng / mL, and one of the following three inhibitors at a concentration of 2 μM-20 μM: Activin, Nodal, and TGFb are added to the expansion medium; during this period, the neural crest cells are cryopreserved using a cryopreservation solution; The neural crest cell expansion medium is composed of the following components: DMEM / F12 medium, L-ascorbic acid at a concentration of 64 mg / L, sodium selenite at a concentration of 14 μg / L, transferrin at a concentration of 10.7 mg / L, sodium bicarbonate at a concentration of 543 mg / L, insulin at a concentration of 19.4 mg / L, fibroblast growth factor 2 at a concentration of 100 μg / L, and transcription growth factor β-3 at a concentration of 2 μg / L; the freezing solution contains dimethyl sulfoxide and serum albumin.

9. The method for preparing mesenchymal stem cells by using neural crest cell line according to claim 1, characterized in that: The step S3 is: The neural crest cell expansion medium was removed, and the cells were resuspended in a specific mesenchymal stem cell differentiation medium, and cultured for 7 days to induce differentiation into mesenchymal stem cells; The specific mesenchymal stem cell differentiation medium consists of the following components: minimum essential culture, 5% serum replacement, 5 ng / mL basic fibroblast growth factor and 1% antibiotics.

10. Use of the mesenchymal stem cells obtained by the method for preparing mesenchymal stem cells from neural crest cell lines according to claims 1 to 9 as a drug for osteoarthritis.

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