A method for promoting in-vitro adhesion and proliferation of umbilical cord mesenchymal stem cells in a simulated in-vivo environment

Abstract

The application provides a method for promoting umbilical cord mesenchymal stem cells to adhere and proliferate in vitro by simulating an in-vivo environment, which comprises the following steps: synthesizing human N-cadherin-Fc fusion protein by genetic engineering technology; and connecting the human N-cadherin-Fc fusion protein to the surface of traditional TCPS cell culture material by hydrophobic force to form a human N-cadherin-Fc fusion protein matrix. The application can overcome the defects in the existing in-vitro expansion technology of umbilical cord mesenchymal stem cells, enhance cell adhesion, shorten the cell proliferation cycle, delay cell aging, expand the production scale of cells and significantly reduce the production cost.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a method for promoting the in vitro adhesion and proliferation of umbilical cord mesenchymal stem cells by simulating the in vivo environment. Technical Background

[0002] Human mesenchymal stem cells (hMSCs) are adult stem cells distributed throughout the body. Umbilical cord tissue is considered a promising source of hMSCs, which possess multiple functions: First, immunomodulatory function is the most fundamental characteristic of hMSCs; they can regulate the body's immune response through a series of mechanisms, thereby regulating and repairing damaged tissues (Christ et al., 2015). Furthermore, hMSCs have low immunogenicity, and injection into the body does not elicit a strong immune response (Gao et al., 2016). Besides immunomodulation, important biological characteristics of hMSCs include homing ability and tissue regeneration potential. They can sense chemokines released from damaged tissue, migrate to target tissues to promote tissue regeneration, or directly replace damaged cells (Savio-Silva et al., 2020, Alfaifi et al., 2018, Han et al., 2012, Rose et al., 2008). These properties of umbilical cord mesenchymal stem cells make them highly promising candidates for regenerative medicine, especially stem cell therapy. Existing preclinical and clinical studies have demonstrated that umbilical cord mesenchymal stem cells exhibit certain efficacy and safety in the treatment of autoimmune diseases, graft-versus-host disease, multiple myeloma, osteoarthritis, cardiac fibrosis, renal fibrosis, pulmonary fibrosis, and end-stage liver disease (Wang et al., 2018).

[0003] For clinical applications, the number of fresh mesenchymal stem cells (MSCs) obtainable from umbilical cords is limited. In vitro expansion of these cells is essential to obtain a sufficient number of functional MSCs. Currently, polystyrene (PS) culture dishes or flasks used for in vitro expansion of MSCs typically only undergo simple tissue culture treatment on their inner surfaces. While these tissue culture treated polystyrene (TC-PS) plates facilitate stem cell adhesion, they lack biomimetic design. This causes MSCs isolated from vivo to gradually lose their original function after in vitro culture, resulting in decreased proliferation efficiency and stem cell expression. This limits the number of usable stem cell passages and restricts the scale of stem cell production. Insufficient stem cell numbers and long expansion times have become bottlenecks restricting the large-scale development of stem cell therapy.

[0004] Given the current challenges in the in vitro expansion of human umbilical cord mesenchymal stem cells, several solutions have been developed to improve expansion efficiency, such as co-culturing with other cells (202010846476.2), adding specific drugs to the culture medium (202010974672.8), growth factors, or activators of certain signaling pathways (202010700822.6). However, these methods still have some problems, such as the introduction of other cell types leading to reduced cell purity, increased difficulty in subsequent separation, the possibility that the addition of drugs may cause cells to deviate from their original cell phenotype and become diseased, and the possibility that signaling pathway activators may reduce the stemness of mesenchymal stem cells.

[0005] Studies have shown that the microenvironment in which stem cells reside determines their fate. Simulating the in vivo microenvironment of stem cells in vitro can not only enhance their proliferation efficiency but also maintain their biological characteristics. The stem cell microenvironment is mainly composed of cytokines, direct cell-cell contact, and the extracellular matrix. Cadets, as core proteins of adhesion junctions—one of the intercellular interactions—are crucial for regulating stem cell fate. Neurocadherins are major intercellular interaction proteins on the surface of human umbilical cord mesenchymal stem cells, playing a significant role in regulating their fate.

[0006] References

[0007] Alfaifi M, Eom YW, Newsome PN, et al. 2018. Mesenchymal stromal cell therapy for liver diseases. J Hepatol[J], 68: 1272-1285.

[0008] Christ B, Bruckner S, Winkler S 2015. The Therapeutic Promise of Mesenchymal Stem Cells for Liver Restoration. Trends Mol Med[J], 21: 673-686.

[0009] Gao F, Chiu SM, Motan DA, et al. 2016. Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell Death Dis[J], 7: e2062.

[0010] Han F, Wang CY, Yang L, et al. 2012. Contribution of murine bone marrowmesenchymal stem cells to pancreas regeneration after partial pancreatectomy in mice. Cell Biol Int[J], 36: 823-831.

[0011] Rose RA, Jiang H, Wang

[0012] Savio-silva C, Beyerstedt S, Soinski-sousa PE, et al. 2020. MesenchymalStem Cell Therapy for Diabetic Kidney Disease: A Review of the Studies Using Syngeneic, Autologous, Allogeneic, and Xenogeneic Cells. Stem Cells Int[J], 2020: 8833725.

[0013] Wang Jianfang, Mo Chunyang, Xu Yanhua, et al. 2018. Clinical application research of bone marrow mesenchymal stem cells. Chinese Journal of Cell Biology [J], 40: 2145-2155. Summary of the Invention

[0014] The purpose of this invention is to provide a method that overcomes the shortcomings of existing in vitro expansion technologies for human umbilical cord mesenchymal stem cells, enhances cell adhesion, shortens the cell proliferation cycle, delays cell senescence, expands the scale of cell production, and significantly reduces its production cost. The specific technical solution is as follows:

[0015] A method for promoting the in vitro adhesion and proliferation of umbilical cord mesenchymal stem cells by simulating the in vivo environment is characterized by modifying traditional cell culture substrates with neurocadherin-Fc fusion protein. By binding the extracellular domain of neurocadherin to human umbilical cord mesenchymal stem cells, cell adhesion is enhanced, the cell proliferation cycle is shortened, and cell senescence is delayed, thereby promoting the in vitro proliferation of human umbilical cord mesenchymal stem cells and increasing cell yield.

[0016] Preferably, the method for modifying traditional cell culture media with the neurocadherin-Fc fusion protein is as follows:

[0017] A neurocadherin-Fc fusion protein matrix was prepared by soaking traditional cell culture media in a solution of neurocadherin-Fc fusion protein. Human umbilical cord mesenchymal stem cells were then cultured on the surface of the prepared neurocadherin-Fc fusion protein matrix and continuously passaged. Cell counting and cell viability assays were performed to determine the effects on human umbilical cord mesenchymal stem cell adhesion, cell senescence, and proliferation.

[0018] In the specific embodiments of the present invention described above, the neurotrophin is human neurotrophin; Fc is the Fc of human IgG (preferably IgG1).

[0019] In the specific embodiments of the present invention described above, the sequence of the neurocadherin is represented by SEQ ID NO: 2, and the sequence of Fc is represented by SEQ ID NO: 3; preferably, the sequence of the neurocadherin-Fc fusion protein is represented by SEQ ID NO: 1.

[0020] In the specific embodiments of the present invention described above, the concentration of the neurocadherin-Fc fusion protein used is 1-20 μg / mL (preferably 5-15 μg / mL).

[0021] In the specific embodiments of the present invention described above, the cell culture medium is selected from PS / TCPS cell culture plates, cell culture dishes, hydrogels, porous scaffolds, films, or microspheres, with TCPS cell culture plates being preferred.

[0022] In the specific embodiments of the present invention described above, the preferred method for cell counting is as follows: Take a small amount of evenly distributed cell suspension to be tested, drop it onto the edge of the counting plate cover, so that the suspension fills the space between the cover and the counting plate, let it stand for 3 minutes, calculate the total number of cells in the four large squares of the counting plate, counting only the cells on the left and top of the lines, and then calculate according to the formula: Cell count = Total number of cells in the four large squares / 4 × 10 4 × Total volume of cell suspension (mL).

[0023] In the specific embodiments of the present invention described above, the preferred method for detecting cell adhesion rate is as follows: fifth-generation human umbilical cord mesenchymal stem cells are prepared at a ratio of 1×10⁻⁶... 4 pcs / cm2 Cells were evenly distributed at a uniform density on the surface of a six-well TCPS culture plate modified with the prepared human neurotrophin-Fc fusion protein and counted as M1. After incubation at 37°C and 5% CO2 (volume concentration) for 4 hours, the culture medium was aspirated, and the plate was washed three times with PBS to remove non-adhering cells. After in situ fixation with 4% paraformaldehyde for 20 minutes, five fields of view were randomly selected from each sample under a microscope and photographed and counted as M2. The cell adhesion rate was then calculated using the formula: Cell adhesion rate = M2 / M1 × 100%.

[0024] In the specific embodiments of the present invention described above, the preferred method for detecting cell viability is as follows: normally cultured cells in the logarithmic growth phase are seeded in a 96-well plate and cultured for 24 hours in an incubator at 37°C and 5% CO2 (volume concentration). 10 μL of CCK-8 solution is added to each well. The cells are then cultured in the incubator for another 2 hours, and the absorbance at 450 nm is measured using a microplate reader.

[0025] In the specific embodiments of the present invention described above, the preferred method for detecting cell doubling time is as follows: Cells in the logarithmic growth phase from normal culture are seeded in a 96-well plate and cultured at 37°C in a 5% CO2 (volume concentration) incubator until the logarithmic growth phase. A portion of the sample is added to each well with 10 μL of CCK-8 solution and cultured for another 2 hours. The absorbance at 450 nm is measured using a microplate reader and recorded as A1. Another portion of the sample is cultured for 10 hours, after which 10 μL of CCK-8 solution is added to each well and cultured for another 2 hours. The absorbance at 450 nm is measured using a microplate reader and recorded as A2. Then, the cell doubling time is calculated using the formula: Cell doubling time = (10 × log2) / log(A2 / A1).

[0026] In the specific embodiments of the present invention described above, the preferred method for detecting cell proliferation is as follows: third-generation human umbilical cord mesenchymal stem cells are prepared at a ratio of 1×102 4 pcs / cm 2 The cells were evenly spread on the surface of a T75 culture flask modified with the prepared human neurotrophin-Fc fusion protein and continuously cultured in an incubator at 37°C and 5% CO2 (volume concentration). When the cell density reached 90%, the cells were digested with trypsin and counted as M3. Then, the cell density was adjusted to 1×10⁻⁶ cells / mL. 4 pcs / cm 2 The cells were evenly spread on the surface of a T75 culture flask modified with the prepared human neurotrophin-Fc fusion protein, and continuously passaged and counted until the cells no longer proliferated. Attached Figure Description

[0027] Figure 1 Construction and expression of human neurocadherin-Fc fusion protein (hN-cad-Fc) plasmid, in which... Figure 1A represents the construction of the hN-cad-Fc expression vector; Figure 1 B represents the expression and detection of the hN-cad-Fc expression vector.

[0028] Figure 2 The adhesion of human umbilical cord mesenchymal stem cells to TCPS, epithelial cadherin-Fc matrix, and human neurocadherin-Fc matrix.

[0029] Figure 3 CCK-8 absorbance assay of human umbilical cord mesenchymal stem cells on TCPS, epithelial cadherin-Fc matrix, and human neurocadherin-Fc matrix.

[0030] Figure 4 The number of passages and cell counts after expansion of human umbilical cord mesenchymal stem cells on TCPS, epithelial cadherin-Fc matrix, and human neurocadherin-Fc matrix. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. It will be understood by those skilled in the art that the following embodiments are for illustrative purposes only and should not be construed as limiting the invention in any way. The scope of protection of the present invention is defined by the appended claims.

[0032] The conventional chemical reagents used in the examples were all purchased from Solarbio Science & Technology Co., Ltd.; the peptides involved were all synthesized by Nanjing Genscript Biotech Co., Ltd.

[0033] Example 1: Construction and expression of human neurocadherin-Fc fusion protein.

[0034] The sequence of the human neurocadherin-Fc fusion protein is shown in SEQ ID NO: 1, where the sequence of SEQ ID NO: 2 represents the sequence of human neurocadherin; and the sequence of SEQ ID NO: 3 represents the sequence of Fc.

[0035] 1.1 Cloning and sequence analysis of the extracellular region gene of neurocadherin in nerve cells

[0036] Based on the human neurocadherin protein sequence and functional regions included in the UniProt database, and combined with the gene sequence (NCBI Reference Sequence: NM_001795.3) in GenBank, specific PCR primers were designed to amplify the extracellular region (EC1-EC5) of neurocadherin protein. The upstream primer (P1) was 5'-CCGGATATCATGCAGAGGCTCATGATGCTCC-3' (SEQ ID NO: 8), introducing an EcoR V restriction site (underlined). The downstream primer was (P2) 5'-AAGCGGCCGCTCTGGGCGGCCATATC-3' (SEQ ID NO: 9), introducing a Not I restriction site (underlined). Primer synthesis and sequencing were performed by Invitrogen Ltd.

[0037] Total mRNA extraction from Scien Cells: mRNA was extracted according to the standard methods described in Molecular Cloning: A Laboratory Manual (3rd Edition). OD values ​​were measured to quantify RNA purity and concentration.

[0038] Reverse transcription was performed using the BD reagent kit purchased from [Company Name]. The reverse transcription system used in MicroRNA Assays is as follows:

[0039]

[0040] The reverse transcription procedure is as follows:

[0041]

[0042] Using mRNA extracted from nerve cells as a template, a fragment of the neurocadherin gene was amplified. The PCR reaction system is as follows:

[0043]

[0044] The amplification conditions were as follows: denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 10 min. 380 μL of ddH2O was added to the reaction solution, followed by extraction once with an equal volume of phenol / chloroform / isoamyl alcohol. 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of anhydrous ethanol were added, and the mixture was incubated at -20℃ for 1 h. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃. The DNA precipitate was washed twice with 70% ethanol, vacuum dried, and dissolved in an appropriate amount of TE buffer.

[0045] 1.2 Construction of pcDNA3.1-human neurocadherin-Fc eukaryotic expression vector

[0046] (1) PCR products purified by double digestion with EcoR V and Not I

[0047] The enzyme digestion system is as follows:

[0048]

[0049] The reaction was carried out overnight at 37°C, followed by enzyme inactivation at 65°C for 15 min. 350 μL of ddH₂O was added to the reaction solution, and the mixture was extracted once with an equal volume of phenol / chloroform / isoamyl alcohol. 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of anhydrous ethanol were added, and the mixture was incubated at -20°C for 1 h. The mixture was then centrifuged at 12000 rpm for 10 min at 4°C. The DNA precipitate was washed twice with 70% ethanol, vacuum dried, and dissolved in 10 μL of TE buffer.

[0050] (2) pcDNA / 3.1 digestion with EcoRV and Not I enzymes;

[0051] The double digestion system (3 × 50 μL) of pcDNA / 3.1 (ThermoFisher) is as follows:

[0052]

[0053] The reaction was carried out overnight at 37°C. The enzyme digestion products were separated by electrophoresis in a 1% agarose gel, the target fragment was excised under UV light, and the fragment was recovered using a DNA agarose gel recovery kit (TaKaRa). The recovered fragment was dissolved in 25 μL ddH2O.

[0054] (3) Ligation and transformation reactions between the vector and the target fragment

[0055] The reaction system is as follows:

[0056]

[0057] The reaction was carried out at 16℃ for 16 hours. Then, competent cells BL21(DE3) were transformed with CaCl2 and cultured overnight at 37℃ for 16–18 hours. Transformants were picked, and plasmids were extracted in small quantities for detection.

[0058] The extracellular region of the target gene, human neurocadherin, and the vector pcDNA3.1 containing the Fc fragment were recovered and double-digested with EcoRV and NotI at 37°C. After electrophoresis, the recovered products were mixed and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, and ampicillin (Amp+) was used for resistance selection. The plasmid was extracted and double-digested for identification. DNA sequence analysis was performed on the preliminarily identified recombinant plasmid. The constructed recombinant plasmid was named pcDNA3.1 / hN-cad-Fc (see...). Figure 1 A) The sequence was verified to be correct by sequencing.

[0059] 1.3 Cell transfection and protein purification

[0060] Transfect pcDNA3.1 / hN-cad-Fc into 293F cells (Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee).

[0061] The target protein was purified using a Hitrap rProtein AFF column manufactured by GE Healthcare by utilizing the specific binding of the immunoglobulin Fc fragment to rProtein A.

[0062] 1.4 Western blotting analysis

[0063] The purified human cadherin-Fc fusion protein was transferred to a PVDF membrane after 10% SDS-PAGE gel electrophoresis. The membrane was blocked with 5% skim milk for 2 h, incubated overnight at 4°C with a primary antibody (rabbit anti-human cadherin extracellular domain monoclonal antibody, RD, 1:400 dilution), and then incubated at room temperature for 1 h with HRP-labeled goat anti-rabbit secondary antibody (abcam, 1:10000 dilution). The membrane was washed with TBST, and then exposed to DAB reagent for development, fixing, and analysis. β-mercaptoethanol was not added to the loading buffer when detecting Fc dimerization. A band was observed at ~240 kDa in the non-reduced state and at ~120 kDa in the reduced state, suggesting that the human cadherin-Fc fusion protein exists in a dimer form. Figure 1 B)

[0064] Example 2: Preparation of human neurotrophin-Fc matrix

[0065] The prepared human cadherin-Fc fusion protein powder was dissolved in sterile PBS to prepare a stock solution with a final concentration of 1 mg / mL. The aliquoted human cadherin-Fc fusion protein stock solution was then diluted with sterile PBS to a working solution with a final concentration of 10 μg / mL. Then, the solution was dispensed at a rate of 100 μL / cm³. 2 The solution was transferred to the TCPS material surface and incubated at 37°C for 2 hours or 4°C overnight. All solution was aspirated, and the cells were washed three times with PBS before cell adhesion and proliferation assays were performed. Human epithelial cadherin-Fc matrix and an equal volume of PBS were prepared and transferred to the TCPS material surface as a control group using the same method.

[0066] Example 3: Detection of Adhesion Ability of Human Umbilical Cord Mesenchymal Stem Cells

[0067] The fifth-generation human umbilical cord mesenchymal stem cells were divided into 1×10 4 pcs / cm 2Cells were evenly distributed at a uniform density on the surface of a prepared six-well TCPS culture plate modified with human neurocadherin-Fc fusion protein and counted as M1. After culturing in a 37℃ 5% CO2 incubator for 4 hours, the culture medium was aspirated, and the cells were washed three times with PBS to remove unadhered cells. Cells were then fixed in situ with 4% paraformaldehyde for 20 minutes. Under a microscope, five fields of view were randomly selected from each sample for imaging, and the cells were counted as M2. The cell adhesion rate was calculated as M2 / M1. Human epithelial cadherin-Fc matrix and unmodified six-well TCPS culture plates served as control groups, with three parallel replicates in each group. The results showed that, compared with traditional TCPS culture plates and human epithelial cadherin-Fc matrix, human neurocadherin-Fc matrix significantly improved the adhesion ability of human umbilical cord mesenchymal stem cells (see [link to study]). Figure 2 ).

[0068] Example 4: Detection of human umbilical cord mesenchymal stem cell viability

[0069] The fifth-generation human umbilical cord mesenchymal stem cells were divided into 1×10 4 pcs / cm 2 The protein was evenly spread on the surface of a prepared 96-well TCPS culture plate modified with human neurocadherin-Fc fusion protein. The plate was incubated at 37°C with 5% CO2 (volume concentration) for 24 hours, with 10 μL of CCK solution added to each well. After incubation for another 2 hours, the absorbance at 450 nm was measured using a microplate reader. Human epithelial cadherin-Fc fusion protein modified and unmodified 96-well TCPS culture plates served as control groups, with five replicates in each group. The results showed that the TCPS culture plate modified with human neurocadherin-Fc fusion protein maintained the in vitro viability of human umbilical cord mesenchymal stem cells (see...). Figure 3 ).

[0070] Example 5: Detection of doubling time of human umbilical cord mesenchymal stem cells

[0071] The fifth-generation human umbilical cord mesenchymal stem cells were divided into 1×10 4 pcs / cm 2Cells were evenly distributed on the surface of a 96-well TCPS culture plate modified with the prepared human cadherin-Fc fusion protein. The plates were incubated at 37°C with 5% CO2 (volume concentration) for 24 hours until the logarithmic growth phase. A portion of the sample was incubated for another 2 hours, with 10 μL of CCK solution added to each well. The absorbance at 450 nm was measured using a microplate reader and recorded as A1. Another portion of the sample was incubated for 10 hours, with 10 μL of CCK solution added to each well. The absorbance at 450 nm was measured using a microplate reader and recorded as A2. Cell doubling time = (10 × log2) / log(A2 / A1). Both modified and unmodified 96-well TCPS culture plates served as control groups, with five replicates in each group. The results showed that, compared with the traditional TCPS culture plate, the TCPS culture plate modified with human neurocadherin-Fc fusion protein shortened the doubling time of human umbilical cord mesenchymal stem cells by 11.19 ± 2.11%, thus reducing the cell culture cycle.

[0072] Example 6: Detection of the proliferation of human umbilical cord mesenchymal stem cells during continuous passage.

[0073] 10 6 Third-generation human umbilical cord mesenchymal stem cells were evenly seeded on the surface of a T75 culture flask modified with prepared human neurocadherin-Fc fusion protein and continuously cultured in an incubator at 37°C and 5% CO2 (volume concentration). When the cell density reached 90%, the cells were digested with trypsin and counted, designated as M3, and the cell density was adjusted to 103. 6 Cells were evenly spread on the surface of a prepared human neurocadherin-Fc fusion protein-modified T75 culture flask and continuously passaged and counted until cell proliferation ceased. Human epithelial cadherin-Fc fusion protein-modified and unmodified T75 culture flasks served as control groups, with three replicates in each group. The results showed that the TCPS culture plate modified with human neurocadherin-Fc fusion protein not only promoted the doubling rate of human umbilical cord mesenchymal stem cells per generation but also delayed cell senescence and extended the usable cell passage to the 14th generation (see [link to study]). Figure 4 ), through modification with human neurocadherin-Fc fusion protein, ultimately 10 6 The initial cells expanded to 1.56 × 10⁻⁶. 12 ±1.91×10 11 Compared to the TCPS group, the cell culture passages were extended by 3 generations, and the total number of cells increased by 44.18-70.93 times. Compared to the epithelial cadherin-Fc fusion protein modification group, the cell culture passages were extended by 2 generations, and the total number of cells increased by 18.32-24.36 times.

Claims

1. The application of the neurocadherin-Fc fusion protein in promoting the adhesion ability of human umbilical cord mesenchymal stem cells, shortening the in vitro doubling time of human umbilical cord mesenchymal stem cells, maintaining the in vitro viability of human umbilical cord mesenchymal stem cells, and delaying the aging of human umbilical cord mesenchymal stem cells, characterized in that: The neurocadherin-Fc fusion protein is immobilized on a cell culture medium, wherein the neurocadherin is human neurocadherin, which has the sequence shown in SEQ ID NO: 2, and the Fc has the sequence shown in SEQ ID NO:

3. The sequence of the neurocadherin-Fc fusion protein is represented by the sequence shown in SEQ ID NO:

1.

2. Application of neurocadherin-Fc fusion protein in increasing the number of human umbilical cord mesenchymal stem cells in vitro, wherein the neurocadherin-Fc fusion protein is immobilized on a cell culture medium, wherein the neurocadherin is human neurocadherin, which is the sequence shown in SEQ ID NO: 2, the Fc is the sequence shown in SEQ ID NO: 3, and the sequence of the neurocadherin-Fc fusion protein is represented by the sequence shown in SEQ ID NO:

1.

3. The method for promoting the in vitro adhesion and proliferation of human umbilical cord mesenchymal stem cells using the neurocadherin-Fc fusion protein as described in any one of claims 1 and 2, characterized in that: The concentration of the neurocadherin-Fc fusion protein used is 1-20 μg / mL.

4. The method according to claim 3, characterized in that: The concentration of the neurocadherin-Fc fusion protein used is 5-15 μg / mL.

5. The method according to claim 3, wherein the cell culture medium is selected from PS / TCPS cell culture plates, cell culture dishes, hydrogels, porous scaffolds, films, or microspheres.

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