Polypeptide supporting in-vitro culture of human pluripotent stem cells and application

Abstract

The invention provides a polypeptide supporting in-vitro culture of human pluripotent stem cells and application, and belongs to the technical field of regenerative medicine. The sequence of the polypeptide is KGGPQVTRGDTFRYI or KGGPQVTRGDTYHSY, and the sequence of the polypeptide is shown as KGGPQVTRGDTYHSY. The polypeptide provided by the invention has excellent cell adhesion capacity, enhances the interaction between cells and an integrin receptor alpha v beta 5, and significantly improves the efficiency and quality of human pluripotent stem cell culture. By using the polypeptide sequence provided by the invention, the dependence on animal source materials is reduced, the biological safety and compatibility of a culture system are improved, and more effective and safer support is provided for clinical application of hPSCs.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology, and in particular to a polypeptide that supports the in vitro culture of human pluripotent stem cells and its applications. Background Technology

[0002] Human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), are a key resource in regenerative medicine, but their in vitro culture technology faces numerous challenges. Traditional culture techniques, such as animal-derived Matrigel or feeder cell culture systems, suffer from risks of animal-derived contamination, batch-to-batch quality fluctuations, and high costs, which can lead to cell senescence and loss of pluripotency. Although culture surfaces modified with integrin receptor ligand proteins such as ligands like tessellatedin and E-adhesin have been shown to support stem cell adhesion and stemness maintenance, these bioactive materials are costly to prepare, cannot be mass-produced, and require aseptic preparation conditions. Multiple studies have shown that peptides containing RGD sequences can effectively support the adhesion and growth of hESCs and hiPSCs. Compared with Matrigel, their performance in cell adhesion and self-renewal needs further improvement. Therefore, exploring novel RGD peptides with stronger integrin binding capacity and hiPSC culture support capabilities is an effective strategy.

[0003] Therefore, improving the efficiency and cost of existing cell culture peptide screening technologies is an urgent technical problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by providing a polypeptide that supports the in vitro culture of human pluripotent stem cells and its applications. The polypeptide provided by this invention can enhance cell adhesion and activity, thereby improving the binding capacity of integrin receptors and the ability to support hPSC culture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a polypeptide that supports the in vitro culture of human pluripotent stem cells, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1; or the amino acid sequence of SEQ ID NO.1 is modified, substituted, deleted or added with one or more amino acids, and has at least 90% homology with the amino acid sequence of SEQ ID NO.1.

[0006] Furthermore, the amino acid sequence of the polypeptide has the amino acid sequence shown in SEQ ID NO.2; or the amino acid sequence shown in SEQ ID NO.2 is modified, substituted, deleted or added with one or more amino acids, and has at least 90% homology with the amino acid sequence shown in SEQ ID NO.2.

[0007] A second objective of the present invention is to provide a cell adhesion reagent containing the aforementioned polypeptide.

[0008] Furthermore, the cell adhesion reagent is a mixture of the polypeptide and DuPont phosphate buffer.

[0009] A third objective of this invention is to provide a coating kit for promoting cell adhesion, the coating kit comprising four containers, each containing the aforementioned cell adhesion reagent, dopamine solution, carboxymethyl chitosan aqueous solution, and polypeptide activation grafting reagent.

[0010] Furthermore, the coating kit contains the aforementioned cell adhesion reagent, gelatin-sodium alginate composite hydrogel precursor solution, calcium chloride aqueous solution, and peptide activation grafting reagent.

[0011] Furthermore, the concentration of the polypeptide in the cell adhesion reagent is 1~1.2 mmol / L, preferably 1 mmol / L.

[0012] Further, the dopamine solution is a mixture of dopamine hydrochloride and Tris-HCl buffer solution adjusted to pH 8.5, with a concentration of 2~2.5 g·L⁻¹. -1 Preferably 2 g·L -1 .

[0013] Furthermore, the mass concentration of the carboxymethyl chitosan is 2.5% to 3.5%, preferably 3%.

[0014] Furthermore, the composite hydrogel precursor solution is an equal-volume mixture of sodium alginate and gelatin, wherein the mass concentration of both sodium alginate and gelatin is 2-2.5%, preferably 2%.

[0015] Further, the calcium chloride aqueous solution has a concentration of 100-110 mmol / L, preferably 100 mmol / L.

[0016] Furthermore, the peptide activation grafting reagent includes morpholine ethanesulfonic acid buffer, N-hydroxysuccinimide solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution.

[0017] A fourth objective of the present invention is to provide a cell culture container having an inner wall for cell adhesion, the inner wall being coated using the coating kit described above.

[0018] The fifth objective of this invention is to provide the application of the above-mentioned polypeptides, cell adhesion reagents, coating kits, and cell culture containers in the culture of human pluripotent stem cells.

[0019] Furthermore, the cells include H1 hESCs and hNF-C1 hiPSCs.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a polypeptide with the sequence KGGPQVTRGDTFRYI or KGGPQVTRGDTYHSY to support the in vitro culture of human pluripotent stem cells (hPSCs). This polypeptide exhibits excellent cell adhesion, enhances cell-integrin receptor αvβ5 interaction, and significantly improves the efficiency and quality of hPSC culture. The use of this polypeptide sequence reduces dependence on animal-derived materials, improves the biosafety and compatibility of the culture system, and provides more effective and safer support for the clinical application of hPSCs. Attached Figure Description

[0021] Figure 1 The structure of integrin αvβ5, as predicted by MolAICal, is shown in blue and yellow, respectively, representing the alpha-v and beta-5 subunits. Figure 2 This is a schematic diagram of peptide design provided by the present invention; Figure 3 The RMSD (root mean square deviation) of integrin αVβ5 and its designated polypeptide complex; Figure 4 This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 1 complex with integrin αVβ5. Figure 5 This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 2 complex with integrin αVβ5; Figure 6 This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 3 complex with integrin αVβ5. Figure 7 This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 4 complex with integrin αVβ5. Figure 8 This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 5 complex with integrin αVβ5. Figure 9This is a residue energy decomposition diagram of the binding free energy of the candidate peptide 6 complex with integrin αVβ5. Figure 10 The results of the CCK8 experiment were obtained after culturing VN peptides and six different candidate peptides on their surfaces for 4 days. Figure 11 The images show the adhesion of H1 hESCs and hNF-C1 hiPSCs on the surfaces of peptide 4, peptide 5, VN peptide, and Matrigel on the fourth day of culture, along with the crystal violet staining results. Figure a shows the statistical data on adhesion, and figure b shows the crystal violet staining results. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0023] definition: In this specification, the expressions “containing” and “comprising” include concepts such as “containing”, “comprising”, “substantially constituted by” and “consisting solely of”.

[0024] Amino acid sequence "homology" refers to the degree of similarity between two or more comparable amino acid sequences. Therefore, the higher the similarity between two amino acid sequences, the greater their identity or similarity. The level of amino acid sequence identity can be determined, for example, using FASTA, a sequence analysis tool, with default parameters. Alternatively, it can be determined using the BLAST algorithm based on Karlin and Altschul. A program called BLASTX has been developed based on such a BLAST algorithm. The specific methods for these resolutions are well-known and can be found on the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ).

[0025] This invention first retrieves the amino acid sequence of the integrin β-5 subunit from the Universal Protein database (UniProt ID: P18084). Using the MolAICal software package, based on the protein amino acid sequence provided in the table, and using AlphaFold model prediction data, a high-precision three-dimensional structure of the β-5 subunit of the target integrin αvβ5 was constructed. Furthermore, by integrating the known structure of the alpha-v subunit of αVβ3 (Protein Data Bank database ID: 1L5G), the complete extracellular three-dimensional structure of the αVβ5 integrin was constructed (see...). Figure 1 The study found that mutating the RGD post-transfer sequence of the VN peptide (KGGPQVTRGDVFTMP) enhances its binding affinity to the integrin receptor αvβ5. Specifically, the X1 site was selected from V, S, and T; the X2 site was selected from aromatic amino acids F, P, Y, or W; the X3 site could be selected from 20 standard amino acids; and the X4 and X5 sites were selected from amino acids with moderate or higher hydrophobicity, such as L, F, V, A, I, M, S, T, Y, W, P, G, or C. This design generated 40,560 different peptide sequence variants (see [link to study]). Figure 2 Further investigation revealed that peptides 4 and 5 have a strong binding affinity to the integrin receptor αvβ5.

[0026] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes them in conjunction with the data, charts and other information of the experimental process.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] Example 1 Synthesize polypeptides.

[0029] In one specific embodiment of the present invention, peptide sequence 4 (KGGPQVTRGDTFRYI) and peptide 5 (KGGPQVTRGDTYHSY) are synthesized using conventional peptide synthesis methods; the synthesized products can be purified using conventional peptide purification methods to prepare cell adhesion reagents.

[0030] In this embodiment, the inventors commissioned Shanghai Qiangyao Biotechnology Co., Ltd. to synthesize an amino acid sequence as shown in SEQ ID NO.1~SEQ ID NO.6 using a standard solid-phase peptide synthesis method, and then purified the peptide to obtain cell adhesion factor.

[0031] Example 2 Preparation of cell adhesion reagent In this embodiment, 1L of cell adhesion reagent was prepared: Take 1000 ml of phosphate buffer, mix well, then add the synthesized polypeptide and stir to dissolve it, thereby obtaining 1 L of cell adhesion reagent, in which the concentration of cell adhesion factor is 1 mmol / L.

[0032] Place the prepared cell adhesion reagent in a brown reagent bottle and store at room temperature, protected from light.

[0033] Example 3 This embodiment provides a coating kit for promoting cell adhesion.

[0034] Cell adhesion reagent, dopamine solution, carboxymethyl chitosan solution, and peptide activation grafting reagent were prepared separately.

[0035] The synthesized peptide was dissolved in Duchenne phosphate buffer to prepare a solution with a peptide concentration of 1-1.2 mmol / L, preferably 1 mmol / L. Dopamine was dissolved in Tris-HCl buffer solution to prepare a solution with a mass concentration of 2–2.5 g·L⁻¹. -1 A solution of 2 g·L is preferred. -1 ; Dissolve carboxymethyl chitosan in water to prepare a solution with a concentration of 2.5% to 3.5% (w / v), preferably 3% (w / v). The peptide-activating grafting reagent comprises a mixture of the following components: (1) The buffer solution was a morpholine ethanesulfonic acid (MES) buffer solution with a pH of 5.6 and a concentration of 19.52 g / L; (2) The activators are N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), both with a concentration of 40 g / L and a volume ratio of 1:1.

[0036] The specific process for obtaining cell-adherent surfaces is as follows: Prepare a 10 mM Tris-HCl buffer solution with a pH of 8.5, and dissolve dopamine in this solution to a final concentration of 2 g·L⁻¹. -1The concentration of the solution was determined. 5 ml of this solution was added to each well of a cell culture plate and incubated at 37°C and 70 rpm for 3 hours. After incubation, the solution was discarded, and the plate was ultrasonically washed with 5 ml of pure water for 5 minutes to remove polydopamine particles from the surface. Next, 5 ml of 3% (w / v) carboxymethyl chitosan (CMC) solution was added to each well and incubated overnight at 37°C with shaking. After thorough rinsing with distilled water and drying, the plate was sterilized with UV light to prepare the PDA-CMC-coated culture plate. A 1 mmol / L peptide solution was prepared for later use. NHS and EDC were dissolved in MES buffer, filtered and sterilized, and then added to the PDA-CMC-coated culture plate. The plate was reacted at room temperature for 40 min. After removing the activation solution, the peptide solution was added, the plate was sealed, and incubated overnight at 4°C. The plate was washed three times with DPBS to obtain the peptide-modified PDA-CMC functionalized surface.

[0037] Example 4 This embodiment provides a coating kit for promoting cell adhesion.

[0038] Cell adhesion reagent, gelatin-sodium alginate composite hydrogel precursor solution, calcium ion crosslinking solution and peptide activation grafting reagent were prepared separately.

[0039] The synthesized polypeptide was dissolved in phosphate buffer to prepare a solution with a polypeptide concentration of 1~1.2 mmol / L, preferably 1 mmol / L. The composite hydrogel precursor solution is an equal volume mixture of sodium alginate and gelatin, wherein the mass concentration of both sodium alginate and gelatin is 2-2.5% (w / v), preferably 2% (w / v). Calcium chloride aqueous solution: Dissolve calcium chloride in water to prepare a calcium chloride aqueous solution of 100~110mmol / L, preferably 100mmol / L; then filter aseptically through a 0.22 μm filter membrane; The peptide-activating grafting reagent comprises a mixture of the following components: (1) The buffer solution was a morpholine ethanesulfonic acid (MES) buffer solution with a pH of 5.6 and a concentration of 19.52 g / L; (2) The activators are N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), both with a concentration of 40 g / L and a volume ratio of 1:1.

[0040] The specific process for obtaining cell-adherent surfaces is as follows: Prepare an SA / Gel precursor solution by mixing equal volumes of 2% (w / v) sodium alginate solution (0.2 g in 10 mL water, stirred at 35°C for 30 min) and 2% gelatin solution (0.2 g in 10 mL water, stirred at 60°C for 30 min), and then heating and stirring for another 30 min. Coat the precursor solution onto the surface of a cell culture plate, sterilize and dry under UV light, then hydrate and crosslink with 100 mmol / L CaCl2 solution. Wash three times with DPBS to obtain a crosslinked SA / Gel surface. Prepare a 1 mmol / L peptide solution for later use. Dissolve NHS and EDC in MES buffer, filter and sterilize, then add to the SA / Gel surface and react at room temperature for 40 min. Remove the activation solution, add the peptide solution, seal the plate, and react overnight at 4°C. Wash three times with DPBS to obtain the SA / Gel-peptide functionalized surface.

[0041] Example 5 This embodiment provides a cell culture container.

[0042] The cell culture container provided in this embodiment has an inner wall for cell adhesion, and the inner wall is coated using the coating kit described in Example 3. The specific process is as follows: The polypeptide sequence synthesized in Example 1 was prepared into a 1 mmol / L Duchenne phosphate buffer (DPBS) solution for later use. Subsequently, a 10 mM Tris-HCl buffer solution with a pH of 8.5 was prepared, and dopamine was dissolved in this solution to a final concentration of 2 g / L. -1 The concentration was determined. This solution was then dispensed into 6-well cell culture plates, 5 ml per well, and incubated at 37°C and 70 rpm for 3 hours. After incubation, the solution was discarded, and the plates were ultrasonically washed with 5 ml of pure water for 5 minutes to remove polydopamine particles from the surface. Next, 5 ml of 3% (w / v) carboxymethyl chitosan (CMC) solution was added to each well, and the plates were incubated overnight at 37°C with shaking. After thorough rinsing with distilled water and drying, the plates were sterilized with ultraviolet light to obtain PDA-CMC-coated culture plates. Furthermore, a 0.1 M MES buffer at pH 5.6 was prepared, in which NHS and EDC were dissolved in a 1:1 ratio. The buffer was sterilely filtered through a 0.22 μm filter, and 3 ml of the activation solution was added to each well of a 6-well culture plate. After activation for 40 minutes, the peptide solution was added, and the plates were incubated overnight at 4°C to finally obtain the peptide-modified PDA-CMC culture surface.

[0043] Cell culture containers generally include: culture flasks (available in various sizes based on bottom area), culture dishes (available in various sizes based on bottom area), and culture plates (e.g., 6-well, 12-well, 24-well, and 96-well plates). Generally, cell expansion is carried out using culture flasks.

[0044] Example 6 Molecular docking and molecular dynamics simulations verified that the peptide has the ability to promote cell adhesion.

[0045] Three-dimensional structures of 40,560 candidate peptides were constructed in the "Peptide docking" module of Schrödinger software, and molecular docking cassettes were set with x, y, and z coordinates of 19.007 Å, 43.544 Å, and 44.490 Å, respectively. The dimensions of the docking cassettes were set to 30.0 Å for length, width, and height. Through two rounds of molecular docking experiments, peptide sequences with high affinity for αVβ5 integrin were screened (as shown in Table 1).

[0046] Table 1. Scoring results of candidate peptides after molecular docking screening.

[0047]

[0048] The selected optimal ligand-receptor complex structure was used for subsequent molecular dynamics simulations performed in NAMD software for 300 nanoseconds to obtain a stable complex structure and explore the binding affinity of the candidate peptide to αVβ5 integrin under alkaline conditions. To assess the equilibrium state of the simulation, the root mean square deviation (RMSD) of the complex backbone atoms relative to their initial structure was monitored; equilibrium was typically reached around 140 nanoseconds. Figure 3 ).

[0049] Finally, the MM / GBSA scores of the equilibrium trajectories were calculated using the MolAICal software package. The highest binding energy was found between peptide 4 and the integrin complex, reaching -46.5031 ± 0.201 kcal·mol⁻¹. -1 (Table 2). Simultaneously, the energy of single amino acids in the peptides of the complex was analyzed, and detailed energy analysis results were shown in... Figures 4-9 This provides a precise energy view of the interaction between the peptide and integrin αVβ5.

[0050] Table 2. Calculated binding free energies of integrin αVβ5 with six candidate peptides and VN peptide over a simulated duration of 300 ns (unit: kcal·mol) -1 ).

[0051]

[0052] Example 7 In vitro, the peptide was verified to have the ability to promote cell adhesion.

[0053] First, the synthesized peptide sequence was prepared into a 1 mmol / L phosphate-buffered saline (DPBS) solution for later use. Then, a 10 mmol / L Tris-HCl buffer solution with a pH of 8.5 was prepared, and dopamine was dissolved in this solution to a final concentration of 2 g / L. -1 The concentration was determined. This solution was then dispensed into 6-well cell culture plates, 5 ml per well, and incubated at 37°C and 70 rpm for 3 hours. After incubation, the solution was discarded, and the plates were ultrasonically washed with 5 ml of pure water for 5 minutes to remove polydopamine particles from the surface. Next, 5 ml of 3% (w / v) carboxymethyl chitosan (CMC) solution was added to each well, and the plates were incubated overnight at 37°C with shaking. After thorough rinsing with distilled water and drying, the plates were sterilized with UV light to obtain PDA-CMC-coated culture plates. Furthermore, a 0.1 M MES buffer at pH 5.6 was prepared, in which NHS and EDC were dissolved in a 1:1 ratio. The buffer was sterilely filtered through a 0.22 μm filter, and 3 ml of the activation solution was added to each well of a 6-well culture plate. After activation for 40 minutes, the peptide solution was added, and the plates were incubated overnight at 4°C to obtain the final peptide-modified PDA-CMC culture surface.

[0054] Next, Matrigel was diluted 1:230 with DMEM / F12 on ice, and 1 ml was added to each well of a 6-well cell culture plate. The plates were incubated at 37°C for at least 3 hours. H1 hESCs and hNF-C1 hiPSCs cells were then cultured at 25,000 / cm². 2 The cells were seeded onto Matrigel surfaces at a density that was adjusted daily. After 3 to 4 days, the cells were treated with 0.5 mmol / L EDTA at 37°C for 4 to 5 minutes, and then inoculated at a density of 25,000 / cm². 2 The density of cells was passaged to new peptide-modified surfaces. After four days of cell culture, cell number and adhesion ability were assessed using a CCK-8 assay to screen for peptide-modified surfaces that support cell adhesion. Figure 10 The experimental results showed that peptides 4 and 5 significantly enhanced the adhesion and proliferation abilities of H1 and HNF-C1 cells, and were the two peptides with the best performance in this experiment. They showed extremely significant differences compared with other groups, and their effects were comparable to those of VN peptide.

[0055] Figure 11The adhesion and growth performance of H1 hESCs and hNF-C1 hiPSCs on peptide-modified surfaces was assessed using CCK-8 assays after inoculation with single cells and clonal clusters. The results showed that all three surfaces effectively supported cell adhesion under both treatment methods, with the surface modified with peptide number 4 (VT) exhibiting superior performance in promoting hiPSC adhesion and growth. p <0.05), and its cell count on day four was comparable to that of the Matrigel group.

[0056] Through the above steps, this invention significantly improves research efficiency, greatly reduces research and development costs, and accelerates the R&D cycle. It provides a novel, efficient, and economical peptide-modified surface for the application of hiPSCs, which helps to accelerate the widespread application of peptide display synthesis substrate surfaces.

[0057] For any points not covered above, existing technologies shall apply.

[0058] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide supporting the in vitro culture of human pluripotent stem cells, characterized in that, The polypeptide has an amino acid sequence as shown in SEQ ID NO.1; or an amino acid sequence as shown in SEQ ID NO.1 that has been modified, substituted, deleted or added with one or more amino acids, and has at least 90% homology with the amino acid sequence shown in SEQ ID NO.

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide has an amino acid sequence as shown in SEQ ID NO. 2; or an amino acid sequence as shown in SEQ ID NO. 2 that has been modified, substituted, deleted, or added with one or more amino acids, and has at least 90% homology with the amino acid sequence shown in SEQ ID NO.

2.

3. A cell adhesion reagent, characterized in that, The cell adhesion reagent contains the polypeptide as described in claim 1 or 2.

4. The cell adhesion reagent according to claim 2, characterized in that, The cell adhesion reagent is a mixture of the polypeptide and DuPont phosphate buffer.

5. A coating kit for promoting cell adhesion, characterized in that, The coating kit comprises four containers, each containing the cell adhesion reagent, dopamine solution, carboxymethyl chitosan aqueous solution, and polypeptide activation grafting reagent as described in claim 3 or 4. Alternatively, it may contain the cell adhesion reagent, gelatin-sodium alginate composite hydrogel precursor solution, calcium chloride aqueous solution, and polypeptide activation grafting reagent as described in claim 3 or 4.

6. The coating reagent kit according to claim 5, characterized in that, The concentration of the peptide in the cell adhesion reagent is 1-1.2 mmol / L; the dopamine solution is a mixture of dopamine hydrochloride and Tris-HCl buffer adjusted to pH 8.5, with a concentration of 2-2.5 g·L⁻¹. -1 The carboxymethyl chitosan has a mass concentration of 2.5% to 3.5%; the composite hydrogel precursor solution is an equal volume mixture of sodium alginate and gelatin, wherein the mass concentrations of sodium alginate and gelatin are both 2% to 2.5%; and the calcium chloride aqueous solution has a concentration of 100 to 110 mmol / L.

7. The coating reagent kit according to claim 6, characterized in that, The peptide activation grafting reagents include morpholine ethanesulfonic acid buffer, N-hydroxysuccinimide solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution.

8. A cell culture container, characterized in that, The cell culture container has an inner wall for cell adhesion, the inner wall being coated using a coating kit as described in any one of claims 6-7.

9. The use of the polypeptide according to claim 1 or 2, the cell adhesion reagent according to any one of claims 3 to 4, the coating kit according to any one of claims 6 to 7, and the cell culture vessel according to claim 8 in culturing human pluripotent stem cells.

10. The application according to claim 9, characterized in that, The cells mentioned include H1 hESCs and hNF-C1hiPSCs.

Images