Application of polypeptide DGEA or derivative thereof in promoting differentiation of pluripotent stem cells into mesenchymal stem cells

Abstract

The invention belongs to the technical field of stem cell development and differentiation, and discloses application of polypeptide DGEA or a derivative thereof in promoting pluripotent stem cells to be differentiated into mesenchymal stem cells. The invention specifically discloses application of the polypeptide and the derivative thereof in promoting differentiation of pluripotent stem cells into pluripotent stem cells or preparing products for promoting differentiation of pluripotent stem cells into pluripotent stem cells. The amino acid sequence of the polypeptide is DGEA. The invention discloses the application of DGEA in promoting differentiation of pluripotent stem cells (embryonic stem cells, embryonic germ cells or induced pluripotent stem cells) into pluripotent stem cells (mesenchymal stem cells) for the first time, and the effect of DGEA is verified through corresponding experiments. The DGEA can obviously improve the efficiency of differentiating the embryonic stem cells, the embryonic germ cells or the induced pluripotent stem cells into the mesenchymal stem cells, and the obtained mesenchymal stem cells are high in safety and high in differentiation capacity.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell development and differentiation, and specifically relates to the application of polypeptide DGEA or its derivatives in promoting the differentiation of pluripotent stem cells into mesenchymal stem cells. Background Art

[0002] Stem cells are broadly classified into pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs). Compared with other types of cells, stem cells have unique properties, such as the potential to differentiate into various lineages and the ability to regenerate damaged tissues. Pluripotency is defined as the intrinsic ability of a cell to differentiate into most or all specialized cell types in the human body. Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from various tissues or organs and exhibit stemness, proliferation, and pluripotency. There are more than 600 clinical trials applying mesenchymal stem cells to treat various diseases, including osteoarthritis, stroke, graft-versus-host disease (GvHD), myocardial infarction, and diabetes. Stem cells naturally reside in a special environment, called a niche, which contains biochemical and biophysical factors that regulate differentiation, fate, and self-renewal capacity. These niches can also maintain the quiescent state of stem cells, which can be further applied in disease modeling and drug screening. In addition to cell-cell interactions, extracellular matrix (ECM) proteins are also one of the key components of the stem cell niche. These proteins not only serve as structural components, but cells bind to ECM proteins through cell adhesion receptors such as integrins, activating several signaling pathways that determine cell fate, morphological changes, proliferation, and differentiation. Typically, xenogeneic or allogeneic ECM molecules are immobilized on tissue culture polystyrene (TCP) flasks and several types of scaffolds, hydrogels, implants, or nanofibers for the adhesion, proliferation, and differentiation of adult and pluripotent stem cells, as well as cells from other tissues.

[0003] Although xenogeneic or allogeneic ECM proteins, such as native or recombinant laminin molecules, are valuable for culturing certain cells, ECM proteins should be produced from human cell lines or E. coli, which makes it difficult to chemically define the molecules mixed with ECM proteins after production. Structural proteins are the main components of the matrix, and the other parts are composed of growth factors, transcription factors, and cytokines. However, the application of some of them in stem cell culture is hampered by short half-life, immune response, contamination with other foreign proteins and tumorigenicity, as well as weak specificity and unexpected toxicity.

[0004] Current ECM protein production methods cannot avoid the risk of contamination with immunogens and pathogens. In addition, ECM proteins have batch-to-batch variability. The development of synthetic ECM protein-derived peptides that mimic the biological and biochemical functions of natural ECM will be beneficial for academic and clinical applications. Peptides derived or stimulated by specific ECM proteins can act as agonists for each ECM protein receptor. Considering that most ECM proteins affect cell adhesion through integrin receptors, many peptides that bind to specific integrin receptors have been developed.

[0005] In recent years, the design of artificial niches based on peptide mimetics to maintain the undifferentiated state of stem cells and promote the proliferation and differentiation of stem cells has become a research hotspot. The application of peptides in biology, medicine, chemistry and other fields has attracted increasing attention. Due to the presence of undefined and xenogeneic components, the use of feeder layers and Matrigel coatings for iPSC expansion poses many challenges, hindering the clinical application of cultured stem cells. Matrigel contains approximately 2,000 different proteins and more than 14,000 unique peptides. Summary of the invention

[0006] The first aspect of the present invention aims to provide the use of polypeptides and their derivatives in promoting the differentiation of pluripotent stem cells into multipotent stem cells or in preparing products that promote the differentiation of pluripotent stem cells into multipotent stem cells.

[0007] The second aspect of the present invention aims to provide a cell culture plate.

[0008] The third aspect of the present invention aims to provide a culture medium.

[0009] The fourth aspect of the present invention aims to provide use of the cell culture plate of the second aspect of the present invention or the culture medium of the third aspect of the present invention in inducing pluripotent stem cells to differentiate into multipotent stem cells.

[0010] The fifth aspect of the present invention aims to provide a method for promoting the differentiation of pluripotent stem cells into multipotent stem cells.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of a polypeptide and its derivatives in promoting the differentiation of pluripotent stem cells into multipotent stem cells or in preparing a product for promoting the differentiation of pluripotent stem cells into multipotent stem cells, wherein the amino acid sequence of the polypeptide is DGEA.

[0013] In some embodiments of the present invention, the pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells.

[0014] In some embodiments of the present invention, the pluripotent stem cells are embryonic stem cells, embryonic germ cells or induced pluripotent stem cells.

[0015] In some embodiments of the present invention, the pluripotent stem cells include adult stem cells.

[0016] In some embodiments of the present invention, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, bone stem cells and hematopoietic stem cells; preferably, they are mesenchymal stem cells.

[0017] In some embodiments of the present invention, the derivative includes a polypeptide containing DGEA and a protein containing DGEA, or a combination of the derivatives.

[0018] In some embodiments of the present invention, the termini of the polypeptide may be modified or unmodified.

[0019] In some embodiments of the present invention, the modification includes amino acid modification (such as cysteine, glutamic acid, alanine, glutamine, aspartic acid, lysine, threonine), glycosylation modification (such as O-β-D-glucose modification), fatty acid modification (such as palmitoylation modification), phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof.

[0020] In some embodiments of the present invention, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

[0021] The second aspect of the present invention provides a cell culture plate, the surface of which is coupled, coated or adsorbed with a polypeptide or a derivative thereof, wherein the amino acid sequence of the polypeptide is DGEA.

[0022] In some embodiments of the invention, the coupling comprises covalent coupling.

[0023] In some embodiments of the present invention, the covalent coupling comprises a reaction between an amino group and a carboxyl group, a reaction between an aldehyde group and an amino group, or a reaction between a thiol group and a maleimide group.

[0024] In some embodiments of the present invention, the cell culture plate includes a currently common cell culture plate, or a modified (such as carboxyl-modified) cell culture plate.

[0025] In some embodiments of the present invention, the derivatives include polypeptides containing DGEA and proteins containing DGEA.

[0026] In some embodiments of the present invention, the termini of the polypeptide may be modified or unmodified.

[0027] In some embodiments of the present invention, the modification includes amino acid modification (such as cysteine, glutamic acid, alanine, glutamine, aspartic acid, lysine, threonine), glycosylation modification (such as O-β-D-glucose modification), fatty acid modification (such as palmitoylation modification), phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof.

[0028] In some embodiments of the present invention, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

[0029] The third aspect of the present invention provides a culture medium containing a polypeptide or a derivative thereof, wherein the amino acid sequence of the polypeptide is DGEA.

[0030] In some embodiments of the present invention, the derivative includes a polypeptide containing DGEA and a protein containing DGEA, or a combination of the derivatives.

[0031] In some embodiments of the present invention, the termini of the polypeptide may be modified or unmodified.

[0032] In some embodiments of the present invention, the modification includes amino acid modification (such as cysteine, glutamic acid, alanine, glutamine, aspartic acid, lysine, threonine), glycosylation modification (such as O-β-D-glucose modification), fatty acid modification (such as palmitoylation modification), phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof.

[0033] In some embodiments of the present invention, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

[0034] In some embodiments of the present invention, the culture medium contains one or more substances that can induce pluripotent stem cells to differentiate into multipotent stem cells or are used for cell culture.

[0035] In some embodiments of the present invention, the substance includes at least one of KOSR, Glutamax, non-essential amino acids (NEAA), ALK5 inhibitors (such as SB431542), bFGF, EGF, FGF9, and ginsenoside monomers.

[0036] In some embodiments of the present invention, the culture medium is any one of RMPI 1640, DMEM / F12 medium (ie, DMEM-Ham's F-12 basal medium), advance RMPI 1640, DMEM, or a combination thereof.

[0037] A fourth aspect of the present invention provides use of the cell culture plate of the second aspect of the present invention or the culture medium of the third aspect of the present invention in inducing pluripotent stem cells to differentiate into multipotent stem cells.

[0038] In some embodiments of the present invention, the pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells.

[0039] In some embodiments of the present invention, the pluripotent stem cells are embryonic stem cells, embryonic germ cells or induced pluripotent stem cells.

[0040] In some embodiments of the present invention, the pluripotent stem cells include adult stem cells.

[0041] In some embodiments of the present invention, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, bone stem cells and hematopoietic stem cells; preferably, they are mesenchymal stem cells.

[0042] The fifth aspect of the present invention provides a method for promoting the differentiation of pluripotent stem cells into multipotent stem cells, comprising the step of using the cell culture plate of the second aspect of the present invention as a container to culture pluripotent stem cells; or, comprising the step of using the culture medium of the third aspect of the present invention to culture pluripotent stem cells.

[0043] In some embodiments of the present invention, the pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells.

[0044] In some embodiments of the present invention, the pluripotent stem cells are embryonic stem cells, embryonic germ cells or induced pluripotent stem cells.

[0045] In some embodiments of the present invention, the pluripotent stem cells include adult stem cells.

[0046] In some embodiments of the present invention, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, skeletal stem cells and hematopoietic stem cells; preferably mesenchymal stem cells,

[0047] In some embodiments of the present invention, the derivative includes a polypeptide containing DGEA and a protein containing DGEA, or a combination of the derivatives.

[0048] In some embodiments of the present invention, the termini of the polypeptide may be modified or unmodified.

[0049] In some embodiments of the present invention, the modification includes amino acid modification (such as cysteine, glutamic acid, alanine, glutamine, aspartic acid, lysine, threonine), glycosylation modification (such as O-β-D-glucose modification), fatty acid modification (such as palmitoylation modification), phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof.

[0050] In some embodiments of the present invention, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

[0051] The beneficial effects of the present invention are:

[0052] Generally, the induced differentiation of pluripotent stem cells uses a gelatin-coated culture dish, but gelatin has the disadvantages of high production cost, high immunogenicity, high probability of nonspecific binding, and easy contamination. The polypeptide provided by the present invention has the advantages of high purity, simple synthesis, large surface area, low production cost, high specificity, and low immunogenicity, making it more suitable for the differentiation of pluripotent stem cells. The present invention discloses for the first time the application of DGEA in promoting the differentiation of pluripotent stem cells (embryonic stem cells, embryonic germ cells or induced pluripotent stem cells) into pluripotent stem cells (mesenchymal stem cells), and verifies its effect through corresponding experiments. DGEA can significantly improve the efficiency of embryonic stem cells, embryonic germ cells or induced pluripotent stem cells to differentiate into mesenchymal stem cells, and the obtained mesenchymal stem cells are highly safe and have strong differentiation ability.

[0053] Specifically, the present invention first couples the polypeptide to a PS cell culture plate modified with a carboxyl group by a standard covalent coupling method sulfo-NHS / EDC method. Then, the culture plate coupled with the peptide is used for the induced differentiation of pluripotent stem cells to obtain differentiated and mature high-purity mesenchymal stem cells. The iMSCs differentiated using the culture container coupled with the peptide have greater clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The expression levels of OCT4, Nanog, CD73, CD90, CD105 and CD44 genes of iMSCs obtained on Day 12 by differentiating H1 ES cells and C27 iPS cells using different methods. In the figure, Laminin521, Gelatin and DGEA correspond to the method of Example 3, the method of Comparative Example 1 and the method of Comparative Example 2, respectively, and * represents P < 0.05.

[0055] Figure 2The iMSC images are obtained by differentiating H1 ES cells and C27 iPS cells on Day 12 using different methods. In the figure, Laminin521, Gelatin and DGEA correspond to the method of Example 3, the method of Comparative Example 1, and the method of Comparative Example 2, respectively. The blank control group is a blank control group in which H1 ES cells and C27 iPS cells were induced to differentiate using ordinary PS culture plates.

[0056] Figure 3 The following are pictures of mature iMSCs on Day 25 obtained from H1 ES cells and C27 iPS cells using the differentiation method of Example 3.

[0057] Figure 4 This is the flow cytometry result of mature iMSCs on Day 25 obtained by using the differentiation method of Example 3 for H1 ES cells. The detection markers are CD90, CD105, CD73 and negative indicators.

[0058] Figure 5 This is the flow cytometry result of mature iMSCs on Day 25 obtained by using the differentiation method of Example 3 for C27 iPS cells. The detection markers are CD90, CD105, CD73 and negative indicators.

[0059] Figure 6 The diagram is a three-lineage differentiation experiment effect diagram of mature iMSCs on Day 25 obtained from H1 ES cells using the differentiation method of Example 3, which are adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation from left to right.

[0060] Figure 7 The diagram shows the effect of three-lineage differentiation experiment of mature iMSCs on Day 25 obtained by using the differentiation method of Example 3 for C27 iPS cells, which are adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation from left to right.

[0061] Figure 8 To test the effects of different concentrations of DGEA and its derivatives, the combination of derived peptides on the gene expression of differentiated mesenchymal stem cells. The figure shows the expression levels of CD90, CD73, CD105, and CD44 genes of iMSC differentiated to Day 12, which were normalized using Laminin521. The blank control group was a carboxylate-based differentiated mesenchymal stem cell without the addition of peptides. In the figure, * represents P < 0.05 DETAILED DESCRIPTION

[0062] The present invention is further described in detail below through specific examples.

[0063] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0064] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0065] The peptides (DGEA and its derivatives) were synthesized at GenScript Biotech Co., Ltd.

[0066] DGEA: Aspartic acid-Glycine-Glutamic acid-Alanine (Asp-Gly-GIu-Ala).

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1 Cultivation of embryonic stem cells and induced pluripotent stem cells

[0069] H1 embryonic stem cells (denoted as H1 ES cells, from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences) and induced pluripotent stem cells (denoted as C27 iPS cells, from Guangzhou Future Homo Sapiens Regenerative Medicine Institute Guangzhou Co., Ltd.) were cultured in Matrigel (BD Biosciences)-coated culture dishes using mTeSR1 medium (StemCell Technologies). The specific operation method is as follows:

[0070] Add 1 mL of Matrigel to a 6-well plate, making sure that Matrigel evenly covers the bottom of the plate. Keep the coated plate in a cell culture incubator at 37°C for 1 hour. Preheat the mTeSR1 medium used for cell growth before use. Before cell seeding, remove Matrigel and immediately add 2 mL of mTeSR1 medium to the culture plate. Inoculate 600,000 cells of the cell suspension into the culture plates. After 24 hours, replace the mTeSR1 medium and continue culturing. Change the medium every day until the cells reach 80% fusion and then passage to obtain H1 ES cells and C27 iPS cells.

[0071] Example 2 Preparation of cell culture plates with covalently coupled polypeptides

[0072] The peptide was coupled to a modified 24-well PS plate with carboxyl groups using a standard EDC / Sulfo-NHS method (24-well carboxyl culture plate purchased from Qingdao Jindian Biochemical Equipment Co., Ltd., model: WP24-5CCCSH).

[0073] Preparation of reagents:

[0074] EDC (sigma, E1769) and Sulfo-NHS (MCE, HY-W002213) were equilibrated to room temperature before opening the vials.

[0075] Preparation of 20 mM EDC stock solution: Add 76.68 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 350.64 mg NaCl (0.3 M) into 20 mL 0.1 M MES Buffer (ECOTOP, R21357), pH 6, filter sterilize, and use immediately after preparation.

[0076] Preparation of 30mM sulfo-NHS stock solution: Add 130.278mg N-hydroxysulfosuccinimide (sulfo-NHS) and 350.64mg NaCl (0.3M) into 20mL 0.1M MES Buffer, pH 6, filter sterilize, and prepare before use.

[0077] 10.0mM EDC / 15.0mM NHS mixture: Mix 20mM EDC and 30mM sulfo-NHS stock solutions in a 1:1 volume ratio and prepare for immediate use.

[0078] 1M, pH=8.5 Ethanolamine preparation (50 mL): Dilute 3.0 mL of ethanolamine (acmec, E83640) to 40 mL with ddH2O, adjust pH to 8.5 with HCl, add ddH2O to 50 mL, and filter sterilize.

[0079] The specific steps for preparing the cell culture plate for covalently coupled peptides are as follows:

[0080] (1) A 24-well carboxyl culture plate was thoroughly washed with distilled water, and then 0.4 mL of a mixture of 10 mM EDC and 15 mM sulfo-NHS was added. The plate was incubated on a clean bench for 2 hours (room temperature), and then washed with water 1 to 2 times to obtain an activated culture plate.

[0081] (2) Add 0.3 mL of 1 mM peptide (DGEA (SEQ ID NO: 1), the peptide is prepared using PBS) to the activated culture plate, seal it with a sealing film, incubate it in a refrigerator at 4°C for 24 hours, and then wash it with water 1 to 2 times.

[0082] (3) The surface of the culture plate was inactivated in 0.3 mL of ethanolamine (1 M, pH = 8.5) for 1 hour (room temperature), and then washed with water 1 to 2 times to obtain a cell culture plate with covalently coupled polypeptides.

[0083] All cell culture plates for covalently coupled peptides were disinfected with 75% ethanol for 30 minutes before cell culture and then washed 8 to 10 times with sterile PBS before being used for cell culture.

[0084] Example 3

[0085] A method for promoting differentiation of pluripotent stem cells into mesenchymal stem cells, comprising the following steps:

[0086] (1) Day-1: 6-well plates were coated with 5 μg / mL Laminin521 (BioLamina) and incubated at 37°C for 30 min, then Laminin521 was discarded and iMSC differentiation medium (composed of DMEM-Ham's F-12 basal medium supplemented with 20% knockout serum replacement (KOSR), 1 mM l-glutamine and 10 mM non-essential amino acids (all from Invitrogen, and supplemented with 10 μM SB431542 (SB) (Sigma-Aldrich)) was added. When the confluence of H1 ES cells and C27 iPS cells reached 80% (Example 1), they were digested with 0.5 mM EDTA (Invitrogen, 15575020) for 10 minutes, centrifuged at 200 g for 5 minutes, and the cells were counted. H1 ES cells and C27 iPS cells were seeded into the treated 6-well plates (300,000 cells / well), each well was seeded, shaken, placed in a 37°C 5% CO2 incubator, cultured, and the medium was changed every other day.

[0087] (2) Day 4: Passaging, 6-well plates were coated with 5 μg / mL Laminin521 (BioLamina), incubated at 37°C for 30 minutes, then Laminin521 was discarded and iMSC differentiation medium (composed of DMEM-Ham's F-12 basal medium, supplemented with 20% knockout serum replacement (KOSR), 1 mM l-glutamine and 10 mM non-essential amino acids, and supplemented with 10 μM SB431542) was added. The cultured H1 ES cells and C27 iPS cells in step (1) were digested with TrypLE (Gibco, A1285901) for 5 minutes, and 2 volumes of differentiation medium were added to terminate the digestion, centrifuged at 200 g for 5 minutes, and the cells were counted. H1 ES cells and C27 iPS cells were inoculated into the treated 6-well plates (400,000 cells / well), shaken, placed in a 37°C 5% CO2 incubator, cultured, and the medium was changed every other day.

[0088] (3) Day 9: Passage, add the culture medium for differentiation of iMSC (composed of DMEM-Ham's F-12 basal medium, supplemented with 20% knockout serum replacement (KOSR), 1mM l-glutamine and 10mM non-essential amino acids, and supplemented with 10μM SB431542) to the 24-well plate covalently coupled with DGEA peptide (i.e., the cell culture plate covalently coupled with polypeptide obtained in Example 2). The H1 ES cells and C27 iPS cells cultured in step (2) were digested with TrypLE for 5 minutes, centrifuged at 200g for 5 minutes, and the cells were counted. H1 ES cells and C27 iPS cells were inoculated into 24-well plates (150,000 cells / well), 3 wells were seeded, shaken, and placed in a 37°C 5% CO2 incubator for culture, and the medium was changed every other day.

[0089] (4) Day 12: Collect cells, half of the cells are used for qPCR verification, and the other half of the cells are further passaged.

[0090] The passage process is as follows: the 24-well plate to which the DGEA peptide has been covalently coupled (i.e., the cell culture plate to which the polypeptide has been covalently coupled obtained in Example 2) is added with a culture medium for differentiation of iMSCs (composed of DMEM-Ham's F-12 basal culture medium, supplemented with 20% knockout serum replacement (KOSR), 1 mM l-glutamine and 10 mM non-essential amino acids, and supplemented with 10 μM SB431542). The H1 ES cells and C27 iPS cells treated in step (3) are digested with TrypLE for 5 minutes, centrifuged at 200 g for 5 minutes, and the cells are counted. The H1 ES cells and C27 iPS cells are seeded into 24-well plates (150,000 cells / well), 3 wells are seeded, shaken, and cultured in a 37°C 5% CO2 incubator. The medium is changed every other day, and passage is performed every 4 days (the passage process is the same as above).

[0091] (5) Day 24: Collect cells and obtain mesenchymal stem cells.

[0092] Comparative Example 1

[0093] A method for differentiating pluripotent stem cells into mesenchymal stem cells, compared with Example 3, the only difference is that the 24-well plate in steps (3) and (4) is replaced with a 24-well plate coated with 5 μg / mL Laminin521 (the coating process is the same as steps (1) to (2) of Example 3).

[0094] Comparative Example 2

[0095] A method for differentiating pluripotent stem cells into mesenchymal stem cells, compared with Example 3, the only difference is that the 24-well plate in steps (3) and (4) is replaced by a 24-well plate coated with 0.1% Gelatin (the coating process is the same as steps (1) to (2) of Example 3).

[0096] Effect Example

[0097] 1.qPCR detection

[0098] qPCR detection was performed on the cells differentiated by the methods of Example 3 and Comparative Examples 1-2 (cells differentiated for 12 days in step (4)) to analyze the differentiation of each cell. The specific process is as follows:

[0099] (1) Extraction of RNA

[0100] 1) Cell lysis: Use 1000 μL RNA isolater (Novozyme, R401-01) to lyse the cultured cells, mix thoroughly with a 1000 μL pipette, and lyse at room temperature for 5 minutes.

[0101] 2) Extract RNA: Take the sample out of the refrigerator, melt it in an ice bath, add 1 / 5 of the RNA isolater volume of chloroform, shake vigorously to mix, and then let it stand at -20℃ for 5 minutes. Centrifuge at 12000g, 4℃, for 15 minutes.

[0102] 3) Precipitate RNA: Transfer the supernatant to a new centrifuge tube, add isopropanol of equal volume to the supernatant, gently invert 6 to 8 times to mix thoroughly (do not use an oscillator to mix), let stand at -20°C for 10 minutes. Centrifuge at 12000g, 4°C, for 10 minutes.

[0103] 4) Wash RNA precipitate: Add 1 mL of 75% ethanol to the precipitate, gently invert 3 times, let stand for 2 minutes, then centrifuge at 12000g, 4°C for 5 minutes. Aspirate and discard the supernatant, and dry on a clean bench (the precipitate turns from white to transparent).

[0104] 5) Dissolve RNA: Add 20-50 μL DEPC water to the precipitate and mix gently using a 200 μL pipette.

[0105] 6) Measure the concentration. Take 1 μL of sample and measure the concentration using an ultra-micro spectrophotometer (NO-ONE).

[0106] (2) RNA reverse transcription (Novozyme, R212-02)

[0107] 1) Genomic DNA removal

[0108] Prepare the mixed solution shown in Table 1 in an RNase-free centrifuge tube, mix gently by pipetting, and incubate at 42°C for 2 minutes.

[0109] Table 1 Mixed liquid preparation system

[0110]

[0111] 2) Prepare the first-strand cDNA synthesis reaction solution

[0112] Prepare the mixture shown in Table 2 in an RNase-free centrifuge tube and mix gently by pipetting.

[0113] Table 2 Mixed liquid preparation system

[0114]

[0115] 3) First-strand cDNA synthesis reaction

[0116] The first-chain cDNA synthesis reaction solution in 2) was subjected to the first-chain cDNA synthesis reaction under the following reaction conditions: 50°C (if the template has a complex secondary structure or a high GC region, the reaction temperature can be increased to 55°C to help increase the yield) for 15 minutes and 85°C for 2 minutes.

[0117] (3) Real-time quantitative PCR

[0118] Prepare the reaction system (10 μL system (384-well plate)): SYBR: 5 μL (Novozyme, Q121), upstream primer: 0.25 μL, downstream primer: 0.25 μL, H2O (DEPC) 3.5 μL and cDNA 1 μL, wherein the upstream and downstream primers are shown in Table 3. Detection on the machine (Roche, Lightcycler 480II), the qPCR reaction procedure is as follows:

[0119] 1. 95.0℃for0:20

[0120] 2. 95.0℃for0:10

[0121] 3. 60℃for0:20

[0122] 4. 70℃for0:10

[0123] +Plate Read

[0124] 5. GO TO2,39more times

[0125] 6. 95.0℃for0:10

[0126] 7.Melt Curve75.0to94.0℃,increment0.5℃,

[0127] For 0.05+Plate Read

[0128] Table 3 QPCR primer sequence list (Synthesized by Shanghai Jierui Bioengineering Co., Ltd.)

[0129]

[0130]

[0131] qPCR results Figure 1 As shown,

[0132] like Figure 1 The gene expression levels of CD90, CD73, CD105, CD44, Oct4, and Nanog in iMSCs progenitor cells on Day 12 induced by differentiation of H1 ES cells and C27 iPS cells are shown. In the experiment of induced differentiation of H1 ES cells, the expression levels of CD73, CD105, and CD44 in the DGEA group were higher than those in the Laminin521 and Gelatin groups, and the difference was statistically significant (p<0.05), and the expression levels of Oct4 and Nanog were lower than those in the Laminin521 and Gelatin groups; in the experiment of induced differentiation of C27 iPS cells, the expression levels of CD73 and CD105 in the DGEA group were higher than those in the Laminin521 and Gelatin groups, and the difference was statistically significant (p<0.05), and the expression levels of Oct4 and Nanog were lower than those in the Laminin521 and Gelatin groups.

[0133] 2. Microscopic examination

[0134] The iMSCs obtained by the methods of Example 3 and Comparative Examples 1-2 (i.e., cells differentiated for 12 days and 25 days) were examined under a microscope. Meanwhile, cells differentiated and cultured using ordinary PS culture plates were used as a blank control group.

[0135] The results are as follows Figure 2-3 shown.

[0136] Figure 2 On day 12, the cells differentiated by Laminin521 and Gelatin groups showed inconsistent cell morphology under the microscope, while the cells in the non-coated group could not attach and grow. Only the iMSC progenitor cells in the DGEA group showed consistent morphology and attached and grew in a spindle shape.

[0137] Figure 3 : On day 25, the mature iMSC cells induced by H1 and C27 were observed to be spindle-shaped and had consistent morphology under the microscope.

[0138] 3. Flow cytometry

[0139] The iMSCs (i.e., cells differentiated for 25 days) obtained by the methods of Example 1 and Comparative Examples 1-2 were subjected to flow cytometry detection. The specific detection process is as follows:

[0140] (1) Washing: Collect the cultured cells, discard the original culture medium, and wash the cells once with 1 mL of PBS.

[0141] (2) Digestion: Discard PBS, add 0.5 mL TrypLE / well, and digest at 37°C for 5-8 minutes.

[0142] (3) Terminate digestion: add 2 mL PBS + 10% FBS and centrifuge at 300 g for 5 min.

[0143] (4) Transfer to 1.5 mL EP tubes (three 1.5 mL EP tubes for each component): 1 mL DPBS / 1.5 mL EP tube, centrifuge at 300 g for 5 minutes.

[0144] (5) Discard the supernatant and resuspend in 100 μL DPBS + 1% FBS.

[0145] (6) Antibodies (BD Biosciences, 562245) were added to the cells in a dark environment, incubated at room temperature for 30-45 minutes in a dark environment, and centrifuged at 300 g for 5 minutes.

[0146] (7) Wash once with 0.5 mL of DPBS and centrifuge at 300 g for 5 minutes.

[0147] (8) Resuspend in 200-300 μL DPBS and analyze by flow cytometry.

[0148] The flow cytometry results are as follows Figure 4-5 shown.

[0149] Figure 4 : Day 25, H1_iMSCs-related MSCs markers were detected by flow cytometry, which met the minimum standard requirements of MSC defined by ISCT. The proportion of cells expressing CD90 was 99.0%, the proportion of cells expressing CD105 was 99.8%, the proportion of cells expressing CD73 was 99.2%, and the proportion of cells expressing negative mixed markers was 1.77%.

[0150] Figure 5 : Day 25, C27_iMSCs-related MSCs markers were detected by flow cytometry, which met the minimum standard requirements of MSC defined by ISCT. More than 99% of the cells (CD90 was 100%, CD105 was 99.9%, and CD73 was 99.9%) expressed MSC positive markers CD90, CD105, and CD73 on their cell surface, and the proportion of cells expressing negative mixed markers was 0.43%.

[0151] 4. Three-lineage differentiation

[0152] The iMSCs obtained by the method of Example 1 (i.e., cells differentiated for 25 days) were tested for tri-lineage differentiation (osteoblastic differentiation, adipogenic differentiation, and chondrogenic differentiation). The specific testing process is as follows:

[0153] Osteogenic differentiation:

[0154] Plating: 0.1% Gelatin was coated on a six-well plate for 0.5 h; 150,000 iMSC cells were seeded in each well, parallel wells were set up, and StemPro TM MSC SFM (Gibco, A1033201), culture.

[0155] Induction: Cell confluence 85% to 90%; change induction solution (formula see Table 4), 3 mL / well, change every 3 days, for 25 days. NC group used StemPro TM MSCs were cultured synchronously in SFM medium.

[0156] Staining: Wash the cells with DPBS, add 1 mL of tissue fixative (Boster, AR1068) to each well and fix at room temperature for 30 minutes, wash the cells with sterile water, add 1 mL of Alizarin Red stain (Oricell, ALIR-10001) to each well and stain for 30 minutes, discard the solution and wash the cells with sterile water, and observe the calcium nodules under a microscope.

[0157] Table 4 Osteogenic differentiation induction fluid formula

[0158]

[0159]

[0160] Adipose-induced differentiation:

[0161] Plating: 0.1% Gelatin coated six-well plates for 0.5 hours; 150,000 iMSC cells were seeded in each well, parallel wells were set, and StemPro TM MSCs were cultured in SFM (Gibco, A1033201).

[0162] Induction: When the confluence is 85% to 90%, change the induction solution (formula see Table 5), 3 mL / well, for 48 to 72 hours;

[0163] Maintenance: After 48 to 72 hours of induction, replace the maintenance solution (formula see Table 5) for 24 hours.

[0164] Alternation: Induction and maintenance were alternating for about 25 days; NC group used StemPro TM MSCs were cultured synchronously in SFM medium.

[0165] Staining: To observe lipid droplets, wash the cells with DPBS, add 1 mL of tissue fixative (Boster, AR1068) to each well and fix at room temperature for 30 minutes, wash the cells with sterile water, add 1 mL of Oil Red O stain (Sigma, 01391) to each well and stain for 30 minutes, discard the solution and wash the cells with sterile water, and observe lipid droplets under a microscope.

[0166] Table 5 Adipogenic differentiation maintenance and induction fluid formula

[0167]

[0168] Chondrogenic differentiation:

[0169] Plating: 12-well plates were coated with 0.1% Gelatin for 0.5 hours; 70,000 iMSC cells were seeded in each well, and parallel wells were set up. StemPro TM MSCs were cultured in SFM (Gibco, A1033201).

[0170] Induction: When the cell confluence reached 85% to 90%, iMSC was replaced with chondrogenic differentiation induction solution (see Table 6 for the formula, 10 ng / mL TGF-β3 was added when used), 2 mL / well, replaced every 3 days, for 25 days. TM Synchronously cultured in MSCSFM medium.

[0171] Staining: Wash the cells with DPBS, add 1 mL of tissue fixative (Boster, AR1068) to each well and fix at room temperature for 30 minutes, wash the cells with sterile water, add 1 mL of Alcian blue stain (Oricell, ALCB-10001) to each well and stain for 30 minutes, discard the solution and wash the cells with sterile water, and observe under a microscope.

[0172] Table 6 Formula of chondrogenic differentiation induction solution

[0173]

[0174] The results of the three-lineage differentiation experiment of H1 cells and C27 cells are shown in Figure 6 and Figure 7 .

[0175] Figure 6 : H1_iMSCs obtained by continuous induction of differentiation for 25 days in the corresponding differentiation medium can differentiate into adipocytes, osteoblasts and chondrocytes.

[0176] Figure 7 : C27_iMSCs obtained by continuous induction of differentiation for 25 days in the corresponding differentiation medium can differentiate into adipocytes, osteoblasts and chondrocytes.

[0177] Further, the use of DGEA derivatives (GGGGDGEASP, GKNGDDGEAGKPGRP, GPAGKDGEAGAQGPP, GKNGDDGEAGKPGRP+GPAGKDGEAGAQGPP) to induce differentiation of H1 cells (testing 1mM and 2mM), the positive control group was Laminin521 and Gelatin, and the blank control group was a carboxyl substrate without oligopeptide treatment. The specific process is the same as Example 2 and Example 3, and the gene expression levels of CD90, CD73, CD105, and CD44 of iMSCs progenitor cells on Day 12 of H1 cell induced differentiation are detected. The results are as follows Figure 8 The experimental groups included DGEA, GGGGDGEASP (SEQ ID NO: 16), GKNGDDGEAGKPGRP (SEQ ID NO: 17), GPAGKDGEAGAQGPP (SEQ ID NO: 18), and GKNGDDGEAGKPGRP+GPAGKDGEAGAQGPP. As shown in the figure, the expression levels of CD73 and CD105 in the experimental group were higher than those in the Laminin521 group and the Gelatin group, and the expression levels of CD73 and CD105 in the GPAGKDGEAGAQGPP group were significantly higher than those in the control group, which was statistically significant (p<0.05).

[0178] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a polypeptide and its derivatives in promoting differentiation of pluripotent stem cells into multipotent stem cells or in preparing a product that promotes differentiation of pluripotent stem cells into multipotent stem cells, wherein the amino acid sequence of the polypeptide is DGEA.

2. The use according to claim 1, characterized in that: The pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells; Preferably, the pluripotent stem cells include adult stem cells; Preferably, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, skeletal stem cells and hematopoietic stem cells.

3. The use according to claim 1, characterized in that: The derivatives include polypeptides containing DGEA and proteins containing DGEA, or a combination of derivatives; Preferably, the termini of the polypeptide may be modified or unmodified; Preferably, the modification comprises amino acid modification, glycosylation modification, fatty acid modification, phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof; Preferably, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

4. A cell culture plate, characterized in that: The surface of the cell culture plate is coupled, coated or adsorbed with a polypeptide or a derivative thereof, and the amino acid sequence of the polypeptide is DGEA.

5. The cell culture plate according to claim 4, characterized in that: The coupling includes covalent coupling; Preferably, the covalent coupling comprises the reaction of an amino group with a carboxyl group, the reaction of an aldehyde group with an amino group, or the reaction of a thiol group with a maleimide group.

6. The cell culture plate according to claim 5, characterized in that: The derivatives include polypeptides containing DGEA and proteins containing DGEA, or a combination of derivatives; Preferably, the termini of the polypeptide may be modified or unmodified; Preferably, the modification comprises amino acid modification, glycosylation modification, fatty acid modification, phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof; Preferably, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

7. A culture medium comprising a polypeptide or a derivative thereof, wherein the amino acid sequence of the polypeptide is DGEA; Preferably, the derivatives include polypeptides containing DGEA and proteins containing DGEA; Preferably, the termini of the polypeptide may be modified or unmodified; Preferably, the modification comprises amino acid modification (e.g., substitution or replacement, deletion, addition, cysteine, glutamic acid, alanine, glutamine, aspartic acid, lysine, threonine), glycosylation modification (O-β-D-glucose modification), fatty acid modification (palmitoylation modification), phosphorylation modification, amidation modification, acetylation modification, PEGylation modification, HESylation modification or a combination thereof; Preferably, any one amino acid in the sequence of DGEA may be deleted or its amino acids may be replaced by other amino acids.

8. The culture medium according to claim 7, characterized in that The culture medium contains one or more substances that can induce pluripotent stem cells to differentiate into multipotent stem cells or are used for cell culture; Preferably, the substance comprises at least one of KOSR, Glutamax, non-essential amino acids, ALK inhibitors, bFGF, EGF, FGF9, and ginsenoside monomers; Preferably, the culture medium is any one of RMPI 1640, DMEM / F12 medium, advance RMPI 1640, DMEM or a combination thereof.

9. Use of the cell culture plate according to any one of claims 4 to 6 or the culture medium according to claim 7 or 8 in inducing pluripotent stem cells to differentiate into multipotent stem cells; Preferably, the pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells; Preferably, the pluripotent stem cells include adult stem cells; Preferably, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, skeletal stem cells and hematopoietic stem cells.

10. A method for promoting differentiation of pluripotent stem cells into multipotent stem cells, comprising the step of using the cell culture plate according to any one of claims 4 to 6 as a container to culture pluripotent stem cells; or, comprising the step of using the culture medium according to claim 7 or 8 to culture pluripotent stem cells; Preferably, the pluripotent stem cells include at least one of embryonic stem cells, embryonic germ cells, embryonic carcinoma cells or induced pluripotent stem cells; Preferably, the pluripotent stem cells include adult stem cells; Preferably, the adult stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, skeletal stem cells and hematopoietic stem cells.

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