Reprogramming culture medium for reprogramming somatic cells into induced pluripotent stem cells and use method of reprogramming culture medium

Abstract

The invention discloses a reprogramming culture medium for reprogramming somatic cells into induced pluripotent stem cells, the reprogramming culture medium comprises a basic culture medium, astragalus polysaccharide, resveratrol, chitosan nano-coated vitamin C and recombinant LIF, the chitosan nano-coated vitamin C is prepared by a specific ionic cross-linking method; the invention also discloses a use method for reprogramming somatic cells into induced pluripotent stem cells by adopting the reprogramming culture medium, which comprises the steps of somatic cell culture, cell inoculation, reprogramming culture and the like, and is suitable for common somatic cells such as human skin fibroblasts, oral mucosa cells, adipocytes and the like. The induced pluripotent stem cells can be obtained by reprogramming somatic cells for 7 days by using the method disclosed by the invention. The culture medium is definite in component and stable in property, the somatic cell reprogramming efficiency can be remarkably improved, the reprogrammed induced pluripotent stem cells are good in dryness, high in hereditary stability and safe, and the culture medium can be widely applied to the fields of regenerative medicine, disease research, medicine development and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell reprogramming, and particularly relates to a reprogramming culture medium for reprogramming somatic cells into pluripotent stem cells and a method for using the same. Background Art

[0002] Pluripotent stem cells have the potential to differentiate into various cell types, showing great application prospects in tissue repair, organ regeneration, and the treatment of intractable diseases, attracting the attention of many researchers. Cell reprogramming technology aims to transform differentiated somatic cells into pluripotent stem cells, and the research results in this field have brought new hope for regenerative medicine, disease modeling, and drug development.

[0003] In 2006, the emergence of induced pluripotent stem cell (iPSC) technology achieved a major breakthrough. Shinya Yamanaka realized the transformation of somatic cells into pluripotent stem cells by introducing specific genes (such as Oct3 / 4 gene, Sox gene, c-Myc gene, and Klf gene) into somatic cells, avoiding the ethical and immune rejection problems faced by embryonic stem cells. However, the traditional iPSC preparation technology has some obvious defects. On the one hand, the retroviral vector used to introduce genes has the risk of random integration into the host genome, which may lead to gene mutations and increase the risk of carcinogenesis. On the other hand, the operation process of introducing multiple genes is complex, costly, and the preparation efficiency is low, making it difficult to meet the needs of large-scale cell production.

[0004] To overcome the above problems, researchers have been continuously exploring new reprogramming methods. Among them, chemical reprogramming technology has gradually emerged. This technology uses small molecule compounds to replace gene introduction, avoiding the risks brought by gene manipulation. For example, CN115003795B uses histone deacetylase inhibitors and other chemical substances to induce cell reprogramming. However, the existing chemical reprogramming methods still have many deficiencies, including low reprogramming efficiency, instability of iPSC stemness and heredity, etc., making it difficult to meet the actual application requirements.

[0005] In terms of culture media, many existing cell reprogramming culture media rely on animal-derived components, such as serum, growth factors of animal origin, etc. These components not only have the problem of large batch-to-batch differences, resulting in difficult reproducibility of experimental results, but also may carry pathogens such as viruses and mycoplasmas that can contaminate cells, affecting cell quality and safety. At the same time, the components of the culture medium are complex and unclear, which is not conducive to in-depth research on the reprogramming mechanism and precise control of the cell culture process.

[0006] In summary, current cell reprogramming technology still faces many challenges in aspects such as reprogramming efficiency, safety, stability, and medium optimization. Developing a highly efficient, safe, stable, and well-defined medium for somatic cell reprogramming into pluripotent stem cells and its usage method is of great practical significance for promoting the development of cell reprogramming technology and realizing its wide application in the field of regenerative medicine and other fields. Summary of the Invention

[0007] In view of the above problems, the present invention provides a reprogramming medium for somatic cell reprogramming into induced pluripotent stem cells and its usage method. This reprogramming medium does not contain animal-derived components such as serum, and by adding specific components such as astragalus polysaccharide, resveratrol, chitosan nanoparticle-encapsulated vitamin C, and recombinant LIF, it provides a good environment for somatic cell reprogramming, improves the reprogramming efficiency, and ensures the quality and stability of the induced pluripotent stem cells after reprogramming.

[0008] In the first aspect of the present invention, there is provided a reprogramming medium for somatic cell reprogramming into induced pluripotent stem cells, which is characterized by comprising the following components: a basal medium, astragalus polysaccharide, resveratrol, chitosan nanoparticle-encapsulated vitamin C, and recombinant LIF. By adding the basal medium, astragalus polysaccharide, resveratrol, chitosan nanoparticle-encapsulated vitamin C, and recombinant LIF, their synergistic effect ensures the nutrient supply and function maintenance during the process of somatic cell reprogramming into induced pluripotent stem cells. Among them, the basal medium provides basic nutrients for somatic cells and a basic environment for cell growth and reprogramming. Recombinant LIF, as an auxiliary factor, plays an important role in maintaining the undifferentiated state of cells and helps somatic cells remain in a state suitable for reprogramming. Astragalus polysaccharide, resveratrol, and chitosan nanoparticle-encapsulated vitamin C promote the process of somatic cell reprogramming from different aspects, and these substances work together to provide a stable and good nutrient and function maintenance environment for somatic cell reprogramming into induced pluripotent stem cells.

[0009] Preferably, for the above reprogramming medium, the final concentrations of each component are as follows: astragalus polysaccharide 80 - 150 μg / mL, resveratrol 2 - 5 μM, chitosan nanoparticle-encapsulated vitamin C 50 - 100 μg / mL, and recombinant LIF 8 - 15 ng / mL. It should be noted that the final concentration described in the present invention refers to the ratio of the mass or amount of substance of each component to the total volume of the prepared solution.

[0010] The reprogramming medium of the present invention can synergistically promote somatic cell reprogramming and maintain the pluripotency of induced pluripotent stem cells by adding astragalus polysaccharide and resveratrol. In the reprogramming medium of the present invention, 80-150 μg / mL of astragalus polysaccharide can activate the Wnt / β-catenin pathway, promote the expression of pluripotency genes such as Oct4 and Sox2, and promote the transformation of somatic cells into induced pluripotent stem cells; 2-5 μM of resveratrol can activate the SIRT1 pathway, effectively extend the telomere length, inhibit cell senescence, and maintain the pluripotency of induced pluripotent stem cells. During the process of somatic cell reprogramming, astragalus polysaccharide and resveratrol cooperate with each other, and act synergistically from two aspects of promoting cell reprogramming and maintaining cell pluripotency, solving the problem of maintaining cell pluripotency during the reprogramming process. At the same time, there is also a certain interaction between the two activated pathways, further enhancing the reprogramming effect and the ability to maintain pluripotency. When cultured until the 7th day, the positive rate of Oct4 > 74%, the positive rate of Sox2 > 72%, and the positive rate of Nanog > 70%.

[0011] The reprogramming of the present invention plays an antioxidant and genomic stability protection role by adding chitosan nanoparticles encapsulated with vitamin C. At the same time, chitosan nanoparticles encapsulated with vitamin C can also act synergistically with astragalus polysaccharide and resveratrol to further optimize the reprogramming environment. Experiments show that the induced pluripotent stem cells after reprogramming show good genetic stability during the passage process. After passage to the 80th generation, the normal karyotype rate is still ≥ 96%.

[0012] More preferably, the above-mentioned reprogramming medium is characterized in that the final concentrations of each component are as follows: astragalus polysaccharide 100 μg / mL, resveratrol 3 μM, chitosan nanoparticles encapsulated with vitamin C 80 μg / mL, and recombinant LIF 10 ng / mL.

[0013] Preferably, the chitosan nanoparticles encapsulated with vitamin C are prepared by the ion cross-linking method. The specific preparation steps are as follows: Weigh deacetylated chitosan and dissolve it in a 1% acetic acid solution (pH 5.5) to obtain a first solution; weigh vitamin C and dissolve it in water to obtain a second solution; slowly mix the first solution and the second solution at a volume ratio of 2:1, and ultrasonically treat the mixed solution to obtain a nano-suspension; centrifuge the nano-suspension at 12000 rpm for 30 min, collect the precipitate, and then freeze-dry the precipitate and fill it with nitrogen to obtain the freeze-dried powder of chitosan nanoparticles encapsulated with vitamin C.

[0014] In the above preparation steps, preferably, the concentration of deacetylated chitosan in the first solution is 1 g / L, and the concentration of vitamin C in the second solution is 1 g / L.

[0015] Preferably, the basal medium is PGM1 medium. The chemical composition of PGM1 medium is clear and feeder layer-independent, and it is closer to the in-vivo cell growth environment, which can provide the necessary material basis for somatic cell reprogramming and the growth of induced pluripotent stem cells.

[0016] In the second aspect of the present invention, a method for reprogramming somatic cells into induced pluripotent stem cells is provided, characterized in that the somatic cells are reprogrammed and cultured using the above-mentioned reprogramming medium, including the following steps: Step 1, somatic cell culture: Select somatic cells as the cell source for reprogramming, culture the selected somatic cells to 80% confluence, digest them with trypsin and count to obtain the digested cells. When the cells are cultured to 80% confluence, the cells are in a good growth state, with active cell metabolism and stable physiological functions at this time. Performing subsequent digestion and counting operations can ensure the consistency of the number and activity of the cells inoculated later. Trypsin can specifically act on the connexins between cells, causing the cells to detach from the surface of the culture dish, facilitating subsequent inoculation operations. After digestion, the cells are counted.

[0017] Step 2, cell inoculation: Inoculate the digested cells obtained in Step 1 at a density of 2×10 4 cells / cm 2 onto a culture dish coated with Matrigel to obtain the post-inoculation cell system. Matrigel is a substance that mimics the extracellular matrix. It is rich in various extracellular matrix components, such as collagen, laminin, etc., and can provide a microenvironment similar to that in the body for the cells, promoting cell adhesion and growth.

[0018] Step 3, reprogramming: Add the above-mentioned reprogramming medium to the post-inoculation cell system obtained in Step 2, and culture it in an incubator at 37°C, 5% CO2, and saturated humidity. Replace the fresh reprogramming medium in full volume every day and culture for 7 days to obtain induced pluripotent stem cells. By replacing the reprogramming medium every day, the nutrients required for cell growth and reprogramming are replenished in a timely manner, and at the same time, the waste products generated by cell metabolism are removed to maintain the stable concentration of each component in the medium. When changing the medium, first carefully aspirate the old medium with a pipette, then gently wash the cells with PBS, and then add the freshly prepared medium. After 7 days of culture, under the synergistic action of each component in the medium, the somatic cells gradually complete the reprogramming process and transform into induced pluripotent stem cells.

[0019] Preferably, the somatic cells in Step 1 are one of human skin fibroblasts and oral mucosal cells. It should be noted that the somatic cells can also be selected from other common and easily obtainable somatic cell types, such as: adipocytes, hair follicle cells, etc.

[0020] In addition, the present invention also provides the application of a reprogramming medium for reprogramming somatic cells into induced pluripotent stem cells in the field of cell reprogramming. The reprogramming efficiency of somatic cells cultured using the reprogramming medium of the present invention and its usage method is significantly improved. The reprogrammed pluripotent stem cells have good stemness, genetic stability, and safety, and can be widely applied in the fields of regenerative medicine, disease research, drug development, and the like.

[0021] In summary, the beneficial effects of the present invention are as follows: (1) The reprogramming medium of the present invention activates the Wnt / β-catenin pathway by adding astragalus polysaccharide and activates the SIRT1 pathway by adding resveratrol. The two act synergistically, and in combination with the chitosan nano-encapsulation of vitamin C to target the scavenging of reactive oxygen species and recombinant LIF to inhibit differentiation, the efficient transformation of somatic cells into induced pluripotent stem cells is achieved, and the pluripotency of the induced pluripotent stem cells is effectively maintained. When cultured until the 7th day, the Oct4 positive rate > 74%, which is significantly higher than that of the traditional technology. At the same time, the reprogrammed induced pluripotent stem cells can stably express stemness genes such as Oct4 and Sox2, solving the problems of low reprogramming efficiency in the prior art and unstable stemness of induced pluripotent stem cells in chemical reprogramming technology.

[0022] (2) The reprogramming medium of the present invention effectively protects genomic stability by utilizing the antioxidant effect of chitosan nano-encapsulating vitamin C and combining with the mechanism of resveratrol to extend telomeres. The normal karyotype rate of the reprogrammed iPSCs is still ≥ 96% after passage to the 80th generation, solving the problems of genetic instability and easy mutation of cells in traditional reprogramming methods.

[0023] (3) The reprogramming medium of the present invention adopts a serum-free formulation and does not contain animal-derived components, solving the problems of high risk of animal-derived component contamination and large batch-to-batch differences. At the same time, the components of the medium are clear, facilitating quality control and research on reprogramming mechanisms.

[0024] (4) During the reprogramming culture process of the present invention, Matrigel coating is used to simulate the in vivo microenvironment, combined with the method of replacing the medium in full volume every day to maintain the concentration of active ingredients, and the seeding density is strictly controlled at 2×10 4 cells / cm 2 , ensuring that the cells enter the reprogramming process synchronously, avoiding excessive cell competition or insufficient signal transduction, and solving the problem that the culture conditions affect the reprogramming efficiency. Description of the Drawings

[0025] Figure 1 It is a DLS result diagram of the prepared chitosan nano-encapsulated vitamin C.

[0026] Figure 2Morphological diagrams of human fibroblasts and their reprogrammed cells. Diagram A shows the morphology of human fibroblasts, and diagrams B - H show the morphology of reprogrammed human fibroblasts. Diagram B shows the morphology after reprogramming with medium 1, diagram C shows the morphology after reprogramming with medium 2, diagram D shows the morphology after reprogramming with medium 3, diagram E shows the morphology after reprogramming with comparative medium 1, diagram F shows the morphology after reprogramming with comparative medium 2, diagram G shows the morphology after reprogramming with comparative medium 3, and diagram H shows the morphology after reprogramming with comparative medium 4.

[0027] Figure 3 Morphological diagrams of human oral mucosal cells and their reprogrammed cells. Diagram A shows the morphology of human oral mucosal cells, and diagram B shows the morphology of human oral mucosal cells after reprogramming with medium 2. Detailed implementation manners

[0028] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0029] In the present invention, the components and reagents involved are all commercially available products or can be obtained by conventional technical means in the art. Unless otherwise stated, the materials, methods and examples of the present invention are merely exemplary and not restrictive.

[0030] Example 1: Chitosan nanoparticles encapsulating vitamin C and its preparation This example provides a method for preparing chitosan nanoparticles encapsulating vitamin C. Chitosan with a deacetylation degree ≥ 90% and a molecular weight of 100 kDa (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.), analytical grade vitamin C (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.), 1% acetic acid solution (self-prepared, pH 5.5) and deionized water are used for preparation. The preparation method includes the following steps: (1) Preparation of chitosan solution: Accurately weigh 100 mg of the chitosan and slowly add it to 100 mL of 1% acetic acid solution (pH 5.5). Place the container containing the solution on a magnetic stirrer and stir at a speed of 300 rpm for 2 h until the chitosan is completely dissolved to form a uniform and transparent chitosan solution.

[0031] (2) Preparation of vitamin C aqueous solution: Accurately weigh 50 mg of vitamin C and add it to 50 mL of deionized water. Use a magnetic stirrer to stir at 200 rpm for 30 min to completely dissolve the vitamin C and obtain a vitamin C aqueous solution.

[0032] (3) Mixing and sonication: Slowly mix the chitosan solution prepared in (1) and the vitamin C aqueous solution at a volume ratio of 2:1, stirring at 250 rpm for 15 min while mixing to ensure full contact between the two solutions. Then transfer the mixed solution to the sample cell of the sonicator, set the sonication power to 200 W, and sonicate for 10 min to form nanoparticles.

[0033] (4) Centrifugal purification: After sonication, transfer the mixed solution to a centrifuge tube, place it in a centrifuge, and centrifuge at 12,000 rpm for 30 min. After centrifugation, the unencapsulated vitamin C and other impurities will be suspended in the supernatant, while the chitosan nanoparticles encapsulating vitamin C will precipitate to the bottom of the centrifuge tube. Carefully discard the supernatant and transfer the precipitate to a new sterile container to obtain preliminarily purified chitosan nanoparticles encapsulating vitamin C.

[0034] (5) Freeze-drying treatment: Wash the precipitate collected by centrifugation in (4) twice with deionized water, then resuspend it with 50 mL of deionized water to form a suspension. Place the suspension in a freeze-dryer, pre-freeze it in a -50°C refrigerator for 4 h, and perform sublimation drying at -40°C for 20 h. After freeze-drying, fill it with nitrogen and seal it to obtain freeze-dried powder of chitosan nanoparticles encapsulating vitamin C.

[0035] Use a dynamic light scattering (DLS) instrument to detect the particle size of the prepared chitosan encapsulating vitamin C. Take an appropriate amount of the nanoparticle sample, dilute it to a suitable concentration with deionized water, add it to the DLS sample cell, and measure it according to the operating procedures of the DLS instrument. The results are as Figure 1 shown. After detection, the particle size of the obtained chitosan nanoparticles encapsulating vitamin C is mainly in the range of 100 - 150 nm, and the average particle size is 123 nm, meeting the usage requirements.

[0036] Use high-performance liquid chromatography (HPLC) to determine the encapsulation efficiency. Accurately weigh 10 mg of the chitosan nanoparticles encapsulating vitamin C, add 1 mL of ethanol-water (1:1, v / v) mixed solvent to dissolve the nanoparticles and release the encapsulated vitamin C. Centrifuge the solution at 12,000 rpm for 5 min, take the supernatant and inject it into the HPLC instrument for analysis. The chromatographic conditions are shown in Table 1. Calculate the content of vitamin C in the sample according to the standard curve of vitamin C and the HPLC peak area of the sample solution, and calculate the encapsulation efficiency according to the following formula: Encapsulation efficiency (%) = (Content of encapsulated vitamin C / Total amount of added vitamin C) × 100%. After detection, the retention time of vitamin C is 7.1 min. According to the formula, the calculated encapsulation efficiency is 85.8%, meeting the requirements of the present invention.

[0037] Table 1 Chromatographic conditions for HPLC detection of vitamin C Example 2: A reprogramming medium for reprogramming somatic cells into induced pluripotent stem cells and its preparation This example provides a reprogramming medium for reprogramming somatic cells into induced pluripotent stem cells and its preparation method. To reflect the influence of components at different concentrations on the effect of the medium, three reprogramming media with different concentration gradients are prepared, and their component contents are shown in Table 2. The three reprogramming media are respectively labeled as Medium 1, Medium 2, and Medium 3.

[0038] The preparation method of the reprogramming medium provided in this example is as follows: Step 1: Accurately measure PGM1 medium (purchased from Beijing Saibei Biotechnology Co., Ltd.) and place it in a sterile container. The chemical composition of PGM1 medium is clear and feeder layer-independent, and it is closer to the in-vivo cell growth environment, which can provide the necessary material basis for the reprogramming of somatic cells and the growth of induced pluripotent stem cells.

[0039] Step 2: Accurately weigh the corresponding mass or molar amount of astragalus polysaccharide, resveratrol, chitosan nanoparticles encapsulated vitamin C, and recombinant LIF respectively. Among them, chitosan nanoparticles encapsulated vitamin C are prepared in Example 1.

[0040] Step 3: Add the weighed astragalus polysaccharide, resveratrol, chitosan nanoparticles encapsulated vitamin C, and recombinant LIF to the PGM1 medium obtained in Step 1 in sequence. After adding each component, stir well to make each component completely dissolve and disperse evenly, obtaining a uniformly mixed medium solution.

[0041] Step 4: Filter and sterilize the prepared medium solution through a 0.22 μm sterile filter membrane to obtain the above-mentioned reprogramming medium for reprogramming somatic cells into induced pluripotent stem cells. Seal the prepared reprogramming medium and store it at 4°C.

[0042] Table 2 Composition table of Medium 1 - 3 Comparative Example 1: A medium for reprogramming somatic cells into induced pluripotent stem cells lacking key components and its preparation To reflect the influence of different key components on the effect of the reprogramming medium, this comparative example provides a medium for reprogramming somatic cells into induced pluripotent stem cells lacking key components and its preparation method. Three media with different concentration gradients are prepared, and their component contents are shown in Table 3. These three media are respectively labeled as Comparative Medium 1, Comparative Medium 2, and Comparative Medium 3. The preparation steps of Comparative Medium 1 - 3 are the same as those in Example 2.

[0043] Table 3 Composition table of Comparative Medium 1 - 3 Comparative Example 2: Medium for Reprogramming Somatic Cells into Induced Pluripotent Stem Cells Containing Ordinary Vitamin C and Its Preparation To reflect the effect of chitosan nanoparticle - encapsulated vitamin C on the reprogramming medium, this comparative example provides a medium for reprogramming somatic cells into induced pluripotent stem cells that does not contain chitosan nanoparticle - encapsulated vitamin C but only contains ordinary vitamin C, and its preparation method. This medium is denoted as Comparative Medium 4. The difference in the composition between Comparative Medium 4 and Medium 2 is that it does not contain chitosan nanoparticle - encapsulated vitamin C and contains 68.64 μg / mL of ordinary vitamin C, and the content of ordinary vitamin C is calculated from the encapsulation efficiency of chitosan - encapsulated vitamin C. The preparation steps of Comparative Medium 4 are the same as those in Example 2.

[0044] Example 3: Reprogramming of Human Skin Fibroblasts into Induced Pluripotent Stem Cells In this example, Medium 1 - 3 and Comparative Medium 1 - 4 were used to reprogram human skin fibroblasts.

[0045] I. Obtaining and Culturing Human Skin Fibroblasts Step 1. Obtaining human skin fibroblasts: Obtain skin tissue samples from healthy volunteers. Under strict aseptic operation conditions, wash the skin tissue 3 times with PBS to remove blood and impurities. Cut the washed skin tissue into small pieces of about 1 mm 3 , then add an appropriate amount of digestive solution containing 0.25% trypsin - 0.02% EDTA, and digest at 37 °C for 30 - 60 min. Gently shake it every 10 min during this period to make the digestion more complete. After digestion, add DMEM medium containing 10% fetal bovine serum to terminate the digestion, and then centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0046] Step 2. Primary culture: Resuspend the cell pellet with fresh DMEM medium, inoculate the cells into a culture flask, and culture them in an incubator at 37 °C, 5% CO2, and saturated humidity. Replace the medium every 3 days. When the cells grow to 80% confluence, conduct subsequent experiments.

[0047] II. Reprogramming of Somatic Cells into Induced Pluripotent Stem Cells Step 1. Somatic cell culture: When the above-mentioned human skin fibroblasts are cultured to 80% confluence, discard the original medium and gently wash the cells with PBS twice. Then add an appropriate amount of 0.25% trypsin-0.02% EDTA digestion solution and digest in a 37°C incubator for 2 - 3 minutes. When the cells begin to round up and gradually detach from the culture flask wall, add DMEM medium containing 10% fetal bovine serum to terminate the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant. Resuspend the cell pellet with fresh DMEM medium, count the cells using a cell counting chamber to obtain the digested cells.

[0048] Step 2. Cell seeding: Seed the digested cells obtained in Step 1 at a density of 2×10 4 cells / cm 2 onto a culture dish coated with Matrigel to obtain the post-seeding cell system. Matrigel is a substance that mimics the extracellular matrix. It is rich in various extracellular matrix components and can provide a microenvironment similar to that in vivo for cells, promoting cell adhesion and growth.

[0049] Step 3. Reprogramming culture: Add Medium 1 - 3 and Control Medium 1 - 4 to the post-seeding cell system obtained in Step 2 respectively, and then place the culture dish in an incubator at 37°C, 5% CO2, and saturated humidity for culture. Replace the corresponding fresh medium in full volume every day for 7 days. When changing the medium, first carefully aspirate the old medium using a pipette, then gently wash the cells with PBS, and then add freshly prepared medium. By changing the medium every day, the nutrients required for cell growth and reprogramming are supplemented in a timely manner, and at the same time, the waste products generated by cell metabolism are removed to maintain the stable concentration of each component in the medium. After 7 days of culture, under the synergistic action of each component in the medium, somatic cells gradually complete the reprogramming process to obtain induced pluripotent stem cells.

[0050] III. Identification of induced pluripotent stem cells This part identifies the induced pluripotent stem cells reprogrammed from the above-mentioned human skin fibroblasts.

[0051] (I) Morphological identification On the 7th day of reprogramming culture, use an inverted microscope to observe the cells in the culture dish and record characteristics such as cell morphology, size, boundary clarity, and cell arrangement pattern.

[0052] The culture results are as Figure 2 shown, Figure 2 A is human skin fibroblasts, Figure 2Cells B - D were cultured using culture media 1 - 3 and showed typical induced pluripotent stem cell clone morphology. The cell clones grew in a tight colony - like manner with clear boundaries. The cells were small in volume with a large nuclear - to - cytoplasmic ratio, the cell nuclei were obvious, and the cells were arranged tightly and orderly. In contrast, for the cells cultured with control media 1 - 3 ( Figure 2 E - G), the number of clone formations was less. Although the cell morphology changed to some extent, most cells still showed a long spindle - shaped fibroblast - like appearance, and the clone boundaries were relatively blurred. For the cells cultured with control medium 4 ( Figure 2 H), induced pluripotent stem cell clone morphology appeared, but both the tightness and the growth number of the cell clones were significantly lower than the culture effects of culture media 1 - 3.

[0053] (2) Detection of stemness gene expression The detection was carried out according to the following steps: Step 1: Cell preparation: On the 7th day of reprogramming culture, the cells cultured with different culture media (culture media 1 - 3, control media 1 - 4) were digested with 0.25% trypsin - 0.02% EDTA digestion solution to detach the cells from the surface of the culture dish. The cell suspension was transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were gently washed twice with PBS, and the supernatant was discarded again after centrifugation to obtain a clean cell pellet.

[0054] Step 2: Fixation and permeabilization: An appropriate amount of 4% paraformaldehyde solution was added to the cell pellet and fixed at room temperature for 15 - 20 min to fix the cell morphology and antigen structure. After fixation, it was centrifuged at 1000 rpm for 5 min, the paraformaldehyde solution was discarded, and the cells were washed twice with PBS. Subsequently, a PBS solution containing 0.1% TritonX - 100 was added and permeabilized at room temperature for 10 - 15 min to enable the antibody to enter the cell and bind to the target antigen. After permeabilization, the cells were washed twice with PBS again.

[0055] Step 3: Antibody incubation: The permeabilized cells were resuspended separately in PBS solutions containing Oct4 primary antibody, Sox2 primary antibody, and Nanog primary antibody, and incubated overnight at 4℃ in the dark. The next day, it was centrifuged at 1000 rpm for 5 min, the primary antibody solution was discarded, and the cells were washed three times with PBS for 5 min each time. Then, AlexaFluor488 fluorescently labeled secondary antibodies corresponding to the primary antibodies were added respectively and incubated at room temperature in the dark for 1 - 2 h. After incubation, the cells were washed three times with PBS to remove the unbound secondary antibodies.

[0056] Step 4: Flow cytometry detection: The washed cells were resuspended in an appropriate amount of PBS, and the cell concentration was adjusted to 1×10 6 ~1×10 7cells / mL, transfer them to a sample tube dedicated for flow cytometry. Detect using a flow cytometer, set the excitation light wavelength to 488 nm, the emission light wavelength to 519 nm, and collect the fluorescence signals at wavelengths around 510 - 530 nm. Analyze the cells one by one through the flow cytometer, record the fluorescence intensity of each cell, and at the same time set unstained cells as negative controls to determine the background fluorescence level. Count the number of positive cells and the total number of detected cells through the flow cytometer software, and calculate the positive cell rate according to the following formula: Positive rate of Oct4 (%) = Number of cells with positive Oct4 gene expression / Total number of detected cells × 100% Positive rate of Sox2 (%) = Number of cells with positive Sox2 gene expression / Total number of detected cells × 100% Positive rate of Nanog (%) = Number of cells with positive Nanog gene expression / Total number of detected cells × 100% Table 4 Positive rates of stemness gene expression in reprogrammed cells The experimental results are shown in Table 4. Compared with control media 1 - 4, the positive rates of expression of the stemness genes Oct4, Sox2, and Nanog in the induced pluripotent stem cells obtained from media 1 - 3 are significantly higher. Medium 2 is particularly prominent, with an Oct4 positive rate of 77.8%, a Sox2 positive rate of 75.8%, and a Nanog positive rate of 73.4%. This indicates that the medium containing components such as astragalus polysaccharide, resveratrol, chitosan - nano - encapsulated vitamin C, and recombinant LIF involved in the invention can efficiently promote the reprogramming of somatic cells into induced pluripotent stem cells and perform excellently in maintaining cell stemness, strongly ensuring a high expression level of stemness genes. From the results of comparing media 1 - 4, the components added in the medium of the present invention play an important role in the reprogramming of somatic cells. With the synergistic cooperation among the components, the medium of the present invention creates an ideal microenvironment for the reprogramming of somatic cells, greatly promoting the expression of stemness genes such as Oct4, Sox2, and Nanog, and effectively maintaining the stemness of induced pluripotent stem cells.

[0057] (III) Karyotype analysis The detection is carried out according to the following steps: Step 1. Cell culture and treatment: Passage the induced pluripotent stem cells cultured using different media. When the cells are passaged to the 80th generation and are in the logarithmic growth phase, add colchicine to the medium at a final concentration of 0.05 μg / mL and continue culturing for 4 h to inhibit the formation of the cell spindle and arrest the cells at the metaphase of mitosis.

[0058] Step 2. Hypotonic treatment: Collect cells and perform hypotonic treatment with 0.075 M KCl solution at 37 °C for 20 - 30 min to expand the cells and disperse the chromosomes.

[0059] Step 3. Fixation and slide preparation: Fix the cells after hypotonic treatment with methanol - glacial acetic acid (3:1) fixative for 3 times, 15 - 20 min each time. Drop the fixed cells onto a pre - cooled glass slide and prepare the slide by air - drying method.

[0060] Step 4. G - banding staining: Appropriately treat the prepared slide with trypsin solution, then stain it with Giemsa stain for 10 - 15 min. After staining, rinse it with water and air - dry it naturally.

[0061] Step 5. Microscopic detection: Observe the morphology and number of chromosomes under a microscope. Select 50 cells in the metaphase for karyotype analysis. According to the characteristics such as the morphology, size, and centromere position of the chromosomes, pair, group, and number the chromosomes to determine whether the karyotype of the cells is normal. Calculate the normal karyotype rate according to the following formula: Normal karyotype rate (%) = Number of cells with normal karyotype / Total number of cells × 100% The detection results are shown in Table 5. After the induced pluripotent stem cells cultured with media 1 - 3 are passaged to the 80th generation, the normal karyotype rate is still ≥96%. In contrast, the normal karyotype rate of the cells cultured with media 1 - 4 is relatively low. It shows that the medium containing multiple specific components in the present invention, including astragalus polysaccharide, resveratrol, chitosan - nanocoated vitamin C, and recombinant LIF, etc., through their synergistic effects, effectively reduce the occurrence of chromosome aberrations by regulating intracellular signaling pathways and maintaining the stability of chromosome structure, and play an important role in the karyotype stability during the long - term passage of induced pluripotent stem cells.

[0062] Table 5 Normal karyotype rate of reprogrammed cells Example 4: Reprogramming and verification of oral mucosal cells In this example, medium 2 is used to reprogram oral mucosal cells.

[0063] I. Obtaining and primary culture of oral mucosal cells Step 1. Acquisition of oral mucosal cells: After strictly following ethical norms and obtaining the informed consent of volunteers, gently scrape the surface tissue of the oral buccal mucosa of healthy volunteers using a sterile spatula. Immediately place the obtained tissue into a sterile centrifuge tube containing PBS. On a sterile operating table, wash the tissue with PBS 5 times to thoroughly remove impurities and residual saliva. Add an appropriate amount of 0.25% trypsin - 0.02% EDTA digestive solution and digest at 80 rpm in a 37°C constant temperature shaker for 45 min. After digestion is completed, add DMEM medium containing 10% fetal bovine serum to terminate digestion, centrifuge at 1200 rpm for 6 min, and discard the supernatant.

[0064] Step 2. Primary culture: Resuspend the cell pellet with freshly prepared DMEM medium containing 15% fetal bovine serum and 1% double antibody (penicillin - streptomycin mixture), inoculate the cells into a culture flask coated with polylysine, and culture in an incubator at 37°C, 5% CO2, and saturated humidity. Replace the medium every 2 days. When the cells grow to 70% - 80% confluence, perform the subsequent reprogramming experiment.

[0065] II. Reprogramming of somatic cells into induced pluripotent stem cells According to the reprogramming operation steps in Example 3, use Medium 2 to reprogram the above - obtained oral mucosal cells.

[0066] III. Identification of induced pluripotent stem cells (I) Morphological identification On the 7th day of reprogramming culture, observe and photograph the cells in the culture dish using an inverted phase - contrast microscope. The results are as Figure 3 shown, Figure 3 A are oral mucosal cells, Figure 3 B are the induced pluripotent stem cells after reprogramming. The cell clones are closely aggregated with clear boundaries. The cells are small in volume, have a large nuclear - to - cytoplasmic ratio, and the cell nuclei are clearly visible. The cells are arranged tightly and orderly, conforming to the morphological characteristics of induced pluripotent stem cell clones, and are similar to the morphology of the induced pluripotent stem cells obtained after reprogramming human skin fibroblasts with Medium 2 in Example 3, indicating that the oral mucosal cells have been successfully reprogrammed into induced pluripotent stem cells.

[0067] (II) Detection of stemness gene expression The detection operation is the same as that in Example 3. After statistical analysis and data calculation by flow cytometry software, the positive rate of Oct4 in the induced pluripotent stem cells obtained by reprogramming is 78.2%, the positive rate of Sox2 is 76.5%, and the positive rate of Nanog is 74.1%. The results show that the oral mucosal cells reprogrammed with Medium 2 have a high level of stemness gene expression, further confirming that the oral mucosal cells have been successfully reprogrammed into induced pluripotent stem cells, and Medium 2 can effectively maintain the cell stemness.

[0068] (III) Karyotype analysis The detection operation was the same as that in Example 3. After statistics, the normal karyotype rate was 96%. The results showed that the induced pluripotent stem cells reprogrammed by Medium 2 could effectively maintain the karyotype stability during long-term passage, demonstrating the significant role of the synergistic effect of each component in Medium 2 in maintaining the genetic stability of cells.

[0069] In summary, the components of the reprogramming medium of the present invention complement each other and act synergistically. The usage method is scientific and standardized, and it makes a significant contribution to the transformation of somatic cells into induced pluripotent stem cells and the maintenance of stemness, providing a solid technical support and broad development prospects for the application of related patented technologies in the biomedical field.

[0070] Meanwhile, the present invention also provides a scientific idea and method for reprogramming somatic cells into induced pluripotent stem cells using this reprogramming medium. This solution covers the entire process from the medium components to cell reprogramming culture. For those of ordinary skill in the art, any improvements and innovations based on the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A reprogramming medium for reprogramming somatic cells into induced pluripotent stem cells, characterized in that, It comprises the following components: basal medium, astragalus polysaccharide, resveratrol, chitosan nanoparticle-encapsulated vitamin C, and recombinant LIF.

2. The reprogramming medium according to claim 1, characterized in that, The final concentrations of each component are as follows: astragalus polysaccharide 80 - 150 μg / mL, resveratrol 2 - 5 μM, chitosan nanoparticle-encapsulated vitamin C 50 - 100 μg / mL, and recombinant LIF 8 - 15 ng / mL.

3. The reprogramming medium according to claim 1, wherein The final concentrations of each component are: astragalus polysaccharide 100 μg / mL, resveratrol 3 μM, chitosan nanoparticle-encapsulated vitamin C 80 μg / mL, and recombinant LIF 10 ng / mL.

4. The reprogramming medium according to claim 1, wherein The chitosan nanoparticle-encapsulated vitamin C is prepared by an ionic cross-linking method, and the specific preparation steps are as follows: Weigh deacetylated chitosan and dissolve it in a 1% acetic acid solution to obtain a first solution; weigh vitamin C and dissolve it in water to obtain a second solution; slowly mix the first solution and the second solution at a volume ratio of 2:1, and ultrasonically treat the mixed solution to obtain a nano-suspension; centrifuge the nano-suspension at 12000 rpm for 30 min, collect the precipitate, and then freeze-dry the precipitate and fill it with nitrogen to obtain the freeze-dried powder of chitosan nanoparticle-encapsulated vitamin C.

5. The reprogramming medium according to claim 1, wherein The basal medium is PGM1 medium.

6. A method for reprogramming somatic cells into induced pluripotent stem cells, characterized in that, The somatic cells are reprogrammed and cultured using the reprogramming medium according to any one of claims 1 - 5, and it includes the following steps: Step 1, somatic cell culture: Select somatic cells cultured to 80% confluence, digest them with trypsin and count to obtain digested cells. Step 2. Cell seeding: Seed the digested cells obtained in Step 1 at a density of 2×10 4 cells / cm 2 onto a Matrigel-coated culture dish to obtain the post-seeding cell system; Step 3, reprogramming culture: Add the reprogramming medium to the cell system after inoculation obtained in Step 2, and culture it in an incubator at 37°C, 5% CO2, and saturated humidity. Replace the fresh reprogramming medium in full volume every day and culture for 7 days to obtain induced pluripotent stem cells.

7. The method according to claim 6, characterized in that, The somatic cells in Step 1 are one of human skin fibroblasts and oral mucosal cells.

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