Method for directionally inducing stem cells to differentiate into cardiac muscle cells

Abstract

The invention provides a method for directionally inducing stem cells to differentiate into heart muscle cells, and belongs to the technical field of cell differentiation. The method comprises the following steps: S1, collecting and primarily treating cells; s2, cell culture; s3, introduction of transcription factors; and S4, differentiating to cardiac muscle cells. By using an elaborately designed culture medium and a step-by-step cell treatment technology, the detailed formula and operation conditions of each step aim at maximizing the efficiency and quality of cell transformation, and efficient and controllable transformation from stem cells to heart muscle cells is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell differentiation, and particularly relates to a method for directionally inducing stem cells to differentiate into cardiac muscle cells. Background Art

[0002] In modern medicine, heart diseases are one of the most major health threats globally, and the injury or death of cardiac muscle cells is a key pathological feature. Due to the very limited regenerative ability of cardiac muscle cells, it makes heart diseases often difficult to be completely cured.

[0003] Stem cells can be induced to differentiate into cardiac muscle cells in vitro, providing a potential treatment approach for heart diseases; however, the efficiency and success rate of this process largely depend on the culture medium used. The application and development of such technologies are limited by the existing cell culture systems. However, the currently available culture media on the market often cannot meet the requirements for effectively inducing the differentiation of stem cells into cardiac muscle cells, and there are some specific problems and challenges:

[0004] Firstly, in the current stem cell culture, the culture medium often lacks the necessary specificity and complexity in composition. Although traditional culture media such as DMEM / F-12 provide basic nutritional support, their efficiency in cell differentiation is not ideal enough;

[0005] Secondly, many existing culture media use animal serum as a nutritional supplement, such as fetal bovine serum (FBS). The use of this component brings various problems, including variability between batches, potential pathogen contamination, etc. These problems not only affect the repeatability and reliability of experimental results but also limit the translation from laboratory to clinic.

[0006] Thirdly, when conducting cell reprogramming experiments, traditional culture media often have some limitations. For example, although the traditional KnockOut DMEM medium basically meets the requirements for cell culture, when performing cell reprogramming, especially in high-demand scenarios involving pluripotency induction, its components cannot fully meet all the nutritional components required for cell growth, limiting the cell proliferation ability; during a long-term culture process, the traditional KnockOut DMEM lacks an adequate buffering system, which may lead to fluctuations in pH value, posing a challenge to the stability of the cell growth environment. The unstable pH value may affect the physiological state and reprogramming efficiency of cells. Under dense culture conditions, waste products (such as ammonia and lactate) generated by cell metabolic activities may accumulate in the culture medium, leading to environmental deterioration and affecting the survival and function of cells;

[0007] Finally, traditional induction differentiation media often lack sufficient signaling molecules to effectively mimic the natural signaling environment of cardiomyocytes during embryonic development. Additionally, the effective differentiation of cardiomyocytes also depends on the appropriate support of the redox state and energy metabolism. Existing media often fail to optimize the selection and ratio of these supplements, resulting in insufficient metabolic status and antioxidant defense ability of cells, thus affecting cell maturation and functional performance and reducing the quality and efficiency of cell differentiation.

[0008] Therefore, there is room for optimization in the design and application of existing technologies for inducing stem cells to differentiate into cardiomyocytes. These problems need to be solved in future research and applications to improve the efficiency and effectiveness of inducing stem cells to differentiate into cardiomyocytes, especially in clinical applications and high-precision biological research, which requires the development of more precise and customized media. Summary of the Invention

[0009] The object of the present invention is to address the above-mentioned problems in the existing technology and propose a method for directionally inducing stem cells to differentiate into cardiomyocytes.

[0010] The object of the present invention can be achieved by the following technical solutions: A method for directionally inducing stem cells to differentiate into cardiomyocytes, the method for directionally inducing stem cells to differentiate into cardiomyocytes includes the following steps:

[0011] S1 Cell Collection and Preliminary Treatment: Use a sterile blood collection needle to draw several milliliters of whole blood from the vein of a subject, and store it in a blood collection tube containing an anticoagulant. Transport the collected whole blood to the laboratory in an environment with a temperature controlled at 4°C.

[0012] Use density gradient centrifugation to separate peripheral blood mononuclear cells: After mixing the whole blood with the survival homeostasis solution, pour it into a centrifuge tube pre-filled with Ficoll-Paque PLUS, and then perform centrifugation operations to form chromatography; Aspirate the middle layer of the ring layer and transfer it to a new sterile centrifuge tube.

[0013] After adding the survival homeostasis solution to the new sterile centrifuge tube, perform washing operations through centrifugation to remove plasma and Ficoll residues.

[0014] The survival homeostasis solution includes pure water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride, and their functions are as follows:

[0015] Pure water: Provides a basic solvent for the solution to ensure that other solutes can be evenly dispersed.

[0016] Sodium chloride: Maintains the osmotic pressure of the solution, keeps the ionic strength similar to that of plasma, and reduces cell damage caused by osmotic pressure differences.

[0017] Sodium dihydrogen phosphate and disodium hydrogen phosphate: These two salts work together to regulate the pH of the solution and maintain the physiological pH environment (about 7.4), which helps to keep the cells active and stable.

[0018] Potassium chloride: Supplements the potassium ion concentration in the extracellular fluid, maintains the electrolyte balance inside and outside the cells, and helps the normal operation of cell functions.

[0019] S2 cell culture: Place the washed cells in a stem cell complex medium and culture them in an environment with a temperature of 37 °C and a carbon dioxide concentration of 5%; the stem cell complex medium includes DMEM / F-12 nutrient solution, KSR serum substitute, non-essential amino acids, L-glutamine, β-sodium thiosulfate, NAC, natural plant extract mixture, N6-(2-hydroxyethyl)-lysine, matrix metalloproteinase inhibitor, bFGF growth factor, antibiotics and antifungal agents; the natural plant extract mixture includes green tea extract and blueberry extract;

[0020] DMEM / F-12 nutrient solution: Provides essential nutrients, including sugars, amino acids and vitamins, and supports the basic metabolic activities of cells.

[0021] KSR serum substitute: Provides animal-free nutrients and growth factors for cells, reducing variations and pathogen contamination caused by animal sera.

[0022] Non-essential amino acids and L-glutamine: Increase the amino acids required for cell growth, and support protein synthesis and cell proliferation.

[0023] β-sodium thiosulfate and NAC: As antioxidants, protect cells from oxidative stress damage.

[0024] Natural plant extracts (green tea extract and blueberry extract): Provide antioxidants and bioactive molecules, enhancing the stress resistance and health status of cells.

[0025] N6-(2-hydroxyethyl)-lysine and matrix metalloproteinase inhibitor: Regulate the composition and degradation of the extracellular matrix, and help to maintain the structural integrity and function of cells.

[0026] bFGF growth factor: A potent cell growth and differentiation factor that promotes cell proliferation and maintains the pluripotency of stem cells.

[0027] Antibiotics and antifungal agents: Prevent bacterial and fungal contamination and ensure the sterility of the culture process.

[0028] Through the combined action of these components, the stem cell complex medium provides an ideal supportive environment for stem cells, enabling them to grow effectively and maintain their pluripotency in in vitro culture, laying the foundation for subsequent differentiation steps. This meticulously designed culture system is the key to achieving efficient cell differentiation and application.

[0029] S3: Introduction of transcription factors: Concentration adjustment: Adjust the concentration of lentiviral particles carrying transcription factors to 10^8 infectious units / mL through an infection unit determination experiment; obtain a lentiviral suspension; the transcription factors are OCT4, SOX2, KLF4, and c-MY;

[0030] Suspension addition: Select cells with normal metabolic activity, count and adjust the cell density using an automatic cell counter; then add the lentiviral suspension to the cells and incubate for more than 24 hours in an environment with a temperature of 37°C and a carbon dioxide concentration of 5%; during the addition of the lentiviral suspension, add polyethyleneimine as a transfection enhancer;

[0031] Replacement of the medium and cell recovery: After incubation, aspirate the stem cell complex medium and replace it with a modified KnockOut DMEM medium, and culture in an environment with a temperature of 37°C and a carbon dioxide concentration of 5%;

[0032] The modified KnockOut DMEM medium specifically includes KnockOut DMEM medium, bFGF growth factor, L-aspartic acid, nicotinamide, serin, ITS, collagen, L-glutamine, HEPES buffer, sodium pyrophosphate;

[0033] The reasons for using OCT4, SOX2, KLF4, and c-MYC as transcription factors are based on the key roles of these factors in cell fate control and reprogramming:

[0034] OCT4: OCT4 is a key factor in maintaining the pluripotency of embryonic stem cells (ES cells) and induced pluripotent stem cells. It regulates the expression of multiple genes involved in the self-renewal of stem cells and the inhibition of differentiation. Appropriate expression of OCT4 is essential in pluripotent cells.

[0035] SOX2: SOX2 is another core pluripotency maintenance factor that acts in concert with OCT4 to jointly promote the establishment and maintenance of the stem cell state. SOX2 is highly expressed in neural progenitor cells and stem cells and is an important factor in regulating the undifferentiated state of cells.

[0036] KLF4: In stem cell reprogramming, KLF4 acts together with OCT4 and SOX2 to promote cell reprogramming and prevent cells from differentiating.

[0037] c-MYC: c-MYC is a known oncogene and also a powerful cell proliferation and growth promoting factor. During the generation of iPSCs, c-MYC increases the cell's metabolism and growth rate, accelerating the reprogramming process.

[0038] The combination of these transcription factors not only effectively reprograms somatic cells into a pluripotent state but also provides a controllable and effective starting point for further cell type-specific cardiomyocytes. Using these factors during the induction of cardiomyocyte differentiation is crucial to ensure that the cells can successfully transform into the desired cell type.

[0039] The modified KnockOut DMEM medium includes multiple specific components. The selection of these components aims to provide an environment suitable for cell growth and differentiation, especially crucial for cell reprogramming and subsequent cell type specialization. Each component has its specific role:

[0040] KnockOut DMEM medium: This is a commonly used, nutrient-rich cell culture medium that provides the basic sugars, vitamins, and inorganic salts required for cell growth. It is designed to support the growth and maintenance of a wide range of cell types, especially for cell types with strict requirements, such as stem cells.

[0041] bFGF growth factor (basic fibroblast growth factor): This is an important growth factor used to maintain cell proliferation and survival, especially for stem cells and other primary cells. bFGF can support cell pluripotency and promote growth during cell differentiation.

[0042] L-Aspartic Acid: This is a non-essential amino acid that is very important for supporting cell metabolism and protein biosynthesis. It can enhance the overall metabolic activity of cells.

[0043] Nicotinamide: A form of vitamin B3 that plays an important role in DNA repair, cell communication, and antioxidant defense, helping to maintain the healthy state of cells.

[0044] Serine: This is an essential amino acid that is very important for cell growth and maintenance, especially in a highly regulated cell environment.

[0045] Collagen: Usually used in the culture medium to improve cell attachment and growth environment, enhancing the structural support of the extracellular matrix.

[0046] ITS (combination of insulin, transferrin, and selenium): This is a supplement often used in serum-free culture conditions to promote cell growth and survival, especially in the culture of stem cells and primary cells.

[0047] L-Glutamine: As an important amino acid, it is indispensable in cell metabolism, especially in rapidly proliferating cell types.

[0048] HEPES buffer: Provides a stable pH environment, which is particularly important for cells that are easily damaged under unstable pH conditions.

[0049] Sodium pyrophosphate: Helps regulate the pH value and osmotic pressure of the culture medium, maintaining the stability of the cell environment.

[0050] These components of the modified KnockOut DMEM medium work together to provide an ideal environment for the normal growth, survival, and functional expression of cells. This medium is designed to maximize the support of the physiological needs of cells to achieve efficient cell culture and differentiation effects.

[0051] Differentiation of S4 into cardiomyocytes: Inductive differentiation culture: Select cells with normal metabolic activity and culture them using an inductive differentiation complex medium. When morphological changes are observed under a microscope, specifically, the cell morphology changes from fibroblast-like to round and clustered, and irregular beating or contraction behaviors are visible at the edge of the cell cluster, the inductive differentiation culture ends;

[0052] Maturation culture: Transfer the cells that have undergone inductive differentiation to a maturation complex medium for culture to obtain cardiomyocytes;

[0053] The inductive differentiation complex medium includes RPMI 1640 medium, Activin A, BMP4, Wnt3a, L-cysteine, niacin, and fibronectin;

[0054] The maturation complex medium includes B27 supplement and RPMI 1640 medium.

[0055] Each component in the inductive differentiation complex medium is carefully selected to provide the necessary biochemical signals and environmental conditions for the specific differentiation of stem cells into cardiomyocytes. The following are the functions of each component:

[0056] RPMI 1640 medium: This is a commonly used cell culture medium that provides essential nutrients, sugars, vitamins, and inorganic salts, supporting the basic metabolic activities of cells and maintaining the conditions required for cell growth.

[0057] Activin A: This is an important growth factor and a member of the TGF-β (transforming growth factor-β) superfamily. It can induce the differentiation of stem cells into endodermal cells, especially the precursor cells of the heart and other visceral organs.

[0058] BMP4: It is also part of the TGF-β family and is crucial for promoting cell fate determination during heart development. BMP4 is used to promote the differentiation of stem cells into cardiac myocytes.

[0059] Wnt3a: This is an important molecule in the Wnt signaling pathway that can regulate the self-renewal and differentiation of stem cells.

[0060] L-Cysteine: It is an important antioxidant amino acid that can help reduce oxidative stress and protect cells from damage during culture. During cell differentiation, reducing oxidative stress is very important for maintaining the differentiation ability and function of cells.

[0061] Niacin: Niacin (vitamin B3) plays an important role in maintaining the energy metabolism and DNA repair of cells. During cell differentiation, niacin helps maintain the energy requirements and genetic stability of cells.

[0062] Fibronectin: This is an extracellular matrix protein that helps cell attachment and migration. During the differentiation of cardiac myocytes, fibronectin can provide a supportive environment that helps cell alignment and the formation of tissue structure.

[0063] These components work together to provide a system that mimics the natural heart development environment for stem cells. By regulating and activating specific signaling pathways, they prompt stem cells to differentiate along the developmental route of cardiac myocytes, which is crucial for generating functional cardiac myocytes in the laboratory.

[0064] Preferably, in step S1, in the survival homeostasis solution, the concentration of sodium chloride is 137 mM; the concentration of sodium dihydrogen phosphate is 1.8 mM; the concentration of disodium hydrogen phosphate is 10 mM; the concentration of potassium chloride is 2.7 mM.

[0065] Preferably, in step S2, in the stem cell complex medium, the concentration of KSR serum substitute is 15 - 20%; the concentration of non-essential amino acids is 0.9 - 1 mM; the concentration of L-glutamine is 1.8 - 2 mM; the concentration of sodium thiosulfate is 0.09 - 0.1 mM; the concentration of NAC is 0.48 - 0.5 mM; the concentration of green tea extract is 0.01 - 0.015 mg / mL; the concentration of blueberry extract is 0.01 - 0.015 mg / mL; the concentration of N6-(2-hydroxyethyl)-lysine is 0.05 - 0.06 mM; the concentration of bFGF growth factor is 20 - 30 ng / mL; the antibiotic is at least one of penicillin and streptomycin, and the antifungal agent is acyclovir; the concentration of the antibiotic is 95 - 100 units / mL; the concentration of the antifungal agent is 0.9 - 1 μg / mL.

[0066] Specific parameter values are set to maximize the promotion of stem cell growth and maintain their undifferentiated state. The following is a detailed analysis of the reasons for parameter settings:

[0067] The concentration of KSR serum substitute is 15 - 20%: KSR (KnockOut Serum Replacement) is a non-animal-derived serum substitute used to maintain the undifferentiated state of stem cells and increase cell growth. This concentration range can effectively support cell proliferation and maintain their pluripotency. Too high or too low a concentration may affect the physiological state of these cells.

[0068] The concentration of non-essential amino acids is 0.9 - 1 mM: Non-essential amino acids are very important in the synthesis of peptides and proteins. This concentration range is sufficient to support the protein synthesis needs of cells without overstimulating cell metabolism.

[0069] The concentration of L-glutamine is 1.8 - 2 mM: L-glutamine is essential for cell growth and maintenance and is a key component of cell energy metabolism. This concentration range helps optimize cell energy utilization and healthy proliferation.

[0070] The concentration of β-mercaptoethanol is 0.09 - 0.1 mM: As an antioxidant for cells, this concentration is sufficient to reduce oxidative stress and protect the integrity of cell DNA and proteins.

[0071] The concentration of NAC is 0.48 - 0.5 mM: NAC is a potent antioxidant. This concentration can effectively protect cells from oxidative damage and improve cell viability and quality.

[0072] The concentrations of green tea extract and blueberry extract are each 0.01 - 0.015 mg / mL: These concentrations of plant extracts are rich in antioxidants, which can help reduce oxidative stress, support cell health, and at the same time provide microenvironment signals to help maintain the undifferentiated state of cells.

[0073] The concentration of N6-(2-hydroxyethyl)-lysine is 0.05 - 0.06 mM: This modified lysine helps stabilize the extracellular matrix and cell attachment, which is beneficial for maintaining the pluripotency of stem cells. This specific concentration helps enhance cell adhesion to the culture medium material, which is conducive to cell growth and expansion.

[0074] The concentration of bFGF growth factor is 20 - 30 ng / mL: bFGF is a key factor for cell growth and maintaining pluripotency. This concentration range optimizes its biological activity and promotes cell proliferation without inducing differentiation.

[0075] The concentrations of antibiotics and antifungal agents are 95 - 100 units / mL and 0.9 - 1 μg / mL respectively: These concentrations are sufficient to prevent bacterial and fungal contamination while minimizing the impact on cell growth and physiological state.

[0076] The setting of these parameters is based on the precise control of the cell growth environment under laboratory conditions, and each concentration has been optimized to ensure maximizing cell survival rate and quality.

[0077] Preferably, in step S2, the preparation process of green tea extract is as follows: Raw material preparation: Select dry green tea leaves;

[0078] Extraction process: Mix the green tea leaf powder with 80% ethanol at a ratio of 1:10 (g / mL); After stirring at room temperature for 6 hours, remove the solid residue by vacuum filtration and collect the extract; Concentration and drying: Use a rotary evaporator to remove ethanol to obtain a concentrated solution, and freeze-dry the concentrated solution to obtain green tea extract powder;

[0079] The preparation process of blueberry extract is as follows: Raw material preparation: Select fresh blueberries, wash them clean and store them frozen;

[0080] Extraction process: Crush the frozen blueberries into fine powder with liquid nitrogen, mix the blueberry powder with 60% ethanol at a ratio of 1:10 (g / mL), and stir for 24 hours under freezing conditions to maintain the stability of active ingredients; Filtration and concentration: Filter the mixture to remove the solid residue, use a rotary evaporator to remove the ethanol solvent and concentrate the extract; Drying: Spray-dry the concentrated liquid to obtain blueberry extract powder.

[0081] Preferably, in step S3, in the modified KnockOut DMEM medium, the concentration of bFGF growth factor is 25 - 30 ng / mL; the concentration of L-aspartic acid is 1.9 - 2 mM; the concentration of nicotinamide is 9 - 10 mM; the concentration of sericin is 0.5 - 0.55 mM; the concentration of ITS is 50 - 55 μg / mL; the concentration of collagen is 50 - 55 μg / mL, the concentration of L-glutamine is 4 - 4.2 mM; the concentration of HEPES buffer is 15 mM; the concentration of sodium pyrophosphate is 1.2 - 1.25 mM.

[0082] The concentration of bFGF growth factor is 25 - 30 ng / mL: Basic fibroblast growth factor (bFGF) is a key regulator of cell proliferation and survival. This concentration range helps to maintain the proliferation activity and pluripotency of stem cells while avoiding over-stimulating cell differentiation.

[0083] The concentration of L-aspartic acid is 1.9 - 2 mM: L-aspartic acid is a non-essential amino acid that is crucial for protein synthesis and cell metabolism. At the specified concentration, it supports cell growth and function, promoting a healthier cell metabolic state.

[0084] The concentration of nicotinamide is 9 - 10 mM: Nicotinamide, as a form of vitamin B3, plays an important role in DNA repair and cell metabolism. This concentration helps enhance the cell's antioxidant defense and energy metabolism, improving the overall viability and quality of the cells.

[0085] The concentration of selenite is 0.5 - 0.55 mM: Selenite is a precursor of selenocysteine and is crucial for the production of carbon monoxide and hydrogen sulfide in cells. An appropriate amount of selenite helps maintain the cell's redox balance and stress resistance.

[0086] The concentration of ITS (Insulin, Transferrin, Selenium) is 50 - 55 μg / mL: ITS is a commonly used cell culture additive for supporting cell growth and function. This concentration range ensures that cells receive sufficient nutrients and growth factors, promoting healthy cell growth.

[0087] The concentration of collagen is 50 - 55 μg / mL: Collagen is one of the main components of the extracellular matrix and is crucial for cell attachment and morphology maintenance. This concentration is beneficial for improving cell attachment and structural integrity, contributing to the stability of cell culture.

[0088] The concentration of L-glutamine is 4 - 4.2 mM: L-glutamine is a key amino acid in cell metabolism, providing energy for cells and supporting protein synthesis. This concentration helps optimize the cell's energy state and growth environment.

[0089] The concentration of HEPES buffer is 15 mM: HEPES is a biocompatible buffer system that helps maintain the pH stability of the culture medium. This concentration range can ensure the acid-base balance of the cell culture environment, promoting the normal operation of cell physiological functions.

[0090] The concentration of sodium pyrophosphate is 1.2 - 1.25 mM: Sodium pyrophosphate, as part of the buffer, helps maintain the pH value and osmotic pressure stability of the culture medium, which is crucial for the healthy growth of cells.

[0091] The setting of these concentrations is aimed at optimizing the cell growth conditions and maintaining good cell viability and physiological state.

[0092] Preferably, in step S4, in the induction differentiation complex medium, the concentration of Activin A is 110 - 120 ng / mL; the concentration of BMP4 is 9 - 10 ng / mL; the concentration of Wnt3a is 25 - 27 ng / mL; the concentration of VEGF is 50 - 54 ng / mL; the concentration of L-cysteine is 200 - 210 μM; the concentration of niacin is 500 - 550 μM; the concentration of fibronectin is 15 - 18 μg / mL; in the maturation complex medium, the concentration of B27 supplement is 2%.

[0093] The setting of each parameter helps to simulate the natural development environment and ensure that cells can differentiate and mature in the expected direction. The reasons for the setting of each parameter are as follows:

[0094] The concentration of Activin A is 110 - 120 ng / mL: Activin A is an important growth factor that can promote the formation of cardiac cells. During heart development, it is involved in regulating the formation and differentiation of cardiomyocytes. This concentration range helps to activate the endogenous signaling pathway and promote the differentiation of cardiomyocytes.

[0095] The concentration of BMP4 is 9 - 10 ng / mL: BMP4 is another key cell differentiation factor, especially in cardiac tissue engineering. BMP4 within this range can stimulate stem cells to differentiate along the cardiomyocyte pathway, and this concentration setting can maximize its effectiveness without overstimulating the cells.

[0096] The concentration of Wnt3a is 25 - 27 ng / mL: Wnt3a is an important molecule in the Wnt signaling pathway, which plays a key role in stem cell differentiation. The concentration of Wnt3a within this range can promote the formation and differentiation of cardiomyocyte precursors and help maintain the characteristics of cardiomyocytes.

[0097] The concentration of VEGF is 50 - 54 ng / mL: This concentration helps to promote angiogenesis in cultured cardiomyocytes, providing better nutrition and oxygen supply, thus supporting the survival and function of newly formed cardiac cells.

[0098] The concentration of L-cysteine is 200 - 210 μM: L-cysteine is an important antioxidant that can protect cells from oxidative stress damage. In cardiac cell culture, this concentration of cysteine helps to maintain the redox state of cells and optimize the differentiation process.

[0099] The concentration of niacin is 500 - 550 μM: Niacin (a form of vitamin B3) is a cofactor for cell metabolism and repair. Increasing the supply of niacin can enhance the energy metabolism and DNA repair ability of cells, helping to support the needs of rapidly differentiating cells.

[0100] Fibronectin concentration is 15 - 18 μg / mL: Fibronectin is a component of the extracellular matrix and is crucial for cell attachment and structural integration. During the differentiation of cardiac myocytes, this concentration of fibronectin helps form structurally intact cardiac tissue and enhance cell - cell connections.

[0101] The concentration of B27 supplement is 2%: The B27 supplement is a commonly used cell - culture additive that provides a range of vitamins, antioxidants, and growth factors, which are extremely important for the maturation and functional maintenance of cardiac myocytes. The use of this supplement helps provide an enriched environment to support the long - term survival and functional performance of cardiac cells.

[0102] These concentrations are set to optimize the culture conditions and ensure a high efficiency of stem - cell - to - cardiac - myocyte transformation.

[0103] Preferably, in step S4, the cut - off conditions for maturation culture are as follows: Verify the expression of cardiac - specific proteins in cells through immunofluorescence staining technology, clearly detect the positive expression of actin and myosin, the cells show continuous and regular beating behavior for at least 7 days, and have the action - potential characteristics of typical cardiac myocytes as confirmed by electrophysiological analysis.

[0104] Compared with the prior art, the beneficial effects of this method for directing the differentiation of stem cells into cardiac myocytes are as follows:

[0105] 1. Special culture - medium formula: All kinds of culture media used in the present invention, including the stem - cell composite culture medium, modified KnockOut DMEM culture medium, induction - differentiation composite culture medium, and maturation composite culture medium, are carefully designed and optimized. These culture media contain specific concentrations of growth factors, amino acids, vitamins, and other supplements such as bFGF, L - glutamine, NAC, ITS, etc. These components are the key to precisely controlling cell growth and differentiation.

[0106] 2. Improve differentiation efficiency and cell survival rate: By adjusting and optimizing the components in the culture medium, such as increasing natural plant extracts and specific growth factors, the differentiation efficiency of stem cells into cardiac myocytes can be significantly improved. For example, the bFGF growth factor is a known promoter of cell proliferation and survival, and specific plant extracts can provide antioxidant protection, thus enhancing the overall health and vitality of cells.

[0107] 3. Control and adjustability: In each step of the present invention, from cell collection, culture to differentiation, there are strict parameter controls, such as the composition and concentration of cell culture media, environmental conditions (temperature, CO2 concentration), etc., ensuring the stability and repeatability of the experimental process. This controllability makes the whole process adjustable and predictable, providing convenience for subsequent applications and research.

[0108] 4. Optimization and introduction of transcription factors: The use of lentiviral vectors to introduce key transcription factors (such as OCT4, SOX2, KLF4, and c-MYC) ensured high-efficiency genetic transduction and uniform cell transformation.

[0109] In summary, through the use of a carefully designed culture medium and step-by-step cell processing techniques, with the detailed formulation and operating conditions of each step aimed at maximizing the efficiency and quality of cell transformation, the present invention achieved efficient and controllable transformation from stem cells to cardiac myocytes. Detailed implementation mode

[0110] The following are specific examples of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these examples.

[0111] A method for directionally inducing the differentiation of stem cells into cardiac myocytes specifically includes the following steps:

[0112] S1 Cell collection and preliminary treatment:

[0113] Subject screening and preparation:

[0114] Ensure that the subject is at least 18 years old, has signed an informed consent form, and has no history of infectious diseases or major chronic diseases.

[0115] The subject needs to fast for 12 hours before blood sampling to reduce the lipid content in the blood and ensure the sample quality.

[0116] Blood sampling procedure:

[0117] Under sterile conditions, a professional medical staff uses a sterile blood collection needle to draw 10 - 20 milliliters of whole blood from the subject's vein.

[0118] Use a blood collection tube pre-filled with an anticoagulant (usually heparin or EDTA) to ensure that the blood sample does not coagulate.

[0119] Density gradient centrifugation separation:

[0120] Pretreatment solution: Mix the whole blood and the survival homeostasis solution in equal proportions. This step is used to dilute the blood, reduce cell adhesion, and improve the subsequent centrifugation effect.

[0121] Centrifuge tube preparation: The centrifuge tube is pre-filled with Ficoll-Paque PLUS, which is a density gradient medium used to effectively separate different cell components in the blood.

[0122] Centrifugation operation: Slowly pour the diluted blood into a centrifuge tube containing Ficoll-Paque, avoiding disturbing the mixed liquid. Set the centrifuge at room temperature, usually at a rotational speed of 400g for 30 minutes. This step aims to form distinct cell layers, where mononuclear cells will be in the ring layer between the Ficoll layer and the plasma layer.

[0123] Cell recovery and washing: Aspirate the ring layer cells: After centrifugation, carefully aspirate the middle ring layer, which contains the target peripheral blood mononuclear cells.

[0124] Transfer the cells: Transfer the ring layer cells to a new sterile centrifuge tube.

[0125] Washing: Add a sufficient amount of survival homeostasis solution to the sterile centrifuge tube and wash the cells by centrifugation (usually 300g for 10 minutes) to remove plasma and Ficoll residues. Repeat the washing step twice to ensure thorough cleaning.

[0126] The survival homeostasis solution includes pure water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride;

[0127] In the survival homeostasis solution, the concentration of sodium chloride is 137 mM; the concentration of sodium dihydrogen phosphate is 1.8 mM; the concentration of disodium hydrogen phosphate is 10 mM; the concentration of potassium chloride is 2.7 mM.

[0128] S2 cell culture: Cell seeding and initial culture: Seed the washed peripheral blood mononuclear cells into a primary culture container, usually a T75 culture flask.

[0129] Add the stem cell complex medium preheated to 37°C, which supports the survival and proliferation of early stem cells. The added amount is adjusted according to the cell density to ensure good cell contact and nutrient absorption.

[0130] Place the culture flask in a temperature-controlled incubator and set the conditions to 37°C and 5% CO 2 .

[0131] Medium optimization and maintenance: Every 24 to 48 hours, observe the cell morphology and growth conditions, and replace the medium if necessary. Remove the aged medium and add fresh medium.

[0132] During the culture process, regularly detect the pH value and cell contamination to ensure the stability of the culture environment and the health of the cells.

[0133] Cell proliferation and subculturing: When the cells reach 80%-90% confluence, perform subculturing. Gently digest the cells with a 0.25% trypsin-EDTA solution, and neutralize the digestion with the survival homeostasis solution after separating the cells.

[0134] Resuspend the cells in fresh medium and distribute them into new culture flasks at an appropriate ratio to maintain cell growth and expansion.

[0135] Cell growth monitoring and quality control: Regularly examine the cell morphology using an optical microscope and pay attention to whether the cells show typical morphological characteristics of stem cells, such as a high nucleus-cytoplasm ratio and a tight cell cluster.

[0136] Verify the stem cell characteristics of the cells by flow cytometry analysis of surface markers, such as using markers like SSEA-4, TRA-1-60, etc.

[0137] Adjustment of cell culture conditions: Adjust the culture conditions according to the cell growth rate and morphological changes, such as the composition of the medium, the adjustment of the gas environment, and the size of the culture vessel.

[0138] In the stem cell complex medium, the concentration of KSR serum replacement is 15 - 20%; the concentration of non-essential amino acids is 0.9 - 1 mM; the concentration of L-glutamine is 1.8 - 2 mM; the concentration of sodium thiosulfate is 0.09 - 0.1 mM; the concentration of NAC is 0.48 - 0.5 mM; the concentration of green tea extract is 0.01 - 0.015 mg / mL; the concentration of blueberry extract is 0.01 - 0.015 mg / mL; the concentration of N6-(2-hydroxyethyl)-lysine is 0.05 - 0.06 mM; the concentration of bFGF growth factor is 20 - 30 ng / mL; the antibiotic is at least one of penicillin and streptomycin, and the antifungal agent is acyclovir; the concentration of the antibiotic is 95 - 100 units / mL; the concentration of the antifungal agent is 0.9 - 1 μg / mL.

[0139] The preparation process of green tea extract is as follows: Raw material preparation: Select dry green tea leaves; Extraction process: Mix the green tea leaf powder with 80% ethanol at a ratio of 1:10 (g / mL); After stirring at room temperature for 6 hours, remove the solid residue by vacuum filtration and collect the extract; Concentration and drying: Remove ethanol using a rotary evaporator to obtain a concentrated solution, and freeze-dry the concentrated solution to obtain green tea extract powder;

[0140] The preparation process of blueberry extract is as follows: Raw material preparation: Select fresh blueberries, wash them clean, and store them frozen;

[0141] Extraction process: Crush the frozen blueberries into fine powder with liquid nitrogen, mix the blueberry powder with 60% ethanol at a ratio of 1:10 (g / mL), and stir for 24 hours under frozen conditions to maintain the stability of the active ingredients; Filtration and concentration: Filter the mixture to remove the solid residue, use a rotary evaporator to remove the ethanol solvent, and concentrate the extract; Drying: Spray-dry the concentrated liquid to obtain blueberry extract powder.

[0142] S3: Introduction of Transcription Factors: Concentration Adjustment: Determine the titer of lentiviral particles carrying transcription factors using an infectivity unit assay (such as real-time PCR quantification analysis), and adjust the concentration to 10^8 infectivity units / mL to ensure sufficient transfection efficiency and cell safety. The transcription factors are OCT4, SOX2, KLF4, and c-MY;

[0143] Cell Preparation and Lentivirus Addition: Before introducing transcription factors, carefully examine the cells to ensure they are in good growth state and metabolic activity. Use an automated cell counter to count the cells and adjust the cell density to approximately 1×10^5 cells / mL.

[0144] Add the lentiviral suspension with adjusted concentration evenly into the culture container. To improve transfection efficiency, polyethyleneimine (PEI) or other non-viral transfection enhancers can be added.

[0145] Incubate the cells at 37°C and 5% CO 2 2. The lentiviral particles will infect the cells and introduce the genes of transcription factors into the cell genome.

[0146] Monitoring and Maintenance after Transfection: Incubate the cells for at least 24 hours after adding the lentivirus, and regularly observe the morphological changes and survival status of the cells during this period. For the lentiviral transfection system, sometimes a mild cytotoxic reaction may be observed.

[0147] After 24 hours of transfection, gently aspirate the medium containing the lentivirus and replace it with pre-warmed modified KnockOut DMEM medium to support cell recovery and further proliferation.

[0148] Evaluation of Transfection Efficiency: Detect the transfection efficiency using a flow cytometer by analyzing the expression of transfected cell surface markers or endogenous fluorescent proteins. For example, lentivirus carrying green fluorescent protein (GFP) tags can be used to monitor the transfection efficiency.

[0149] Adjust the parameters of subsequent experiments according to the results of transfection efficiency, such as the concentration of lentivirus, the seeding density of cells, or the transfection time.

[0150] In the improved KnockOut DMEM medium, the concentration of bFGF growth factor is 25 - 30 ng / mL; the concentration of L-aspartic acid is 1.9 - 2 mM; the concentration of nicotinamide is 9 - 10 mM, the concentration of sericin is 0.5 - 0.55 mM, the concentration of ITS is 50 - 55 μg / mL, the concentration of collagen is 50 - 55 μg / mL, the concentration of L-glutamine is 4 - 4.2 mM, the concentration of HEPES buffer is 15 mM, and the concentration of sodium pyrophosphate is 1.2 - 1.25 mM.

[0151] S4: Differentiation into cardiomyocytes:

[0152] Induction differentiation stage:

[0153] Cell selection and preparation: Select cells that have been successfully transformed with transcription factors in stage S3 and exhibit normal metabolic activity. Under sterile conditions, use an automatic cell counter to measure the cell density and adjust the cells to the required concentration, usually about 100,000 cells per well.

[0154] Differentiation induction: After cell seeding, add the induction differentiation medium. This step is particularly important because components in the medium such as Activin A, BMP4, and Wnt3a are key factors for inducing cardiomyocyte differentiation. These factors play a role in activating the cell fate determination pathway in the initial few days of culture.

[0155] Observation and monitoring: In the early stage of induction differentiation, the changes in cell morphology need to be carefully monitored. From the 2nd to 4th day of cell culture, the cell morphology changes from fibroblast-like to a more rounded and clustered arrangement. Use an inverted microscope to observe the changes in cell morphology and the formation of clusters every day.

[0156] Functional evaluation: From the 4th to 7th day of differentiation, look for beating or contracting behavior at the edge of cell clusters, which is the initial manifestation of cardiomyocyte functionality. At this time, specific staining or labeling techniques (such as calcium imaging) can be used to evaluate the electrophysiological properties of the cells.

[0157] Mature culture stage: Medium replacement: After the induction differentiation is completed, that is, after the cells show beating or contracting behavior, aspirate the old differentiation medium and add the mature complex medium, which is rich in B27 supplement, to support the maturation and long-term survival of cardiomyocytes.

[0158] Long-term culture: Continue to culture the cells in the mature medium for at least 7 to 14 days. During this period, the cardiomyocytes will continue to mature and exhibit more stable and synchronous beating behavior. Regularly change the medium to maintain constant nutritional and environmental conditions.

[0159] The cut-off conditions for maturation culture are as follows: Verify the expression of cardiomyocyte-specific proteins in cells through immunofluorescence staining technology, clearly detect the positive expression of actin and myosin, the cells show continuous and regular beating behavior for at least 7 days, and the action potential characteristics of typical cardiac muscle cells are confirmed through electrophysiological analysis.

[0160] Functional and morphological evaluation: Observe and record the morphological changes and functional beating of cells using a microscope. The ion channel function and synchronous beating ability of cells can be further evaluated through electrophysiological techniques such as patch clamp technique.

[0161] Data recording and analysis: Record all observed data in detail, including morphological changes, beating frequency, and synchrony. These data are crucial for evaluating differentiation efficiency and optimizing the protocol.

[0162] In the induction differentiation complex medium, the concentration of Activin A is 110 - 120 ng / mL; the concentration of BMP4 is 9 - 10 ng / mL; the concentration of Wnt3a is 25 - 27 ng / mL; the concentration of VEGF is 50 - 54 ng / mL; the concentration of L-cysteine is 200 - 210 μM; the concentration of niacin is 500 - 550 μM; the concentration of fibronectin is 15 - 18 μg / mL;

[0163] In the maturation complex medium, the concentration of B27 supplement is 2%.

[0164] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar ways to substitute, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for inducing stem cells to differentiate into cardiac myocytes, characterized in that: The method for directing the differentiation of stem cells into cardiac muscle cells comprises the following steps: S1 Cell collection and preliminary processing: Use a sterile blood collection needle to draw several milliliters of whole blood from the subject's vein, store it in a blood collection tube containing an anticoagulant, and transport the collected whole blood to the laboratory in an environment controlled at 4°C; Peripheral blood mononuclear cells are separated using density gradient centrifugation: whole blood is mixed with a survival steady-state solution, poured into a centrifuge tube pre-filled with Ficoll-Paque PLUS, and then centrifuged to form a chromatogram; the middle ring layer is aspirated and transferred to a new sterile centrifuge tube; stem cells in the blood are screened and collected using a flow cytometer and specific cell surface markers (such as CD34+). After adding the survival steady-state solution to a new sterile centrifuge tube, wash the tube by centrifugation to remove the plasma and Ficoll residues; The survival steady-state solution includes pure water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride; In the survival steady-state solution, the concentration of sodium chloride is 137 mM; the concentration of sodium dihydrogen phosphate is 1.8 mM; the concentration of sodium dihydrogen phosphate is 10 mM; and the concentration of potassium chloride is 2.7 mM. S2 cell culture: The washed cells are placed in a stem cell complex medium and cultured in an environment with a temperature of 37°C and a carbon dioxide concentration of 5%; the stem cell complex medium includes DMEM / F-12 nutrient solution, KSR serum replacement, non-essential amino acids, L-glutamine, β-sodium thiosulfate, NAC, a natural plant extract mixture, N6-(2-hydroxyethyl)-lysine, a matrix metalloproteinase inhibitor, bFGF growth factor, antibiotics and antifungal agents; the natural plant extract mixture includes green tea extract and blueberry extract; S3: Introduction of transcription factors: Concentration adjustment: The concentration of lentiviral particles carrying transcription factors was adjusted to 10^8 infectious units / mL through an infectious unit determination experiment; a lentiviral suspension was obtained; the transcription factors were OCT4, SOX2, KLF4 and c-MY; Suspension addition: Select cells showing normal metabolic activity, count and adjust cell density using an automatic cell counter; then add lentiviral suspension to the cells. During the addition process, add polyethyleneimine as a transfection enhancer to improve transfection efficiency; incubate at a temperature of 37°C and a carbon dioxide concentration of 5% for more than 24 hours; Replacement of culture medium and cell recovery: After the incubation, remove the stem cell complex culture medium and replace it with modified KnockOut DMEM culture medium, and culture in an environment with a temperature of 37°C and a carbon dioxide concentration of 5%; S4 differentiation into cardiac myocytes: Induced differentiation culture: cells showing normal metabolic activity are selected and cultured using an induced differentiation complex medium. When morphological changes are observed under a microscope, specifically, the cell morphology changes from fiber-like to round, clustered, and irregular beating or contraction behavior is observed at the edge of the cell cluster, the induced differentiation culture is terminated; Maturation culture: The induced differentiated cells are transferred to a maturation complex culture medium for culture to obtain cardiac myocytes. The differentiation-inducing complex medium includes RPMI 1640 medium, Activin A, BMP4, Wnt3a, L-cysteine, niacin, and fibronectin; The maturation complex medium includes B27 supplement and RPMI 1640 medium.

2. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S1, in the survival steady-state solution, the concentration of sodium chloride is 137 mM; the concentration of sodium dihydrogen phosphate is 1.8 mM; the concentration of sodium dihydrogen phosphate is 10 mM; and the concentration of potassium chloride is 2.7 mM.

3. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S2, in the stem cell complex culture medium, the concentration of KSR serum replacement is 15-20%; the concentration of non-essential amino acids is 0.9-1mM; the concentration of L-glutamine is 1.8-2mM; the concentration of β-sodium thiosulfate is 0.09-0.1mM; the concentration of NAC is 0.48-0.5mM; the concentration of green tea extract is 0.01-0.015mg / mL; the concentration of blueberry extract is 0.01-0.015mg / mL; the concentration of N6-(2-hydroxyethyl)-lysine is 0.05-0.06mM; the concentration of bFGF growth factor is 20-30ng / mL; the antibiotic is at least one of penicillin and streptomycin, and the antifungal agent is acyclovir; the antibiotic concentration is 95-100units / mL; and the antifungal agent concentration is 0.9-1μg / mL.

4. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S2, the green tea extract preparation process is as follows: raw material preparation: selecting dry green tea leaves; extraction process: mixing green tea leaf powder with 80% ethanol at a ratio of 1:10 (g / mL); after stirring at room temperature for 6 hours, removing solid residues by vacuum filtration and collecting the extract; concentration and drying: using a rotary evaporator to remove ethanol to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain green tea extract powder; The preparation process of blueberry extract is as follows: Raw material preparation: select fresh blueberries, clean them, and freeze them; Extraction process: Frozen blueberries were crushed into fine powder using liquid nitrogen, and the blueberry powder was mixed with 60% ethanol at a ratio of 1:10 (g / mL) and stirred for 24 hours under freezing conditions to maintain the stability of the active ingredients; Filtration and concentration: The mixture was filtered to remove solid residues, and the ethanol solvent was removed using a rotary evaporator to concentrate the extract; Drying: The concentrated liquid is spray-dried to obtain blueberry extract powder.

5. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S3, in the modified KnockOut DMEM medium, the bFGF growth factor concentration is 25-30 ng / mL; the L-aspartame acid concentration is 1.9-2 mM; the nicotinamide concentration is 9-10 mM, the serline concentration is 0.5-0.55 mM, the ITS concentration is 50-55 μg / mL, the collagen concentration is 50-55 μg / mL, the L-glutamine concentration is 4-4.2 mM, the HEPES buffer concentration is 15-17 mM, and the sodium pyrophosphate concentration is 1.2-1.25 mM.

6. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S4, in the differentiation induction complex medium, the concentration of Activin A is 110-120 ng / mL; the concentration of BMP4 is 9-10 ng / mL; the concentration of Wnt3a is 25-27 ng / mL; the concentration of VEGF is 50-54 ng / mL; the concentration of L-cysteine ​​is 200-210 μM; the concentration of niacin is 500-550 μM; and the concentration of fibronectin is 15-18 μg / mL; In the maturation complex medium, the concentration of B27 supplement was 2%.

7. The method for directed induction of stem cell differentiation into cardiac myocytes according to claim 1, characterized in that: In step S4, the cutoff conditions for mature culture are as follows: the expression of myocardial-specific proteins in the cells is verified by immunofluorescence staining, the positive expression of actin and myosin is clearly detected, the cells exhibit sustained and regular beating behavior for at least 7 days, and the action potential characteristics of typical cardiac myocytes are confirmed by electrophysiological analysis.