Preparation method, purification method and application of myocardial cells derived from induced pluripotent stem cells

Abstract

The invention provides a preparation method, a purification method and a culture medium combination of myocardial cells derived from induced pluripotent stem cells, the prepared myocardial cells and application of the myocardial cells, the preparation method comprises the following steps: respectively treating the induced pluripotent stem cells by using M1, M2A, M2B, M3 and M4 culture mediums, the M2A culture medium comprises the following steps: adding a first Wnt pathway activator into the M1 culture medium, adding a second Wnt pathway activator into the M3 culture medium, and adding a second Wnt pathway activator into the M4 culture medium; the M2B culture medium is prepared by adding a second Wnt pathway activator into the M1 culture medium; the purification method comprises the step of treating a myocardial cell culture with an M5 culture medium, wherein the M5 culture medium comprises L-sodium lactate. The myocardial cells can be efficiently and stably differentiated from the induced pluripotent stem cells by using the preparation method and the purification method disclosed by the invention, and the myocardial cells prepared by using the method or the culture medium combination disclosed by the invention are relatively high in quality and high in purity, and can fully support downstream research and application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method, a purification method and an application of cardiomyocytes derived from induced pluripotent stem cells. Background Art

[0002] In recent years, induced pluripotent stem cell (iPSC) technology has rapidly developed and has played a significant role in research into the pathogenesis, pathological processes, and drug screening of a wide range of diseases. One advantage of iPSCs is that they can be generated without the need for human embryos, thus avoiding the ethical issues and controversies surrounding embryonic stem cells. Furthermore, CRISPR / Cas9 gene editing technology allows for the convenient repair or insertion of mutations, generating control cell lines with the same genotype as iPSCs, thus enhancing the rigor of scientific research. The advent of iPSCs and the rapid development of this field have opened our eyes to the endless possibilities of using human cells to establish novel disease models for research.

[0003] Cardiovascular disease poses a serious threat to human health. Limited cardiomyocyte regenerative capacity is a major contributor to the high morbidity and mortality rates associated with heart disease. While clinical efforts seek reliable sources of cardiomyocytes, researchers are also committed to exploring the pathological mechanisms of cardiomyopathy and conducting reliable drug screening. In recent years, establishing disease models and conducting scientific research using cardiomyocytes derived from iPS cells has become a hot topic in the stem cell and cardiovascular fields.

[0004] However, despite the huge potential of iPSC in the study of cardiomyocyte differentiation and cardiovascular diseases, current technology still faces a series of challenges that restrict its widespread application in clinical and laboratory research. First, the differentiation efficiency of iPSC into cardiomyocytes is still low, and the cell purity and stability during the differentiation process are poor, resulting in suboptimal function and morphology of the obtained cardiomyocytes, and low cardiomyocyte preparation yield. Secondly, the process of iPSC differentiation into cardiomyocytes usually takes a long time, and there are still certain difficulties in cell yield and quality control, which limits its efficient application in disease models and drug screening. Furthermore, since incomplete or immature myocardial phenotypes often appear during the differentiation of iPSC into cardiomyocytes, these incompletely differentiated cells may not provide sufficient biological validity in practical applications, thereby affecting the authenticity of the disease model and the effectiveness of drug screening. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a preparation method, a purification method, a culture medium combination, the prepared cardiomyocytes and applications thereof derived from induced pluripotent stem cells.

[0006] One of the objectives of the present invention is to provide a method for preparing cardiomyocytes derived from induced pluripotent stem cells, comprising the following steps:

[0007] (1) expanding and culturing induced pluripotent stem cells to obtain a culture containing the expanded induced pluripotent stem cells;

[0008] (2) inducing cardiomyocyte differentiation of the expanded induced pluripotent stem cells obtained in step (1), comprising:

[0009] (2-1) Discard the culture medium obtained in step (1), add M2A medium, and culture for 22 to 26 hours;

[0010] (2-2) Discard the culture medium from step (2-1), add M2B culture medium, and culture for 44 to 52 hours, changing the medium every 22 to 26 hours;

[0011] (2-3) Discard the culture medium from step (2-2), add M3 culture medium, and culture for 44 to 52 hours, changing the medium every 22 to 26 hours;

[0012] (2-4) Discard the culture medium from step (2-3), add M1 culture medium, and culture for 66 to 78 hours, changing the medium every 22 to 26 hours;

[0013] (2-5) Discard the culture medium from step (2-4), add M4 culture medium, and culture for 22 to 26 hours;

[0014] (2-6) Discard the culture medium from step (2-5), perform digestion and termination treatment, inoculate into new wells, and culture with resurfacing medium for 22 to 26 hours;

[0015] (2-7) Obtaining induced differentiated cardiomyocytes;

[0016] Among them, M1 medium includes RPMI 1640 medium and B-27 supplement without insulin;

[0017] M2A medium includes the addition of a first Wnt pathway activator to M1 medium;

[0018] M2B medium consists of adding a second Wnt pathway activator to M1 medium;

[0019] M3 medium includes the addition of a first Wnt pathway inhibitor to M1 medium;

[0020] M4 medium consists of RPMI 1640 medium and B27 serum-free supplement;

[0021] wherein the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):1;

[0022] Preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1.8-20):1;

[0023] More preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (2.25-4.5):1;

[0024] More preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is 3:1.

[0025] Furthermore, the final concentration of the first Wnt pathway activator is 5 to 10 μM, and the final concentration of the second Wnt pathway activator is 0.5 to 5 μM;

[0026] Preferably, the final concentration of the first Wnt pathway activator is 8 to 10 μM, and the final concentration of the second Wnt pathway activator is 0.5 to 5 μM;

[0027] More preferably, the final concentration of the first Wnt pathway activator is 9 μM, and the final concentration of the second Wnt pathway activator is 2 to 4 μM;

[0028] More preferably, the final concentration of the first Wnt pathway activator is 9 μM, and the final concentration of the second Wnt pathway activator is 3 μM;

[0029] Furthermore, the first Wnt pathway activator and the second Wnt pathway activator each include any one selected from CHIR 99021, BML-284, HLY78, SKL2001, and CP21R7;

[0030] Preferably, the first Wnt pathway activator is CHIR 99021, and the second Wnt pathway activator is CHIR 99021.

[0031] Furthermore, the first Wnt pathway inhibitor is IWR-1; preferably, the final concentration of IWR-1 is 5 to 15 μM, more preferably 10 μM.

[0032] Furthermore, induced pluripotent stem cells include human induced pluripotent stem cells.

[0033] Furthermore, in the above step (2-1), the culture is carried out for about 1 day, preferably, 22 to 26 hours; more preferably, 23 to 25 hours; more preferably, 24 hours;

[0034] Furthermore, in the above step (2-2), the culture is carried out for about 2 days, preferably, 44 to 52 hours, more preferably, 46 to 50 hours; more preferably, 48 hours;

[0035] Furthermore, in the above step (2-3), the culture is carried out for about 2 days, preferably, 44 to 52 hours, more preferably, 46 to 50 hours; more preferably, 48 hours;

[0036] Furthermore, in the above steps (2-4), the culture is carried out for about 3 days, preferably, for 66 to 78 hours, more preferably for 69 to 75 hours, and more preferably for 72 hours;

[0037] Furthermore, in the above steps (2-2), (2-3) and (2-4), the liquid is changed every approximately 1 day, preferably every 22 to 26 hours; more preferably, every 23 to 25 hours; more preferably, every 24 hours.

[0038] Furthermore, in the above steps (2-5), the culture is carried out for about 1 day, preferably, for 22 to 26 hours; more preferably, for 23 to 25 hours; more preferably, for 24 hours;

[0039] Furthermore, in the above steps (2-6), the culture is carried out for about 1 day, preferably, for 22 to 26 hours; more preferably, for 23 to 25 hours; more preferably, for 24 hours.

[0040] Furthermore, the above step (1) specifically includes:

[0041] (1-1) When the cell confluence of the induced pluripotent stem cells in normal subculture reaches 80-90%, digestion treatment is performed and plating is performed using a plating medium, which includes E8 medium and Y27632;

[0042] (1-2) Discard the plating medium from step (1-1) and add E8 medium;

[0043] (1-3) After culturing for 26 to 28 hours, when the cell confluence of the induced pluripotent stem cells reaches 90 to 100%, a culture containing the expanded induced pluripotent stem cells is obtained, and the cell confluence of the expanded induced pluripotent stem cells obtained at this time is used as the starting cell confluence;

[0044] Preferably, the cell density corresponding to the initial cell confluence is 3×10^5 cells / cm 2 ~4×10^5cells / cm 2 ;

[0045] More preferably, the cell density corresponding to the initial cell confluence is 3×10^5 cells / cm2 .

[0046] A second object of the present invention is to provide a method for purifying cardiomyocytes derived from induced pluripotent stem cells, comprising:

[0047] 1) Discard all culture medium from the culture containing cardiomyocytes;

[0048] 2) Add M5 medium and continue culturing, changing the medium every 44 to 52 hours. Observe cell morphology 22 to 26 hours after each medium change.

[0049] The M5 medium includes sodium L-lactate, and the final concentration of sodium L-lactate is 5 to 12 mM;

[0050] Preferably, the final concentration of sodium L-lactate is 5 to 8 mM;

[0051] More preferably, the final concentration of sodium L-lactate is 5 mM.

[0052] Furthermore, the culture containing cardiomyocytes in the above step 1) is the induced differentiated cardiomyocytes obtained in step (2-7) of the above method for preparing cardiomyocytes derived from induced pluripotent stem cells.

[0053] Furthermore, the above step 1) specifically includes: discarding all the culture medium of the induced differentiated cardiomyocytes obtained in steps (2-7).

[0054] Furthermore, in the above step 2), the liquid is changed every 2 days, preferably, every 44 to 52 hours; more preferably, every 46 to 50 hours; more preferably, every 48 hours;

[0055] Furthermore, in the above step 2), the cell morphology is observed about 1 day after each medium change. Preferably, the cell morphology is observed 22 to 26 hours after each medium change; more preferably, the cell morphology is observed 23 to 25 hours after each medium change; more preferably, the cell morphology is observed 24 hours after each medium change.

[0056] Furthermore, the M5 medium is any one of the following ① to ③:

[0057] ①M5 medium includes RPMI 1640 sugar-free medium and sodium L-lactate;

[0058] ②M5 culture medium includes DMEM sugar-free medium and sodium L-lactate;

[0059] ③M5 culture medium includes DMEM sugar-free and glutamine-free medium and sodium L-lactate;

[0060] Preferably, the M5 culture medium comprises DMEM sugar-free and glutamine-free culture medium and sodium L-lactate.

[0061] Furthermore, the method for purifying cardiomyocytes provided by the present invention further comprises step 3) identifying cardiomyocytes and / or step 4) freezing cardiomyocytes.

[0062] Furthermore, induced pluripotent stem cells include human induced pluripotent stem cells.

[0063] A third object of the present invention is to provide a culture medium composition for preparing cardiomyocytes from induced pluripotent stem cells, comprising:

[0064] M1 medium;

[0065] M2A medium;

[0066] M2B medium;

[0067] M3 medium;

[0068] and M4 medium;

[0069] Among them, M1 medium includes RPMI 1640 medium and B-27 supplement without insulin;

[0070] M2A medium includes the addition of a first Wnt pathway activator to M1 medium;

[0071] M2B medium consists of adding a second Wnt pathway activator to M1 medium;

[0072] M3 medium includes the addition of a first Wnt pathway inhibitor to M1 medium;

[0073] M4 medium consists of RPMI 1640 medium and B27 serum-free supplement;

[0074] wherein the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):1;

[0075] Preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1.8-20):1;

[0076] More preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (2.25-4.5):1;

[0077] More preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is 3:1.

[0078] Furthermore, the final concentration of the first Wnt pathway activator is 5 to 10 μM, and the final concentration of the second Wnt pathway activator is 0.5 to 5 μM;

[0079] Preferably, the final concentration of the first Wnt pathway activator is 8 to 10 μM, and the final concentration of the second Wnt pathway activator is 0.5 to 5 μM;

[0080] More preferably, the final concentration of the first Wnt pathway activator is 9 μM, and the final concentration of the second Wnt pathway activator is 2 to 4 μM;

[0081] More preferably, the final concentration of the first Wnt pathway activator is 9 μM, and the final concentration of the second Wnt pathway activator is 3 μM;

[0082] Furthermore, the first Wnt pathway activator and the second Wnt pathway activator each include any one selected from CHIR 99021, BML-284, HLY78, SKL2001, and CP21R7;

[0083] Preferably, the first Wnt pathway activator is CHIR 99021, and the second Wnt pathway activator is CHIR 99021.

[0084] Furthermore, the first Wnt pathway inhibitor is IWR-1; preferably, the final concentration of IWR-1 is 5 to 15 μM, more preferably 10 μM.

[0085] Furthermore, the culture medium combination provided by the present invention also includes M5 culture medium;

[0086] Wherein, M5 medium includes sodium L-lactate, and the final concentration of sodium L-lactate is 5-12 mM;

[0087] Preferably, the final concentration of sodium L-lactate is 5 to 8 mM;

[0088] More preferably, the final concentration of sodium L-lactate is 5 mM;

[0089] Furthermore, the M5 medium is any one of the following ① to ③:

[0090] ①M5 medium includes RPMI 1640 sugar-free medium and sodium L-lactate;

[0091] ②M5 culture medium includes DMEM sugar-free medium and sodium L-lactate;

[0092] ③M5 culture medium includes DMEM sugar-free and glutamine-free medium and sodium L-lactate;

[0093] Preferably, the M5 culture medium comprises DMEM sugar-free and glutamine-free culture medium and sodium L-lactate.

[0094] Furthermore, induced pluripotent stem cells include human induced pluripotent stem cells.

[0095] A fourth object of the present invention is to provide a cardiomyocyte prepared using the aforementioned cardiomyocyte preparation method, the aforementioned purification method, or the aforementioned culture medium combination.

[0096] A fifth object of the present invention is to provide a method for preparing cardiomyocytes, a method for purifying cardiomyocytes, or a use of the above-mentioned culture medium combination in preparing cardiomyocytes;

[0097] Preferably, the cardiomyocytes are cardiomyocytes derived from induced pluripotent stem cells;

[0098] Preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells.

[0099] A sixth object of the present invention is to provide a method for preparing the aforementioned cardiomyocytes, a purification method, a culture medium combination, or a use of the aforementioned cardiomyocytes in preparing a reagent for lentiviral infection or ASO transfection.

[0100] The preparation method, purification method, culture medium combination, prepared cardiomyocytes and their applications for induced pluripotent stem cell-derived cardiomyocytes provided by the present invention have the following advantages:

[0101] 1. The present invention has developed a preparation method for efficiently differentiating induced pluripotent stem cells into cardiomyocytes. Through in-depth research on the Wnt signaling pathway, the provided cardiomyocyte preparation method uses a combined dose of the Wnt pathway activator CHIR99021 to continuously treat iPSCs. Compared with the existing technology, it can stably and efficiently differentiate cardiomyocytes; the differentiated cardiomyocytes can be seen beating as early as the sixth day of differentiation, and the beating gradually increases with the passage of differentiation time.

[0102] 2. The cardiomyocyte preparation method provided by the present invention begins to add differentiation medium when the cell confluence rate is close to 100%, which can improve the phenomenon of cell shedding during differentiation; the cells are in a high-density state at the beginning of differentiation. In the present invention, the corresponding differentiation medium is replaced every day to ensure that the cells have sufficient nutrient sources. Therefore, the relative density of the cardiomyocytes finally harvested is high, and the cell density of a single well after re-plating can reach 1-2×10^5cell / cm 2 , that is, obtaining cardiomyocytes with large yield and high purity.

[0103] 3. In the cardiomyocyte preparation and purification methods provided herein, the proportion of cTnT-positive cells can reach at least 70% before purification, and this percentage can be increased to over 90% within just 3-5 days of purification. Conversely, if the purity before purification is only 30%, the purification process will likely take longer than 5 days. The cardiomyocyte preparation and purification methods provided herein save time and cost, enabling efficient harvesting of cardiomyocytes.

[0104] 4. In the cardiomyocyte preparation and purification method provided by the present invention, sodium lactate with a final concentration of 5 to 12 mM is used in the purification step, which can not only achieve the purpose of removing foreign cells, but also ensure the healthy growth state and strong beating ability of cardiomyocytes.

[0105] 5. The proportion of cTnT-positive cells in the cardiomyocytes prepared by the method of the present invention was as high as over 95% by flow cytometry, and cTnT and α-actinin2 were also significantly expressed by immunofluorescence. Patch clamp results showed that the cardiomyocytes had good electrophysiological effects. In summary, the cardiomyocytes prepared by the method of the present invention are of high quality and can fully support downstream research and applications.

[0106] 6. The cardiomyocytes prepared by the method of the present invention can achieve good experimental results in applications such as lentiviral infection, siRNA, and ASO transfection, and the infected or transfected cardiomyocytes can still achieve good beating, which further demonstrates that the prepared cardiomyocytes have high activity, uniformity, and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Schematic diagram of the myocardial cell differentiation and purification process of the present invention

[0108] Figure 2 Induction effect of a single dose of CHIR 99021 (A. Group 1-1, 3 μM CHIR 99021; B. Group 1-2, 4 μM CHIR 99021; C. Group 1-3, 5 μM CHIR 99021; D. Group 1-4, 6 μM CHIR 99021; E. Group 1-5, 7 μM CHIR 99021; F. Group 1-6, 8 μM CHIR 99021; G. Group 1-7, 9 μM CHIR 99021; H. Group 1-8, 10 μM CHIR 99021)

[0109] Figure 3 Induction effect of combined dosages of CHIR 99021 (A. Group 1-9, CHIR 99021 at 9 μM and 0.5 μM; B. Group 1-10, CHIR 99021 at 9 μM and 1 μM; C. Group 1-11, CHIR 99021 at 9 μM and 2 μM; D. Group 1-12, CHIR 99021 at 9 μM and 3 μM; E. Group 1-13, CHIR 99021 at 9 μM and 4 μM; F. Group 1-14, CHIR 99021 at 9 μM and 5 μM)

[0110] Figure 4Effects of different purification media on myocardial cell purification (A. Before purification; B. Purification medium 1; C. Purification medium 2; D. Purification medium 3; E. Purification medium 9)

[0111] Figure 5 Comparison of cardiomyocyte purity obtained from different starting cell confluences (A. low confluence density; B. medium confluence density; C. high confluence density)

[0112] Figure 6 Bright field image of the cardiomyocytes prepared by the present invention

[0113] Figure 7 Immunofluorescence identification results of cardiomyocytes prepared by the present invention (A. using cTnT as a cardiomyocyte marker; B. using α-Actinin 2 as a cardiomyocyte marker)

[0114] Figure 8 Cell purity test results of the cardiomyocytes prepared by the present invention

[0115] Figure 9 Immunofluorescence imaging results of cardiomyocytes prepared by the present invention one week after lentivirus infection (A. Inverted fluorescence microscope imaging results (bright field); B. Inverted fluorescence microscope imaging results)

[0116] Figure 10 Immunofluorescence imaging results of cardiomyocytes prepared by the present invention 48 hours after ASO-FAM transfection (A. Inverted fluorescence microscope imaging results (bright field); B. Inverted fluorescence microscope imaging results)

[0117] Figure 11 Comparison of the Differentiation Effects of the Differentiation Method of the Present Invention and the Prior Art (A. to E. Differentiation Method of the Present Invention; F. to J. Differentiation Method of the Prior Art) DETAILED DESCRIPTION

[0118] The technical solution of the present invention is further described below based on specific embodiments. The protection scope of the present invention is not limited to the following embodiments, which are listed for illustrative purposes only and do not limit the present invention in any way.

[0119] The experimental methods or parameters without specific conditions in the examples are generally conventional means in the art, or are in accordance with the methods or conditions recommended or disclosed by the reagent manufacturers.

[0120] Glossary

[0121] iPSC: induced pluripotent stem cells, induced pluripotent stem cells, refer to the introduction of specific exogenous transcription factors into somatic cells to induce them to reprogram into pluripotent stem cells, which have proliferation and differentiation capabilities similar to embryonic stem cell lines.

[0122] Y27632: A small molecule inhibitor of Rho-associated protein kinases ROCK1 and ROCK2 that can increase the survival and growth of human embryonic stem cells and is widely used in stem cell research.

[0123] B-27 additive: an antioxidant that can maintain long-term in vitro culture of neuronal cells. Its main function is to inhibit the proliferation and growth of glial cells and can be used for myocardial cell differentiation.

[0124] CHIR 99021: A Wnt activator that can affect the process of stem cell differentiation by regulating the Wnt / β-catenin signaling pathway.

[0125] IWR-1: IWR-1 is a Wnt inhibitor that can downregulate the Wnt / β-catenin signaling pathway, thereby promoting the differentiation of iPSCs into mesoderm cells.

[0126] ASOs (antisense oligonucleotides) are also known as ASONs, ASODNs, and other similar terminology. ASOs are single-stranded deoxyribonucleic acids (DNAs), typically consisting of 15 to 25 nucleotides. They bind to complementary target RNAs and modulate their function. ASO transfection involves the introduction of ASOs into cells, which can further inhibit mRNA translation or degrade mRNA via RNases. ASOs are widely used in in vitro cell-based assays.

[0127] In the present invention, the "final concentration" of a substance refers to the final concentration of the substance in a prepared preparation (e.g., culture medium). For example, the phrase "add CHIR 99021 to a final concentration of 9 μM" during the preparation of M2A culture medium means that the concentration of CHIR 99021 in the prepared M2A culture medium is 9 μM, i.e., the concentration of the substance in the final preparation.

[0128] In the present invention, “cell confluence” is expressed as a percentage and “cell density” is expressed as cells / cm 2 As a unit. When the cell confluence reaches 80-90%, the corresponding cell density is about 2×10^5cells / cm 2 The cell density corresponding to a cell confluence of 90-100% is about 3×10^5 cells / cm 2 .

[0129] In this context, "fluid exchange" refers to the process of discarding old culture medium and adding new culture medium of the same type. For example, the phrase "add M2B culture medium, culture for 44-52 hours, and perform fluid exchange every 22-26 hours" refers to the process of discarding old M2B culture medium and adding new M2B culture medium.

[0130] The present invention provides a method for preparing cardiomyocytes derived from induced pluripotent stem cells, comprising the following steps: (1) amplifying and culturing induced pluripotent stem cells to obtain a culture containing amplified induced pluripotent stem cells; (2) inducing cardiomyocyte differentiation of the amplified induced pluripotent stem cells obtained in step (1), comprising: (2-1) discarding the culture medium in the culture obtained in step (1), adding M2A culture medium, and culturing for 22 to 26 hours; (2-2) discarding the culture medium in step (2-1), adding M2B culture medium, and culturing for 44 to 52 hours, during which the medium is replaced every 22 to 26 hours; (2-3) discarding the culture medium in step (2-4) and adding M2B culture medium. (2-2) medium, add M3 medium, culture for 44 to 52 hours, and change the medium every 22 to 26 hours; (2-4) discard the medium of step (2-3), add M1 medium, culture for 66 to 78 hours, and change the medium every 22 to 26 hours; (2-5) discard the medium of step (2-4), add M4 medium, and culture for 22 to 26 hours; (2-6) discard the medium of step (2-5), perform digestion and termination treatment, and inoculate into new wells, and culture with re-plating medium for 22 to 26 hours; (2-7) obtain induced differentiated cardiomyocytes; wherein, M1 medium includes RPMI 1640 medium and B-27 supplement without insulin; M2A medium includes M1 medium plus a first Wnt pathway activator; M2B medium includes M1 medium plus a second Wnt pathway activator; M3 medium includes M1 medium plus a first Wnt pathway inhibitor; M4 medium includes RPMI 1640 medium and B27 serum-free supplement.

[0131] In a specific embodiment, the first Wnt pathway activator and the second Wnt pathway activator each include any one selected from CHIR 99021, BML-284, HLY78, SKL2001, and CP21R7.

[0132] In a specific embodiment, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):1; preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is 18:1, 9:1, 4.5:1, 3:1, 2.25:1, or 1.8:1.

[0133] In a specific embodiment, the final concentration of the first Wnt pathway activator is 5 to 10 μM; more preferably, the final concentration of the first Wnt pathway activator is 10 μM, 9 μM, 8 μM, 7 μM, 6 μM, or 5 μM;

[0134] In a specific embodiment, the final concentration of the second Wnt pathway activator is 0.5 to 5 μM; more preferably, the final concentration of the second Wnt pathway activator is 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM;

[0135] In a specific embodiment, the first Wnt pathway activator is CHIR 99021, and the second Wnt pathway activator is CHIR 99021.

[0136] In a specific embodiment, the first Wnt pathway inhibitor is IWR-1; preferably, the final concentration of IWR-1 is 5-15 μM, more preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.

[0137] In a specific embodiment, the culture time in the above step (2-1), step (2-5), or step (2-6) is 22 to 26 hours; preferably, the culture time is 22 hours, 23 hours, 24 hours, 25 hours or 26 hours;

[0138] In a specific embodiment, the culture time in the above step (2-2) or step (2-3) is 44 to 52 hours; preferably, the culture time is 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, or 52 hours;

[0139] In a specific embodiment, the culture time in the above steps (2-4) is 66 to 78 hours; preferably, the culture time is 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, or 78 hours;

[0140] In a specific embodiment, in the above step (2-2), step (2-3), or step (2-4), the liquid is changed every 22 to 26 hours; preferably, the liquid is changed every 22 hours, 23 hours, 24 hours, 25 hours or 26 hours.

[0141] The present invention further provides a method for purifying cardiomyocytes derived from induced pluripotent stem cells, comprising: 1) discarding all culture medium from a culture medium containing the cardiomyocytes; 2) adding M5 culture medium and continuing the culture, during which the medium is replaced every 44 to 52 hours, and the cell morphology is observed 22 to 26 hours after each replacement; the M5 culture medium includes sodium L-lactate, and the final concentration of the sodium L-lactate is 5 to 12 mM; preferably, the final concentration of the sodium L-lactate is 5 mM, 6 mM, 7 mM, or 8 mM.

[0142] M5 medium is any one of the following ① to ③: ① M5 medium includes RPMI 1640 sugar-free medium and L-sodium lactate; ② M5 medium includes DMEM sugar-free medium and L-sodium lactate; ③ M5 medium includes DMEM sugar-free and glutamine-free medium and L-sodium lactate;

[0143] In a specific embodiment, the M5 medium includes DMEM sugar-free and glutamine-free medium and sodium L-lactate.

[0144] In a specific embodiment, the above step 2) is performed once every 44 to 52 hours; preferably, the solution is changed once every 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, or 52 hours;

[0145] In a specific embodiment, in the above step 2), the cell morphology is observed 22 to 26 hours after each medium change; preferably, the cell morphology is observed 22 hours, 23 hours, 24 hours, 25 hours or 26 hours after each medium change.

[0146] The present invention further provides a culture medium combination for preparing cardiomyocytes from induced pluripotent stem cells, comprising: M1 culture medium; M2A culture medium; M2B culture medium; M3 culture medium; and M4 culture medium; wherein the M1 culture medium comprises RPMI1640 culture medium and B-27 additive without insulin; the M2A culture medium comprises adding a first Wnt pathway activator to the M1 culture medium; the M2B culture medium comprises adding a second Wnt pathway activator to the M1 culture medium; the M3 culture medium comprises adding a first Wnt pathway inhibitor to the M1 culture medium; and the M4 culture medium comprises RPMI 1640 culture medium and B27 serum-free additive.

[0147] In a specific embodiment, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):1; preferably, the ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is 18:1, 9:1, 4.5:1, 3:1, 2.25:1, or 1.8:1.

[0148] In a specific embodiment, the above-mentioned culture medium combination further includes M5 culture medium, wherein the M5 culture medium includes sodium L-lactate, and the final concentration of sodium L-lactate is 5-12 mM; preferably, the final concentration of sodium L-lactate is 5 mM, 6 mM, 7 mM, or 8 mM.

[0149] The present invention further provides a cardiomyocyte prepared using the aforementioned cardiomyocyte preparation method, the aforementioned purification method, or the aforementioned culture medium combination.

[0150] The present invention further provides the above-mentioned cardiomyocyte preparation method, purification method, or use of the above-mentioned culture medium combination in preparing cardiomyocytes; preferably, the cardiomyocytes are cardiomyocytes derived from induced pluripotent stem cells; preferably, the cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells.

[0151] The present invention further provides a method for preparing the aforementioned cardiomyocytes, a purification method, a culture medium combination, or use of the aforementioned cardiomyocytes in preparing a reagent for lentiviral infection or ASO transfection.

[0152] Unless otherwise defined, the terms used in the present invention have the meanings commonly understood by those skilled in the art. The present invention will be further described in detail below through examples.

[0153] Example

[0154] Example 1: Effects of Different CHIR 99021 Treatments on the Differentiation of Cardiomyocytes Derived from Induced Pluripotent Stem Cells

[0155] Experimental Materials:

[0156] Induced Pluripotent Stem Cells: iPSC-5, an induced pluripotent stem cell line developed in our laboratory, is derived from human peripheral blood (blood-derived subjects have signed informed consent). This induced pluripotent stem cell line was constructed using conventional methods.

[0157] Reagents: RPMI 1640 medium (gibco, 11875093), B-27 supplement without insulin (invitrogen, A1895601), B27 serum-free supplement (invitrogen, 17504044), CHIR 99021 (stemgent, 04-0004-10), IWR-1 (MCE, HY-12238), DMEM (Gibco, 11966025), DMEM (Gibco, A1443001), Sodium L-lactate (Sigma, L7022), Y27632 (stemcell, 72304), E8 medium (Gibco, A1517001), Ascorbic acid (Sigma, 49752), Glutamine (Gibco, 35050061), Non-essential amino acid solution (Gibco, 11140050), Trypsin-EDTA (Thermo, 25200056), FBS (Corning, 35-081-CV).

[0158] Experimental steps:

[0159] 1. Prepare culture medium

[0160] M1 medium: RPMI 1640 medium and insulin-free B-27 supplement, prepared at a volume ratio of 98:2 for a final concentration of 1x insulin-free B-27 supplement.

[0161] Cardiomyocyte Differentiation Medium:

[0162] The preparation of differentiation medium for a single CHIR 99021 cell is as follows:

[0163] RPMI 1640 medium and insulin-free B-27 supplement were prepared at a volume ratio of 98:2, and CHIR 99021 was added at final concentrations of 3, 4, 5, 6, 7, 8, 9, and 10 μM, respectively.

[0164] The differentiation medium for CHIR 99021, including M2A medium and M2B medium, is prepared as follows:

[0165] M2A medium: Prepare RPMI 1640 medium with insulin-deficient B-27 supplement at a volume ratio of 98:2, and add CHIR 99021 to a final concentration of 9 μM;

[0166] M2B medium: RPMI 1640 medium and insulin-deficient B-27 supplement were prepared at a volume ratio of 98:2, and CHIR 99021 was added at final concentrations of 0.5, 1, 2, 3, 4, and 5 μM, respectively.

[0167] M3 medium: includes RPMI 1640 medium, insulin-deficient B-27 supplement, and IWR-1. It is prepared at a volume ratio of 98:2 between RPMI 1640 medium and insulin-deficient B-27 supplement. IWR-1 is added to a final concentration of 10 μM.

[0168] M4 medium: includes RPMI 1640 medium and B27 serum-free supplement, prepared at a volume ratio of 98:2. The final concentration of B27 serum-free supplement in the prepared M4 medium is 1×.

[0169] Resewing medium: comprising RPMI 1640 medium, B27 serum-free supplement, Y27632, ascorbic acid, glutamine, and non-essential amino acid solution. The volume ratio of RPMI 1640 medium, B27 serum-free supplement, Y27632 (stock concentration 10 μM), ascorbic acid (stock concentration 100 mg / ml), glutamine (stock concentration 100X), and non-essential amino acid solution (stock concentration 100X) is 87.8:10:0.1:0.1:1:1. The final concentrations of B27 serum-free supplement, Y27632, ascorbic acid, glutamine, and non-essential amino acid solution in the prepared resewing medium are 5×, 0.01 μM, 0.1 mg / ml, 1×, and 1×, respectively.

[0170] M5 medium: contains DMEM (sugar-free and glutamine-free) supplemented with sodium L-lactate at a final concentration of 5 mM.

[0171] 2. iPSC Preparation

[0172] When the cell confluence of induced pluripotent stem cells reaches 80-90% (the cell density is about 2×10^5 cells / cm 2 ), digestion treatment was performed and plating was performed using a plating medium. A six-well plate was used for plating. The plating medium included E8 medium and Y27632 at a final concentration of 10 μM, 2 ml per well;

[0173] After culturing for 24 h, discard the old culture medium and add 2 ml of E8 culture medium per well, and continue culturing for 26 to 28 h.

[0174] 3. Cardiomyocyte differentiation

[0175] Differentiation medium containing different final concentrations of CHIR 99021 and different culture times were used to induce cardiomyocyte differentiation.

[0176] The specific grouping is shown in Table 1 below. Groups 1-1 to 1-8 were cultured in differentiation medium with a single CHIR 99021 dosage for 2 days; Groups 1-9 to 1-14 were cultured in differentiation medium with different combinations of CHIR 99021 dosages, where the cells were cultured in differentiation medium M2A for 1 day and then in differentiation medium M2B for 2 days.

[0177] Table 1 CHIR 99021 dosage and culture time of differentiation medium in each group

[0178]

[0179] The specific cardiomyocyte differentiation preparation process is as follows:

[0180] (1) Expansion and culture of induced pluripotent stem cells, specifically including:

[0181] When the cell confluence of induced pluripotent stem cells in normal subculture reaches 80% to 90% (the cell density is about 2×10^5 cells / cm 2 ), digested and plated using a six-well plate containing E8 medium and Y27632 at a final concentration of 10 μM, 2 ml per well;

[0182] After culturing for 24 h, discard the old culture medium and add 2 ml of E8 culture medium to each well;

[0183] Culture for 26-28 hours and culture induced pluripotent stem cells until the cell confluence reaches 90-100% (the cell density corresponding to the cell confluence of 90-100% is about 3×10^5 cells / cm 2 ) to obtain a culture containing expanded induced pluripotent stem cells, and use the cell fusion degree of the expanded induced pluripotent stem cells obtained at this time as the starting cell fusion degree for cardiomyocyte differentiation. The day of adding cardiomyocyte differentiation medium is recorded as DAY0.

[0184] (II) Cardiomyocyte differentiation, culture, and purification

[0185] Differentiation culture using a single dose of CHIR 99021, specifically including:

[0186] On day 0, discard the old culture medium and add a single amount of differentiation medium for CHIR 99021, 2 ml per well;

[0187] DAY1: After 24 hours of culture, discard the old culture medium and add a new amount of differentiation medium for CHIR 99021, 2 ml per well;

[0188] DAY2: After 24 hours of culture, discard the old culture medium and add 2 ml of M1 culture medium to each well;

[0189] DAY3: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0190] DAY4: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0191] DAY5, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0192] DAY6: After 24 hours of culture, discard the old culture medium and add 2 ml of M1 culture medium to each well;

[0193] DAY7, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0194] DAY8, after 24 hours of culture, discard the old culture medium and add M4 culture medium (2 ml per well);

[0195] On day 9, after 24 hours of culture, discard the old medium and add 1 ml of Trypsin-EDTA for digestion for 5-10 min. Then add 5 ml of stop solution (prepared with a volume ratio of RPMI 1640 medium and FBS of 90:10) to terminate the culture. Then, inoculate into new wells and culture with re-plating medium.

[0196] DAY10, observe cell morphology;

[0197] DAY11, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0198] DAY12, observe cell morphology;

[0199] DAY13, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0200] DAY14, observe cell morphology;

[0201] DAY15, cardiomyocyte identification (microscope observation, immunofluorescence detection of cardiomyocyte markers, flow cytometry detection of cell purity, application testing, etc.), and cryopreservation.

[0202] Differentiation culture of CHIR 99021 dosage combination, specifically including:

[0203] On day 0, discard the old culture medium and add 2 ml of M2A culture medium to each well;

[0204] DAY1: After 24 hours of culture, discard the old culture medium and add 2 ml of M2B culture medium to each well;

[0205] DAY2: After 24 hours of culture, discard the old culture medium and add 2 ml of M2B culture medium to each well;

[0206] DAY3: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0207] DAY4: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0208] DAY5, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0209] DAY6: After 24 hours of culture, discard the old culture medium and add 2 ml of M1 culture medium to each well;

[0210] DAY7, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0211] DAY8, after 24 hours of culture, discard the old culture medium and add M4 culture medium (2 ml per well);

[0212] On day 9, after 24 hours of culture, discard the old medium and add 1 ml of Trypsin-EDTA for digestion for 5-10 min. Then add 5 ml of stop solution (prepared with a volume ratio of RPMI 1640 medium and FBS of 90:10) to terminate the culture. Then, inoculate into new wells and culture with re-plating medium.

[0213] DAY10, observe cell morphology;

[0214] DAY11, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0215] DAY12, observe cell morphology;

[0216] DAY13, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0217] DAY14, observe cell morphology;

[0218] DAY15, cardiomyocyte identification (microscope observation, immunofluorescence detection of cardiomyocyte markers, flow cytometry detection of cell purity, application testing, etc.), and cryopreservation.

[0219] Experimental results:

[0220] From DAY 6 to DAY 9 of cell differentiation, cardiomyocyte beating can be seen.

[0221] 1. Results of different CHIR 99021 modes of action inducing cardiomyocyte differentiation

[0222] As shown in the following table and Figure 2 、 Figure 3 As shown, when cardiomyocytes were cultured to DAY 15, flow cytometry was used to detect cell purity. The cells were transferred to a 1.5 ml EP tube and fixed with 4% PFA for 15 minutes, permeabilized with 0.2% Triton for 15 minutes, and stained with BD cTnT flow cytometry antibody for 15 minutes at room temperature using a Beckman flow cytometer and CytExpert analysis software.

[0223] Table 2 The proportion of cTnT+ cardiomyocytes obtained in each group

[0224] Group Proportion of cTnT+ myocardial cells (%) 1-1 45.60 1-2 48.78 1-3 52.24 1-4 92.99 1-5 86.69 1-6 86.16 1-7 77.47 1-8 73.17 1-9 57.14 1-10 90.05 1-11 94.28 1-12 96.37 1-13 94.59 1-14 67.65

[0225] As shown in the table above and Figure 2 、 Figure 3 As shown, among the groups using a single CHIR 99021 dosage, only groups 1-4 obtained a cardiomyocyte cTnT-positive cell ratio of more than 90%;

[0226] In the groups with different CHIR 99021 dosage combinations, the proportion of cTnT-positive cardiomyocytes obtained in groups 1-10, 1-11, 1-12, and 1-13 was greater than 90%, and the proportion of cTnT-positive cardiomyocytes obtained in group 1-12 was greater than 95%.

[0227] It can be seen that the present invention can induce cardiomyocytes with higher purity by using different CHIR 99021 dosage combinations and different action times to successively treat induced pluripotent stem cells.

[0228] Furthermore, in the groups using different CHIR 99021 dosage combinations, the culture time in each step of the above differentiation culture and purification was 22 hours, 23 hours, 25 hours, and 26 hours, and cardiomyocytes with higher purity were obtained.

[0229] Example 2: Effects of different purification media on myocardial cell purification

[0230] Experimental steps:

[0231] To compare the purification effects of different purification media on cardiomyocytes, cardiomyocytes differentiated from Groups 1-12 to DAY11 in Example 1 were purified using different purification media. The purification method was the same as the steps from Day 11 to Day 15 in Example 1, except that the M5 medium used the purification medium formula shown in Table 3 below. The proportion of cTnT-positive cells was detected by flow cytometry on Day 15.

[0232] Table 3 Composition of different purification media

[0233]

[0234] Experimental results:

[0235] like Figure 4 As shown in A, the purity of cardiomyocytes before purification was 86.38%; Figure 4 As shown in B, C, and D, after 4 days of treatment with purification medium 1, purification medium 2, and purification medium 3, the purity of cardiomyocytes reached 91.65%, 92.03%, and 96.34%, respectively. It can be seen that purification medium 3 has the best purification effect. Figure 4 As shown in Figure E, using L-lactic acid to replace sodium L-lactate in purification medium 3 also leads to a decrease in the purity of the purified cardiomyocytes (76.93%).

[0236] Table 4 Comparison of the purity of cardiomyocytes prepared using purification medium containing different concentrations of sodium L-lactate

[0237] Purification medium serial number Final concentration of sodium L-lactate (mM) DAY15 cardiomyocyte purity (cTnT+%) 3 5 96.34 4 2 82.04 5 3 89.56 6 8 95.01 7 10 92.82 8 12 92.65

[0238] Cardiac myocytes can utilize lactate and ultimately convert it into acetyl-CoA, which enters the tricarboxylic acid cycle (TCA cycle) to provide energy for the cells. However, myocardial cells cannot effectively metabolize lactate. Therefore, in an environment without glucose and glutamine, where only lactate is present, myocardial cells find it difficult to maintain a good state of survival.

[0239] As shown in Table 4, if the concentration of sodium L-lactate in the purification medium is too low, the cell state will deteriorate and the purity will decrease. If the concentration of sodium L-lactate is too high, the purification effect will not be improved.

[0240] Example 3: Effects of different starting cell confluences on cardiomyocyte purity

[0241] Experimental steps:

[0242] In order to compare the effects of different initial cell fusion degrees on the purity of differentiated cardiomyocytes, for groups 1-12 in Example 1, the cells were cultured at a density of 2×10^5 cells / cm 2 、3×10^5cells / cm2 , 4×10^5cells / cm 2 At 4 hr, cardiomyocyte differentiation was started. The differentiation and purification steps and the culture medium used were the same as those in the differentiation culture using the CHIR 99021 dosage combination in Groups 1-12 of Example 1. The culture time in each differentiation and purification step was 24 h.

[0243] like Figure 5 As shown in Figure 2, too high or too low initial cell confluence will affect the purity of differentiated cardiomyocytes. The optimal initial cell confluence is a medium confluence density (cell confluence 90-100%), corresponding to a cell density of approximately 3×10^5 cells / cm 2 .

[0244] Example 4: Design of a culture process for the differentiation of cardiomyocytes derived from induced pluripotent stem cells

[0245] Through Examples 1, 2, and 3, the optimal differentiation and purification process of cardiomyocytes derived from induced pluripotent stem cells of the present invention was determined as follows: Figure 1 However, this schematic diagram is only used to illustrate the present invention and does not limit the present invention. The specific culture process is as follows:

[0246] 1. Prepare culture medium

[0247] M1 medium: RPMI 1640 medium and insulin-free B-27 supplement, prepared at a volume ratio of 98:2 for a final concentration of 1x insulin-free B-27 supplement.

[0248] M2A medium: Prepare RPMI 1640 medium with insulin-deficient B-27 supplement at a volume ratio of 98:2, and add CHIR 99021 to a final concentration of 9 μM;

[0249] M2B medium: RPMI 1640 medium and insulin-free B-27 supplement were prepared at a volume ratio of 98:2, and CHIR 99021 was added to a final concentration of 3 μM.

[0250] M3 medium: includes RPMI 1640 medium, insulin-deficient B-27 supplement, and IWR-1. It is prepared at a volume ratio of 98:2 between RPMI 1640 medium and insulin-deficient B-27 supplement. IWR-1 is added to a final concentration of 10 μM.

[0251] M4 medium: includes RPMI 1640 medium and B27 serum-free supplement, prepared at a volume ratio of 98:2. The final concentration of B27 serum-free supplement in the prepared M4 medium is 1×.

[0252] Resewing medium: comprising RPMI 1640 medium, B27 serum-free supplement, Y27632, ascorbic acid, glutamine, and non-essential amino acid solution. The volume ratio of RPMI 1640 medium, B27 serum-free supplement, Y27632 (stock concentration 10 μM), ascorbic acid (stock concentration 100 mg / ml), glutamine (stock concentration 100X), and non-essential amino acid solution (stock concentration 100X) is 87.8:10:0.1:0.1:1:1. The final concentrations of B27 serum-free supplement, Y27632, ascorbic acid, glutamine, and non-essential amino acid solution in the prepared resewing medium are 5×, 0.01 μM, 0.1 mg / ml, 1×, and 1×, respectively.

[0253] M5 medium: contains DMEM (sugar-free and glutamine-free) supplemented with sodium L-lactate at a final concentration of 5 mM.

[0254] 2. iPSC Preparation

[0255] When the cell confluence of induced pluripotent stem cells reaches 80-90% (the cell density is about 2×10^5 cells / cm 2 ), digested and plated using a six-well plate containing E8 medium and Y27632 at a final concentration of 10 μM, 2 ml per well;

[0256] After culturing for 24 h, discard the old culture medium and add 2 ml of E8 culture medium to each well;

[0257] Culture for 26-28 hours and culture induced pluripotent stem cells until the cell confluence reaches 90-100% (the cell density corresponding to the cell confluence of 90-100% is about 3×10^5 cells / cm 2 ) to obtain a culture containing expanded induced pluripotent stem cells, and use the cell fusion degree of the expanded induced pluripotent stem cells obtained at this time as the starting cell fusion degree for cardiomyocyte differentiation. The day of adding cardiomyocyte differentiation medium is recorded as DAY0.

[0258] 3. Cardiomyocyte differentiation, culture and purification

[0259] On day 0, discard the old culture medium and add 2 ml of M2A culture medium to each well;

[0260] DAY1: After 24 hours of culture, discard the old culture medium and add 2 ml of M2B culture medium to each well;

[0261] DAY2: After 24 hours of culture, discard the old culture medium and add 2 ml of M2B culture medium to each well;

[0262] DAY3: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0263] DAY4: After 24 hours of culture, discard the old culture medium and add 2 ml of M3 culture medium to each well;

[0264] DAY5, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0265] DAY6: After 24 hours of culture, discard the old culture medium and add 2 ml of M1 culture medium to each well;

[0266] DAY7, after 24 hours of culture, discard the old culture medium and add M1 culture medium, 2 ml per well;

[0267] DAY8, after 24 hours of culture, discard the old culture medium and add M4 culture medium (2 ml per well);

[0268] On day 9, after 24 hours of culture, discard the old medium and add 1 ml of Trypsin-EDTA for digestion for 5-10 min. Then add 5 ml of stop solution (prepared with a volume ratio of RPMI 1640 medium and FBS of 90:10) to terminate the culture. Then, inoculate into new wells and culture with re-plating medium.

[0269] DAY10, observe cell morphology;

[0270] DAY11, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0271] DAY12, observe cell morphology;

[0272] DAY13, after 24 hours of culture, discard the old culture medium and add M5 culture medium (2 ml per well);

[0273] DAY14, observe cell morphology;

[0274] DAY15, cardiomyocyte identification (microscope observation, immunofluorescence detection of cardiomyocyte markers, flow cytometry detection of cell purity, application testing, etc.), and cryopreservation.

[0275] Example 5: Cell Identification of Cardiomyocytes Obtained by the Present Invention

[0276] The cardiomyocyte culture process in Example 4 was used to differentiate and purify the iPSC-5 cell line, and the obtained cardiomyocytes were identified.

[0277] ①Microscope observation

[0278] The obtained cardiomyocytes were observed under a fluorescence microscope.

[0279] like Figure 6 As shown, the differentiated cells can be observed to contract spontaneously under a microscope, and the differentiated cardiomyocytes are in good condition.

[0280] ②Immunofluorescence identification

[0281] The cardiomyocyte markers cTnT and α-Actinin2 in the obtained cardiomyocytes were detected by immunofluorescence.

[0282] like Figure 7 As shown in A and B, the obtained cardiomyocytes can express the cardiac-specific proteins cTnT and α-Actinin 2.

[0283] ③Cell purity detection

[0284] The purity of the obtained cardiomyocytes was assessed by flow cytometry. The cells were transferred to a 1.5 ml EP tube and fixed with 4% PFA for 15 minutes. The membranes were permeabilized with 0.2% Triton for 15 minutes and stained with BD cTnT flow cytometry antibody for 15 minutes at room temperature. The flow cytometer was used, and the analysis software was CytExpert.

[0285] like Figure 8 As shown, among the obtained cardiomyocytes, the proportion of cells expressing cTnT is above 90%, indicating that the cardiomyocytes are of high purity and good quality, which can well support subsequent experimental research.

[0286] Example 6: Lentiviral infection and ASO transfection of cardiomyocytes obtained by the present invention

[0287] The cardiomyocyte culture process in Example 4 was used to differentiate the iPSC-5 cell line into cardiomyocytes, and the obtained cardiomyocytes were infected with lentivirus and transfected with ASO, respectively.

[0288] ① Lentivirus infection

[0289] The obtained cardiomyocytes were infected with lentivirus (GFP marker), and immunofluorescence imaging was performed one week after infection.

[0290] like Figure 9As shown in A and B, the fluorescence ratio exceeds 90%. The iPSC-derived cardiomyocytes prepared by the present invention can be efficiently transfected with lentiviruses. Therefore, they can serve as a good model for drug screening and disease research.

[0291] ②ASO transfection

[0292] The obtained cardiomyocytes were transfected with ASO-FAM and detected by immunofluorescence imaging 48 hours later.

[0293] like Figure 10 As shown in A and B, the fluorescence ratio exceeds 90%. The iPSC-derived cardiomyocytes prepared by the present invention can be transfected with ASO with high efficiency and have wide applications.

[0294] In summary, the cardiomyocytes prepared using the method of the present invention are in good condition, high quality, and high purity, which can well support subsequent experimental research, and can be subjected to operations such as lentiviral transfection and ASO transfection. They can serve as a good drug screening and disease research model and have the potential for wide application.

[0295] Example 7: Stability test of the cardiomyocyte preparation method of the present invention

[0296] Experimental Materials:

[0297] Induced Pluripotent Stem Cells: Ten induced pluripotent stem cell lines, iPSC-1, iPSC-2, iPSC-3, iPSC-4, iPSC-5, iPSC-6, iPSC-7, iPSC-8, iPSC-9, and iPSC-10, are in-house-developed cell lines in our laboratory, derived from human peripheral blood (blood-derived subjects have signed informed consent). These induced pluripotent stem cell lines were constructed using conventional methods.

[0298] Experimental steps:

[0299] To verify the stability of the cardiomyocyte preparation method of the present invention, 10 iPSC clones were differentiated using the cardiomyocyte preparation method of the present invention as in Example 4, namely iPSC-1, iPSC-2, iPSC-3, iPSC-4, iPSC-5, iPSC-6, iPSC-7, iPSC-8, iPSC-9, and iPSC-10, and the proportion of cTnT-positive cells was detected by flow cytometry on DAY15.

[0300] Ten iPSC clones were differentiated using the existing technical method (see document DOI: 10.1016 / j.stem.2020.06.001, in which 6.0 μM CHIR99021 was selected on DAY0-2, and the other steps were described in the document) and were identified as iPSC-1, iPSC-2, iPSC-3, iPSC-4, iPSC-5, iPSC-6, iPSC-7, iPSC-8, iPSC-9, and iPSC-10. The proportion of cTnT-positive cells was detected by flow cytometry on DAY15.

[0301] Experimental results:

[0302] like Figure 11 As shown in Figures A. to E. and Table 5, the cardiomyocyte purity obtained using the cardiomyocyte preparation method provided by the present invention all reached over 90%. Calculations show that the coefficient of variation (coefficient of variation = standard deviation / mean) of the cardiomyocyte purity data obtained using the present method is lower than that obtained using the prior art method (0.012 < 0.052), demonstrating that the cardiomyocyte preparation method provided by the present invention is more stable than prior art methods.

[0303] The data were subjected to a paired sample T test, and the results showed that the difference was statistically significant (p<0.01). The purity of the cardiomyocytes prepared by the method of the present invention was significantly higher than that of the prior art method.

[0304] Table 5 Comparison of the purity of cardiomyocytes prepared by the method of the present invention and the prior art method

[0305] iPSC cell lines Myocardial cell purity (cTnT+%) of the method of the present invention Prior art method Myocardial cell purity (cTnT+%) iPSC-1 93.29 89.86 iPSC-2 93.93 82.00 iPSC-3 93.96 77.47 iPSC-4 94.16 85.93 iPSC-5 90.86 93.29 iPSC-6 93.02 88.70 iPSC-7 93.60 85.24 iPSC-8 94.72 90.22 iPSC-9 92.05 85.90 iPSC-10 93.80 85.45

[0306] Those skilled in the art should note that the embodiments described in the present invention are merely exemplary and that various other substitutions, changes, and improvements may be made within the scope of the present invention. Therefore, the present invention is not limited to the above embodiments, but is only limited by the claims.

Claims

1. A method for preparing cardiomyocytes derived from induced pluripotent stem cells, comprising the following steps: (1) expanding and culturing induced pluripotent stem cells to obtain a culture containing the expanded induced pluripotent stem cells; (2) inducing cardiomyocyte differentiation of the amplified induced pluripotent stem cells obtained in step (1), comprising: (2-1) discarding the culture medium obtained in step (1), adding M2A medium, and culturing for 22 to 26 hours; (2-2) Discarding the culture medium from step (2-1), adding M2B culture medium, and culturing for 44 to 52 hours, during which the medium was changed every 22 to 26 hours; (2-3) Discarding the culture medium from step (2-2), adding M3 culture medium, and culturing for 44 to 52 hours, during which the medium was changed every 22 to 26 hours; (2-4) Discarding the culture medium from step (2-3), adding M1 culture medium, and culturing for 66 to 78 hours, during which the medium was changed every 22 to 26 hours; (2-5) Discarding the culture medium from step (2-4), adding M4 culture medium, and culturing for 22 to 26 hours; (2-6) Discarding the culture medium from step (2-5), performing digestion and termination treatment, inoculating into new wells, and culturing with resurfacing medium for 22 to 26 hours; (2-7) obtaining the induced differentiated cardiomyocytes; Wherein, the M1 culture medium comprises RPMI 1640 culture medium and B-27 supplement without insulin; The M2A culture medium comprises adding a first Wnt pathway activator to the M1 culture medium; The M2B culture medium includes adding a second Wnt pathway activator to the M1 culture medium; The M3 culture medium comprises adding a first Wnt pathway inhibitor to the M1 culture medium; The M4 medium includes RPMI 1640 medium and B27 serum-free supplement; The ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):

1.

2. The preparation method according to claim 1, wherein The first Wnt pathway activator and the second Wnt pathway activator are each selected from any one of CHIR 99021, BML-284, HLY78, SKL2001, and CP21R7; Preferably, the first Wnt pathway activator is CHIR 99021; the second Wnt pathway activator is CHIR99021.

3. A method for purifying cardiomyocytes, comprising the following steps: 1) Discard all culture medium from the culture containing cardiomyocytes; 2) Add M5 medium and continue culturing; Characterized in that the M5 culture medium includes sodium L-lactate, and the final concentration of the sodium L-lactate is 5-12 mM.

4. The purification method according to claim 3, wherein The M5 culture medium is any one of the following ① to ③: ① The M5 culture medium includes RPMI 1640 sugar-free culture medium and sodium L-lactate; ② The M5 culture medium includes DMEM sugar-free culture medium and sodium L-lactate; ③ The M5 culture medium includes DMEM sugar-free and glutamine-free culture medium and sodium L-lactate; Preferably, the M5 culture medium comprises DMEM sugar-free and glutamine-free culture medium and sodium L-lactate.

5. A culture medium composition for preparing cardiomyocytes from induced pluripotent stem cells, characterized in that: include: M1 medium; M2A medium; M2B medium; M3 medium; and M4 medium; The M1 culture medium includes RPMI 1640 culture medium and B-27 supplement without insulin; The M2A culture medium comprises adding a first Wnt pathway activator to the M1 culture medium; The M2B culture medium includes adding a second Wnt pathway activator to the M1 culture medium; The M3 culture medium comprises adding a first Wnt pathway inhibitor to the M1 culture medium; The M4 medium includes RPMI 1640 medium and B27 serum-free supplement; The ratio of the final concentration of the first Wnt pathway activator to the final concentration of the second Wnt pathway activator is (1-20):

1.

6. The culture medium combination according to claim 5, wherein The first Wnt pathway activator and the second Wnt pathway activator are each selected from any one of CHIR 99021, BML-284, HLY78, SKL2001, and CP21R7; Preferably, the first Wnt pathway activator is CHIR 99021; the second Wnt pathway activator is CHIR99021.

7. The culture medium combination according to any one of claims 5 to 6, characterized in that Also included is M5 medium; The M5 culture medium includes sodium L-lactate, and the final concentration of the sodium L-lactate is 5 to 12 mM.

8. Cardiomyocytes prepared by the preparation method according to any one of claims 1 to 2, the purification method according to any one of claims 3 to 4, or the culture medium combination according to any one of claims 5 to 7.

9. Use of the preparation method according to any one of claims 1 to 2, the purification method according to any one of claims 3 to 4, or the culture medium combination according to any one of claims 5 to 7 in preparing cardiomyocytes derived from induced pluripotent stem cells.

10. Use of the preparation method according to any one of claims 1 to 2, the purification method according to any one of claims 3 to 4, the culture medium combination according to any one of claims 5 to 7, or the cardiomyocytes according to claim 8 in preparing a reagent for lentiviral infection or ASO transfection.

Images