Myocardial differentiation kit with screening and purifying functions and use method thereof

Abstract

The invention discloses a myocardial differentiation kit with screening and purifying functions and a using method thereof.The myocardial differentiation kit comprises a basic culture medium, a differentiation inducer and a screening and purifying reagent, the basic culture medium is used for providing basic nutrient substances for cell growth and differentiation, the differentiation inducer comprises BMP4, Activin A, CHIR99021 and VEGF, and the screening and purifying reagent is used for screening and purifying the screening and purifying reagent. Compared with the prior art, the myocardial differentiation kit disclosed by the invention has the following advantages that the myocardial differentiation kit can simulate a key signal channel in an embryonic development process by reasonably matching a differentiation inducer combination, the differentiation efficiency of pluripotent stem cells to myocardial cells is remarkably improved, the differentiation rate can reach 80% or above, and the myocardial differentiation effect is good. The screening and purifying reagent realizes rapid and efficient screening and purification of myocardial cells by utilizing the characteristic that a magnetic bead coupling antibody is combined with a specific marker cTNT on the surface of the myocardial cells and combining a magnetic separation technology, the purity can reach 95% or above, the damage to the cells is small, and the kit integrates differentiation and screening and purifying functions into a whole.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a myocardial differentiation kit with screening and purification functions and a method for using the same. Background Art

[0002] Cardiomyocytes play a key role in the physiological functions of the heart and have broad application prospects in the fields of cardiovascular disease research, drug screening, and cell therapy. Pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells), which have the ability to proliferate infinitely and differentiate into various cell types, are important sources for obtaining cardiomyocytes.

[0003] Currently, the methods for differentiating pluripotent stem cells into cardiomyocytes mainly rely on mimicking the cellular microenvironment during embryonic development and inducing pluripotent stem cells to gradually differentiate into cardiomyocytes by adding various cytokines, growth factors, and small molecule compounds. However, these traditional differentiation methods have some obvious defects: on the one hand, multiple cell types are often produced during the differentiation process, and the purity of cardiomyocytes is low, seriously affecting subsequent research and applications; on the other hand, there is a lack of effective screening and purification means, and existing screening methods (such as centrifugal separation based on physical properties, fluorescence-activated cell sorting based on surface markers, etc.) have problems such as complex operation, high cost, and great cell damage, making it difficult to meet the needs of large-scale and efficient acquisition of high-purity cardiomyocytes. Therefore, it is of great practical significance to develop a myocardial differentiation technology that can simultaneously achieve efficient differentiation and rapid screening and purification. Thus, a myocardial differentiation kit with screening and purification functions is provided to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to provide a myocardial differentiation kit with screening and purification functions, which can achieve the efficient differentiation of pluripotent stem cells into cardiomyocytes, rapidly screen and purify cardiomyocytes after differentiation, simplify the operation process, improve the purity and quality of cardiomyocytes, and at the same time provide a method for using the kit to ensure the standardization and effectiveness of the operation.

[0005] To solve the problems raised in the above background art, the technical solution adopted by the present invention to solve the technical problems is: a myocardial differentiation kit with screening and purification functions and its use method, which includes: a basal medium, a differentiation inducer, and a screening and purification reagent. The basal medium is used to provide basic nutrients for cell growth and differentiation. The differentiation inducer includes BMP4, Activin A, CHIR99021, and VEGF. BMP4 and Activin A are used to initiate cell differentiation into the mesoderm. CHIR99021 can activate the Wnt signaling pathway and promote the differentiation of mesoderm cells into myocardial progenitor cells. VEGF helps the further maturation and vascularization of myocardial cells. The screening and purification reagent is a magnetic bead-conjugated antibody that specifically binds to myocardial cell surface markers and a magnetic bead separation buffer.

[0006] As a preferred technical solution of the present invention, the concentration of BMP4 is 5-10 ng / mL, the concentration of Activin A is 50-100 ng / mL, the concentration of CHIR99021 is 3-5 μM, and the concentration of VEGF is 10-20 ng / mL.

[0007] As a preferred technical solution of the present invention, the basal medium contains DMEM / F12 medium, L-glutamine, non-essential amino acids, β-mercaptoethanol, and fetal bovine serum.

[0008] As a preferred technical solution of the present invention, the myocardial cell surface marker is cTNT, the concentration of the magnetic bead-conjugated antibody is 1-5 μg / mL, and the magnetic bead separation buffer is PBS buffer, 0.5% BSA, and 2 mM EDTA.

[0009] As a preferred technical solution of the present invention, the magnetic bead-conjugated antibody can specifically bind to cTNT on the surface of myocardial cells, and the rapid separation and purification of myocardial cells can be achieved through a magnetic separation device.

[0010] A use method of a myocardial differentiation kit with screening and purification functions includes the following steps:

[0011] Step 1: First, perform cell seeding. Seed pluripotent stem cells at an appropriate density in a cell culture plate containing a basal medium, and culture them in an incubator at 37°C and 5% CO2. When the cell confluence reaches 70-80%, perform differentiation induction.

[0012] Step 2: In the differentiation induction of Step 1, aspirate the basal medium and add a differentiation medium containing a combination of differentiation inducers to induce pluripotent stem cells to gradually differentiate into myocardial cells.

[0013] Step 3: Screen and purify the cardiomyocytes in Step 3. After the differentiation culture is completed, discard the differentiation medium, gently rinse the cells 2 - 3 times with PBS buffer, add an appropriate amount of the magnetic bead-conjugated antibody in the screening and purification reagent, and incubate to allow the magnetic bead-conjugated antibody to fully bind to cTNT on the surface of cardiomyocytes;

[0014] Step 4: After the incubation is completed, add magnetic bead separation buffer, digest the cells with trypsin, collect the cell suspension, transfer the cell suspension to a magnetic separation device, perform magnetic separation on it, and separate the cardiomyocytes bound with the magnetic bead-conjugated antibody, which are the purified cardiomyocytes.

[0015] As a preferred technical solution of the present invention, in Step 1, the pluripotent stem cells have a suitable density of 5×10 4 -1×10 5 cells per square centimeter.

[0016] As a preferred technical solution of the present invention, in Step 2, the cells are cultured in an incubator at 37°C and 5% CO2. From the 1st to the 3rd day of culture, a differentiation medium containing BMP4 and Activin A is used. On the 4th to the 6th day, it is replaced with a differentiation medium containing CHIR99021. On the 7th to the 10th day, a differentiation medium containing VEGF is used.

[0017] As a preferred technical solution of the present invention, in Step 3, the incubation is carried out at 4°C for 30 - 60 min.

[0018] The present invention has the following advantages: The cardiomyocyte differentiation kit of the present invention can simulate the key signal pathways during embryonic development by reasonably matching the differentiation inducer combination, significantly improving the differentiation efficiency of pluripotent stem cells into cardiomyocytes, and the differentiation rate can reach more than 80%;

[0019] The screening and purification reagent utilizes the property that the magnetic bead-conjugated antibody binds to the specific marker cTNT on the surface of cardiomyocytes, combines with the magnetic separation technology, realizes the rapid and efficient screening and purification of cardiomyocytes, the purity can reach more than 95%, and the operation is simple with little damage to cells;

[0020] This kit integrates the functions of differentiation and screening and purification, provides necessary consumables, simplifies the experimental operation process, reduces the experimental cost and operation difficulty, and is suitable for the preparation of a large number of cardiomyocytes. Brief Description of the Drawings

[0021] Figure 1 It is a schematic diagram of the usage method of the cardiomyocyte differentiation kit with screening and purification functions in the preferred embodiment of the present invention. Detailed Embodiments

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. In the following description, many specific details are set forth to facilitate a thorough understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0023] In order to more clearly understand the above objects, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Please refer to Figure 1 , a myocardial differentiation kit with screening and purification functions of the present invention includes: a basal medium, a differentiation inducer, and a screening and purification reagent. The basal medium is used to provide basic nutrients for cell growth and differentiation. The differentiation inducer includes BMP4 (bone morphogenetic protein), Activin A (activin A), CHIR99021 (inhibitor), and VEGF (vascular endothelial growth factor). BMP4 and Activin A are used to initiate cell differentiation into the mesoderm. CHIR99021 can activate the Wnt signaling pathway and promote the differentiation of mesodermal cells into myocardial progenitor cells. VEGF helps the further maturation and vascularization of myocardial cells. The screening and purification reagent is a magnetic bead-conjugated antibody that specifically binds to the myocardial cell surface marker and a magnetic bead separation buffer.

[0025] Among them, the concentration of BMP4 is 5-10 ng / mL, the concentration of Activin A is 50-100 ng / mL, the concentration of CHIR99021 is 3-5 μM, the concentration of VEGF is 10-20 ng / mL. The basal medium contains DMEM / F12 medium, L-glutamine, non-essential amino acids, β-mercaptoethanol, and fetal bovine serum. The myocardial cell surface marker is cTNT (cardiac troponin T). The concentration of the magnetic bead-conjugated antibody is 1-5 μg / mL. The magnetic bead separation buffer is PBS buffer, 0.5% BSA, and 2 mM EDTA. The magnetic bead-conjugated antibody can specifically bind to cTNT on the surface of myocardial cells, and rapid separation and purification of myocardial cells can be achieved through a magnetic separation device.

[0026] Example 1

[0027] The usage method of the myocardial differentiation kit with screening and purification functions includes the following steps:

[0028] Cell seeding: Take induced pluripotent stem cells and seed them at 8×10 per square centimeter 4Cells were seeded at a density of [number of cells] per well in a 6-well cell culture plate, and 2 mL of basal medium was added to each well. The cells were cultured in an incubator at 37°C and 5% CO2. After 2 days of culture, the cell confluence reached 75%, and differentiation induction was carried out.

[0029] Differentiation induction: Days 1 - 3: Aspirate the basal medium, and add 2 mL of differentiation medium containing BMP4 (8 ng / mL) and Activin A (80 ng / mL) to each well. Continue to culture in an incubator at 37°C and 5% CO2; Days 4 - 6: Discard the original differentiation medium, and add 2 mL of differentiation medium containing CHIR99021 (4 μM) to each well, and continue the culture; Days 7 - 10: Replace with differentiation medium containing VEGF (15 ng / mL), and culture until day 10 to complete the differentiation process of pluripotent stem cells into cardiomyocytes.

[0030] Screening and purification: After the differentiation culture is completed, discard the differentiation medium, and gently wash the cells 3 times with PBS buffer. Add 0.5 mL of magnetic bead-conjugated antibody (against cTNT) with a concentration of 3 μg / mL to each well, and incubate at 4°C for 45 min; after incubation, add 1 mL of magnetic bead separation buffer to each well, digest the cells with trypsin, gently pipette to detach the cells from the culture plate, and collect the cell suspension into a centrifuge tube; place the centrifuge tube in a magnetic separation device and let it stand for 5 min to allow the cardiomyocytes bound with the magnetic bead-conjugated antibody to adsorb on the wall of the magnetic separation device. Carefully aspirate the supernatant, wash the cells 2 times with magnetic bead separation buffer, let it stand for 3 min after each wash, and aspirate the supernatant to obtain purified cardiomyocytes.

[0031] Detection and analysis: The expressions of cardiomyocyte markers cTNT and α-actinin were detected by immunofluorescence staining. The results showed that 96% of the cells were positive for cTNT and α-actinin, indicating that highly pure cardiomyocytes were obtained; at the same time, the expression levels of cardiomyocyte-related genes (such as NKX2.5, GATA4, etc.) were detected by real-time quantitative PCR to verify the differentiation effect of cardiomyocytes.

[0032] Example 2

[0033] Human embryonic stem cell line and mouse induced pluripotent stem cell line were selected. Respectively, according to the procedure in Example 1, cell seeding: Take the human embryonic stem cell line and mouse induced pluripotent stem cell line, and seed them at a density of 8×10 4 cells per square centimeter in the corresponding 6-well cell culture plate. Add 2 mL of basal medium to each well, and culture in an incubator at 37°C and 5% CO2. After 2 days of culture, the cell confluence reached 75%, and differentiation induction was carried out.

[0034] Differentiation induction: Days 1 - 3: Aspirate the basal medium, add 2 mL of differentiation medium containing BMP4 (8 ng / mL) and Activin A (80 ng / mL) to each well, and continue to culture in an incubator at 37°C and 5% CO2; Days 4 - 6: Discard the original differentiation medium, add 2 mL of differentiation medium containing CHIR99021 (4 μM) to each well, and continue to culture; Days 7 - 10: Replace with differentiation medium containing VEGF (15 ng / mL), and culture until Day 10 to complete the differentiation process of human embryonic stem cells and mouse induced pluripotent stem cells into cardiomyocytes.

[0035] Screening and purification: After the differentiation culture is completed, discard the differentiation medium, and gently rinse the cells 3 times with PBS buffer. Add 0.5 mL of magnetic bead-conjugated antibody (against cTNT) with a concentration of 3 μg / mL to each well, and incubate at 4°C for 45 min; after incubation, add 1 mL of magnetic bead separation buffer to each well, digest the cells with trypsin, gently pipette to detach the cells from the culture plate, and collect the cell suspension into a centrifuge tube; place the centrifuge tube in a magnetic separation device, let it stand for 5 min to allow the cardiomyocytes conjugated with the magnetic bead-conjugated antibody to adsorb on the wall of the magnetic separation device. Carefully aspirate the supernatant, wash the cells 2 times with magnetic bead separation buffer, let it stand for 3 min after each wash, and aspirate the supernatant to obtain purified cardiomyocytes for operation. As shown in Table 1

[0036] Table 1

[0037] Differentiation efficiency Purity of cardiomyocytes after purification Human embryonic stem cells 87％ 95.8％ Mouse induced pluripotent stem cells 83％ 94.5％

[0038] As shown in Table 1, the results show that the differentiation efficiency of human ESCs is 87%, and the purity of cardiomyocytes after purification is 95.8%; the differentiation efficiency of mouse iPSCs is 83%, and the purity is 94.5%. This verifies the applicability of the kit to pluripotent stem cells from different species and different sources, and proves that the kit of the present invention has good generality and is applicable to the differentiation and purification of pluripotent stem cells from different sources into cardiomyocytes.

[0039] The foregoing shows and describes the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes within the meaning and scope of the equivalent elements of the claims in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0040] Other parts not described in detail in the present invention belong to the prior art and will not be elaborated herein.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A myocardial differentiation kit with screening and purification functions, characterized in that It includes: a basal medium, a differentiation inducer, and a screening and purification reagent. The basal medium is used to provide basic nutrients for cell growth and differentiation. The differentiation inducer includes BMP4, Activin A, CHIR99021, and VEGF. BMP4 and Activin A are used to initiate the differentiation of cells into mesoderm. CHIR99021 can activate the Wnt signaling pathway and promote the differentiation of mesoderm cells into cardiac progenitor cells. VEGF helps the further maturation and vascularization of cardiomyocytes. The screening and purification reagent is a magnetic bead-conjugated antibody that specifically binds to a cardiac cell surface marker and a magnetic bead separation buffer.

2. The myocardial differentiation kit with screening and purification functions according to claim 1, wherein, The concentration of BMP4 is 5 - 10 ng / mL, the concentration of Activin A is 50 - 100 ng / mL, the concentration of CHIR99021 is 3 - 5 μM, and the concentration of VEGF is 10 - 20 ng / mL.

3. The myocardial differentiation kit with screening and purification functions according to claim 1, characterized in that, The basal medium contains DMEM / F12 medium, L-glutamine, non-essential amino acids, β-mercaptoethanol, and fetal bovine serum.

4. A myocardial differentiation kit with screening and purification functions according to claim 1, characterized in that, The cardiac cell surface marker is cTNT. The concentration of the magnetic bead-conjugated antibody is 1 - 5 μg / mL, and the magnetic bead separation buffer is PBS buffer, 0.5% BSA, and 2 mM EDTA.

5. A myocardial differentiation kit with screening and purification functions according to claim 1, characterized in that, The magnetic bead-conjugated antibody can specifically bind to cTNT on the surface of cardiac cells, and rapid separation and purification of cardiac cells can be achieved through a magnetic separation device.

6. The method of using a myocardial differentiation kit with screening and purification functions according to any one of claims 1-5, characterized in that, It includes the following steps: Step 1: First, perform cell seeding. Seed pluripotent stem cells at an appropriate density in a cell culture plate containing the basal medium, and culture them in an incubator at 37°C and 5% CO2. When the cell confluence reaches 70 - 80%, perform differentiation induction. Step 2: In the differentiation induction of Step 1, aspirate the basal medium, and add a differentiation medium containing a combination of differentiation inducers to induce the gradual differentiation of pluripotent stem cells into cardiomyocytes. Step 3: Screen and purify the cardiomyocytes in Step 3. After the differentiation culture is completed, discard the differentiation medium, gently rinse the cells 2 - 3 times with PBS buffer, add an appropriate amount of the magnetic bead-conjugated antibody in the screening and purification reagent, and incubate to allow the magnetic bead-conjugated antibody to fully bind to cTNT on the surface of cardiac cells. Step 4: After the incubation is completed, add the magnetic bead separation buffer, digest the cells with trypsin, collect the cell suspension, transfer the cell suspension to a magnetic separation device, perform magnetic separation on it, and separate the cardiomyocytes bound to the magnetic bead-conjugated antibody, which are the purified cardiomyocytes.

7. The method for using a myocardial differentiation kit with screening and purification functions according to claim 6, characterized in that, In step one, the pluripotent stem cells are at an appropriate density of 5×10 4 -1×10 5 cells per square centimeter.

8. The usage method of a myocardial differentiation kit with screening and purification functions as described in claim 6, characterized in that, In Step 2, culture in an incubator at 37°C and 5% CO2. On the 1st - 3rd day of culture, use a differentiation medium containing BMP4 and Activin A. On the 4th - 6th day, change to a differentiation medium containing CHIR99021. On the 7th - 10th day, use a differentiation medium containing VEGF.

9. The method for using a myocardial differentiation kit with screening and purification functions according to claim 6, characterized in that, In Step 3, the incubation is carried out at 4°C for 30 - 60 min.

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