Method for preparing myocardial cells through induced differentiation

Abstract

The invention discloses a method for preparing myocardial cells through induced differentiation, and relates to the technical field of biotechnology and cytobiology, the method for preparing myocardial cells through induced differentiation comprises the following steps: S1, pretreatment: treating a culture interface of a culture container, and coating by using a specific matrix or protein; s2, inoculating iPSC: inoculating into the pretreated culture container according to a specific cell suspension concentration, and waiting for cell adherence; s3, a first induced differentiation stage: discarding all the culture medium in the step S2, and adding a proper amount of a first-stage induced culture medium for treatment; s4, a second induced differentiation stage: adding a proper amount of a second-stage induction culture medium, treating and harvesting cells; the purpose of directionally differentiating the myocardial cells by the iPSC is achieved through a staged culture mode, a new culture medium formula is used, a new thought and method for stem cell differentiation and directional culture are provided, the iPSC is effectively differentiated into the myocardial cells, and corresponding gene markers are expressed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and cell biology, in particular to a method for preparing myocardial cells by inducing differentiation. Background Art

[0002] Induced pluripotent stem cells (iPSCs) are artificially induced stem cells that are highly similar to embryonic stem cells (ESCs) in terms of cell morphology, growth characteristics, and expression of stem cell markers. They are also almost completely identical in terms of DNA methylation patterns, gene expression profiles, chromatin states, and the formation of chimeric animals.

[0003] iPSCs have strong self-renewal and multidirectional differentiation potentials, and can differentiate into almost all 210 types of human cells in vitro, bringing broad prospects for disease treatment. Compared with ESCs, using iPSCs for tissue or organ transplantation can avoid rejection by the immune system, while also avoiding the ethical controversy faced by ESCs. Therefore, iPSCs have become an important source of stem cells that have attracted much attention in the field of regenerative medicine.

[0004] The breakthrough development of stem cell technology will change the future medical treatment. It is no longer limited to chemical synthetic drugs, but uses small molecule proteins, antibodies or stem cells with biological activity to deliver them into the body in a targeted manner to achieve disease treatment. In particular, the 2012 Nobel Prize in Physiology or Medicine was awarded to Japanese scientist Shinya Yamanaka, who successfully reversed adult cells into iPSCs, which greatly promoted the development and application of iPSC technology. Induced pluripotent stem cell (iPSC) technology has shown broad application prospects in the field of regenerative medicine. Currently, 9 indications have entered the clinical trial stage, including corneal transplantation, Parkinson's disease, retinal macular degeneration, heart disease, spinal cord disease, transfusion disease, arthritis, diabetes and tumor-related diseases.

[0005] In my country, related iPSC-derived cardiomyocyte products have also been approved for clinical trials. Cardiomyocytes differentiated from iPSCs are used for the treatment of severe chronic ischemic heart failure through intravenous injection. Compared with traditional treatments, this method is not only safer, but also can significantly improve patients' cardiac function and quality of life. In addition, the chemical induction method has further enriched the somatic cell "reprogramming" technology. The use of specific chemical small molecules can promote the remodeling of somatic cells into pluripotent stem cells and further differentiate into the desired cell types, such as cardiomyocytes. This method is simple to operate, efficient, and does not require gene editing, avoiding related ethical disputes. More importantly, the transplantation of iPSC-derived cells prepared from the patient's own cells can avoid immune rejection and improve the safety and effectiveness of treatment.

[0006] In general, iPSC-differentiated cardiomyocytes show broad application prospects in the treatment of heart failure, but the relevant research is not sufficient, and the optional alternative products are not yet mature, and further research and development and clinical verification are still needed; the method of inducing differentiation to prepare cardiomyocytes in the prior art does not achieve the purpose of directed differentiation of iPSC into cardiomyocytes through staged culture, and does not adopt the culture medium formula provided in this application to effectively differentiate iPSC into cardiomyocytes and express corresponding gene markers; therefore, in view of this situation, there is an urgent need to develop a method for inducing differentiation to prepare cardiomyocytes to meet the needs of actual use. Summary of the invention

[0007] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a method for inducing differentiation to prepare cardiomyocytes. Through a staged culture approach, the purpose of directed differentiation of iPSC into cardiomyocytes is achieved. A new culture medium formula is used to provide new ideas and methods for stem cell differentiation and directed culture, which effectively differentiates iPSC into cardiomyocytes and expresses corresponding gene markers.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A method for inducing differentiation to prepare cardiomyocytes comprises the following steps:

[0010] S1. Pretreatment: Treat the culture interface of the culture container and coat it with a specific matrix or protein;

[0011] S2, iPSC inoculation: Inoculate cells into a pre-treated culture container at a specific cell suspension concentration and wait for the cells to adhere to the wall. The culture medium used in this stage is iPSC culture medium, and the culture time is 1-3 days;

[0012] S3, first induction differentiation stage: discard all the culture medium in step S2, add an appropriate amount of the first stage induction medium, and treat for 2-3 days without changing the medium;

[0013] S4, second induction differentiation stage: discard all the culture medium in step S3, add an appropriate amount of second stage induction medium, and treat for 10 days, wherein the medium is completely replaced every 2 days. UM171 is added from the eighth day until the cells are harvested on the 10th day.

[0014] As a preferred embodiment: the first stage induction medium used in step S3 includes stem34pro medium and a first factor additive, wherein the first factor additive is: 5-30 ng / ml VEGF, 5-30 ng / ml SCF, 3-10 nM A83-01, 2-10 nM BRD5648, 5-30 ng / ml BMP4 and 2 nM glutamine.

[0015] As a preferred embodiment: the second stage induction medium used in step S4 includes stem34pro medium and a second factor additive, and the second factor additive is: 25-60ng / ml VEGF, 25-60ng / ml SCF, 25-60ng / ml Flt-3, 5-30ng / ml TPO, 5-30ng / ml IL-3, 5-30ng / ml GM-SCF, 75-120ng / ml UM171, and 20-100nM LDN-212854.

[0016] As a preferred embodiment: in step S1, the specific matrix or protein includes one or more combinations of matrigel, placental decellularized matrix, laminin-521 protein and fibronectin.

[0017] As a preferred solution: in step S1, the specific matrix or protein includes laminin-521 protein with a concentration of 3 μg / ml and fibronectin with a concentration of 2 μg / ml.

[0018] As a preferred solution: during the iPSC inoculation process in step S2, the cell suspension concentration used is 20,000-80,000 cells / ml.

[0019] As a preferred solution: the concentration of the cell suspension used in step S2 is 50,000 cells / ml; the iPSC culture medium used in step S2 is E8 culture medium or mTseR culture medium.

[0020] As a preferred solution: in the second differentiation induction stage in step S4, the cells are harvested by gently washing the cells with D-PBS buffer, adding an appropriate amount of accutase to digest for 2 minutes to dissociate the cells, collecting the digested cell suspension, centrifuging, collecting the cell precipitate, and obtaining cardiomyocytes.

[0021] As a preferred solution: the pretreatment in step S1 is specifically: using a Ca-free 2+ Mg 2+ The culture container was rinsed with ion-free PBS solution, and after discarding the rinse solution, 3μg / ml laminin-521 and 2μg / ml fibronectin were added to the culture container, and then placed in a 37℃ incubator containing 5% CO2 for coating for at least 1 hour.

[0022] As a preferred solution: in step S2, iPSCs are revived before iPSC inoculation: iPSCs are taken out from the deep cryogenic environment, quickly thawed at 37°C, and centrifuged at 200g for 3-5 minutes using D-PBS.

[0023] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that by adopting a method for inducing differentiation to prepare cardiomyocytes provided by the present application, the purpose of directed differentiation of iPSC into cardiomyocytes is achieved through staged culture. Compared with the prior art, this method uses a new culture medium formula and provides a new idea and method for stem cell differentiation and directed culture; the present application constructs a complete set of methods for preparing cardiomyocytes, which are easy to operate and highly practical; the present application clarifies that under this method for inducing differentiation to prepare cardiomyocytes, iPSCs can be effectively differentiated into cardiomyocytes and express corresponding gene markers.

[0024] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A process flow chart of a method for inducing differentiation and preparing cardiomyocytes according to the present invention;

[0026] Figure 2 This is an observation picture of the second day of culture of iPSC-directed induced differentiation of cardiomyocytes of the present invention;

[0027] Figure 3 This is an observation picture of the iPSC directed induction differentiation of cardiomyocytes cultured on the 4th day of the present invention;

[0028] Figure 4 This is an observation picture of the iPSC directed induction differentiation of cardiomyocytes cultured on the 7th day of the present invention;

[0029] Figure 5 This is an observation picture of the iPSC directed induction differentiation of cardiomyocytes cultured on the 12th day of the present invention;

[0030] Figure 6 This is a real-time quantitative detection diagram of characteristic genes at different culture time points of iPSC-directed induced cardiomyocyte differentiation of the present invention;

[0031] Figure 7 Schematic diagram showing the effects of different BRD5648 concentrations on the directed induction and differentiation of iPSC into cardiomyocytes according to the present invention;

[0032] Figure 8 Schematic diagram showing the effects of different A83-01 concentrations on the directed induction and differentiation of iPSCs into cardiomyocytes according to the present invention;

[0033] Fig. 9 Schematic diagram of the effects of different LDN-212854 concentrations on the directed induction and differentiation of iPSCs into cardiomyocytes according to the present invention. DETAILED DESCRIPTION

[0034] The present invention Figures 1 to 9 As shown, a method for inducing differentiation to prepare cardiomyocytes comprises the following steps:

[0035] S1. Pretreatment: Treat the culture interface of the culture container and coat it with a specific matrix or protein;

[0036] S2, iPSC inoculation: Inoculate cells into a pre-treated culture container at a specific cell suspension concentration and wait for the cells to adhere to the wall. The culture medium used in this stage is iPSC culture medium, and the culture time is 1-3 days;

[0037] S3, first induction differentiation stage: discard all the culture medium in step S2, add an appropriate amount of the first stage induction medium, and treat for 2-3 days without changing the medium;

[0038] S4, second induction differentiation stage: discard all the culture medium in step S3, add an appropriate amount of second stage induction medium, and treat for 10 days, wherein the medium is completely replaced every 2 days. UM171 is added from the eighth day until the cells are harvested on the 10th day.

[0039] The first stage induction medium used in step S3 includes stem34pro medium and the first factor additive, and the first factor additive is: 5-30ng / ml VEGF, 5-30ng / ml SCF, 3-10nM A83-01, 2-10nM BRD5648, 5-30ng / ml BMP4 and 2nM glutamine.

[0040] The second stage induction medium used in step S4 includes stem34pro medium and a second factor additive, wherein the second factor additive is: 25-60 ng / ml VEGF, 25-60 ng / ml SCF, 25-60 ng / ml Flt-3, 5-30 ng / ml TPO, 5-30 ng / ml IL-3, 5-30 ng / ml GM-SCF, 75-120 ng / ml UM171, and 20-100 nM LDN-212854.

[0041] In step S1, the specific matrix or protein includes one or more combinations of matrigel, placental decellularized matrix, laminin-521 protein and fibronectin.

[0042] In step S1, the specific matrix or protein includes laminin-521 protein at a concentration of 3 μg / ml and fibronectin at a concentration of 2 μg / ml.

[0043] During the iPSC inoculation process in step S2, the cell suspension concentration used is 20,000-80,000 cells / ml.

[0044] The cell suspension concentration used in step S2 is 50,000 cells / ml; the iPSC culture medium used in step S2 is E8 culture medium or mTseR culture medium.

[0045] In the second differentiation induction stage in step S4, the cells are harvested by gently washing the cells with D-PBS buffer, adding an appropriate amount of accutase to digest for 2 minutes to dissociate the cells, collecting the digested cell suspension, centrifuging, collecting the cell precipitate, and obtaining cardiomyocytes.

[0046] The pretreatment in step S1 is specifically as follows: using a Ca-free 2+ Mg 2+ The culture container was rinsed with ion-free PBS solution, and after discarding the rinse solution, 3μg / ml laminin-521 and 2μg / ml fibronectin were added to the culture container, and then placed in a 37℃ incubator containing 5% CO2 for coating for at least 1 hour.

[0047] In step S2, iPSCs are revived before iPSC inoculation: iPSCs are taken out from the deep cryogenic environment, quickly thawed at 37° C., and centrifuged at 200 g for 3-5 minutes using D-PBS.

[0048] Example 1: A method for inducing differentiation to prepare cardiomyocytes

[0049] S1. Preprocessing

[0050] Coating of culture surfaces

[0051] Rinse the T75 culture flask with PBS solution that does not contain Ca2+ and Mg2+ ions, discard the rinse solution, add 3μg / ml laminin-521 and 2μg / ml fibronectin into the culture container, and then place it in a 37℃ incubator containing 5% CO2 for coating for at least 1 hour. Aspirate the solution in the culture flask before use.

[0052] S2. iPSC recovery, iPSC digestion and plating

[0053] The iPSCs were taken out from the deep cryogenic environment, quickly thawed in a 37°C water bath, centrifuged at 200 g for 3 minutes using D-PBS, washed once, resuspended in an appropriate amount of E8 complete medium, and sampled for counting.

[0054] iPSCs were digested with accutase. After iPSCs were digested, the cell pellets were collected by centrifugation, resuspended at 50,000 cells / ml using iPSC culture medium, and CEPT was added. The cells were then seeded into T75 culture flasks and cultured for 24 hours before entering the next stage. CEPT included Chroman1, Emricasan, Polyamine, and Trans-ISRIB.

[0055] S3, the first induction differentiation stage

[0056] All the culture medium in the T75 culture flask was discarded and replaced with the first stage induction medium, and cultured for 48 hours without any treatment during this period.

[0057] S4, the second induction differentiation stage

[0058] All the medium in the T75 culture flask was discarded and replaced with the second stage induction medium. The medium was changed every 48 hours, and UM171 was added on the eighth day of culture until the end of the tenth day of culture.

[0059] Harvesting cells

[0060] The differentiated cardiomyocytes are digested with accutase, and the precipitate is collected by centrifugation to obtain a cardiomyocyte suspension. The present invention can obtain cardiomyocytes within 12 days, and is suitable for the scenario of quickly obtaining cardiomyocytes for application.

[0061] Detection of myocardial related gene marker expression

[0062] RNA was extracted from iPSCs and differentiated cells at different time points and reverse transcribed to obtain cDNA libraries. Real-time fluorescence quantitative detection was used to detect cells before and after differentiation, and myocardial-related gene markers such as S100A1, Myl2, cTNT, Mef2c, HEY1, and CD36 were detected.

[0063] The results showed that the directed differentiation was correct and successful, and iPSCs could effectively differentiate into cardiomyocytes and express corresponding gene markers such as S100A1, Myl2, cTNT, Mef2c, HEY1, CD36 and other gene markers of successful cardiomyocyte differentiation; Figure 6 shown.

[0064] Example 2: Effects of different concentrations of BRD5648 on cardiomyocyte differentiation

[0065] As a factor that has an important influence on myocardial differentiation, this example introduces the specific screening of preferred concentrations:

[0066] A concentration gradient of BRD5648 was set in the first stage induction medium, namely 2, 4, 8, and 10 nM, while other conditions remained unchanged, for cardiomyocyte differentiation, and qPCR was used to detect the expression of cardiomyocyte marker genes.

[0067] The differentiation step is as described in Example 1. The qPCR detection method is as follows: extracting cell RNA before and after differentiation and performing reverse transcription to obtain a cDNA library. The cells before and after differentiation are detected using real-time fluorescence quantitative detection to detect myocardial-related gene markers such as S100A1, cTNT, and CD36.

[0068] The experimental results show that when the concentration of BRD5648 is 8nM, the expression of S100A1, cTNT, and CD36 can achieve the best effect, so 8nM BRD5648 is the preferred concentration; Figure 7 shown.

[0069] Example 3: Effects of different concentrations of A83-01 on cardiomyocyte differentiation

[0070] As a factor that has an important influence on myocardial differentiation, this example introduces the specific screening of the preferred concentration

[0071] A concentration gradient of A83-01 in the first stage induction medium was set to 3, 4, 7, and 10 nM, respectively, while other conditions remained unchanged for cardiomyocyte differentiation, and qPCR was used to detect the expression of cardiomyocyte marker genes.

[0072] The differentiation step is as described in Example 1. The qPCR detection method is as follows: extracting cell RNA before and after differentiation and performing reverse transcription to obtain a cDNA library. The cells before and after differentiation are detected using real-time fluorescence quantitative detection to detect myocardial-related gene markers such as S100A1, cTNT, and CD36.

[0073] The experimental results show that when the concentration of A83-01 is 7nM, the expression of S100A1, cTNT, and CD36 can achieve the best effect, so 7nM A83-01 is the preferred concentration; Figure 8 shown.

[0074] Example 4: Effects of different concentrations of LDN-212854 on cardiomyocyte differentiation

[0075] As a factor that has an important influence on myocardial differentiation, this example introduces the specific screening of preferred concentrations:

[0076] The concentration gradient of LDN-212854 in the first stage induction medium was set to 20, 40, 80, and 100 nM, respectively, and the other conditions remained unchanged for cardiomyocyte differentiation. The expression of cardiomyocyte marker genes was finally detected by qPCR.

[0077] The differentiation step is as described in Example 1. The qPCR detection method is as follows: extracting cell RNA before and after differentiation and performing reverse transcription to obtain a cDNA library. The cells before and after differentiation are detected using real-time fluorescence quantitative detection to detect myocardial-related gene markers such as S100A1, cTNT, and CD36.

[0078] The experimental results show that when the concentration of LDN-212854 is 40nM, the expression of S100A1, cTNT, and CD36 can achieve the best effect, so 40nM LDN-212854 is the preferred concentration; Fig. 9 shown.

[0079] The design focus of the present invention is that by adopting a method for inducing differentiation to prepare cardiomyocytes provided by the present application, the purpose of directed differentiation of iPSC into cardiomyocytes is achieved through a staged culture method. Compared with the existing technology, this method uses a new culture medium formula and provides a new idea and method for stem cell differentiation and directed culture; the present application constructs a complete set of methods for preparing cardiomyocytes, which are easy to operate and highly practical; the present application clarifies that under this method for inducing differentiation to prepare cardiomyocytes, iPSCs can be effectively differentiated into cardiomyocytes and express corresponding gene markers.

[0080] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for inducing differentiation to prepare cardiomyocytes, characterized in that: The steps include: S1. Pretreatment: Treat the culture interface of the culture container and coat it with a specific matrix or protein; S2, iPSC inoculation: Inoculate cells into a pre-treated culture container at a specific cell suspension concentration and wait for the cells to adhere to the wall. The culture medium used in this stage is iPSC culture medium, and the culture time is 1-3 days; S3, first induction differentiation stage: discard all the culture medium in step S2, add an appropriate amount of the first stage induction medium, and treat for 2-3 days without changing the medium; S4, second induction differentiation stage: discard all the culture medium in step S3, add an appropriate amount of second stage induction medium, and treat for 10 days, wherein the medium is completely replaced every 2 days. UM171 is added from the eighth day until the cells are harvested on the 10th day.

2. The method for preparing cardiomyocytes by inducing differentiation according to claim 1, characterized in that: The first stage induction medium used in step S3 includes stem34pro medium and a first factor additive, wherein the first factor additive is: 5-30 ng / ml VEGF, 5-30 ng / ml SCF, 3-10 nM A83-01, 2-10 nM BRD5648, 5-30 ng / ml BMP4 and 2 nM glutamine.

3. The method for preparing cardiomyocytes by inducing differentiation according to claim 1, characterized in that: The second stage induction medium used in step S4 includes stem34pro medium and a second factor additive, wherein the second factor additive is: 25-60ng / ml VEGF, 25-60ng / ml SCF, 25-60ng / ml Flt-3, 5-30ng / ml TPO, 5-30ng / ml IL-3, 5-30ng / ml GM-SCF, 75-120ng / ml UM171, and 20-100nM LDN-212854.

4. The method for preparing cardiomyocytes by inducing differentiation according to claim 1, characterized in that: In step S1, the specific matrix or protein includes one or more combinations of matrigel, placental decellularized matrix, laminin-521 protein and fibronectin.

5. The method for preparing cardiomyocytes by inducing differentiation according to claim 4, characterized in that: In the step S1, the specific matrix or protein includes laminin-521 protein with a concentration of 3 μg / ml and fibronectin with a concentration of 2 μg / ml.

6. The method for inducing differentiation and preparing cardiomyocytes according to claim 1, characterized in that: During the iPSC inoculation process in step S2, the cell suspension concentration used is 20,000-80,000 cells / ml.

7. The method for preparing cardiomyocytes by inducing differentiation according to claim 6, characterized in that: The cell suspension concentration used in step S2 is 50,000 cells / ml; the iPSC culture medium used in step S2 is E8 culture medium or mTseR culture medium.

8. The method for preparing cardiomyocytes by inducing differentiation according to claim 1, characterized in that: In the second differentiation induction stage in step S4, the cells are harvested by gently washing the cells with D-PBS buffer, adding an appropriate amount of accutase to digest for 2 minutes to dissociate the cells, collecting the digested cell suspension, centrifuging, collecting the cell precipitate, and obtaining cardiomyocytes.

9. The method for preparing cardiomyocytes by inducing differentiation according to claim 5, characterized in that: The pretreatment in step S1 is specifically: using a Ca-free 2+ Mg 2+ The culture container was rinsed with ion-free PBS solution, and after discarding the rinse solution, 3μg / ml laminin-521 and 2μg / ml fibronectin were added to the culture container, and then placed in a 37℃ incubator containing 5% CO2 for coating for at least 1 hour.

10. The method for preparing cardiomyocytes by inducing differentiation according to claim 1, characterized in that: In step S2, iPSCs are revived before iPSC inoculation: iPSCs are taken out from the deep cryogenic environment, quickly thawed at 37° C., and centrifuged at 200 g for 3-5 minutes using D-PBS.

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