CN108913655B - Method for establishing human-derived myocardial hypertrophy model based on pluripotent stem cell technology - Google Patents

Abstract

The invention discloses a method for establishing a "human-derived" myocardial hypertrophy model based on pluripotent stem cell technology. In this method, cardiomyocytes differentiated from pluripotent stem cells that have been cultured for about 30 days are resuspended and then seeded into a 12-well plate, with 1.5×10 ⁶ cells per well. About 24 hours after the cells adhere to the wall, the phorbol-12-myristate-13-acetate dosing experiment can be started, and the induction is carried out at a concentration of 1 μM for 4 days. The invention can establish a stable human-derived myocardial hypertrophy model based on the pluripotent stem cell technology, and the shape and function conform to the characteristics of myocardial cell hypertrophy, the operation is convenient, and the modeling success rate is high.

Description

A method for establishing a "human-derived" cardiac hypertrophy model based on pluripotent stem cell technology

Technical field:

The invention belongs to the field of biomedical technology, and relates to a method for establishing a "human-derived" myocardial hypertrophy model based on pluripotent stem cell technology, which can be used for mechanism research of myocardial hypertrophy and research and development of therapeutic and preventive drugs.

Background technique:

Hypertrophic cardiomyopathy is one of the most common heart diseases, with an incidence rate of 0.2-0.5% in the general population. There are at least two million patients with hypertrophic cardiomyopathy in my country, and the main pathological manifestation is myocardial hypertrophy. Cardiac hypertrophy is caused by various diseases that trigger increased myocardial workload, such as hypertension, valvular insufficiency, myocardial infarction, hereditary diseases, endocrine disorders, etc. Failure, malignant arrhythmia and even sudden cardiac death, seriously endanger human health. Models currently used in studies of cardiac hypertrophy include mouse and rat models, Langendorff models of isolated hearts, and cardiomyocyte models isolated from non-primate or immortalized cell lines. However, these non-human disease models differ greatly from humans in various physiological systems (e.g., human cardiomyocytes beat at 60 beats/min, whereas mice beat at 400-600 beats/min), Many findings cannot be directly applied to humans. Therefore, how to establish a "human-derived" cardiomyocyte model is very important for the study of cardiac hypertrophy.

The emergence of human pluripotent stem cells (including human embryonic stem cells and human induced pluripotent stem cells) has provided a powerful impetus and a revolutionary paradigm shift for translational medicine and regenerative medicine, and has shortened the distance of stem cells for clinical disease treatment. It has great potential value in model establishment, disease molecular mechanism research, and new drug development. Compared with human adult stem cells, an obvious advantage of human pluripotent stem cells is their pluripotency, and it is this pluripotency that gives them the ability to differentiate into any cell in the human body, such as cardiomyocytes, Blood vessel cells, nerve cells, liver cells, etc. For cardiovascular disease research, human primary cardiomyocytes are the most ideal cell resource, but it is difficult to obtain and the in vitro culture conditions are harsh, so their application is greatly limited. The application of myocardial differentiation technology can differentiate human pluripotent stem cells into cardiomyocytes. These pluripotent stem cell-derived cardiomyocytes carry the human genome, have a "human-derived" physiological internal environment, are relatively easy to obtain and can be cultured in vitro for a long time. The most similar cell resource known to human primary cardiomyocytes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to establish a method for establishing a "human-derived" myocardial hypertrophy model based on pluripotent stem cell technology, so as to solve the problem of the lack of a "human-derived" myocardial hypertrophy model in the existing modeling technology, which is the mechanism of myocardial hypertrophy. Provides reliable human-derived models for research and development of therapeutic and preventive drugs.

For achieving the above object, technical scheme of the present invention is as follows:

A method for establishing a "human-derived" myocardial hypertrophy model based on pluripotent stem cell technology includes the following steps:

1) directional differentiation of pluripotent stem cells to obtain cardiomyocytes;

2) Aspirate the old cardiomyocyte medium, wash with Dulbecco's Phosphate Buffered Saline (DPBS), add 1 ml of trypsin-EDTA digestion solution to each well, digest in a 37°C incubator, and use DMEM/F12 medium to neutralize the digestion After centrifugation, cardiomyocyte culture medium (RPMI+B27+Insulin) was added, and the cells were inoculated into Matrigel-coated 12-well plates at a ratio of 1:8-1:12. 1.5×10 ⁶ cells per well, adherent for 24 hours;

3) PMA dosing experiment was carried out, 1.2 ml of PMA solution with a final concentration of 1 μM was added to each well for 4 days to obtain a myocardial hypertrophy model.

Preferably, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

Preferably, the described step 1) is specifically:

The 2D monolayer differentiation scheme induced by small molecule compounds is adopted. The basic technical route is to complete the induction and transformation of embryonic stem cells to mesoderm on days 0-1, and complete the induction and transformation of mesoderm to cardiac mesoderm on days 1-3. After -7 days, the induced transformation of mesoderm to cardiomyocytes was completed, and after the 7th day, it was mainly the maturation of cardiomyocytes. First, pluripotent stem cells such as H9 embryonic stem cells were inoculated into 6-well plates at a ratio of 1:8-1:12, and the medium was changed every day. When the cell density reached about 80%, the differentiation operation could be performed. Use up the old medium, wash the myocardial differentiation medium (RPMI+B27-Insulin) once, add 2 ml of differentiation medium containing 8 μM CHIR to each well, replace it with myocardial differentiation medium after 2 days, 2 ml per well, and on the 3rd day, each well. 2 ml of myocardial differentiation medium containing 5 μM IWR was added to the wells, and the culture was continued for 2 days. Change to myocardial differentiation medium on day 5. After the 7th day, the medium was changed to cardiomyocyte medium. At this time, if the differentiation effect was good, sheets of beating cardiomyocytes could be seen.

Preferably, in the myocardial differentiation medium (RPMI+B27-Insulin), B27-Insulin accounts for 2% of the total amount of the medium.

Preferably, in the cardiomyocyte culture medium (RPMI+B27+Insulin), B27+Insulin accounts for 2% of the total amount of the culture medium.

Preferably, the step 2) centrifugation conditions are: 1000rpm/min, centrifugation for 5 minutes.

Description of drawings

Figure 1 is a schematic diagram of the method steps for establishing a myocardial hypertrophy model based on pluripotent stem cell technology.

Figure 2 is a bar graph comparing the size of cardiomyocytes in the untreated group and the PMA-induced group.

Figure 3 is a bar graph comparing the proportion of multinucleated cells in cardiomyocytes of the untreated group and the PMA-induced group.

Figure 4 is a bar graph comparing the fluorescence intensity of ANF, a marker of cardiac hypertrophy, in cardiomyocytes of the untreated group and the PMA-induced group.

Fig. 5 is a bar graph comparing the gene expression of cardiac hypertrophy marker ANF in cardiomyocytes of untreated group and PMA-induced group.

Detailed ways

1) Directed differentiation of pluripotent stem cells to obtain cardiomyocytes: First, pluripotent stem cells such as H9 embryonic stem cells were seeded into 6-well plates (Corning) at a ratio of 1:8-1:12, and the stem cell medium mTeSR (STEM CELL Technology) was replaced daily. ), when the cell density reaches about 80%, differentiation can be performed. On day 0, the old medium was aspirated, the myocardial differentiation medium (RPMI+B27-Insulin) (Gibco) was washed once, and 2 ml of differentiation medium containing 8 μM CHIR (Medchem Axon) was added to each well. After 2 days, it was replaced with myocardial differentiation medium. Differentiation medium, 2 ml per well, on the third day, 2 ml of myocardial differentiation medium containing 5 μM IWR (Medchem Axon) was added to each well, and the culture was continued for 2 days. Change to myocardial differentiation medium on day 5. After the 7th day, the medium was changed to cardiomyocyte medium (RPMI+B27+Insulin) (Gibco). At this time, if the differentiation effect was good, sheets of beating cardiomyocytes could be seen.

2) Establishment of myocardial hypertrophy model: select cardiomyocytes cultured for about 30 days after differentiation, exhaust the old medium, wash with Dulbecco's phosphate buffered saline (DPBS) (Gibco), and add 1 ml of trypsin to each well. -EDTA digestion solution (Gibco), digested in a 37°C incubator, use DMEM/F12 medium (Gibco) to neutralize the digestion solution and collect the cell suspension into a centrifuge tube, add cardiomyocyte culture medium (RPMI+B27+Insulin) after centrifugation ) (Gibco), seeded into Matrigel (BD)-coated 12-well plates (Corning) at a ratio of 1:8-1: ¹² , 1.5 x 106 cells per well, adherent for 24 hours, and carried out In the PMA dosing experiment, 1.2 ml of PMA (Sigma Aldrich) solution with a final concentration of 1 μM was added to each well for 4 days to obtain a myocardial hypertrophy model (Figure 1).

3) Identification of cardiac hypertrophy model: The morphology and function of cardiomyocytes induced by PMA were identified by immunostaining and quantitative PCR experiments. Cardiomyocyte immunostaining was performed using the myocardial specific markers TNNT2 (Abcam) and α-actinin (Abcam), and the results showed that compared with the untreated group, the size of cardiomyocytes in the PMA-induced group was significantly increased (Figure 2), and the multinucleated cells were significantly increased. increased (Fig. 3); the myocardial specific marker TNNT2 (Abcam) and the myocardial hypertrophy specific marker atrial natriurelic factor (ANF) were co-stained, and the results showed that the ANF signal in the cardiomyocytes of the PMA-induced group was significantly enhanced (Fig. 4); the results of the real-time quantitative PCR experiment showed that the ANF gene expression in the cardiomyocytes of the PMA-induced group was significantly up-regulated (Fig. 5). The above experimental results confirmed that the cardiomyocytes induced by PMA had a clear characteristic of myocardial hypertrophy.

4) Application of myocardial hypertrophy model: The research results of the present invention show that a reliable "human-derived" myocardial hypertrophy model can be established by applying pluripotent stem cell technology, and the research of the present invention can solve the lack of "human-derived" in the existing modeling technology. The problem of cardiac hypertrophy model provides a reliable human-derived model for the mechanism study of cardiac hypertrophy and the development of therapeutic and preventive drugs.

Claims (1)

1. a method based on pluripotent stem cell technology to set up " human-derived " myocardial hypertrophy model, is characterized in that, comprises the steps: 1) Directed differentiation of pluripotent stem cells to obtain cardiomyocytes: First, H9 embryonic stem cells were seeded into 6-well plates at a ratio of 1:8-1:12, and the stem cell medium mTeSR was replaced daily. When the cell density reached 80%, differentiation was performed. Operation, on the 0th day, the old stem cell medium mTeSR was aspirated, the myocardial differentiation medium was washed once, and 8 μM CHIR and 2 ml of myocardial differentiation medium were added to each well. After 2 days, it was replaced with myocardial differentiation medium, 2 ml per well. , on the 3rd day, 5 μM IWR and 2 ml of myocardial differentiation medium were added to each well, and the culture was continued for 2 days. On the 5th day, the cardiomyocyte differentiation medium was replaced, and after the 7th day, the cardiomyocyte medium was replaced. 2) Establishment of myocardial hypertrophy model: Select cardiomyocytes cultured for 30 days after differentiation, drain the old medium, wash with Dulbecco's phosphate buffered saline (DPBS), and add 1 ml of trypsin-EDTA to each well for digestion Digested in a 37°C incubator, neutralized the digested solution with DMEM/F12 medium and collected the cell suspension into a centrifuge tube, added cardiomyocyte medium after centrifugation, and inoculated onto Matrigel at a ratio of 1:8-1:12 Matrigel-coated 12-well plate, 1.5×10 ⁶ cells per well, adherent for 24 hours, PMA dosing experiment was carried out, each well was added with phorbol-12-myristate-13- at a final concentration of 1 μM Acetate (Phorbol-12-myristate-13-acetate, PMA) solution 1.2 ml, acting for 4 days, to obtain myocardial hypertrophy model; The myocardial differentiation medium used in the steps 1) and 2) is basal medium RPMI plus insulin-free B27, wherein the insulin-free B27 accounts for 2% of the total amount of the total medium; The cardiomyocyte culture medium used in the steps 1) and 2) is the basal medium RPMI plus insulin-containing B27, and the insulin-containing B27 accounts for 2% of the total medium.

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