Method for obtaining high-purity myocardial cells by inducing differentiation of pluripotent stem cells

Abstract

The invention relates to a method for producing myocardial cells through differentiation induction of iPSC (induced pluripotent stem cells). Specifically, LAA is added in the differentiation process to improve the cell viability and the myocardial cell conversion rate, by means of accurate adjustment of an agonist CHIR99021 and an inhibitor IWR-1 of a Wnt / beta-catenin signal channel, continuous activation of the Wnt signal channel is maintained in the early stage to enable iPSC to be stably converted into mesoderm cells, the success rate of myocardial differentiation is improved, and finally, the success rate of myocardial differentiation is increased. The high-purity myocardial cells are obtained through treatment of a sugar-free purification reagent. The method for differentiating the myocardial cells from the iPSC is high in universality and repeatability, can be well applied to a disease source difficult to differentiate or an iPSC cell line subjected to gene editing, and can be widely applied to the field of myocardial cell differentiation. And the obtained high-purity myocardial cells can be widely applied to heart safety evaluation of drugs, high-throughput screening of cardiovascular treatment drugs, and application of the myocardial cells as 2D and 3D research models of cardiovascular diseases to cardiovascular pathogenesis research.

Description

Technical Field

[0001] The present invention relates to a method for differentiating cardiomyocytes from induced pluripotent stem cells (iPSCs), and in particular to a method for obtaining high-purity cardiomyocytes through differentiation. Background Art

[0002] Human iPSC-differentiated cardiomyocytes have similar contractile functions, action potentials, and ion channels as primary cardiomyocytes, and can be mass-produced in vitro, making them the best in vitro model for cardiovascular disease research, and can address a large number of unmet needs in drug development and preclinical research. Currently, domestic and foreign research institutions and pharmaceutical companies have widely used them for drug cardiac safety evaluation, high-throughput screening of cardiovascular disease therapeutic drugs, and as 2D and 3D research models for cardiovascular disease, and for the study of cardiovascular pathogenesis.

[0003] As the application of iPSC-differentiated cardiomyocytes in scientific research and industrial fields continues to expand, the methods of iPSC-differentiated cardiomyocytes are also constantly updated. Among them, the most common and classic differentiation method is to use different combinations of small molecule additives to regulate the Wnt signaling pathway to induce iPSC differentiation into cardiomyocytes. Commonly used small molecule additives include Chir99021, IWR-1, IWP-2, IWP-4, Wnt-C59, etc. However, the differentiation efficiency of different small molecule additives varies greatly, and the same small molecule additives with different concentrations and different durations of action also have an impact on the differentiation efficiency. Most of the methods of iPSC-differentiated cardiomyocytes that are already well known to everyone are not efficient, and the differentiation efficiency of different batches and different samples is extremely unstable and has poor reproducibility. At present, the differentiation of iPSC-derived cardiomyocytes has become a problem that plagues many scientific researchers.

[0004] The differentiation of iPSC into cardiomyocytes goes through four stages. First, iPSC differentiates into mesendoderm cells, then the proliferation of mesoderm cells, followed by the transformation of mesoderm cells into early cardiomyocytes, and finally the maturation of cardiomyocytes, during which spontaneous beating of cardiomyocytes is observed. What is more important in the whole process is the state of iPSC before the differentiation begins, which determines the final conversion efficiency of cardiomyocytes. In most cases, the efficiency of iPSC differentiation into mesoderm cannot reach 100%, and the conversion efficiency of mesoderm into early cardiomyocytes is not complete, resulting in the final cardiomyocyte purity often being lower than expected. We can further improve the purity of cardiomyocytes by treating them with purification reagents. Summary of the invention

[0005] At present, the methods for differentiating cardiomyocytes from iPSCs generally have the problems of low efficiency, weak universality and poor repeatability. The present invention mainly provides a method for differentiating cardiomyocytes from iPSCs with improved differentiation efficiency, strong universality and high repeatability, and the method can further improve the purity of cardiomyocytes differentiated from iPSCs.

[0006] For the above invention, the specific technical solution is:

[0007] This protocol uses the precise regulation of CHIR99021, an agonist of the Wnt / β-catenin signaling pathway, and IWR-1, an inhibitor, during the differentiation of iPSC into cardiomyocytes, so that iPSCs differentiate into mesendoderm cells after the initial activation of the Wnt signaling pathway, and stably transform into mesoderm cells after the continuous activation of the Wnt signaling pathway, while promoting the proliferation of mesoderm cells; then, through the inhibition of the Wnt signaling pathway, mesoderm cells are directed to differentiate into cardiomyocytes. During the differentiation process, ascorbic acid (LAA) is added throughout the process to improve the survival rate of cells during differentiation and the conversion rate of iPSC into cardiomyocytes. In the later stage, the purity of differentiated cardiomyocytes is further improved by treatment with sugar-free purification reagents.

[0008] Specific steps for implementing the plan:

[0009] Culture of iPSCs before cardiomyocyte differentiation: iPSCs are passaged 3-5 times, and the confluence reaches 70-85% and is ready for re-plating. According to the difference in the growth rate of different iPSCs, iPSCs are digested with Tryple for 3-5 minutes, centrifuged and discarded, and then resuspended with Stemflex+10uM Y-27632, and iPSCs are re-plated at a ratio of 1:2-1:4. After re-plating, continue to culture for 48 hours, and use Stemflex to replace the medium once every 24 hours. iPSCs form a good tight monolayer state, and the confluence is greater than 90%, ready for cardiomyocyte differentiation.

[0010] Steps for cardiomyocyte differentiation (taking 6-well plate as an example):

[0011] The formula of cardiomyocyte differentiation basal solution is: RPMI 1640, 2% B27 minus insulin, 50-200uM LAA; the formula of cardiomyocyte culture medium is: RPMI 1640, 2% FBS, 2% B27, 1% Pen Strep;

[0012] The formula of myocardial cell purification solution is: DMEM (no glucose), HEPES, L-Sodium Lactate;

[0013] Step S1, Day 0: Add 2 ml of cardiomyocyte differentiation medium to each well, add 5-10 uM CHIR99021, and maintain culture for 24 hours;

[0014] Step S2, Day 1-2: Add 2 ml of cardiomyocyte differentiation medium to each well, add 0.5-2 uM CHIR99021, and change the medium every 24 hours;

[0015] Step S3, Day 3: Add 3 ml of cardiomyocyte differentiation medium to each well, add 5-10 uM IWR-1, and maintain culture for 48 hours;

[0016] Step S4, Day 5-8: Add 2 ml of cardiomyocyte differentiation medium to each well and change the medium every 24 hours;

[0017] Step S5, Day 9: Digest the cardiomyocytes with 0.25% Trypsin-EDTA for 3-5 minutes, terminate the digestion with DMEM F12 containing 10% FBS, discard the supernatant after centrifugation, resuspend with cardiomyocyte culture medium + 10uM Y-27632, inoculate into a 10cm dish coated with Matrigel, and change the medium every 48 hours;

[0018] Step S6, Day 11: Discard the supernatant and add 10 ml of myocardial cell purification solution to a 10 cm dish for 48 hours;

[0019] Step S7, Day 13-20: Discard the supernatant, add 10 ml of cardiomyocyte culture medium to a 10 cm dish, and change the medium every 48 hours.

[0020] Furthermore, DMEM (no glucose) in the formula of cardiomyocyte purification solution can be replaced by RPMI 1640 (no glucose);

[0021] Furthermore, in step S1, after adding 5-10uM CHIR99021 to the differentiation medium for 24 hours, some cells will die, which is a normal phenomenon;

[0022] Furthermore, in step S2, the cell confluence can reach more than 90% after adding 0.5-2uM CHIR99021 to the differentiation medium for 48 hours;

[0023] Furthermore, in step S3, 5-10uM IWR-1 can be replaced by 2-5uM Wnt-C59 with similar differentiation effects;

[0024] Furthermore, in step S4, cardiomyocyte beating can be seen on the 7th or 8th day of differentiation;

[0025] Furthermore, in step S5, according to 6 to 8×10 6 The cells were seeded into 10 cm dishes.

[0026] Beneficial effects of the present invention:

[0027] The present invention is a cardiomyocyte differentiation method widely applicable to various iPSC samples, including healthy samples, disease samples and gene-edited samples. On the 0th day of differentiation, after CHIR99021 is added to the differentiation medium for 24 hours, the Wnt signaling pathway is activated to convert iPSC into mesendoderm cells, and different iPSC cell lines have different numbers of cell deaths in this step. On the 1st and 2nd days of differentiation, a small amount of CHIR99021 is continued to be added to the differentiation medium to maintain the activation of the Wnt signaling pathway and stably transform into mesoderm cells, while promoting the proliferation of mesoderm cells, so that the cell confluence is restored to a higher level. On the 3rd to 4th day of differentiation, IWR-1 is added to the differentiation medium to convert the mesoderm into early cardiomyocytes, and the beating of cardiomyocytes is observed on the 7th or 8th day through the subsequent maintenance culture of the differentiation medium. During the differentiation process, by continuously adding LAA to the differentiation medium, the confluence of surviving cells during the cardiomyocyte transformation process is increased, and the yield of cardiomyocytes is increased. In addition, considering the differences in cardiomyocyte conversion efficiency of different iPSC cell lines, in order to obtain high-purity cardiomyocytes for downstream experiments, a digestion and re-plating step was added on the 9th day, and a step of sugar-free purification solution treatment on the 11th day, so that high-purity cardiomyocytes can be obtained by differentiation of different iPSC samples. The present invention uses iPSC to differentiate cardiomyocytes in a simple step, and each step has a clear role. By adding a small amount of CHIR99021 on the first and second days of differentiation, iPSC can be stably converted into mesoderm cells and the number of cells can be restored, solving the problem of difficult cardiomyocyte differentiation of iPSCs in certain diseases and iPSC cell lines after gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Flow chart for differentiation of iPSCs into cardiomyocytes;

[0029] Figure 2 The bright field image of iPSCs reaching a density of more than 90% after 48 hours of re-plating (including the bright field images of re-plating for 24 hours and 48 hours);

[0030] Figure 3 The difference between adding LAA and not adding LAA during differentiation (bright field images from day 2 to day 8 of differentiation with and without LAA, with more surviving cells and converted myocardium after adding LAA);

[0031] Figure 4This is a bright field image before and after myocardial cell purification (obvious non-myocardial cells exist before purification, and non-myocardial cell-specific death can be observed 48 hours after purification);

[0032] Figure 5 The figure is the flow cytometry graph of cTnT expression in cardiomyocytes before and after purification (the purity of cardiomyocytes before purification was less than 90%, and the purity of cardiomyocytes after purification was more than 95%).

Claims

1. A method for differentiating high-purity cardiomyocytes: During the process of iPSC differentiation into cardiomyocytes, 50-200uM ascorbic acid (LAA) is added throughout the process to improve the cell survival rate during the differentiation process and the conversion rate of iPSC into cardiomyocytes. After differentiation begins (Day 0), the differentiation solution containing 5-10uM CHIR99021 is used for 24 hours. On Day 1, the reagent is replaced with a differentiation solution containing 0.5-2uM CHIR99021 for 48 hours, and the solution is changed every 24 hours. On Day 3, the reagent is replaced with a differentiation solution containing 5-10uM IWR-1 for 48 hours. Starting from Day 5, the differentiation solution is used for 4 consecutive days, and the solution is changed every 24 hours. On Day 9, the digested cardiomyocytes are re-plated with cardiomyocyte culture medium + 10uM Y-27632 and cultured for 48 hours. Starting from Day 11, the cardiomyocyte purification solution is used for 48 hours. From Day 13 to 20, the culture is continued with cardiomyocyte culture medium, and the solution is changed every 48 hours. In the process of differentiating cardiomyocytes from iPSCs as described in claim 1, 50-200 uM LAA is added to the differentiation solution to improve the survival rate of cells during differentiation and the conversion rate of iPSCs to cardiomyocytes. The differentiation solution as claimed in claim 1, comprising RPMI 1640, 2% B27 minus insulin, and 50-200uM LAA. The cardiomyocyte culture medium according to claim 1, comprising RPMI 1640, 2% FBS, 2% B27, and 1% PenStrep. The myocardial cell purification solution as claimed in claim 1, which comprises DMEM (no glucose), HEPES, and L-Sodium Lactate, and utilizes the sugar-free basic solution to remove non-myocardial cell debris and increase the purity of myocardial cells to more than 95%. The differentiation solution added on Day 0 during the differentiation of cardiomyocytes from iPSCs as claimed in claim 1 contains 5-10 uM CHIR99021, which activates the Wnt signaling pathway to transform iPSCs into mesendoderm cells. The differentiation solution added on Day 1-2 during the iPSC differentiation of cardiomyocytes as claimed in claim 1 contains 0.5-2uM CHIR99021 to ensure that the Wnt signaling pathway is continuously activated and then stably transformed into mesoderm cells. The differentiation solution added on Day 3-4 during the iPSC differentiation of cardiomyocytes as claimed in claim 1 contains 5-10 uM IWR-1, which inhibits the Wnt signaling pathway and directs the transformation of mesoderm cells into early cardiomyocytes. The digestion and resurfacing of cardiomyocytes on Day 9 as described in claim 1 is to enable the cardiomyocytes to form a monolayer of cells, so that the cardiomyocyte purification solution added on Day 11 can fully interact with the cardiomyocytes and improve the efficiency of purification. The addition of the cardiomyocyte purification solution on Day 11 as described in claim 1 for 48 hours is to utilize a sugar-free culture system to remove non-cardiomyocytes and improve the purity of the produced cardiomyocytes. The cardiomyocytes themselves have a certain tolerance to the sugar-free culture system.

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