A method for inducing pluripotent stem cells to differentiate into cardiomyocytes using small molecules

Abstract

The present invention relates to the field of stem cell biotechnology, and discloses a reagent and technical system for obtaining cardiomyocytes by inducing pluripotent stem cells (PSC) using small molecules. The induction system of the present invention involves small molecules such as bFGF, RA, XAV939, Activin A, Chir99021, BMP4, FGF 10, and cytokines, which are added at fixed ratios at different times to induce expanded pluripotent stem cells (EPSC) and embryonic stem cells (ESC) to differentiate into cardiomyocytes. The present invention realizes 2D differentiation of pluripotent stem cells into cardiomyocytes, and cardiomyocytes can be obtained within 20 days, providing a stable research platform for the construction of heart disease models and drug screening.

Description

Technical Field

[0001] The present invention relates to the field of stem cell biotechnology, and in particular to the application of small molecules such as bFGF, RA, XAV939, Activin A, Chir99021, BMP4, FGF 10 and cytokines in inducing the differentiation of pluripotent stem cells such as EPSC into cardiomyocytes. Background Art

[0002] According to the World Health Statistics Report 2022 released by the World Health Organization, heart disease, dementia and stroke are still the number one killers of human death. In China, the high incidence of heart disease has become a trend, with an average of 2 out of every 5 deaths due to cardiovascular disease. Because cardiovascular disease has the characteristics of high morbidity, high disability rate and high mortality rate, even with the most advanced and complete treatment methods, the number of patients who die from heart failure after myocardial infarction is still as high as 7.4 million each year. With the development of society and the progress of science and technology, the incidence of ischemic heart disease is increasing, and the cases tend to be younger, which has attracted people's great attention. At present, the effective way to treat myocardial necrosis is mainly heart transplantation. However, due to the shortcomings of heart transplantation, such as donor shortage, low organ adaptability and complex immune response, most patients cannot receive heart transplantation in clinical practice. To this end, clinical and experimental studies have attempted to find a new treatment for myocardial necrosis.

[0003] Current studies have confirmed that stem cell transplantation can improve cardiac function in animal models of myocardial infarction. The differentiated cardiomyocytes can participate in the coordinated beating of the entire heart, effectively improve cardiac function, and provide a new idea for the treatment of heart disease.

[0004] As the most common economic animal in China, pigs have sufficient sources and diverse breeds. Because they have a high degree of similarity with humans in gross anatomy and organ function, they are often used as clinical models for disease research and experimental animals for regenerative medicine research. They are the preferred organ donors for human xenotransplantation. After using genetic engineering technology to knock out pigs' own retroviruses and specific antigen genes, xenotransplantation technology has made great progress. Among them, pig heart valves have long been used as raw materials for human heart valve replacement. In 2022, the Griffith research team at the University of Maryland reported the world's first pig heart transplant surgery. The patient survived for two months after the operation, bringing hope to xenotransplantation. The emergence of induced pluripotent stem cells and extended pluripotent stem cell lines has resolved the ethical debate on the acquisition and use of embryonic stem cells. The use of pig pluripotent stem cells to construct a stem cell differentiation system into cardiomyocytes is conducive to the generation of cardiomyocytes using pig pluripotent stem cells as raw materials, promotes the study of mammalian myocardial development process, and can provide a reference for human cardiomyocyte induction and transplantation therapy. Summary of the invention

[0005] The purpose of the present invention is to establish an effective 2D induction culture system and provide a method for inducing pluripotent stem cells to differentiate into cardiomyocytes.

[0006] In order to achieve the above-mentioned object, the present invention provides, in a first aspect, pluripotent stem cells such as pig EPSC and human H9 cell line, wherein the stem cells have the potential to differentiate into three germ layers.

[0007] The second aspect of the present invention provides a method for inducing differentiation of pig EPSC and human ESC into mesoderm using small molecules such as Chir99021, XAV939, BMP4, Activin A, etc.

[0008] The third aspect of the present invention provides a method for inducing the differentiation of mesodermal cells into cardiomyocyte progenitor cells using small molecules such as bFGF, VEGF, VC, IWR-1, RA, etc.

[0009] The fourth aspect of the present invention provides a method for inducing the differentiation of porcine myocardial progenitor cells into myocardial cells using small molecules such as bFGF, VEGF, VC, BMP4, FGF10, etc.

[0010] In the aforementioned method, the optimal concentration of each small molecule is as follows:

[0011] 10 ng / mL BMP4, 20 ng / mL Activin A, 10 ng / mL bFGF, 5 ng / mL VEGF, 5 μM IWR-1, 650 μg / mL VC, 1 μM RA, 5 ng / mL bFGF, 5 ng / mL VEGF, 975 μg / mL VC, 10 ng / mL BMP4, 25 ng / mL FGF10. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Verification of the pluripotency of porcine EPSCs

[0013] Figure 2 Alkaline phosphatase (AP) staining of porcine EPSC

[0014] Figure 3 The timeline of the induction process and the morphology and gene expression detection of the mesoderm stage

[0015] Figure 4 This is a system optimization of the combination of different concentrations of BMP4 and Activin A.

[0016] Figure 5 The morphology and qRT-PCR results of mesoderm cells induced to differentiate into cardiomyocytes

[0017] Figure 6 The results of immunofluorescence staining and Western Blotting of induced pig cardiomyocytes

[0018] Figure 7 The transcriptome analysis results of induced porcine cardiomyocytes and original undifferentiated porcine EPSCs

[0019] Figure 8 It is the detection of pluripotency genes and marker genes of human embryonic stem cell line H9 at different stages under the same induction system.

[0020] Fig. 9 This is the result of immunofluorescence staining of the myocardial marker gene TNNT2 on the 10th day in the human embryonic stem cell line H9 under the same induction system. DETAILED DESCRIPTION

[0021] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] Unless otherwise specified, the technical means involved in the present invention are all conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0023] The formula of the basic stem cell induction medium used in the following examples is RPMI 1640 basic culture medium, 2.5% FBS, N2 Supplement (50×), B27 Supplement (100×), 0.1 mmol / L NEAA, 1 mmol / L Glu, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0024] The following is a specific example of the configuration of the differentiation medium used in this example to induce differentiation of PSCs into cardiac mesoderm cells, induce differentiation of mesoderm cells into cardiac progenitor cells, and induce differentiation of cardiac progenitor cells into cardiomyocytes.

[0025] Example 1 Culture medium for induction of PSC into cardiomyocytes under 2D culture conditions

[0026] This example uses the above-mentioned basic induction medium as the induction basal liquid, supplemented with small molecules and cytokines, and the specific composition is as follows:

[0027] (1) Inducing differentiation from PSCs to cardiac mesoderm:

[0028] Basic stem cell induction medium, 10 ng / mL BMP4, 20 ng / mL Activin A, 6 μM chir99021, 5 ng / mL VEGF, gently invert and mix

[0029] (2) Inducing differentiation from cardiac mesoderm cells to cardiomyocyte progenitor cells:

[0030] Basal stem cell induction medium, 10 ng / mL bFGF, 5 ng / mL VEGF, 5 μM IWR-1, 1 μM RA, gently invert to mix

[0031] (3) Culture medium for inducing differentiation from cardiac progenitor cells to cardiac myocytes:

[0032] Basal stem cell induction medium, 15 ng / mL bFGF, 5 ng / mL VEGF, 10 ng / mL BMP4, 25 ng / mL FGF10, mix gently by inverting.

[0033] Example 2 Method for inducing porcine EPSCs to differentiate into cardiomyocytes in vitro under 2D culture conditions

[0034] When the cell density of EPSCs reaches about 80% for normal passage, a factor induction experiment is performed on them using the differentiation induction medium in Example 1. The induction is divided into three stages:

[0035] (1) EPSCs to cardiac mesoderm stage. Spread 0.1% gelatin on the culture plate and place the culture plate in a 37°C incubator for 15-60 min in advance. Use PSCs to cardiac mesoderm stage induction differentiation medium, place in a 37°C constant temperature incubator, and change the culture medium every 24 h for 2 days.

[0036] (2) Cardiac mesoderm to cardiomyocyte progenitor cell stage. The obtained cardiac mesoderm cells were washed twice with 1×PBS, and the culture medium for the cardiac mesoderm to cardiomyocyte progenitor cell stage was replaced. The cells were cultured in a 37°C constant temperature incubator, and the culture medium was changed every 24 hours for 2 days.

[0037] (3) Cardiac progenitor cell to cardiomyocyte stage: The obtained mesoderm cells were washed twice with 1× PBS, replaced with the cardiomyocyte progenitor cell to cardiomyocyte differentiation medium, and cultured in a 37°C constant temperature incubator, changing the culture medium every 24 hours for 6-16 days.

[0038] Example 3 Method for inducing differentiation of human embryonic stem cell line H9 into cardiomyocytes in vitro under 2D culture conditions

[0039] (1) Induce H9 cells to the mesoderm stage. Spread Corning ® Matrigel ® Matrix was coated on the culture plate at 4°C overnight. PSCs were cultured in the mesoderm stage induction differentiation medium in a 37°C incubator, and the culture medium was changed every 24 hours for 2 days.

[0040] (2) Mesoderm to cardiac mesoderm stage. The obtained mesoderm cells were washed twice with 1× PBS, replaced with the mesoderm to cardiac mesoderm stage induction differentiation medium, and cultured in a 37°C constant temperature incubator, changing the culture medium every 24 hours for 2 days.

[0041] (3) Transformation of cardiac mesoderm into cardiomyocytes. The obtained mesoderm cells were washed twice with 1× PBS, replaced with cardiac mesoderm to cardiomyocyte differentiation medium, and cultured in a 37°C constant temperature incubator, changing the culture medium every 24 hours for 8-20 days.

[0042] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A chemical reagent, characterized in that: The chemical reagent is divided into three stages according to the period of use, namely, stage I culture medium, stage II culture medium, and stage III culture medium; Phase I culture medium consists of basal stem cell induction medium, Chir99021, XAV939, BMP4, and ActivinA small molecule, and for porcine EPSCs, the working concentrations and ratios are as follows: BMP4 concentration is 10-20 ng / mL, ActivinA concentration is 10-20 ng / mL; BMP4:ActivinA ratio is 1:2; Phase II medium consisted of basal stem cell induction medium, bFGF, VEGF, VC, IWR-1, and RA small molecules, and for porcine EPSCs, the working concentrations and ratios were as follows: 10-20 ng / mL bFGF, 5-10 ng / mL VEGF, 1-5 μM IWR-1, 50-650 μg / mL VC, 1-10 μM RA; Phase III culture medium consisted of basal stem cell induction medium, bFGF, VEGF, VC, BMP4, and FGF10 small molecules, and for porcine EPSCs, the working concentrations and ratios were as follows: 5 ng / mL bFGF, 5 ng / mL VEGF, 975 μg / mL VC, 10 ng / mL BMP4, and 25 ng / mL FGF10; The formula of the basic stem cell induction medium is RPMI 1640 basic culture medium, 2.5% FBS, N2 Supplement, B27 Supplement, 0.1 mmol / L NEAA, 1 mmol / L Glu, 100 units / mL penicillin, and 100 μg / mL streptomycin.

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