CN117448263A - Inducer for inducing pluripotent stem cells to differentiate into pulsating myocardial cells and application thereof - Google Patents
Inducer for inducing pluripotent stem cells to differentiate into pulsating myocardial cells and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of cell biology, and discloses an inducer for inducing pluripotent stem cells to differentiate into pulsating myocardial cells, which comprises the following components: bone morphogenic protein-2, RY-Box transcription factor 9 and Kruppel-like factor 4. The inducer for inducing the differentiation of the pluripotent stem cells into the beating myocardial cells and the application thereof are characterized in that the differentiation of the pluripotent stem cells into the myocardial cells is induced by adopting an inducer formed by combining a plurality of factors including bone morphogenetic protein-2, RY-Box transcription factor 9 and Kruppel like factor 4, and the bone morphogenetic protein-2 plays a role in promoting myocardial gene expression and myocardial feature formation in myocardial cell differentiation; RY-Box transcription factor 9 can activate the expression of the specific gene of the myocardial cells and participate in key regulation, kruppel-like factor 4 can inhibit cell proliferation, and the differentiation of the pluripotent stem cells to the myocardial cells can be regulated more comprehensively and accurately through the combined action of multiple factors.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to an inducer for inducing pluripotent stem cells to differentiate into pulsating myocardial cells and application thereof.
Background
Cardiomyocytes are the most abundant structural cell group in the heart, do not have self-renewal ability, and in the event of sudden myocardial infarction, a large number of cardiomyocytes die and cannot regenerate due to ischemia-reperfusion injury, resulting in heart failure, and stem cells have self-renewal ability and can differentiate into "primitive cells" of various functional cells such as cardiomyocytes, nerve cells and the like.
The latest international heart disease treatment concept is to inject mixed stem cell-derived cardiomyocytes, and once myocardial infarction occurs, the mixed cells with self-renewal capacity can replace dead cardiomyocytes to repair the heart, while in the research process for the cardiomyocytes, an inducer plays a key role in inducing the differentiation of Pluripotent Stem Cells (PSCs) into specific cell types, and the occurrence and maturation of the cardiomyocytes are induced by regulating mechanisms such as gene expression, extracellular matrix generation and remodeling, cell cycle regulation and the like.
However, many inducers currently used generally use only one factor to induce the iPSC to differentiate toward myocardial cells, the single factor often has poor effect, the myocardial cell differentiation is difficult to meet the actual requirement, the complex process is difficult to realize by the combined action of multiple factors, if only one factor is used, the multiple conditions are difficult to meet simultaneously, so that the myocardial cell differentiation is affected, and no reliable model system can directly observe the situation of the iPSC to differentiate toward myocardial cells, which is important for evaluating the effect of the inducer, so we propose an inducer for inducing the differentiation of pluripotent stem cells into pulsating myocardial cells and the application thereof.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the inducer for inducing the pluripotent stem cells to differentiate into the beating myocardial cells and the application thereof provided by the invention have the advantages of improving the induction effect of the pluripotent stem cells by combining a plurality of factors, and the like, and solve the problems that a plurality of inducers used at present usually only use one factor to induce the i PSC to differentiate towards the myocardial cells, the single factor often has poor effect, the actual requirement is difficult to meet, the myocardial cell differentiation is a complex process and can be realized by the combined action of a plurality of factors, and if only one factor is used, a plurality of conditions are difficult to meet simultaneously, thereby influencing the myocardial cell differentiation, and a reliable model system does not exist at present to directly observe the i PSC to differentiate towards the myocardial cells, which is very important for evaluating the effect of the inducers.
(II) technical scheme
In order to achieve the purpose of improving the induction effect of the pluripotent stem cells by combining a plurality of factors, the invention provides the following technical scheme:
an inducer for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes, comprising the following components: bone morphogenic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4.
Preferably, the concentration ratio between the bone morphogenic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4 is 1:1:1, and is 50ng/mL.
A culture medium for inducing pluripotent stem cells to differentiate into beating myocardial cells comprises the following raw materials in parts by weight: 10% of fetal bovine serum, 1% of penicillin, 1% of non-essential amino acids, 1% of glutamine, 1% of streptomycin and 86% of saline.
A method of inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes comprising an inducer that induces differentiation of pluripotent stem cells into pulsating cardiomyocytes and a medium that induces differentiation of pluripotent stem cells into pulsating cardiomyocytes, further comprising the steps of:
s1, dissolving an inducer into a culture medium;
s2, sterilizing a culture medium;
s3, separating the mouse embryonic stem cells and the endothelial progenitor cells to obtain induced pluripotent stem cells;
s4, transferring the obtained induced pluripotent stem cells into a culture medium, and controlling environmental factors;
s5, periodically collecting cell samples, and detecting and marking myocardial cell specific proteins;
s6, injecting the obtained induced pluripotent stem cells into a mouse body, and performing histological analysis.
Preferably, the medium is heated to about 65℃and sterilized.
Preferably, the mouse embryonic stem cells are isolated by using a mouse embryonic stem cell isolation reagent, and the induced pluripotent stem cells are isolated from the mouse endothelial progenitor cells by an endothelial progenitor cell isolation reagent.
Preferably, the environmental factors include temperature, humidity, and oxygen concentration.
Preferably, the detection of cardiomyocyte-specific proteins includes cell counting, chromosome analysis, and protein expression.
Preferably, the cardiomyocyte-specific protein is labeled with fluorescein-3-carboxylic acid ethyl ester.
Preferably, the cardiomyocyte-specific protein is detected by flow cytometry; histological analysis was performed by fluorescence microscopy.
(III) beneficial effects
Compared with the prior art, the invention provides an inducer for inducing the differentiation of pluripotent stem cells into pulsating myocardial cells and application thereof, and has the following beneficial effects:
1. the inducer for inducing the differentiation of the pluripotent stem cells into the beating myocardial cells and the application thereof are characterized in that the inducer which is formed by combining a plurality of factors including bone morphogenetic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4 is adopted to induce the differentiation of the pluripotent stem cells towards the myocardial cells, and the bone morphogenetic protein-2 has important differentiation induction and bone formation effects and plays roles in promoting myocardial gene expression and myocardial feature formation in myocardial cell differentiation; RY-Box transcription factor 9 is a transcription factor, can activate the expression of specific genes of cardiac myocytes and participate in key regulation in the process of cardiac myocyte differentiation, kruppe l-like factor 4 can inhibit cell proliferation and play an important role in the process of cardiac myocyte differentiation, and through the combined action of multiple factors, the differentiation of pluripotent stem cells into cardiac myocytes can be regulated more comprehensively and accurately.
2. The inducer for inducing the differentiation of the pluripotent stem cells into the beating myocardial cells and the application thereof evaluate the differentiation effect of the myocardial cells through a series of steps, including dissolution of the inducer, sterilization of a culture medium, separation of the pluripotent stem cells, control of environmental factors and the like, periodically collect cell samples, detect and mark myocardial cell specific proteins, detect cell characteristics and carry out histological analysis through tools such as a flow cytometer, a fluorescence microscope and the like, can directly observe and evaluate the differentiation condition of the cells in vivo tissues, reflect the characteristics and functions of the cells more truly, provide a reliable model system to observe and evaluate the differentiation condition of i PSC towards the myocardial cells, comprehensively and comprehensively enable researchers to understand the differentiation process of the cells more deeply, and provide powerful support for related researches and applications.
Drawings
FIG. 1 is a schematic diagram of the flow chart of the inducer application for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes according to the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1, an inducer for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes comprises the following components: bone morphogenic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4.
Furthermore, bone morphogenic protein-2 is a TGF-beta superfamily factor with a great number of members, has important differentiation induction and bone formation effects, and plays a role in promoting myocardial gene expression and myocardial feature formation in myocardial cell differentiation; RY-Box transcription factor 9 is a transcription factor, can activate the expression of specific genes of myocardial cells and is involved in key regulation and control in the myocardial cell differentiation process; kruppe l-like factor 4 is a member of the Kruppe l-like transcription factor family, is capable of inhibiting cell proliferation and plays an important role in the process of cardiac muscle cell differentiation.
Specifically, the concentration ratio between the bone morphogenetic protein-2, the RY-Box transcription factor 9 and the Kruppe l-like factor 4 is 1:1:1, and is 50ng/mL.
Furthermore, three factors, namely bone morphogenetic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4, are used in the inducer at a specific concentration ratio and are all 50ng/mL, and by the combined action of the multiple factors, the complex environment in the myocardial cell differentiation process can be simulated, the induction effect is enhanced, and the actual research requirement is better met.
A culture medium for inducing pluripotent stem cells to differentiate into beating myocardial cells comprises the following raw materials in parts by weight: 10% of fetal bovine serum, 1% of penicillin, 1% of non-essential amino acids, 1% of glutamine, 1% of streptomycin and 86% of saline.
Further, the fetal bovine serum can provide nutrients and growth factors, which are helpful for cell growth and differentiation; penicillin and streptomycin can kill bacteria, prevent bacterial pollution and ensure the clean purification of the culture environment; non-essential amino acids and glutamine are important cell nutrients, which are beneficial to metabolism and protein synthesis of cells; the osmotic pressure of the culture medium can be regulated by the saline, so that the cells can be kept to grow and differentiate under the proper osmotic pressure environment; the components are prepared by adopting proper weight percentage ratio, balanced nutrition support can be provided for cells, the induced pluripotent stem cells can be efficiently and pertinently differentiated into pulsating myocardial cells, the design components of the culture medium are simple and practical, the operation is convenient, the repeatability is good, and the culture medium is suitable for clinical application.
A method of inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes comprising an inducer that induces differentiation of pluripotent stem cells into pulsating cardiomyocytes and a medium that induces differentiation of pluripotent stem cells into pulsating cardiomyocytes, further comprising the steps of:
s1, dissolving an inducer into a culture medium;
s2, sterilizing a culture medium;
s3, separating the mouse embryonic stem cells and the endothelial progenitor cells to obtain induced pluripotent stem cells;
s4, transferring the obtained induced pluripotent stem cells into a culture medium, and controlling environmental factors;
s5, periodically collecting cell samples, and detecting and marking myocardial cell specific proteins;
s6, injecting the obtained induced pluripotent stem cells into a mouse body, and performing histological analysis.
Furthermore, by using specific inducers and culture mediums, the multipotent stem cells can be effectively promoted to differentiate towards the pulsating cardiomyocytes, the multipotent stem cells have high pertinence, and by controlling the concentration of the inducers in the culture mediums and environmental factors and periodically detecting and marking the specific proteins of the cardiomyocytes, the multipotent stem cells can be ensured to be effectively differentiated into the pulsating cardiomyocytes, and the effect thereof can be evaluated by histological analysis.
Specifically, the medium is heated to about 65℃and sterilized.
Further, the culture medium is heated to about 65 ℃ so that the sterilization has the advantages that microorganisms in the culture medium, including bacteria, fungi, viruses and the like, can be effectively killed or inhibited, the operation can eliminate potential pollution sources, ensure the purity of the culture medium, provide a sterile environment for cell culture, and are crucial to a method for inducing the differentiation of pluripotent stem cells into pulsating cardiomyocytes, because any exogenous microbial pollution can influence the growth and differentiation of cells and even cause inaccuracy of experimental results, the reliability and reproducibility of experiments can be improved by heating the culture medium for sterilization, and the differentiation of pluripotent stem cells into pulsating cardiomyocytes can be effectively induced.
Specifically, the mouse embryonic stem cells are isolated by using a mouse embryonic stem cell isolation reagent, and the induced pluripotent stem cells are isolated from the mouse endothelial progenitor cells by an endothelial progenitor cell isolation reagent.
Further, a murine embryonic stem cell separation reagent is a reagent for separating and extracting embryonic stem cells from a mouse embryo, and generally comprises a series of chemicals and enzymes for treating and manipulating embryonic tissues to separate and extract embryonic stem cells; the endothelial progenitor cell separation reagent is a reagent for separating and extracting endothelial progenitor cells from tissues or cell samples, the endothelial progenitor cells are early-stage endothelial cell precursors, have potential differentiation capability and can further develop into mature endothelial cells, the endothelial progenitor cells play an important role in angiogenesis and tissue reconstruction processes, and the endothelial progenitor cell separation reagent generally comprises specific antibodies, staining agents or other bioactive molecules and is used for marking and separating the endothelial progenitor cells so as to further study functions and applications thereof.
In particular, environmental factors include temperature, humidity, and oxygen concentration.
Furthermore, the proper temperature can provide a warm environment required by cell culture, is favorable for cell growth and differentiation, and can simulate an in-vivo environment in the process of inducing the differentiation of the pluripotent stem cells into the pulsating cardiomyocytes, and the constant temperature provides the most suitable conditions for the differentiation and functional development of the cardiomyocytes; moderate humidity can maintain the stability of moisture in the culture medium, prevent cell dehydration and death, and the cells need sufficient moisture to maintain biochemical reactions and cell functions in the cells, especially during cell differentiation, the control of humidity is critical to maintaining cell health and development; oxygen is a key factor in cellular respiration and energy metabolism, and proper oxygen concentration can provide sufficient oxygen supply in the process of inducing the differentiation of pluripotent stem cells into beating myocardial cells, support the growth and metabolic demands of the myocardial cells, maintain proper oxygen level can promote the differentiation of cells into beating myocardial cells, and contribute to cell maturation and the formation of myocardial functions.
Specifically, the detection of cardiomyocyte-specific proteins includes cell counting, chromosome analysis, and protein expression.
Further, cell counting can determine the proliferation of cells during the induction of pluripotent stem cells to beating cardiomyocytes, and by cell counting, the efficiency and success of the induction process, as well as the growth and expansion of cells, can be assessed; chromosome analysis allows examination of the chromosome structure and number of cells, and determination of whether the cells have normal chromosome composition, and the normality of the chromosome of the cells is an important factor for ensuring the stability and genetic stability of differentiation products during induction; protein expression analysis can determine whether a cell successfully differentiated into a beating cardiomyocyte and evaluate its expression level of cardiomyocyte-specific proteins, which can provide quantitative and qualitative information about the differentiation process, as well as evidence of whether a cell has cardiomyocyte characteristics.
Specifically, cardiomyocyte-specific proteins were labeled with fluorescein-3-carboxylic acid ethyl ester.
Further, fluorescein-3-carboxylic acid ethyl ester is a commonly used fluorescent dye that can selectively bind to cardiomyocyte-specific proteins; this means that it can accurately label cardiomyocytes without interfering with other cell types; a bright fluorescent signal can be generated, which enables it to be easily detected and observed under a microscope or on a flow cytometer or the like, thereby enabling rapid and accurate identification of cardiomyocytes; and because the fluorescein-3-carboxylic acid ethyl ester can mark the myocardial cell specific protein, researchers can use the mark to monitor the change and activity of myocardial cells in real time, which is very valuable in research on development, function, drug response and the like of myocardial cells.
Specifically, detecting cardiomyocyte-specific proteins by flow cytometry; histological analysis was performed by fluorescence microscopy.
Furthermore, the flow cytometer is a highly accurate instrument, the cell type can be identified and analyzed by measuring the expression level of specific proteins in cells, and the expression condition of the specific proteins of the myocardial cells can be accurately detected by the flow cytometer in the process of inducing the differentiation of pluripotent stem cells into pulsating myocardial cells, so that the differentiation state and purity of the cells can be confirmed; the fluorescence microscope is a high-resolution microscope capable of observing and analyzing fluorescent markers in cells and tissues, and in a method of inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes, the morphology, structure and function of cells can be observed and analyzed using the fluorescence microscope by labeling the cardiomyocytes with fluorescent dyes or fluorescent proteins, thereby evaluating the effect of cell differentiation and the characteristics of the cardiomyocytes.
In summary, the inducer for inducing the differentiation of the pluripotent stem cells into the beating myocardial cells and the application thereof induce the differentiation of the pluripotent stem cells into the myocardial cells by adopting the inducer formed by combining a plurality of factors including bone morphogenetic protein-2, RY-Box transcription factor 9 and Kruppe l-like factor 4, and the bone morphogenetic protein-2 has important differentiation induction and bone formation effects and plays a role in promoting myocardial gene expression and myocardial feature formation in myocardial cell differentiation; RY-Box transcription factor 9 is a transcription factor, can activate the expression of specific genes of cardiac myocytes and participate in key regulation in the process of cardiac myocyte differentiation, kruppe l-like factor 4 can inhibit cell proliferation and play an important role in the process of cardiac myocyte differentiation, and through the combined action of multiple factors, the differentiation of pluripotent stem cells into cardiac myocytes can be regulated more comprehensively and accurately.
In addition, the inducer for inducing the pluripotent stem cells to differentiate into the beating myocardial cells and application thereof evaluate the effect of myocardial cell differentiation through a series of steps, including dissolution of the inducer, sterilization of a culture medium, separation of the pluripotent stem cells, control of environmental factors and the like, periodically collect cell samples, detect and mark myocardial cell specific proteins through tools such as a flow cytometer and a fluorescence microscope, detect and histological analysis of cell characteristics, can directly observe and evaluate the differentiation condition of cells in vivo tissues, reflect the characteristics and functions of the cells more truly, provide a reliable model system to observe and evaluate the differentiation condition of the i PSC to the myocardial cells, comprehensively and comprehensively enable researchers to know the differentiation process of the cells deeply, provide powerful support for related researches and applications, solve the problem that a plurality of inducers used at present usually only use one factor to induce the differentiation of the i PSC to the myocardial cells, the single factor often has poor effect, is difficult to meet the actual requirement, myocardial cell differentiation is a complex process, and can be realized through the combined action of a plurality of factors, if the factor is used, and the effect of the myocardial cell differentiation condition is difficult to directly evaluate the myocardial cell differentiation condition is difficult to be achieved, if the current differentiation condition of the myocardial cell is difficult to be directly evaluated, and the differentiation condition of the myocardial cell is difficult to be directly and the differentiation condition is difficult to be evaluated.
The related modules involved in the system are all hardware system modules or functional modules in the prior art combining computer software programs or protocols with hardware, and the computer software programs or protocols involved in the functional modules are all known technologies for those skilled in the art and are not improvements of the system; the system is improved in interaction relation or connection relation among the modules, namely, the overall structure of the system is improved, so that the corresponding technical problems to be solved by the system are solved.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An inducer for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes, comprising the following components: bone morphogenic protein-2, RY-Box transcription factor 9 and Kruppel-like factor 4.
2. The inducer of claim 1 wherein the concentration ratio of bone morphogenic protein-2, RY-Box transcription factor 9 and kruppel-like factor 4 is 1:1:1 and is 50ng/mL.
3. The culture medium for inducing the differentiation of the pluripotent stem cells into the pulsating myocardial cells is characterized by comprising the following raw materials in parts by weight: 10% of fetal bovine serum, 1% of penicillin, 1% of non-essential amino acids, 1% of glutamine, 1% of streptomycin and 86% of saline.
4. A method for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes, comprising the inducer for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes according to claim 1 and the medium for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes according to claim 3, further comprising the steps of:
s1, dissolving an inducer into a culture medium;
s2, sterilizing a culture medium;
s3, separating the mouse embryonic stem cells and the endothelial progenitor cells to obtain induced pluripotent stem cells;
s4, transferring the obtained induced pluripotent stem cells into a culture medium, and controlling environmental factors;
s5, periodically collecting cell samples, and detecting and marking myocardial cell specific proteins;
s6, injecting the obtained induced pluripotent stem cells into a mouse body, and performing histological analysis.
5. The method for inducing differentiation of pluripotent stem cells into pulsating cardiomyocytes according to claim 4, wherein in S2, the culture medium is heated to about 65 ℃ and sterilized.
6. The method of claim 4, wherein in S3, the mouse embryonic stem cells are isolated by using a mouse embryonic stem cell isolation reagent, and the induced pluripotent stem cells are isolated from the mouse endothelial progenitor cells by using an endothelial progenitor cell isolation reagent.
7. The method according to claim 4, wherein the environmental factors in S4 include temperature, humidity and oxygen concentration.
8. The method according to claim 4, wherein the detecting content of the cardiomyocyte-specific protein in S5 comprises cell count, chromosome analysis and protein expression.
9. The method of inducing differentiation of pluripotent stem cells into beating cardiomyocytes according to claim 4, wherein the cardiomyocyte-specific protein is labeled in S5 with fluorescein-3-carboxylic acid ethyl ester.
10. The method of inducing differentiation of pluripotent stem cells into beating cardiomyocytes according to claim 4, wherein in S5, the cardiomyocyte-specific protein is detected by a flow cytometer; in S6, the histological analysis is performed by fluorescence microscopy.
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