CN111117951A - Method for regulating hPSC differentiation direction through IGF/insulin pathway and application - Google Patents
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Abstract
A method for adjusting hPSC differentiation direction through IGF/insulin pathway and application thereof relate to the technical field of cell differentiation. Methods of modulating the direction of differentiation of hpscs via the IGF/insulin pathway include: exogenously activating CK2 signal channel to promote differentiation of hPSC into epicardial cell and inhibit differentiation of hPSC into myocardial cell; exogenous inhibition of the IGF/insulin downstream pathway to promote differentiation of hPSCs into cardiomyocytes and inhibition of hPSCs differentiation into epicardial cells influences the efficiency of epicardial and cardiomyocyte differentiation by interfering with the insulin/IGF-mediated CK2 pathway. The application of the method for changing the differentiation of the pluripotent stem cells to the myocardial or epicardial direction by influencing the IGF/insulin pathway specifically comprises the following steps: the application of a substance for activating a CK2 signal channel or a substance for inhibiting an IGF/insulin downstream channel in promoting differentiation of hPSC into epicardial cells or myocardial cells.
Description
Technical Field
The invention relates to the technical field of cell differentiation, in particular to a method for regulating hPSC differentiation direction through an IGF/insulin pathway and application thereof.
Background
Human pluripotent stem cells (hpscs) have the potential to differentiate into all somatic cells of the human body, and the ultimate differentiation fate is often influenced by various specific growth factors. The hPSC can be used for in vitro research on the influence of the interaction of various signal pathways on the final fate of cells, and has important guiding effect on the development of related medicaments.
Related studies have shown that Insulin (Insulin) and Insulin-like growth factors (IGFs) are critical for hPSC maintenance, and they are also essential components of all stem cell culture media, cell survival or proliferation is greatly affected in the absence of Insulin, in the maintenance of cellular pluripotency, Insulin and IGF activate mTOR by activating the PI3K/AKT pathway, and affect the expression of β -catenin-dependent genes by interacting with MAPK/ERK, thereby assisting in the maintenance of pluripotency, in the process of cellular differentiation, Insulin/IGFs activate PI3 2, which is also required for the direction of neural differentiation, when PI3K is inhibited, leading to differentiation in the mesoderm direction, PI3K inhibitors, such as LY294002 and wortmannin, are both commonly used to regulate signaling pathways for the purpose of achieving specific directional endoderm, in addition to PI K pathway, Insulin/CK 63 is also one of the downstream igck 67 2 signaling pathways, however, akck 6778 is also implicated in the role of hPSC.
In addition, under the condition of no interference of exogenous drugs, part of hPSC-induced mesodermal cells can spontaneously differentiate into myocardial cells, so that the final yield of the myocardial cells can be remarkably improved through the interference of different cytokines and small molecular compounds on intracellular signal channels.
Therefore, there is a need for a method for stably promoting differentiation of hPSC into cardiomyocytes or promoting differentiation of epicardial cells.
Disclosure of Invention
The present invention aims to provide a method for regulating the differentiation direction of hPSC through IGF/insulin pathway, which influences the differentiation efficiency of epicardium and cardiomyocytes through interference with the insulin/IGF-mediated CK2 pathway.
The invention aims to provide an application of changing differentiation of pluripotent stem cells to the myocardial or epicardial direction by a method for influencing IGF/insulin pathway, which specifically comprises the following steps: the application of a substance for activating a CK2 signal channel or a substance for inhibiting an IGF/insulin downstream channel in the preparation of a medicament for promoting the differentiation of hPSC into epicardial cells or myocardial cells.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a method for regulating hPSC differentiation direction through IGF/insulin pathway, exogenously activates CK2 signal pathway to promote hPSC to differentiate into epicardial cell and inhibit hPSC from differentiating into myocardial cell; exogenous inhibition of the IGF/insulin downstream pathway promotes differentiation of hPSCs into cardiomyocytes and inhibits differentiation of hPSCs into epicardial cells.
Further, in the preferred embodiment of the present invention, the method for activating the CK2 signal path includes: by activating the insulin receptor or IGF receptor.
Further, in a preferred embodiment of the invention, the method of activating the insulin receptor or IGF receptor is: insulin, IGF family growth factors, or other biologicals, chemically synthesized substances, or naturally extracted substances that activate the CK2 signaling pathway by activating insulin receptors or IGF receptors are used.
Further, in the preferred embodiment of the present invention, the IGF/insulin downstream pathway includes the PI3K signaling pathway and the CK2 signaling pathway; a method of inhibiting IGF/insulin downstream pathways comprising: inhibit the PI3K signal path, or inhibit the CK2 signal path.
Further, in the preferred embodiment of the present invention, the method for suppressing the PI3K signal path is: wortmannin, PI103, BEZ235, LY294002, or other biological, chemical synthetic or naturally extracted substances with similar properties are used.
Further, in the preferred embodiment of the present invention, the method for suppressing the CK2 signal path is: CX4945, PI103, Apigenin, LY294002, or other biological products, chemical synthetic substances or natural extracts having similar properties are used.
Further, in the preferred embodiment of the invention, methods of inhibiting the CK2 signaling pathway include methods of inhibiting Dvl and pGSK3 β levels by blocking insulin, blocking IGF receptors, blocking CK2, and the like.
Further, in the preferred embodiment of the invention, blocking of the insulin receptor is accomplished by antagonizing the insulin receptor using naturally occurring or artificially modified products that bind free insulin, blocking of the IGF receptor is accomplished by antagonizing IGF receptor using naturally occurring or artificially modified products that bind free IGF, and blocking of CK2 and other similar compounds that inhibit Dvl and pGSK3 β levels by adding an effective amount of a CK2 inhibitor to the hPSC differentiation process.
The application of the substance for activating CK2 signal channel in preparing medicine for promoting differentiation of hPSC into epicardial cell.
The application of the substance for inhibiting IGF/insulin downstream passage in the preparation of the medicine for promoting the differentiation of hPSC into myocardial cells.
The method for regulating the hPSC differentiation direction through the IGF/insulin pathway and the application have the beneficial effects that: the method for regulating the hPSC differentiation direction through an IGF/insulin pathway comprises the following steps: exogenously activating CK2 signal channel to promote differentiation of hPSC into epicardial cell and inhibit differentiation of hPSC into myocardial cell; exogenous inhibition of the IGF/insulin downstream pathway to promote differentiation of hPSCs into cardiomyocytes and inhibition of hPSCs differentiation into epicardial cells influences the efficiency of epicardial and cardiomyocyte differentiation by interfering with the insulin/IGF-mediated CK2 pathway. The application of the embodiment of the invention for changing the differentiation of the pluripotent stem cells to the myocardial or epicardial direction by the method for influencing the IGF/insulin pathway specifically comprises the following steps: the application of a substance for activating a CK2 signal channel or a substance for inhibiting an IGF/insulin downstream channel in the preparation of a medicament for promoting the differentiation of hPSC into epicardial cells or myocardial cells.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is an immunofluorescence map of cells in mesodermal spontaneous differentiation, Hoechst marks the nucleus, CTNT marks the cardiomyocytes, WT1 marks the epicardial cells;
FIG. 2 is a flow-induced cytogram of epicardial cells with insulin at different concentrations, WT1 marking the epicardial cells;
FIG. 3 is a photograph of IGF antibody and LY294002 on cardiomyocytes in an induced flow format, CTNT marking cardiomyocytes and WT1 marking epicardial cells;
FIG. 4 is a graph showing the results of expression of cardiomyocyte genes NKX2.5 and TNNT2 under LY294002 treatment;
FIG. 5 is an immunofluorescence plot under LY294002 treatment, Hoechst marks nuclei, NKX2.5 and TNNT2 marks cardiomyocytes;
FIG. 6 is a structural diagram of LY294002 inducing cardiomyocytes;
FIG. 7 is a graph showing the results of IGF antibody inhibiting CK2 protein levels;
FIG. 8 is a graph of LY294002, the results of PI103 inhibiting CK2 protein levels, Wortmannin, BEZ235 is a PI3K inhibitor that does not have an effect on CK2, and PI103 is a PI3K inhibitor that also inhibits CK 2;
FIG. 9 is a graph showing the results of the expression of cardiomyocyte markers NKX2.5 and CTNT, MEF2C under PI103 treatment;
FIG. 10 is a graph showing the results of the expression of cardiomyocyte markers NKX2.5 and CTNT, MEF2C, under the treatment of CK2 inhibitor CX 4945;
FIG. 11 is a graph showing the results of expression of cardiomyocyte markers NKX2.5 and CTNT, MEF2C under treatment with CK2 inhibitor Apigenin;
FIG. 12 is a flow cytogram of CK2 inhibitor Apigenin treated, CTNT labeling cardiomyocytes;
FIG. 13 is a graph showing the results of the expression of the metabolism-related genes OXCT1, ECH1, NDUFB10 and IDH3G by adding LY294002, Wortmannin and CX4945 during induction of cardiomyocyte differentiation using a WNT inhibitor.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following is a detailed description of the method and application of the present invention for regulating the differentiation direction of hPSC via IGF/insulin pathway.
The embodiment of the invention provides a method for regulating the differentiation direction of hPSC through an IGF/insulin pathway, wherein the IGF/insulin pathway strength is exogenously increased, the differentiation of hPSC into epicardial cells is promoted mainly by activating a CK2 signal pathway, and the differentiation of hPSC into myocardial cells is inhibited; exogenously inhibits the IGF/insulin downstream pathway, mainly by inhibiting the PI3K signaling pathway or the CK2 signaling pathway to promote differentiation of hPSCs into cardiomyocytes and inhibit differentiation of hPSCs into epicardial cells.
In this example, specific methods for increasing IGF/insulin pathway strength include: by activating insulin receptor or IGF receptor, CK2 signaling pathway is activated.
Preferably, the method of activating the insulin receptor or IGF receptor is: insulin is usually used, either as insulin, an IGF family growth factor, or other biological, chemical or natural extracts that activate the CK2 signaling pathway by activating the insulin receptor and the IGF receptor, at concentrations of 100ng/mL to 1. mu.g/mL.
Preferably, exogenous addition of insulin promotes mesoderm development to the epicardium and inhibits cardiac differentiation.
In order to understand the spontaneous differentiation trend of mesoderm cells and research the endogenous signal pathway of the mesoderm cells, the GSK3 β inhibitor CHIR99021 is used for opening the Wnt/β -catenin pathway to induce the differentiation of hPSCs into mesoderm, and the downstream spontaneous differentiation trend is observed.
The application detects whether the effect of inhibiting the myocardium by adding insulin in a differentiation system can be achieved, and finds that the expression of myocardial characteristic genes NKX2.5 and TNNT2 can be remarkably reduced by adding insulin (Stage 1) in the first two days of differentiation, and the result is also realized in the IGF1 treatment. However, the addition of insulin (Stage 2) during the differentiation of mesoderm into myocardium did not have a significant effect on myocardial differentiation. In the detection of the epicardial orientation, the application finds that the epicardial tropism of the cells treated by the Stage 1 insulin is enhanced, and the treatment of the insulin in the Stage 1 does not have a significant influence on the myocardial induction of the IWP 2.
In this example, when an insulin/IGF downstream pathway inhibitor is used to inhibit the IGF/insulin downstream pathway, the metabolic function, as well as the maturation of cardiomyocytes can be altered. Inhibiting IGF/insulin downstream pathways that alter cardiomyocyte metabolic function includes the PI3K signaling pathway and the CK2 signaling pathway, and accordingly, methods of inhibiting IGF/insulin downstream pathways include: inhibiting the PI3K signal path, or inhibiting the CK2 signal path; the insulin/IGF downstream pathway inhibitor comprises a substance capable of inhibiting PI3K signaling pathway and a substance capable of inhibiting CK2 signaling pathway.
The method for inhibiting the PI3K signal path comprises the following steps: substances that inhibit the PI3K signaling pathway are used, including Wortmannin, PI103, BEZ235, LY294002, or other biologicals, chemically synthesized substances or naturally extracted substances with similar properties.
The method for inhibiting the CK2 signal path is as follows: substances that inhibit the CK2 signaling pathway are used, including CX4945, PI103, Apigenin, LY294002, or other biologicals, chemically synthesized substances or naturally extracted substances with similar properties.
Specific methods for inhibiting the CK2 signaling pathway include inhibition of the CK2 signaling pathway by blocking insulin, blocking IGF receptors, blocking CK2, and other similar methods that inhibit the Dvl and pGSK3 β levels, and use of chemicals and biologics that inhibit the CK2 signaling pathway.
In general, the methods for blocking insulin receptors are: examples of the biological agent include natural or artificial modified products capable of binding to free insulin and natural or artificial modified products capable of antagonizing insulin receptor, and preferably include insulin antibodies and insulin receptor-blocking antibodies.
Generally, the methods of blocking IGF receptors are: biologicals such as natural or artificial modified products which bind free IGF and natural or artificial modified products which antagonize IGF receptor are used, preferably IGF antibodies, and IGF receptor blocking antibodies.
It is generally preferred that the CK2 and other similar methods of inhibiting the levels of Dvl and pGSK3 β involve contacting hPSC with an effective amount of a CK2 inhibitor, CK2 inhibitors include LY294002, PI103, CX4945, Apigenin, Apigenin and other chemically synthesized and naturally extracted substances that inhibit CK2 activity, preferably LY294002, Apigenin.
Preferably, inhibition of downstream endogenous IGFs promotes differentiation of the mesoderm towards the myocardium.
Since insulin showed much greater effect in Stage 1 than Stage 2, the present application reviewed changes in micro-array with respect to IGF-related genes over time in spontaneous differentiation, the results of which were re-validated by qPCR. The results indicate that either IGF1 or IGF2 exhibited high expression as differentiation progressed.
WB measurements of the levels of IGF protein present in the cells at the first 5 days were performed, which also showed a trend of increasing gradually with the progress. In addition, part of Insulin-like growth factor binding proteins (IGFBPs) also show a tendency to increase gradually, which has an important role in extracellular stabilization of IGF. Phosphorylation of AKT was shown to be inhibited when IGF antibodies were added the day after differentiation, which also suggests that endogenous IGF secretion has an effect on intracellular signaling pathways.
To block the effects of endogenous IGFs, this application found that Stage 2 treated with the classical PI3K inhibitor LY294002 significantly promoted myocardial-directed differentiation while epicardial differentiation was inhibited, to verify the feasibility of LY294002 to induce myocardial differentiation, this application performed experiments on two groups of embryonic stem cells (H1 and H9) and one group of induced pluripotent stem cells (NL-4) and all showed that LY294002 had a promoting effect on cardiomyocyte differentiation, after which the present application performed gene expression analysis of the entire differentiation process, the early markers PDGFR α, KDR, TAL1 that mediated myocardial and hematopoietic were not significantly affected, differentiation of HHEX early endoderm markers, late markers hematopoietic progenitor cells, influenced by 294002, showed a trend of ascending and descending, the myocardial differentiation markers NKX2.5, TNNT2, MEF2C all showed a sustained increase under 29ex 2 treatment, the epicardial marker, and the smooth tissue marker WT 40028, and the trend of extracellular marker WT 4002 after 2920 and smooth muscle cell passage (ctn 40020).
The application finds that LY294002 blocks the downstream of endogenous IGF to promote myocardial differentiation mainly through CK2 pathway.
The application examines the expression of non-phosphorylated β -catenin (activated β -catenin) after exogenously adding insulin or LY 29400224 h, and the result shows that the insulin does not show an enhancement effect, but LY294002 can obviously inhibit the amount of the non-phosphorylated β -catenin, and the effect is similar to that of Wnt inhibitor IWP 2.
To confirm whether the β -catenin pathway changes were due to activation of the PI3K pathway, the present application used multiple PI3K inhibitors (Wortmannin, PI103, BEZ235) and included a concentration gradient (10nM, 100nM, 1 μ M) to attempt to induce myocardial differentiation, however, at non-lethal concentrations, only PI103 could achieve similar effects to LY294002, while Wortmannin and BEZ235 did not have the ability to induce myocardial differentiation, in order to look for the presence of other PI3K inhibitors between LY294002 and PI103, the present application noted CK2, one of the targets widely reported in previous studies related to Wnt/β -catenin pathway regulation, further studies showed that the protein level of CK2 α would also down-regulate when the effects of endogenous differentiation were blocked using IGF antibodies, of four PI3K inhibitors, only PI 29103 and another two of these showed good inhibition of CK 4002 and another apvjk 3 inhibitor of IGF 3 addi 4933, which induced good inhibition of cd 3.
Based on the above-mentioned effects of IGF/insulin pathway on myocardial or epicardial differentiation, chemical substances or biological products that activate or inhibit the pathway can be searched for, and the chemical substances or biological products can be used for promoting the differentiation of myocardial cells or epicardial cells, and the specific applications include: the application of the substance for activating CK2 signal channel in preparing the medicine for promoting the differentiation of hPSC into epicardial cells, wherein the substance for activating CK2 signal channel comprises insulin, IGF family growth factors, other chemical synthetic substances and natural extracted substances capable of activating insulin receptor and IGF receptor.
The application of the substance for inhibiting IGF/insulin downstream pathway in preparing the medicine for promoting hPSC to differentiate into myocardial cells, wherein the substance for inhibiting IGF/insulin downstream pathway comprises LY294002, PI103, Apigenin, CX4945 and other chemosynthesis substances and natural extraction substances capable of inhibiting CK2 activity; also included are biologicals such as natural or artificial modifications that bind free IGF and insulin, and natural or artificial modifications that antagonize insulin receptor and IGF receptor.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example aims to study the effect of exogenous modest increases in IGF/insulin pathway strength on epicardial cell differentiation, the course and results of which are as follows:
it is known that IGF and insulin act through the same downstream pathways, either via the insulin receptor or the IGF receptor. Adding a proper amount of insulin into a differentiation medium, detecting the induction effect of the insulin on the epicardial cells by flow cytometry, wherein figure 1 is an immunofluorescence chart of the cells in spontaneous differentiation of mesoderm, figure 2 is a result chart of the induction of the insulin on the epicardial cells under different concentrations, the concentration of the insulin is 100ng/mL-10 mu g/mL, and WT1 is an epicardial marker.
As can be seen from FIG. 1, after differentiation of pluripotent stem cells into mesoderm, cell fate spontaneously progressed to cardiomyocytes and epicardial cells in a large number. As can be seen from FIG. 2, insulin can properly induce epicardial cell differentiation when it is at a medium or low concentration (100 ng/mL-1. mu.g/mL); while the insulin concentration continues to increase (1 ng/mL-10. mu.g/mL), it will rather inhibit the differentiation of epicardial cells. It is known that insulin stimulation at a certain concentration increases the strength of the IGF/insulin pathway, thereby promoting epicardial cell differentiation.
Example 2
This example aims to study the effect of exogenous inhibition of IGF/insulin downstream pathway on cardiomyocyte differentiation, and the study procedure and results are as follows:
A. the induction effect of IGF antibody and LY294002 on cardiomyocytes was detected by flow cytometry, FIG. 3 is a graph of the induction results of IGF antibody and LY294002 on cardiomyocytes, CTNT marks cardiomyocytes, and WT1 marks epicardium.
B. qPCR was used to detect the expression of cardiomyocyte markers NKX2.5 and TNNT2 under LY294002 treatment, and FIG. 4 is a graph showing the results of expression of cardiomyocyte markers NKX2.5 and TNNT2 under LY294002 treatment.
C. The expression of cardiomyocyte markers NKX2.5 and CTNT under LY294002 treatment was detected by immunofluorescence staining, and fig. 5 is a graph showing the results of the expression of cardiomyocyte markers NKX2.5 and CTNT under LY294002 treatment.
D. Immunofluorescence staining was used to detect the structures of LY 294002-induced cardiomyocytes, fig. 6 is a structural diagram of LY 294002-induced cardiomyocytes, and ACTN and CTNT are α actinin and cardiac troponin T.
As can be seen from fig. 3-6, when IGF antibodies were used to bind free secreted IGF, there was some induction of cardiomyocyte differentiation; when a more potent downstream inhibitor LY294002 was further used, a significant effect of cardiomyocyte differentiation was observed (fig. 2). At the same time, a significant increase in the cardiomyocyte markers NKX2.5 and TNNT2 was observed (fig. 3); NKX2.5 and CTNT double positive cells were also detected in LY294002 treated cells (fig. 4). When LY 294002-induced cardiomyocytes were passaged and cultured for a further 20 days, a clear myocardial structure was observed (fig. 5). Apigenin, a CK2 inhibitor, also induced cardiomyocyte differentiation (figure 6). Binding to IGF/insulin/LY 294002 mediates epicardial/cardiomyocyte differentiation via CK2 (see all), and it is thus known that inhibition of the IGF/insulin downstream pathway by biologicals or chemicals promotes cardiomyocyte differentiation.
Example 3
The purpose of this example is to study the effect of different chemicals and biological products for inhibiting CK2 signal pathway on cardiomyocyte differentiation, and the study process and results are as follows:
as can be seen in fig. 7, by inhibiting IGF, CK2 α levels decreased, indicating a role for endogenous IGF in cellular pathway regulation, whereas exogenous addition of chemicals that inhibited IGF/insulin pathway downstream showed that only two of LY294002 and PI103 resulted in inhibition of CK2 α (fig. 8), further studies showed that PI103 induced myocardial differentiation (fig. 9), CK2 inhibitors CX4945 and Apigenin also induced myocardial differentiation (fig. 10-12).
A. FIG. 7 shows Western blot to detect protein level of CK2 α after IGF antibody treatment, with GAPDH as internal control.
B. FIG. 8 shows the protein level of CK2 α after Western blot detection of PI3K inhibitor treatment, and GAPDH is internal reference.
C. qPCR was used to detect the expression of cardiomyocyte markers NKX2.5 and TNNT2, MEF2C under PI103 treatment, and FIG. 9 is a graph showing the expression results of cardiomyocyte markers NKX2.5 and TNNT2, MEF2C under PI103 treatment.
D. qPCR was used to detect the expression of cardiomyocyte markers NKX2.5 and TNNT2, MEF2C under CX4945 treatment, and FIG. 10 is a graph showing the expression results of cardiomyocyte markers NKX2.5 and TNNT2, MEF2C under CX4945 treatment.
E. qPCR was used to detect the expression of cardiomyocyte markers NKX2.5, TNNT2 and MEF2C under Apigenin treatment, and FIG. 11 is a graph showing the expression results of cardiomyocyte markers NKX2.5, TNNT2 and MEF2C under Apigenin treatment.
F. The expression of the cardiomyocyte marker CTNT under the treatment of Apigenin is detected by flow cytometry, and FIG. 12 is a flow result graph of the cardiomyocyte marker CTNT under the treatment of Apigenin.
Example 4
The purpose of this example was to investigate whether inhibition of the insulin/IGF downstream signaling pathway would lead to cardiomyocyte differentiation when used in combination with the classical WNT inhibitors. As can be seen from FIG. 3, when LY294002 was used to inhibit PI3K and CK2 signaling pathways downstream of insulin/IGF, increased expression of the metabolism-related genes OXCT1, ECH1, NDUFB10, IDH3G was seen. Similar effects were achieved when either Wortmannin inhibited PI3K pathway alone or CX4945 inhibited CK2 pathway alone (FIG. 13).
G. qPCR detection of the expression of the metabolism-related genes OXCT1, ECH1, NDUFB10 and IDH3G by addition of LY294002, Wortmannin and CX4945 during induction of cardiomyocyte differentiation using a WNT inhibitor, and FIG. 13 is a graph showing the expression results of the metabolism-related genes OXCT1, ECH1, NDUFB10 and IDH3G by addition of LY294002, Wortmannin and CX4945 during induction of cardiomyocyte differentiation using a WNT inhibitor.
CK2 is a target in the IGF/insulin downstream pathway. CK2 was inhibited when IGF antibodies were used to bind free endogenous IGFs (fig. 7). Whereas insulin and IGF are widely reported for the activation of the PI3K pathway. Thus with the PI3K pathway inhibitors Wortmannin, PI103, BEZ235, only PI103 consistently showed inhibition of CK2 with LY294002 (fig. 8). And PI103 induced an increase in the cardiac markers NKX2.5, TNNT2, MEF2C (fig. 9). This example continued to be extended to other CK2 inhibitors that showed that the CK2 inhibitor CX4945 induced an increase in the cardiac markers NKX2.5, TNNT2, MEF2C (fig. 10). The CK2 inhibitor Apigenin induced an increase in the cardiac markers NKX2.5, TNNT2, MEF2C (fig. 11). The yield of CTNT positive cardiomyocytes was significantly improved under treatment with Apigenin (fig. 12). Further studies show that LY294002 can change the metabolic characteristics of cardiomyocytes induced by WNT inhibitors, and the expression of metabolism-related genes OXCT1, ECH1, NDUFB10 and IDH3G can be observed to be increased. Similar effects were achieved by either Wortmannin blocking the insulin/IGF downstream PI3K pathway or CX4945 blocking the insulin/IGF downstream CK2 pathway (FIG. 13).
In conclusion, the method for regulating the differentiation direction of hPSC via IGF/insulin pathway according to the embodiments of the present invention affects the differentiation efficiency of epicardium and cardiomyocytes via interference with the insulin/IGF-mediated CK2 pathway; the application of the embodiment of the invention for changing the differentiation of the pluripotent stem cells to the myocardial or epicardial direction by the method for influencing the IGF/insulin pathway specifically comprises the following steps: the application of a substance for activating a CK2 signal channel or a substance for inhibiting a CK2 signal channel in preparing a medicament for promoting the differentiation of hPSC into epicardial cells or myocardial cells.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A method for regulating the differentiation direction of hPSC through IGF/insulin pathway, characterized in that CK2 signal pathway is exogenously activated to promote the differentiation of hPSC into epicardial cells and inhibit the differentiation of hPSC into cardiomyocytes; exogenous inhibition of the IGF/insulin downstream pathway promotes differentiation of hPSCs into cardiomyocytes and inhibits differentiation of hPSCs into epicardial cells.
2. The method of modulating the direction of differentiation of hpscs via the IGF/insulin pathway of claim 1, wherein said method of activating the CK2 signaling pathway comprises: by activating the insulin receptor or IGF receptor.
3. The method of modulating the direction of differentiation of hpscs via the IGF/insulin pathway according to claim 2, characterized in that the method of activating the insulin receptor or IGF receptor is: insulin, IGF family growth factors, or other biologicals, chemically synthesized substances, or naturally extracted substances that activate the CK2 signaling pathway by activating insulin receptors or IGF receptors are used.
4. The method of modulating the direction of differentiation of hpscs via the IGF/insulin pathway of claim 1, wherein the IGF/insulin downstream pathway comprises the PI3K signaling pathway and the CK2 signaling pathway; the method of inhibiting IGF/insulin downstream pathways comprises: inhibit the PI3K signal path, or inhibit the CK2 signal path.
5. The method of modulating the direction of differentiation of hPSC via the IGF/insulin pathway as claimed in claim 4, wherein the method of inhibiting PI3K signaling pathway is: wortmannin, PI103, BEZ235, LY294002, or other biological, chemical synthetic or naturally extracted substances with similar properties are used.
6. The method of modulating the direction of differentiation of hPSC via the IGF/insulin pathway as claimed in claim 4, wherein the method of inhibiting CK2 signaling pathway is: CX4945, PI103, Apigenin, LY294002, or other biological products, chemical synthetic substances or natural extracts having similar properties are used.
7. The method of claim 4 in which inhibition of CK2 signaling pathway comprises inhibition of Dvl and pGSK3 β levels by blocking insulin, IGF receptors, CK2 and other similar means.
8. The method of claim 7 in which the blocking of the insulin receptor is by antagonizing natural or artificial modifications of the insulin receptor using natural or artificial modifications that bind free insulin, the blocking of the IGF receptor is by antagonizing natural or artificial modifications of the IGF receptor using natural or artificial modifications that bind free IGF, and the blocking of CK2 and other similar means that inhibit the levels of Dvl and pGSK3 β is by the addition of an effective amount of a CK2 inhibitor during hPSC differentiation.
9. The application of the substance for activating CK2 signal channel in preparing medicine for promoting differentiation of hPSC into epicardial cell.
10. The application of the substance for inhibiting IGF/insulin downstream passage in the preparation of the medicine for promoting the differentiation of hPSC into myocardial cells.
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