Application of MK2206 in regulating cell fate transition during early embryonic development

Abstract

The present invention discloses an application of MK2206 in regulating the transition of cell fate in early embryonic development, and belongs to the field of biotechnology. The present invention reprograms pluripotent stem cells into a totipotent cell by adding MK2206. The present invention regulates the metabolic pathway method through the action of the MK2206 small molecule, and can effectively regulate the transition of cell fate from pluripotency to totipotency without gene editing. Specifically: MK2206 inhibits the AKT signaling pathway, thereby increasing the expression of the 2C gene. At the same time, MK2206 also inhibits glycolysis, promotes the accumulation of TCA cycle intermediates, and promotes the transition from pluripotency to totipotency. The present invention provides a new method to accurately regulate cell fate transition and metabolic regulation, and provides a new technical means for regenerative medicine, stem cell research and optimization of embryonic development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of MK2206 in regulating cell fate transition in early embryonic development. Background Art

[0002] Early embryonic development is a complex and delicate process involving cell division, differentiation, and fate determination. In mammals, the fertilized egg possesses totipotency, capable of developing into a complete individual. As the embryo develops, cells gradually lose totipotency and transition to pluripotency, ultimately giving rise to distinct cell lineages and tissues and organs. This article reviews the molecular mechanisms and regulatory factors that govern the transition from totipotency to pluripotency during early embryonic development.

[0003] Totipotency refers to the ability of a single cell to develop into a complete individual. In mammals, cells derived from the fertilized egg and its early divisions (such as those at the 2-cell and 4-cell stages) are totipotent. Pluripotency refers to the ability of a cell to differentiate into multiple cell types, but not to develop into a complete individual. Embryonic stem cells (ESCs) are a typical example of pluripotent cells, capable of differentiating into various cell types within the embryo but unable to form extraembryonic tissues.

[0004] The transition from pluripotency to totipotency during early embryonic development is a complex process involving multiple molecular mechanisms and regulatory factors. The coordinated actions of ZGA (zygotic genome activation), epigenetic reprogramming, transcription factors, and signaling pathways ensure the smooth progress of this process. However, there are no reports that MK2206 can regulate cell fate transitions during early embryonic development. Summary of the Invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of MK2206 in regulating cell fate transition in early embryonic development, thereby improving the totipotency state of early embryonic cells by adding MK2206.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing an application of MK2206 in regulating cell fate transition in early embryonic development; the cell fate transition is the transition from pluripotent embryonic stem cells to totipotent stem cells.

[0007] Furthermore, the early embryonic cells are mouse embryonic stem cells.

[0008] Furthermore, the cell fate transformation is achieved by adding MK2206 to inhibit the AKT signaling pathway, thereby increasing the expression of the 2C gene.

[0009] Furthermore, the cell fate transformation is achieved by adding MK2206 to increase the proportion of MERVL-tdTomato-positive cell subpopulations in mouse embryonic stem cells.

[0010] Furthermore, the cell fate transition is achieved by adding MK2206 to increase the expression of zygote-activated genes.

[0011] Furthermore, the cell fate transition is achieved by adding MK2206 to inhibit glycolysis and promote the accumulation of TCA cycle intermediates.

[0012] An agent for regulating cell fate transition in early embryonic development, comprising MK2206.

[0013] A culture medium for regulating cell fate transitions during early embryonic development, comprising MK2206.

[0014] A method for regulating cell fate transition in early embryonic development, comprising the following steps:

[0015] MK2206 was added to the culture medium of embryonic stem cells, and the culture was continued until the cell fate changed.

[0016] The present invention has the following beneficial effects:

[0017] Mouse embryonic stem cells are pluripotent stem cells. This invention reprograms these pluripotent stem cells into totipotent cells by adding MK2206. By using the small molecule MK2206 to regulate metabolic pathways, this method effectively modulates the transition from pluripotency to totipotency without requiring gene editing. Specifically, MK2206 inhibits the AKT signaling pathway, thereby increasing 2C gene expression. MK2206 also inhibits glycolysis, promoting the accumulation of TCA cycle intermediates, thereby promoting the transition from pluripotency to totipotency during early embryonic development.

[0018] The present invention can significantly increase the expression of 2C gene in a mouse embryonic stem cell model after treatment with MK2206, and induce cells to form a 2C-like state.

[0019] The metabolic analysis results of the present invention show that metabolic reprogramming of cells treated with MK2206 plays an important role, especially in enhancing the TCA cycle.

[0020] In summary, the present invention provides a new method to precisely regulate cell fate transition and metabolic regulation, providing a new technical means for regenerative medicine, stem cell research and optimization of embryonic development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results show the expression levels of pluripotency genes in mouse embryonic stem cells treated with DMSO and MK2206.

[0022] Figure 2 Flow cytometry was used to evaluate the MERVL-tdTomato-positive subpopulation in mouse embryonic stem cells treated with DMSO and MK2206.

[0023] Figure 3 The results show the differentially expressed genes in mouse embryonic stem cells treated with DMSO and MK2206.

[0024] Figure 4 The expression results of ZGA pathway in mouse embryonic stem cells treated with DMSO and MK2206.

[0025] Figure 5 This is the dynamic distribution diagram of differential metabolites after mouse embryonic stem cells were treated with MK2206.

[0026] Figure 6 The results show the expression level of pluripotency genes after adding succinic acid and disodium succinate to the culture system.

[0027] Figure 7 The results are shown for differentially expressed genes in late 2C stage embryos treated with DMSO and MK2206.

[0028] Figure 8 Figure 2 shows the expression of ZGA gene in late 2C stage embryos treated with DMSO and MK2206.

[0029] Figure 9 This is a diagram of blastocyst development after cells were cultured for 4.5 days.

[0030] Figure 10 The results show the blastocyst development rate after the cells were cultured for 4.5 days. DETAILED DESCRIPTION

[0031] After adding MK2206 to the culture system of mouse embryonic stem cells in the present invention, the cell fate changes from pluripotency to totipotency, which is specifically manifested as follows:

[0032] (1) Changes in gene expression: The expression of pluripotency-related genes is significantly downregulated. The transcriptional downregulation of these genes causes cells to exit the pluripotent state. At the same time, the expression of totipotency-specific genes and recombinant sequences such as Dux, Zscan4, Gm4340, Tcstv3 genes and endogenous retrovirus MERVL is significantly upregulated. The activation of these genes is an important sign that cells acquire totipotency.

[0033] (2) The proportion of MERVL-tdTomato-positive subpopulations: MERVL-tdTomato is a reporter gene that is specifically expressed in mouse two-cell stage embryos. In mouse embryonic stem cells, the MERVL-tdTomato-positive cell subpopulation refers to those cells that express the reporter gene, and these cells have a higher pluripotent characteristic. Mouse embryonic stem cells are usually in a pluripotent state, but there is also a certain proportion of MERVL-tdTomato-positive subpopulations. The presence of these positive cells indicates that mouse embryonic stem cells contain cell subpopulations with higher developmental potential, which are closer to the pluripotent state. In the present invention, by treating with MK2206, more cells in mouse embryonic stem cells are induced to transform into a MERVL-tdTomato-positive state, thereby obtaining cells that are closer to pluripotent stem cells.

[0034] (3) Changes in the expression of zygotic activation genes: In mouse embryos, the zygotic genome is initially silent. As the embryo develops to the 2-cell stage, the zygotic genome becomes activated and the ZGA gene begins to express, causing embryonic development to gradually shift to zygotic genome regulation. In the present invention, after treating mouse embryonic stem cells with MK2206, the expression of zygotic activation genes expressed in early embryos was significantly increased, indicating that MK2206 can promote the transition of embryonic stem cells to an earlier embryonic state, closer to the state of fertilized eggs and 2-cell stage blastomere totipotent cells.

[0035] (4) Changes in metabolic characteristics: During early embryonic development in mice, the metabolic characteristics of two-cell embryos (2C) and blastocysts (BC) differ significantly. In the early stages of embryonic development, metabolic remodeling is a key factor in maintaining totipotency and embryonic development. In the present invention, after treating mouse embryonic stem cells with MK2206, the intermediate products of glycolysis decreased and the intermediate products of TCA increased, which prompted the embryonic stem cells to transition to a state closer to totipotent cells. This metabolic change may promote the transition of cells from pluripotency to totipotency by affecting the redox state and energy metabolism of cells.

[0036] During early mouse embryonic development, the accumulation of succinate helps maintain cellular totipotency. High succinate levels can promote the transition from pluripotency to totipotency by affecting the cell's redox state and energy metabolism. This metabolic remodeling is a key factor in maintaining totipotency and embryonic development. The present invention, by regulating the metabolic state of embryonic stem cells using MK2206, specifically increasing succinate levels, could provide a more totipotent cell source for regenerative medicine and facilitate the development of new cell therapy strategies.

[0037] (5) Blastocyst development: The present invention treated mouse fertilized eggs with MK2206, which increased the expression of the 2C gene in the late 2-cell embryos. This locked the cells in a totipotent state, preventing them from exiting the state and undergoing subsequent differentiation. After culturing the embryos for 4.5 days, the blastocyst formation rate was significantly reduced, suggesting that overexpression of the 2C gene may have disrupted these processes.

[0038] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0039] Example 1:

[0040] 1. Experimental Methods

[0041] 1. Cell culture and treatment

[0042] Mouse embryonic stem cells (mESC, C57Bl / 6J origin) were seeded into culture plates and cultured in embryonic stem cell medium (containing LIF and fetal bovine serum). Once the cells had attached and grown to 50-60% confluency, MK2206 was added at a final concentration of 1 μM and cultured for 7 days. During this time, cells were passaged at a ratio of 1:4-1:6 depending on cell density, and the medium was changed every two days to ensure a constant MK2206 concentration.

[0043] The culture medium is DMEM medium. 500 mL of DMEM medium contains 15% fetal bovine serum, 5 mL of GlutaMax, 5 mL of non-essential amino acids (Gibco, 11140050), 5 mL of penicillin / streptomycin, and 100 μM 2-mercaptoethanol, and is supplemented with 50 μL of LIF (Millipore, ESG1106).

[0044] 2. RNA Extraction and Quantitative PCR Analysis

[0045] After 7 days of treatment with MK2206, mouse embryonic stem cells were collected and total RNA was extracted using the TRlzol method. The RNA was reverse transcribed into cDNA using ABScript III RT Master Mix (Abclonal, RK20429) and amplified using 2xRealStar Green Power Mix (Genestar, A311-10). The expression level of the 2C gene was detected, using β-actin as an internal control. Cells treated with dimethyl sulfoxide (DMSO) were used as a control group, and the expression of pluripotency genes ( Dux 、 Zscan4 、 Gm4340 、 Tcstv3 ) relative expression changes.

[0046] 3. Establishment of MERVL-tdTomato reporter cell line

[0047] The MERVL promoter-driven tdTomato fluorescent gene was inserted into the Rosa26 locus of mouse embryonic stem cells by site-directed integration to monitor the proportion of 2C state cells in real time. The specific process is as follows:

[0048] A conserved 730-bp fragment of the MERVL gene was amplified from the mouse genome. This fragment was used in the promoter of a reporter system to activate the expression of a red fluorescent protein, thereby indicating the proportion of totipotent stem cells through the expression level of red fluorescence. Using CRISPR combined with HMEJ, the CAS9 protein and sgRNA targeting the Rosa26 locus were used to generate site-specific DNA breaks, which were then repaired using exogenous fragments, allowing for precise insertion of the reporter system.

[0049] After 7 days of MK2206 treatment, the changes in the proportion of MERVL-tdTomato-positive cells were analyzed by flow cytometry.

[0050] 4. Transcriptome Sequencing Analysis

[0051] RNA was extracted from cells treated with MK2206 and then analyzed using NEBNext Ultra TM RNA libraries were obtained using an RNA library preparation kit and subsequently sequenced with paired-end 150 base pairs on the Illumina platform. Clean reads were aligned to the mm10 reference genome using HISAT2, gene expression was calculated using FeatureCounts, and DESeq2 was used for differentially expressed gene analysis.

[0052] 5. Metabolomics analysis

[0053] The intracellular metabolite content was analyzed using the LC-MS (liquid chromatography-mass spectrometry) platform, and the metabolite changes in the experimental group treated with MK2206 for 7 days were compared with those in the control group.

[0054] 6. Succinic acid supplementation experiment

[0055] The basal culture medium (serum LIF medium) was supplemented with succinic acid or disodium succinate at a final concentration of 5 mM, and then mouse embryonic stem cells were cultured in the culture medium for 24 hours to detect changes in 2C gene expression.

[0056] 7. Embryo experiments

[0057] Mouse fertilized eggs were cultured in the presence of MK2206 (1 μM) until the late 2-cell stage, and the embryos were collected for RNA sequencing to analyze the expression of 2C genes. The embryos were then cultured for 4.5 days to assess the blastocyst formation rate.

[0058] 2. Experimental Results

[0059] 1. After treating mouse embryonic stem cells with MK2206 for 7 days, cell pellets were collected and RNA was extracted. Real-time quantitative PCR analysis was performed on the expression levels of pluripotency genes in mouse embryonic stem cells treated with dimethyl sulfoxide (DMSO) and MK2206. The results are shown in Figure 1 .

[0060] Depend on Figure 1 It can be seen that after treating mouse embryonic stem cells with MK2206 for 7 days, the expression level of cell pluripotency genes was significantly increased.

[0061] 2. After 7 days of MK2206 treatment, flow cytometry was used to evaluate the MERVL-tdTomato positive subpopulation in mouse embryonic stem cells treated with dimethyl sulfoxide (DMSO) and MK2206. The results are shown in Figure 2 .

[0062] Depend on Figure 2 It can be seen that constructing a MERVL-tdTomato embryonic stem cell line can effectively increase the proportion of totipotent cells in stem cells.

[0063] 3. Transcriptome sequencing results are shown in Figure 3 and Figure 4 . Figure 3 Figure 3 shows the differentially expressed genes in mouse embryonic stem cells treated with DMSO and MK2206. Genes with a log2 fold change (FC) > 1 and P < 0.05 are represented by red dots, while genes with a log2 fold change (FC) less than -1 and P < 0.05 are represented by blue dots. Figure 4 The expression results of ZGA gene in mouse embryonic stem cells treated with DMSO and MK2206.

[0064] Depend on Figure 3 and Figure 4 It can be seen that compared with DMSO, the expression level of zygote-activated genes expressed in early embryos was significantly increased after mouse embryonic stem cells were treated with MK2206.

[0065] 4. The present invention also conducted a metabolome analysis. After treating mouse embryonic stem cells with MK2206, the dynamic distribution of differential metabolites was shown in Figure 5; Among them, red dots indicate differential metabolites with log2FC>0.5, and green dots indicate differential metabolites with log2FC<-0.5. UMP is uridine monophosphate, AMP is adenine monophosphate, IMP is inosinic monophosphate, dCMP is deoxycytidylic monophosphate, L-citrulline is L-citrulline, Succinic Acid is succinic acid, dTMP is thymidine deoxynucleotide, Citric-acid is citric acid, L-Aspartate is L-arginine, Serine is serine, Ornithine is ornithine, Pyruvic-acid is pyruvic acid, Guanosine-diphosphate is guanosine diphosphate, cis-Aconitic-acid is cis-aconitic acid, Cyclic-AMP is cyclic adenosine monophosphate, Phosphoenolpyruvic-acid is phosphoenolpyruvate, 3-phosphoglycerate is 3-phosphoglyceric acid, 2-Phospho-D-glyceric acid is 2-phospho-D-glyceric acid, Guanosine is guanosine nucleoside, and 3-phenyllactic-acid is 3-phenyllactic acid.

[0066] Depend on Figure 5 It can be seen that in cells treated with MK2206, the intermediate products of glycolysis were significantly reduced, while the intermediate products of TCA were increased.

[0067] 5. After adding 5mM succinate or 60mM disodium succinate (Succinate Na) to the culture medium, the expression changes of 2C gene were detected after culturing for 24 hours. Figure 6 .

[0068] As shown in Table 6, the addition of succinic acid and disodium succinate to the culture system can improve the totipotency of stem cells.

[0069] 6. Treat mouse fertilized eggs with DMSO and MK2206, and take late 2-cell embryos for sequencing. The differentially expressed genes in late 2C embryos are shown in Figure 7 The expression of ZGA gene is shown in Figure 8 Continue to culture cells for 4.5 days, and the blastocyst development diagram is shown in Figure 9 The results of blastocyst development rate are shown in Figure 10 .

[0070] Figure 7 In the figure, genes with log2 fold change (FC) > 0.5 and P < 0.05 are represented by red dots, while genes with log2 FC less than -0.5 and P < 0.05 are represented by blue dots.

[0071] Depend on Figure 7-10 Sequencing of late 2-cell embryos obtained from fertilized eggs treated with the small molecule MK2206 revealed increased expression of the 2C gene in the MK2206-treated group, resulting in an inability to exit the totipotent state. Further culturing of the embryos for 4.5 days revealed an arrest in embryonic development, with a significant decrease in the blastocyst formation rate.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of MK2206 in the preparation of a reagent for increasing the expression of pluripotency genes in embryonic stem cells; the pluripotency genes are Dux, Zscan4, Gm4340, and Tcstv3.

2. The use according to claim 1, characterized in that Transformation of embryonic stem cells into totipotent stem cells.

3. The use according to claim 1 or 2, characterized in that The embryonic stem cells are mouse embryonic stem cells.

4. The use according to claim 3, characterized in that By adding MK2206, the AKT signaling pathway was inhibited, thereby increasing the expression of the 2C gene.

5. The use according to claim 3, characterized in that The proportion of MERVL-tdTomato positive cell subpopulation in mouse embryonic stem cells was increased by adding MK2206.

6. The use according to claim 3, characterized in that The expression of zygote-activated genes was increased by adding MK2206.

7. The use according to claim 5, characterized in that Glycolysis was inhibited by adding MK2206, which promoted the accumulation of TCA cycle intermediates.

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