Mode for promoting generation of totipotent stem cells of mice

Abstract

The invention provides a culture method for promoting conversion of mouse embryonic stem cells into totipotent stem cells. The cells can be kept in a brand new form and subcultured under the condition. According to the method adopted by the invention, an inhibitor WM-3825 of 2 mu M H4K16ac is added into a DMEM (Dulbecco Modified Eagle Medium) with a culture medium composition of 10% fetal calf serum according to a proportion of 1: 1000, the mouse embryonic stem cells can be normally cultured and passaged, the change of the totipotent gene is detected through real-time fluorescent quantitative PCR (Polymerase Chain Reaction), the expression of the totipotent related gene Dux is found to be obviously increased, and the expression of the mouse embryonic stem cells is obviously improved. Flow cytometry detection shows that the proportion of the totipotent stem cells is remarkably increased, and AP dyeing shows that the pluripotency of the mouse embryonic stem cells is reduced. The method starts from cell culture, and the method is possibly suitable for research on conversion from pluripotency to totipotency of embryonic stem cells of other mammals including human. By adding a small-molecule inhibitor WM-3825 of H4K16ac, a brand-new method for promoting conversion from the mouse pluripotent stem cells to the totipotent stem cells is established, and the 2-cell period of mouse embryonic development can be simulated in vitro through the culture system. The invention provides a new method for the generation of the totipotent stem cells, deepens the understanding of people on different states of the stem cells, and provides a new perspective for understanding an epigenetics mechanism between the conversion of the totipotent stem cells and the conversion of the pluripotent stem cells.

Description

Technical Field

[0001] This invention relates to a method for promoting the generation of mouse pluripotent stem cells. Background Technology

[0002] Embryonic stem cells (ESCs) originate from the inner cell mass of the preimplantation blastocyst and possess the ability to continuously self-renew and maintain an undifferentiated state under specific culture conditions. Furthermore, ESCs are pluripotent, capable of differentiating into various cell types under appropriate induction conditions, thus serving as an ideal model for studying key biological questions such as cell differentiation, embryonic development mechanisms, and signaling network regulation during animal development. Pluripotent stem cells have the ability to develop into all three germ layers but cannot develop into extraembryonic tissues, thus failing to form a complete organism. During the culture of mouse embryonic stem cells, approximately 1% of cell types distinct from pluripotent stem cells spontaneously emerge. Their most fundamental characteristic is their ability to independently develop into a complete, healthy organism, including all tissues constituting the embryo itself and extraembryonic tissues (such as the placenta and yolk sac), exhibiting totipotency. Because their gene expression profile resembles that of the 2-cell stage, they are called 2-cell-like cells (2CLCs). This type of cell model is crucial for studying key events in early embryonic development. The mechanism by which mouse pluripotent stem cells (2CLCs) are maintained has not been fully understood. Therefore, studying the effects of small molecule inhibitors on the transformation of mouse embryonic stem cell pluripotency and the differentiation of embryonic stem cells will help open up new avenues for stem cell therapy and promote the clinical application of pluripotent stem cells.

[0003] Leukemia inhibitory factor (LIF) is a key factor that can maintain the self-renewal of mouse embryonic stem cells in vitro, also known as leukemia inhibitory factor.

[0004] WM-3825 is a highly effective, selective, and reversible competitive inhibitor of acetyl-CoA. Its structure mimics a portion of acetyl-CoA, allowing it to bind tightly to the acetyl-CoA binding pocket of KAT8, preventing the transfer of the acetyl group to lysine residue 16 of histone H4, thereby reducing H4K16ac levels.

[0005] When the small molecule inhibitor WM-3825 was added to the culture system of mouse embryonic stem cells, we found that under this condition, mouse embryonic stem cells could grow normally and the phenotype tended to be totipotent. Further testing proved that under this condition, the expression of cells totipotency-related genes increased. Summary of the Invention

[0006] The present invention aims to explore new culture methods for mouse embryonic stem cells and provide a novel in vitro induction method for the transformation of pluripotent stem cells into totipotent stem cells.

[0007] The key technical solution adopted in this invention is to add 2μM of the small molecule inhibitor WM-3825 to the mouse embryonic stem cell culture system, which resulted in a significant increase in the production of pluripotent stem cells.

[0008] Detection of H4K16ac protein content after using WM-3825: (1) Take mouse embryonic stem cells 46C with a growth density of 80-90%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.5 mL of 0.05% trypsin and digest the cells for about 30 seconds. Gently pipette the cells to collect the cell suspension and transfer it to a 1.5 mL centrifuge tube. Add 1 mL of DMEM containing serum, mix well, and stop the digestion. (3) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 150 μL of RIPA lysis buffer to lyse the cells, then add 50 μL of 4× loading buffer and mix well. Boil the sample at 100℃ for 15-20 minutes to obtain the protein sample. (4) Prepare a 15% protein gel and determine the protein concentration in the sample using a BCA kit to determine the loading amount, then load the sample on ice; (5) 30 μg of total protein was separated by SDS-PAGE gel electrophoresis, and then transferred to a PVDF membrane by wet transfer. After the transfer, the membrane was blocked with TBST solution containing 5% skim milk at room temperature for 4 hours. Subsequently, the membrane was incubated overnight at 4°C with a specific primary antibody (dilution ratio of 1:1000), using mouse H4 as an internal control. (6) After washing three times with TBST, incubate with the corresponding secondary antibody (diluted 1:8000) at room temperature for 1 hour. Develop using an enhanced chemiluminescence (ECL) substrate under a chemiluminescence imaging system.

[0009] Detection of pluripotent stem cell production: (1) The mouse embryonic stem cell line with the green fluorescence expression tracing system was induced. The induction was observed to be successful by fluorescence microscopy. The cells were taken, the culture medium was discarded, and the cells were washed three times with PBS buffer solution. (2) Digest the cells (as described above), centrifuge and discard the supernatant, then resuspend in PBS; (3) After resuspending, centrifuge again to clean the cells, and wash 2-3 times; (4) Transfer the resuspension to a flow cytometer tube, place it on ice, turn on the flow cytometer, and resuspend the cells in pre-cooled PBS before detection. After detection, analyze the results.

[0010] The present invention has the following advantages: (1) The use of the small molecule inhibitor WM-3825 promoted the transformation of mouse embryonic stem cells into pluripotent stem cells and formed a stable cell line. Under this condition, mouse embryonic stem cells could grow normally and their phenotype could be maintained in a certain state. (2) This invention provides a new approach to obtaining pluripotent stem cells, starting from the cells themselves. (3) The pluripotent stem cells established in this invention can open up new avenues for studying the activation of mammalian zygote genomes and the molecular mechanisms of embryonic development. (4) This method may be applicable to the transformation of totipotent cells in other mammals, including humans. Attached Figure Description

[0011] Figure 1 Figure A shows the expression level of H4K16ac in mouse embryonic stem cells after culturing for 48 hours with the addition of 2 μM small molecule inhibitor WM-3825, detected by Western blotting. Real-time quantitative PCR revealed a significant increase in the expression of genes related to pluripotency in mouse embryonic stem cells. Figure 1 B.

[0012] Figure 2 As shown in A and 2B, mouse embryonic stem cells with Msl3 knocked out under DMEM culture conditions of 10% FBS showed mild differentiation by AP staining.

[0013] Figure 3 As shown, mouse embryonic stem cells with green fluorescent groups, cultured in DMEM with 10% FBS and 2 μM of the small molecule inhibitor WM-3825, showed a significantly increased proportion of pluripotent stem cells under flow cytometry analysis. Detailed Implementation

[0014] Example The mouse embryonic stem cells used in the experiment were provided by the University of Southern California.

[0015] The effects of WM-3825 and the detection of totipotency-related genes after treatment were assessed. (1) Western blot was used to detect the expression of H4K16ac in the cell line treated with WM-3825. The specific method is as follows: a. Once the knockout cell line has grown to a confluence in a six-well plate, wash once with PBS, add 0.5 mL of 0.05% trypsin, digest the cells for about 2 minutes, pipette the cells, aspirate the cell suspension, transfer it to a 1.5 mL centrifuge tube containing 1 mL of culture medium, continue pipetting, and stop digestion. b. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 150 uL of protein lysis buffer and 50 uL of 4x loading buffer, collect the protein, and boil the protein in hot water for 15 minutes. c. Prepare the upper and lower gels, electrophoresis for 2 hours, transfer membrane for 2 hours, block with 5% skim milk for 4 hours, wash the membrane with TBST every 15 minutes for three times, apply Msl3 antibody, incubate overnight at 4°C, wash with primary antibody, wash the membrane with TBST every 15 minutes for three times, apply the secondary antibody corresponding to Msl3, incubate on a shaker at room temperature for 1 hour, wash with secondary antibody, wash the membrane with TBST every 15 minutes for a total of three times, turn on the exposure machine, and expose. d. The results showed that the addition of the small molecule WM-3825 affected histone acetylation, thereby influencing the stemness changes in mouse embryonic stem cells, such as... Figure 1 A. (2) Real-time quantitative PCR was used to detect totipotency-related genes in cell lines with added inhibitors. The specific method is as follows: a. Once the cell line has grown to confluence in a six-well plate, wash once with PBS, add 0.5 mL of 0.05% trypsin, digest the cells for about 2 minutes, pipette the cells, aspirate the cell suspension, transfer it to a 1.5 mL centrifuge tube containing 1 mL of culture medium, continue pipetting, and stop the digestion. b. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 350 μL of cell lysis buffer (LB), mix by pipetting, and lyse the cells; c. Following the RNA extraction kit procedure, extract RNA, measure RNA concentration, and reverse transcriptase kit procedure to convert RNA to cDNA. Add cDNA, primers, SYBR fluorescent dye, and water to a dedicated 96-well plate according to the real-time quantitative PCR reaction system. Set the reaction program and analyze the data. Figure 3 As shown in A; d. According to Figure 1 B showed that after inhibitor treatment, the totipotency-related genes Dux and Merv1 in mouse embryonic stem cells were significantly increased.

[0016] Cell self-renewal status detection: (1) Morphological observation: The above cell lines were observed using a Leica DMIL inverted microscope. The results showed that mouse embryonic stem cells differentiated when 2 μM of the small molecule inhibitor WM-3825 was added to 10% FBS DMEM medium, and the phenotype maintained a certain morphology, allowing for passage culture. Figure 2 As shown in B. (2) Alkaline phosphatase (AP) staining was used to detect the self-renewal status. The specific method is as follows: a. Inoculate an appropriate amount of KO.Msl3 cell line and culture for a certain period of time before performing AP staining; b. Prepare the BCIP / NBT staining working solution according to the AP staining kit instructions; c. Discard the culture medium in the culture plate, wash with PBS 3-5 times, 3-5 minutes each time, add 500μl of 4% paraformaldehyde to fix the cells for 1-2 minutes, discard the fixative, and wash with PBS 3-5 times, 3-5 minutes each time. d. After the final wash, remove the washing solution and add an appropriate amount of BCIP / NBT staining working solution to ensure that the cells are fully covered; e. Incubate at room temperature in the dark for 30 minutes or longer (up to 24 hours) until the color develops to the desired shade; f. Remove the BCIP / NBT staining working solution and wash with PBS 1-2 times to terminate the colorimetric reaction; g. Finally, after adding an appropriate amount of PBS, place the culture plate under a Leica DMIL inverted microscope to observe cell staining and determine the cell self-renewal status. Figure 2 As shown.

[0017] Detection of pluripotent stem cell production (1) Flow cytometry (FACS) was used to detect the production of pluripotent stem cells in the WM-3825-treated cell line. The specific method is as follows: a. Induce the cell line with the green fluorescent expression tracing system driven by the Mervl gene promoter. Observe the successful induction by fluorescence microscopy, take the cells, discard the culture medium, and wash three times with PBS buffer solution. b. Digest the cells (as described above), centrifuge, discard the supernatant, and resuspend in PBS; c. After resuspending, centrifuge again to clean the cells, repeating the process 2-3 times. d. Transfer the resuspension to a flow cytometry tube, place it on ice, turn on the flow cytometer. Before detection, resuspend the cells in pre-chilled PBS. After detection, analyze the results as follows: Figure 3 As shown; e. Analysis showed that the content of pluripotent stem cells increased in cells treated with inhibitors.

Claims

1. A method for promoting the generation of mouse pluripotent stem cells, characterized in that, Adding 2 μM WM-3825 to DMEM medium containing 10% FBS reduced H4K16ac levels to promote the generation of pluripotent stem cells.

2. The method according to claim 1, characterized in that, The mouse embryonic stem cell line 46C was subjected to Western blot experiments after treatment with WM-3825: (1) Take mouse embryonic stem cells 46C with a growth density of 80-90%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (2) Add 0.5 mL of 0.05% trypsin and digest the cells for about 30 seconds. Gently pipette the cells to collect the cell suspension and transfer it to a 1.5 mL centrifuge tube. Add 1 mL of DMEM containing serum, mix well, and stop the digestion. (3) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 150 μL of RIPA lysis buffer to lyse the cells, then add 50 μL of 4× loading buffer and mix well. Boil the sample at 100℃ for 15-20 minutes to obtain the protein sample. (4) Prepare a 15% protein gel and determine the protein concentration in the sample using a BCA kit to determine the loading amount, then load the sample on ice; (5) 30 μg of total protein was separated by SDS-PAGE gel electrophoresis, and then transferred to a PVDF membrane by wet transfer. After the transfer, the membrane was blocked with TBST solution containing 5% skim milk at room temperature for 4 hours. Subsequently, the membrane was incubated overnight at 4°C with a specific primary antibody (dilution ratio of 1:1000), using mouse H4 as an internal control. (6) After washing three times with TBST, incubate with the corresponding secondary antibody (diluted 1:8000) at room temperature for 1 hour. Develop using an enhanced chemiluminescence (ECL) substrate under a chemiluminescence imaging system.

3. The method according to claim 2, characterized in that, The mouse embryonic stem cell line with the green fluorescence expression tracing system was subjected to flow cytometry experiments after treatment with WM-3825 small molecule: (1) The mouse embryonic stem cell line with the green fluorescence expression tracing system was induced. The induction was observed to be successful by fluorescence microscopy. The cells were taken, the culture medium was discarded, and the cells were washed three times with PBS buffer solution. (2) Digest the cells (as described above), centrifuge and discard the supernatant, then resuspend in PBS; (3) After resuspending, centrifuge again to clean the cells, and wash 2-3 times; (4) Transfer the resuspension to a flow cytometer tube, place it on ice, turn on the flow cytometer, and resuspend the cells in pre-cooled PBS before detection. After detection, analyze the results.

4. The method according to claims 2 and 3, characterized in that, The addition of 2 μM WM-3825 significantly increased the proportion of mouse pluripotent stem cells, and they were able to be cultured and passaged normally in DMEM medium containing 10% FBS, which is expected to provide a model for in vitro pluripotent stem cell research.

Images