Application method of chemical small molecule combination for promoting pluripotent to totipotent transformation of embryonic stem cells

Abstract

The invention provides a chemical small molecule combination application method for promoting pluripotent to totipotent transformation of embryonic stem cells. The cells can improve the proportion of 2CLCs under the condition. The components of the culture medium are a culture medium added with 10% of serum, and TTNPB with the final concentration of 2.5 [mu] M and CRT0044876 with the final concentration of 5 [mu] M are added at the same time. By establishing the brand new culture method for converting the mouse embryonic stem cells from pluripotent to totipotent, the culture conditions of the totipotent stem cells can be better optimized, people can be helped to better understand the mechanisms of life development and cell differentiation, a new insight can be provided for researching the molecular mechanism of converting the mammals from pluripotent to totipotent, and the method has good application prospects. The application of the totipotent stem cells in the medical field is expanded.

Description

Technical Field

[0001] The present invention relates to a method for applying a combination of chemical small molecules for promoting the transformation of embryonic stem cells from pluripotency to totipotency. Background Art

[0002] Embryonic stem cells (ESCs) are derived from the inner cell mass of the pre-implantation blastocyst stage. Under certain conditions, ESCs can proliferate indefinitely (self-renew) in an undifferentiated state. Their cells are small in size, similar in morphology to early embryonic cells, with large nuclei. In vitro differentiation culture is inhibited, and the cells are tightly arranged, with clear cell edges and grow in colonies. At the same time, they maintain the developmental potential to produce endoderm, mesoderm, and ectoderm cells. Under certain conditions, they can be induced into a variety of cells (pluripotency). Therefore, embryonic stem cells are often used as a very useful tool for basic research on developmental biology such as animal cell tissue differentiation, embryogenesis, and cell signaling network regulation. Under natural culture conditions, mouse embryonic stem cells (mESCs) will produce about 1% transient cell populations, which have a large number of co-expressed genes at the transcriptional level with mouse fertilized eggs that develop to the 2-cell stage. Therefore, such cells are called 2-cell-like cells (2C-like cells, 2CLCs). This type of cell has the ability to differentiate into intraembryonic and extraembryonic tissues, so 2CLCs have the potential to differentiate into all cells and tissues of the body or are called totipotent. In recent years, studies have shown that under certain conditions, embryonic stem cells can exit pluripotency and enter a totipotent state, showing differentiation potential similar to that of fertilized eggs. However, existing methods usually rely on gene editing or reprogramming technology, which has problems such as complex operation, low efficiency, and possible genetic mutation. Therefore, developing a method of promoting the transformation of embryonic stem cells from pluripotency to totipotency with a small chemical molecule is both simple and efficient, and has important clinical application value. In the preliminary experiments, we screened various small molecules and conducted cell induction experiments. We finally found that when TTNPB and CRT0044876 were added, the proportion of 2CLCs in mouse embryonic stem cells increased. Under this condition, other mammals, including human ESCs, may also be successfully induced into totipotent stem cells. Therefore, by adding small molecules TTNPB and CRT0044876, the proportion of 2CLCs in embryonic stem cells can be better increased, and the mechanisms of life development and cell differentiation can be better understood. It can provide new insights into the molecular mechanism of the transformation of mammalian pluripotency to totipotency and expand the application of totipotent stem cells in the medical field.

[0003] TTNPB is a potent RAR agonist that enhances the reprogramming efficiency of chemically induced pluripotent stem cells (CiPSCs).

[0004] CRT0044876 is a highly potent and selective inhibitor of purine / pyrimidine nucleoside endonuclease 1 (APE1) with an IC50 of approximately 3 μM. CRT0044876 inhibits the AP endonuclease, 3′-phosphodiesterase, and 3′-phosphatase activities of APE and is also a specific inhibitor of the exonuclease III family to which APE1 belongs. CRT0044876 enhances the cytotoxicity of several DNA base-targeted compounds.

[0005] By adding two small molecules, TINPB and CRT0044876, to the culture system of mouse embryonic stem cells containing serum, we observed their phenotypes and real-time fluorescence quantitative PCR to prove that the proportion of 2CLCs increased under this condition. This can provide new insights into the molecular mechanism of the transformation from pluripotency to totipotency in mammals and expand the application of totipotent stem cells in the clinical field. Summary of the invention

[0006] The present invention aims to explore new culture conditions for inducing mouse pluripotent stem cells and provide a chemical method for increasing the conversion ratio of embryonic stem cells from pluripotency to pluripotency.

[0007] The technical solution adopted by the present invention to solve the above technical problems is to explore the types and concentrations of small molecules on the basis of culture without LIF. By adding two small molecules, TTNPB and CRT0044876, mouse embryonic stem cells are induced to increase the proportion of 2CLCs.

[0008] The specific operation of the present invention comprises the following steps: (1) Take 0.5 ml of 0.1% gelatin to coat the cell culture plate and place it in a cell culture incubator at 37°C and 5% CO2 for 30 minutes; (2) Take ZSCAN4-GFP-traced mouse embryonic stem cells with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (3) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (4) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (5) Take out the wrapped culture plate, discard the gelatin, and add 1 ml of DMEM culture medium containing 10% FBS to each well of the culture plate; (6) Add 6.0×10 3 cells, shake them horizontally in a cross shape to make the cells evenly distributed, and place them in an incubator for 24 hours until the cells are completely attached to the wall; (7) Add the following small molecules to the cell culture plate: 2.5 μM TINPB, 5 μM CRT0044876; (8) Place the cell culture dish in a cell culture incubator at 37°C and 5% CO2 concentration.

[0009] Cell pluripotency status detection (9) A Leica DMIL inverted microscope was used to observe the cells under the above culture conditions ①-⑧, and cell RNA samples were collected for real-time fluorescence quantitative PCR. The results showed that when the small molecule 2.5μM TINPB + 5μM CRT0044876 was added to the culture conditions, the proportion of mouse embryonic stem cell 2CLCs increased, the expression of pluripotency-related genes decreased, and the expression of totipotency-related genes increased.

[0010] The present invention has the following advantages: (1) Two small molecules, TTNPB and CRT0044876, were added to the serum-containing DMEM culture medium to promote the transformation of mouse embryonic stem cells from pluripotency to totipotency. Under this condition, the proportion of 2CLCs can be increased, promoting the transformation of mouse embryonic stem cells to totipotency. (2) Different from other methods for inducing embryonic stem cells to transform from pluripotency to totipotency, the present invention uses completely new induction conditions, and the induced cells are highly stable and can be continuously passaged. (3) The present invention adopts a method of adding chemical small molecules, and thus by adding small molecules TTNPB and CRT0044876, it is possible to better increase the proportion of 2CLCs in embryonic stem cells, better understand the mechanism of life development and cell differentiation, and provide new insights into the molecular mechanism of the transformation from pluripotency to totipotency in mammals. (4) The novel method for converting pluripotent stem cells to multipotent stem cells established by the present invention can open up a new approach for studying the molecular mechanism of the conversion of mammalian pluripotency to totipotency, and expand the application of totipotent stem cells in the clinical field. (5) This method may be applicable to the induction of embryonic stem cells from other mammals, including humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown is the result of real-time fluorescence quantitative PCR of cells cultured under this condition. The results show that the expression level of pluripotency marker genes is significantly downregulated, while the expression of totipotency-related genes is significantly upregulated, indicating the transformation of pluripotent stem cells into totipotent stem cells.

[0012] Figure 2 Shown is the fluorescence observation result of cells cultured under this condition. The result shows that the proportion of totipotent stem cells increases under this condition, and more pluripotent stem cells undergo the de-pluripotency process and enter the totipotent state. DETAILED DESCRIPTION

[0013] Example The mouse embryonic stem cells used in the experiment were provided by the University of Southern California.

[0014] Explore new culture conditions for mouse embryonic stem cells and provide a method for the combined application of small chemical molecules to transform embryonic stem cells from pluripotency to totipotency: (1) Take 0.5 ml of 0.1% gelatin to coat the cell culture plate and place it in a cell culture incubator at 37°C and 5% CO2 for 30 minutes; (2) Take ZSCAN4-GFP-traced mouse embryonic stem cells with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (3) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (4) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (5) Take out the wrapped culture plate, discard the gelatin, and add 1 ml of DMEM culture medium containing 10% FBS to each well of the culture plate; (6) Add 6.0×103 cells to each culture well, shake horizontally in a cross shape to evenly distribute the cells, and place in an incubator for 24 h until the cells are completely attached; (7) After the cells were completely attached, the following small molecules were added to the cell culture plate: 2.5 μM TTNPB + 5 μM CRT0044876; (8) Place the cell culture dish in a cell culture incubator at 37°C and 5% CO2 concentration.

[0015] Cell pluripotency status detection: (1) Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of pluripotent stem cell marker genes. The specific method is as follows: a. When the knockout cell line is full in the six-well plate, wash it once with PBS, add 0.5mL of 0.05% trypsin, digest the cells for about 2 minutes, blow the cells with a pipette, aspirate the cell suspension, transfer it to a 1.5mL centrifuge tube with 1mL of culture medium, and continue blowing to terminate the digestion; b. Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 350 uL of cell lysis buffer LB, mix by pipetting, and lyse the cells; c. Extract RNA according to the RNA extraction kit, measure RNA concentration, reverse RNA to cDNA according to the reverse transcription kit, add cDNA, primers, SYBR fluorescent dye, water to a dedicated 96-well plate according to the real-time fluorescence quantitative PCR reaction system, set the reaction program, and analyze the data, such as Figure 1 As shown, it can be seen that the expression levels of pluripotency marker genes (Nanog, Oct4, etc.) were significantly downregulated, while the expression of totipotency-related genes (Dux, Zscan4, etc.) was significantly upregulated. (2) Fluorescence microscopy was used to detect the omnipotence status. The specific method is as follows: The cells under the above culture conditions were observed using a Leica DMIL inverted microscope. When ZSCAN4-GFP-traced mouse embryonic stem cells appeared in 2CLCs, GFP was expressed and green light was visible. Figure 2 As shown, it can be seen that the luminescence ratio of mESCs is increased, and pluripotent stem cells are converted into totipotent stem cells.

Claims

1. A method for applying a combination of chemical small molecules to promote the transformation of embryonic stem cells from pluripotency to totipotency, characterized in that: Two small molecules, TTNPB and CRT0044876, were added to the culture medium to induce mouse embryonic stem cells and increase the proportion of totipotent stem cells.

2. The method according to claim 1, characterized in that Passaging and culturing of the mouse embryonic stem cells under the conditions of two small molecules, TTNPB and CRT0044876: (1) Take 0.5 ml of 0.1% gelatin to coat the cell culture plate and place it in a cell culture incubator at 37°C and 5% CO2 for 30 min. (2) Take ZSCAN4-GFP-traced mouse embryonic stem cells with a growth density of 70-80%, discard the culture medium, wash once with PBS buffer to remove the residual culture medium; (3) Add 0.3 ml of 0.05% trypsin and digest the cells for about 2 min until the edges of the cells float. Use a pipette to blow the cells, aspirate the cell suspension, and transfer it to a 1.5 ml centrifuge tube containing 1 ml of DMEM serum culture medium. Continue to blow and mix to terminate the digestion. (4) After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, 1 ml of DMEM culture medium containing 10% FBS was added to resuspend the cells, and the cells were counted using a cell counting plate to calculate the cell density; (5) Take out the wrapped culture plate, discard the gelatin, and add 1 ml of DMEM culture medium containing 10% FBS to each well of the culture plate; (6) Add 6.0×10 3 cells, and add 1 μL LIF, shake horizontally in a cross shape to make the cells evenly distributed, and culture in an incubator for 24 hours until the cells are completely attached to the wall; (7) After the cells were completely attached, the following small molecules were added to the cell culture plate: 2.5 μM TTNPB and 5 μM CRT0044876; (8) Place the cell culture dish in a cell culture incubator at 37°C and 5% CO2 concentration.

3. The method according to claim 2, characterized in that When 2.5μM TTNPB and 5μM CRT0044876 were added, the proportion of 2CLCs in cells increased.

Images