Method and application for guiding pluripotent stem cells to become developmentally arrested cells

Abstract

The present invention discloses a method for guiding pluripotent stem cells to become developmentally arrested cells and its application, relating to the technical field of stem cells. The method for guiding pluripotent stem cells to become developmentally arrested cells includes: continuously culturing in a medium for culturing pluripotent stem cells with rapamycin added thereto. By adding rapamycin in the culture of pluripotent stem cells (such as human embryonic stem cells), it is possible to guide pluripotent stem cells to become cells that are developmentally arrested in a natural state, which is beneficial to preventing the excessive proliferation of pluripotent stem cells in special circumstances, provides new ideas for more applications of human pluripotent stem cells, and can improve the efficiency of cell production; it is beneficial to obtain pluripotent stem cells with a specific degree of development as needed, providing more possibilities for the application of human pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular, to a method for guiding pluripotent stem cells to become developmentally arrested cells and its applications. Background Art

[0002] Human pluripotent stem cells (hPSCs) can differentiate into all cell types in our body and are an important model system for studying human embryonic development. Specific cell types generated by hPSC differentiation play important roles in cell therapy, drug screening, and disease model research. Evidently, human pluripotent stem cells provide materials for the regeneration and repair of various tissues and drug screening.

[0003] Cells at different developmental degrees of human pluripotent stem cells all have therapeutic application values, and it is very difficult to obtain human pluripotent stem cells at a specific developmental degree.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] An object of the present invention is to provide a method for guiding pluripotent stem cells to become developmentally arrested cells, aiming to make pluripotent stem cells become cells in a developmentally arrested state similar to the natural state, providing more possibilities for the application of pluripotent stem cells.

[0006] Another object of the present invention is to provide a method for obtaining pluripotent stem cells, which can obtain pluripotent stem cells at a specific developmental degree.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for guiding pluripotent stem cells to become developmentally arrested cells, including: adding rapamycin to a culture medium for culturing pluripotent stem cells.

[0009] In an optional embodiment, the concentration of rapamycin in the culture medium is 20 - 200 nM; preferably 50 - 100 nM.

[0010] In an optional embodiment, the pluripotent stem cells are human pluripotent stem cells.

[0011] In an optional embodiment, the human pluripotent stem cells are selected from human embryonic stem cells or human induced pluripotent stem cells.

[0012] In an optional embodiment, the culture of human pluripotent stem cells is carried out in an E8 culture medium added with rapamycin.

[0013] In an alternative embodiment, the components of the E8 medium include DMEM / F12 medium, L-ascorbic acid-2-phosphate magnesium, sodium selenite, transferrin, insulin, basic fibroblast growth factor, and transforming growth factor-β.

[0014] In an alternative embodiment, in the E8 medium, the concentration of L-ascorbic acid-2-phosphate magnesium is 60-70 mg / L, the concentration of sodium selenite is 12-16 μg / L, the concentration of transferrin is 8-12 mg / L, the concentration of insulin is 18-22 mg / L, the concentration of basic fibroblast growth factor is 90-110 μg / L, and the concentration of transforming growth factor-β is 1-3 μg / L.

[0015] In a second aspect, the present invention provides a method for obtaining pluripotent stem cells, which uses the method of any one of the foregoing embodiments to direct human pluripotent stem cells to become developmentally arrested cells.

[0016] In an alternative embodiment, after stopping the culture, the obtained cells are rinsed with a medium.

[0017] In an alternative embodiment, the obtained cells are rinsed with the E8 medium at least 2 times.

[0018] The present invention has the following beneficial effects: By adding rapamycin to the culture of pluripotent stem cells (such as human pluripotent stem cells), the inventor can direct pluripotent stem cells to become cells that are developmentally arrested in a natural state, which is beneficial to preventing the over-proliferation of pluripotent stem cells in special circumstances, provides a new idea for more applications of human pluripotent stem cells, and can improve the efficiency of cell production; it is beneficial to obtain pluripotent stem cells with a specific degree of development according to needs, providing more possibilities for the application of human pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0020] Figure 1 It is a test result graph of the growth rate of rapamycin-treated human pluripotent stem cells;

[0021] Figure 2 It is a test result graph of the expression of rapamycin-treated human pluripotent stem cells;

[0022] Figure 3Test chart of the cell morphology of rapamycin-treated human pluripotent stem cells;

[0023] Figure 4 Test result chart of the growth of the culture group and the control group of human pluripotent stem cells continuously treated with rapamycin. Detailed implementation manners

[0024] To make the objectives, 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0025] Mammalian target of rapamycin (mTOR) is an atypical serine / threonine kinase. The mTOR signaling pathway has the functions of promoting material metabolism, participating in apoptosis and autophagy, and plays a non-negligible role in various diseases. Rapamycin is a specific inhibitor of mTOR. It is also the earliest discovered mTOR inhibitor and is a macrolide antibiotic isolated from bacteria in the 1970s.

[0026] The embodiments of the present invention provide a method for guiding pluripotent stem cells to become developmentally arrested cells, including: adding rapamycin to the culture medium for culturing pluripotent stem cells. The inventors unexpectedly found that adding rapamycin to the culture of pluripotent stem cells can make the pluripotent stem cells become cells in a developmentally arrested state similar to the natural state, which is beneficial to preventing the over-proliferation of pluripotent stem cells in special cases, provides new ideas for more applications of human pluripotent stem cells, and can improve the efficiency of cell production.

[0027] Specifically, the pluripotent stem cells can be human pluripotent stem cells or pluripotent stem cells of other animals. The human pluripotent stem cells can be common human pluripotent stem cells such as human embryonic stem cells or human induced pluripotent stem cells.

[0028] To better control the human pluripotent stem cells in a developmentally arrested state, the inventors optimized the dosage of rapamycin. In the culture medium, the concentration of rapamycin in the culture medium is 20-200 nM; preferably 50-100 nM.

[0029] Specifically, the concentration of rapamycin can be 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 200 nM, etc., or any value between the above concentration values.

[0030] In some embodiments, human pluripotent stem cells are cultured in E8 medium supplemented with rapamycin. The E8 medium is suitable for the culture of human pluripotent stem cells and for the embodiments of the present invention.

[0031] In other embodiments, instead of using E8 medium, other existing media suitable for the culture of human pluripotent stem cells can be used.

[0032] Furthermore, in the E8 medium, the concentration of L-ascorbic acid-2-phosphate magnesium is 60 - 70 mg / L, the concentration of sodium selenite is 12 - 16 μg / L, the concentration of transferrin is 8 - 12 mg / L, the concentration of insulin is 18 - 22 mg / L, the concentration of basic fibroblast growth factor is 90 - 110 μg / L, and the concentration of transforming growth factor-β is 1 - 3 μg / L.

[0033] It should be noted that by controlling the components in the E8 medium, the growth of human pluripotent stem cells can be better promoted. The concentrations of L-ascorbic acid-2-phosphate magnesium, sodium selenite, transferrin, insulin, basic fibroblast growth factor, and transforming growth factor-β can be controlled within the specified ranges above.

[0034] The embodiments of the present invention provide a method for obtaining pluripotent stem cells, which uses the method in the foregoing embodiments to guide pluripotent stem cells into developmentally arrested cells, and thus specific developmentally advanced pluripotent stem cells can be obtained, having very good application prospects.

[0035] In some embodiments, after stopping the culture, the obtained cells are rinsed with a medium, and through rinsing, impurities adhering to the cell surface can be removed. The medium used for rinsing is not limited, and the E8 medium can be used to rinse the obtained cells, and the number of rinsing times is preferably at least 2 times.

[0036] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0037] It should be noted that for the E8 medium used in the following embodiments, the other components except the inhibitor include: the concentration of L-ascorbic acid-2-phosphate magnesium is 64 mg / L, the concentration of sodium selenite is 14 μg / L, the concentration of transferrin is 10 mg / L, the concentration of insulin is 20 mg / L, the concentration of basic fibroblast growth factor is 100 μg / L, and the concentration of transforming growth factor-β is 2 μg / L.

[0038] Example 1

[0039] This embodiment provides a method for guiding pluripotent stem cells into developmentally arrested cells, which is specifically implemented as follows:

[0040] Human pluripotent stem cells H1 (i.e., H1 human embryonic stem cells) were cultured in E8 medium, and the fresh medium was changed daily. Passage was carried out when the cell density reached 70%-80%. First, wash twice with DPBS-EDTA, then incubate at room temperature for 5 minutes. Aspirate DPBS-EDTA for the third time and add E8 medium containing 5 μM ROCK inhibitor. After resuspending the cells, passage them at a density of 1:6 to 1:12 and add them to a 12-well plate pre-coated with Matrigel. One day after the cells adhered to the plate, add E8 medium containing rapamycin.

[0041] After the cells were passaged three times, the cells were harvested for quantitative PCR and immunofluorescence experiments to detect the expression of pluripotent genes. And the cells were injected subcutaneously into nude mice. After 6-8 weeks, the teratoma tissues were isolated, fixed, paraffin-embedded and sectioned for staining to observe the differentiation morphology of the three germ layers. The test results are shown in Figures 1-3 . In the figure, control refers to the blank test without adding rapamycin.

[0042] From Figure 1 It can be seen that the growth rate of human pluripotent stem cells continuously treated with rapamycin significantly slows down.

[0043] Figure 2 This is the pluripotent gene detection of H1 after being treated with rapamycin for 3 generations. The results show that it can still be highly expressed and has the characteristics of stem cells. From Figure 2 It can be seen that human pluripotent stem cells continuously treated with rapamycin highly express OCT4 and NANOG.

[0044] From Figure 3 It can be seen that human pluripotent stem cells continuously treated with rapamycin can obtain teratomas with the morphology of the three germ layers.

[0045] Example 2

[0046] Human pluripotent stem cells H1 were cultured in E8 medium, and the fresh medium was changed daily until the cell density reached 70%-80%. First, wash twice with DPBS-EDTA, then incubate at room temperature for 5 minutes. Remove DPBS-EDTA for the third time and resuspend the cells with E8 medium containing 5 μM Rock inhibitor. Then passage them at a density of 1:6 to 1:12 and add them to a 12-well plate pre-coated with Matrigel, and add E8 medium containing rapamycin inhibitor.

[0047] When the cells continuously grew in the same well for about 8 days, even without changing the E8 medium, the rapamycin-treated cells could still survive well, as shown in Figure 4As shown in A. During the culture of human pluripotent stem cells continuously treated with rapamycin, the E8 medium was not renewed, and it gradually exceeded the control group in the later stage of growth compared with the control group.

[0048] When the E8 medium was changed daily, rapamycin could prevent the overgrowth of stem cells, as Figure 4 shown in B. During the culture of human pluripotent stem cells continuously treated with rapamycin, the E8 medium was renewed daily, and it gradually reached and exceeded the control group in the later stage of growth.

[0049] From Figure 4 this, it can be seen that culturing human pluripotent stem cells with a medium containing rapamycin can effectively prevent the overgrowth of human pluripotent stem cell clones.

[0050] In summary, the present invention provides a method and application for guiding pluripotent stem cells to become developmentally arrested cells. By adding rapamycin to the culture of pluripotent stem cells (such as human pluripotent stem cells), pluripotent stem cells can be guided to become cells in a developmentally arrested state similar to the natural state, which is beneficial to preventing the over-proliferation of pluripotent stem cells in special cases, provides new ideas for more applications of human pluripotent stem cells, and can improve the efficiency of cell production.

[0051] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for guiding pluripotent stem cells to become developmentally arrested cells, characterized in that, Comprising: Adding rapamycin to the medium for culturing pluripotent stem cells; The pluripotent stem cells are human pluripotent stem cells; The human pluripotent stem cells are selected from human embryonic stem cells or human induced pluripotent stem cells, and the human embryonic stem cells are H1 human embryonic stem cells; The concentration of rapamycin in the medium is 50 - 100 nM; the culturing of the human pluripotent stem cells is carried out in E8 medium supplemented with rapamycin; The components of the E8 medium include DMEM / F12 medium, L-ascorbic acid-2-phosphate magnesium, sodium selenite, transferrin, insulin, basic fibroblast growth factor, and transforming growth factor-β.

2. The method according to claim 1, characterized in that In the E8 medium, the concentration of L-ascorbic acid-2-phosphate magnesium is 60 - 70 mg / L, the concentration of sodium selenite is 12 - 16 μg / L, the concentration of transferrin is 8 - 12 mg / L, the concentration of insulin is 18 - 22 mg / L, the concentration of basic fibroblast growth factor is 90 - 110 μg / L, and the concentration of transforming growth factor-β is 1 - 3 μg / L.

3. A method for obtaining pluripotent stem cells, characterized in that, It uses the method described in any one of claims 1 - 2 to guide pluripotent stem cells to become developmentally arrested cells.

4. The method according to claim 3, wherein After stopping the culturing, the obtained cells are rinsed with the medium.

5. The method according to claim 4, wherein The obtained cells are rinsed with E8 medium at least 2 times.

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