Method for efficiently obtaining human definitive endoderm cells in vitro
By combining pretreatment culture medium and shaping endoderm differentiation culture medium, the problem of limited differentiation efficiency and purity of shaping endoderm in existing technologies has been solved, realizing the acquisition of human shaping endoderm cells with high efficiency and high purity, simplifying the operation process, and improving the reproducibility and reliability of experiments.
Patent Information
- Application Number
- CN202511778278.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-28
- Publication Date
- 2026-02-27
AI Technical Summary
Existing methods for defining endoderm differentiation suffer from limitations in differentiation efficiency and cell population purity, as well as batch-to-batch variability, which affects experimental reproducibility and data reliability.
Human pluripotent stem cells were pretreated and cultured in a pretreatment medium for two days, and then induced to differentiate using a fixed endoderm differentiation medium containing fetal bovine serum and activin A. This avoided the addition of specific growth factors or small molecule inhibitors during differentiation. Dimethyl sulfoxide was used to alter cell membrane permeability and inhibit the cell cycle, thereby promoting signal transduction and ensuring differentiation efficiency and purity.
This method enables the efficient and high-purity acquisition of human endoderm cells, simplifies the operation process, reduces the impact of batch-to-batch variation, improves the reproducibility of experiments and the reliability of data, and is conducive to large-scale application and industrialization.
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Figure CN121574907A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biotechnology, in particular to a method for obtaining human difinitive endoderm cells in vitro. BACKGROUND
[0002] Difinitive endoderm (DE) is one of the three main germ layers formed in the early stage of embryonic development, and is the common precursor cell source of all endoderm-derived organs (such as liver, pancreas, lung, thyroid, thymus, and gastrointestinal lining epithelium). Successfully obtaining high-quality and high-purity DE cells is the necessary starting point and basis for further differentiation into functional cells with therapeutic potential. Without reliable DE differentiation, it is impossible to realize the construction of complex endoderm organ tissues for disease modeling, drug screening, and cell replacement therapy using stem cells. Therefore, it is of great significance to efficiently and accurately induce pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) to differentiate into difinitive endoderm.
[0003] The existing difinitive endoderm differentiation method is mainly based on the in vitro induction and differentiation technology of pluripotent stem cells. By simulating the signal pathways of endoderm formation during embryonic development, combining growth factors, small molecule compounds, and optimizing the culture conditions, the most commonly used differentiation scheme is to simulate the Nodal signal during the formation of the embryonic primitive gut embryo, and to combine the culture medium containing fetal bovine serum or serum substitutes to induce difinitive endoderm differentiation in human pluripotent stem cells. The differentiation cycle is generally 3-4 days. Although the induction efficiency of difinitive endoderm in this way can reach about 80%, there are still many impurities. In order to improve the differentiation efficiency, the existing optimization scheme generally adds specific growth factors or small molecule inhibitors within 1-1.5 days of differentiation, which can increase the difinitive endoderm differentiation efficiency to 90-95%. In order to ensure the differentiation effect, the use time range of growth factors or small molecule compounds has a high requirement, and the difference between different batches of growth factors and small molecule compounds will also affect the differentiation efficiency, which seriously affects the repeatability of the experiment and the reliability of the data, and hinders the transformation and application of research results. SUMMARY
[0004] In view of the above defects, the purpose of the present application is to provide a method for obtaining human difinitive endoderm cells in vitro, which solves the problem that the existing difinitive endoderm differentiation efficiency and cell population purity are limited and affected by batch-to-batch variation.
[0005] To achieve this purpose, the technical scheme adopted by the present application is as follows: A method for obtaining human difinitive endoderm cells in vitro, comprising the following steps: S1, inoculating human pluripotent stem cells in Matrigel and using stem cell maintenance medium for maintenance culture for 16-24h; S2, Day-2 to Day-1, aspirate the stem cell maintenance medium, add the pretreatment medium for culture and replace the pretreatment medium every day; The pretreatment medium is the stem cell maintenance medium added with dimethyl sulfoxide; S3, Day 0 to Day 2, aspirate the pretreatment medium, add the definitive endoderm differentiation medium for induction culture and replace the definitive endoderm differentiation medium every day; The definitive endoderm differentiation medium is RPMI1640 medium added with fetal bovine serum and activin A; S4, Day 3, obtain the desired human definitive endoderm cells.
[0006] Preferably, in the pretreatment medium, the concentration of dimethyl sulfoxide is 0.2-3%.
[0007] Preferably, in step S1, the six-well plate is coated with Matrigel and incubated at 37℃ for more than 30 minutes; The human pluripotent stem cells in good condition and with a confluence of 80-90% are inoculated into the Matrigel.
[0008] Preferably, in steps S1, S2 and S3, the culture condition is 37℃, 5% CO2 incubator culture.
[0009] Preferably, the stem cell maintenance medium is mTeSR1 TM Culture medium.
[0010] Preferably, in the definitive endoderm differentiation medium, the concentration of fetal bovine serum is 0.5-2% and the concentration of activin A is 20-200 ng / mL.
[0011] Preferably, in steps S2 and S3, the volume of the pretreatment medium or the definitive endoderm differentiation medium added each time is 1.5-3 mL / well.
[0012] Preferably, in step S1, the inoculation amount of human pluripotent stem cells is 0.1×10 6 -1×10 6 / well.
[0013] Preferably, in step S2, before step S3, the cell density needs to reach 70-80%.
[0014] The technical solution provided by the present application can include the following beneficial effects: The application uses a pretreatment medium to pretreat human pluripotent stem cells after maintenance culture, and then induces differentiation after two days of culture, and only a single definitive endoderm differentiation medium is used in the induction differentiation to realize the differentiation of human pluripotent stem cells into human definitive endoderm cells, which is simple in steps, effectively avoids the influence of the difference of growth factors and small molecule compounds between different batches on the differentiation efficiency, and at the same time, the pretreatment medium is matched with the definitive endoderm differentiation medium, which significantly improves the efficiency and purity of the definitive endoderm differentiation. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 is the schematic diagram of the in vitro acquisition process of the application.
[0016] Figure 2 is the cell counting result diagram of Example 1 and Comparative Example 1 of the application.
[0017] Figure 3 is the immunofluorescence detection result diagram of Example 1 and Comparative Example 1 of the application. DETAILED DESCRIPTION
[0018] The technical solutions of the application will be further described below through specific embodiments.
[0019] In order to facilitate the understanding of the application, the application will be described more fully below. The application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the application more thorough and comprehensive.
[0020] Unless specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. Unless the manufacturer is specified, all reagents or instruments used are conventional products that can be obtained on the market.
[0021] A method for efficiently acquiring human definitive endoderm cells in vitro, comprising the following steps: S1, inoculating human pluripotent stem cells in Matrigel and using stem cell maintenance medium for maintenance culture for 16-24h; S2, from Day-2 to Day-1, aspirate the stem cell maintenance medium, add the pretreatment medium for culture and replace the pretreatment medium every day; The pretreatment medium is a stem cell maintenance medium added with dimethyl sulfoxide; S3, from Day0 to Day2, aspirate the pretreatment medium, add the definitive endoderm differentiation medium for induction culture and replace the definitive endoderm differentiation medium every day; The definitive endoderm differentiation medium is an RPMI1640 medium added with fetal bovine serum and activin A; S4, Day 3, obtain the desired human definitive endoderm cells.
[0022] Cell differentiation is complex and needs to be adapted to the corresponding culture process, the existing method for obtaining human definitive endoderm cells in vitro is to simulate the Nodal signal of the embryonic primitive gut blastula formation period, and the medium combined with fetal bovine serum is used for endoderm differentiation, but the differentiation efficiency reached by the method is about 80%, and there are many mixed cells, in order to improve the in vitro acquisition efficiency, specific growth factors or small molecule inhibitors are added within 1-1.5 days of differentiation, but this operation is more complex, there are batch-to-batch variation problems, which affect the repeatability of the experiment and the reliability of the data, and is not conducive to subsequent disease modeling, drug screening and other applications. In order to solve the problems existing in the prior art, the present application proposes a method for obtaining human definitive endoderm cells in vitro with high efficiency, the specific process is as shown in Figure 1 Based on the basic induction method of simulating the Nodal signal of the embryonic primitive gut blastula formation period, the pre-treatment medium is used for culture for two days before differentiation, and then the endoderm differentiation medium containing fetal bovine serum and activin A is used for culture, and during the differentiation period, i.e. Day 0-Day 3, no specific growth factor or small molecule inhibitor needs to be added, the whole operation is simple, only the medium needs to be replaced, to a certain extent, the influence of the difference of growth factors and small molecule compounds between different batches on the differentiation efficiency is avoided.
[0023] In addition, the pre-treatment medium contains dimethyl sulfoxide, which can change the permeability of the cell membrane, promote signal transduction, inhibit the transition of cells from G1 phase to S phase, induce cell population to arrest in G1 phase, reduce the difference in differentiation efficiency caused by different cell cycles, and prepare cell state for differentiation. In addition, dimethyl sulfoxide can also effectively remodel the epigenetic state, regulate key signaling pathways, and create a cell internal environment conducive to differentiation rather than maintaining pluripotency, so as to realize the differentiation of human pluripotent stem cells in vitro into definitive endoderm by using only the definitive endoderm differentiation medium during the differentiation period. The purity of the obtained definitive endoderm cells is high, and the proportion of successfully transformed human definitive endoderm cells is high. By using the pre-treatment medium and the definitive endoderm differentiation medium, the steps are simple, no specific growth factor or small molecule inhibitor needs to be added at a specific time during the differentiation period, the influence of growth factors or small molecule inhibitors between different batches on the differentiation efficiency is avoided, and the efficient acquisition of high-purity human definitive endoderm cells is completed only by using a single definitive endoderm differentiation medium during the differentiation process. The problems of limited differentiation efficiency and cell population purity and batch-to-batch variation are solved, which is conducive to large-scale application and industrialization.
[0024] The definitive endoderm differentiation medium is RPMI1640 medium added with fetal bovine serum and activin A, the RPMI1640 medium is a basic culture medium without endogenous differentiation interference factors, which ensures that the activin A can directly start the expression of definitive endoderm specific transcription factors and inhibit other pathways, ensuring the accuracy of the differentiation direction, and the fetal bovine serum can effectively reduce the apoptosis of cells during differentiation. The definitive endoderm differentiation medium has wide adaptability, high stability, and comprehensive nutritional support, and can meet the differentiation needs after being treated by the pretreatment medium, thereby realizing efficient and high-purity acquisition of human definitive endoderm cells. The anti-apoptotic effect of dimethyl sulfoxide and the nutritional support and anti-toxicity effect of fetal bovine serum form a double guarantee, and dimethyl sulfoxide induces cell cycle synchronization, so that the cells are in a cell cycle phase sensitive to activin A. From the logical dependence and functionality, the pretreatment medium can make the cells enter the preparation stage of differentiation in advance, and the subsequent definitive endoderm differentiation medium considers the low cost and effectiveness of differentiation, and only in combination with the pretreatment medium before differentiation can efficient definitive endoderm differentiation be realized.
[0025] Preferably, the matrix glue used can be Matrigel matrix glue, and a pretreatment of matrix glue coating is required before step S1.
[0026] Preferably, the concentration of dimethyl sulfoxide in the pretreatment medium is 0.2-3%.
[0027] Specifically, the concentration of dimethyl sulfoxide is 0.2-3%, and dimethyl sulfoxide can produce cytotoxicity, which affects cell culture and induction differentiation. Therefore, within this range, it is ensured that dimethyl sulfoxide can produce sufficient biological effects, cooperate with the definitive endoderm differentiation medium to meet the efficient and high-purity in vitro acquisition of human definitive endoderm cells, and avoid producing strong cytotoxicity to affect cell culture and induction differentiation and causing a large number of cell deaths.
[0028] Although some cells are sensitive to the culture conditions of 1% dimethyl sulfoxide and may exhibit heavy cytotoxicity, the use of 1%-3% dimethyl sulfoxide in human pluripotent stem cells basically has no obvious effect on the cells, and the cooperation with the subsequent definitive endoderm differentiation medium can also have a significant improvement effect on the definitive endoderm differentiation.
[0029] Preferably, in step S1, the six-well plate is coated with matrix glue and incubated at 37°C for more than 30 minutes. The human pluripotent stem cells in good condition and with a confluence of 80-90% are inoculated into the matrix glue.
[0030] Specifically, before the pre-treatment and induction differentiation, the human pluripotent stem cells need to be maintained in culture, keep the pluripotency of the human pluripotent stem cells, and select the human pluripotent stem cells in good condition and at a confluence of 80-90% to avoid spontaneous differentiation of the human pluripotent stem cells, ensure the uniformity of the cell state, and improve the synchronization of the subsequent induction differentiation.
[0031] Preferably, in steps S1, S2 and S3, the culture conditions are 37°C and 5% CO2 incubator culture.
[0032] Specifically, the culture environment maximally simulates the environment of in vivo growth and meets the needs of in vitro preparation of human pluripotent stem cells.
[0033] Preferably, the stem cell maintenance medium is mTeSR1 TM culture medium.
[0034] Specifically, mTeSR1 TM The culture medium contains recombinant proteins and specific nutrients, which can meet the needs of the maintenance culture of human pluripotent stem cells, effectively inhibit the spontaneous differentiation of human pluripotent stem cells, and meet the needs of subsequent induction differentiation.
[0035] Preferably, in the definitive endoderm differentiation medium, the concentration of fetal bovine serum is 0.5-2%, and the concentration of activin A is 20-200 ng / mL.
[0036] Specifically, limiting the concentration of activin A can ensure that activin A stably activates the corresponding pathway, meet the needs of the initiation of expression of definitive endoderm-specific transcription factors, ensure differentiation in the desired direction, meet the differentiation needs, and use a lower concentration of fetal bovine serum to meet the needs of nutrition and anti-apoptosis while not interfering with the specific signal of activin A, ensuring the normal differentiation of human pluripotent stem cells into human definitive endoderm cells.
[0037] Preferably, in steps S2 and S3, the volume of the pre-treatment medium or the definitive endoderm differentiation medium added each time is 1.5-3 mL / well.
[0038] Specifically, the culture area of a six-well plate is adapted to form a uniform liquid environment, ensuring that the cells are in sufficient contact with the nutrients and differentiation signal molecules in the pre-treatment medium or the definitive endoderm differentiation medium, and ensuring that the cells can normally metabolize and differentiate.
[0039] Preferably, in step S1, the inoculation amount of human pluripotent stem cells is 0.1×10 6 -1×10 6 cells / well.
[0040] Specifically, at this inoculation amount, the pluripotent stem cells are effectively maintained in an undifferentiated state, ensuring that the pluripotent stem cells form dense and uniform colonies on the Matrigel, facilitating subsequent culture of the cells in a medium with consistent effects and reducing the heterogeneity of differentiated cells.
[0041] Preferably, in step S2, the cell density needs to reach 70-80% before step S3 is performed.
[0042] Specifically, before induction of differentiation, the human pluripotent stem cells need to maintain normal pluripotent stem cell morphology, maintain pluripotency, and the cell density needs to reach 70-80% before induction, which is the quality control standard for cells before differentiation.
[0043] The technical solutions of the present application are further illustrated by specific embodiments.
[0044] Example 1 S1, use Matrigel to coat a six-well plate, incubate at 37°C for 30 minutes or more; Select human induced pluripotent stem cells iPS-DYR0100 in good condition and with a confluence of 80-90%, inoculate 0.1 x 10 6 -1 x 10 6 cells per hole into the above-mentioned Matrigel-coated six-well plate, use stem cell maintenance medium for maintenance culture, and culture in a 37°C, 5% CO2 incubator until the next day; wherein the stem cell maintenance medium is commercial mTeSR1 TM medium; S2, Day-2: aspirate the stem cell maintenance medium, add 3mL of pretreatment medium per well, and culture in a 37°C, 5% CO2 incubator until the next day; The pretreatment medium is commercial mTeSR1TM medium containing 2% dimethyl sulfoxide; Day-1: aspirate the pretreatment medium, add 3mL of pretreatment medium per well again, and culture in a 37°C, 5% CO2 incubator until the next day; S3, Day0: aspirate the pretreatment medium, add 3mL of definitive endoderm differentiation medium per well, and culture in a 37°C, 5% CO2 incubator until the next day; The definitive endoderm differentiation medium is RPMI1640 medium containing 1.5% fetal bovine serum and 100ng / mL of activin A; Day1: aspirate the definitive endoderm differentiation medium, add 3mL of definitive endoderm differentiation medium per well, and culture in a 37°C, 5% CO2 incubator until the next day; Day 2: Aspirate the pre-treatment medium, add 3 mL of definitive endoderm differentiation medium to each well, and incubate at 37°C in a 5% CO2 incubator until the next day; S4, Day 3: Marked as the end point of differentiation, and the desired human definitive endoderm cells were obtained. This Example 1 was marked as +DMSO (dimethyl sulfoxide). The bright field field-of-view shooting, cell counting, and immunofluorescence detection of definitive endoderm-specific markers were performed on the differentiated definitive endoderm cells, and the results are shown in FIGS. Figure 2 and Figure 3
[0045] Comparative Example 1 S1, use Matrigel to coat the six-well plate, and incubate at 37°C for more than 30 minutes; Select human induced pluripotent stem cells iPS-DYR0100 in good condition and with a cell confluence of 80-90%, and inoculate into the above Matrigel-coated six-well plate at a cell amount of 0.1 x 10 6 -1 x 10 6 cells per well, and use stem cell maintenance medium for maintenance culture, and incubate at 37°C in a 5% CO2 incubator until the next day; wherein the stem cell maintenance medium is a commercial mTeSR1 TM medium. S2, Day 0: Aspirate the pre-treatment medium, add 3 mL of definitive endoderm differentiation medium to each well, and incubate at 37°C in a 5% CO2 incubator until the next day; wherein the definitive endoderm differentiation medium is RPMI1640 medium containing 1.5% fetal bovine serum and 100 ng / mL of activin A; Day 1: Aspirate the definitive endoderm differentiation medium, add 3 mL of definitive endoderm differentiation medium to each well, and incubate at 37°C in a 5% CO2 incubator until the next day; Day 2: Aspirate the definitive endoderm differentiation medium, add 3 mL of definitive endoderm differentiation medium to each well, and incubate at 37°C in a 5% CO2 incubator until the next day; S3, Day 3: Marked as the end point of differentiation, and the human definitive endoderm cells were obtained. This Comparative Example 1 was marked as -DMSO. The bright field field-of-view shooting, cell counting, and immunofluorescence detection of definitive endoderm-specific markers were performed on the differentiated definitive endoderm cells, and the results are shown in FIGS. Figure 2 and Figure 3
[0046] Figure 2 The cell morphology of the human definitive endoderm cells obtained by the embodiment 1 and the comparative example 1 at the differentiation end point under the bright field view shooting can be known by the cell counts, and the cell amount obtained under the same condition after the dimethyl sulfoxide pretreatment is much more than that without the dimethyl sulfoxide pretreatment, and it can be seen that the differentiation efficiency can be effectively improved by using the pretreatment medium.
[0047] Figure 3 The immunofluorescence detection result diagram of the definitive endoderm specific markers is shown, wherein DAPI is a cell nucleus dye, corresponding to blue fluorescence, marking the cell nucleus of all cells, SOX17 and FOXA2 are specific markers of human definitive endoderm, and the induced differentiated human definitive endoderm cells are marked by SOX17 and FOXA2, and present green and red respectively, so that the column chart of the cell proportion of the marker expression cells can be obtained, and only SOX17 + is counted, and it can be seen that the proportion of the corresponding marker expression cells of the embodiment 1 is greater than that of the comparative example 1, and is higher than 80%, but only SOX17 expression is single positive cell, and there are partial differentiation incomplete or potential false positive cells, and by counting SOX17 + and FOXA2 + , it can be seen that the double positive cells, that is, the content of the truly applicable human definitive endoderm cells, the purity of the human definitive endoderm cells of the embodiment 1 is much greater than that of the comparative example 1. Therefore, there are a large number of false positive cells in the comparative example 1, and the purity is much lower than that of the embodiment 1, that is, a large number of high-purity stable human definitive endoderm cells can be obtained by the embodiment 1.
[0048] It can be known from Figure 2 and Figure 3 that by the in vitro acquisition method of the present application, the morphology of the obtained human pluripotent stem cells is normal and uniform, and the differentiated cells are more, the differentiation efficiency is high, and the purity of the corresponding human definitive endoderm cells is high, without adding specific small molecule inhibitors or growth factors during differentiation, reducing the material cost brought by a large amount of use of growth factors or small molecule compounds, improving the stability and accessibility of the in vitro acquisition of human definitive endoderm cells, and promoting the standardization and industrialization of the production.
[0049] The technical principles of the present application are described above in combination with specific embodiments. These descriptions are only for explaining the principles of the present application, and cannot be explained as limitations on the protection scope of the present application in any way. Based on the explanations here, other specific embodiments of the present application can be conceived by those skilled in the art without creative labor, and these embodiments will fall within the protection scope of the present application.
Claims
1. A method for efficiently obtaining human fixed-shape endoderm cells in vitro, characterized in that, Includes the following steps: S1. Human pluripotent stem cells are seeded into matrix gel and maintained in stem cell maintenance medium for 16-24 hours. S2, Day-2 to Day-1: Discard the stem cell maintenance medium, add the pretreatment medium for culture, and change the pretreatment medium once a day; The pretreatment culture medium is a stem cell maintenance culture medium supplemented with dimethyl sulfoxide; S3, Day0 to Day2: Discard the pretreatment medium, add the shaping endoderm differentiation medium for induction culture, and change the shaping endoderm differentiation medium once a day. The endodermal differentiation medium for shaping was RPMI 1640 medium supplemented with fetal bovine serum and activin A. S4, Day 3, to obtain the desired human shaped endoderm cells.
2. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: The concentration of dimethyl sulfoxide in the pretreatment medium is 0.2-3%.
3. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: In step S1, a six-well plate is coated with a matrix adhesive and incubated at 37°C for at least 30 minutes; Human pluripotent stem cells in good condition with a confluence of 80-90% were seeded into the matrix gel.
4. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: In steps S1, S2 and S3, the culture conditions are 37℃ and 5% CO2 incubator.
5. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: The stem cell maintenance culture medium is mTeSR1 TM Culture medium.
6. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: In the culture medium for endodermal differentiation, the concentration of fetal bovine serum is 0.5-2%, and the concentration of activin A is 20-200 ng / mL.
7. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 3, characterized in that: In steps S2 and S3, the volume of pretreatment culture medium or shaping endoderm differentiation culture medium added each time is 1.5-3 mL / well.
8. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 3, characterized in that: In step S1, the seeding amount of human pluripotent stem cells is 0.1 × 10⁻⁶. 6 -1×10 6 / hole.
9. The method for efficiently obtaining human fixed-shape endoderm cells in vitro according to claim 1, characterized in that: In step S2, the cell density needs to reach 70-80% before proceeding to step S3.