A method for promoting the differentiation of induced pluripotent stem cells into the three germ layers and its application in differentiation potential detection.

Abstract

This invention provides a method for promoting the differentiation of induced pluripotent stem cells (iPSCs) into three germ layers and its application in differentiation potential detection, relating to the field of biotechnology. The method includes seeding iPSCs into a culture medium containing 5 μM~20 μM E-cadherin activator 1, shaking culture to form embryoids, adding differentiation medium for differentiation culture, obtaining embryoids with activated three germ layer differentiation potential; seeding the embryoids with activated three germ layer differentiation potential into a container coated with a cell adhesion matrix, adding differentiation medium for further differentiation culture, inducing differentiation into three germ layer cells. This invention solves the technical problems in existing technologies where induction of iPSC differentiation into three germ layers via the embryoid pathway results in poor embryoid formation, poor embryoid state, poor long-term maintenance, and poor adhesion after embryoid transfer, leading to poor endoderm differentiation and migration abilities.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for promoting the differentiation of induced pluripotent stem cells into the three germ layers and its application in the detection of differentiation potential. Background Technology

[0002] Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that are transformed into embryonic stem cells by introducing reprogramming factors into terminally differentiated cells. iPSCs have the potential to self-renew and differentiate into all cells of the three germ layers.

[0003] Existing techniques for validating the three germ layer differentiation of iPSCs commonly include STEMdiff. TM The Trilineage Differentiation Kit (Catalog #05230) provides culture media for differentiation into the three germ layers. However, due to the varying proliferative capacities of different iPS cell lines, the adherent cell density is often too high or too low after seeding according to the instructions. This leads to cell death and differentiation failure during subsequent differentiation due to nutrient deprivation or lack of intercellular communication. Furthermore, the differentiation pathway in this kit is based on single cells, resulting in single-cell cells for all three germ layers. This state is more suitable for qPCR detection and is not applicable for obtaining differentiated cell morphology through immunofluorescence staining after differentiation. The morphology of the three germ layers presented by this kit is poor, failing to exhibit the typical characteristics of the different germ layers.

[0004] Existing techniques for inducing iPS differentiation into three germ layers via the embryoid pathway often suffer from problems such as poor embryoid formation ability, poor embryoid state, poor long-term maintenance ability, poor adhesion ability of embryoids after transfer to the plate, poor endoderm differentiation and migration ability, and poor endoderm staining effect.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for promoting the differentiation of induced pluripotent stem cells into the three germ layers, in order to solve the technical problems of poor embryomorphic body formation ability, poor embryomorphic body state, poor long-term maintenance ability, and poor adhesion ability of embryomorphic bodies after transfer to the plate, which leads to poor endoderm differentiation and crawling ability in the existing method of inducing iPS to differentiate into the three germ layers through the embryomorphic body pathway.

[0007] The second objective of this invention is to provide the application of the above-mentioned method in detecting the three-layer differentiation potential of induced pluripotent stem cells or in preparing products for detecting the three-layer differentiation potential of induced pluripotent stem cells.

[0008] The third objective of this invention is to provide a method for detecting the three germ layer differentiation potential of induced pluripotent stem cells.

[0009] The fourth objective of this invention is to provide a kit for detecting the differentiation potential of induced pluripotent stem cells into three germ layers.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] In a first aspect, the present invention provides a method for promoting the differentiation of induced pluripotent stem cells into the three germ layers, comprising the following steps:

[0012] A. Induced pluripotent stem cells were seeded in a culture medium containing 5 μM~20 μM E-cadherin activator 1, and cultured with shaking to form embryoid bodies. Differentiation culture medium was added for differentiation culture to obtain embryoid bodies with activated differentiation potential of the three germ layers.

[0013] B. The embryoids with activated trilaminar differentiation potential are seeded into a container coated with cell adhesion matrix, and differentiation culture medium is added to continue differentiation culture to induce differentiation into trilaminar cells.

[0014] Furthermore, the concentration of E-cadherin activator 1 in the culture medium was 10 μM;

[0015] The seeding amount of the induced pluripotent stem cells is (0.1~10)×10 per 2 mL of culture medium. 6 cell.

[0016] Furthermore, the rotation speed of the oscillation culture is 50~80 rpm;

[0017] The shaking culture time is 16-18 hours.

[0018] Furthermore, the time for obtaining the embryoid body with activated trilaminar differentiation potential is 5 to 8 days after adding the differentiation medium;

[0019] The differentiation medium comprises the following components by volume percentage: 75-80% basal medium, 18-22% serum substitute or fetal bovine serum, 0.05-0.15% β-mercaptoethanol, 0.8-1.2% L-glutamine, 0.8-1.2% non-essential amino acids, and 0.8-1.2% antibiotic solution.

[0020] Furthermore, the basal culture medium includes DMEM high-glucose medium;

[0021] The serum alternatives include Knockout™ SR

[0022] The non-essential amino acids include Gibco™ MEM non-essential amino acids.

[0023] The dual-antibody solution includes penicillin and streptomycin.

[0024] Furthermore, the inoculation amount of embryoids is 1-2 embryoids per 0.5 mL of differentiation medium;

[0025] In step A of inducing differentiation into three germ layer cells, the differentiation medium is added on days 16-18;

[0026] The cell adhesion matrix includes any one of extracellular matrix, gelatin, Vitronectin, iMatrix511, Laminin, or PEG hydrogel.

[0027] Furthermore, the differentiation culture includes replacing the differentiation medium with a fresh one every day;

[0028] The three germ layers include the endoderm, ectoderm, and mesoderm.

[0029] Secondly, the present invention provides the application of the above-described method in detecting the three-layer differentiation potential of induced pluripotent stem cells or in preparing products for detecting the three-layer differentiation potential of induced pluripotent stem cells.

[0030] Thirdly, the present invention provides a method for detecting the differentiation potential of induced pluripotent stem cells into three germ layers. The method described above is used to obtain induced pluripotent stem cell differentiation into three germ layer cells, and the three germ layer cells are subjected to immunofluorescence staining and / or detection of the expression level of three germ layer markers to analyze the differentiation potential of induced pluripotent stem cells into three germ layers.

[0031] Fourthly, the present invention provides a kit for detecting the differentiation potential of induced pluripotent stem cells into three germ layers, comprising the above-mentioned culture medium containing 5 μM~20 μM E-cadherin activator 1, differentiation culture medium, and a container coated with cell adhesion matrix.

[0032] This invention provides a method for promoting the differentiation of induced pluripotent stem cells (iPSCs) into the three germ layers. Adding E-cadherin activator 1 to the culture medium maintains iPSC cell viability and enhances the ability to form embryoid bodies. During shaking culture, it promotes the formation of uniform, compact, and smooth-edged embryoid bodies, increasing their number and structural stability. This improves the adhesion rate of embryoid bodies after seeding into containers coated with cell adhesion matrix, as well as their ability to migrate and differentiate after adhesion. It also increases the success rate of immunofluorescence staining for iPSC differentiation into the endoderm. This method solves the technical problems of existing methods that induce iPSC differentiation into the three germ layers via the embryoid body pathway, such as poor embryoid body formation, poor embryoid body condition, poor long-term maintenance, and poor adhesion after transfer to a culture plate, leading to poor endoderm differentiation and migration. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is an example diagram of the inoculation of embryoids provided in Embodiment 1 of the present invention into a Matrigel-pre-coated 24-well plate;

[0035] Figure 2 This is an observation diagram of aggregates during the differentiation of the three germ layers provided in Experiment 1 of the present invention;

[0036] Figure 3 This is a statistical chart of the embryoid body adhesion rate during the three-layer differentiation process provided in Experiment 2 of the present invention;

[0037] Figure 4 This is an observation image of a single embryoid body during the differentiation of the three germ layers provided in Experiment 2 of the present invention;

[0038] Figure 5 This is an observation image of immunofluorescence staining of three germ layer cells provided in Example 2 of the present invention. Detailed Implementation

[0039] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0040] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] This invention provides a method for promoting the differentiation of induced pluripotent stem cells into the three germ layers, comprising the following steps:

[0043] A. Induced pluripotent stem cells were seeded in a culture medium containing 5 μM~20 μM E-cadherin activator 1, and cultured with shaking to form embryoid bodies. Differentiation culture medium was added for differentiation culture to obtain embryoid bodies with activated differentiation potential of the three germ layers.

[0044] B. The embryoids with activated trilaminar differentiation potential are seeded into a container coated with cell adhesion matrix, and differentiation culture medium is added to continue differentiation culture to induce differentiation into trilaminar cells.

[0045] Adding E-cadherin activator 1 to the culture medium can maintain iPSC cell viability and improve embryoid formation ability. During shaking culture, it promotes the formation of uniform, compact, and smooth-edged embryoids, increasing the number of embryoids. It also enhances structural stability, improves the adhesion rate of embryoids after seeding into containers coated with cell adhesion matrix, and improves the crawling and differentiation ability of embryoids after adhesion. This also improves the success rate of immunofluorescence staining for iPSC differentiation into endoderm. It solves the technical problems of poor embryoid formation ability, poor embryoid state, poor long-term maintenance ability, and poor adhesion ability of embryoids after transfer to the plate, which leads to poor endoderm differentiation and crawling ability in the existing method of inducing iPS to differentiate into three germ layers through the embryoid pathway.

[0046] In some specific embodiments, the concentration of E-cadherin activator 1 in the culture medium is 10 μM;

[0047] In some specific embodiments, the seeding amount of the induced pluripotent stem cells is (0.1~10) × 10 per 2 mL of culture medium. 6 Cells. Ensuring sufficient intercellular interactions to initiate aggregate formation while preventing excessive density from leading to local nutrient depletion or the formation of a necrotic core.

[0048] To achieve the optimal dynamic suspension environment, in some specific embodiments, the rotation speed of the oscillation culture is 50-80 rpm; in some specific embodiments, the oscillation culture time is 16-18 hours.

[0049] In some specific embodiments, the embryoids with activated trilaminar differentiation potential are obtained 5-8 days after the addition of differentiation medium. After suspension induction culture (Day 5 to Day 8), the embryoids form an activated state with further differentiation potential.

[0050] In some specific embodiments, the differentiation medium comprises the following components by volume percentage: 75-80% basal medium, 18-22% serum substitute or fetal bovine serum, 0.05-0.15% β-mercaptoethanol, 0.8-1.2% L-glutamine, 0.8-1.2% non-essential amino acids, and 0.8-1.2% antibiotic solution.

[0051] In some specific embodiments, the basal culture medium includes DMEM high glucose medium; the serum substitute is Knockout™ SR; the non-essential amino acids are Gibco™ MEM non-essential amino acids; and the dual antibiotic solution includes penicillin and streptomycin.

[0052] In some specific implementations, the inoculum size is 1-2 embryoids per 0.5 mL of differentiation medium. Single-clonal independent culture and observation facilitate subsequent image acquisition and statistical analysis, and avoid signal confusion caused by overlapping of multiple embryoids.

[0053] In some specific embodiments, the differentiation culture medium is added on days 16-18 after step A of inducing differentiation into three germ layer cells. By days 16-18 of differentiation, the cells of each germ layer have completed migration, expansion, and expression of functional proteins.

[0054] In some specific embodiments, the cell adhesion matrix includes any one of extracellular matrix, gelatin, Vitronectin, iMatrix511, Laminin, or PEG hydrogel.

[0055] In some specific implementations, a serum substitute is used in the differentiation medium, and the cell adhesion matrix is ​​an extracellular matrix (Matrigel matrix). Using a serum substitute instead of fetal bovine serum, and seeding the embryoids onto the Matrigel matrix on the seventh day of embryoid differentiation culture, can improve the efficiency of iPS embryoid formation, adhesion, and trilaminar differentiation.

[0056] In some specific embodiments, the differentiation culture includes replacing the differentiation medium with a fresh one every day, replenishing depleted nutrients, and maintaining a stable culture environment.

[0057] In some specific embodiments, the three germ layers include the endoderm, ectoderm, and mesoderm.

[0058] The above-described method can significantly improve the ability of embryoid bodies to migrate and differentiate after attachment, and increase the success rate of immunofluorescence staining for iPSC differentiation into the endoderm. According to another aspect of the present invention, the above-described method is also provided for the application of detecting the three-layer differentiation potential of induced pluripotent stem cells or for preparing products for detecting the three-layer differentiation potential of induced pluripotent stem cells.

[0059] According to another aspect of the present invention, a method for detecting the differentiation potential of induced pluripotent stem cells into three germ layers is also provided. The method described above is used to obtain induced pluripotent stem cell differentiation into three germ layer cells, and the three germ layer cells are subjected to immunofluorescence staining and / or detection of the expression level of three germ layer markers to analyze the differentiation potential of induced pluripotent stem cells into three germ layers.

[0060] According to another aspect of the present invention, a kit for detecting the differentiation potential of induced pluripotent stem cells in three germ layers is also provided, comprising the above-mentioned culture medium containing 5 μM~20 μM E-cadherin activator 1, differentiation culture medium, and a container coated with a cell adhesion matrix.

[0061] The standardized kit integrates culture medium containing E-cadherin activator 1, premixed differentiation system, and coating plate, greatly simplifying the operation process.

[0062] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0063] Reagents:

[0064] The chemical structure of E-cadherin activator 1 is shown in Formula I:

[0065]

[0066] Formula I

[0067] E-cadherin activator 1: Purchased from MCE, product number: HY-164712;

[0068] ROCK inhibitor Y27632: purchased from Stemgent, catalog number: 04-0012-10

[0069] Essential 8 culture medium: purchased from Gibco, catalog number: A1517001

[0070] mTeSR1 medium: purchased from StemCell, catalog number: #85850

[0071] DEME high-glucose medium: purchased from Gibco, SH30243.01

[0072] iPSC test cells: Using umbilical cord blood as the starting material, mononuclear cells were isolated and then induced into erythroid progenitor cells. The expanded erythroid progenitor cells were then electroporated for reprogramming. An additional plasmid expressing the Yamanaka reprogramming factor was transduced into the cells using an electroporator. Single colonies were cultured in Essential 8 complete medium. After single colony selection and expansion, iPS cell preparation was completed.

[0073] Example 1: Trilayer Differentiation

[0074] 1. Resuscitation and expansion of iPSC sample cells

[0075] Take one vial of frozen iPSC cells to be tested, thaw in a 37°C water bath for 2-3 minutes, then transfer to Essential 8 complete medium and centrifuge at 300g for 5 minutes. Discard the supernatant, resuspend the cells in Essential 8 complete medium containing 10 μM E-cadherin activator 1, and count them. Count the cells at 3 × 10⁻⁶ cells / mL. 4 live cells / cm 2 The iPSCs were seeded at a density of 2 × 10⁻⁶ cells per T25 culture flask pre-coated with Vitronectin (VTN-N) and cultured. When the iPSC confluence reached approximately 60-80%, they were passaged at a rate of 2 × 10⁻⁶ cells per flask. 4 live cells / cm 2 The culture was carried out in a T75 culture flask pre-coated with Vitronectin (VTN-N).

[0076] 2. Trilayer differentiation culture

[0077] (1) Formation of embryoid bodies

[0078] iPSC cells were harvested when their confluence reached approximately 60-80%. The supernatant was discarded, and the cells were washed with 7.5 mL of 1× DPBS. Then, 7.5 mL of 0.5 mM EDTA solution was added, and the cells were incubated at 37 ℃ for 6 min. After resuspending the cells in 10 mL of mTeSR1 complete culture medium containing 10 μM cadherin activator 1, samples were taken for counting. 3.0 × 10⁶ cells were collected. 6 One live cell was seeded into one well of a 6-well ultra-low adsorption plate using mTeSR1 complete medium containing 10 μM E-cadherin activator 1, and the volume was increased to a final volume of 2 mL. The plate was placed in a shaker at 70 rpm / min in a 37°C, 5% CO2 incubator and cultured for 18 hours.

[0079] (2) Embryomorph differentiation culture

[0080] Tilt the 6-well plate to allow the iPSC cell aggregates (embryomorphs) to settle under gravity. Carefully aspirate the supernatant and add 4 mL of fresh differentiation medium to each well. Incubate statically at 37°C in a 5% CO2 incubator. Record this as Day 0. Change the medium every other day.

[0081] The differentiation medium was prepared as shown in Table 1. The non-essential amino acids were Gibco™ MEM non-essential amino acids, the serum substitute was Knockout™ SR, and the antibiotic solution included penicillin at a concentration of 100 units / mL and streptomycin at a concentration of 100 μg / mL.

[0082] Table 1

[0083]

[0084] (3) Embryomorph transfer and adherent culture

[0085] Day 7: Inoculate one embryoid into one well of a Matrigel pre-coated 24-well plate, adding 0.5 mL of differentiation medium to each well. Inoculate 12 wells in a 24-well plate, as shown in the inoculation diagram. Figure 1 As shown, gray indicates the inoculation wells. Continue differentiation culture, changing the medium every other day, replacing 1 mL of fresh differentiation medium in each well. Differentiation culture continues until Day 18, when three germ layer cells have formed.

[0086] Comparative Example 1

[0087] Unlike Example 1, the ROCK inhibitor Y27632 was used instead of E-cadherin activator 1.

[0088] Experiment 1

[0089] The formation of iPSC cell aggregates during Day 0, Day 2, Day 4, Day 6, and Day 7 of embryoid differentiation culture in Example 1 and Comparative Example 1 was observed, and the results are as follows: Figure 2 As shown.

[0090] This method, which induces iPSC differentiation into three germ layers via the embryoid pathway, involves harvesting iPS cells for trigerm layer differentiation testing. A bioreactor is used to mediate embryoid formation. The iPSCs to be tested are seeded into 6-well ultra-low adsorption plates and placed on a shaker in a 37°C, 5% CO2 incubator. The plates are then horizontally shaken at 70 rpm / min for 16–18 hours (this process is defined as shaking the plate) to mediate embryoid formation. During this process, 10 μM E-cadherin activator 1 is added to promote iPS cell adhesion and survival in suspension. Compared to adding the ROCK inhibitor Y27632, the addition of E-cadherin activator 1 significantly improves the embryoid formation ability, resulting in more iPS aggregates with tighter, rounder, and smoother edges, and better proliferation (increased embryoid diameter).

[0091] Experiment 2

[0092] The embryoids were observed during plate transfer and adherence culture in Example 1 and Comparative Example 1, and the embryoid formation on Day 9 was compared. The results are as follows: Figure 3 and Figure 4 As shown.

[0093] Data showed that using this differentiation medium induced the differentiation of the aggregates into embryoids. During this process, a serum substitute was used instead of fetal bovine serum, and key components required for differentiation were added to mediate the differentiation of iPS cells into three germ layers. After 7 days of induced differentiation, the resulting embryoids were seeded into 24-well plates coated with Matrigel. Embryoids formed with Y27632 during pelleting often exhibited problems such as poor adhesion and crawling ability after adhesion after plate transfer. Example 1 showed that adding E-cadherin activator 1 significantly improved the adhesion rate of embryoids after plate transfer, as well as their crawling and differentiation abilities after adhesion.

[0094] Example 2: Detection of differentiation potential of iPSC test cells

[0095] Immunofluorescence staining was performed on triple germ layer cells cultured to Day 18 as described in Example 1. Figure 1In the 24-well plate shown, two wells from A1 to A3 were selected for AFP staining, two wells from A4 to A6 were selected for NESTIN staining, two wells from B1 to B3 were selected for α-SMA staining, and two wells from B4 to B5 were selected for the negative control group staining. The antibody reagents for the negative control group and the experimental group were prepared as shown in Table 2.

[0096] Table 2

[0097]

[0098] Perform immunofluorescence staining according to the following steps:

[0099] 1. Cell fixation:

[0100] Discard the original culture medium and wash each well once with 500 μL of DPBS. Add 300 μL of 4% paraformaldehyde to each well and fix the cells at room temperature for 10 min. Discard the fixative and wash each well twice with 500 μL of DPBS.

[0101] 2. Cell permeability:

[0102] Add 300 μL of 0.1% Triton X-100 working solution to each well, permeate cells at room temperature for 10 min, aspirate the permeate, and wash each well twice with 500 μL of DPBS.

[0103] 3. Enclosed:

[0104] For endoderm AFP staining, add 300 μL of 1% BSA working solution to each well; for ectoderm NESTIN staining, add 300 μL of 3% BSA solution to each well; for mesoderm α-SMA staining, add 300 μL of 2% BSA solution to each well; and incubate at room temperature for 30 min.

[0105] 4. Fluorescent staining:

[0106] Primary antibody binding: Discard the blocking solution. For endoderm staining, add 300 μL of prepared AFP antibody working solution to each well and incubate at 4°C for 18 h in the dark. For ectoderm staining, add 300 μL of prepared NESTIN antibody working solution to each well and incubate at 4°C for 18 h in the dark. For mesoderm staining, add 300 μL of prepared α-SMA primary antibody working solution to each well and incubate at 4°C for 18 h in the dark.

[0107] Mesodermal α-SMA secondary antibody conjugation: Wash twice with 500 μL DPBS in each well, then add 300 μL of the prepared α-SMA secondary antibody working solution and incubate at room temperature in the dark for 1 h. Discard the secondary antibody solution and wash twice with 500 μL DPBS in each well. The endoderm AFP staining wells and ectoderm NESTIN staining wells are then placed at room temperature in the dark without any further treatment.

[0108] DAPI staining: For all experimental and control groups, wash twice with 500 μL of DPBS per well, then counterstain the cell nuclei with 300 μL of diluted DAPI solution and incubate at room temperature for 15 min. Discard the DAPI solution and wash twice with 500 μL of DPBS per well.

[0109] 5. Observation and photography using a fluorescence microscope:

[0110] Add 500 μL of DPBS (1×) containing 50% glycerol to each well, observe using an inverted fluorescence microscope, and capture images with 20x and 40x objectives.

[0111] Comparative Example 2

[0112] Unlike Example 2, three germ layer cells differentiated and cultured to Day 18 in Comparative Example 1 were selected for immunofluorescence staining.

[0113] After embryoid bodies were transferred to a plate and induced to differentiate for 18 days, morphological staining images of the three germ layers were obtained using immunofluorescence, as shown below. Figure 5 As shown, data indicates that embryoids formed by adding Y27632 during embryoid formation often exhibit problems such as failure of the endoderm to emerge, poor differentiation ability, and staining failure. Adding E-cadherin activator 1 during embryoid formation can significantly improve the emergence and differentiation ability of embryoids after adhesion, and increase the success rate of immunofluorescence staining for iPSC differentiation into the endoderm.

[0114] It should be noted that the trigerm cells induced to differentiate in Example 1 can also be used to extract RNA and use qPCR or array methods to detect the expression levels of trigerm cell markers in order to assess the differentiation potential of the iPSC cells to be tested.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for promoting the differentiation of induced pluripotent stem cells into three germ layers, characterized in that, Includes the following steps: A. Induced pluripotent stem cells were seeded in a culture medium containing 5 μM~20 μM E-cadherin activator 1, and cultured with shaking to form embryoid bodies. Differentiation culture medium was added for differentiation culture to obtain embryoid bodies with activated differentiation potential of the three germ layers. B. The embryoid bodies with activated trilaminar differentiation potential were seeded into a container coated with cell adhesion matrix, and differentiation medium was added to continue differentiation culture to induce differentiation into trilaminar cells. The chemical structure of the E-cadherin activator 1 is shown in Formula I: Formula I.

2. The method according to claim 1, characterized in that, The concentration of E-cadherin activator 1 in the culture medium was 10 μM; The seeding amount of the induced pluripotent stem cells is (0.1~10)×10 per 2 mL of culture medium. 6 cell.

3. The method according to claim 1, characterized in that, The rotation speed of the oscillation culture is 50~80 rpm; The shaking culture time is 16-18 hours.

4. The method according to claim 1, characterized in that, The time to obtain embryoids with activated trilaminar differentiation potential is 5 to 8 days after adding differentiation medium; The differentiation medium comprises the following components by volume percentage: 75-80% basal medium, 18-22% serum substitute, 0.05-0.15% β-mercaptoethanol, 0.8-1.2% L-glutamine, 0.8-1.2% non-essential amino acids, and 0.8-1.2% antibiotic solution.

5. The method according to claim 4, characterized in that, The basal culture medium includes DMEM high glucose medium; The serum alternatives include Knockout™ SR; The non-essential amino acids include Gibco™ MEM non-essential amino acids; The dual-antibody solution includes penicillin and streptomycin.

6. The method according to claim 1, characterized in that, The inoculation amount of embryoids is 1-2 embryoids per 0.5 mL of differentiation medium; In step A of inducing differentiation into three germ layer cells, the differentiation medium is added on days 16-18; The cell adhesion matrix is ​​an extracellular matrix.

7. The method according to any one of claims 1 to 6, characterized in that, The differentiation culture includes changing the differentiation medium once every 1 day; The three germ layers include the endoderm, ectoderm, and mesoderm.

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