Method for preparing mesoderm progenitor cells from human pluripotent stem cells and induced differentiation culture medium of mesoderm progenitor cells

Abstract

The invention relates to the field of cell biology, and particularly discloses a method for preparing mesoderm progenitor cells from human pluripotent stem cells and an induced differentiation culture medium of the mesoderm progenitor cells. The method comprises the following steps: inoculating human pluripotent stem cells in a single cell state into a culture dish covered with matrigel; and inducing the human pluripotent stem cells to be differentiated into the mesoderm progenitor cells within 1-3 days by using the mesoderm progenitor cell induced differentiation culture medium. Wherein the mesoderm progenitor cell induced differentiation culture medium is composed of a basic culture medium and an additive, and the additive contains a B-27 additive, non-essential amino acid, glutamine, ascorbic acid, polyvinyl alcohol, a GSK-3 inhibitor and a CDK8 inhibitor. According to the method, the human pluripotent stem cells are subjected to induced differentiation by using the mesoderm progenitor cell induced differentiation culture medium with specific composition, the method does not depend on cloning of the pluripotent stem cells, a large number of high-uniformity mesoderm progenitor cells can be obtained through induced differentiation within 1-3 days, the induction efficiency is high, and the stability is high.

Description

Technical Field

[0001] The present application relates to the field of cell biology, and more specifically, to a method for preparing mesodermal progenitor cells from human pluripotent stem cells and an induction differentiation culture medium thereof. Background Art

[0002] Mesodermal progenitor cells (MPCs) refer to a type of cells with specific differentiation potential in the mesoderm that differentiates between the ectoderm and the endoderm during embryonic development. These cells have been designated as specific mesoderm lineages, such as extraembryonic, lateral, intermediate, paraaxial and notochordal mesoderm, in the early stages of embryonic development, and further differentiate into a variety of cell types, such as muscle cells, bone cells, blood and lymphatic system cells, etc. in the subsequent development process. The characteristic genes of mesodermal progenitor cells mainly include TBXT (also known as Brachyury), MIXL1, etc. Mesodermal progenitor cells are widely used in tissue regeneration and repair. Derivatives of mesodermal progenitor cells, such as endothelial cells, muscle cells, hematopoietic cells, etc., can be transplanted into recipients to promote the recovery of damaged muscles, bones or blood systems. In drug development, mesodermal progenitor cells and their derivatives can help discover new drug targets, evaluate drug effects and safety, and optimize drug design by simulating disease environments.

[0003] The acquisition of primary mesodermal progenitor cells faces the problem of limited donor materials. Human pluripotent stem cells, including human embryonic stem cells and human induced pluripotent stem cells, have strong self-renewal and unlimited proliferation capabilities, and can be induced to differentiate into mesodermal progenitor cells under specific conditions, providing a large number of cell sources for the application of mesodermal progenitor cells.

[0004] There are currently various methods for inducing the differentiation of human pluripotent stem cells into mesodermal progenitor cells, including the embryoid body method and the monolayer cell induction method. The embryoid body method produces a multilayer structure containing multiple cell types such as mesodermal progenitor cells by suspending and culturing human pluripotent stem cells to self-organize into a three-dimensional cell aggregate (embryoid body); the limitations of this method are that the large variability in the embryoid body formation process will affect the stability and consistency of the yield of mesodermal progenitor cells, and the dense aggregation state of embryoid body cells poses a challenge to the real-time monitoring of cell morphology, affecting the fine control and evaluation of the differentiation process. The monolayer cell induction method is to inoculate human pluripotent stem cells in matrigel or vitronectin, and then induce them to differentiate into mesodermal progenitor cells through an induction medium containing small molecule compounds or protein factors; the advantages of this method are that the chemical composition of the induction medium is clear, and it is simple to operate, easy to control, and does not require the help of a complex culture system, but the uniformity and stability of the induction are relatively low, which is reflected in the large differences in the expression of the mesodermal progenitor cell-specific gene TBXT.

[0005] In view of this, there is a need to improve the method for preparing mesodermal progenitor cells by inducing differentiation of human pluripotent stem cells, so as to increase the induction efficiency and uniformity of mesodermal progenitor cells. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems of low induction efficiency and uniformity in the prior art of preparing mesodermal progenitor cells by inducing differentiation of human pluripotent stem cells, and to provide a method for preparing mesodermal progenitor cells from human pluripotent stem cells and an induction differentiation medium thereof.

[0007] This application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for preparing mesodermal progenitor cells from human pluripotent stem cells, comprising the following steps:

[0009] The human pluripotent stem cells in a single-cell state are seeded in a culture dish coated with matrix gel;

[0010] Inducing human pluripotent stem cells to differentiate into mesodermal progenitor cells within 1-3 days using a mesodermal progenitor cell induction differentiation medium;

[0011] Among them, the mesodermal progenitor cell induction differentiation medium consists of a basal medium and an additive, and the additive contains a CDK8 inhibitor.

[0012] Furthermore, the CDK8 inhibitor is any one of BI-1347, AS2863619 and MSC2530818.

[0013] Furthermore, the additive consists of the following components: B-27 additive, non-essential amino acids, glutamine, ascorbic acid, polyvinyl alcohol, GSK-3 inhibitor, and CDK8 inhibitor.

[0014] Preferably, the above-mentioned additives are composed of the following components:

[0015] B-27 supplement 1-4%, non-essential amino acids 0.5-2%, glutamine 1-5 mM, ascorbic acid 25-75 μM, polyvinyl alcohol 0.02%-0.18%, GSK-3 inhibitor 2-8 μM, and CDK8 inhibitor 1-4 μM.

[0016] Preferably, the GSK-3 inhibitor is CHIR99021, and the B-27 additive is a B-27 additive without vitamin A.

[0017] More preferably, the above-mentioned additives are composed of the following components:

[0018] B-27 supplement (minus vitamin A) (2%), non-essential amino acids (1%), glutamine (2 mM), ascorbic acid (50 μM), polyvinyl alcohol (0.06%), GSK-3 inhibitor (6 μM) and CDK8 inhibitor (2 μM).

[0019] Preferably, the above-mentioned basal culture medium is any one of RPMI1640, DMEM, IMDM and DMEM / F12.

[0020] Preferably, the process of inoculating human pluripotent stem cells into a culture dish covered with matrix gel comprises:

[0021] Human pluripotent stem cells were dissociated into single cells, resuspended in a human pluripotent stem cell culture medium containing 5-10 μM ROCK inhibitor, and then seeded into a culture dish covered with matrigel and cultured at 35-40° C. and 3-7% CO 2 .

[0022] In a second aspect, the present application provides a mesodermal progenitor cell differentiation induction medium, which is composed of a basal medium and additives;

[0023] The basal culture medium was any one of RPMI1640, DMEM, IMDM and DMEM / F12;

[0024] The additive consists of the following components: B-27 additive 1-4%, non-essential amino acid 0.5-2%, glutamine 1-5mM, ascorbic acid 25-75μM, polyvinyl alcohol 0.02%-0.18%, GSK-3 inhibitor 2-8μM, and CDK8 inhibitor 1-4μM.

[0025] In a third aspect, the present application provides a kit for preparing mesodermal progenitor cells, which comprises the above-mentioned mesodermal progenitor cell induction differentiation medium.

[0026] In a fourth aspect, the present application provides a mesodermal progenitor cell obtained by the method of any one of claims 1-7.

[0027] In summary, this application has the following beneficial effects:

[0028] The method for preparing mesodermal progenitor cells from human pluripotent stem cells provided in the present application uses a mesodermal progenitor cell induction differentiation medium of a specific composition to induce differentiation of human pluripotent stem cells in a single-cell state, does not rely on the cloning of pluripotent stem cells, can induce differentiation within 1-3 days to obtain a large number of highly uniform mesodermal progenitor cells, and has high induction efficiency and strong stability.

[0029] The present application also provides a mesodermal progenitor cell induction differentiation medium, which regulates the induction differentiation of mesodermal progenitor cells by adding a variety of cell culture additives and cell signaling pathway inhibitors. Among them, the CDK8 inhibitor is the most special. Experiments show that the mesodermal progenitor cell induction differentiation medium containing CDK8 inhibitors can significantly increase the expression values ​​of mesodermal progenitor cell-specific genes TBXT, MIXL1 and TBX6 relative to the mesodermal progenitor cell induction differentiation medium without CDK8 inhibitors, thereby indicating that CDK8 inhibitors have a significant enhancing effect on the induction differentiation of mesodermal progenitor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a morphological diagram of the process of directing the induction of mesodermal progenitor cell differentiation from human pluripotent stem cells in Example 1;

[0031] Figure 2 This is an immunofluorescence analysis of Brachyury expression in mesodermal progenitor cells differentiated from Example 1;

[0032] Figure 3 RT-qPCR analysis results of mesodermal progenitor cells differentiated in Example 1 and Comparative Example 1;

[0033] Figure 4 This is a graph showing the results of flow cytometry analysis of the mesodermal progenitor cells differentiated in Example 1 and Comparative Example 1 using anti-Brachyury antibodies. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0035] The technical solution of the present invention is:

[0036] The present embodiment provides a method for preparing mesodermal progenitor cells from human pluripotent stem cells, which comprises the following steps:

[0037] S1, seeding: single-cell human pluripotent stem cells are seeded in a culture dish coated with matrix gel.

[0038] Wherein, "human pluripotent stem cells" include human embryonic stem cells or human induced pluripotent stem cells. Human embryonic stem cells are commercially available human embryonic stem cells, preferably cells of any of the following NIH numbered cell lines: BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ​​ES06, TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14.

[0039] The method for dissociating human pluripotent stem cells into a single cell state is: incubating human pluripotent stem cells with a cell digestion solution at 35-40° C. and 3-7% CO2 for 3-5 minutes to obtain human pluripotent stem cells in a single cell state. Preferably, the cell digestion solution is any one of Accutase, EDTA and ReLeSR; more preferably, Accutase is selected as the cell digestion solution.

[0040] The process of inoculating human pluripotent stem cells in a culture dish coated with matrix gel includes: centrifuging the human pluripotent stem cells in a single cell state to remove the supernatant, adding them to a human pluripotent stem cell culture medium containing 5-10 μM ROCK inhibitor to resuspend them, and then inoculating them in a culture dish coated with matrix gel (1:50-1:100 dilution), and culturing them at 35-40°C and 3-7% CO2.

[0041] Preferably, the human pluripotent stem cell culture medium is any one of Essential 8, TeSR and PGM1, preferably Essential 8.

[0042] ROCK inhibitor refers to a substance that inhibits Rho kinase (ROCK), and may be a substance that inhibits either ROCK I or ROCK II. There is no particular limitation on the ROCK inhibitor as long as it has the above-mentioned function. Preferably, the ROCK inhibitor is Y-27632, and the chemical formula of its dihydrochloride is C 14 H 21 N3O.2HCl, molecular weight is 320.26.

[0043] S2, inducing differentiation: using a mesodermal progenitor cell induction differentiation medium to induce the human pluripotent stem cells to differentiate into mesodermal progenitor cells within 1-3 days.

[0044] More specifically, step S2 is: preparing a mesodermal progenitor cell induction differentiation medium, switching the human pluripotent stem cell culture medium to a mesodermal progenitor cell induction differentiation medium, and culturing at 35-40° C. and 3-7% CO 2 for 1-3 days.

[0045] Among them, the mesoderm progenitor cell induction differentiation medium consists of two parts:

[0046] (I) Basic culture medium:

[0047] There are four options for the basic culture medium, namely, RPMI1640 culture medium, DMEM culture medium, IMDM or DMEM / F12. It can be any culture medium, or a culture medium formed by mixing two different culture media in a certain mass ratio, for example, a mixture of 1:0.5-1.5.

[0048] (ii) Additives:

[0049] The additive consists of the following components: B-27 additive, non-essential amino acids, glutamine, ascorbic acid, polyvinyl alcohol, GSK-3 inhibitor, and the CDK8 inhibitor.

[0050] Among them, B-27 additive is a cell culture additive, which is an optimized serum-free additive. After adding B-27 additive to the mesodermal progenitor cell induction differentiation medium, it provides nutrients such as lipids and proteins necessary for cell survival. Preferably, the B-27 additive can be selected from the B-27 additive without vitamin A, or the ordinary B-27 additive can be selected.

[0051] Non-essential amino acids are a mixture of non-essential amino acids that can be directly used in cell culture experiments, including, for example, L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine. Glutamine and non-essential amino acids are added to the mesodermal progenitor cell differentiation induction medium to provide the cells with the necessary amino acid support to meet their growth and differentiation needs.

[0052] Ascorbic acid, L-ascorbic acid, L-ascorbic acid phosphate or other ascorbic acid derivatives. Adding an appropriate amount of ascorbic acid to the mesodermal progenitor cell induction differentiation medium can promote the proliferation and differentiation of human pluripotent stem cells, significantly increase the in vitro proliferation multiple of human pluripotent stem cells, and affect their differentiation direction.

[0053] Polyvinyl alcohol: Adding an appropriate amount of polyvinyl alcohol to the mesoderm progenitor cell differentiation medium can serve as a slow-release pool for signal molecules (such as small molecules, proteins, etc.), maintain the stability of the concentration of signal molecules, and enhance the stability of cell differentiation.

[0054] GSK-3 inhibitors promote the expression of mesoderm characteristic genes by upregulating the Wnt signaling pathway, providing an effective regulatory means for the induction and differentiation of mesoderm progenitor cells. By controlling its concentration and culture conditions, a better differentiation effect can be achieved. Preferably, the GSK-3 inhibitor is CHIR99021, SB216763 or CHIR-98014; preferably, the GSK-3 inhibitor is CHIR99021.

[0055] CDK8 inhibitor. The applicant has found that adding CDK8 inhibitor to the mesodermal progenitor cell induction differentiation medium can significantly increase the proportion of cells expressing the specific gene Brachyury, and improve the uniformity and purity of mesodermal progenitor cells. The CDK8 inhibitor can be any one of BI-1347, AS2863619 and MSC2530818; preferably, the CDK8 inhibitor is BI-1347. The content of CDK8 inhibitor is 1 to 4 μM; more preferably, the content of CDK8 inhibitor is 1.5 to 3 μM; more preferably, the content of CDK8 inhibitor is 2 μM.

[0056] Preferably, the additive consists of the following components:

[0057] B-27 supplement (without vitamin A) 1-4%, non-essential amino acids 0.5-2%, glutamine 1-5mM, ascorbic acid 25-75μM, polyvinyl alcohol 0.02%-0.18%, GSK-3 inhibitor 2-8μM, and CDK8 inhibitor 1-4μM.

[0058] Further preferably, the additive consists of the following components:

[0059] B-27 supplement (without vitamin A) 2-3%, non-essential amino acids 0.8-1.5%, glutamine 1.5-3mM, ascorbic acid 35-55μM, polyvinyl alcohol 0.05%-0.11%, GSK-3 inhibitor 3-6μM, and CDK8 inhibitor 1.5-3μM.

[0060] More preferably, the additive consists of the following components:

[0061] B-27 supplement (without vitamin A) 2%, non-essential amino acids 1%, glutamine 2mM, ascorbic acid 50μM, polyvinyl alcohol 0.06%, GSK-3 inhibitor 6μM, and CDK8 inhibitor 2μM.

[0062] It should be noted that the content of each component of the above additives refers to the concentration in the neural stem cell induction differentiation medium.

[0063] Some of the reagents and sources used in this embodiment are shown in Table 1:

[0064] Table 1.

[0065] Reagents brand Part Number Matrigel Corning 354277 DMEM / F-12 1:1 HyClone SH30023.01 PGM1 Pluripotent Stem Cell Medium (with supplements) Saibei Biotechnology CA1007500 Y27632 Pottery T1870 PBS (Without Ca2+ / Mg2+) SPERIKON SP002040500 50 x B-27 Supplement (minus vitamin A) gibco 12587-010 100× Glutamine gibco 35050-61 Nonessential amino acids Solarbio N1250 CHIR99021 Pottery T2301 Polyvinyl alcohol Sigma 900289-5 Accutase Thermo 00-4555-56 Ascorbic acid-2-phosphate Sigma 49752-10G BI-1347 MCE HY-120350

[0066] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0067] Example 1

[0068] This embodiment provides a method for preparing mesodermal progenitor cells from human pluripotent stem cells, comprising:

[0069] (1) Human pluripotent stem cells (H9 cells) were incubated with Accutase digestion solution at 37°C and 5% CO2 for 4 minutes to dissociate into single cells; after centrifugation to remove the supernatant, human pluripotent stem cell culture medium PGM1 containing 10 μM ROCK inhibitor (Y-27632) was added and resuspended.

[0070] (2) Single-cell human pluripotent stem cells were seeded in a 12-well plate covered with Matrigel (1:100 dilution) at a cell number of 30,000 cells / well.

[0071] (3) Add 1 mL of human pluripotent stem cell culture medium (PGM1 + 10 μM Y27632) to each well and culture at 37° C., 5% CO 2 for 1 day.

[0072] (4) Begin inducing mesodermal progenitor cell differentiation: aspirate the consumed medium, add 1 mL of pre-warmed mesodermal progenitor cell differentiation induction medium to each well, and culture at 37° C., 5% CO 2 for 2 days.

[0073] (5) Aspirate the culture medium, wash the residual culture medium with phosphate buffered saline (PBS), add Accutase, digest at 37°C for 5 minutes, centrifuge, and harvest the mesodermal progenitor cells.

[0074] In the above step (4), the formula of the mesodermal progenitor cell differentiation induction medium is as follows:

[0075] RPMI1640 medium, 1% non-essential amino acids, 2% B27 (without vitamin A), 2 mM glutamine, 50 ng / mL L-ascorbic acid phosphate, 0.06% polyvinyl alcohol, 5 μM GSK-3 inhibitor, 2 μM CDK8 inhibitor.

[0076] Among them, the GSK-3 inhibitor is CHIR99021; the CDK8 inhibitor is BI-1347.

[0077] Figure 1 The cell morphology at different stages during the directed induction differentiation of human embryonic stem cells (H9) is shown in the figure. As can be seen from the figure, the cell morphology rapidly changes from the pluripotent stem cell morphology to the mesodermal progenitor cell morphology. Scattered mesodermal progenitor cells can be seen on the second day.

[0078] Example 2

[0079] The difference between this embodiment and embodiment 1 is that the formula of the mesodermal progenitor cell induction differentiation medium is:

[0080] RPMI1640 medium, 0.5% non-essential amino acids, 1% B27 (without vitamin A), 5 mM glutamine, 25 ng / mL L-ascorbic acid phosphate, 0.18% polyvinyl alcohol, 2 μM GSK-3 inhibitor, 1 μM CDK8 inhibitor.

[0081] Among them, the GSK-3 inhibitor is CHIR99021; the CDK8 inhibitor is BI-1347.

[0082] Example 3

[0083] The difference between this embodiment and embodiment 1 is that the formula of the mesodermal progenitor cell induction differentiation medium is:

[0084] IMDM medium, 2% non-essential amino acids, 4% B27 (without vitamin A), 1 mM glutamine, 75 ng / mL L-ascorbyl phosphate, 0.02% polyvinyl alcohol, 8 μM GSK-3 inhibitor, 4 μM CDK8 inhibitor.

[0085] Example 4

[0086] The difference between this embodiment and embodiment 1 is that the formula of the mesodermal progenitor cell induction differentiation medium is:

[0087] DMEM / F12 medium, 0.8% non-essential amino acids, 2% B27 (without vitamin A), 1.5 mM glutamine, 55 ng / mL L-ascorbic acid, 0.05% polyvinyl alcohol, 6 μM GSK-3 inhibitor, 3 μM CDK8 inhibitor.

[0088] Among them, the GSK-3 inhibitor is CHIR-98014; the CDK8 inhibitor is BI-1347.

[0089] Example 5

[0090] The difference between this embodiment and embodiment 1 is that the formula of the mesodermal progenitor cell induction differentiation medium is:

[0091] DMEM medium, 1.5% non-essential amino acids, 3% B27 (without vitamin A), 3 mM glutamine, 35 ng / mL L-ascorbic acid, 0.11% polyvinyl alcohol, 3 μM GSK-3 inhibitor, 1.5 μM CDK8 inhibitor.

[0092] Among them, the GSK-3 inhibitor is SB216763; the CDK8 inhibitor is BI-1347.

[0093] Example 6

[0094] The difference between this embodiment and embodiment 1 is that:

[0095] The CDK8 inhibitor in the mesodermal progenitor cell differentiation induction medium is AS2863619, and the concentration is 2 μM.

[0096] Example 7

[0097] The difference between this embodiment and embodiment 1 is that:

[0098] The CDK8 inhibitor in the mesodermal progenitor cell differentiation induction medium is MSC2530818, and the concentration is 2 μM.

[0099] Example 8

[0100] The difference between this embodiment and embodiment 1 is that step S1:

[0101] (1) Human pluripotent stem cells (TE03 cells) were incubated with EDTA digestion solution at 37°C and 5% CO2 for 5 min to dissociate into single cells; after centrifugation to remove the supernatant, human pluripotent stem cell culture medium Essential 8 containing 8 μM ROCK inhibitor (Y-27632) was added to resuspend the cells.

[0102] (2) Single-cell human pluripotent stem cells were seeded in a 12-well plate covered with Matrigel (1:80 dilution) at a cell number of 30,000 cells / well.

[0103] (3) Add 1 mL of human pluripotent stem cell culture medium (PGM1 + 10 μM Y27632) to each well and culture at 37° C., 5% CO 2 for 1 day.

[0104] Example 9

[0105] The difference between this embodiment and embodiment 1 is that step S1, inoculation:

[0106] (1) Human pluripotent stem cells (BG01 cells) were incubated with ReLeSR digestion solution at 37°C and 5% CO2 for 3 min to dissociate into single cells; after centrifugation to remove the supernatant, human pluripotent stem cell culture medium TeSR containing 6 μM ROCK inhibitor (Y-27632) was added to resuspend the cells.

[0107] (2) Single-cell human pluripotent stem cells were seeded in a 12-well plate covered with Matrigel (1:50 dilution) at a cell number of 30,000 cells / well.

[0108] (3) Add 1 mL of human pluripotent stem cell culture medium (TeSR+6 μM Y27632) to each well and culture at 37°C and 5% CO2 for 1 day.

[0109] Example 10

[0110] The difference between this embodiment and embodiment 1 is that step S2, inducing differentiation:

[0111] (4) Begin inducing mesodermal progenitor cell differentiation: aspirate the consumed medium, add 1 mL of pre-warmed mesodermal progenitor cell differentiation induction medium to each well, and culture at 37° C., 5% CO 2 for 1 day.

[0112] (5) Aspirate the culture medium, wash the residual culture medium with phosphate buffered saline (PBS), add Accutase, digest at 37°C for 5 minutes, centrifuge, and harvest the mesodermal progenitor cells.

[0113] Embodiment 11

[0114] The difference between this embodiment and embodiment 1 is that step S2, inducing differentiation:

[0115] (4) Begin inducing mesodermal progenitor cell differentiation: aspirate the consumed medium, add 1 mL of pre-warmed mesodermal progenitor cell differentiation induction medium to each well, and culture at 37° C., 5% CO 2 for 3 days.

[0116] (5) Aspirate the culture medium, wash the residual culture medium with phosphate buffered saline (PBS), add Accutase, digest at 37°C for 5 minutes, centrifuge, and harvest the mesodermal progenitor cells.

[0117] Comparative Example 1

[0118] The difference between this comparative example and Example 1 is that in step S2, during differentiation induction, the mesodermal progenitor cell differentiation induction medium does not contain a CDK8 inhibitor.

[0119] Performance testing

[0120] The mesodermal progenitor cells collected from Example 1 (the culture medium contains a CDK8 inhibitor, named MPC with BI-1347) and Comparative Example 1 (the culture medium does not contain a CDK8 inhibitor, named MPC w / o BI-1347) were used as experimental objects to compare the differences in the expression of marker genes of neural stem cells obtained under the above two groups of conditions:

[0121] 1. Immunofluorescence detection

[0122] 1. Experimental process:

[0123] (1) Fixation: The culture medium in the culture plate (provided in Example 1) obtained after differentiation induction was aspirated, washed once with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 min.

[0124] (2) Permeabilization: Aspirate the fixative and add PBS containing 0.1% Triton-X at room temperature for 15 to 20 minutes.

[0125] (3) Blocking: Aspirate the permeabilization solution and add PBS containing 3% bovine serum albumin (BSA) at room temperature for 30 minutes.

[0126] (4) Primary antibody incubation: dilute Brachyury (CST, catalog number: 81694S, species: Rabbit) 1:200 with 3% BSA in PBS and incubate at 4°C overnight (12 h-16 h).

[0127] (5) Secondary antibody incubation: remove the primary antibody, wash with PBS three times, 5 min / time, add 3% BSA and 1:500 dilution of donkey anti-rabbit IgG H&L (Alexa Fluor 594)(abcam, catalog number: ab150076) in PBS, protect from light, incubate at room temperature for 1 hour, remove the secondary antibody by aspiration, and wash with PBS three times, 5 min each time.

[0128] (6) DAPI staining of cell nuclei.

[0129] 2. Experimental results:

[0130] Immunofluorescence test results Figure 2 As shown in Table 2:

[0131] Table 2.

[0132]

[0133]

[0134] Depend on Figure 2As can be seen from Table 2, the results of immunofluorescence detection showed that NSC with BI-1347 expressed the mesodermal progenitor cell-specific gene Brachyury (TBXT), and the expression ratio and fluorescence intensity of the specific gene Brachyury in NSC with BI-1347 were significantly higher than those in NSC w / o BI-1347, indicating that the mesodermal progenitor cells obtained by induced differentiation in a culture medium containing a CDK8 inhibitor had higher uniformity and purity.

[0135] 2. RT-qPCR detection:

[0136] 1. Experimental process:

[0137] The cells were collected and RNA was extracted using RNA isolater Total RNA Extraction Reagent (Novozyme, Catalog No.: R401-01). Reverse transcription kit (Novozyme, Catalog No.: R412-02) was used for cDNA synthesis. ChamQ Universal SYBR qPCR Master Mix (Novozyme, Catalog No.: Q711-02) was used as the reaction reagent. A 20 μl reaction system was prepared according to the instructions. Raw data collection and quality inspection were completed on the BIO-RAD CFX Connect "Real-Time PCR Detection System (Bio-Rad, Catalog No.: 1855201) instrument.

[0138] The specific procedure of RT-qPCR is as follows: first, 95℃, 30 seconds; then 95℃, 10 seconds; 60℃, 30 seconds; 40 cycles of reaction; and finally the dissolution procedure. The expression values ​​of different gene mRNAs are normalized to the GAPDH gene. The primer information used is shown in Table 3:

[0139] Table 3.

[0140]

[0141]

[0142] 2. Experimental results

[0143] The results are as follows Figure 3As shown, H9, MPC w / o BI-1347 and MPC with BI-1347 indicate the human pluripotent stem cell line H9, mesodermal progenitor cells induced by mesodermal progenitor cell medium without CDK8 inhibitor BI-1347, and mesodermal progenitor cells induced by complete mesodermal progenitor cell medium containing CDK8 inhibitor BI-1347, respectively. Although the expression of mesodermal progenitor cells specific genes of the two types of conditional inductions is significantly upregulated relative to that of H9, the expression values ​​of mesodermal progenitor cell specific genes TBXT, MIXL1 and TBX6 of the conditional induction of mesodermal progenitor cells (MPC with BI-1347) containing CDK8 inhibitors are significantly higher than those of the conditional induction of mesodermal progenitor cells (MPC w / o BI-1347) without CDK8 inhibitors. This shows that the addition of CDK8 inhibitors to the mesodermal progenitor cell induction differentiation medium can effectively enhance the induction and differentiation of mesodermal progenitor cells, which is unpredictable.

[0144] 3. Flow Cytometry Analysis

[0145] 1. Experimental process:

[0146] (1) Fixation / membrane permeability

[0147] Cells were digested with accutase for 5 min and centrifuged at 200 × g to prepare single cells. TM Transcription Factor Buffer Set Kit Fixation / Permeabilization (BD Pharmingen TM , item number: 562574).

[0148] (2) Primary antibody incubation

[0149] Dilute the anti-Brachyury antibody (CST, Catalog No.: 81694S, Species: Rabbit) 1:1000 in PBS buffer containing 0.5% BSA, add 100 μL to each tube of cells, incubate at room temperature for 1 hour; add 1 volume of PBS, centrifuge at 200×g, wash twice with PBS, and discard the supernatant.

[0150] (3) Secondary antibody incubation

[0151] Resuspend the cells in 100 μL of 1:2000 dilution of donkey anti-rabbit IgG H&L (Alexa Fluor 647)(abcam, catalog number: ab150075), incubate at room temperature in the dark for 30 min; add 1 volume of PBS, centrifuge at 200×g, wash twice with PBS, and discard the supernatant.

[0152] (4) Resuspend the cells in 200-500 μL PBS and analyze on a flow cytometer.

[0153] 2. Experimental results:

[0154] The results are as follows Figure 4 As shown, MPC w / o BI-1347 and MPC with BI-1347 indicate mesodermal progenitor cells induced by mesodermal progenitor cell medium without CDK8 inhibitor BI-1347 and mesodermal progenitor cells induced by complete mesodermal progenitor cell medium containing CDK8 inhibitor BI-1347, respectively. Consistent with the RT-qPCR results, the flow cytometry analysis results showed that the expression intensity of Brachyury (TBXT) in mesodermal progenitor cells induced by conditions containing CDK8 inhibitors (MPC with BI-1347) was significantly higher than that in mesodermal progenitor cells induced by conditions without CDK8 inhibitors (MPC w / o BI-1347). The above results indicate that the CDK8 inhibitor BI-1347 is an essential component for enhancing the induction effect of mesodermal progenitor cells.

[0155] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A method for preparing mesodermal progenitor cells from human pluripotent stem cells, characterized in that: The following steps are involved: The human pluripotent stem cells in a single-cell state are seeded in a culture dish coated with matrix gel; Inducing the human pluripotent stem cells to differentiate into mesodermal progenitor cells within 1-3 days using a mesodermal progenitor cell induction differentiation medium; Wherein, the mesodermal progenitor cell induction differentiation medium consists of a basal medium and an additive, and the additive contains a CDK8 inhibitor.

2. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 1, characterized in that: The CDK8 inhibitor is any one of BI-1347, AS2863619 and MSC2530818.

3. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 1 or 2, characterized in that: The additive consists of the following components: B-27 additive, non-essential amino acids, glutamine, ascorbic acid, polyvinyl alcohol, GSK-3 inhibitor, and the CDK8 inhibitor.

4. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 3, characterized in that: The additives are composed of the following components: composition: B-27 additive 1-4%, non-essential amino acids 0.5-2%, glutamine 1-5mM, ascorbic acid 25-75μM, polyvinyl alcohol 0.02%-0.18%, GSK-3 inhibitor 2-8μM, and the CDK8 inhibitor 1-4μM.

5. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 3, characterized in that: The GSK-3 inhibitor is CHIR99021, and the B-27 additive is a B-27 additive with vitamin A removed.

6. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 1, characterized in that: The basic culture medium is any one of RPMI1640, DMEM, IMDM and DMEM / F12.

7. The method for preparing mesodermal progenitor cells from human pluripotent stem cells according to claim 1, characterized in that: The process of inoculating the human pluripotent stem cells into a culture dish covered with matrix gel comprises: The human pluripotent stem cells are dissociated into single cells, resuspended in a human pluripotent stem cell culture medium containing 5-10 μM ROCK inhibitor, and then inoculated into a culture dish covered with matrix gel and cultured at 35-40° C. and 3-7% CO 2 .

8. A mesodermal progenitor cell differentiation induction medium, characterized in that: It consists of a basal medium and additives; The basal culture medium is any one of RPMI1640, DMEM, IMDM and DMEM / F12; The additive consists of the following components: 1-4% B-27 additive, 0.5-2% non-essential amino acids, 1-5mM glutamine, 25-75μM ascorbic acid, 0.02%-0.18% polyvinyl alcohol, 2-8μM GSK-3 inhibitor, and 1-4μM CDK8 inhibitor.

9. A kit for preparing mesodermal progenitor cells, characterized in that: It comprises the mesodermal progenitor cell differentiation induction medium as claimed in claim 8.

10. A mesodermal progenitor cell, characterized in that: It is obtained by the method described in any one of claims 1 to 7.

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