Method for promoting differentiation of human pluripotent stem cells to hematopoietic endothelial cells by regulating WNT signal and application

Abstract

The invention provides a method for promoting differentiation of human pluripotent stem cells to hematopoietic endothelial cells by regulating WNT signals and application. The inventor creatively finds that the yield of hematopoietic endothelial cells can be effectively increased by adding a Wnt signal channel inhibitor IWR1 into a hematopoietic endothelial specialized culture medium in a specific time period, and the problems that in-vitro differentiation efficiency of the hematopoietic endothelial cells is low and regulation and control difficulty is large in the prior art are solved. An efficient, controllable and stable method is provided for obtaining the hematopoietic endothelial cells in vitro.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and specifically relates to a method and application of promoting the differentiation of human pluripotent stem cells into hematopoietic endothelial cells by regulating WNT signals. Background Art

[0002] Hemogenic endothelial cells (HE) are a type of specialized endothelial cells with potential hematopoietic ability. Through the process of endothelial to hematopoietic transition (EHT), hematopoietic endothelial cells can generate hematopoietic stem and progenitor cells (HSPC). These HSPCs play a vital role in embryonic development. They not only have the ability to self-renew, but can also differentiate into various blood cell lineages, including red blood cells, white blood cells and platelets. The generation of HSPCs is closely related to the maintenance of the body's lifelong hematopoietic function. Therefore, hematopoietic endothelial cells and the process of their transformation into HSPCs have important scientific and clinical value in understanding the hematopoietic mechanism, the development of the blood system, the occurrence and treatment of hematopoietic diseases.

[0003] Although hematopoietic endothelial cells have important biological functions during embryonic development, their extremely rare number and very short development process make their study in vivo extremely difficult. In order to gain a deeper understanding of this process, scientists have begun to try to use human pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells, iPSCs) to induce differentiation into hematopoietic endothelial cells in vitro. This method provides a new platform for studying the developmental mechanism of hematopoietic endothelial cells. However, since the in vitro differentiation process is affected by multiple factors, the current differentiation efficiency is still low, and there are great difficulties in reproducing the fine spatiotemporal regulation during embryonic development. These problems not only restrict the basic research of hematopoietic endothelial cells, but also affect their potential for application in clinical treatment.

[0004] At present, the main challenges faced by the in vitro differentiation of hematopoietic endothelial cells include the following aspects: (1) Hematopoietic endothelial cells are extremely short-lived during embryonic development and are difficult to stably culture and expand in vitro; (2) Existing differentiation methods have low efficiency, resulting in a limited number of differentiated hematopoietic endothelial cells, which cannot meet scientific research and clinical needs; (3) The regulatory mechanism of in vitro differentiation is not yet fully understood, and there is a lack of strategies to accurately control the differentiation process. Therefore, how to improve the efficiency of human pluripotent stem cell differentiation of hematopoietic endothelial cells, especially how to simulate and accurately regulate the EHT process in vitro, has become a key issue that needs to be solved in this field.

[0005] Research on improving the differentiation efficiency of hematopoietic endothelial cells not only helps to deeply understand the molecular mechanism of the hematopoietic process, but also provides potential clinical applications for the treatment of hematopoietic diseases. Therefore, developing a method for efficiently, controllably and stably differentiating hematopoietic endothelial cells in vitro has important scientific research significance and clinical application value. Summary of the Invention

[0006] In order to make up for the deficiencies of the prior art, the present invention aims to solve the problems of low differentiation efficiency and great difficulty in regulation in the current technology, so as to provide an effective solution for the research and application of hematopoietic endothelial cells.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a method for preparing hematopoietic endothelial cells is provided.

[0009] Furthermore, the method includes:

[0010] 1) Providing hematopoietic mesoderm cells or a cell culture containing hematopoietic mesoderm cells;

[0011] 2) Culturing the hematopoietic mesoderm cells or the cell culture containing hematopoietic mesoderm cells in a hematopoietic endothelial cell specialization medium;

[0012] 3) Adding a WNT signaling pathway inhibitor to the above-mentioned hematopoietic endothelial cell specialization medium.

[0013] Furthermore, step 3) is carried out on the 1st - 4th day, 1st - 7th day or 4th - 7th day of the culture in step 2).

[0014] Those skilled in the art can understand that although the preparation process of hematopoietic endothelial cells is described step by step in this embodiment, step 3) (adding a WNT signaling pathway inhibitor to the above-mentioned hematopoietic endothelial cell specialization medium) is not carried out after step 2), but during the culture process of step 2). For example, step 2) is carried out for 10 days, but step 3) is carried out on its 1st day to 4th day or 1st day to 7th day.

[0015] Furthermore, the WNT signaling pathway inhibitor includes IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59 and IWP2.

[0016] Preferably, the Wnt signaling pathway inhibitor is IWR1.

[0017] Preferably, the added concentration of IWR1 is 0.01 μM - 50 μM.

[0018] More preferably, the added concentration of IWR1 is 2.5 μM - 10 μM.

[0019] Furthermore, the hematopoietic endothelial cell specialization medium contains VEGF, bFGF, SCF, IL-3, TPO, Flt-3L, and BMP4.

[0020] Preferably, the hematopoietic endothelial cell specialization medium is STEMdiff TM APEL TM 2 medium supplemented with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L, and BMP4.

[0021] Furthermore, the hematopoietic mesoderm cells or the cell culture containing hematopoietic mesoderm cells are obtained by culturing mesoderm cells or the cell culture containing mesoderm cells in a hematopoietic mesoderm cell specialization medium.

[0022] Furthermore, the mesoderm cells or the cell culture containing mesoderm cells are cultured in the hematopoietic mesoderm cell specialization medium for 2 days to obtain the hematopoietic mesoderm cells or the cell culture containing hematopoietic mesoderm cells.

[0023] Furthermore, the hematopoietic mesoderm cell specialization medium contains VEGF and bFGF.

[0024] Preferably, the hematopoietic mesoderm cell specialization medium is STEMdiff TM APEL TM 2 medium supplemented with VEGF and bFGF.

[0025] Furthermore, the mesoderm cells or the cell culture containing mesoderm cells are obtained by inducing mesoderm from pluripotent stem cells.

[0026] Furthermore, the mesoderm induction is carried out in a mesoderm induction medium containing CHIR99021;

[0027] Preferably, the mesoderm induction medium is STEMdiff TM APEL TM 2 medium supplemented with CHIR99021.

[0028] Furthermore, the pluripotent stem cells are induced pluripotent stem cells.

[0029] Preferably, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0030] Furthermore, the hematopoietic mesoderm cells are KDR + .

[0031] Furthermore, the hematopoietic endothelial cells are CD34 + and KDR + .

[0032] In the present invention, "stem cell" refers to an undifferentiated or insufficiently differentiated cell, which can self-renew on the one hand, that is, produce more cells identical to itself, and on the other hand, can differentiate into two or more mature cell types. According to the source of stem cells, stem cells can be divided into embryonic stem cells (ES cells) and adult stem cells. Embryonic stem cells can come from early animal embryos, such as the inner cell mass of blastocysts (i.e., early embryos), and have the ability to differentiate into each cell type of the body (omnipotence). Adult stem cells are present in various organs and tissues of adults, and have the ability to differentiate and replace the cells of their tissues (pluripotency). Hematopoietic stem cells (HSC) belong to adult stem cells, are present in the bone marrow, and have the ability to differentiate into various blood cells. Hematopoietic stem cells (HSC) can produce both myeloid and lymphoid progenitor cells, and then produce myeloid cells (such as monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (such as T cells, B cells, NK cells, etc.). The ability of stem cells to self-replicate and differentiate into multiple or specific cell types makes them central to cell replacement therapy.

[0033] In the present invention, "induced pluripotent stem cells (iPSCs)" refer to stem cells with totipotency or pluripotency obtained by artificially inducing the expression of certain genes from certain adult cells (such as fibroblasts). In some methods known in the art, iPSCs can be obtained by transfecting certain stem cell-related genes into non-pluripotent cells such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. In some methods, the transfected genes may include the transcription factors Oct4, Sox2, Klf4, and c-Myc, although co-transfection of other genes may potentially improve the induction efficiency. In other methods, somatic cells can be transformed using the Oct4, Sox2, Nanog, and Lin28 genes with a lentiviral system. Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4; certain members of the Sox gene family (such as Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (such as Klf1, Klf2, Klf4, and Klf5); certain members of the Myc family (such as C-myc, L-myc, and N-myc); Nanog, Lin28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-Catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof. Currently, various reagents for preparing iPSCs, such as reprogramming vectors, expression cassettes, culture media, etc., and even commercialized iPSCs, are available on the market. hiPSCs refer to iPSCs induced from human cells.

[0034] In the present invention, "mesoderm cells" refer to the cell layer located between the ectoderm and the endoderm at the late gastrula stage during the embryonic development of triploblastic animals. Mesoderm cells can develop into the dermis, muscles, bones, and other connective tissues of the body, as well as the circulatory system, including the heart, blood vessels, bone marrow, lymph nodes, lymphatic vessels, etc.; the serosa and mesentery of the body cavity end and internal organs, as well as the connective tissues, blood vessels, and smooth muscles in the internal organs; the kidneys, ureters, gonads (excluding germ cells), genital ducts, the cortical part of the adrenal glands, etc. In this article, mesoderm cells refer to the cells with mesoderm cell markers (such as Braychury) produced after induced pluripotent stem cells (iPSCs) are cultured in a mesoderm induction medium. Correspondingly, the process of inducing iPSCs to differentiate into mesoderm cells is called "mesoderm induction". Methods for generating mesoderm cells from induced pluripotent stem cells (iPSCs) are known in the art. For example, there are already commercial mesoderm induction media, such as STEMdiff™ Mesoderm Induction Medium. In a specific example provided in this article, mesoderm cells are obtained by culturing monolayer adherent iPSCs in a mesoderm induction medium for 1 day (about 24 hours). It can be expected that this mesoderm induction stage can be longer. For example, 1.5 days, 2 days, 3 days, etc., as long as the desired mesoderm cells can be obtained.

[0035] "Hematopoietic mesoderm specification" refers to the process of inducing mesoderm cells to differentiate into "hematopoietic mesoderm cells" in this article. "Hematopoietic mesoderm cells" can be considered as the precursor cells of hematopoietic endothelial cells, and their cell marker is KDR. + . In an example in this article, hematopoietic mesoderm cells can be obtained by continuously culturing mesoderm cells in a mesoderm induction medium (also called hematopoietic mesoderm specification medium) supplemented with VEGF and bFGF for about 2 days and detecting the expression of cell markers.

[0036] "Hematopoietic endothelium specialization" refers to the process of inducing the differentiation of hematopoietic mesoderm cells into "hemogenic endothelium cells" in this article. Currently, researchers have considered that hematopoietic stem cells in vivo are derived from hematopoietic endothelium cells. In one example of this article, hematopoietic mesoderm cells can be continuously cultured in a mesoderm induction medium supplemented with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L, and BMP4 (when used for preparing hematopoietic endothelium cells, it is also referred to as hematopoietic endothelium specialization medium in this article; when used for preparing hematopoietic stem cells, it is also referred to as hematopoietic endothelium specialization and endothelium-hematopoietic transition medium) for several days (such as 3 to 12 days or more days, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, etc.) to obtain hematopoietic endothelium cells. Markers CD34 and KDR can be used for the isolation or identification of hematopoietic endothelium cells.

[0037] The second aspect of the present invention provides the application of WNT signaling pathway inhibitors in promoting the in vitro differentiation of pluripotent stem cells into hematopoietic endothelium cells.

[0038] Furthermore, the WNT signaling pathway inhibitors include IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59, and IWP2.

[0039] Preferably, the Wnt signaling pathway inhibitor is IWR1.

[0040] Preferably, the concentration range of IWR1 is 0.01 μM - 50 μM.

[0041] More preferably, the concentration range of IWR1 is 2.5 μM - 10 μM.

[0042] Furthermore, the pluripotent stem cells are induced pluripotent stem cells.

[0043] Preferably, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0044] The third aspect of the present invention provides a culture system for preparing hematopoietic endothelium cells.

[0045] Furthermore, the culture system contains a WNT signaling pathway inhibitor.

[0046] Furthermore, the WNT signaling pathway inhibitors include IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59, and IWP2.

[0047] Furthermore, the Wnt signaling pathway inhibitor is IWR1.

[0048] Furthermore, the concentration range of IWR1 is 0.01 μM - 50 μM.

[0049] Furthermore, the concentration range of IWR1 is 2.5 μM - 10 μM.

[0050] Furthermore, the culture system further comprises the hematopoietic endothelial specialization medium, hematopoietic mesoderm specialization medium, and mesoderm induction medium described in the first aspect of the present invention.

[0051] Advantages and beneficial effects of the present invention:

[0052] The present invention provides a method for preparing hematopoietic endothelial cells. The inventors of the present application found that adding the Wnt signaling pathway inhibitor IWR1 to the hematopoietic endothelial specialization medium at a specific time period can effectively increase the yield of hematopoietic endothelial cells, solving the problems of low differentiation efficiency and high regulation difficulty of hematopoietic endothelial cells in vitro, and providing an efficient, controllable, and stable method for obtaining hematopoietic endothelial cells in vitro. Brief Description of the Drawings

[0053] Figure 1 Flow chart showing the regulation of WNT signaling on the differentiation of human pluripotent stem cells into hematopoietic endothelial cells and hematopoietic stem and progenitor cells; the differentiation process of human pluripotent stem cells into hematopoietic cells mainly includes monolayer cell formation, mesoderm induction, hematopoietic mesoderm specialization, hematopoietic endothelial specialization, and endothelial-hematopoietic transition; Day-1-0: single-cell formation, using TeSR-E8 medium, cell density of 8000 cells / cm 2 , adding 10 μM Y-27632; Day0-1: using mesoderm induction medium containing 9 μM CHIR99021 (CHIR); Day1-3: using hematopoietic mesoderm specialization medium containing 20 ng / mL VEGF and 20 ng / mL bFGF; Day3-12: using hematopoietic endothelial specialization and endothelial-hematopoietic transition medium containing 20 ng / mL VEGF, 20 ng / mL bFGF, 50 ng / mL SCF, 10 ng / mL IL-3, 30 ng / mL TPO, 10 ng / mL Flt-3L, and 10 ng / mL BMP4, where cell passage is performed on Day 3, and the cell seeding density is 2×10 4 cells / cm 2 , additionally adding 10 μM Y-27632, changing the medium after 24 hours, removing Y-27632, and performing treatments with IWR and CHIR within this time period, including testing of concentration and action time windows;

[0054] Figure 2It shows that on the 3rd day of induced differentiation, the expressions of hematopoietic-related markers CD34, KDR, and CD235a during the differentiation process of human pluripotent stem cells into hematopoietic mesoderm were detected by flow cytometry;

[0055] Figure 3 It shows that on the 6th day of induced differentiation, the effects of treating with 5 μM IWR1 or 3 μM CHIR on the differentiation and cell morphology of hematopoietic endothelial cells from the 3rd to the 6th day were observed under a microscope. The CTL was the control group, which was treated with the same volume of DMSO solvent;

[0056] Figure 4 It shows the effects of treating with WNT signal on the differentiation of hematopoietic endothelial cells from the 3rd to the 6th day. Among them, Figure A shows that on the 12th day of induced differentiation, the expressions of hematopoietic endothelial cell-related markers CD34, KDR, and CD235a after treating with 5 μM IWR1 or 3 μM CHIR from the 3rd to the 6th day (D3-6) were analyzed by flow cytometry; Figure B is the bar chart corresponding to the statistical results of Figure A;

[0057] Figure 5 It shows that on the 9th day of induced differentiation, the effects of treating with 5 μM IWR1 or 3 μM CHIR on the differentiation and cell morphology of hematopoietic endothelial cells from the 3rd to the 6th day (D3-6), from the 3rd to the 9th day (D3-9), and from the 6th to the 9th day (D6-9) were observed under a microscope. The CTL was the control group, which was treated with the same volume of DMSO solvent;

[0058] Figure 6 It shows the effects of WNT signal on the differentiation of hematopoietic endothelial cells; among them, Figure A shows that on the 9th day of induced differentiation, the expressions of hematopoietic endothelial cell-related markers CD34, KDR, CD45, and CD235a after treating with 5 μM IWR1 or 3 μM CHIR from the 3rd to the 6th day (D3-6), from the 3rd to the 9th day (D3-9), and from the 6th to the 9th day (D6-9) were analyzed by flow cytometry, and Figure B is the bar chart corresponding to the statistical results of Figure A;

[0059] Figure 7 It shows the effects of WNT signal on the differentiation of hematopoietic endothelial cells and hematopoietic stem and progenitor cells; among them, Figure A shows that on the 12th day of induced differentiation, the expressions of hematopoietic endothelial cell-related markers CD34, KDR, and CD45 after treating with 5 μM IWR1 or 3 μM CHIR from the 3rd to the 6th day (D3-6), from the 3rd to the 9th day (D3-9), from the 3rd to the 12th day (D3-12), from the 6th to the 9th day (D6-9), from the 6th to the 12th day (D6-12), and from the 9th to the 12th day (D9-12) were analyzed by flow cytometry, and Figure B is the bar chart corresponding to the statistical results of Figure A;

[0060] Figure 8It was shown that on the 6th day of induced differentiation, flow cytometry was used to analyze the effects of different concentrations of IWR1 (0.01 - 50 μM) on the expression of hematopoietic endothelial cell-related markers CD34 and KDR; CTL was the control group, treated with the same volume of DMSO (0.01%); the numbers shown in the figure (0.01 - 50) represent the corresponding concentrations of IWR1 (μM). Detailed implementation manners

[0061] The following further elaborates the present invention in conjunction with specific embodiments. The embodiments are only used to explain the present invention and cannot be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0062] For the experimental methods without specific conditions noted in the following embodiments, they are usually implemented according to conventional conditions or the conditions recommended by the manufacturers. For the materials, reagents, etc. used in the following embodiments, unless otherwise specified, they can all be obtained from commercial channels.

[0063] The reagent information used in the following embodiments is shown in Table 1.

[0064] Table 1 Reagent information

[0065] The steps of flow cytometry detection used in the following embodiments are as follows:

[0066] 1. Reagents and antibodies required for FACS detection:

[0067] 1) Washing reagent: Buffer A (PBS + 4% FBS)

[0068] 2) Directly labeled primary antibodies: FITC anti-human CD34 antibody, APC anti-human KDR antibody, APC anti-human CD235a antibody, APC anti-human CD45 antibody.

[0069] 2. Preparation of samples to be tested:

[0070] 1) Prepare TrypLE working solution: Pipette an appropriate amount of DPBS into a new 15 mL centrifuge tube, add the corresponding volume of TrypLE stock solution in a 1:1 ratio, mix well to obtain the working solution, and preheat it in a 37°C water bath for 10 minutes.

[0071] 2) Take out the differentiated cells from the incubator, aspirate and discard the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash them twice with DPBS (the amount of DPBS used each time is not less than the amount of the original culture medium), 1 minute each time (when washing, place the DBPS in the plate / bottle for 30 - 45 seconds and then aspirate it).

[0072] 3) Add TrypLE working solution (add 1 mL of TrypLE working solution to each well of a 6 - well culture plate), make it evenly cover the bottom of the plate, and place it in the incubator for incubation for 2 - 5 minutes. During this period, you can observe under the microscope. The cells shrink, become round and disperse.

[0073] 4) Gently tap the culture bottle / plate to make the cells detach from the bottom of the plate, then gently pipette several times, and finally add an equal volume of Buffer A to terminate digestion. After cell counting, take 1×10 6 cells. (For suspension cells, the cell digestion step is not required. Directly collect the suspension cells for subsequent operations).

[0074] 5) After balancing, centrifuge at 200 g for 5 min. After centrifugation, aspirate and discard the supernatant, gently flick the bottom of the centrifuge tube to fully disperse the cells, add an appropriate amount of Buffer A to resuspend, centrifuge at 200 g for 5 minutes, and discard the supernatant.

[0075] 6) Wash the cells twice with Buffer A, 3 mL of Buffer A each time, centrifuge at 200 g for 5 minutes, and discard the supernatant.

[0076] 7) Incubate with directly labeled primary antibody: After resuspending the cells with 100 µL of Buffer A, add 1 test of directly labeled primary antibody to each tube, incubate at 4°C for 30 minutes, and gently flick the centrifuge tube every 10 minutes to allow the cells to fully bind to the antibody.

[0077] 8) Wash the cells three times with Buffer A, 3 mL of Buffer A each time, centrifuge at 200 g for 5 minutes, and discard the supernatant.

[0078] 9) Add 200 µL of DPBS to each tube to resuspend the cells, and filter the cells through a 70 - µm pore - size filter to remove undigested cell clumps, transfer them to a 96 - well culture plate, and store them at 4°C in the dark, waiting for detection on the machine.

[0079] 3. Flow cytometry detection on the machine:

[0080] 1) Turn on the flow cytometer Guava easyCyte HT and the computer.

[0081] 2) Set up the flow cytometer; open the flow cytometry software and set various parameters.

[0082] 3) After the machine becomes ready, clean the machine.

[0083] 4) First, through the isotype control sample, set the voltages and gains of FSC and SSC to place the discrete cell population in the appropriate position in the quadrant. Generally, cell debris is in the lower left corner, and larger cell clumps are in the upper right corner. Circle the target cell population, set the Gate, and proceed to the next step of analysis.

[0084] 5) Select the appropriate detection channels according to the fluorophore conjugated to the antibody. By adjusting the voltages and compensations of the corresponding channels, the negative and positive cell populations can be clearly distinguished, and then the experimental samples are detected in turn.

[0085] 6) After the detection is completed, clean the flow cytometer and turn off the flow cytometer and the computer.

[0086] Example 1 Process of inducing hematopoietic endothelial cells and hematopoietic stem cells from human pluripotent stem cells

[0087] 1. Formation of monolayer adherent cells

[0088] Experimental operation: Day - 1

[0089] 1) Take an appropriate amount of TrypLE working solution and preheat it in a 37°C water bath for 10 minutes;

[0090] 2) According to the amount of medium required for passage, prepare TeSR - E8 medium containing 10 μM Y - 27632. Add 1 μL of Y - 27632 (10 mM) stock solution to each milliliter of TeSR - E8 medium and preheat it in a 37°C water bath for 10 minutes;

[0091] 3) Take out the hiPSC - 001 - 5 cells to be passaged (cell confluence 70% - 80%) from the incubator, aspirate the original medium, and wash twice with DPBS (the amount of DPBS used each time is not less than the amount of the original medium), 1 minute each time (when washing, place the DBPS in the well / flask for 30 - 45 seconds and then aspirate);

[0092] 4) After adding TrypLE working solution (add about 1 mL of TrypLE working solution to a six - well plate and about 2 mL to a T25 flask), make it evenly cover the bottom of the plate, and incubate in the incubator for 2 - 5 minutes. During this period, observe under the microscope. The cells shrink, become round and disperse;

[0093] 5) Gently tap the culture flask / plate to make the cells detach from the bottom of the plate, then gently pipette several times, and finally add an equal volume of digestion stop solution to terminate the digestion;

[0094] 6) After equilibration, centrifuge at 200 g for 5 min. After centrifugation, aspirate and discard the supernatant. Gently flick the bottom of the centrifuge tube to fully disperse the cells. Resuspend the cells in an appropriate amount of TeSR-E8 medium containing 10 μM Y-27632. After cell counting, adjust to the appropriate cell density.

[0095] 7) Take out the Matrigel-coated culture plate / flask, remove the remaining coating solution, and wash once with DPBS. Inoculate the well-mixed cell suspension into the coated culture plate / flask at a density of 8000 cells / cm 2 . Mark information such as the passage date, cell type, and cell passage number. Place the culture plate / flask in an incubator at 37°C and 5% CO 2 for static culture.

[0096] Note: Control the cell seeding density at 8000 - 10000 cells / cm 2 . Do not shake the culture plate / flask after inoculation to prevent the cells from aggregating in the center of the culture plate / dish.

[0097] 2. Mesoderm Induction

[0098] Experimental operation: Day0

[0099] 1) Take an appropriate amount of mesoderm induction medium and preheat it in a 37°C water bath for 10 minutes.

[0100] 2) After 24 hours of monolayer adherent cell formation, take out the cells to be differentiated from the incubator, aspirate and discard the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash twice with DPBS (the amount of DPBS used each time is not less than the amount of the original medium), 1 minute each time (when washing, place the DBPS in the plate / flask for 30 - 45 seconds before aspirating).

[0101] 3) Add the mesoderm induction medium, and then place it in an incubator at 37°C and 5% CO 2 for static culture for 24 hours (add 2 mL of culture medium to each well of a 6-well culture plate).

[0102] 3. Hematopoietic Mesoderm Specification

[0103] Experimental operation: Day1

[0104] 1) Take an appropriate amount of prepared hematopoietic mesoderm specification medium and preheat it in a 37°C water bath for 10 minutes.

[0105] 2) After 24 hours of mesoderm induction, take the differentiated cells from the incubator, aspirate the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash them twice with DPBS (the amount of DPBS used each time is not less than the amount of the original culture medium), 1 minute each time (when washing, place the DBPS in the plate / flask for 30 - 45 seconds and then aspirate).

[0106] 3) Add the hematopoietic mesoderm specification medium, and then place it in an incubator at 37°C and 5% CO 2 and incubate statically for 48 hours (add 2 mL of culture medium to each well of a 6-well culture plate).

[0107] 4. Hematopoietic & Endothelial Specification and Endothelial-to-Hematopoietic Transition

[0108] Experimental operation: Day3

[0109] 1) Take an appropriate amount of the hematopoietic endothelial specification and endothelial-to-hematopoietic cell conversion medium, place it in a 37°C water bath and preheat for 10 minutes;

[0110] 2) Take an appropriate amount of TrypLE working solution, place it in a 37°C water bath and preheat for 10 minutes;

[0111] 3) After 48 h of hematopoietic mesoderm specification, take the differentiated cells from the incubator, aspirate the original culture medium, add an appropriate amount of DPBS to wash the cells, and wash them twice with DPBS (the amount of DPBS used each time is not less than the amount of the original culture medium), 1 minute each time (when washing, place the DBPS in the plate / flask for 30 - 45 seconds and then aspirate);

[0112] 4) Add TrypLE working solution (add 1 mL of TrypLE working solution to each well of a 6-well culture plate), make it evenly cover the bottom of the plate, place it in the incubator and incubate for 2 - 5 minutes. During this period, observe under the microscope, and it is okay when the cells shrink, become round and disperse;

[0113] 5) Gently tap the culture flask / plate to make the cells detach from the bottom of the plate, then gently pipette several times, and finally add an equal volume of the digestion termination solution to terminate the digestion;

[0114] 6) After balancing, centrifuge at 200 g for 5 min. After centrifugation, aspirate the supernatant, gently flick the bottom of the centrifuge tube to fully disperse the cells, add an appropriate amount of the hematopoietic endothelial specification and endothelial-to-hematopoietic cell conversion medium containing 10 μM Y-27632 to resuspend. After cell counting, adjust to an appropriate cell density;

[0115] 7) Take out the culture plate / flask coated with Matrigel, remove the remaining coating solution, and wash it once with DPBS. Inoculate the well-mixed cell suspension into the coated culture plate / flask at an inoculation density of 2×10 4 cells / cm 2 , mark information such as the passage date, cell type, and cell passage number, and then place it in an incubator at 37°C and 5% CO 2 for static culture (add 2 mL of culture medium to each well of a 6-well culture plate).

[0116] Experimental operation: Day4

[0117] 1) Take an appropriate amount of the hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium prepared, and preheat it in a 37°C water bath for 10 minutes;

[0118] 2) Take out the differentiated cells from the incubator, aspirate the original culture medium, replace it with fresh hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium, and then place it in an incubator at 37°C and 5% CO 2 for static culture (add 2 mL of culture medium to each well of a 6-well culture plate).

[0119] Experimental operation: Day6

[0120] 1) Take an appropriate amount of the hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium prepared, and preheat it in a 37°C water bath for 10 minutes;

[0121] 2) Take out the differentiated cells from the incubator, aspirate the original culture medium, replace it with fresh hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium, and then place it in an incubator at 37°C and 5%CO 2 for static culture (add 2 mL of culture medium to each well of a 6-well culture plate).

[0122] Experimental operation: Day8

[0123] 1) Take an appropriate amount of the hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium prepared, and preheat it in a 37°C water bath for 10 minutes;

[0124] 2) Take out the differentiated cells from the incubator, aspirate the original culture medium, replace it with fresh hematopoietic endothelium specialization and endothelium-hematopoietic cell conversion culture medium, and then place it in an incubator at 37°C and 5% CO 2 for static culture (add 2 mL of culture medium to each well of a 6-well culture plate);

[0125] 3) Observe the cell morphology on Day9, and hematopoietic endothelial cells migrate to form hematopoietic centers.

[0126] Experimental operation: Day10

[0127] 1) Take an appropriate amount of prepared hematopoietic endothelial specialization and endothelial-hematopoietic cell conversion medium, and place it in a 37°C water bath for preheating for 10 minutes;

[0128] 2) Take the differentiated cells from the incubator, collect the original culture medium into a 15 mL centrifuge tube, balance and centrifuge at 200 g for 5 minutes. After centrifugation, aspirate and discard the supernatant, gently flick the bottom of the centrifuge tube to fully disperse the cells, and resuspend them with an appropriate amount of hematopoietic endothelial specialization and endothelial-hematopoietic cell conversion medium;

[0129] 3) Re-inoculate the resuspended cells into a culture plate / flask, and then place it in a 37°C, 5% CO 2 incubator for static culture (add 2 mL of culture medium to each well of a 6-well culture plate).

[0130] Experimental operation: Day12

[0131] Take the differentiated cells from the incubator, collect the original culture medium into a 15 mL centrifuge tube, balance and centrifuge at 200 g for 5 minutes. After centrifugation, aspirate and discard the supernatant, gently flick the bottom of the centrifuge tube to fully disperse the cells, and use the obtained hematopoietic stem cells for subsequent experiments or cryopreservation.

[0132] Example 2 Effects of WNT Signaling on the Differentiation of Hematopoietic Endothelial Cells and Hematopoietic Stem and Progenitor Cells

[0133] To further explore the effect of the WNT signaling pathway on the differentiation of hematopoietic endothelial cells, we established a process system for the differentiation of human pluripotent stem cells into hematopoietic endothelial cells and hematopoietic stem and progenitor cells, and explored its effect on the differentiation of hematopoietic endothelial cells by adding the WNT signaling pathway activator CHIR99021 (CHIR) or inhibitor IWR1 at different time windows between days 3 and 12. For details, see Figure 1 .

[0134] To ensure the consistency of the obtained hematopoietic mesoderm cells, we detected the expression of the mesoderm marker KDR in the cells on day 3 by flow cytometry. At the same time, we also detected the expression of the hematopoietic endothelial cell and hematopoietic stem and progenitor cell marker CD34, as well as the primary hematopoietic marker CD235a. The flow cytometry results showed that the proportion of KDR + cells in the cells induced to differentiate on day 3 was 89.77% - 91.54%, while the proportions of CD34 + and CD235a + cells were extremely low. For details, see Figure 2 . The above results indicate that most of the cells induced to differentiate on day 3 are hematopoietic mesoderm cells and have not yet been transformed into hematopoietic endothelial cells.

[0135] To investigate the effect of the WNT signaling pathway on the differentiation of hematopoietic endothelial cells, we added 5 μM IWR1 or 3 μM CHIR to the hematopoietic endothelial specification and endothelial-hematopoietic cell conversion medium additionally at days 3-6, 3-9, 3-12, 6-9, 6-12, and 9-12 during induced differentiation (the addition phase means adding IWR1 or CHIR to the medium every day; for example, adding IWR1 at 3-6 days means adding IWR1 to the medium every day on days 3, 4, 5, and 6) to explore the effect of activating or inhibiting the WNT signaling pathway on the differentiation of hematopoietic endothelial cells and hematopoietic stem and progenitor cells.

[0136] On day 6 of induced differentiation, we observed the changes in cell morphology. Microscopic photographs showed that compared with the control group (CTL), the IWR1-treated group had more spindle-shaped hematopoietic endothelial-like cells with distinct nuclei and cytoplasm; while the CHIR-treated group had more polygonal heterogeneous cells, as shown in Figure 3 . To further detect the proportion of hematopoietic endothelial cells in each experimental group, we used flow cytometry to detect the co-expression of the hematopoietic endothelial cell markers CD34 and KDR, as well as the co-expression of the primary hematopoietic markers CD34 and CD235a. Flow cytometry results showed that compared with the control group and the CHIR (3 μM)-treated group, treatment with IWR1 (5 μM) at 3-6 days significantly increased the proportion of CD34 + KDR + hematopoietic endothelial cells (78.6% ± 2.2% vs. 55.6% ± 1.8% or 46.9.6% ± 1.7%); while treatment with CHIR (3 μM) at 3-6 days significantly decreased the proportion of CD34 + KDR + hematopoietic endothelial cells (46.9.6% ± 1.7% vs. 55.6% ± 1.8%), as shown in Figure 4 A-B. Flow cytometry results also showed that treatment with IWR1 (5 μM) or CHIR (3 μM) at 3-6 days significantly decreased the proportion of CD34 + CD235a + primary hematopoietic cells, as shown in Figure 4 A-B.

[0137] On day 9 of induced differentiation, we further analyzed the effects of adding IWR1 (5 μM) or CHIR (3 μM) at 3-6 days (D3-6), 3-9 days (D3-9), and 6-9 days (D6-9) on hematopoietic endothelial cells, hematopoietic stem and progenitor cells, and primary hematopoietic cells. Microscopic pictures showed that round and floating blood cells were produced in each group, but adding CHIR at 3-9 days (D3-9 CHIR) significantly inhibited the production of floating blood cells, as shown in Figure 5To further detect the proportion of hematopoietic endothelial cells and the generation of hematopoietic stem and progenitor cells in each experimental group, we used flow cytometry to detect the co-expression of the hematopoietic endothelial cell markers CD34 and KDR, and the hematopoietic stem and progenitor cell marker CD34 + CD45 + , as well as the co-expression of the primary hematopoietic markers CD34 and CD235a. Flow cytometry results showed that compared with the control group, adding IWR1 (5 μM) at day 3-6 (D3-6) and day 3-9 (D3-9) significantly increased the proportion of CD34 + KDR + hematopoietic endothelial cells (70.5% ± 3%, 67.3% ± 1.1% vs. 55.5% ± 0.2%), significantly decreased the proportion of CD34 + CD235a + primary hematopoietic cells, but did not significantly affect the generation of CD34 + CD45 + hematopoietic stem and progenitor cells; adding CHIR (3 μM) at day 3-6 (D3-6) and day 3-9 (D3-9) significantly decreased the proportion of CD34 + KDR + hematopoietic endothelial cells (40.3% ± 0.5%, 27.8% ± 0.2% vs. 55.5% ± 0.2%), CD34 + CD45 + hematopoietic stem and progenitor cells (0.7% ± 0.2%, 0.3% ± 0.3% vs. 5.3% ± 0.7%) and CD34 + CD235a + primary hematopoietic cells (2.4% ± 0.7%, 0.3% ± 0.1% vs. 11.4% ± 0.3%); adding IWR1 (5 μM) or CHIR (3 μM) at day 6-9 (D6-9) both significantly increased the proportion of CD34 + KDR + hematopoietic endothelial cells, significantly decreased the proportion of CD34 + CD45 + hematopoietic stem and progenitor cells and CD34 + CD235a + primary hematopoietic cells, as shown in Figure 6 A - B.

[0138] On the 12th day of induced differentiation, we further analyzed the effects of adding IWR1 (5 μM) or CHIR (3 μM) on hematopoietic endothelial cells and hematopoietic stem and progenitor cells at days 3-6 (D3-6), days 3-9 (D3-9), days 3-12 (D3-12), days 6-9 (D6-9), days 6-12 (D6-12), and days 9-12 (D9-12). Flow cytometry results on the 12th day showed that, compared with the control group, adding IWR1 at different time points did not significantly affect the proportion of + KDR + hematopoietic endothelial cells. This result may be due to the fact that hematopoietic endothelial cells gradually differentiated into hematopoietic stem and progenitor cells in the late stage of differentiation. Flow cytometry results further showed that adding IWR1 at days 3-6 significantly increased the proportion of + CD45 + hematopoietic stem and progenitor cells; while adding CHIR at different time windows significantly inhibited the generation of + CD45 + hematopoietic stem and progenitor cells, as shown in Figure 7 A - B.

[0139] In summary, the WNT signaling pathway inhibitor IWR1 can promote the differentiation of induced pluripotent stem cells into hematopoietic endothelial cells and further promote the generation of hematopoietic stem and progenitor cells. The addition stage can be days 3-6, days 3-9, or days 6-9 of differentiation.

[0140] Example 3 Effects of Different Concentrations of IWR1 on the Differentiation Efficiency of Hematopoietic Endothelial Cells

[0141] In addition, to explore whether the concentration of IWR1 affects the differentiation efficiency of hematopoietic endothelial cells, we chose to add different concentrations of IWR1 (0.01 - 50 μM) at days 3-6. Flow cytometry analysis results showed that within the concentration range of 0.01 - 50 μM, IWR1 could promote the differentiation of + KDR + hematopoietic endothelial cells. Among them, the proportions of + KDR + cells in the treatment groups with 2.5 μM, 5 μM, and 10 μM IWR1 were significantly higher than those in other concentration groups, as shown in Figure 8 . The above experimental results show that the effective concentration range of IWR1 is relatively wide, and the optimal concentration range is 2.5 - 10 μM.

[0142] In summary, the results show that inhibiting the WNT signaling pathway can significantly promote the differentiation of hematopoietic endothelial cells and / or hematopoietic stem and progenitor cells at specific time windows during the differentiation of hematopoietic mesoderm cells into hematopoietic endothelial cells and / or hematopoietic stem and progenitor cells.

[0143] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing hemogenic endothelial cells, characterized in that: The method comprises: 1) Providing hematopoietic mesoderm cells or cell cultures comprising hematopoietic mesoderm cells; 2) culturing the hematopoietic mesoderm cells or the cell culture comprising the hematopoietic mesoderm cells in a hematopoietic endothelial specialized medium; 3) Add WNT signaling pathway inhibitors to the above hemogenic endothelial specialization culture medium.

2. The method according to claim 1, characterized in that: The step 3) is performed on day 1-4, day 1-7 or day 4-7 of the cultivation in step 2).

3. The method according to claim 1, characterized in that The WNT signaling pathway inhibitors include IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59 and IWP2; Preferably, the Wnt signaling pathway inhibitor is IWR1; Preferably, the added concentration of IWR1 is 0.01 μM-50 μM; More preferably, the added concentration of IWR1 is 2.5 μM-10 μM.

4. The method according to claim 1, characterized in that: The hemogenic endothelial specialized culture medium contains VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4; Preferably, the hemogenic endothelial specialized culture medium is STEMdiff supplemented with VEGF, bFGF, SCF, IL-3, TPO, Flt-3L and BMP4. TM APEL TM 2 Culture medium.

5. The method according to claim 1, characterized in that The hematopoietic mesodermal cells or cell cultures comprising hematopoietic mesodermal cells are obtained by culturing mesodermal cells or cell cultures comprising mesodermal cells in a hematopoietic mesodermal specialized medium; Preferably, the mesodermal cells or cell cultures comprising mesodermal cells are cultured in the hematopoietic mesoderm-specific medium for 2 days to obtain the hematopoietic mesoderm cells or cell cultures comprising hematopoietic mesoderm cells; Preferably, the hematopoietic mesoderm specialized culture medium contains VEGF and bFGF; Preferably, the hematopoietic mesoderm specialized medium is STEMdiff supplemented with VEGF and bFGF. TM APEL TM 2 Culture medium.

6. The method according to claim 5, characterized in that The mesodermal cells or cell culture comprising mesodermal cells are obtained by inducing mesoderm from pluripotent stem cells; Preferably, the mesoderm induction is performed in a mesoderm induction medium containing CHIR99021; Preferably, the mesoderm induction medium is STEMdiff supplemented with CHIR99021. TM APEL TM 2 culture medium; Preferably, the pluripotent stem cells are induced pluripotent stem cells; More preferably, the induced pluripotent stem cells are human induced pluripotent stem cells.

7. The method according to any one of claims 1 to 5, characterized in that: The hematopoietic mesoderm cells are KDR + ; Preferably, the hemogenic endothelial cells are CD34 + and KDR + .

8. Application of WNT signaling pathway inhibitors in promoting the differentiation of pluripotent stem cells into hematopoietic endothelial cells in vitro.

9. The use according to claim 8, characterized in that: The WNT signaling pathway inhibitors include IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59 and IWP2; Preferably, the Wnt signaling pathway inhibitor is IWR1; Preferably, the concentration of IWR1 is in the range of 0.01 μM-50 μM. More preferably, the concentration range of IWR1 is 2.5 μM-10 μM; Preferably, the pluripotent stem cells are induced pluripotent stem cells; More preferably, the induced pluripotent stem cells are human induced pluripotent stem cells.

10. A culture system for preparing hemogenic endothelial cells, characterized in that: The culture system comprises a WNT signaling pathway inhibitor; Preferably, the WNT signaling pathway inhibitors include IWR1, XAV939, DKK-1, sFRP-1, Wnt-C59 and IWP2; Preferably, the Wnt signaling pathway inhibitor is IWR1; Preferably, the concentration range of IWR1 is 0.01 μM-50 μM; More preferably, the concentration range of IWR1 is 2.5 μM-10 μM; Preferably, the culture system further comprises the hemogenic endothelial specialized medium according to any one of claims 1 to 4, the hemogenic mesoderm specialized medium according to claim 5, and the mesoderm induction medium according to claim 6.

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