Nouritrophoblast-free method for inducing human pluripotent stem cells to differentiate to hematopoiesis in vitro

Abstract

The invention discloses a trophoblast-free method for inducing human pluripotent stem cells to differentiate into hematopoietic cells in vitro, and belongs to the technical field of biology. The method comprises the following steps: firstly, culturing human pluripotent stem cells, digesting the cells, adding an embryoid to form a culture medium to prepare a cell suspension, inoculating the cell suspension to an agarose template with an inverted pyramid-shaped groove, culturing until an embryoid is formed, then adding an embryoid adherent culture medium, transferring to a culture dish which is bottomed by an extracellular matrix in advance, culturing until the embryoid adheres to the wall, and finally obtaining the human pluripotent stem cells. And a hematopoietic differentiation culture medium is used for inducing hematopoietic differentiation and generating ancestor cells of blood. The method disclosed by the invention has the advantages of simplicity, rapidness, economy and the like, can be used for rapidly and efficiently differentiating to obtain a large number of hematopoietic progenitor cells, is suitable for detection of hematopoietic differentiation potential and clinical application of differentiation to functional blood cells, can also be used for high-throughput large-scale screening of blood-related drugs, and has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an economical and practical method for inducing human pluripotent stem cells to differentiate into hematopoiesis in vitro without a trophoblast. Background Art

[0002] Blood is vital to maintaining human health. With the rapid development of medical technology, major operations usually require a large amount of blood products. Major traffic or disaster events also require a large amount of blood, and blood supply is often tight.

[0003] Human pluripotent stem cells (including embryonic stem cells and human induced pluripotent stem cells) have the ability to be cultured for a long time in vitro and maintain self-renewal. They have the potential for multidirectional differentiation and can differentiate in different directions, including nerves, vascular endothelium, vascular smooth muscle, blood, myocardium, bones, muscles, etc. The process of directional induction and differentiation of human pluripotent stem cells into blood cells such as hematopoietic progenitor cells is usually called hematopoietic differentiation. At present, the methods for directional hematopoietic differentiation of human pluripotent stem cells include: co-culture with stromal cells, EB formation method, extracellular matrix protein method, and adding a large number of various types of cytokines and small molecule compounds to promote hematopoietic differentiation during the hematopoietic differentiation process. However, these existing methods generally have the problems of complex differentiation methods, high cost of reagents and consumables (key culture media and culture consumables are highly dependent on imports), low differentiation efficiency, and inability to obtain a large number of hematopoietic progenitor cells on a large scale, which limits the clinical treatment application of the in vitro hematopoietic differentiation system of human pluripotent stem cells and the application of high-throughput large-scale screening of blood disease therapeutic drugs, and is not conducive to the use of in vitro hematopoietic differentiation systems for theoretical research and exploration.

[0004] In order to meet the huge demand for artificial blood cells and blood products in my country, the demand for large-scale screening of blood-related drugs, and the demand for mechanism theoretical research, it is particularly important to develop an in vitro method with independent intellectual property rights that is efficient, convenient, economical, and capable of large-scale and low-cost induction of human pluripotent stem cells to differentiate into hematopoiesis. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an economical and practical method for inducing human pluripotent stem cells to differentiate into hematopoiesis in vitro without a trophoblast. The induction hematopoietic differentiation medium of the present invention has unique components, requires few types of cytokines and low concentrations (only hVEGF needs to be added as cytokines), and significantly reduces the cost of inducing differentiation. Other in vitro hematopoietic differentiation systems often require the addition of a large number of various types of cytokines and small molecule compounds, and / or the purchase of expensive imported commercial culture media (typically, TW109139975A requires the addition of up to 12 types of cytokines and expensive imported StemPro-34 culture medium, and CN201510664655 requires the addition of up to 5 types of cytokines and expensive imported StemlineⅡ culture medium); since the types of added cytokines are simple, it is very easy to control the stability of the culture system, and hPSC can be efficiently induced to differentiate into blood; the present invention adopts a mold-turning method to mass-produce cheap inverted pyramid-shaped groove agarose templates, and then can differentiate and obtain hematopoietic progenitor cells on a large scale at low cost, which has not been reported in previous patents and has very good application prospects.

[0006] In order to achieve the above object, the specific technical solution of the present invention is:

[0007] The present invention provides a novel in vitro method for inducing hematopoietic differentiation of human pluripotent stem cells without a trophoblast, comprising the following steps:

[0008] (1) culturing human pluripotent stem cells (hPSCs) in a culture dish treated with Matrigel, digesting the cells, adding embryoid bodies (EBs) to form a culture medium, and preparing a cell suspension; the human pluripotent stem cells (hPSCs) include human embryonic stem cell lines (hESCs) and human induced pluripotent stem cell lines (hiPSCs);

[0009] (2) inoculating the cell suspension obtained in step (1) into an agarose template with an inverted pyramid-shaped groove, and culturing in an incubator at 37° C. and 5% CO 2 (V / V) until embryoid bodies (EBs) are formed;

[0010] (3) resuspending the embryoid bodies (EBs) obtained in step (2) in an embryoid body attachment culture medium, transferring the EBs to a culture dish pre-coated with an extracellular matrix, and culturing the EBs until they adhere to the wall;

[0011] (4) Change to hematopoietic differentiation medium to induce differentiation into blood progenitor cells.

[0012] Based on the above technical solution, further, the embryoid formation medium described in step (1) is mTeSR1 basal medium containing 0.5-1.5 μM Thiazovivin.

[0013] Based on the above technical solution, further, in step (2), the embryoid body formation time is 1 to 3 days, and the medium is replaced by half every 1-2 days.

[0014] Based on the above technical solution, further, the embryoid body attachment culture medium described in step (3) is composed of mTeSR1 basal culture medium containing 9-11 μM Thiazovivin, 2-4 ng / ml hBMP4, and 40-60 ng / ml hVEGF.

[0015] Based on the above technical solution, further, the extracellular matrix described in step (3) includes Vitronectin.

[0016] Based on the above technical solution, further, the time for culturing until adhesion in step (3) is 24 to 72 hours.

[0017] Based on the above technical solution, further, in step (3), the seeding density of the embryoid bodies is controlled at 100 to 200 cells / cm 2 Or 200-400 cells / well (24-well plate).

[0018] Based on the above technical scheme, further, the composition of the hematopoietic differentiation medium described in step (4) is 500-550 ml IMDM medium, 8-12% by volume of fetal bovine serum (FBS, decomplemented), 50-150 μM of each non-essential amino acid (NEAA), 1-3 mM L-glutamine, 45-55 μg / ml ascorbic acid, 30-50 μl transferrin, 15-25 ng / ml vascular endothelial growth factor (VEGF), and 4-6 μl β-mercaptoethanol.

[0019] Based on the above technical solution, further, the time for inducing differentiation in step (4) is 15 to 19 days.

[0020] Based on the above technical solution, further, in step (4), during the differentiation induction process, half the volume of the medium is replaced every 1-2 days.

[0021] Based on the above technical solution, further, the blood progenitor cells described in step (4) include hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells.

[0022] The beneficial effects of the present invention compared with the prior art are as follows:

[0023] 1. Compared with the existing trophoblast in vitro hematopoiesis induction method, the present invention can efficiently induce hPSC to differentiate into blood, and obtain good results without the participation of mouse stromal cells (such as AGM-S3 or OP9). The method of the present invention is relatively simple.

[0024] 2. Compared with other existing EB-formation-based in vitro hematopoietic induction methods without a trophoblast layer, the present invention adopts a mold-turning method to mass-produce cheap agarose templates, and does not need to import expensive culture dishes for EB formation experiments, thereby reducing costs; the culture medium used in each stage contains a small amount of cytokines, and the hematopoietic differentiation culture medium with the largest usage can be prepared by itself, with low cost. Therefore, there is no need to use commercialized expensive imported culture medium in the hematopoietic differentiation stage, which greatly reduces the cost of hematopoietic differentiation and has the potential for large-scale application.

[0025] 3. The test results show that the hematopoietic differentiation method of the present invention can efficiently produce hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells, and the hematopoietic progenitor cells have good morphology and comprehensive hematopoietic differentiation potential. If combined with the differentiation method of downstream lineages, it may have considerable potential for differentiating various functional lineage blood cells / immune cells. The combination of these advantages shows the unique potential of the present invention in the clinical application of cell therapy and the establishment of a high-throughput screening system, and can also be used as an in vitro system for the study of the theoretical mechanism of hematopoietic differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0027] Figure 1 The flow cytometer detection and hematopoietic clone formation detection results in Example 1, wherein A is the flow cytometer detection of hematopoietic progenitor cells (i), myeloid progenitor cells (ii) and erythroid progenitor cells (iii) differentiated after D17 days of induction of hematopoietic differentiation; B is the hematopoietic clone detection result of CFU-GM (granulocyte and macrophage colony-forming unit), CFU-E (erythrocyte colony-forming unit), BFU-E (burst erythroid colony-forming unit), CFU-MIX (mixed colony-forming unit) and other types of cells produced by in vitro hematopoietic differentiation without trophoblast at D17, wherein i is the counting result diagram of various types of hematopoietic clones, and ii is the typical morphological diagram of various types of hematopoietic clones.

[0028] Figure 2 This is the result of flow cytometry analysis of hematopoietic progenitor cells at a seeding density of 50 EBs / well, using 6 combinations of extracellular matrix and culture medium cytokines to investigate the hematopoietic differentiation efficiency.

[0029] Figure 3The figure shows the flow cytometry results of myeloid progenitor cells at a seeding density of 50 EBs / well, investigating the hematopoietic differentiation efficiency using 6 combinations of extracellular matrix and culture medium cytokines.

[0030] Figure 4 The figure shows the flow cytometry results of erythroid progenitor cells at a seeding density of 50 EBs / well, investigating the hematopoietic differentiation efficiency using 6 combinations of extracellular matrix and culture medium cytokines.

[0031] Figure 5 Bar graph showing the number of different cell types at a seeding density of 50 EBs / well and the effect of 6 extracellular matrix and culture medium cytokine combinations on hematopoietic differentiation efficiency.

[0032] Figure 6 The effect of four different planting densities on hematopoietic differentiation efficiency under the optimal combination conditions, where A is hematopoietic progenitor cells (CD34 + / CD43 + group as an indicator), myeloid progenitor cells (with CD34 + / CD45 + group as an indicator) and erythroid progenitor cells (GPA + / CD71 + A is a flow cytometry ratio diagram of the number of cells of different types. B is a bar graph of the number of cells of different types. DETAILED DESCRIPTION

[0033] In order to make the invention objectives, technical solutions and advantages of the application more clear, the present invention is further described in detail below in conjunction with specific implementation methods, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0034] The kits, reagents and other related materials in the following examples are all commercially available products;

[0035] Example 1

[0036] 1. Materials

[0037] 1. Main instruments and reagents:

[0038] (1) Main instruments and consumables

[0039]

[0040] (2) Main reagents

[0041]

[0042]

[0043] (3) Flow cytometry antibodies and 7-AAD dye were purchased from BD Biosciences, USA:

[0044] APC-conjugated mouse anti-human CD34 flow cytometry antibody BD 555824 PE-conjugated mouse anti-human CD43 flow cytometry antibody BD 560199 PE-Cy7 conjugated mouse anti-human CD45 flow cytometry antibody BD 557748 APC-conjugated mouse anti-human GPA flow cytometry antibody BD 551336 PE-conjugated mouse anti-human CD71 flow cytometry antibody BD 555537 7-AAD dye BD 559925

[0045] 2. Preparation of main solution

[0046] (1) Preparation of humanized hPSC culture medium

[0047] mTeSR TM 1 Basal Medium 400ml

[0048] mTeSR TM 1 5×Supplement 100ml

[0049] A total of 500 ml, mix well, and place in a 4℃ refrigerator for later use.

[0050] (2) Preparation of EB formation medium

[0051] Humanized hPSC basal medium [see (1)]

[0052] 1μM Thiazovivin

[0053] Mix well and place in a 4°C refrigerator for later use.

[0054] (3) Preparation of EB Adhesion Culture Medium

[0055] Humanized hPSC basal medium [see (1)]

[0056] 10μM Thiazovivin

[0057] 3ng / ml hBMP4

[0058] 50ng / ml hVEGF

[0059] Mix well and place in a 4°C refrigerator for later use.

[0060] (4) Preparation of hematopoietic differentiation medium

[0061] IMDM medium 500～550ml FBS (fetal bovine serum, decomplemented) 8～12％15 NEAA (non-essential amino acids) 50~150μM L-Glutamine 1~3mM Ascorbic acid 45~55μg / ml Transferrin 30~50μl stock solution hVEGF (human vascular endothelial growth factor) 15~25ng / ml β-mercaptoethanol 4~6μl stock solution 20

[0062] A total of 600 ml was mixed and filtered through a 0.22 μm filter and placed in a 4°C refrigerator for later use.

[0063] (5) Preparation of SM buffer

[0064] D-PBS

[0065] 2% FBS (V / V)

[0066] Mix well and place in a 4°C refrigerator for later use.

[0067] 3. Cell lines

[0068] H1 hESCs were provided by the State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences;

[0069] 2. Experimental steps

[0070] 1. Cell culture and passaging of human pluripotent stem cell lines

[0071] The cells used in this example are human embryonic stem cell line H1 hESC, and the culture medium is mTeSR1 medium. The cells are cultured in an incubator at 37°C and 5% CO2 (V / V). The culture dish is a 35 mm dish. After the cells grow to 80% confluence, the cells are washed once with D-PBS, digested with ReleSR at 37°C for 3 minutes, and then the hESCs are blown into appropriate small cell clusters with mTeSR1 medium, and then subcultured to Matrigel-bottomed culture dishes at an area ratio of 1:3 for further culture.

[0072] 2. Human pluripotent stem cells (hPSCs) form embryoid bodies (EBs) and differentiate into hematopoiesis

[0073] For convenience, this experiment is described using a 24-well plate as an example.

[0074] A. Template Preparation

[0075] i) Use laser to cut out the inverted pyramid groove template and make the first mold with polymer material.

[0076] ii) Add 3% agarose aqueous solution (prepared with deionized water) into the polymer material mold, preferably with a flat surface, and place it at room temperature for 4 minutes or at 4°C for 4 minutes to fully cool and solidify.

[0077] iii) Use flat-tipped tweezers to pick up the solidified agarose template and carefully place it into the well plate with the template side facing up, and fill the empty space at the edge with 100 μl of melted 1% agarose (prepared with deionized water) to fix the agarose template;

[0078] iv) Open the lid and irradiate with ultraviolet light for 1-2 hours to sterilize, then put the lid back on and irradiate for another 30 minutes, then wrap with sealing film and set aside.

[0079] B. hPSCs form EBs

[0080] This implementation is described using H1 hESC as an example:

[0081] i) When the H1 hESCs in the 35 mm culture dish reached 80% confluence, the medium was discarded and the cells were washed once with D-PBS.

[0082] ii) Digest with ReleSR at 37°C for 3 minutes, observe under a microscope and discard the enzyme solution after the cells are properly digested.

[0083] iii) Prepare freshly about 10 ml of EB formation medium.

[0084] iv) hESCs were blown into appropriate small cell clusters using mTeSR1 medium, resuspended in 6 ml EB formation medium, and seeded into 6 wells of a 24-well plate prepared with agarose templates at a ratio of 1:6 (1 ml per well). After shaking, the cells were cultured in an incubator at 37°C and 5% CO2 (V / V).

[0085] v) Carefully remove the cells the next day and check the EB formation efficiency under a microscope. If everything is normal, carefully aspirate half of the culture medium supernatant, fill with fresh culture medium, and return the cells to the incubator for continued culture.

[0086] C. EB attachment and mesoderm differentiation

[0087] i) Carefully remove the cells on the third day and check the EB formation efficiency under a microscope. If everything is normal, take photos and prepare to transfer them to the extracellular matrix for further growth.

[0088] ii) 24 μl of Vitronectin was aliquoted and frozen at -80°C, diluted to 3000 μl with D-PBS, and 250 μl was dispensed into one well of a 24-well plate, for a total of 12 wells, and stored at 4°C, or placed directly at room temperature for 1 hour for later use.

[0089] iii) Prepare 12 ml of fresh EB attachment medium.

[0090] iv) For each well where EBs are formed, carefully use a 1 ml pipette tip to suck away the old culture medium along the edge gap, then add 500 μl EB adhesion culture medium, and pipette and hit for 3 times to blow the EBs off the agarose template. Transfer all of them into a 1.5 ml centrifuge tube. Check under a microscope to see if the EBs are transferred cleanly, then wash them once with 500 μl EB adhesion culture medium and transfer them into the centrifuge tube, making a total volume of 1 ml.

[0091] v) Discard the Vitronectin in the 24-well plate, pipette and evenly distribute the EB suspension, and dispense 500 μl of EB into each well of the Vitronectin-coated 24-well plate, for a total of 12 wells. Shake vigorously, check under a microscope, and place at room temperature for 5-10 minutes after shaking. Then, culture in an incubator at 37°C, 5% CO2 (V / V) overnight.

[0092] vi) On the fourth day, carefully remove the culture dish and check the EB adhesion under a microscope. If all EBs are attached, discard the culture medium and add 0.5 ml of fresh EB adhesion medium to each well. If some EBs are not attached, carefully remove 0.25 ml of the culture medium supernatant and add fresh EB adhesion medium up to 0.5 ml. Shake well and return to the culture medium for further culture. The day when EBs adhere is defined as D0, and the same applies to the following days.

[0093] D. Induce hematopoietic differentiation

[0094] i) On the fifth day, the EB adhesion was checked again under a microscope and 750 μl of hematopoietic differentiation medium was replaced.

[0095] ii) On the sixth day, 750 μl of hematopoietic differentiation medium was added.

[0096] iii) Start half-medium change on the seventh day: remove half of the supernatant and add half of fresh hematopoietic differentiation medium.

[0097] 3. Detection of hematopoietic differentiation efficiency

[0098] A. Flow cytometry

[0099] i) Differentiated cells were observed under a microscope. Vesicle-like structures gradually appeared from D3 to D6, followed by more cobblestone-like granular cells. The time point for flow cytometry analysis was determined between D13 and D20 based on the growth status. Generally, D17 was the peak of hematopoietic differentiation and was used as the time point for flow cytometry analysis.

[0100] ii) The medium remains in a 15 ml tube and 100 μl of regular FBS is added for the final trypsin neutralization.

[0101] iii) Each culture well was washed once with 1 ml D-PBS and merged into the 15 ml tube, treated with 0.25% Trypsin / EDTA (Wt / V) at 37°C for 3 min, 2-3 times the volume of ice-cold D-PBS was added to dilute trypsin, and the cells were repeatedly blown with a 1 ml pipette tip to a state close to single cells, merged into the tube, and finally the remaining cells were rinsed with D-PBS and merged into the centrifuge tube, centrifuged at 300×g at room temperature for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μl SM buffer.

[0102] iv) After mixing, pipette 30 μl of cells into a 0.6 ml eppendorff tube for cell counting.

[0103] v) The remaining cells were filtered through a 70 μm filter membrane, and 10 μl of normal rabbit serum was added to every 0.5 ml volume, mixed, and placed on ice for 20 minutes to block the non-specific binding sites of the antibody.

[0104] vi) Centrifuge at 300×g for 5 min at room temperature, discard the supernatant, and resuspend in 100 μl SM buffer.

[0105] vii) Add 1 μl of the corresponding flow cytometry antibodies and 0.5 μl of 7-AAD dye, shake to mix, and incubate on ice in the dark for 30-40 min.

[0106] viii) Wash once with 1 ml D-PBS, centrifuge at 300 g for 5 min at room temperature, discard the supernatant, resuspend in 200 μl SM buffer, and transfer to a flow cytometry loading tube.

[0107] ix) Beckman Coulter CytoFlex LX flow cytometer was used for detection, and the experimental data were analyzed using Flowjo10 software and GraphPad Prism software for statistical analysis.

[0108] B. Colony culture to detect hematopoietic differentiation potential

[0109] At D17 of hematopoietic differentiation, 5 × 10 4 The hematopoietic colony formation test was performed on each cell. CFU-E (erythrocyte colonies) were counted on the 7th day after colony culture, and CFU-MIX (mixed colonies), CFU-GM (neutrophil colonies), and BFU-E (blast erythrocyte colonies) were counted on the 12th to 14th day. The process is described using a 24-well plate as an example:

[0110] i) Prepare a 15 ml centrifuge tube, draw out the old hematopoietic differentiation medium and add it to the 15 ml centrifuge tube (reserved for terminating cell digestion).

[0111] ii) Add 1 ml of D-PBS to the 24-well plate and wash once. After washing, transfer the plates to a centrifuge tube.

[0112] iii) Add 100 μl of 0.25% trypsin and spread it evenly on the cell surface. Use a 200 μl pipette tip to pierce the hematopoietic clones and blow them apart. After digestion at 37°C for 3 minutes, observe the release of cobblestone-like hematopoietic stem / progenitor cells under a microscope.

[0113] iv) 300 μl of ice-cold D-PBS was added to the wells to terminate the reaction. The cells were repeatedly beaten with a 1 ml pipette tip to form uniform cells and then merged into a centrifuge tube.

[0114] v) Wash each well twice with 0.5 ml D-PBS and transfer the cells to a centrifuge tube.

[0115] vi) Take 25 μl of the cell suspension and count it to calculate the cell concentration.

[0116] vii) Take 5×10 4The cell suspension of the differentiated cells was centrifuged at 300 × g for 5 min and the supernatant was removed.

[0117] viii) Resuspend the cells in 300μl IMDM medium, transfer the cell suspension to a 14ml polypropylene round bottom tube, add 180μl IMDM medium to resuspend the remaining cells, and transfer them to the medium tube that has been added with methylcellulose, cytokines and double antibodies. The formula is detailed in the table below.

[0118]

[0119] ix) Mix the semi-solid culture medium horizontally, shake the cap and the residual culture medium on the side wall downward, vortex for three times (about 7 seconds each time), and let it stand for 20 minutes until the bubbles disappear.

[0120] x) Use a 2 ml syringe to draw out the mixed semi-solid culture medium, expel the bubbles in the syringe, and seed it into the center of 4 Petri Dishes, 1 ml / dish, and shake the culture medium to make it evenly distributed.

[0121] xi) Divide the four 35 mm dishes into two culture dishes with a diameter of 10 cm, and use a 35 mm dish with sterile water to maintain humidity. Incubate in a 37°C incubator, preferably inside. Do not move for the first three days to avoid affecting clone formation.

[0122] xii) The number of CFU-E was observed after 7 days, and the number of CFU-MIX, CFU-GM and BFU-E was observed after 12-14 days. The four dishes were treated as four replicates and statistically analyzed.

[0123] The results of flow cytometry and hematopoietic clone formation assays were as follows Figure 1 As shown, Figure 1 A is a flow cytometry test result diagram, where i, ii, and iii are the flow cytometry test results after the differentiated cells were incubated with anti-CD34 / CD43, anti-CD34 / CD45, and anti-GPA / CD71 antibodies after induction of hematopoietic differentiation D17 days. It can be seen from the figure that the trophoblast-free in vitro hematopoietic differentiation method can efficiently obtain hematopoietic progenitor cells (with CD34 + / CD43 + group as an indicator), myeloid progenitor cells (with CD34 + / CD45 + group as an indicator) and erythroid progenitor cells (GPA + / CD71 + group as an indicator); Figure 1B is a result diagram of colony culture detection of hematopoietic clone differentiation potential, where i is the counting result diagram of various types of hematopoietic clones, and ii is the typical morphology diagram of various types of hematopoietic clones. It can be seen from the figure that cells differentiated by the trophoblast-free in vitro hematopoietic differentiation system D17 can effectively produce hematopoietic clones of types such as CFU-GM, CFU-E, BFU-E, and CFU-MIX. The above test results show that the in vitro differentiation method can produce a certain number of hematopoietic progenitor cells, myeloid progenitor cells, and erythroid progenitor cells.

[0124] Comparative Example 1

[0125] The experimental process of this comparative example is the same as that of Example 1. The main difference lies in the extracellular matrix (ECM) components used for EB adhesion, EB formation culture medium components and adhesion culture medium components under the condition of planting EBs at a density of 50 EBs / well. A total of 6 groups of experiments were set up, and Experiment 4 was exactly the same as Example 1 for comparison.

[0126] 1. Setting of extracellular matrix (ECM) components used for EB attachment:

[0127] Experiments (1)-(3) were primed with Matrigel, and experiments (4)-(6) were primed with Vitronectin;

[0128] 2. The following are the settings for different combinations of culture media:

[0129] Experiments (1), (4)

[0130] EB formation medium: mTeSR1 + 1 μM Thiazovivin

[0131] EB attachment culture medium: mTeSR1+10μM Thiazovivin+3ng / ml hBMP4+50ng / ml hVEGF

[0132] Experiments (2), (5)

[0133] EB formation medium: mTeSR1+1μM Thiazovivin+3ng / ml hBMP4

[0134] EB attachment culture medium: mTeSR1+10μM Thiazovivin+20ng / ml hBMP4+100ng / ml hSCF+100ng / ml hFL+50ng / ml hVEGF

[0135] Experiments (3), (6)

[0136] EB formation medium: mTeSR1+1μM Thiazovivin+3ng / ml hBMP4

[0137] EB attachment culture medium: mTeSR1+10μM Thiazovivin+3ng / ml hBMP4+50ng / ml hVEGF

[0138] The results of 6 extracellular matrix and culture medium cytokine combination experiments to investigate the efficiency of hematopoietic differentiation are shown in the figure below. Figure 2-Figure 5 As shown, differentiated cells at D17 were detected with a combination of anti-CD34 / CD43, anti-CD34 / CD45, and anti-GPA / CD71 antibodies for hematopoietic progenitor cells ( Figure 2 ), myeloid progenitor cells ( Figure 3 ) and erythroid progenitor cells ( Figure 4 ) ratio, and calculate the bar graph of the number of different types of cells ( Figure 5 ).Depend on Figure 2-Figure 5 It can be seen that the experiment with the highest hematopoietic differentiation efficiency is experiment (4), namely, base material: Vitronectin, EB formation medium: mTeSR1+1μM Thiazovivin, EB adhesion medium: mTeSR1+10μM Thiazovivin+3ng / mlhBMP4+50ng / ml hVEGF.

[0139] Comparative Example 2

[0140] Exploration of EB planting density conditions

[0141] After determining the culture medium and extracellular matrix components, the EB planting density was optimized. The experimental process was basically the same as that in Example 1. The main difference was that the hematopoietic differentiation efficiency produced by the planting density of 50, 100, 150, and 300 EBs / well (24-well plate) was tested respectively.

[0142] The results of the effects of four different implantation densities on hematopoietic differentiation efficiency are as follows Figure 6 As shown, 50, 100, 150, and 300 EBs / well of a 24-well plate were added, and hematopoietic differentiated cells at D17 were detected by a combination of anti-CD34 / CD43, anti-CD34 / CD45, and anti-GPA / CD71 antibodies to detect hematopoietic progenitor cells (with CD34 + / CD43 + group as an indicator), myeloid progenitor cells (with CD34 + / CD45 + group as an indicator) and erythroid progenitor cells (GPA + / CD71 + The proportion of the cell population was calculated (A), and the number of different cell types was calculated as a bar graph (B). Figure 6 It can be seen that the planting density of 300 EBs / well achieved the highest hematopoietic differentiation efficiency.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 inducing hematopoietic differentiation of human pluripotent stem cells in vitro without a trophoblast, characterized in that: The following steps are involved: (1) culturing human pluripotent stem cells (hPSCs) in a culture dish treated with Matrigel, digesting the cells, adding embryoid bodies (EBs) to form a culture medium, and preparing a cell suspension; the human pluripotent stem cells (hPSCs) include human embryonic stem cell lines (hESCs) and human induced pluripotent stem cell lines (hiPSCs); (2) inoculating the cell suspension obtained in step (1) into an agarose template with an inverted pyramid-shaped groove, and culturing in an incubator at 37° C. and 5% CO 2 (V / V) until embryoid bodies (EBs) are formed; (3) resuspending the embryoid bodies (EBs) obtained in step (2) in an embryoid body attachment culture medium, transferring the EBs to a culture dish pre-coated with an extracellular matrix, and culturing the EBs until they adhere to the wall; (4) Change to hematopoietic differentiation medium to induce differentiation into blood progenitor cells.

2. The method according to claim 1, characterized in that The embryoid formation medium described in step (1) is mTeSR1 basal medium containing 0.5-1.5 μM Thiazovivin.

3. The method according to claim 1, characterized in that The embryoid formation time in step (2) is 1 to 3 days, and the medium is replaced by half every 1-2 days.

4. The method according to claim 1, characterized in that: The embryoid body attachment culture medium described in step (3) is composed of mTeSR1 basal culture medium containing 9-11 μM Thiazovivin, 2-4 ng / ml hBMP4, and 40-60 ng / ml hVEGF; the extracellular matrix includes Vitronectin; and the culture time until attachment is 24-72 hours.

5. The method according to claim 1, characterized in that In step (3), the seeding density of embryoid bodies is controlled at 100 to 200 cells / cm 2 Or 200-400 cells / well (24-well plate).

6. The method according to claim 1, characterized in that The hematopoietic differentiation medium described in step (4) is composed of 500-550 ml IMDM medium, 8-12% by volume of fetal bovine serum (FBS, decomplemented), 50-150 μM of each non-essential amino acid (NEAA), 1-3 mM L-glutamine, 45-55 μg / ml ascorbic acid, 30-50 μl transferrin, 15-25 ng / ml vascular endothelial growth factor (hVEGF), and 4-6 μl β-mercaptoethanol.

7. The method according to claim 1, characterized in that The time for inducing differentiation in step (4) is 15 to 19 days, and the medium is replaced by half every 1 to 2 days during the differentiation induction process.

8. The method according to claim 1, characterized in that The blood progenitor cells described in step (4) include hematopoietic progenitor cells, myeloid progenitor cells and erythroid progenitor cells.

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