Use of leukotrienes in inducing stem cells to differentiate into hematopoietic stem / progenitor cells and uses thereof

Abstract

The present application provides the use of leukotrienes in inducing stem cells to differentiate into hematopoietic stem / progenitor cells and the application thereof. According to specific embodiments of the present application, the addition of appropriate amounts of leukotrienes during the process of inducing stem cells to differentiate into hematopoietic stem / progenitor cells can significantly improve the differentiation efficiency of the stem cells into hematopoietic stem / progenitor cells, and significantly increase the number of erythroid cells, megakaryocytic cells, granulocytic cells and mononuclear cells obtained after the differentiation of the stem cells through hematopoietic stem / progenitor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell engineering, in particular, the present application relates to the use of leukotrienes in inducing stem cells to differentiate into hematopoietic stem / progenitor cells and the application thereof. BACKGROUND

[0002] Whether in war trauma treatment, or in clinical treatment, the timely supply of blood is the key to successful treatment. However, the world is currently facing a shortage of blood supply, making it difficult to ensure the supply of blood.

[0003] Stem cells are primitive, undifferentiated cells, and have the characteristics of continuous self-renewal, maintaining continuous proliferation, etc., and can differentiate into various tissue cells under certain conditions to provide new cells for tissues. Among them, hematopoietic stem cells (HSCs) are a group of multipotent stem cells with self-renewal ability and potential to differentiate into all mature blood cell types. HSC transplantation is the most commonly used stem cell treatment method in clinical practice, and is widely used in the treatment of hematopoietic failure in various blood system diseases, immune system diseases, tumors, and radiation damage. Currently, the HSCs used for clinical transplantation mainly come from three sources: umbilical cord blood, bone marrow, and peripheral blood. However, the shortage of sources, low matching rate, and other problems still pose obstacles to the clinical application of HSCs. Therefore, there is an urgent need to find a widely available, more cost-effective, and safer HSC resource. The emergence of human pluripotent stem cells (hPSCs) is expected to solve this problem. hPSCs include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), which have the ability of non-differentiated proliferation and developmental totipotency, and can be induced to differentiate into cells of the three germ layers in vitro. It is currently a research hotspot and difficulty in the field of stem cells. Because PSCs can be cultured and expanded in vitro for a long time while maintaining their multi-directional differentiation ability, they have become a seed cell with great application prospects. After nearly 20 years of research, it is still a challenge to obtain HSCs with complete hematopoietic function in vitro by efficiently inducing hPSCs. In 2013, the laboratories of Amabile and Suzuki first established a hiPSC hematopoietic differentiation system in vivo using teratoma formation. They injected hiPSCs directly into NSG mice, allowing the mice to form teratomas, and detected human CD34 + CD45 +Cells, which have continuous multilineage engraftment ability. These studies first demonstrated that hiPSCs have the ability to differentiate into HSCs with long-term engraftment function. However, how to efficiently realize the in vitro preparation of HSCs and various functional blood cells downstream thereof initiated from pluripotent stem cells is still a difficult problem.

[0004] Taking the preparation of red blood cells as an example, the preparation of red blood cells from pluripotent stem cells usually undergoes mesoderm, hematopoietic endothelium, hematopoietic stem / progenitor cells, erythroid-megakaryocyte common progenitor cells, erythroid progenitor cells, and mature red blood cells. The induction is usually a staged induction scheme, and the successful preparation of hematopoietic stem / progenitor cells is a key node in the preparation process. As early as 2008, two reports confirmed that red blood cells could be prepared by inducing hESCs. Improving the efficiency of the production of intermediate states in the induction will promote the preparation of functional blood cells in the final stage of the induction. Therefore, finding and determining the regulatory factors that can effectively improve hematopoietic differentiation and erythroid differentiation and utilizing them will improve the yield of red blood cells derived from pluripotent stem cells and meet the demand for blood supply. SUMMARY

[0005] The present application is based on the discovery and understanding of the inventors on the fact and problem that there is a large contradiction between supply and demand of hematopoietic stem / progenitor cells and mature blood cells, and exploring a method that can effectively improve hematopoietic differentiation and erythroid differentiation will improve the yield of blood cells derived from stem cells. After a large number of experimental studies, the inventors found that the addition of leukotrienes at a suitable stage in the differentiation and culture process of pluripotent stem cells can effectively promote the differentiation of hematopoietic stem / progenitor cells and further promote the differentiation of erythroid cells, significantly improving the differentiation efficiency of pluripotent stem cells.

[0006] Therefore, in a first aspect of the present application, the present application provides a use of leukotrienes in inducing stem cells to differentiate into hematopoietic stem / progenitor cells. According to an embodiment of the present application, the leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, improving the efficiency of the differentiation of stem cells into hematopoietic stem / progenitor cells.

[0007] According to an embodiment of the present application, the above use can further include at least one of the following additional technical features:

[0008] According to an embodiment of the present application, the leukotrienes include at least one of the following: LTB4, LTC4, LTD4, and LTE4.

[0009] According to an embodiment of the present application, the stem cells include at least one of pluripotent stem cells and induced pluripotent stem cells.

[0010] According to an embodiment of the present application, the pluripotent stem cells include at least one of human pluripotent stem cells and human induced pluripotent stem cells.

[0011] According to embodiments of the present application, the pluripotent stem cells comprise human embryonic stem cells.

[0012] According to embodiments of the present application, the pluripotent stem cells comprise human embryonic stem cell line-H1.

[0013] According to some embodiments of the present application, the hematopoietic stem cells, hematopoietic progenitor cells can significantly increase the content of red blood cell lineage cells, such as red blood cells, after further differentiation. The hematopoietic stem / progenitor cells obtained by the present application can be used for treating blood diseases.

[0014] In a second aspect of the present application, the present application provides a use of leukotrienes in the preparation of a reagent. According to embodiments of the present application, the reagent is used for inducing differentiation of stem cells into hematopoietic stem / progenitor cells. As described above, leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and improve the efficiency of differentiation of stem cells into hematopoietic stem / progenitor cells. Therefore, the reagent containing leukotrienes also has the effect of promoting the differentiation of stem cells into hematopoietic stem / progenitor cells and improving the efficiency of differentiation of stem cells into hematopoietic stem / progenitor cells.

[0015] According to embodiments of the present application, the use described above can further include at least one of the following additional technical features:

[0016] According to embodiments of the present application, the leukotrienes comprise at least one of the following: LTB4, LTC4, LTD4, and LTE4.

[0017] According to embodiments of the present application, the stem cells comprise at least one of pluripotent stem cells and induced pluripotent stem cells.

[0018] According to embodiments of the present application, the pluripotent stem cells comprise at least one of human pluripotent stem cells and human induced pluripotent stem cells.

[0019] According to embodiments of the present application, the pluripotent stem cells comprise human embryonic stem cells.

[0020] According to embodiments of the present application, the pluripotent stem cells comprise human embryonic stem cell line-H1.

[0021] In a third aspect of the present application, the present application provides a culture medium system. According to embodiments of the present application, the culture medium system comprises a first stage culture medium and / or a second stage culture medium, wherein the first stage culture medium and / or the second stage culture medium comprises leukotrienes. As described above, leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and improve the efficiency of differentiation of stem cells into hematopoietic stem / progenitor cells. Therefore, the culture medium containing leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and improve the efficiency of differentiation of stem cells into hematopoietic stem / progenitor cells.

[0022] According to embodiments of the present application, the culture medium system described above can further comprise at least one of the following additional technical features:

[0023] According to embodiments of the present application, the leukotriene comprises at least one of the following: LTB4, LTC4, LTD4 and LTE4.

[0024] According to embodiments of the present application, the leukotriene is LTB4.

[0025] According to embodiments of the present application, the final concentration of the leukotriene in the first-stage culture medium and / or the second-stage culture medium is 1-320 nM. According to some specific embodiments of the present application, the inventors screened and optimized the concentration of the leukotriene, and found that when the final concentration is 1-320 nM, the stem cells can be efficiently induced to differentiate into hematopoietic stem / progenitor cells.

[0026] According to embodiments of the present application, the first-stage culture medium further comprises: AA2P, bFGF, BMP4, Activin A, Glutamax and P / S.

[0027] According to embodiments of the present application, the second-stage culture medium comprises: AA2P, bFGF, VEGF, SB431542, Glutamax and P / S. The inventors further optimized and screened the concentration of each component in the first-stage culture medium and the second-stage culture medium.

[0028] According to embodiments of the present application, in the first-stage culture medium, the final concentration of the AA2P is 45-55 μg / mL.

[0029] According to embodiments of the present application, in the first-stage culture medium, the final concentration of the bFGF is 20-30 ng / mL.

[0030] According to embodiments of the present application, in the first-stage culture medium, the final concentration of the BMP4 is 20-30 ng / mL.

[0031] According to embodiments of the present application, in the first-stage culture medium, the final concentration of the Activin A is 20-30 ng / mL.

[0032] According to embodiments of the present application, in the second-stage culture medium, the final concentration of the AA2P is 45-55 μg / mL.

[0033] According to embodiments of the present application, in the second-stage culture medium, the final concentration of the bFGF is 20-30 ng / mL.

[0034] According to an embodiment of the present application, the final concentration of VEGF in the second stage medium is 45-50 ng / mL.

[0035] According to an embodiment of the present application, the final concentration of SB431542 in the second stage medium is 2-8 μM.

[0036] According to an embodiment of the present application, the first stage medium and the second stage medium further comprise Advanced D / F12 basal medium.

[0037] In a fourth aspect of the present application, a method for inducing stem cells to differentiate into hematopoietic stem / progenitor cells is provided. According to an embodiment of the present application, the method comprises: 1) culturing the stem cells in the first stage medium of the medium system as described above to obtain first stage induced cells; and 2) culturing the first stage induced cells in the second stage medium of the medium system as described above to obtain the hematopoietic stem / progenitor cells. As described above, leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and thus the method according to an embodiment of the present application can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells.

[0038] According to an embodiment of the present application, the method can further comprise at least one of the following additional technical features:

[0039] According to an embodiment of the present application, the stem cells comprise at least one of pluripotent stem cells and induced pluripotent stem cells.

[0040] According to an embodiment of the present application, the pluripotent stem cells comprise at least one of human pluripotent stem cells and human induced pluripotent stem cells.

[0041] According to an embodiment of the present application, the pluripotent stem cells comprise human embryonic stem cells.

[0042] According to an embodiment of the present application, the pluripotent stem cells comprise human embryonic stem cell line-H1.

[0043] In a fifth aspect of the present application, the use of leukotrienes in the preparation of a medicament for treating or preventing hematopoietic support therapy related diseases is provided. As described above, leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and thus the medicament comprising leukotrienes can effectively promote the differentiation of stem cells into hematopoietic stem / progenitor cells, and can also promote the differentiation of hematopoietic stem / progenitor cells into erythroid cells, thereby effectively treating or preventing hematopoietic support therapy related diseases.

[0044] According to the embodiments of the present application, the above pharmaceutical use can further include at least one of the following additional technical features:

[0045] According to the embodiments of the present application, the hematopoietic support therapy related disease is at least one of blood system tumor, solid tumor and immune system disease.

[0046] According to the embodiments of the present application, the blood system tumor includes at least one of chronic myelocytic leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome, aplastic anemia, Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, paroxysmal nocturnal hemoglobinuria and amegakaryocytic thrombocytopenia.

[0047] According to the embodiments of the present application, the solid tumor includes at least one of breast cancer, ovarian cancer, testicular cancer, neuroblastoma and small cell lung cancer.

[0048] According to the embodiments of the present application, the immune system disease includes at least one of severe combined immunodeficiency and severe autoimmune disease.

[0049] Additional aspects and advantages of the present application will be given in part in the following description, become apparent from the following description, or be learned by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0050] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0051] Figure 1 is a result chart of hematopoietic differentiation level detection of pluripotent stem cell culture induction to Day 6 according to embodiments of the present application;

[0052] Figure 2 is a result chart of hematopoietic differentiation level detection of pluripotent stem cell culture induction to Day 6 according to embodiments of the present application;

[0053] Figure 3 is a result chart of hematopoietic differentiation level detection of pluripotent stem cell culture induction to Day 6 according to embodiments of the present application;

[0054] Figure 4 is a result chart of hematopoietic differentiation level detection of pluripotent stem cell culture induction to Day 6 according to embodiments of the present application;

[0055] Figure 5 is a result chart of the hematopoietic differentiation and erythroid differentiation level of pluripotent stem cells according to an embodiment of the present application when the pluripotent stem cells are induced for 18 days;

[0056] Figure 6 is a result chart of the survival efficiency of red blood cells and the number of red blood cells when pluripotent stem cells are induced for 18 days according to an embodiment of the present application;

[0057] Figure 7 is a photograph of red blood cells when pluripotent stem cells are induced for 18 days according to an embodiment of the present application;

[0058] Figure 8 is a result chart of the hematopoietic differentiation level of pluripotent stem cells when the pluripotent stem cells are induced for 6 days according to an embodiment of the present application, wherein different concentrations of LTB4 are added. DETAILED DESCRIPTION

[0059] Embodiments of the present application are described in detail below with reference to examples shown in the accompanying drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and cannot be understood as limiting the present application.

[0060] In the present application, the "leukotriene" is a group of substances produced by the metabolism of arachidonic acid through the 5-lipoxygenase pathway, and the leukotriene is produced by leukocytes and has a conjugated triene structure, which includes leukotriene A4 (LTA4), leukotriene B4 (LTB4), leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4), wherein the molecular formula of LTA4 is C 20 H 30 O3, and the molar mass is 318.45; the molecular formula of LTB4 is C 20 H 32 O4, and the molar mass is 336.46; the molecular formula of LTC4 is C 30 H 47 N3O9S, and the molar mass is 625.775; the molecular formula of LTD4 is C 25 H 40 N2O6S, and the molar mass is 496.66.

[0061] In the present application, the "first stage of differentiation of pluripotent stem cells into erythroid cells" refers to the stage of differentiation of pluripotent stem cells into mesoderm cells; the "second stage of differentiation of pluripotent stem cells into erythroid cells" refers to the stage of differentiation of mesoderm cells into hemogenic endothelial cells; the "third stage of differentiation of pluripotent stem cells into erythroid cells" refers to the stage of differentiation of hemogenic endothelial cells into erythroblasts; and the "fourth stage of differentiation of pluripotent stem cells into erythroid cells" refers to the stage of differentiation of erythroblasts into red blood cells.

[0062] As used herein, "erythroid cells" include at least one of mature red blood cells, young red blood cells, early, middle, and late red blood cells.

[0063] As used herein, "megakaryocytic cells" refer to a type of cells in bone marrow that are differentiated from hematopoietic stem cells, with large nuclei, including at least one of primitive megakaryocytes, young megakaryocytes, and megakaryocytes and platelets.

[0064] As used herein, "granulocytic cells" include at least one of eosinophils, neutrophils, and basophils.

[0065] As used herein, "monocytic cells" include at least one of primitive monocytes, young (pre-) monocytes, and monocytes.

[0066] The following examples will be described in detail. In the following examples, the experimental methods used are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0067] Example 1 Effect of LTB4 on the induced differentiation of hESC-H1 into hematopoietic stem / progenitor cells and red blood cells

[0068] In this example, the inventors added small molecules or cytokines at different stages to simulate the process of red blood cell development in the human body, to promote the differentiation of hESC-H1 into red blood cells through hemogenic endothelium, hematopoietic stem / progenitor cells, erythroid progenitor cells, and erythroblasts, and the induction system is as shown in Figure 1 The effect of LTB4 on the induced differentiation of hESC-H1 into erythroid cells was detected by adding LTB4 at the second stage of the induction system, and the specific experimental process is as follows:

[0069] 1.1 Induction culture of hESC-H1

[0070] 1) hESC-H1 (purchased from WiCell Corporation, USA) was resuscitated in a 6-well plate and cultured using mTeSR Plus medium for 2-3 passages. When the cells grew to the appropriate density, the subsequent in vitro induction could be performed;

[0071] 2) When the undifferentiated wild-type H1 in the 6-well plate was grown to 70%-80% of the bottom area of the plate, the old culture medium was aspirated, and the cells were washed once with 1 mL of PBS;

[0072] 4) 500 μL of Accutase was added to each well of the 6-well plate containing cells obtained in step 2), and incubated at 37°C for 3 min;

[0073] 5) After the digestion was completed, the edge of the 6-well culture plate was tapped gently to promote the cells to fall off the plate bottom;

[0074] 6) Add the cells obtained in step 5) into 3 mL of mTeSR Plus medium to terminate the digestion;

[0075] 7) Transfer the cell-containing mixture obtained in step 6) into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min at room temperature to obtain the cells;

[0076] 8) Meanwhile, prepare mTeSR Plus medium containing 10 μM Y27632;

[0077] 9) Resuspend the cells obtained in step 7) by adding 1 mL of mTeSR Plus containing 10 μM Y27632 and count them;

[0078] 10) Seed the cells obtained in step 9) in a low-attachment 6-well plate at a density of 2 x 10 5 / mL, add 3 mL of mTeSR Plus containing 10 μM Y27632 per well, mark as Day-1, and place in a constant-temperature incubator at 37°C and 5% CO2 for 24 h of culture;

[0079] 11) After the culture in step 10) is completed, centrifuge the cells at 1000 rpm for 5 min at room temperature to obtain them;

[0080] 12) Replace the first-stage induction medium with the cells obtained in step 11) to mark Day 0;

[0081] 13) After culturing the cells in the first-stage induction medium for two days, centrifuge the cells at 1200 rpm for 5 min at room temperature to obtain them, mark as Day 2;

[0082] 14) Replace the second-stage induction medium with the cells obtained in step 13) every day, and after four days of culture, centrifuge the cells at 1400 rpm for 5 min at room temperature to obtain them, mark as Day 6;

[0083] 15) Replace the third-stage medium with the cells obtained in step 14) every other day, add 2 mL of medium on the second day of replacement, and after nine days of culture, filter the cells with a 100-μm mesh, discard the remaining EBs, centrifuge the cells at 1600 rpm for 5 min at room temperature to obtain them, mark as Day 15;

[0084] 16) Seed the cells obtained in step 15) in a general 6-well plate at a density of 2 x 10 5 / mL, add 3 mL of fourth-stage medium per well, and add medium as needed, and after three days of culture, mark as D18.

[0085] In the experiment, the experimental groups (LTB4-S2, H1-LTB4 +The culture medium used for each culture stage is as follows:

[0086] Stage 1 induction medium: Advanced D / F12 base medium, AA2P (50 pg / mL), bFGF (25 ng / mL), BMP4 (25 ng / mL), Activin A (25 ng / mL), Glutamax (1X), P / S (1X).

[0087] Stage 2 induction medium: Advanced D / F12 base medium, AA2P (50 pg / mL), bFGF (25 ng / mL), VEGF (50 ng / mL), SB431542 (5 mM), Glutamax (1X), P / S (1X), and LTB4 (300 nM).

[0088] Stage 3 induction medium: BEL base medium (200 mL), SCF (50 ng / mL), TPO (20 ng / mL), Flt3L (20 ng / mL), IL-3 (20 ng / mL), VEGF (20 ng / mL), SB431542 (10 mM), EPO (PeproTech) (5 u / mL), Transferrin (100 pg / mL); wherein the BEL base medium comprises: IMDM (91 mL), F12 (91 mL), 10% Deionized BSA (5 mL), Lindeic acid (20 pL), Linolenic acid (20 pL), AA2P (2 mL), Synthe chol (400 pL), a-MTG (7.8 pL), GlutaMax (2 mL), P / S (1 mL), Protein-free hybHdonta mix (PFHM) (10 mL), Insulin-transferrin selenium (ITS) (2 mL).

[0089] Stage 4 induction medium: Basic medium (200 mL), EPO (pepro Tech) (5 u / mL), heparin (3 u / mL); wherein the Basic medium comprises: IMDM (91 mL), F12 (91 mL), AB serum (5 mL), Linoleic acid (20 μL), Linolenic acid (20 μL), AA2P (2 mL), Synthechol (400 μL), a-MTG (7.8 μL), GlutaMAX (2 mL), P / S (1 mL), PFHM (10 mL), ITS (2 mL).

[0090] In this embodiment, a control group (CON, H1-CON) without LTB4 added in the second stage induction medium is also provided. The components and concentrations of the first, third and fourth stage induction medium and other components in the second stage induction medium are the same as those of the experimental group, except that no LTB4 is added.

[0091] 1.2 Flow cytometry detection

[0092] The cells collected on day 6 (Day 6), day 12 (Day 12) and day 18 (Day 18) during the induction process in step 1.1 (experimental group, H1-LTB4 + ) and the cells without the addition of LTB4 (control group, H1-CON) during the induction process were subjected to flow cytometry detection, and the specific experimental operation was as follows:

[0093] 1) After the cell mass collected on day 6 (Day 6), day 12 (Day 12) and day 18 (Day 18) was blown evenly, 1 mL of culture medium containing cells was taken, centrifuged at room temperature at 1000 rpm for 5 min, and the supernatant was discarded;

[0094] 2) The cell mass obtained in step 1) was added to 500 μL of Accutase digestion solution, mixed evenly by beating, and digested in a 37°C incubator for 3 min;

[0095] 3) After the above digestion operation, the cells were blown again to facilitate the digestion of the cell mass into single cells;

[0096] 4) 1 mL of medium was added to the single cells to terminate digestion, and the cells were transferred to a 15 mL centrifuge tube containing 8 mL of PBS, centrifuged at room temperature at 1000 rpm for 5 min;

[0097] 5) After centrifugation of the product obtained in step 4), the supernatant was discarded, and the cells were resuspended in 100 μL of PBS in a 1.5 mL EP tube;

[0098] 6) Label the corresponding flow antibody, incubate at 4°C refrigerator for 30 min, after incubation, add 1 mL PBS to wash the cells twice, and resuspend the cells with 300 μL PBS;

[0099] 7) Put the resuspended cells of step 6) through a screen and into a flow tube, and detect the cell marker proteins and other indicators on the machine.

[0100] 1.3 Cell expansion detection

[0101] The human embryonic stem cells H1 were induced for erythroid differentiation in stages, and cell counting was performed at day 18 to detect the expansion of the cells in the experimental group and the control group.

[0102] 1.4 Experimental result analysis

[0103] The specific experimental results are shown in Figures 2-8 , in which the expression of the blood-endothelial markers CD144 and CD31, the hematopoietic progenitor cell markers CD43 and CD45, and the erythrocyte-specific markers CD71 and CD235a was analyzed by flow cytometry at different stages of the induction of the human embryonic stem cells H1. Figure 2 It can be seen that, at day 6 of the culture, the proportion of the blood-endothelial cells expressing CD31 / CD144, CD31 / CD34, and CD34 / CD44 in the experimental group was significantly increased compared with the control group. Figure 3 It can be seen that, at day 9 of the culture, the number of the erythroid colonies CFU-E (erythroid burst-forming unit) and BFU-E (erythroid colony-forming unit) was significantly higher than that of the control group. Figure 4 It can be seen that, at day 12 of the culture, the number of the hematopoietic progenitor cells expressing CD43 + and CD45 - , the hematopoietic progenitor cells with a higher maturity expressing CD43 + and CD45 + , and the erythroblasts expressing CD71 + and CD235a + in the experimental group was significantly increased compared with the control group. Figure 5 The experimental results shown in Figure 6 and Figure 7 indicate that, at day 18 of the induction and culture of the human embryonic stem cells H1, the induced erythrocytes obtained in the experimental group and the control group efficiently expressed CD235a + .

[0104] Effect of LTB4 concentration on the induced differentiation of hESC-H1 into hematopoietic stem / progenitor cells and red blood cells

[0105] In this example, the hESC-H1 (purchased from WiCell Research Institute, USA) was induced to culture with different concentrations (0, 1, 50, 150, 300 nM) of LTB4 in the second culture stage, and the expression of cell markers of the cells obtained on the 6th day of culture was detected by flow cytometry, wherein the first, second, third and fourth stage induction culture of the hESC-H1 and the detection step of flow cytometry refer to Example 1.

[0106] The experimental results are shown in Table 2. Figure 8 As shown in Table 2, the addition of different concentrations of LTB4 in the second induction stage can effectively promote the expression of the hematopoietic endothelial cell markers CD31 / CD144 / CD34 / CD44 on the 6th day of culture.

[0107] In summary, the results of Example 1 and Example 2 show that the addition of 1-300 nM LTB4 in the second stage of the existing pluripotent stem cell erythroid induction culture system can significantly promote the differentiation and expansion efficiency of the pluripotent stem cells into red blood cells.

[0108] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0109] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. Use of leukotrienes in inducing stem cell differentiation into hematopoietic stem / progenitor cells, wherein the leukotriene is LTB4; The stem cells mentioned are human embryonic stem cells.

2. The use according to claim 1, characterized in that, The stem cells mentioned are human embryonic stem cell line-H1.

3. Use of leukotrienes in the preparation of reagents used to induce stem cells to differentiate into hematopoietic stem / progenitor cells, wherein the leukotrienes are LTB4; The stem cells mentioned are human embryonic stem cells.

4. The use according to claim 3, characterized in that, The stem cells mentioned are human embryonic stem cell line-H1.

5. A method for inducing stem cells to differentiate into hematopoietic stem / progenitor cells, characterized in that, include: 1) The stem cells are cultured in a first-stage culture medium in a culture medium system to obtain first-stage induced cells; 2) The cells induced in the first stage are cultured in the second stage culture medium of the culture medium system to obtain the hematopoietic stem / progenitor cells; The stem cells include at least one of human pluripotent stem cells and human induced pluripotent stem cells; The culture medium system includes: a first-stage culture medium and / or a second-stage culture medium, wherein the first-stage culture medium and / or the second-stage culture medium contains leukotrienes, and the leukotrienes are LTB4. The stem cells mentioned are human embryonic stem cells.

6. The method according to claim 5, characterized in that, The stem cells mentioned are human embryonic stem cell line-H1.

7. The method according to claim 5, characterized in that, The final concentration of the leukotrienes in the first-stage culture medium and / or the second-stage culture medium is 1-320 nM.

8. The method according to claim 5, characterized in that, The culture medium for the first stage further includes: AA2P, bFGF, BMP4, Activin A, Glutamax, and P / S; Optionally, the culture medium for the second stage includes: AA2P, bFGF, VEGF, SB431542, Glutamax and P / S; Optionally, in the culture medium of the first stage, the final concentration of AA2P is 45-55 µg / mL; Optionally, in the culture medium of the first stage, the final concentration of bFGF is 20-30 ng / mL; Optionally, in the culture medium of the first stage, the final concentration of BMP4 is 20-30 ng / mL; Optionally, in the culture medium of the first stage, the final concentration of ActivinA is 20-30 ng / mL; Optionally, in the culture medium of the second stage, the final concentration of AA2P is 45-55 µg / mL; Optionally, in the culture medium of the second stage, the final concentration of bFGF is 20-30 ng / mL; Optionally, in the culture medium of the second stage, the final concentration of VEGF is 45-50 ng / mL; Optionally, in the culture medium of the second stage, the final concentration of SB431542 is 2-8 µM.

9. The method according to claim 5, characterized in that, The first-stage culture medium and the second-stage culture medium also include AdvancedD / F12 basal medium.

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