Method for promoting hematopoietic differentiation of stem cells and application thereof

Abstract

The invention provides a method for promoting hematopoietic differentiation of stem cells and application of the method. The method comprises the following steps: carrying out hematopoietic differentiation culture on the stem cells by adopting culture mediums containing histone deacetylase inhibitors in sequence; the culture medium containing the histone deacetylase inhibitor comprises a culture medium I, a culture medium II and a culture medium III, wherein the culture medium I, the culture medium II and the culture medium III are culture mediums containing a histone deacetylase inhibitor; the histone deacetylase inhibitor is selected from sodium butyrate or sodium valproate. The histone deacetylase inhibitor is added into the culture medium for hematopoietic differentiation, so that the efficiency of obtaining the hematopoietic precursor cells through hematopoietic differentiation of the human pluripotent stem cells can be remarkably improved, and hematopoietic differentiation can be effectively promoted.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for promoting hematopoietic differentiation of stem cells and an application thereof. Background Art

[0002] Hematopoietic stem cells (HSCs) are adult stem cells in the blood system. They are a heterogeneous group with the ability of long-term self-renewal and the potential to differentiate into various mature blood cells. Due to their ability to reconstruct hematopoiesis, hematopoietic stem cells are widely used in the clinic to treat various blood diseases, including hereditary diseases (such as thalassemia, sickle cell anemia, aplastic anemia or Fanconi anemia, etc.) and non-hereditary diseases (such as leukemia, etc.). However, due to the limited sources of hematopoietic stem cells, difficult matching, and obstacles to in vitro expansion, their clinical application is greatly limited.

[0003] The differentiation pathway of hematopoietic stem cells is very complex, and the hematopoietic system is generally divided into two major branches: myeloid and lymphoid. The myeloid lineage is responsible for three major functions: oxygen transport, hemostasis, and innate immunity; the lymphoid lineage produces B, T, and natural killer (NK) cells, which are responsible for adaptive immunity.

[0004] CN112226409A provides an improved embryoid body (EB) differentiation system, which can differentiate embryoid bodies (EB) into CD34 + Differentiation of hematopoietic progenitor cells. The invention also provides a method for differentiating and culturing the embryoid bodies using an improved embryoid body differentiation system, thereby achieving efficient formation of embryoid bodies and overcoming the problem of easy adhesion of embryoid body differentiation spheres.

[0005] Although the prior art discloses a variety of hematopoietic differentiation methods, the efficiency of the existing hematopoietic differentiation methods still needs to be improved. Therefore, it is necessary to provide a hematopoietic differentiation method that improves the efficiency of hematopoietic differentiation and promote the clinical application of human pluripotent stem cells (hPSCs). Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a method for promoting hematopoietic differentiation of stem cells and its application. The method provided by the present invention can greatly improve the efficiency of hematopoietic differentiation and effectively promote the induction and differentiation of human pluripotent stem cells into CD31 + CD34 + Endothelial cells and CD31 + CD43 +The inventors found that the addition or non-addition of sodium butyrate or sodium valproate has a great influence on the efficiency of hematopoietic differentiation of stem cells. When sodium butyrate or sodium valproate is added, the differentiation efficiency of stem cells is higher and the cell yield is higher; when sodium butyrate or sodium valproate is not added, the differentiation efficiency of stem cells is reduced.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for promoting hematopoietic differentiation of stem cells, comprising adding a sirtuin inhibitor to a hematopoietic differentiation medium.

[0009] Preferably, the deacetylase inhibitor is a histone deacetylase inhibitor.

[0010] Preferably, the histone deacetylase inhibitor is selected from sodium butyrate or sodium valproate.

[0011] In the present invention, experiments were conducted on different types of histone deacetylase inhibitors, and the results showed that compared with other histone deacetylase inhibitors, sodium butyrate or sodium valproate had significant effects on promoting hematopoietic differentiation of stem cells. The inventors found that for the hematopoietic differentiation of stem cells, the addition and non-addition of sodium butyrate or sodium valproate had a great influence on the efficiency of hematopoietic differentiation of stem cells. When sodium butyrate or sodium valproate was added, the differentiation efficiency of stem cells was higher and the cell yield was higher; while when sodium butyrate or sodium valproate was not added, the differentiation efficiency of stem cells was reduced.

[0012] Preferably, the method comprises: sequentially using culture media containing histone deacetylase inhibitors to culture stem cells for hematopoietic differentiation; the culture media containing histone deacetylase inhibitors comprises culture media I, culture media II and culture media III;

[0013] Wherein, the culture medium I comprises a basal culture medium, a histone deacetylase inhibitor, BMP4 and ACTIVINA;

[0014] The culture medium II comprises a basal culture medium, a histone deacetylase inhibitor, bFGF and VEGF;

[0015] The culture medium III includes a basal medium, a histone deacetylase inhibitor, bFGF, VEGF and SB431542.

[0016] Preferably, the final concentration of the histone deacetylase inhibitor in medium I, medium II and medium III is independently 1-1000 μM, for example, 1 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM or 1000 μM, etc.

[0017] Preferably, when the histone deacetylase inhibitor is sodium butyrate, the final concentration of sodium butyrate in culture medium I, culture medium II and culture medium III is independently 100-500 μM, for example, 100 μM, μM, 200 μM, 300 μM, 400 μM or 500 μM, etc.

[0018] Preferably, when the histone deacetylase inhibitor is sodium valproate, the final concentration of sodium valproate in culture medium I, culture medium II and culture medium III is independently 1-1000 μM, for example, it can be 1 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM or 1000 μM, etc.

[0019] In the present invention, histone deacetylase inhibition can significantly increase the hematopoietic differentiation efficiency of stem cells, especially when sodium butyrate or sodium valproate is used, it has a better effect of promoting hematopoietic differentiation, wherein the concentration range of sodium butyrate is 100-500 μM, or the concentration range of sodium valproate is 1-1000 μM, the hematopoietic differentiation promoting effect is the best; when the addition amount of the histone deacetylase inhibitor is too low, its effect of promoting differentiation is weakened, and when the addition amount of the histone deacetylase inhibitor is too high, it is not conducive to cell differentiation, so it is necessary to control the histone deacetylase inhibitor to be added within an appropriate range.

[0020] In the present invention, the culture medium I comprises a basal culture medium, a histone deacetylase inhibitor, BMP4 (human bone morphogenetic protein-4) and ACTIVIN A (activin A).

[0021] Preferably, the basal medium in the culture medium I is E6 medium.

[0022] Preferably, the culture medium I comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A.

[0023] In the present invention, the concentration of the histone deacetylase inhibitor is 1-1000 μM, for example, it can be 1 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM or 1000 μM, etc. The concentration of the BMP4 is 2-100 ng / mL, for example, it can be 2 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc. The concentration of the ACTIVIN A is 10-100 ng / mL, for example, it can be 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc.

[0024] In the present invention, when the histone deacetylase inhibitor is sodium butyrate, the culture medium I comprises, in terms of final concentrations: 100-500 μM sodium butyrate, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A.

[0025] In the present invention, when the histone deacetylase inhibitor is sodium valproate, the culture medium I comprises, in terms of final concentrations: 1-1000 μM sodium valproate, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A.

[0026] In the present invention, the culture medium I can effectively promote mesoderm differentiation. Among them, the E6 culture medium can be used for lineage differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells; BMP-4 is a pleiotropic ligand protein, belonging to the TGFβ family, involved in vascular system circulation, can activate receptors on vascular cells, and can promote induced differentiation; Activin A is a multifunctional cytokine, a member of the TGF-β superfamily, and can regulate hematopoietic cell proliferation.

[0027] In the present invention, the culture medium II comprises a basal culture medium, a histone deacetylase inhibitor, bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor).

[0028] Preferably, the basal medium in the culture medium II is E6 medium.

[0029] Preferably, the culture medium II comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF and 5-100 ng / mL VEGF.

[0030] In the present invention, the concentration of the histone deacetylase inhibitor is 1-1000 μM, for example, it can be 1 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM or 1000 μM, etc. The concentration of the bFGF is 5-100 ng / mL, for example, it can be 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc. The concentration of the VEGF is 5-100 ng / mL, for example, it can be 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc.

[0031] In the present invention, when the histone deacetylase inhibitor is sodium butyrate, the culture medium II comprises, in terms of final concentrations: 100-500 μM sodium butyrate, 5-100 ng / mL bFGF and 5-100 ng / mL VEGF.

[0032] In the present invention, when the histone deacetylase inhibitor is sodium valproate, the culture medium II comprises, in terms of final concentrations: 1-1000 μM sodium valproate, 5-100 ng / mL bFGF and 5-100 ng / mL VEGF.

[0033] In the present invention, the culture medium II can effectively promote endothelial cell differentiation.

[0034] In the present invention, the culture medium III comprises a basal culture medium, a histone deacetylase inhibitor, bFGF, VEGF and SB431542.

[0035] Preferably, the basal medium in the culture medium III is E6 medium.

[0036] Preferably, the culture medium III comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF and 2-50 μM SB431542.

[0037] In the present invention, the concentration of the histone deacetylase inhibitor is 1-1000 μM, for example, it can be 1 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM or 1000 μM, etc. The concentration of the bFGF is 5-100 ng / mL, for example, it can be 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc. The concentration of the VEGF is 5-100 ng / mL, for example, it can be 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL, etc. The concentration of SB431542 is 2-50 μM, for example, 2 μM, 10 μM, 20 μM, 30 μM, 40 μM or 50 μM.

[0038] In the present invention, when the histone deacetylase inhibitor is sodium butyrate, the culture medium III comprises, by final concentration, 100-500 μM sodium butyrate, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF and 2-50 μM SB431542.

[0039] In the present invention, when the histone deacetylase inhibitor is sodium valproate, the culture medium III comprises, by final concentration, 1-1000 μM sodium valproate, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF and 2-50 μM SB431542.

[0040] In the present invention, the culture medium III can effectively promote the generation of hematopoietic precursor cells.

[0041] Preferably, the method for promoting hematopoietic differentiation of stem cells comprises:

[0042] On day 0-1 of culture, the stem cells are cultured using medium I;

[0043] On the 2nd to 3rd day of culture, the stem cells are cultured using medium II;

[0044] On day 4 of culture, the stem cells were cultured using medium III.

[0045] Preferably, the stem cells comprise embryonic stem cells or induced pluripotent stem cells.

[0046] Preferably, the induced pluripotent stem cells are induced pluripotent stem cells derived from human urine cells.

[0047] Preferably, before culturing in medium I, the stem cells are seeded in a culture plate in advance, and the seeding density of the stem cells is (1-1.5)×10 5 Pieces / mL.

[0048] Preferably, the seeded stem cells are pre-cultured in mTeSR1 medium containing 8-10 μM Y27632 for 10-12 h.

[0049] The concentration of Y27632 in the present invention is 8-10 μM, for example, 8 μM, 9 μM or 10 μM.

[0050] In a second aspect, the present invention provides a culture medium for promoting hematopoietic differentiation of stem cells, the culture medium comprising culture medium I, culture medium II and culture medium III.

[0051] The culture medium I comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A;

[0052] The medium II includes, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, and 5-100 ng / mL VEGF;

[0053] The culture medium III includes, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF, and 2-50 μM SB431542.

[0054] As a preferred technical solution of the present invention, the culture medium I comprises: 100-500 μM sodium butyrate, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A in terms of final concentration.

[0055] The culture medium II includes, by final concentration, 100-500 μM sodium butyrate, 5-100 ng / mL bFGF, and 5-100 ng / mL VEGF.

[0056] The culture medium III includes, by final concentration, 100-500 μM sodium butyrate, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF, and 2-50 μM SB431542.

[0057] As a preferred technical solution of the present invention, the culture medium I comprises, in terms of final concentrations: 1-1000 μM sodium valproate, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A.

[0058] The culture medium II includes, by final concentration, 1-1000 μM sodium valproate, 5-100 ng / mL bFGF, and 5-100 ng / mL VEGF.

[0059] The culture medium III includes, by final concentration, 1-1000 μM sodium valproate, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF, and 2-50 μM SB431542.

[0060] In a third aspect, the present invention provides the use of the method for promoting hematopoietic differentiation of stem cells described in the first aspect and / or the culture medium for promoting hematopoietic differentiation of stem cells described in the second aspect in directed hematopoietic differentiation.

[0061] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The present invention adds a histone deacetylase inhibitor to the culture medium for hematopoietic differentiation, which can significantly improve the efficiency of obtaining hematopoietic precursor cells through hematopoietic differentiation of human pluripotent stem cells and effectively promote hematopoietic differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is a flow cytometry result of human induced pluripotent stem cells differentiating into hematopoietic precursor cells.

[0065] Figure 2 CD31 is produced by differentiation of human induced pluripotent stem cells + CD34 + Endothelial cells and CD31 + CD43 + Comparison of flow cytometry results of hematopoietic precursor cells. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0068] Example 1

[0069] This embodiment provides a method for promoting hematopoietic differentiation of stem cells, wherein the stem cells are induced pluripotent stem cells derived from human urine cells, comprising the following steps:

[0070] The induced pluripotent stem cells were seeded in a 12-well culture plate (the culture plate was pre-coated with GrowthFactor Reduced Matrigel (BD) overnight) at a seeding density of 1.5×10 5 The seeded embryonic stem cells were cultured in mTeSR1 medium (containing 10 μM Y27632) for 10-12 hours to obtain adherent cells in a 37° C., 5% CO 2 incubator.

[0071] On the 0th to 1st day of culture, the obtained adherent cells were cultured in medium I; the medium I was obtained by adding sodium butyrate, BMP4 (peprotech Inc.) and ACTIVIN A (Sino Biological Inc.) to the basal medium E6, wherein the final concentration of sodium butyrate was 250 μM, the final concentration of BMP4 was 50 ng / mL, and the final concentration of ACTIVIN A was 30 ng / mL.

[0072] On the 2nd to 3rd day of culture, culture medium II was used to continue culture; the culture medium II was composed of basal culture medium E6 supplemented with sodium butyrate, VEGF (Sino Biological Inc.) and bFGF (Sino Biological Inc.), wherein the final concentration of sodium butyrate was 250 μM, the final concentration of VEGF was 50 ng / mL, and the final concentration of bFGF was 50 ng / mL.

[0073] On the 4th day of culture, culture medium III was used to continue culture; culture medium III was composed of basal culture medium E6 supplemented with sodium butyrate, bFGF (Sino Biological Inc.), VEGF (Sino Biological Inc.) and SB431542, wherein the final concentration of sodium butyrate was 250 μM, the final concentration of bFGF was 50 ng / mL, the final concentration of VEGF was 50 ng / mL, and the final concentration of SB431542 (CAS: 301836-41-9) was 10 μM.

[0074] On the 7th day of differentiation, the formation of blood cells can be detected. The proportion of cells in each part of PE-CD31 (BD), FITC-CD34 (BD), and APC-CD43 (BD) was detected by flow cytometry.

[0075] Comparative Example 1

[0076] This comparative example provides a method for promoting hematopoietic differentiation of stem cells. The only difference between this comparative example and Example 1 is that the culture media I, II and III used do not contain sodium butyrate, and the remaining culture steps are carried out with reference to Example 1.

[0077] The test results of Example 1 and Comparative Example 1 are as follows Figure 1-2 As shown, Example 1 is recorded as the experimental group, and Comparative Example 1 is recorded as the control group. Figure 1 This is a flow cytometry result of human induced pluripotent stem cells differentiating into hematopoietic precursor cells. Figure 1 CD43 produced by the control group (control, without sodium butyrate) and the experimental group (NaB, with sodium butyrate) with or without sodium butyrate + The situation of hematopoietic precursor cells (HPCs); Figure 1 It can be seen that adding histone deacetylase inhibitors to the hematopoietic differentiation culture medium can significantly improve the efficiency of obtaining hematopoietic precursor cells through hematopoietic differentiation of human pluripotent stem cells and can effectively promote hematopoietic differentiation.

[0078] Figure 2 CD31 produced by differentiation of human induced pluripotent stem cells + CD34 + Endothelial cells and CD31 + CD43 + Comparison of flow cytometry results of hematopoietic progenitor cells; Figure 2 A is CD31 produced by differentiation of human induced pluripotent stem cells + CD34 + A is the flow cytometry results of endothelial cells, B is the statistical comparison of human induced pluripotent stem cells differentiating into hematopoietic precursor cells. Figure 2 The results also further demonstrated that adding histone deacetylase inhibitors to the hematopoietic differentiation medium can produce more CD31 + CD34 + Endothelial cells and produce more CD31 + CD43 + of hematopoietic precursor cells.

[0079] Example 2

[0080] This embodiment provides a method for promoting hematopoietic differentiation of stem cells. The difference between this embodiment and embodiment 1 is that:

[0081] On the 0th to 1st day of culture, the adherent cells were cultured in medium I; the medium I was obtained by adding sodium butyrate, BMP4 (peprotech Inc.) and ACTIVIN A (Sino Biological Inc.) to the basal medium E6, wherein the final concentration of sodium butyrate was 100 μM, the final concentration of BMP4 was 50 ng / mL, and the final concentration of ACTIVIN A was 30 ng / mL.

[0082] On the 2nd to 3rd day of culture, culture medium II was used to continue culture; the culture medium II was composed of basal culture medium E6 supplemented with sodium butyrate, VEGF (Sino Biological Inc.) and bFGF (Sino Biological Inc.), wherein the final concentration of sodium butyrate was 100 μM, the final concentration of VEGF was 50 ng / mL, and the final concentration of bFGF was 50 ng / mL.

[0083] On the 4th day of culture, culture medium III was used to continue culture; culture medium III was composed of basal culture medium E6 supplemented with sodium butyrate, bFGF (Sino Biological Inc.), VEGF (Sino Biological Inc.) and SB431542, wherein the final concentrations of sodium butyrate were 100 μM, bFGF were 50 ng / mL, VEGF were 50 ng / mL, and SB431542 were 10 μM.

[0084] The remaining steps refer to Example 1.

[0085] Example 3

[0086] This embodiment provides a method for promoting hematopoietic differentiation of stem cells. The difference between this embodiment and embodiment 1 is that:

[0087] On the 0th to 1st day of culture, the adherent cells were cultured in medium I; the medium I was obtained by adding sodium butyrate, BMP4 (peprotech Inc.) and ACTIVIN A (Sino Biological Inc.) to the basal medium E6, wherein the final concentration of sodium butyrate was 500 μM, the final concentration of BMP4 was 50 ng / mL, and the final concentration of ACTIVIN A was 30 ng / mL.

[0088] On the 2nd to 3rd day of culture, culture medium II was used to continue culture; the culture medium II was composed of basal culture medium E6 supplemented with sodium butyrate, VEGF (Sino Biological Inc.) and bFGF (Sino Biological Inc.), wherein the final concentration of sodium butyrate was 500 μM, the final concentration of VEGF was 50 ng / mL, and the final concentration of bFGF was 50 ng / mL.

[0089] On the 4th day of culture, culture medium III was used to continue culture; culture medium III was composed of basal culture medium E6 supplemented with sodium butyrate, bFGF (Sino Biological Inc.), VEGF (Sino Biological Inc.) and SB431542, wherein the final concentrations of sodium butyrate were 500 μM, bFGF were 50 ng / mL, VEGF were 50 ng / mL, and SB431542 were 10 μM.

[0090] The remaining steps refer to Example 1.

[0091] Example 4

[0092] This example provides a method for promoting hematopoietic differentiation of stem cells. The difference between this example and Example 1 is that the histone deacetylase inhibitor used in culture media I, II and III is sodium valproate, and the final concentration of the sodium valproate is 500 μM. The remaining steps refer to Example 1.

[0093] Example 5

[0094] This example provides a method for promoting hematopoietic differentiation of stem cells. The difference between this example and Example 1 is that the histone deacetylase inhibitor used in culture media I, II and III is sodium valproate, and the final concentration of the sodium valproate is 100 μM. The remaining steps refer to Example 1.

[0095] Example 6

[0096] This example provides a method for promoting hematopoietic differentiation of stem cells. The difference between this example and Example 1 is that the histone deacetylase inhibitor used in culture media I, II and III is sodium valproate, and the final concentration of the sodium valproate is 1000 μM. The remaining steps refer to Example 1.

[0097] Example 7

[0098] This example provides a method for promoting hematopoietic differentiation of stem cells. The difference between this example and Example 4 is that the final concentration of sodium valproate is 1200 μM. The remaining steps refer to Example 1.

[0099] Example 8

[0100] This embodiment provides a method for promoting hematopoietic differentiation of stem cells. The difference between this embodiment and embodiment 1 is that the concentration of sodium butyrate in the culture media I, II and III is 50 μM. The remaining steps refer to embodiment 1.

[0101] Example 9

[0102] This embodiment provides a method for promoting hematopoietic differentiation of stem cells. The difference between this embodiment and embodiment 1 is that the concentration of sodium butyrate in the culture media I, II and III is 600 μM. The remaining steps refer to embodiment 1.

[0103] In the methods for promoting hematopoietic differentiation of stem cells in Examples 1-9 and Comparative Example 1, the results of blood cell formation are shown in Table 1.

[0104] Table 1

[0105] method <![CDATA[CD31 + CD43 + Statistical results of hematopoietic precursor cells%]]> Example 1 59.97±3.96 Example 2 33.4±1.51 Example 3 22.91±1.63 Example 4 41.78±1.56 Example 5 23.48±1.2 Example 6 21.53±1.76 Example 7 2.6±0.49 Example 8 9.96±1.28 Example 9 5.5±0.64 Comparative Example 1 10.7±0.46

[0106] By comparing Examples 1-4 with Comparative Example 1, it can be seen that adding histone deacetylase inhibitors to the hematopoietic differentiation culture medium can significantly improve the efficiency of obtaining hematopoietic precursor cells through hematopoietic differentiation of human pluripotent stem cells, and can effectively promote hematopoietic differentiation.

[0107] By comparing Example 1 with Examples 2-9 and Comparative Example 1, it can be seen that histone deacetylase inhibition can significantly increase the hematopoietic differentiation efficiency of stem cells, especially when sodium butyrate or sodium valproate is used, it has a better effect of promoting hematopoietic differentiation, wherein the concentration range of sodium butyrate is 100-500 μM, or the concentration range of sodium valproate is 1-1000 μM, the effect of promoting hematopoietic differentiation is best, when the addition amount of the histone deacetylase inhibitor is too low, its promoting effect is weakened, when the addition amount of the histone deacetylase inhibitor is too high, it is not conducive to cell differentiation, so it is necessary to control the histone deacetylase inhibitor within an appropriate addition range.

[0108] In summary, the method for promoting hematopoietic differentiation of stem cells provided by the present invention can improve the efficiency of obtaining hematopoietic precursor cells by hematopoietic differentiation of human pluripotent stem cells, and can effectively promote hematopoietic differentiation and increase CD31 + CD34 + Endothelial cells and CD31 + CD43 + The addition of histone deacetylase inhibitors, especially sodium butyrate or sodium valproate, to the hematopoietic differentiation medium can significantly improve the efficiency of hematopoietic differentiation of human pluripotent stem cells.

[0109] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for promoting hematopoietic differentiation of stem cells, characterized in that: Add sirtuin inhibitors to the hematopoietic differentiation medium.

2. The method for promoting hematopoietic differentiation of stem cells according to claim 1, characterized in that: The deacetylase inhibitor is a histone deacetylase inhibitor; Preferably, the histone deacetylase inhibitor is selected from sodium butyrate or sodium valproate.

3. The method for promoting hematopoietic differentiation of stem cells according to claim 2, characterized in that: The method comprises: sequentially using culture media containing histone deacetylase inhibitors to perform hematopoietic differentiation culture on stem cells; the culture media containing histone deacetylase inhibitors comprises culture media I, culture media II and culture media III; Wherein, the culture medium I comprises a basal culture medium, a histone deacetylase inhibitor, BMP4 and ACTIVIN A; The culture medium II comprises a basal culture medium, a histone deacetylase inhibitor, bFGF and VEGF; The culture medium III includes a basal medium, a histone deacetylase inhibitor, bFGF, VEGF and SB431542.

4. The method for promoting hematopoietic differentiation of stem cells according to claim 3, characterized in that: The final concentration of the histone deacetylase inhibitor in medium I, medium II and medium III is independently 1-1000 μM; Preferably, when the histone deacetylase inhibitor is sodium butyrate, the final concentration of sodium butyrate in medium I, medium II and medium III is independently 100-500 μM; Preferably, when the histone deacetylase inhibitor is sodium valproate, the final concentration of sodium valproate in culture medium I, culture medium II and culture medium III is independently 1-1000 μM.

5. The method for promoting hematopoietic differentiation of stem cells according to claim 3 or 4, characterized in that: The basal medium in the culture medium I is E6 medium; Preferably, the culture medium I comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A; Preferably, the basal medium in the medium II is E6 medium; Preferably, the culture medium II comprises, by final concentration: 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF and 5-100 ng / mL VEGF; Preferably, the basal medium in the medium III is E6 medium; Preferably, the culture medium III comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF and 2-50 μM SB431542.

6. The method for promoting hematopoietic differentiation of stem cells according to any one of claims 3 to 5, characterized in that: The method comprises: On day 0-1 of culture, the stem cells are cultured using medium I; On the 2nd to 3rd day of culture, the stem cells are cultured using medium II; On the 4th day of culture, the stem cells are cultured using medium III; Preferably, the stem cells include embryonic stem cells or induced pluripotent stem cells; Preferably, the induced pluripotent stem cells are induced pluripotent stem cells derived from human urine cells.

7. The method for promoting hematopoietic differentiation of stem cells according to any one of claims 3 to 6, characterized in that: Before culturing in medium I, the stem cells were seeded in a culture plate at a seeding density of (1-1.5)×10 5 Pieces / mL.

8. The method for promoting hematopoietic differentiation of stem cells according to claim 7, characterized in that: The seeded stem cells were pre-cultured in mTeSR1 medium containing 8-10 μM Y27632 for 10-12 h.

9. A culture medium for promoting hematopoietic differentiation of stem cells, characterized in that: The culture medium includes culture medium I, culture medium II and culture medium III; The culture medium I comprises, by final concentration, 1-1000 μM histone deacetylase inhibitor, 2-100 ng / mL BMP4 and 10-100 ng / mL ACTIVIN A; The medium II includes, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, and 5-100 ng / mL VEGF; The culture medium III includes, by final concentration, 1-1000 μM histone deacetylase inhibitor, 5-100 ng / mL bFGF, 5-100 ng / mL VEGF, and 2-50 μM SB431542.

10. Use of the method for promoting hematopoietic differentiation of stem cells according to any one of claims 1 to 8 and / or the culture medium for promoting hematopoietic differentiation of stem cells according to claim 9 in directed hematopoietic differentiation.

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