Methods for co-culturing to induce stem cell differentiation into hematopoietic progenitor cells

Abstract

This invention discloses a method for co-culturing and inducing stem cell differentiation into hematopoietic progenitor cells. The method includes the following steps: culturing pluripotent stem cells to obtain embryoid bodies; culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and co-culturing the hematopoietic endothelial cells with mesenchymal stem cells to obtain hematopoietic progenitor cells. Through optimization of the culture system, the differentiation efficiency and the number of hematopoietic progenitor cells obtained are significantly improved; by co-culturing mesenchymal stem cells with hematopoietic endothelial cells, the number of hematopoietic progenitor cells is further increased while maintaining high differentiation efficiency and uniform and stable expression of various markers.

Description

Technical Field

[0001] This invention belongs to the field of cell technology, and more specifically, this invention relates to a method for co-culturing and inducing stem cells to differentiate into hematopoietic progenitor cells. Background Technology

[0002] Hematologic disorders are diseases originating in the hematopoietic system or affecting the hematopoietic system, resulting in abnormal changes in the blood. They often manifest as anemia, bleeding, and fever. The incidence of childhood malignant cancers in my country is on the rise. Data from 2014 shows that leukemia is the leading cause of childhood malignant tumors, accounting for approximately one-third. For malignant hematologic disorders, chemotherapy is often not very effective. Since Professor Thomas first pioneered hematopoietic stem cell (HSC) transplantation in the mid-20th century, HSC transplantation has been widely used in the clinical treatment of leukemia and has become one of the effective methods for treating acute leukemia, malignant lymphoma, and severe aplastic anemia.

[0003] Currently, HSCs are mainly derived from umbilical cord blood, bone marrow, and peripheral blood. HSC transplantation is mainly divided into autologous and allogeneic HSC transplantation. Although autologous transplantation has the advantages of no transplant rejection and no graft-versus-host disease, the supply of autologous HSCs stored in umbilical cord blood banks is insufficient to meet demand, limiting its clinical application in diseases. Although allogeneic transplantation has better long-term efficacy and a lower relapse rate than autologous transplantation, its matching efficiency is extremely low and the source is limited, thus restricting the clinical application of allogeneic HSC transplantation.

[0004] Therefore, there is an urgent need in this field to find safer, lower-cost, and more stable hematopoietic stem / progenitor cell resources. Pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, can differentiate into various tissues in the body. They can be used to create disease models, conduct drug toxicity tests, and, through cell transplantation, replace damaged or diseased cells, promoting wound repair and treating diseases. Hematopoietic stem cells exist in the body throughout life and can differentiate into various cells of the blood system, including erythrocytes, granulocytes, macrophages, monocytes, microglia, dendritic cells, B lymphocytes, T lymphocytes, and NK lymphocytes, and have significant value in the clinical treatment of hematological diseases and cancer.

[0005] Hematopoietic stem cells can rebuild the hematopoietic system, differentiate into various lineages of hematopoietic cells, and maintain their own stemness. However, it is difficult to expand hematopoietic stem cells in large quantities at the single-cell level in vitro. Furthermore, it is difficult to obtain hematopoietic stem cells that can be reconstructed in vivo for a long time through in vitro stem cell induction differentiation. Only hematopoietic progenitor cells that can be reconstructed in vivo for a short time can be obtained. Hematopoietic progenitor cells have the characteristics of hematopoietic stem cells and can differentiate into various lineages of blood cells, which can be used to solve clinical hematological diseases.

[0006] Currently, the main methods for inducing human pluripotent stem cells to differentiate into hematopoietic progenitor cells are embryomorphic differentiation and stromal cell co-culture. These methods also have some drawbacks: embryomorphic differentiation generally requires a large number of pluripotent stem cells, and the inconsistency in their differentiation stages leads to generally low differentiation efficiency and long processing times; stromal cell co-culture is inefficient, introduces animal-derived components, or uses serum-containing culture systems or feeder cells, making it unsuitable for subsequent clinical-grade cell preparations. Therefore, there is an urgent need in this field to develop a method for the efficient and rapid preparation of stably differentiated hematopoietic progenitor cells under serum-free conditions with a defined chemical composition. Summary of the Invention

[0007] The purpose of this invention is to develop a method for the efficient and rapid preparation of differentiated hematopoietic progenitor cells with a defined chemical composition under serum-free conditions.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A first aspect of the present invention provides a method for preparing hematopoietic progenitor cells, comprising the following steps:

[0010] (1) Culture pluripotent stem cells to obtain embryoid bodies;

[0011] (2) The embryoid body is subjected to mesodermal differentiation culture to obtain mesodermal cells;

[0012] (3) The mesodermal cells are subjected to hematopoietic endothelial differentiation culture to obtain hematopoietic endothelial cells;

[0013] (4) The hematopoietic endothelial cells are co-cultured with mesenchymal stem cells to obtain hematopoietic progenitor cells.

[0014] Preferably, the mesenchymal stem cells are selected from at least one of the following: endothelial mesenchymal stem cells, myocardial mesenchymal stem cells, bone mesenchymal stem cells, cartilage mesenchymal stem cells, tendon mesenchymal stem cells, skeletal muscle mesenchymal stem cells, bone mesenchymal stem cells, cartilage mesenchymal stem cells, tendon mesenchymal stem cells, adipose mesenchymal stem cells, neural mesenchymal stem cells, endocrine mesenchymal stem cells, hematopoietic mesenchymal stem cells, hematopoietic mesenchymal stem cells, liver mesenchymal stem cells, hepatocyte precursor mesenchymal stem cells, uterine blood mesenchymal stem cells, dental pulp mesenchymal stem cells, umbilical cord mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0015] Preferably, the culture system in step (1) contains a ROCK inhibitor. Preferably, the ROCK inhibitor includes, but is not limited to, at least one selected from the following: Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, and Narciclasine. Preferably, the concentration of the ROCK inhibitor is 1-50 μM; more preferably 5-20 μM; and even more preferably 10 μM. Preferably, the ROCK inhibitor is Y-27632, with a concentration of 1-50 μM; more preferably 5-20 μM; and even more preferably 10 μM.

[0016] Preferably, the culture system in step (1) is a pluripotent stem cell culture medium containing a ROCK inhibitor. Preferably, the pluripotent stem cell culture medium includes, but is not limited to, the following: E8 medium, mTESR medium, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF medium, StemMACS iPS-Brew medium, Stem-Partner ACF medium, TeSR-AOF medium, and TeSR2 medium.

[0017] Preferably, the culture time in step (1) is 12-30 hours, more preferably 15-24 hours; and even more preferably 20-24 hours.

[0018] Preferably, the culture system in step (2) contains BMP4 and / or a GSK-3β inhibitor. Preferably, the culture system in step (2) contains a GSK-3β inhibitor.

[0019] Preferably, the concentration of BMP4 is 0-100 ng / ml; more preferably, the concentration of BMP4 is 5-50 ng / ml; and even more preferably, the concentration of BMP4 is 10-20 ng / ml.

[0020] Preferably, the GSK-3β inhibitor includes, but is not limited to, at least one selected from the following: B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHI R-99021. Preferably, the concentration of the GSK-3β inhibitor is 0.5-20 μM; more preferably 1-10 μM; and even more preferably 3-5 μM. Preferably, the GSK-3β inhibitor is CHIR-99021, and its concentration is 0.5-20 μM; more preferably 1-10 μM; and even more preferably 3-5 μM.

[0021] Preferably, the culture system in step (2) does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0022] Preferably, no monothioglycerol (MTG) is added to the culture system in step (2).

[0023] Preferably, the culture system in step (2) is a basal culture medium containing BMP4 and / or GSK-3β inhibitors.

[0024] Preferably, the culture system or the basal culture medium in step (2) contains at least one, at least two, at least three, or four of the following: B27 additive, non-essential amino acids, glutamine, and vitamin C. Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (such as a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of the glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages are by weight-volume ratios, and the concentration of vitamin C is 10-100 ug / ml, more preferably 20-50 ug / ml, and even more preferably 50 ug / ml.

[0025] Preferably, the culture time in step (2) is 18-54 hours, more preferably 20-48 hours; and even more preferably 24-48 hours.

[0026] Preferably, the culture system of step (3) contains at least one, at least two, at least three or four of the following: BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGFβ / ALK inhibitor.

[0027] Preferably, the concentration of BMP4 is 1-50 ng / ml; more preferably, the concentration is 2-20 ng / ml; and even more preferably, the concentration is 5-10 ng / ml.

[0028] Preferably, the vascular endothelial growth factor (VEGF) includes, but is not limited to, at least one selected from the following: VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor. Preferably, the concentration of the VEGF is 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml. Preferably, the VEGF is VEGF-165 or VEGF-A, with a concentration of 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml.

[0029] Preferably, the fibroblast growth factor (FGF) is a polypeptide composed of about 150-200 amino acids, existing in two closely related forms: basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF), wherein the concentration of the FGF (acidic fibroblast kinase and / or basic fibroblast kinase) is 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml. Preferably, the FGF is FGF-2 (bFGF), with a concentration of 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml.

[0030] Preferably, the TGFβ / ALK inhibitor includes, but is not limited to, at least one selected from the following: SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761. Preferably, the concentration of the TGFβ / ALK inhibitor is 1-50 μM; more preferably 5-20 μM; and even more preferably 5-10 μM. Preferably, the TGFβ / ALK inhibitor is SB431542, and its concentration is 1-50 μM; more preferably 5-20 μM; and even more preferably 5-10 μM.

[0031] Preferably, the culture system in step (3) does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0032] Preferably, no monothioglycerol (MTG) is added to the culture system in step (3).

[0033] Preferably, the culture system in step (3) is a basal culture medium containing at least one, at least two, at least three or four of BMP4, vascular endothelial growth factor, fibroblast growth factor and TGFβ / ALK inhibitor.

[0034] Preferably, the culture system or basal culture medium in step (3) contains at least one, at least two, three, or four of the following: B27 additive, non-essential amino acids, glutamine, and vitamin C. Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (such as a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of the glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages are by weight-volume ratios, and the concentration of vitamin C is 10-100 ug / ml, more preferably 20-50 ug / ml, and even more preferably 50 ug / ml.

[0035] Preferably, the culture time in step (3) is 2-6 days, more preferably 3-5 days; and even more preferably 4 days.

[0036] Preferably, step (4) of co-culturing the hematopoietic endothelial cells and mesenchymal stem cells includes: mixing the hematopoietic endothelial cells differentiated in step (3) with the mesenchymal stem cells for co-culturing. Preferably, the mixing is: seeding the hematopoietic endothelial cells into the mesenchymal stem cells.

[0037] Preferably, in the mixing step, the growth density of mesenchymal stem cells is 60-80%; more preferably, it is around 70%.

[0038] Preferably, the mesenchymal stem cells used in step (4) are pretreated mesenchymal stem cells. Preferably, the pretreatment step involves: seeding mesenchymal stem cells and passage culturing them until the growth density reaches 60-80% (preferably around 70%) before use. Preferably, the initial seeding quantity of mesenchymal stem cells from different sources can be 4-10 × 10⁻⁶, depending on their growth rate. 4 / cm 2 (e.g. 5×10) 4 / cm 2 6×10 4 / cm 2 8×10 4 / cm 2 10×10 4 / cm 2 ).

[0039] Preferably, the culture system of step (4) contains at least one, at least two, or three of the following: BMP4, vascular endothelial growth factor, and stem cell factor.

[0040] Preferably, the concentration of BMP4 is 1-50 ng / ml; more preferably, the concentration is 2-20 ng / ml; and even more preferably, the concentration is 5-10 ng / ml.

[0041] Preferably, the vascular endothelial growth factor (VEGF) includes, but is not limited to, at least one selected from the following: VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor. Preferably, the concentration of the VEGF is 1-50 ng / ml; more preferably, the concentration is 5-20 ng / ml; even more preferably, the concentration is 10 ng / ml. Preferably, the VEGF is VEGF-165 or VEGF-A, with a concentration of 1-50 ng / ml; more preferably, the concentration is 5-20 ng / ml; even more preferably, the concentration is 10 ng / ml.

[0042] Preferably, the concentration of the stem cell growth factor (SCF) is 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; and even more preferably, the concentration is 20-50 ng / ml.

[0043] Preferably, the culture system in step (4) does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0044] Preferably, no monothioglycerol (MTG) is added to the culture system in step (4).

[0045] Preferably, the culture system in step (4) is a basic culture medium containing at least one, at least two, or three of BMP4, vascular endothelial growth factor, and stem cell factors.

[0046] Preferably, the culture system or basal culture medium in step (4) comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven of the following: B27 additive, non-essential amino acids, glutamine, vitamin C, N-acetyl-L-cysteine ​​(NAC), minocycline hydrochloride, and insulin-transferrin-selenium (ITS-G). Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (such as a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of the glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages mentioned above are mass-volume ratios. The concentration of vitamin C added is 10-100 μg / ml, more preferably 20-50 μg / ml, and even more preferably 50 μg / ml. The concentration of N-acetyl-L-cysteine ​​added is 5-100 μM, more preferably 10-50 μM, and even more preferably 30 μM. The concentration of minocycline hydrochloride added is 0.1-20 μM, more preferably 1-5 μM, and even more preferably 2 μM. The volume percentage of insulin-transferrin-selenium added is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%.

[0047] Preferably, the culture time in step (4) is 4-8 days, more preferably 5-7 days; and even more preferably 6 days.

[0048] Preferably, the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under serum-free conditions.

[0049] Preferably, the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under vitamin A-free conditions.

[0050] Preferably, the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under antibiotic-free conditions.

[0051] Preferably, the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under thioglycerol-free conditions.

[0052] Preferably, the method for preparing hematopoietic progenitor cells does not require purification and / or enrichment steps.

[0053] Preferably, the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under conditions without a trophoblast layer.

[0054] Preferably, the culture in any of steps (1) to (4) is a suspension culture or an adherent culture.

[0055] A second aspect of the present invention provides a kit for preparing hematopoietic progenitor cells, comprising at least one, at least two, at least three, or four of the following: an embryoid culture system, a mesodermal differentiation culture system, a hematopoietic endothelial differentiation culture system, and a mesenchymal stem cell co-culture differentiation system. Preferably, the kit comprises a mesodermal differentiation culture system, a hematopoietic endothelial differentiation culture system, and a mesenchymal stem cell co-culture differentiation system. Preferably, the kit further comprises an embryoid culture system.

[0056] Preferably, the kit is used in the method for preparing hematopoietic progenitor cells according to the first aspect of the present invention.

[0057] Preferably, the embryoid culture system contains a ROCK inhibitor. Preferably, the ROCK inhibitor includes, but is not limited to, at least one selected from the following: Blebbistatin, HA-100, Y-27632, HA-1077, KD-025, Y-33075, and Narciclasine. Preferably, the concentration of the ROCK inhibitor is 1-50 μM; more preferably 5-20 μM; and even more preferably 10 μM. Preferably, the ROCK inhibitor is Y-27632, with a concentration of 1-50 μM; more preferably 5-20 μM; and even more preferably 10 μM.

[0058] Preferably, the embryoid culture system is a pluripotent stem cell culture medium containing a ROCK inhibitor. Preferably, the pluripotent stem cell culture medium includes, but is not limited to, the following: E8 medium, mTESR medium, StemFit Basic 03, StemFit Basic 04, NutriStem hPSC XF medium, StemMACS iPS-Brew medium, Stem-PartnerACF medium, TeSR-AOF medium, and TeSR2 medium.

[0059] Preferably, the mesodermal differentiation culture system contains BMP4 and / or a GSK-3β inhibitor.

[0060] Preferably, the concentration of BMP4 is 0-100 ng / ml; more preferably, the concentration of BMP4 is 5-50 ng / ml; and even more preferably, the concentration of BMP4 is 10-20 ng / ml.

[0061] Preferably, the GSK-3β inhibitor includes, but is not limited to, at least one selected from the following: B216763, TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, and CHI R-99021. Preferably, the concentration of the GSK-3β inhibitor is 0.5-20 μM; more preferably 1-10 μM; and even more preferably 3-5 μM. The GSK-3β inhibitor is CHIR-99021, and its concentration is 0.5-20 μM; more preferably 1-10 μM; and even more preferably 3-5 μM.

[0062] Preferably, the mesodermal differentiation culture system does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0063] Preferably, the mesodermal differentiation culture system does not contain monothioglycerol (MTG).

[0064] Preferably, the mesodermal differentiation culture system is a basal culture medium containing BMP4 and / or GSK-3β inhibitors.

[0065] Preferably, the mesodermal differentiation culture system comprises at least one, at least two, at least three, or four of the following: B27 additive, non-essential amino acids, glutamine, and vitamin C. Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (e.g., a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of the glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages are by weight / volume ratios. The concentration of vitamin C is 10-100 ug / ml, more preferably 20-50 ug / ml, and even more preferably 50 ug / ml.

[0066] Preferably, the hematopoietic endothelial differentiation culture system comprises at least one, at least two, at least three, or four of the following: BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGFβ / ALK inhibitor.

[0067] Preferably, the concentration of BMP4 is 1-50 ng / ml; more preferably, the concentration is 2-20 ng / ml; and even more preferably, the concentration is 5-10 ng / ml.

[0068] Preferably, the vascular endothelial growth factor (VEGF) includes, but is not limited to, at least one selected from the following: VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor. Preferably, the concentration of the VEGF is 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml. Preferably, the VEGF is VEGF-165 or VEGF-A, with a concentration of 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml.

[0069] Preferably, the fibroblast growth factor (FGF) is a polypeptide composed of about 150-200 amino acids, existing in two closely related forms: basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF), wherein the concentration of the FGF (acidic fibroblast kinase and / or basic fibroblast kinase) is 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml. Preferably, the FGF is FGF-2 (bFGF), with a concentration of 5-100 ng / ml; more preferably, the concentration is 10-50 ng / ml; even more preferably, the concentration is 20-50 ng / ml.

[0070] Preferably, the TGFβ / ALK inhibitor includes, but is not limited to, at least one selected from the following: SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947 (HTS-466284), LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, LY2109761. Preferably, the concentration of the TGFβ / ALK inhibitor is 1-50 μM; more preferably 5-20 μM; and even more preferably 5-10 μM. Preferably, the TGFβ / ALK inhibitor is SB431542, and its concentration is 1-50 μM; more preferably 5-20 μM; and even more preferably 5-10 μM.

[0071] Preferably, the hematopoietic endothelial differentiation culture system does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0072] Preferably, the hematopoietic endothelial differentiation culture system does not contain monothioglycerol (MTG).

[0073] Preferably, the hematopoietic endothelial differentiation culture system is a basal culture medium containing at least one, at least two, at least three, or four of the following: BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGFβ / ALK inhibitors.

[0074] Preferably, the hematopoietic endothelial differentiation culture system comprises at least one, at least two, three, or four of the following: B27 additive, non-essential amino acids, glutamine, and vitamin C. Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (e.g., a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages are by weight / volume ratios. The concentration of vitamin C is 10-100 ug / ml, more preferably 20-50 ug / ml, and even more preferably 50 ug / ml.

[0075] Preferably, the mesenchymal stem cell co-culture differentiation system further includes mesenchymal stem cells.

[0076] Preferably, the mesenchymal stem cells are pretreated mesenchymal stem cells. Preferably, the pretreatment step includes: seeding mesenchymal stem cells and passage culturing them until the growth density reaches 60-80% (preferably around 70%) before use. Preferably, the initial seeding quantity of mesenchymal stem cells from different sources can be 4-10 × 10⁻⁶, depending on their growth rate. 4 / cm 2 (e.g. 5×10) 4 / cm 2 6×10 4 / cm 2 8×10 4 / cm 2 10×10 4 / cm 2 ).

[0077] Preferably, the mesenchymal stem cell co-culture differentiation system comprises at least one, at least two, or three of the following: BMP4, vascular endothelial growth factor, and stem cell factor.

[0078] Preferably, the concentration of BMP4 is 1-50 ng / ml; more preferably, the concentration is 2-20 ng / ml; and even more preferably, the concentration is 5-10 ng / ml.

[0079] Preferably, the vascular endothelial growth factor (VEGF) includes, but is not limited to, at least one selected from the following: VEGF-A, VEGF-165, VEGF-183, VEGF-110, VEGF-121, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor. Preferably, the concentration of the VEGF is 1-50 ng / ml; more preferably, the concentration is 5-20 ng / ml; even more preferably, the concentration is 10 ng / ml. Preferably, the VEGF is VEGF-165 or VEGF-A, with a concentration of 1-50 ng / ml; more preferably, the concentration is 5-20 ng / ml; even more preferably, the concentration is 10 ng / ml.

[0080] Preferably, the concentration of the stem cell growth factor (SCF) is 5-100 ng / ml; more preferably, the concentration is 10-50; and even more preferably, the concentration is 20-50 ng / ml.

[0081] Preferably, the mesenchymal stem cell co-culture differentiation system does not contain antibiotics. Antibiotics include, but are not limited to: amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin. Preferably, penicillin and streptomycin are not added to the culture system in step (2). Preferably, the antibiotic is penicillin and / or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.

[0082] Preferably, the mesenchymal stem cell co-culture differentiation system does not contain monothioglycerol (MTG).

[0083] Preferably, the mesenchymal stem cell co-culture differentiation system is a basal culture medium containing at least one, at least two, or three of BMP4, vascular endothelial growth factor, and stem cell factors.

[0084] Preferably, the mesenchymal stem cell co-culture differentiation system comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven of the following: B27 additive, non-essential amino acids, glutamine, vitamin C, N-acetyl-L-cysteine ​​(NAC), minocycline hydrochloride, and insulin-transferrin-selenium. Preferably, the B27 additive is a vitamin A-free B27 additive. Preferably, the concentration of the B27 additive (such as a vitamin A-free B27 additive) is 0.5-10%, more preferably 1-5%, and even more preferably 2%. The concentration of the non-essential amino acids is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. The concentration of glutamine is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%. All percentages mentioned above are mass-volume ratios. The concentration of vitamin C added is 10-100 μg / ml, more preferably 20-50 μg / ml, and even more preferably 50 μg / ml. The concentration of N-acetyl-L-cysteine ​​added is 5-100 μM, more preferably 10-50 μM, and even more preferably 30 μM. The concentration of minocycline hydrochloride added is 0.1-20 μM, more preferably 1-5 μM, and even more preferably 2 μM. The volume percentage of insulin-transferrin-selenium added is 0.2-10%, more preferably 0.5-2%, and even more preferably 1%.

[0085] A third aspect of the present invention provides the preparation method described in the first aspect of the present invention, or hematopoietic cells prepared using the kit described in the second aspect of the present invention.

[0086] Preferably, the hematopoietic cells have a CD34+ phenotype.

[0087] Preferably, the hematopoietic cells have a CD43+ phenotype.

[0088] Preferably, the hematopoietic cells have a CD45+ phenotype.

[0089] Preferably, the hematopoietic progenitor cells have the CD117+ phenotype.

[0090] Preferably, the hematopoietic cells have a CD34+CD45+ phenotype.

[0091] Preferably, the hematopoietic cells have a CD34+CD43+ phenotype.

[0092] Preferably, the hematopoietic cells have a CD43+CD45+ phenotype.

[0093] Preferably, the hematopoietic cells have a CD34+CD117+ phenotype.

[0094] Preferably, the hematopoietic cells have a CD45+CD117+ phenotype.

[0095] Preferably, the hematopoietic cells have the CD43+CD117+ phenotype.

[0096] Preferably, the hematopoietic progenitor cell is a human hematopoietic progenitor cell.

[0097] Preferably, the hematopoietic progenitor cells are a population of hematopoietic progenitor cells.

[0098] Preferably, the hematopoietic progenitor cells have one, two, three, four, five, or six characteristics selected from group (A):

[0099] (i) More than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells have hematopoietic progenitor cell surface antigen CD34+.

[0100] (ii) More than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells have the hematopoietic progenitor cell surface antigen combination CD45+.

[0101] (iii) More than 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the cells have the hematopoietic progenitor cell surface antigen combination CD117+.

[0102] (iv) More than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells have the hematopoietic progenitor cell surface antigen combination CD34+CD45+.

[0103] (v) More than 50%, 55%, 60%, 65%, or 70% of cells possess the hematopoietic progenitor cell surface antigen combination CD34+CD117+; and

[0104] (vi) More than 40%, 45%, 50%, 55%, 60%, 65%, or 70% of cells have the hematopoietic progenitor cell surface antigen combination CD45+CD117+.

[0105] Preferably, the hematopoietic progenitor cells have the following characteristics: 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more of the cells have the hematopoietic progenitor cell surface antigen combination CD43+.

[0106] Preferably, the method described in the first aspect of the present invention can obtain hematopoietic progenitor cells with the above characteristics without purification and / or enrichment steps.

[0107] Preferably, the hematopoietic progenitor cells have the ability to differentiate into CD34+CD45+ blood precursor cells.

[0108] Preferably, the hematopoietic progenitor cells have the ability to differentiate into erythroid blood cells.

[0109] Preferably, the hematopoietic progenitor cells have the ability to differentiate into myeloid blood cells.

[0110] Preferably, the hematopoietic progenitor cells also have the ability to differentiate into lymphocytes.

[0111] A fourth aspect of the present invention provides a product comprising the hematopoietic progenitor cells described in the third aspect of the present invention.

[0112] Preferably, the product is a pharmaceutical composition comprising: the hematopoietic progenitor cells described in the third aspect of the present invention, and a pharmaceutically acceptable carrier.

[0113] Preferably, the pharmaceutical composition is a liquid formulation. Preferably, the pharmaceutical composition is a cell-based formulation. Preferably, the pharmaceutical composition is an intravenous injection reagent. Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer solution, glucose, water, DMSO, and combinations thereof. Preferably, the concentration of hematopoietic progenitor cells in the pharmaceutical composition is 1 × 10⁻⁶. 3 pcs / ml - 1×10 7 The number of cells / ml is preferably 1×10⁻⁶. 4 -1×10 6 1 x 10^6 cells / ml, more preferably 1 x 10^6 cells / ml. 5 9.9 × 10⁻⁶ cells / ml 5 per ml.

[0114] A fifth aspect of the invention provides the application of the cells described in the third aspect of the invention.

[0115] Preferably, the application is in the preparation of medicaments for the treatment and / or prevention of blood diseases.

[0116] Preferably, a method for treating blood diseases is provided, comprising the steps of: administering hematopoietic progenitor cells as described in the third aspect of the present invention to a subject in need, or administering a pharmaceutical composition as described in the fourth aspect of the present invention.

[0117] Preferably, the object is a mammal, more preferably a primate, and even more preferably a human.

[0118] Preferably, the application site is the vein or bone marrow of the subject.

[0119] The hematologic diseases mentioned refer to diseases related to abnormal cell function in the blood, including but not limited to: anemia, thrombocytopenia, leukemia, lymphoma, severe aplastic anemia, multiple myeloma, or combinations thereof.

[0120] The beneficial effects of this invention are:

[0121] 1. The differentiation method of the present invention can be used for the preparation, in vitro expansion and maintenance of hematopoietic progenitor cells; it can not only prepare hematopoietic progenitor cells quickly and efficiently, but also the prepared hematopoietic progenitor cells have a stable differentiation ability to differentiate into a variety of different blood cells (including those that simultaneously have erythroid, myeloid and lymphoid cells).

[0122] 2. The differentiation method of the present invention significantly improves the differentiation efficiency and the number of hematopoietic progenitor cells obtained by optimizing the culture system. The expression ratio of each marker is not only high and stable, but also the expression of each marker is uniform in the target cells.

[0123] 3. By co-culturing mesenchymal stem cells with hematopoietic endothelial cells, the number of hematopoietic progenitor cells can be further increased by 3-8 times while maintaining high differentiation efficiency and uniform and stable expression of various markers. Attached Figure Description

[0124] Figure 1 This is a microscopic observation of the starting stem cells from Example 1.

[0125] Figure 2 is a flow cytometry analysis of stem cell stemness markers in Example 1; Figure 2a It is a surface dry fine mark detection; Figure 2b It is an intramembrane correlation marker detection.

[0126] Figure 3 This is a microscopic observation of D12 cells in Example 1.

[0127] Figure 4 This is a flow cytometry analysis of D12 cells from Example 1.

[0128] Figure 5 This is a microscopic observation of the human uterine blood mesenchymal stem cell group derived from blood cells, as verified in Example 2.

[0129] Figure 6 This is a microscopic observation of the human dental pulp mesenchymal stem cell group derived from blood cells, as verified in Example 2.

[0130] Figure 7 This is a microscopic observation of the human umbilical cord mesenchymal stem cell group derived from blood cells, as verified in Example 2.

[0131] Figure 8 This is a microscopic observation of a human bone marrow mesenchymal stem cell group derived from blood cells, as verified in Example 2.

[0132] Figure 9 This is a flow cytometry analysis diagram from Example 2 verifying the blood cell origin.

[0133] Figure 10 This is a flow cytometry analysis of hematopoietic progenitor cells obtained from the co-culture group of human uterine blood mesenchymal stem cells in Example 2.

[0134] Figure 11 This is a flow cytometry analysis of hematopoietic progenitor cells obtained from the co-culture group of human dental pulp mesenchymal stem cells in Example 2.

[0135] Figure 12 This is a flow cytometry analysis of hematopoietic progenitor cells obtained from the human umbilical cord mesenchymal stem cell co-culture group in Example 2.

[0136] Figure 13 This is a flow cytometry analysis of hematopoietic progenitor cells obtained from the human bone marrow mesenchymal stem cell co-culture group in Example 2. Detailed Implementation

[0137] The present invention will be further described in detail below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0138] definition

[0139] The term "pluripotency" refers to stem cells that have the potential to differentiate into all types of cells that make up one or more tissues or organs, such as any of the three germ layers: endoderm (internal stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital organs), or ectoderm (epidermal tissue and nervous system).

[0140] "Pluripotent stem cells" refer to cells capable of producing cells from all three germinal layers: the endoderm, mesoderm, and ectoderm. While theoretically pluripotent stem cells can differentiate into any cell type in the body, pluripotency assays are typically based on the differentiation of pluripotent cells into several cell types within each germinal layer. Preferably, the pluripotent stem cells are derived from mammals, more preferably from primates, and even more preferably from humans. The pluripotent stem cells include, but are not limited to, embryonic stem cells and / or induced pluripotent stem cells. Preferably, the human pluripotent stem cells (PSCs) are human embryonic stem cells (hESCs) (e.g., H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (e.g., WC50, IMR90). Preferably, the human embryonic stem cells are commercially available human embryonic stem cell lines. In some embodiments, the human embryonic stem cells are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0141] The term "induced pluripotent stem cells," often abbreviated as iPS cells or iPSCs, refers to a type of pluripotent stem cell, such as muscle cells, neurons, and epidermal cells, that is artificially prepared by introducing or exposing non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (e.g., fibroblasts, hematopoietic cells), through the introduction or exposure to reprogramming factors.

[0142] The term "embryonic stem cells" is often abbreviated as ES cells or ESCs, and refers to pluripotent stem cells derived from early embryos.

[0143] The term "mesenchymal stem cells" (MSCs) refers to a type of adult stem cell that exists in various tissues (such as bone marrow, umbilical cord blood and tissue, placental tissue, and adipose tissue), possesses multi-lineage differentiation potential, and is not a hematopoietic stem cell. These stem cells have the potential to differentiate into various mesenchymal cell lineages (such as osteoblasts, chondrocytes, and adipocytes) or non-mesenchymal cell lineages, and possess unique cytokine secretion functions. Cell models are used in research on proliferation, transplantation, and differentiation, as well as in the identification of in vitro immune responses.

[0144] The term "differentiation" is a process by which less specialized cells form offspring of at least one more specialized new cell type.

[0145] The term "embryoid body," also known as an embryoid body or aggregate, refers to a homogeneous or heterogeneous cluster of cells containing differentiated cells, partially differentiated cells, and / or pluripotent stem cells in suspension culture. To summarize some of the inherent clues of differentiation in vivo, certain aspects of the invention can use three-dimensional embryoid bodies as an intermediate step. Differentiation can be initiated at the beginning of cell aggregation, and cells can begin to reproduce embryonic development to a limited extent. While they cannot form trophoblastic ectoderm tissue, almost all other cell types present in the organism can develop. The invention can further promote hematopoietic progenitor cell differentiation after embryoid body formation.

[0146] The term "hematopoietic progenitor cell" refers to a hematopoietic progenitor cell formed by directed differentiation from pluripotent stem cells, as described in the first aspect of this invention. The hematopoietic progenitor cells of this invention are hematopoietic progenitor cells that simultaneously possess the differentiation capabilities of erythroid, myeloid, and lymphoid lineages.

[0147] The term "basal medium" refers to a culture medium with a defined chemical composition, including but not limited to basic cell culture media such as Iscove's modified Dulbecco's medium (IMDM), Eagle's Basal Medium (BME), Eagle MEM, DMEM, Ham, RPMI 1640, and Fischer medium.

[0148] The term "vitamin C" includes vitamin C or its various salts or derivatives thereof.

[0149] The term "glutamine" refers to L-glutamine, an amino acid that encodes proteins. It is a non-essential amino acid for mammals and is used as an essential additive for cell culture in this invention.

[0150] The term "SB431542" also includes SB431542 and its salts, especially pharmaceutically acceptable salts.

[0151] The term "CHIR99021" includes CHIR99021 and its salts, especially pharmaceutically acceptable salts.

[0152] The term "Y-27632" also includes Y-27632 and its salts, especially pharmaceutically acceptable salts. A preferred pharmaceutically acceptable salt is Y-27632 2HCl.

[0153] The term "thioglycerol" is Monothioglycerol (MTG), which is a reducing agent necessary for culturing stem cells and is equivalent to β-mercaptoethanol.

[0154] The term "bone morphogenetic protein-4" (BMP4) plays a regulatory role in the proliferation and differentiation of various cells during embryonic development.

[0155] The term "vitamin A-free B27 supplement" is custom-made. This product contains no added vitamin A. Vitamin A (retinol) can be converted into retinoic acid, which can induce stem cells to differentiate into nerve cells. A vitamin A-free formula is ideal for stem cell culture.

[0156] Those skilled in the art can use, process, and administer hematopoietic progenitor cells using conventional methods. For example, each batch of hematopoietic progenitor cells must undergo sterility, endotoxin, and mycoplasma testing, as well as DNA identity verification, before distribution or use. Each batch of cells must meet the following requirements: cell viability ≥95% and cell purity (positive indicators ≥95%, negative indicators <2%). Acute toxicity and allergy tests on the hematopoietic progenitor cells must be negative.

[0157] Cell detection markers

[0158] The surface stem cell markers of this invention are SSEA4, TRA-1-81, and TRA-1-60; the intramembrane stem cell markers are Nanog, Oct4, and Sox2. Mesodermal cells are identified using KDR. Hematopoietic endothelial cells are identified using CD31, CD34, CD43, and CD309. Hematopoietic progenitor cells prepared using the method of this invention can be verified by detecting cell surface antigens CD34, CD43, CD45, CD90, and CD117.

[0159] CD34 antigen is a highly glycosylated single-pass transmembrane protein that is selectively expressed on the surface of hematopoietic progenitor cells (HPCs) and vascular endothelial cells (ECs). In this invention, no purification and / or enrichment steps are required, and the proportion of CD34-expressing hematopoietic progenitor cells in the total cell population obtained from differentiation in step (4) is preferably ≥90%.

[0160] KDR antigen, or CD309, is a vascular endothelial growth factor (VEGF) receptor that is widely expressed in various mesodermal tissues during development, including vascular endothelial cells in the embryonic stage. In vitro and in vivo data show that KDR is crucial for the development and formation of vascular endothelial cells and hematopoietic cells. In this invention, no purification and / or enrichment steps are required, and the proportion of hematopoietic progenitor cells expressing KDR in the total cell population obtained from differentiation in step (4) is preferably ≥90%.

[0161] CD43 antigen is a glycoprotein encoded by an SNP gene, also known as leukocyte sialic acid glycoprotein or sialic acid protein, expressed on the surface of most blood leukocytes such as B cells, T cells, NK cells, and granulocytes. In this invention, CD43 positivity is used as a marker to identify hematopoietic progenitor cells differentiated from iPSCs. In this invention, no purification and / or enrichment steps are required; preferably, in the total cell population obtained from differentiation in step (4), the proportion of hematopoietic progenitor cells expressing CD43 is ≥90%.

[0162] CD45 antigen is composed of a class of structurally similar, large-molecule transmembrane proteins that are widely present on the surface of leukocytes. Its cytoplasmic segment acts as a protein tyrosine phosphatase, activating the substrates P56lck and P59fyn by dephosphorylating tyrosine residues, playing an important role in cellular signal transduction. CD45 is a surface marker of mature blood progenitor cells. In this invention, no purification and / or enrichment steps are required. In the total cell population obtained from differentiation in step (4), the proportion of hematopoietic progenitor cells expressing CD45 is preferably ≥90%.

[0163] The purity and differentiation degree of the hematopoietic progenitor cells of this invention can be detected using common methods, such as flow cytometry. During detection, different specific antibodies targeting corresponding cell surface antigens are added. These antibodies can be complete monoclonal or polyclonal antibodies, or immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; single-chain Fv molecules (scFV); or chimeric antibodies. After the added antibodies bind to the antigens on the cell surface for a certain period, the cells can be automatically analyzed and / or sorted using flow cytometry.

[0164] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0165] The following are the sources of the reagents used in this invention:

[0166]

[0167]

[0168] Example 1: Preparation of hematopoietic progenitor cells through non-co-culture

[0169] The pluripotent stem cells used were ESCs, specifically the H1 cell line.

[0170] 1. Preparation of hematopoietic progenitor cells

[0171] 1.1 Pluripotent stem cell passage and differentiation (H1 cell culture conditions: adherent culture in six-well plates without feeder layer; H1 cell differentiation: suspension culture in six-well plates with low adhesion)

[0172] D-1 medium: E8 medium + 10 μM Y27632

[0173] Remove pluripotent stem cells with approximately 80% confluence from the incubator, discard the culture medium, wash once with DPBS, and discard the DPBS. Add 0.5 ml of Accutase to each well and incubate at 37°C for 2 min. After 2 min, add DMEM / F12 medium to each well to stop the enzyme activity, pipette the cells, collect them in centrifuge tubes, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the cells in 1 ml of E8 medium. Perform AO / PI cell counting, and transfer the required cell volume to D-1 medium at a rate of 8 × 10⁶ cells / well. 4 Cells at a rate of 1 cell / well are seeded into low-attach six-well plates and labeled as differentiation D-1. The cells are mixed thoroughly by rubbing them up, down, left, and right (using a cross-hatching method) and then incubated at 37°C in a 5% CO2 incubator for 20-24 hours.

[0174] 1.2 Stage I: Mesodermal Differentiation Induction (D0-D1)

[0175] Stage I culture medium: RPM1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50ug / ml vitamin C + 20ng / ml BMP4 + 3uM CHIR-99021.

[0176] Remove the D-1 seeded cells from the incubator and observe them under a microscope (photographs are taken). The cells appear spherical. Tilt the low-adhesion six-well plate, aspirate some of the supernatant, leaving approximately 500 μl of supernatant. Add 2 ml of Stage I medium to each well, gently mix the cells, and return the plate to the incubator. After two days of differentiation, collect the cells for flow cytometry analysis to determine the expression ratio of KDR+ in endothelial cells. The culture time is 42-48 hours.

[0177] 1.3 Stage II: Induction of hematopoietic endothelial differentiation (D2-D5)

[0178] Stage II: RPM 1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50 μg / ml vitamin C + 5 ng / ml BMP4 + 50 ng / ml VEGF-165 + 50 ng / ml FGF2 + 10 μM SB431542

[0179] Remove cells differentiated to day 2 from the incubator and observe them under a microscope (photographs for record-keeping). Tilt and let the low-attachment six-well plate stand still, aspirate some of the supernatant, leaving approximately 500 μl of supernatant in each well, add 2 ml of Stage II medium per well, gently mix the cells, and return to the incubator. When differentiation reaches day 4, remove the cells from the incubator, tilt and let the low-attachment six-well plate stand still, aspirate the supernatant, add 2 ml of Stage II medium per well, mix the cells, and return to the incubator. When differentiation reaches day 6, collect the cells for flow cytometry analysis to detect the expression ratio of the hematopoietic endothelial phenotype CD31+CD34+.

[0180] 1.4 Stage III: Induction of hematopoietic progenitor cell differentiation (D6-D12)

[0181] Stage III medium: IMDM medium + 2% vitamin A-free B27 supplement + 1% NEAA + 1% GlutaMax + 50ug / ml vitamin C + 5ng / ml BMP4 + 10ng / ml VEGF-165 + 20ng / ml SCF + 30μM NAC (N-acetyl-L-cysteine) + 2μM Minocycline hydrochloride

[0182] Cells differentiated to day 6 were removed from the incubator and observed under a microscope (photographs recorded). Cell processing was as follows: Cells were placed at an angle in a low-attached six-well plate, the supernatant was aspirated, leaving approximately 500 μl of supernatant in each well, and 2 ml of Stage III medium was added to each well. The cells were gently mixed and returned to the incubator. At day 8, cells were removed from the incubator, the low-attached six-well plate was tilted and allowed to stand, the medium was aspirated, and 2 ml of Stage III medium was added to each well. The cells were gently mixed and returned to the incubator. At day 10, the same procedure as day 8 was followed for medium change. When differentiated to D12, the secreted cells were collected by centrifugation (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 5 ml of Stage III medium. The cell suspension was then filtered through a 40 μm sieve. The filtered cells were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 1 ml of Stage III medium for cell counting and flow cytometry analysis of the proportion of CD34+CD45+ markers on the surface of hematopoietic progenitor cells.

[0183] 2. Differentiation efficiency detection

[0184] The flow cytometry analysis method is as follows:

[0185] 1) Collect the cells and centrifuge at 1500 rpm for 3 minutes;

[0186] 2) Discard the supernatant, resuspend the cells in PBS, pass through a 40-mesh sieve, count the cells AO / PI, centrifuge for 5 minutes at 1500 rpm;

[0187] 3) After discarding the supernatant, add PBS, centrifuge at 1500 rpm for 3 minutes, and then discard the PBS;

[0188] 4) Add 200 μl of PBS containing 0.5% BSA, resuspend the cells, add the required flow cytometry antibodies, and protect the antibodies from light when handling them. After mixing, place in a 4°C refrigerator for 30 minutes.

[0189] 5) After 30 minutes, remove the cells from the 4-degree refrigerator, add 1 ml of 0.5% BSA, centrifuge the cells at 1500 rpm for 5 minutes;

[0190] 6) Discard the supernatant, wash the cells with 500 μl PBS, and centrifuge the cells at 1500 rpm for 5 minutes each time;

[0191] 7) Discard the supernatant, resuspend the cells in 200 μl PBS, and perform flow cytometry analysis (NovoCyte 3000, injector: NS200, manufacturer: Agilent Technologies).

[0192] The starting stem cells from Example 1 were taken and observed under a microscope; their morphologies are as follows: Figure 1 As shown. Figure 1 The left image shows cell morphology under a 4x microscope. Figure 1 The image on the right shows cell morphology under a 10x microscope.

[0193] Flow cytometry analysis was performed on the initiating stem cells to detect their stem cell markers. The results of the stem cell surface stem cell markers (SSEA4, TR A-1-81, TRA-1-60) are as follows: Figure 2a As shown, the results of stem cell membrane marker (Nanog, Oct4, Sox2) detection are as follows: Figure 2b As shown. Figure 2a As shown, the expression rate of SSEA4 was 99.85%; the expression rate of TRA-1-81 was 99.61%; and the expression rate of TRA-1-60 was 99.83%. Figure 2b As shown, the expression rates of Nanog and Oct4 were 79.33%, Oct4 was 93.09%, and Sox2 was 94.33%. This indicates that the initial embryonic stem cells showed no differentiation trend and were normal, qualified ESCs.

[0194] D12 cells obtained from steps 1.1-1.4 of Example 1 were observed under a microscope, and their morphology was as follows: Figure 3 As shown. Figure 3 The cell morphology under a 10x microscope shows a cell progenitor cell that has been differentiated for 12 days. It is about 8 μm in size and is a round, mononuclear single cell with a large nucleus.

[0195] D12 cells obtained from steps 1.1-1.4 of Example 1 were subjected to flow cytometry analysis to detect the expression of their biomarkers, such as... Figure 4 As shown, the flow cytometry results for differentiated D12 are as follows: CD34+ proportion is 96.58%, CD45+ proportion is 88.78%, CD117+ proportion is 69.26%, CD34+CD45+ proportion is 85.71%, CD34+CD117+ proportion is 58.18%, and CD45+CD117+ proportion is 44.26%.

[0196] 3. Detection of hematopoietic progenitor cell count

[0197] (1) Remove the cells differentiated to D12 from the incubator, observe the cells under a microscope, and take pictures for record-keeping;

[0198] (2) Place the cells in a clean bench, gently shake the cell plate, gently blow down the blood cells at the bottom, and then collect the cells into a centrifuge tube.

[0199] (3) Centrifuge the cells in the centrifuge tube at 1500 rpm for 5 min;

[0200] (4) Discard the supernatant, resuspend the cells in 3 ml of stage III medium, and then filter the cell suspension through a 40 μm sieve;

[0201] (5) Centrifuge the filtered cell suspension again at 1500 rpm for 5 min;

[0202] (6) Discard the supernatant, resuspend the cells in 1 ml of stage III medium, then mix 15 μl of cell suspension with 15 μl of AO / PI dye, add 20 μl of the liquid to a cell counting plate, and then count the cells.

[0203] (7) Perform flow cytometry on the cells. Multiply the ratio of CD34+CD45+ after flow cytometry by the AO / PI count result and record it as the number of CD34+CD45+ cells, i.e. the number of hematopoietic progenitor cells.

[0204] D12 cell count under non-co-culture conditions: AO / PI cell count: 1.8 × 10⁻⁶ 6 (Number of cells secreted per well of a six-well plate); CD34+CD45+ cell count: 1.8 × 10⁻⁶ 6 ×85.71%=1.54×10 6 .

[0205] Example 2: Co-culture preparation of hematopoietic progenitor cells

[0206] 1. Co-culture preparation of hematopoietic progenitor cells

[0207] 1.1 Pretreatment of MSC

[0208] Preparation of human uterine blood mesenchymal stem cells: Menstrual blood from women was collected, separated with lymphatic separation fluid, centrifuged to obtain the white membrane layer, and filtered.

[0209] Preparation of human dental pulp mesenchymal stem cells: obtained by enzyme digestion of dental pulp from cleaned teeth.

[0210] Human umbilical cord mesenchymal stem cells were purchased from Zhongqiao Xinzhou; human bone marrow mesenchymal stem cells were purchased from Zhongqiao Xinzhou.

[0211] HIV-1-derived lentiviral vectors mediate the infection of MSCs with the green fluorescent protein (GFP) gene:

[0212] (1) Seed the cells onto the plate according to experimental requirements (1×10⁶ cells / year). 5 (Each cell) into a six-well plate, the cell count should be about 40-50% on day 2, and incubate overnight at 37°C;

[0213] (2) Before infection, remove the GFP virus from the -80℃ freezer and place it on ice to thaw.

[0214] (3) Before infection, replace the culture medium with Polybrene (usually 5-10 μg / mL), 2 ml per well, then add the virus solution evenly to each well, shake gently, and incubate at 37°C overnight;

[0215] (4) After overnight infection, the cells were replaced with normal culture medium and cultured at 37°C. Fluorescence was observed 48-72 hours after infection. 5. When the cell density reached about 90%, the medium was replaced with fresh culture medium containing an appropriate concentration of Puromycin, and MSC cells stably carrying the Puromycin resistance gene were screened.

[0216] 1.2 Co-culture

[0217] Cells differentiated to D6 in step 1.3 of Example 1 were removed from the incubator. Cells differentiated to D6 from different wells of the same batch of low-attach six-well plates were seeded into MS C cells prepared in step 1.1 of Example 2, where the cell confluence reached approximately 70%. Cell processing was as follows: The cells in the low-attach six-well plate were tilted, the supernatant was aspirated, leaving approximately 500 μl of supernatant in each well. Stage III medium was added to each well (2 ml), and the cells were gently mixed. Then, the cells were aspirated and seeded into lentivirus-infected GFP mesenchymal stem cells (different wells of the low-attach six-well plate corresponded to human uterine blood mesenchymal stem cells / human dental pulp mesenchymal stem cells / human umbilical cord mesenchymal stem cells / human bone marrow mesenchymal stem cells, respectively). The cells were gently mixed and returned to the incubator. At D8, the cells were removed from the incubator, the medium was aspirated, 2 ml of the Stage III medium described in Example 1 was added, and the cells were gently mixed and returned to the incubator. At D10, the same procedure as D8 was followed for medium replacement. When differentiated to D12, the secreted cells were aspirated into centrifuge tubes, and the blood cells at the bottom were gently blown down for collection, taking care not to blow up the mesenchymal stem cells at the bottom. The cells were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 5 ml of Stage III medium. The cell suspension was then filtered through a 40 μm sieve. The filtered cells were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 1 ml of Stage III medium for cell counting and flow cytometry analysis of the proportion of CD34+CD45+ markers on the surface of hematopoietic progenitor cells.

[0218] 2. Verify the origin of blood cells

[0219] MSC cells treated according to step 1.1 of Example 2 but not inoculated with cells from step 1.3 of Example 1 were used as a negative control. Co-cultured cells inoculated with cells from step 1.3 of Example 1 were used as the experimental group. Under a microscope, the morphology was as follows: Figure 5-8 As shown in (10×). Figure 5 The images show human uterine blood mesenchymal stem cell groups. The left image is the negative control group, and the right image is the co-culture group (experimental group). Figure 6 These are human dental pulp mesenchymal stem cell groups. The left image is the negative control group, and the right image is the co-culture group (experimental group). Figure 7 The images show human umbilical cord mesenchymal stem cell groups. The left image is the negative control group, and the right image is the co-culture group (experimental group). Figure 8 The images show human bone marrow mesenchymal stem cell groups. The left image represents the negative control group, and the right image represents the co-culture group (experimental group). It can be seen that after 6 days of co-culture with cells differentiated to D6 in step 1.3 of Example 1, the co-culture group showed a large amount of blood cell secretion. The MSC negative control group, which was not inoculated with cells differentiated to D6 in step 1.3 of Example 1, showed no blood cell secretion under the same treatment conditions.

[0220] MSC-GFP cells inoculated with D6 cells from step 1.3 of Example 1 were analyzed, and the GFP / CD34CD45CD117 ratio of MSC-GFP cells and supernatant blood cells was measured. Figure 9 As shown, no GFP+ CD34+CD45+ blood cells were detected in the supernatant.

[0221] It is known that the large number of blood cells obtained from co-culture originate from the initial pluripotent stem cells, not from the MSCs added during co-culture.

[0222] 3. Flow cytometry analysis of differentiation efficiency

[0223] D12 cells obtained from co-culture in Example 2 were subjected to flow cytometry analysis according to the method described in Example 1 to detect the expression of their biomarkers. Figure 10 Corresponding to human uterine blood mesenchymal stem cells, the flow cytometry results were as follows: CD34+ proportion was 98.82%, CD45+ proportion was 93.75%, CD117+ proportion was 82.72%, CD34+CD45+ proportion was 91.88%, CD34+CD117+ proportion was 64.28%, and CD45+CD117+ proportion was 63.23%.

[0224] Figure 11 Corresponding to human dental pulp mesenchymal stem cells, the flow cytometry results were as follows: CD34+ proportion was 99.29%, CD45+ proportion was 87.03%, CD117+ proportion was 74.32%, CD34+CD45+ proportion was 85.67%, CD34+CD117+ proportion was 60.48%, and CD45+CD117+ proportion was 50.09%.

[0225] Figure 12 Corresponding to human umbilical cord mesenchymal stem cells, the flow cytometry results were as follows: CD34+ proportion was 99.09%, CD45+ proportion was 92.64%, CD117+ proportion was 76.06%, CD34+CD45+ proportion was 91.13%, CD34+CD117+ proportion was 55.89%, and CD45+CD117+ proportion was 58.06%.

[0226] Figure 13 Corresponding to human bone marrow mesenchymal stem cells, the flow cytometry results were as follows: CD34+ proportion was 98.90%, CD45+ proportion was 90.17%, CD117+ proportion was 76.48%, CD34+CD45+ proportion was 89.17%, CD34+CD117+ proportion was 63.46%, and CD45+CD117+ proportion was 58.92%.

[0227] It is evident that co-culturing different mesenchymal stem cells can effectively increase the expression rate of biomarkers.

[0228] 4. Detection of hematopoietic progenitor cell count

[0229] The number of hematopoietic progenitor cells was detected according to the method described in Example 1.

[0230] Co-cultured with human uterine MSC cells, AO / PI cell count: 5.6 × 10⁶ 6 (Number of cells secreted per well of a six-well plate); CD34+CD45+ cell count: 5.6 × 10⁻⁶ 6 ×91.88%=5.15×10 6 .

[0231] Co-culture with human dental pulp MSC cells: AO / PI cell count: 6.2 × 10⁻⁶ 6 (Number of cells secreted per well of a six-well plate); CD34+CD45+ cell count: 6.2×10⁻⁶ 6 ×85.67%=5.31×10 6 .

[0232] Co-culture with human bone marrow MSC cells: AO / PI cell count: 5.69 × 10⁻⁶ 6 (Number of cells secreted per well of a six-well plate); CD34+CD45+ cell count: 5.69×10⁻⁶ 6 ×91.13%=5.19×10 6 .

[0233] Co-culture with human umbilical cord blood MSC cells: AO / PI cell count: 5.25 × 10⁻⁶ 6 (Number of cells secreted per well of a six-well plate); CD34+CD45+ cell count: 5.25×10⁻⁶ 6 ×89.17%=4.68×10 6 .

[0234] The results showed that using different mesenchymal stem cells, compared with not co-culturing, greatly increased the number of hematopoietic progenitor cells obtained while maintaining high differentiation efficiency and stable expression of various markers.

[0235] Example 3: Preparation of hematopoietic progenitor cells

[0236] The operation of this embodiment is largely the same as that of this embodiment 2, except that the initial stem cells used are hiPSCs (purchased from Shisze Biotechnology, catalog number XS-iPS).

[0237] Example 4: Preparation of hematopoietic progenitor cells

[0238] The operation of this embodiment is largely the same as that of Example 2, except that the culture medium for each stage is as follows:

[0239] 1.1 D-1 medium: E8 medium + 10 μM Y27632

[0240] 1.2 Stage I: Mesodermal Differentiation Induction (D0-D1)

[0241] Stage I culture medium: RPM1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50ug / ml vitamin C + 10ng / ml BMP4 + 5uM CHIR-99021.

[0242] 1.3 Stage II: Induction of hematopoietic endothelial differentiation (D2-D5)

[0243] Stage II: RPM 1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50 μg / ml vitamin C + 10 ng / ml BMP4 + 20 ng / ml VEGFA + 20 ng / ml FGF2 + 5 μM SB431542

[0244] 1.4 Stage III: Induction of hematopoietic progenitor cell differentiation (D6-D12)

[0245] Stage III medium: IMDM medium + 2% vitamin A-free B27 supplement + 1% NEAA + 1% GlutaMax + 50ug / ml vitamin C + 10ng / ml BMP4 + 10ng / ml VEGFA + 50ng / ml SCF + 30μM NAC (N-acetyl-L-cysteine) + 2μM Minocycline hydrochloride

[0246] Example 5: Preparation of hematopoietic progenitor cells

[0247] The operation of this embodiment is largely the same as that of Example 2, with the only differences being the culture medium and culture time at each stage, as follows:

[0248] 1.1 D-1 medium: mTESR medium + 10 μM Y27632

[0249] 1.2 Stage I: Mesodermal Differentiation Induction (D0)

[0250] Stage I culture medium: RPM1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50ug / ml vitamin C + 5uM CHIR-99021.

[0251] 1.3 Stage II: Induction of hematopoietic endothelial differentiation (D1-D4)

[0252] Stage II: RPM 1640 medium + 2% vitamin A-free B27 supplement + 1% non-essential amino acids + 1% glutamine + 50 μg / ml vitamin C + 5 ng / ml BMP4 + 50 ng / ml VEGFA + 50 ng / ml FGF2 + 10 μM SB431542

[0253] 1.4 Stage III: Induction of hematopoietic progenitor cell differentiation (D5-D12)

[0254] Stage III medium: α-MEM medium + 2% vitamin A-free B27 supplement + 1% NEAA + 1% GlutaMax + insulin-transferrin-selenium (ITS-G) (100X) + 50ug / ml vitamin C + 5ng / ml BMP4 + 10ng / ml VE GFA + 50ng / ml SCF + 30μM NAC (N-acetyl-L-cysteine) + 2μM Minocycline hydrochloride.

[0255] Comparative Example 1

[0256] The method for preparing hematopoietic cells is the same as that in Example 1, except that 1% (by weight / volume) of penicillin-streptomycin is added to Stage I, Stage II and Stage III culture media.

[0257] The number of hematopoietic progenitor cells and the expression of biomarkers were detected in Comparative Example 1 according to the method described in Example 1.

[0258] The experimental results above show that the absence of penicillin-streptomycin in the culture medium at each stage can significantly increase the number of hematopoietic progenitor cells and their differentiation efficiency.

[0259] Comparative Example 2

[0260] The method for preparing hematopoietic cells was the same as that in Example 1, except that 0.1 mM thioglycerol was added to Stage I, Stage II and Stage III culture media.

[0261] The number of hematopoietic progenitor cells and the expression of biomarkers were detected in Comparative Example 2 according to the method described in Example 1.

[0262] The experimental results above show that not adding thioglycerol to the culture medium at each stage can significantly increase the number of hematopoietic progenitor cells and the differentiation efficiency.

[0263] Comparative Example 3

[0264] The method for preparing hematopoietic cells was the same as that in Example 1, except that 0.1 mM thioglycerol and 1% penicillin-streptomycin were added to Stage I, Stage II and Stage III culture media.

[0265] The number of hematopoietic progenitor cells and the expression of biomarkers were detected in Comparative Example 3 according to the method described in Example 1.

[0266] The experimental results above show that the absence of penicillin-streptomycin and thioglycerol in the culture medium at each stage can significantly increase the number of hematopoietic progenitor cells and their differentiation efficiency.

[0267] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing hematopoietic progenitor cells, comprising the following steps: (1) Culture pluripotent stem cells to obtain embryoid bodies; (2) The embryoid body is subjected to mesodermal differentiation culture to obtain mesodermal cells; (3) The mesodermal cells are subjected to hematopoietic endothelial differentiation culture to obtain hematopoietic endothelial cells; (4) The hematopoietic endothelial cells and mesenchymal stem cells are mixed and co-cultured to obtain hematopoietic progenitor cells; The preparation method prepares hematopoietic progenitor cells under antibiotic-free and thioglycerol-free conditions; The mixing process involves seeding hematopoietic endothelial cells into mesenchymal stem cells. In the mixing step, the growth density of mesenchymal stem cells is 60%-80%; The culture medium in step (4) is IMDM medium + 2% vitamin A-free B27 additive + 1% NEAA + 1% GlutaMax + 50 μg / mL vitamin C + 5 ng / mL BMP4 + 10 ng / mL VEGF + 20 ng / mL SCF + 30 μM NAC + 2 μM Minocycline hydrochloride; or the culture medium in step (4) is IMDM medium + 2% vitamin A-free B27 additive + 1% NEAA + 1% GlutaMax + 50 μg / mL vitamin C + 10 ng / mL BMP4 + 10 ng / mL VEGFA + 50 ng / mL SCF + 30 μM NAC + 2 μM Minocycline hydrochloride; or the culture medium in step (4) is α-MEM medium + 2% vitamin A-free B27 additive + 1% NEAA + 1% GlutaMax + ITS-G 100X + 50 μg / mL vitamin C + 5 ng / mL BMP4 + 10 ng / mL VEGFA +50ng / mL SCF+30μM NAC+2μM Minocycline hydrochloride; The mesenchymal stem cells are selected from at least one of the following: endothelial mesenchymal stem cells, myocardial mesenchymal stem cells, tendon mesenchymal stem cells, skeletal muscle mesenchymal stem cells, bone mesenchymal stem cells, adipose mesenchymal stem cells, neural mesenchymal stem cells, endocrine mesenchymal stem cells, hematopoietic mesenchymal stem cells, liver mesenchymal stem cells, hepatocyte precursor mesenchymal stem cells, uterine blood mesenchymal stem cells, dental pulp mesenchymal stem cells, umbilical cord mesenchymal stem cells, and bone marrow mesenchymal stem cells.

2. The preparation method according to claim 1: the culture system in step (1) contains a ROCK inhibitor.

3. The preparation method according to claim 1 or 2: the culture system of step (2) contains BMP4 and / or GSK-3β inhibitor.

4. The preparation method according to claim 1 or 2, characterized in that: The culture system of step (3) contains at least one of the following: BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGFβ / ALK inhibitor.

5. The use of the preparation method according to any one of claims 1-4 in the preparation of a medicament for treating and / or preventing hematological diseases.

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