WO2023125972A1 - 干细胞诱导分化为造血祖细胞的方法 - Google Patents

干细胞诱导分化为造血祖细胞的方法 Download PDF

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WO2023125972A1
WO2023125972A1 PCT/CN2022/144143 CN2022144143W WO2023125972A1 WO 2023125972 A1 WO2023125972 A1 WO 2023125972A1 CN 2022144143 W CN2022144143 W CN 2022144143W WO 2023125972 A1 WO2023125972 A1 WO 2023125972A1
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cells
hematopoietic progenitor
hematopoietic
differentiation
cell
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李翔
李�雨
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士泽生物医药(苏州)有限公司
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Definitions

  • the invention belongs to the field of cell technology, and more specifically, the invention relates to a method for inducing stem cells to differentiate into hematopoietic progenitor cells.
  • Hematological diseases are diseases that originate in the hematopoietic system, or affect the hematopoietic system accompanied by abnormal blood changes, which often manifest as anemia, bleeding, fever and other symptoms.
  • the incidence of malignant cancer in children in my country is on the rise. According to the data as of 2014, among the malignant tumors in children, the incidence of leukemia ranks first, accounting for about one-third. For malignant hematological diseases, the clinical effect of chemotherapy is often not ideal.
  • HSC transplantation Since Professor Thomas first carried out hematopoietic stem cell (HSC) transplantation in the middle of the 20th century, HSC transplantation has been widely used in the clinical treatment of leukemia, and has become the first choice for the treatment of acute leukemia, malignant lymphoma, severe aplastic anemia and other diseases. one of the effective means.
  • HSCs are mainly derived from umbilical cord blood, bone marrow and peripheral blood.
  • HSC transplantation is mainly divided into autologous and allogeneic HSC transplantation.
  • autologous transplantation has the advantages of no graft rejection, no graft-versus-host disease and other complications, the number of autologous HSCs stored in cord blood banks is in short supply, which limits its clinical application in diseases.
  • the long-term curative effect of allogeneic transplantation is better than that of autologous transplantation and the recurrence rate is low, the matching efficiency is extremely low and the source is limited, which limits the clinical application of allogeneic HSC transplantation.
  • Pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells, which can differentiate into various tissues in the body. They can be used to make disease models, conduct drug toxicity tests, and replace damaged and diseased cells through cell transplantation to promote body wound repair and treatment. disease.
  • Hematopoietic stem cells exist in the body for life and can differentiate into various cells of the blood system, including red blood cells, granulocytes, macrophages, monocytes, microglia, dendritic cells, B-lymphocytes, and T-lymphocytes , NK-lymphocytes, etc., are of great value in the clinical treatment of blood diseases, cancer, etc.
  • Hematopoietic stem cells can rebuild the hematopoietic system, can differentiate into various lineages of hematopoietic cells and maintain their own stemness, but it is difficult to isolate hematopoietic stem cells at the single-cell level in vitro to expand in large quantities, and it is difficult to obtain long-term reconstruction in vivo by inducing differentiation of stem cells in vitro. Hematopoietic stem cells can only obtain short-term reconstruction of hematopoietic progenitor cells in vivo, and hematopoietic progenitor cells have the characteristics of hematopoietic stem cells and can differentiate blood cells of various lineages to solve clinical blood diseases.
  • the main ways to induce the differentiation of human pluripotent stem cells into hematopoietic progenitor cells are: embryoid body differentiation method and stromal cell co-culture method. These methods also have some defects: the embryoid body method generally needs to consume a large number of pluripotent stem cells, and the inconsistency of their differentiation stages leads to their generally low differentiation efficiency and time-consuming; the efficiency of the stromal cell co-culture method is unstable, and animal sources ingredients; or the use of serum-containing culture system or trophoblast cells, which is not suitable for the subsequent production of clinical-grade cell preparations. Therefore, there is an urgent need in this field to develop a method for preparing hematopoietic progenitor cells with defined chemical composition, high efficiency, and rapid differentiation and stability under serum-free conditions.
  • the purpose of the present invention is to develop a method for preparing differentiated and stable hematopoietic progenitor cells in a highly efficient and rapid manner under serum-free conditions.
  • a first aspect of the present invention provides a method for preparing hematopoietic progenitor cells, comprising the following steps:
  • the culture system in the step (1) contains a ROCK inhibitor.
  • 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.
  • the concentration of the ROCK inhibitor is 1-50 ⁇ M; more preferably 5-20 M; still more preferably 10 ⁇ M.
  • the ROCK inhibitor is Y-27632, and its concentration is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 10 ⁇ M.
  • the culture system in the step (1) is a pluripotent stem cell culture medium containing a ROCK inhibitor.
  • the pluripotent stem cell culture medium includes but 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 culture base, TeSR-AOF medium and TeSR2 medium.
  • the culture time of the step (1) is 12-30 hours, more preferably 15-24 hours; still more preferably 20-24 hours.
  • the culture system in step (2) contains BMP4 and/or GSK-3 ⁇ inhibitors.
  • the culture system in the step (2) includes a GSK-3 ⁇ inhibitor.
  • the concentration of BMP4 is 0-100 ng/ml; more preferably, the concentration of BMP4 is 5-50 ng/ml; even more preferably, the concentration of BMP4 is 10-20 ng/ml.
  • the GSK-3 ⁇ 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 CHIR-99021.
  • the concentration of the GSK-3 ⁇ inhibitor is 0.5-20 ⁇ M; more preferably 1-10 ⁇ M; still more preferably 3-5 ⁇ M.
  • the GSK-3 ⁇ inhibitor is CHIR-99021, and its concentration is 0.5-20 ⁇ M; more preferably 1-10 ⁇ M; still more preferably 3-5 ⁇ M.
  • the culture system in step (2) does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • no thioglycerol (Monothioglycerol, MTG) is added to the culture system in the step (2).
  • the culture system in the step (2) is a basal medium containing BMP4 and/or GSK-3 ⁇ inhibitors.
  • the culture system of the step (2) or the basal medium comprises at least one, at least two, at least three or four of the following: B27 supplements, non-essential amino acids, glutamine, vitamins c.
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • the culture time of the step (2) is 18-54 hours, more preferably 20-48 hours; still more preferably 24-48 hours.
  • the culture system of the step (3) comprises at least one, at least two, at least three or four of the following: BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGF ⁇ /ALK inhibitory agent.
  • the concentration of BMP4 is 1-50ng/ml; more preferably, the concentration is 2-20ng/ml; still more preferably, the concentration is 5-10ng/ml.
  • the vascular endothelial growth factor 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.
  • the concentration of VEGF is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; still more preferably, the concentration is 20-50ng/ml.
  • the VEGF is VEGF-165 or VEGF-A, and its concentration is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; still more preferably, the concentration is 20-50ng /ml.
  • the fibroblast growth factor is a polypeptide consisting of about 150-200 amino acids, existing in two closely related forms, i.e. basic fibroblast growth factor (bFGF) and acidic Fibroblast growth factor (aFGF), the concentration of said FGF (acidic fibroblast factor and/or basic fibroblast factor) is 5-100 ng/ml; more preferably, said concentration is 10-50 ng/ml; Even more preferably, the concentration is 20-50 ng/ml.
  • the FGF is FGF-2 (bFGF), and its concentration is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; more preferably, the concentration is 20-50ng/ml ml.
  • the TGF ⁇ /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.
  • the concentration of the TGF ⁇ /ALK inhibitor is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 5-10 ⁇ M.
  • the TGF ⁇ /ALK inhibitor is SB431542, and its concentration is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 5-10 ⁇ M.
  • the culture system in step (3) does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • no thioglycerol (Monothioglycerol, MTG) is added to the culture system in the step (3).
  • the culture system of the step (3) comprises at least one, at least two, at least three or four selected from BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGF ⁇ /ALK inhibitor. cultured in basal medium.
  • the culture system or basal medium in step (3) contains at least one, at least two, three or four of the following: B27 supplement, non-essential amino acids, glutamine, and vitamin C.
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • the culture time of the step (3) is 2-6 days, more preferably 3-5 days; still more preferably 4 days.
  • the culture system in the step (4) comprises at least one, at least two, or three selected from the following: BMP4, vascular endothelial growth factor and stem cell factor.
  • the concentration of BMP4 is 1-50ng/ml; more preferably, the concentration is 2-20ng/ml; still more preferably, the concentration is 5-10ng/ml.
  • the vascular endothelial growth factor 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.
  • the concentration of VEGF is 1-50ng/ml; more preferably, the concentration is 5-20ng/ml; still more preferably, the concentration is 10ng/ml.
  • the VEGF is VEGF-165 or VEGF-A, and its concentration is 1-50ng/ml; more preferably, the concentration is 5-20ng/ml; more preferably, the concentration is 10ng/ml .
  • the concentration of the stem cell growth factor is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; more preferably, the concentration is 20-50ng /ml.
  • the culture system in step (4) does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • no thioglycerol (Monothioglycerol, MTG) is added to the culture system in the step (4).
  • the culture system in the step (4) is a basal medium comprising at least one, at least two, or three selected from BMP4, vascular endothelial growth factor and stem cell factor.
  • the culture system or basal 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 selected from the following: B27 supplements, non-essential amino acids, glutamine, vitamin C, N-acetyl-L-cysteine (NAC), minocycline hydrochloride, and insulin-transferrin-selenium (ITS- G).
  • B27 supplements non-essential amino acids
  • glutamine glutamine
  • vitamin C N-acetyl-L-cysteine
  • minocycline hydrochloride minocycline hydrochloride
  • ITS- G insulin-transferrin-selenium
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • the added concentration of N-acetyl-L-cysteine is 5-100uM, more preferably 10-50uM, and even more preferably 30uM.
  • the added concentration of the minocycline hydrochloride is 0.1-20uM, more preferably 1-5uM, even more preferably 2uM.
  • the volume percentage of the insulin-transferrin-selenium added is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the culture time of the step (4) is 4-8 days, more preferably 5-7 days; even more preferably 6 days.
  • the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under serum-free conditions.
  • the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under vitamin A-free conditions.
  • the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells without antibiotics.
  • the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under thioglycerol-free conditions.
  • the preparation method of hematopoietic progenitor cells does not need to undergo purification and/or enrichment steps.
  • the method for preparing hematopoietic progenitor cells is a method for preparing hematopoietic progenitor cells under trophoblast-free conditions.
  • the culture in any one of steps (1) to (4) is suspension culture or adherent culture.
  • the second aspect of the present invention provides a kit for preparing hematopoietic progenitor cells, comprising at least one of an embryoid body culture system, a mesoderm differentiation culture system, a hematopoietic endothelial differentiation culture system, and a hematopoietic progenitor cell differentiation culture system, At least two, at least three, four.
  • the kit includes a mesoderm differentiation culture system and a hematopoietic endothelial differentiation culture system.
  • the kit also includes a hematopoietic progenitor cell differentiation culture system.
  • the kit also includes an embryoid body culture system.
  • the kit is used in the method for preparing hematopoietic progenitor cells described in the first aspect of the present invention.
  • the embryoid body culture system comprises a ROCK inhibitor.
  • 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.
  • the concentration of the ROCK inhibitor is 1-50 ⁇ M; more preferably 5-20 M; still more preferably 10 ⁇ M.
  • the ROCK inhibitor is Y-27632, and its concentration is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 10 ⁇ M.
  • the embryoid body culture system is a pluripotent stem cell culture medium containing a ROCK inhibitor.
  • the pluripotent stem cell culture medium includes but 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 culture base, TeSR-AOF medium and TeSR2 medium.
  • the mesoderm differentiation culture system comprises BMP4 and/or GSK-3 ⁇ inhibitors.
  • the mesoderm differentiation culture system comprises a GSK-3 ⁇ inhibitor
  • the concentration of BMP4 is 0-100 ng/ml; more preferably, the concentration of BMP4 is 5-50 ng/ml; even more preferably, the concentration of BMP4 is 10-20 ng/ml.
  • the GSK-3 ⁇ 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 CHIR-99021.
  • the concentration of the GSK-3 ⁇ inhibitor is 0.5-20 ⁇ M; more preferably 1-10 ⁇ M; still more preferably 3-5 ⁇ M.
  • the GSK-3 ⁇ inhibitor is CHIR-99021, and its concentration is 0.5-20 ⁇ M; more preferably 1-10 ⁇ M; still more preferably 3-5 ⁇ M.
  • the mesoderm differentiation culture system does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • the mesoderm differentiation culture system does not contain thioglycerol (Monothioglycerol, MTG).
  • the mesoderm differentiation culture system is a basal medium containing BMP4 and/or GSK-3 ⁇ inhibitors.
  • the mesoderm differentiation culture system comprises at least one, at least two, at least three or four of the following: B27 supplements, non-essential amino acids, glutamine, and vitamin C.
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • 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 TGF ⁇ /ALK inhibitor.
  • the concentration of BMP4 is 1-50ng/ml; more preferably, the concentration is 2-20ng/ml; still more preferably, the concentration is 5-10ng/ml.
  • the vascular endothelial growth factor 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.
  • the concentration of VEGF is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; still more preferably, the concentration is 20-50ng/ml.
  • the VEGF is VEGF-165 or VEGF-A, and its concentration is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; still more preferably, the concentration is 20-50ng /ml.
  • the fibroblast growth factor is a polypeptide consisting of about 150-200 amino acids, existing in two closely related forms, i.e. basic fibroblast growth factor (bFGF) and acidic Fibroblast growth factor (aFGF), the concentration of said FGF (acidic fibroblast factor and/or basic fibroblast factor) is 5-100 ng/ml; more preferably, said concentration is 10-50 ng/ml; Even more preferably, the concentration is 20-50 ng/ml.
  • the FGF is FGF-2 (bFGF), and its concentration is 5-100ng/ml; more preferably, the concentration is 10-50ng/ml; more preferably, the concentration is 20-50ng/ml ml.
  • the TGF ⁇ /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.
  • the concentration of the TGF ⁇ /ALK inhibitor is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 5-10 ⁇ M.
  • the TGF ⁇ /ALK inhibitor is SB431542, and its concentration is 1-50 ⁇ M; more preferably 5-20 ⁇ M; still more preferably 5-10 ⁇ M.
  • the hematopoietic endothelial differentiation culture system does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • the hematopoietic endothelial differentiation culture system does not contain thioglycerol (Monothioglycerol, MTG).
  • the hematopoietic endothelial differentiation culture system comprises at least one, at least two, at least three or four of BMP4, vascular endothelial growth factor, fibroblast growth factor, and TGF ⁇ /ALK inhibitor Basal medium.
  • the hematopoietic endothelial differentiation culture system comprises at least one, at least two, three or four of the following: B27 supplement, non-essential amino acid, glutamine, vitamin C.
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • the hematopoietic progenitor cell differentiation culture system comprises at least one, at least two, or three selected from the following: BMP4, vascular endothelial growth factor and stem cell factor.
  • the concentration of BMP4 is 1-50ng/ml; more preferably, the concentration is 2-20ng/ml; still more preferably, the concentration is 5-10ng/ml.
  • the vascular endothelial growth factor 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.
  • the concentration of VEGF is 1-50ng/ml; more preferably, the concentration is 5-20ng/ml; still more preferably, the concentration is 10ng/ml.
  • the VEGF is VEGF-165 or VEGF-A, and its concentration is 1-50ng/ml; more preferably, the concentration is 5-20ng/ml; more preferably, the concentration is 10ng/ml . .
  • the concentration of the stem cell growth factor is 5-100ng/ml; more preferably, the concentration is 10-50; more preferably, the concentration is 20-50ng/ml .
  • the hematopoietic progenitor cell differentiation culture system does not contain antibiotics.
  • Antibiotics include, but are not limited to: Amphotericin, Nystatin, Gentamicin, Tetracycline, Erythromycin, Penicillin, Streptomycin.
  • no penicillin or streptomycin is added to the culture system of the step (2).
  • the antibiotic is penicillin and/or streptomycin. More preferably, the antibiotic is penicillin-streptomycin.
  • the hematopoietic progenitor cell differentiation culture system does not contain thioglycerol (Monothioglycerol, MTG).
  • the hematopoietic progenitor cell differentiation culture system is a basal medium comprising at least one, at least two, or three selected from BMP4, vascular endothelial growth factor and stem cell factor.
  • the hematopoietic progenitor cell differentiation culture 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 additives, non-essential amino acids, Glutamine, vitamin C, N-acetyl-L-cysteine (NAC), minocycline hydrochloride, and insulin-transferrin-selenium.
  • B27 additives non-essential amino acids
  • Glutamine Glutamine
  • vitamin C N-acetyl-L-cysteine (NAC), minocycline hydrochloride
  • insulin-transferrin-selenium insulin-transferrin-selenium
  • the B27 additive is a vitamin A-free B27 additive.
  • the added concentration of the B27 additive eg B27 additive without vitamin A
  • the added concentration of the non-essential amino acid is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the added concentration of glutamine is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the above percentages are mass-volume ratios, and the added concentration of the vitamin C is 10-100 ug/ml, more preferably 20-50 ug/ml, even more preferably 50 ug/ml.
  • the added concentration of N-acetyl-L-cysteine is 5-100uM, more preferably 10-50uM, and even more preferably 30uM.
  • the added concentration of the minocycline hydrochloride is 0.1-20xx, more preferably 1-5uM, even more preferably 2uM.
  • the volume percentage of the insulin-transferrin-selenium added is 0.2-10%, more preferably 0.5-2%, even more preferably 1%.
  • the third aspect of the present invention provides the preparation method described in the first aspect of the present invention, or the hematopoietic group cells prepared by using the kit described in the second aspect of the present invention.
  • the cells of the hematopoietic group have a CD34+ phenotype.
  • the cells of the hematopoietic group have a CD43+ phenotype.
  • the cells of the hematopoietic group have a CD45+ phenotype.
  • the cells of the hematopoietic group have a CD34+CD45+ phenotype.
  • the cells of the hematopoietic group have a CD34+CD43+ phenotype.
  • the cells of the hematopoietic group have a CD43+CD45+ phenotype.
  • the hematopoietic cells have a CD34+CD117+ phenotype.
  • the cells of the hematopoietic group have a CD45+CD117+ phenotype.
  • the hematopoietic cells have a CD43+CD117+ phenotype
  • the hematopoietic progenitor cells are human hematopoietic progenitor cells.
  • the hematopoietic progenitor cells are a population of hematopoietic progenitor cells.
  • the hematopoietic progenitor cells have 1, 2, 3, 4, 5, 6, 7, 8 or 9 characteristics selected from the group (A):
  • the method according to the first aspect of the present invention can obtain 1, 2, 3, 4, 5, Hematopoietic progenitor cells with 6, 7, 8 or 9 characteristics.
  • the hematopoietic progenitor cells have the ability to differentiate into CD34+CD45+ blood precursor cells.
  • the hematopoietic progenitor cells have the ability to differentiate into erythroid blood cells.
  • said hematopoietic progenitor cells have the ability to differentiate into myeloid blood cells.
  • the hematopoietic progenitor cells also have the ability to differentiate into lymphocytes.
  • the fourth aspect of the present invention provides a product comprising the hematopoietic progenitor cells described in the third aspect of the present invention.
  • the product is a pharmaceutical composition
  • the pharmaceutical composition includes: the hematopoietic progenitor cells described in the third aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is a cell preparation.
  • the pharmaceutical composition is an intravenous injection reagent.
  • the pharmaceutically acceptable carrier includes, but is not limited to: saline, buffer, glucose, water, DMSO, and combinations thereof.
  • the concentration of hematopoietic progenitor cells in the pharmaceutical composition is 1 ⁇ 10 3 cells/ml-1 ⁇ 10 7 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 6 cells/ml, more preferably The ground is 1 ⁇ 10 5 /ml-9.9 ⁇ 10 5 /ml.
  • the fifth aspect of the present invention provides the use of the cells described in the third aspect of the present invention.
  • the application is in the preparation of medicines for treating and/or preventing blood diseases.
  • a method for treating hematological diseases comprising the step of: administering the hematopoietic progenitor cells described in the third aspect of the present invention, or administering the pharmaceutical composition described in the fourth aspect of the present invention to a subject in need.
  • the subject is a mammal, more preferably a primate, and even more preferably a human.
  • the site of administration is within the subject's vein or bone marrow.
  • the blood diseases refer to diseases related to cell lesions in the blood, including but not limited to: anemia, thrombocytopenia, leukemia, lymphoma, severe aplastic anemia, multiple myeloma or a combination thereof.
  • the differentiation method of the present invention can be used for the preparation, in vitro expansion and maintenance of hematopoietic progenitor cells; not only can hematopoietic progenitor cells be prepared quickly and efficiently, but also the prepared hematopoietic progenitor cells have stable differentiation into various blood cells (including At the same time, it has the differentiation ability of erythroid, myeloid and lymphoid cells).
  • 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 high and stable; and the expression of each marker is uniform in the target cells.
  • Fig. 1 is a microscope observation diagram of the starting stem cells in Example 1.
  • Figure 2 is a flow cytometric analysis diagram of the stemness markers of the starting stem cells in Example 1;
  • Figure 2a is the detection of surface stemness markers;
  • Figure 2b is the detection of membrane stemness markers.
  • Figure 3 is a microscopic view of D0 cells and the mesoderm stage after 2 days of differentiation in Example 1; the top of Figure 3 is the cell morphology when D0 begins to differentiate; the bottom of Figure 3 is the cell morphology of the mesoderm stage after two days of differentiation.
  • FIG. 4 is a diagram of flow cytometric analysis of mesoderm cells differentiated for two days in Example 1.
  • FIG. 4 is a diagram of flow cytometric analysis of mesoderm cells differentiated for two days in Example 1.
  • Fig. 5 is a microscopic view of the cells in Example 1, which continued to differentiate for 4 days at the hematopoietic endothelial stage.
  • Fig. 6 is a diagram of flow cytometry analysis of cells differentiated in the hematopoietic endothelial stage for 4 days in Example 1.
  • FIG. 7 is a microscopic view of the cells in Example 1 that continued to differentiate at the stage of hematopoietic progenitor cells for 6 days.
  • Fig. 8 is a diagram of flow cytometric analysis of the cells in Example 1 that continued to differentiate at the stage of hematopoietic progenitor cells for 6 days.
  • FIG. 9 is a graph of the QPCR detection results of cell-related genes at various stages in Example 1.
  • Example 10 is a morphological diagram of CFU cells in the hematopoietic group in Example 1 (the upper left image is CFU-E, the upper right image is CFU-GEMM, the lower left image is BFU-E, and the lower right image is CFU-GM).
  • FIG. 11 is a graph showing the results of CFU detection experiments in Example 1 and Example 2.
  • Fig. 12 is a microscope observation diagram of the starting stem cells in Example 2.
  • Figure 13 is a flow cytometric analysis diagram of the stemness markers of the starting stem cells in Example 2;
  • Figure 13a is the detection of surface stemness markers;
  • Figure 10b is the detection of stemness markers in the membrane.
  • FIG. 14 is a microscopic view of cells at the mesoderm stage differentiated for 2 days in Example 2.
  • FIG. 15 is a diagram of flow cytometric analysis of mesoderm cells differentiated for two days in Example 2.
  • Fig. 16 is a microscopic view of the cells in Example 2, which continued to differentiate for 4 days at the hematopoietic endothelial stage.
  • Fig. 17 is a diagram of flow cytometry analysis of cells differentiated in the hematopoietic endothelial stage for 4 days in Example 2.
  • FIG. 18 is a microscopic view of the cells in Example 2 that continued to differentiate at the stage of hematopoietic progenitor cells for 6 days.
  • Fig. 19 is a diagram of flow cytometric analysis of cells differentiated at the stage of hematopoietic progenitor cells for 6 days in Example 2.
  • FIG. 20 is a diagram of the QPCR detection results of cell-related genes at various stages in Example 2.
  • Fig. 21 is a microscopic view of hematopoietic cells obtained from cell differentiation for 12 days in Example 3.
  • Fig. 22 is a diagram of flow cytometric analysis of the hematopoietic group cells obtained from cell differentiation for 12 days in Example 3.
  • Fig. 23 is a diagram of flow cytometric analysis of the hematopoietic group cells obtained from cell differentiation for 12 days in Example 4.
  • Fig. 24 is a diagram of flow cytometric analysis of the hematopoietic group cells obtained from cell differentiation for 12 days in Example 5.
  • Fig. 25 is a diagram of flow cytometric analysis of hematopoietic cells obtained from H1-P6 cells differentiated for 12 days in Example 6.
  • Fig. 26 is a diagram of flow cytometric analysis of the hematopoietic group cells obtained from H1-P7 cells differentiated for 12 days in Example 6.
  • Fig. 27 is a diagram of flow cytometric analysis of the hematopoietic group cells obtained from H1-P8 cells differentiated for 12 days in Example 6.
  • pluripotent refers to stem cells that have the potential to differentiate into all cells of one or more tissues or organs, for example, any of the three germ layers; endoderm (inner stomach lining, gastrointestinal tract, lungs) , mesoderm (muscle, bone, blood, genitourinary) or ectoderm (skin tissue and nervous system).
  • pluripotent stem cells refers to cells capable of giving rise to cells of all three germinal layers, namely endoderm, mesoderm and ectoderm. Although pluripotent stem cells can theoretically differentiate into any cell in the body, experimental assays of pluripotency are often based on the differentiation of pluripotent cells into several cell types per germinal layer. Preferably, the pluripotent stem cells are derived from mammals, more preferably from primates, and still more preferably from humans. The pluripotent stem cells include, but are not limited to, embryonic stem cells and/or induced pluripotent stem cells.
  • the human pluripotent stem cells are human embryonic stem cells (hESCs) (eg H1, H9) and/or human induced pluripotent stem cells (hiPSCs) (eg WC50, IMR90).
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • the human embryonic stem cells are commercial human embryonic stem cell lines.
  • 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.
  • iPS cells induced pluripotent stem cells
  • iPSCs non-pluripotent cells (usually adult somatic cells) or terminally differentiated cells (such as fibroblasts, Hematopoietic cells) artificially prepared pluripotent stem cells, such as muscle cells, neurons, epidermal cells, etc.
  • embryonic stem cells are pluripotent stem cells derived from early embryos.
  • differentiation is the process by which less specialized cells form the progeny of at least one new, more specialized cell type.
  • embryoid body ie embryoid body or aggregate, refers to a homogeneous or heterogeneous cluster of cells comprising differentiated cells, partially differentiated cells and/or pluripotent stem cells in suspension culture.
  • embryoid body ie embryoid body or aggregate, refers to a homogeneous or heterogeneous cluster of cells comprising differentiated cells, partially differentiated cells and/or pluripotent stem cells in suspension culture.
  • certain aspects of the invention may use three-dimensional embryoid bodies as an intermediate step. At the onset of cell aggregation, differentiation can be initiated and cells can initiate to a limited extent to recapitulate embryonic development. Although they cannot form trophectoderm tissue, virtually every other type of cell present in the organism can develop.
  • the invention can further promote the differentiation of hematopoietic progenitor cells after embryoid body formation.
  • hematopoietic progenitor cell refers to the hematopoietic progenitor cell as described in the first aspect of the present invention formed by directed differentiation from pluripotent stem cells.
  • the hematopoietic progenitor cells of the present invention are hematopoietic progenitor cells having the ability to differentiate into erythroid, myeloid, and lymphoid lines.
  • basic medium belongs to chemically defined media, including but not limited to basic cell culture such as Iscove's modified Dulbecco's medium (IMDM), Eagle's Basal Medium (BME), Eagle MEM, DMEM, Ham, RPMI1640 and Fischer medium base.
  • IMDM Iscove's modified Dulbecco's medium
  • BME Eagle's Basal Medium
  • Eagle MEM DMEM
  • Ham RPMI1640
  • Fischer medium base Fischer medium base
  • vitamin C includes vitamin C or salts thereof in various forms or derivatives thereof in various forms.
  • glutamine refers to L-glutamine (L-Glutamine), which is an encoded amino acid in protein synthesis, a non-essential amino acid for mammals, and is used as an essential supplement for cell culture in the present invention.
  • SB431542 also includes SB431542 and salts thereof, especially pharmaceutically acceptable salts.
  • CHIR99021 includes CHIR99021 and salts thereof, especially pharmaceutically acceptable salts.
  • Y-27632 also includes Y-27632 and salts thereof, especially pharmaceutically acceptable salts.
  • a preferred pharmaceutically acceptable salt is Y-27632 2HCL.
  • thioglycerol means Monothioglycerol, MTG, which is a reducing agent necessary for culturing stem cells, and is equivalent to ⁇ -mercaptoethanol.
  • bone morphogenetic protein-4" refers to bone morphogenetic protein-4, BMP4, which regulates the proliferation and differentiation of various cells during embryonic development.
  • B27 supplement without vitamin A is custom Additives, no vitamin A.
  • Vitamin A retinol
  • retinoic acid can be converted into retinoic acid, which induces the differentiation of stem cells into nerve cells.
  • the vitamin A-free formula is ideal for stem cell culture.
  • each batch of hematopoietic progenitor cells must pass sterility, endotoxin, and mycoplasma inspections, as well as DNA identity verification, before distribution or use.
  • Each batch of released cells must comply with cell viability ⁇ 95% and cell purity (positive index ⁇ 95%, negative index ⁇ 2%). Hematopoietic progenitor cell acute toxicity and allergy test results were negative.
  • the surface stem cell markers of the present invention are SSEA4, TRA-1-81, TRA-1-60; the membrane stem cell markers are Nanog, Oct4, Sox2.
  • Cells detected at the mesoderm stage are labeled KDR.
  • the detection markers of cells in the hematopoietic endothelial stage are CD31, CD34, CD43, and CD309.
  • the hematopoietic progenitor cells prepared by the method of the present invention can be verified by detecting the cell surface antigens CD34, CD43, CD45, CD90 and CD117.
  • CD34 antigen is a highly glycosylated single transmembrane protein, which is selectively expressed on the surface of human hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC) and vascular endothelial cells (EC).
  • HSC human hematopoietic stem cells
  • HPC hematopoietic progenitor cells
  • EC vascular endothelial cells
  • the proportion of CD34-expressing hematopoietic progenitor cells in the total cell population is preferably ⁇ 90%.
  • the KDR antigen namely CD309
  • CD309 is a vascular endothelial growth factor (VEGF) receptor that is widely expressed in various mesoderm tissues during development and expressed in vascular endothelial cells at the embryonic stage.
  • VEGF vascular endothelial growth factor
  • In vitro and in vivo data show that KDR is essential for the development and formation of vascular endothelial cells and hematopoietic cells.
  • no purification and/or enrichment steps are required, and in the total cell population differentiated in step (4), the proportion of hematopoietic progenitor cells expressing KDR in the total cell population is preferably ⁇ 90%.
  • CD43 antigen is a glycoprotein encoded by the SNP gene, also known as leukocyte sialoglycoprotein or sialoprotein, expressed on the surface of most blood leukocytes such as B cells, T cells, NK cells, and granulocytes.
  • CD43 positivity is used to mark hematopoietic progenitor cells differentiated from iPSCs.
  • no purification and/or enrichment steps are required, and in the total cell population differentiated in step (4), the proportion of CD43-expressing hematopoietic progenitor cells in the total cell population is preferably ⁇ 90%.
  • CD45 antigen is composed of a class of transmembrane proteins with similar structure and large molecular weight, which are widely present on the surface of leukocytes. Activated by phosphorylation, it plays an important role in cell information transduction.
  • CD45 is a surface marker of mature blood progenitor cells. In the present invention, no purification and/or enrichment steps are required, and in the total cell population differentiated in step (4), the proportion of CD45-expressing hematopoietic progenitor cells in the total cell population is preferably ⁇ 90%.
  • the purity and degree of differentiation of the hematopoietic progenitor cells of the present invention can be detected by general methods, such as flow cytometry.
  • different specific antibodies targeted to the corresponding cell surface antigens are added, and the antibody can be a complete monoclonal or polyclonal antibody, or an antibody fragment with immunological activity, such as Fab' or (Fab)2 fragment; single chain Fv molecules (scFV); or chimeric antibodies.
  • Antibodies are added to bind to antigens on the cell surface for a certain period of time, and cells can be automatically analyzed and/or sorted by flow cytometry.
  • Reagent name Company Name hiPSCs Shize Bio H1 cell/H9 cell Zhongqiao Xinzhou DMEM/F12 medium Gibco E8 medium Gibco RPM1640 medium Gibco IMDM medium Gibco Accutase Sizhengbai
  • the pluripotent stem cells used are ESCs, specifically the H1 cell line.
  • D-1 medium E8 medium + 10 ⁇ M Y27632
  • Stage I medium RPM1640 medium + 2% B27 supplement without vitamin A + 1% non-essential amino acid + 1% glutamine + 50ug/ml vitamin C + 20ng/ml BMP4 + 3uM CHIR-99021.
  • StageIII medium IMDM medium + 2% B27 supplement without vitamin A + 1% NEAA + 1% GlutaMax + 50ug/ml vitamin C + 5ng/ml BMP4 + 10ng/ml VEGF-165 + 20ng/ml SCF + 30 ⁇ M NAC(N-acetyl-L-cysteine)+2 ⁇ M Minocycline hydrochloride
  • the cells differentiated to D6 were taken out of the incubator, and the cells were observed under a microscope (photographed and recorded). Place the cells in the low-attachment six-well plate at an angle, suck off the supernatant, leave about 500 ⁇ l supernatant in each well, add StageIII medium, 2ml in each well, mix the cells gently, and put them back into the incubator.
  • StageIII medium 2ml in each well
  • differentiated to D8 take the cells out of the incubator, place the low-attachment six-well plate tilted, absorb the medium, add Stage III medium, 2ml per well, mix gently, and return to the incubator.
  • differentiated to D10 perform the same operation as D8 and change half of the solution.
  • the secreted cells were collected by centrifugation (1500rpm, 5min), the supernatant was discarded, resuspended with 5ml of Stage III medium, and then the cell suspension was filtered through a 40 ⁇ m mesh, and the filtered cells were centrifuged (1500rpm , 5 min), the supernatant was discarded, and resuspended with 1 ml of Stage III medium for cell counting and flow cytometric detection of the ratio of CD34+CD45+ markers on the surface of hematopoietic progenitor cells.
  • Flow cytometry detection method is as follows:
  • Example 1 The starting stem cells in Example 1 were taken and observed under a microscope, and their morphologies are shown in Figure 1.
  • the left side of Figure 1 is the morphology of cells under a 4x microscope, and the right side of Figure 1 is the morphology of cells under a 10x microscope.
  • stem cell markers SSEA4, TRA-1-81, TRA-1-60
  • stem cell markers Nag, Oct4, Sox2
  • the expression ratio of SSEA4 is 99.85%
  • the expression ratio of TRA-1-81 is 99.61%
  • the expression ratio of TRA-1-60 is 99.83%
  • the expression ratio of Nanog is 79.33%
  • the expression ratio of Oct4 was 93.09%
  • the expression ratio of Sox2 was 94.33%. It shows that the initial embryonic stem cells have no tendency to differentiate and are normal and qualified ESCs.
  • the D0 cells before differentiation in Example 1 and the D2 cells that had not yet entered the hematopoietic endothelial stage after two days of differentiation were taken and observed under a microscope.
  • the morphology is shown in FIG. 3 respectively.
  • the upper left image of Figure 3 is the cell morphology (4x) when the ES cells D0 start to differentiate, and the upper right image of Figure 3 is the cell morphology (10x) when the ES cells D0 begin to differentiate.
  • the lower left image of Figure 3 is the morphology of mesoderm cells when ES cells were differentiated for two days (4x), and the lower right image of Figure 3 is the morphology of mesoderm cells when ES cells were differentiated for two days (10x).
  • the mesoderm stage cells differentiated for two days have a diameter of about 100-200 ⁇ m.
  • Flow cytometric analysis was performed on the mesoderm stage cells differentiated for two days to detect the expression of -KDR+ (CD309), as shown in Figure 4, for the cells differentiated at the stage I mesoderm stage for two days, the proportion of KDR+ cells was as high as 96.23 %, indicating that differentiated cells are at the mesoderm-endothelial stage.
  • Example 1 the D6 cells that continued to differentiate for 4 days in the Stage II hematopoietic endothelial induction differentiation stage and had not yet entered the hematopoietic progenitor cell induction differentiation stage were observed under a microscope, and the morphology was shown in Figure 5.
  • the left image of Figure 5 is the cell morphology under a 4x microscope, and the right image is the cell morphology under a 10x microscope.
  • the hematopoietic endothelial stage cells differentiated for 6 days have a diameter of about 150-300 ⁇ m.
  • Flow cytometric analysis was performed on the cells in the hematopoietic endothelial induction stage of Stage II differentiation for 4 days, and the expression of markers CD31, CD34, CD43 and KDR (CD309) were detected.
  • the expression of FITC-CD31 was positive
  • the proportion of positive expression of PE-Cy7-CD34 was 48.08%
  • the proportion of positive expression of PE-Cy7-CD34 was 62.68%
  • the proportion of positive expression of APC-CD43 was 43.22%
  • the proportion of positive expression of PE-CD309 was 53.05%.
  • the D12 cells differentiated for 6 days in the Stage III hematopoietic endothelial induction differentiation stage in Example 1 were taken and observed under a microscope, and the morphologies are shown in FIG. 7 .
  • Figure 7 is the cell morphology under a 4x microscope.
  • the cell-forming progenitor cells differentiated for 12 days, the size is about 8 ⁇ m, and they are round mononuclear monocytes with large nuclei.
  • the cell count results of H1 cell differentiation D12 are: AO/PI cell count: 2.08 ⁇ 10 6 (the amount of cells secreted by one well of a six-well plate); cell viability: 90.25%.
  • Reagents FastPure Cell/Tissue Total RNA Isolation Kit V2 (Novizan), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Novizan), ChamQ Universal SYBR qPCR Master Mix (Novizan); fluorescence quantitative PCR instrument Light Cycle 480 II 96/384 Roche.
  • FIG. 9 shows the detection effect of the following genes: FLK1, SOX17, GATA1, CDX2, CXCL12, GATA2, RUNX1, KITLG, GFI1B, GFI1, MYB, HOXA10, HOXA5, HOXA6, HOXA9. Visible, hematopoietic cell-related genes were highly expressed.
  • CFU colony-forming unit
  • CFU-E erythroid cell colony-forming unit
  • BFU-E burst erythrocyte colony-forming unit
  • CFU-GM granulocyte/macrophage
  • CFU-GEMM mixed cell line colony forming unit
  • Example 1 After 14 days of culture, colony counting was performed. The test results are shown in Figure 10(4x) and Figure 11. The results show that the hematopoietic cells differentiated in Example 1 have the ability to form CFU-E, BFU-E, CFU-GM and CFU-GEMM, indicating that the hematopoietic progenitor cells obtained in Example 1 are strong in stemness, good in quality, and capable of differentiation The ability to be a variety of blood cells such as erythroid and myeloid.
  • Example 2 The preparation of hematopoietic progenitor cells in Example 2 is roughly the same as in Example 1, except that the embryonic stem cells used are H9cell cell lines.
  • Example 2 flow cytometric analysis was performed on the cells of each stage in Example 2.
  • Example 2 The starting stem cells in Example 2 were taken and observed under a microscope, and their morphologies are shown in Figure 12.
  • the left side of Figure 12 is the cell morphology under the 4x microscope, and the right side of Figure 12 is the cell morphology under the 10x microscope.
  • the starting stem cells of Example 2 were analyzed by flow cytometry to detect their stemness markers.
  • the detection results of stem cell markers (SSEA4, TRA-1-81, TRA-1-60) on the surface of stem cells are shown in Figure 13a, and the detection results of stem cell markers (Nanog, Oct4, Sox2) in the membrane of stem cells are shown in Figure 13b.
  • Figure 13 shows that the initial embryonic stem cells have no tendency to differentiate and are normal qualified ESCs.
  • the D2 (mesoderm) cells that had not yet entered the hematopoietic endothelial stage after two days of differentiation in Example 2 were taken and observed under a microscope.
  • the morphology is shown in FIG. 14 .
  • the left and right panels of Fig. 14 are the morphological diagrams (10x) of mesoderm cells when H9 cells were differentiated for two days.
  • Flow cytometric analysis was performed on the mesoderm stage cells differentiated for two days to detect the expression of -KDR+ (CD309), as shown in Figure 15, for the cells differentiated at the Stage I mesoderm stage for two days, the proportion of KDR+ cells was as high as 95.96 %, indicating that differentiated cells are at the mesoderm-endothelial stage.
  • Example 2 D6 cells that continued to differentiate for 4 days at the Stage II hematopoietic endothelial induction differentiation stage and had not yet entered the induction differentiation stage of hematopoietic progenitor cells were observed under a microscope, and their morphologies were shown in Figure 16.
  • the left image of Figure 16 is the cell morphology under a 4x microscope, and the right image is the cell morphology under a 10x microscope.
  • the hematopoietic endothelial stage cells differentiated for 6 days have a diameter of about 150-300 ⁇ m.
  • Flow cytometric analysis was performed on the cells in the hematopoietic endothelial induction stage of Stage II differentiation for 4 days, and the expression of the markers CD31, CD34, CD43 and KDR (CD309) was detected, as shown in FIG. 17 .
  • the D12 cells differentiated for 6 days in the Stage III hematopoietic endothelial induction differentiation stage in Example 2 were taken and observed under a microscope.
  • the morphology is shown in FIG. 18 .
  • Figure 18 is the cell morphology under a 4x microscope.
  • the cell progenitor cells differentiated for 12 days, the size is about 8 ⁇ m, and they are round mononuclear monocytes with large nuclei.
  • Example 2 According to the method in Example 1, the number of target cells finally obtained in Example 2 was detected.
  • the cell count results of H9cell differentiation D12 are: AO/PI cell count: 1.8 ⁇ 10 6 (the amount of cells secreted by one well of a six-well plate); cell viability: 91.38%.
  • FIG. 20 shows the detection effect of the following genes: FLK1, SOX17, GATA1, CDX2, CXCL12, GATA2, RUNX1, KITLG, GFI1B, GFI1, MYB, HOXA10, HOXA5, HOXA6, HOXA9. Visible, hematopoietic cell-related genes were highly expressed.
  • Example 2 According to the method in Example 1, the colony-forming unit detection experiment was performed on the hematopoietic progenitor cells finally differentiated in Example 2.
  • CFU-E erythroid colony forming unit
  • BFU-E burst erythroid colony forming unit
  • CFU-GM granulocyte/macrophage
  • CFU-GEMM mixed cell line colony forming unit
  • Example 2 The operation of this example is roughly the same as that of Example 1, except that hiPSCs (purchased from Shize Biology, Cat. No. XS-iPS) are used.
  • hiPSCs purchased from Shize Biology, Cat. No. XS-iPS
  • the final differentiated hematopoietic progenitor cells were observed under a microscope, the morphology is shown in Figure 21 (4x).
  • the final differentiated hematopoietic progenitor cells were analyzed by flow cytometry, and the marker expression of the final differentiated hematopoietic progenitor cells was detected.
  • CD34+CD45+ ratio 97.30%
  • CD34+CD43+ ratio 91.55%
  • CD45+CD43+ ratio 89.20%
  • CD34+CD117+ ratio 62.18%
  • CD45+CD117+ ratio 53.84%
  • +CD117+ ratio 57.05%.
  • Stage I medium RPM1640 medium + 2% B27 supplement without vitamin A + 1% non-essential amino acid + 1% glutamine + 50ug/ml vitamin C + 10ng/ml BMP4 + 5uM CHIR-99021.
  • StageIII medium IMDM medium + 2% B27 supplement without vitamin A + 1% NEAA + 1% GlutaMax + 50ug/ml vitamin C + 10ng/ml BMP4 + 10ng/ml VEGFA + 50ng/ml SCF + 30 ⁇ M NAC ( N-acetyl-L-cysteine)+2 ⁇ M Minocycline hydrochloride
  • the hematopoietic progenitor cells continued to differentiate for 6 days, that is, the flow cytometric results on D12 were: the ratio of CD34+CD45+ was CD34+CD43+ ratio: 87.73%; CD45+CD43+ ratio: 91.23%; CD34+CD117+ ratio: 42.10%; CD45+CD117+ ratio: 43%; CD43+CD117+ ratio: 41.87%.
  • Stage I medium RPM1640 medium + 2% B27 supplement without vitamin A + 1% non-essential amino acid + 1% glutamine + 50ug/ml vitamin C + 5uM CHIR-99021.
  • StageIII medium ⁇ -MEM medium + 2% B27 supplement without vitamin A + 1% NEAA + 1% GlutaMax + insulin-transferrin-selenium (ITS-G) (100X) + 50ug/ml vitamin C + 5ng /ml BMP4+10ng/ml VEGFA+50ng/ml SCF+30 ⁇ M NAC (N-acetyl-L-cysteine)+2 ⁇ M Minocycline hydrochloride.
  • ITS-G insulin-transferrin-selenium
  • the final differentiated hematopoietic progenitor cells were analyzed by flow cytometry to detect the expression of their markers.
  • the hematopoietic progenitor cells continued to differentiate for 6 days, that is, the flow cytometry result of D12: CD34+CD45+ ratio was: 96.99%; CD34+CD43+ ratio: 94.02%; CD45+CD43+ ratio: 94.43%; CD34+CD117+ ratio: 50.94%; CD45+CD117+ ratio: 51.47%; CD43+CD117+ ratio: 50.92%.
  • Figures 25-27 show the flow cytometric analysis of the final differentiated hematopoietic progenitor cells. According to the results, the differentiation method of the present invention has good repeatability, the differentiation effect is very stable, the differentiation efficiency can be maintained above 90%, and a large number of hematopoietic progenitor cells can be obtained.
  • the method for preparing hematopoietic cells was the same as in Example 1, except that 0.1 mM thioglycerol and 1% penicillin-streptomycin were added to the Stage I, Stage II and Stage III medium.
  • Example 1 According to the method described in Example 1, the number of hematopoietic progenitor cells and the expression of markers were detected in Comparative Example 1. The result shows that the differentiation efficiency and the number of cells of the hematopoietic progenitor cells of Comparative Example 1 are significantly lower than those of the examples; it is shown that the present invention can significantly improve the hematopoietic progenitor cells by not adding penicillin-streptomycin and thioglycerol in the medium of each stage. quantity, and differentiation efficiency.

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Abstract

本发明公开了一种干细胞诱导分化为造血祖细胞的方法。所述方法包括以下步骤:将多能干细胞进行培养,获得拟胚体;将所述拟胚体进行中胚层分化培养,获得中胚层细胞;将所述中胚层细胞进行生血内皮分化培养,获得生血内皮细胞;将所述生血内皮细胞进行造血祖细胞分化培养,获得造血祖细胞。本发明不仅可以快速、高效地制备造血祖细胞,而且所制备的造血祖细胞具有稳定的分化为多种不同血液细胞(包括同时具有红系、髓系及淋巴系细胞)的分化能力;通过对培养体系的优化,显著提高了分化效率以及获得造血祖细胞的数量。

Description

干细胞诱导分化为造血祖细胞的方法 技术领域
本发明属于细胞技术领域,更具体地,本发明涉及干细胞诱导分化为造血祖细胞的方法。
背景技术
血液病是原发于造血系统的疾病,或影响造血系统伴发血液异常改变,其常表现为贫血、出血、发热等症状。我国儿童恶性癌症的发病率呈上升趋势,截止2014年的数据显示,儿童恶性肿瘤中,白血病的发病率居第一位,约占三分之一。对于恶性血液系统疾病而言,临床上的化疗效果往往不甚理想。自从二十世纪中期Thomas教授首先开展了造血干细胞(hematopoieticstem cell,HSC)移植以来,HSC移植广泛地被应用于白血病的临床治疗中,已经成为治疗急性白血病、恶性淋巴瘤、重型再障等病的有效手段之一。
目前HSC主要来源于脐带血、骨髓和外周血。HSC移植主要分为自体和异体HSC移植两种。尽管自体移植具有无移植排斥、无移植物抗宿主病等并发症的优点,但脐血库储存的自体HSC数量供不应求,使其在疾病中的临床应用中受到限制。异体移植虽然远期疗效优于自体移植且复发率低,但是配型效率极低,来源有限,从而限制了异基因HSC移植的临床应用。
因此,目前本领域迫切需要寻求更为安全、成本较低、来源稳定的造血干/祖细胞资源。多能干细胞包括胚胎干细胞和诱导多能干细胞,能分化为体内各种组织,可以用来制作疾病模型、进行药物毒性检验,并可通过细胞移植,取代损伤病变的细胞,促进机体创伤修复和治疗疾病。造血干细胞终身存在于生物体内,能够分化为血液系统的各类细胞,包括红细胞、粒细胞、巨噬细胞、单核细胞、小胶质细胞、树突细胞、B-淋巴细胞、T-淋巴细胞、NK-淋巴细胞等,在临床治疗血液类疾病、癌症等方面有重要价值。
造血干细胞能够重建造血系统,能分化为各种谱系的造血细胞并且维持自身的干性,但是体外在单细胞水平上分离造血干细胞难以大量扩增,而体外通过干细胞诱导分化难以获得体内长期重建的造血干细胞,只能获得体内短期重建的造血祖细胞,而造血祖细胞具有造血干细胞的特性,可以分化各谱系血细胞,用于解决临床血液类疾病。
目前诱导人类多能干细胞向造血祖细胞分化的途径主要有:拟胚体分化法和基质细胞共培养法。这些方法也存在一些缺陷:拟胚体法一般需要消耗大量多能干细胞,其分化阶段的不一致导致其分化效率普遍较低且耗时较长;基质细胞共培养法效率不稳定,会引入动物源成分;或者使用了含有血清的培养体系或滋养层细胞,因而不适于后续临床级细胞制剂的生产。因此,本领域迫切需要开发在无血清条件下,化学成分确定的、高效、快速制备分化稳定的造血祖细胞的方法。
发明内容
本发明的目的在于开发在无血清条件下,化学成分确定的、高效、快速制备分化稳定的造血祖细胞的方法。
为了实现上述目的,本发明所采取的技术方案是:
本发明的第一个方面,提供造血祖细胞的制备方法,包括以下步骤:
(1)将多能干细胞进行培养,获得拟胚体;
(2)将所述拟胚体进行中胚层分化培养,获得中胚层细胞;
(3)将所述中胚层细胞进行生血内皮分化培养,获得生血内皮细胞;
(4)将所述生血内皮细胞进行造血祖细胞分化培养,获得造血祖细胞。
优选的,所述步骤(1)的培养体系包含ROCK抑制剂。优选的,所述ROCK抑制剂包括但不限于选自以下的至少一种:Blebbistatin、HA-100、Y-27632、HA-1077、KD-025、Y-33075和Narciclasine。优选的,所述ROCK抑制剂的浓度为1-50μM;更优选为5-20M;更进一步优选为10μM。优选的,所述ROCK抑制剂是Y-27632,其浓度为1-50μM;更优选为5-20μM;更进一步优选为10μM。
优选的,所述步骤(1)的培养体系是包含ROCK抑制剂的多能干细胞培养基。优选的,所述多能干细胞培养基包括但不限于以下:E8培养基、mTESR培养基、StemFit Basic 03、StemFit Basic 04、NutriStem hPSC XF培养基、StemMACS iPS-Brew培养基、Stem-Partner ACF培养基、TeSR-AOF培养基和TeSR2培养基。
优选的,所述步骤(1)的培养时间为12-30小时,更优选为15-24小时;更进一步优选为20-24小时。
优选的,所述步骤(2)的培养体系中,包含BMP4和/或GSK-3β抑制剂。优选的,所述步骤(2)的培养体系中,包含GSK-3β抑制剂。
优选的,所述BMP4的浓度是0-100ng/ml;更优选的,所述BMP4的浓度是5-50ng/ml;更进一步优选的,所述BMP4的浓度是10-20ng/ml。
优选的,所述GSK-3β抑制剂包括但不限于选自以下的至少一种:B216763、TWS119、NP031112、SB216763、CHIR-98014、AZD2858、AZD1080、SB415286、LY2090314和CHIR-99021。优选的,所述GSK-3β抑制剂的浓度是0.5-20μM;更优选为1-10μM;更进一步优选为3-5μM。优选的,所述GSK-3β抑制剂是CHIR-99021,其浓度是0.5-20μM;更优选为1-10μM;更进一步优选为3-5μM。
优选的,所述步骤(2)的培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述步骤(2)的培养体系中不添加硫代甘油(Monothioglycerol,MTG)。
优选的,所述步骤(2)中的培养体系是包含BMP4和/或GSK-3β抑制剂的基础培养基。
优选的,所述步骤(2)的培养体系或所述基础培养基包含选自以下的至少一种、至少二种、至少三种或四种:B27添加剂,非必需氨基酸,谷氨酰胺,维生素C。优选的,所述B27添加剂是不含维生素A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素 C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。
优选的,所述步骤(2)的培养时间为18-54小时,更优选为20-48小时;更进一步优选为24-48小时。
优选的,所述步骤(3)的培养体系包含选自以下的至少一种、至少二种、至少三种或四种:BMP4、血管内皮生长因子、成纤维细胞生长因子、和TGFβ/ALK抑制剂。
优选的,所述BMP4的浓度是1-50ng/ml;更优选的,所述浓度是2-20ng/ml;更进一步优选的,所述浓度是5-10ng/ml。
优选的,所述血管内皮生长因子(vascular endothelial growth factor,VEGF)包括但不限于选自以下的至少一种:VEGF-A、VEGF-165、VEGF-183、VEGF-110、VEGF-121、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子。优选的,所述VEGF的浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。优选的,所述VEGF是VEGF-165或VEGF-A,其浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述成纤维细胞生长因子(fibroblast growth factor,FGF)由约150-200氨基酸组成的多肽,以两种密切相关的形式存在,即碱性成纤维细胞生长因子(bFGF)与酸性成纤维细胞生长因子(aFGF),所述FGF(酸性成纤维细胞因子和/或碱性成纤维细胞因子)的浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。优选的,所述FGF是FGF-2(bFGF),其浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述TGFβ/ALK抑制剂包括但不限于选自以下的至少一种: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。优选的,所述TGFβ/ALK抑制剂的浓度是1-50μM;更优选为5-20μM;更进一步优选为5-10μM。优选的,所述TGFβ/ALK抑制剂是SB431542,其浓度是1-50μM;更优选为5-20μM;更进一步优选为5-10μM。
优选的,所述步骤(3)的培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述步骤(3)的培养体系中不添加硫代甘油(Monothioglycerol,MTG)。
优选的,所述步骤(3)的培养体系是包含选自BMP4、血管内皮生长因子、成纤维细胞生长因子、和TGFβ/ALK抑制剂中的至少一种、至少二种、至少三种或四种的基础培养基中培养。
优选的,所述步骤(3)的培养体系或基础培养基包含选自以下的至少一种、至少二种、三种或四种:B27添加剂,非必须氨基酸,谷氨酰胺,维生素C。优选的,所述B27添加剂是不含维生素A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度 为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。
优选的,所述步骤(3)的培养时间为2-6天,更优选为3-5天;更进一步优选为4天。
优选的,所述步骤(4)的培养体系包含选自以下的至少一种、至少二种、或三种:BMP4、血管内皮生长因子和干细胞因子。
优选的,所述BMP4的浓度是1-50ng/ml;更优选的,所述浓度是2-20ng/ml;更进一步优选的,所述浓度是5-10ng/ml。
优选的,所述血管内皮生长因子(vascular endothelial growth factor,VEGF)包括但不限于选自以下的至少一种:VEGF-A、VEGF-165、VEGF-183、VEGF-110、VEGF-121、VEGF-B、VEGF-C、VEGF-D、VEGF-E和胎盘生长因子。优选的,所述VEGF的浓度是1-50ng/ml;更优选的,所述浓度是5-20ng/ml;更进一步优选的,所述浓度是10ng/ml。优选的,所述VEGF是VEGF-165或VEGF-A,其浓度是1-50ng/ml;更优选的,所述浓度是5-20ng/ml;更进一步优选的,所述浓度是10ng/ml。
优选的,所述干细胞生长因子(stem cell factor,SCF)的浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述步骤(4)的培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述步骤(4)的培养体系中不添加硫代甘油(Monothioglycerol,MTG)。
优选的,所述步骤(4)中的培养体系是包含选自BMP4、血管内皮生长因子和干细胞因子的至少一种、至少二种、或三种的基础培养基。
优选的,所述步骤(4)中的培养体系或基础培养基,包含选自以下的至少一种、至少二种、至少三种、至少四种、至少五种、至少六种或七种:B27添加剂,非必须氨基酸,谷氨酰胺,维生素C,N-乙酰基-L-半胱氨酸(NAC),盐酸米诺环素(Minocycline hydrochloride),和胰岛素-转铁蛋白-硒(ITS-G)。优选的,所述B27添加剂是不含维生素A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。所述N-乙酰基-L-半胱氨酸的添加浓度为5-100uM,更优选为10-50uM,更进一步优选为30uM。所述盐酸米诺环素的添加浓度为0.1-20uM,更优选为1-5uM,更进一步优选为2uM。所述胰岛素-转铁蛋白-硒的添加的体积百分比为0.2-10%,更优选为0.5-2%,更进一步优选为1%。
优选的,所述步骤(4)的培养时间为4-8天,更优选为5-7天;更进一步优选为6天。
优选的,所述造血祖细胞的制备方法是在无血清条件下制备造血祖细胞的方法。
优选的,所述造血祖细胞的制备方法是在无维生素A条件下制备造血祖细胞的方法。
优选的,所述造血祖细胞的制备方法是在无抗生素条件下制备造血祖细胞的方法。
优选的,所述造血祖细胞的制备方法是在无硫代甘油条件下制备造血祖细胞的方法。
优选的,所述造血祖细胞的制备方法无需经过纯化和/或富集步骤。
优选的,所述造血祖细胞的制备方法是在无滋养层条件下制备造血祖细胞的方法。
优选的,步骤(1)至(4)的任一步骤的所述培养是悬浮培养或贴壁培养。
本发明的第二个方面,提供用于制备造血祖细胞的试剂盒,包含拟胚体培养体系,中胚层分化培养体系,生血内皮分化培养体系,和造血祖细胞分化培养体系中至少一种、至少二中、至少三种、四种。优选的,所述试剂盒包括中胚层分化培养体系和生血内皮分化培养体系。优选的,所述试剂盒还包含造血祖细胞分化培养体系。优选的,所述试剂盒还包含拟胚体培养体系。
优选的,所述试剂盒用于本发明第一方面所述的造血祖细胞的制备方法。
优选的,所述拟胚体培养体系包含ROCK抑制剂。优选的,所述ROCK抑制剂包括但不限于选自以下的至少一种:Blebbistatin、HA-100、Y-27632、HA-1077、KD-025、Y-33075和Narciclasine。优选的,所述ROCK抑制剂的浓度为1-50μM;更优选为5-20M;更进一步优选为10μM。优选的,所述ROCK抑制剂是Y-27632,其浓度为1-50μM;更优选为5-20μM;更进一步优选为10μM。
优选的,所述拟胚体培养体系是包含ROCK抑制剂的多能干细胞培养基。优选的,所述多能干细胞培养基包括但不限于以下:E8培养基、mTESR培养基、StemFit Basic 03、StemFit Basic 04、NutriStem hPSC XF培养基、StemMACS iPS-Brew培养基、Stem-Partner ACF培养基、TeSR-AOF培养基和TeSR2培养基。
优选的,所述中胚层分化培养体系包含BMP4和/或GSK-3β抑制剂。优选的,所述中胚层分化培养体系包含GSK-3β抑制剂
优选的,所述BMP4的浓度是0-100ng/ml;更优选的,所述BMP4的浓度是5-50ng/ml;更进一步优选的,所述BMP4的浓度是10-20ng/ml。
优选的,所述GSK-3β抑制剂包括但不限于选自以下的至少一种:B216763、TWS119、NP031112、SB216763、CHIR-98014、AZD2858、AZD1080、SB415286、LY2090314和CHIR-99021。优选的,所述GSK-3β抑制剂的浓度是0.5-20μM;更优选为1-10μM;更进一步优选为3-5μM。所述GSK-3β抑制剂是CHIR-99021,其浓度是0.5-20μM;更优选为1-10μM;更进一步优选为3-5μM。
优选的,所述中胚层分化培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述中胚层分化培养体系不包含硫代甘油(Monothioglycerol,MTG)。
优选的,所述中胚层分化培养体系是包含BMP4和/或GSK-3β抑制剂的基础培养基。
优选的,所述中胚层分化培养体系包含选自以下的至少一种、至少二种、至少三种或四 种:B27添加剂,非必需氨基酸,谷氨酰胺,维生素C。优选的,所述B27添加剂是不含维生素A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。
优选的,所述生血内皮分化培养体系包含选自以下的至少一种、至少二种、至少三种或四种:BMP4、血管内皮生长因子、成纤维细胞生长因子、和TGFβ/ALK抑制剂。
优选的,所述BMP4的浓度是1-50ng/ml;更优选的,所述浓度是2-20ng/ml;更进一步优选的,所述浓度是5-10ng/ml。
优选的,所述血管内皮生长因子(vascular endothelial growth factor,VEGF)包括但不限于选自以下的至少一种:VEGF-A,VEGF-165,VEGF-183,VEGF-110,VEGF-121,VEGF-B,VEGF-C,VEGF-D,VEGF-E和胎盘生长因子。优选的,所述VEGF的浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。优选的,所述VEGF是VEGF-165或VEGF-A,其浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述成纤维细胞生长因子(fibroblast growth factor,FGF)由约150-200氨基酸组成的多肽,以两种密切相关的形式存在,即碱性成纤维细胞生长因子(bFGF)与酸性成纤维细胞生长因子(aFGF),所述FGF(酸性成纤维细胞因子和/或碱性成纤维细胞因子)的浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。优选的,所述FGF是FGF-2(bFGF),其浓度是5-100ng/ml;更优选的,所述浓度是10-50ng/ml;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述TGFβ/ALK抑制剂包括但不限于选自以下的至少一种: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。优选的,所述TGFβ/ALK抑制剂的浓度是1-50μM;更优选为5-20μM;更进一步优选为5-10μM。优选的,所述TGFβ/ALK抑制剂是SB431542,其浓度是1-50μM;更优选为5-20μM;更进一步优选为5-10μM。
优选的,所述生血内皮分化培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述生血内皮分化培养体系不包含硫代甘油(Monothioglycerol,MTG)。
优选的,所述生血内皮分化培养体系是包含选自BMP4、血管内皮生长因子、成纤维细胞生长因子、和TGFβ/ALK抑制剂中的至少一种、至少二种、至少三种或四种的基础培养基。
优选的,所述生血内皮分化培养体系包含选自以下的至少一种、至少二种、三种或四种:B27添加剂,非必须氨基酸,谷氨酰胺,维生素C。优选的,所述B27添加剂是不含维生素 A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。
优选的,所述造血祖细胞分化培养体系包含选自以下的至少一种、至少二种、或三种:BMP4、血管内皮生长因子和干细胞因子。
优选的,所述BMP4的浓度是1-50ng/ml;更优选的,所述浓度是2-20ng/ml;更进一步优选的,所述浓度是5-10ng/ml。
优选的,所述血管内皮生长因子(vascular endothelial growth factor,VEGF)包括但不限于选自以下的至少一种:VEGF-A,VEGF-165,VEGF-183,VEGF-110,VEGF-121,VEGF-B,VEGF-C,VEGF-D,VEGF-E和胎盘生长因子。优选的,所述VEGF的浓度是1-50ng/ml;更优选的,所述浓度是5-20ng/ml;更进一步优选的,所述浓度是10ng/ml。优选的,所述VEGF是VEGF-165或VEGF-A,其浓度是1-50ng/ml;更优选的,所述浓度是5-20ng/ml;更进一步优选的,所述浓度是10ng/ml。。
优选的,所述干细胞生长因子(stem cell factor,SCF)的浓度是5-100ng/ml;更优选的,所述浓度是10-50;更进一步优选的,所述浓度是20-50ng/ml。
优选的,所述造血祖细胞分化培养体系不包含抗生素。抗生素包括但不限于:两性霉素、制霉菌素、庆大霉素、四环素、红霉素、青霉素、链霉素。优选的,所述步骤(2)的培养体系中不添加青霉素、链霉素。优选的,所述抗生素为青霉素和/或链霉素。更优选的,所述抗生素为青霉素-链霉素。
优选的,所述造血祖细胞分化培养体系不包含硫代甘油(Monothioglycerol,MTG)。
优选的,所述造血祖细胞分化培养体系是包含选自BMP4、血管内皮生长因子和干细胞因子的至少一种、至少二种、或三种的基础培养基。
优选的,所述造血祖细胞分化培养体系包含选自以下的至少一种、至少二种、至少三种、至少四种、至少五种、至少六种或七种:B27添加剂,非必须氨基酸,谷氨酰胺,维生素C,N-乙酰基-L-半胱氨酸(NAC),盐酸米诺环素(Minocycline hydrochloride),和胰岛素-转铁蛋白-硒。优选的,所述B27添加剂是不含维生素A的B27添加剂。优选的,所述B27添加剂(如不含维生素A的B27添加剂)的添加浓度为0.5-10%,更优选为1-5%,更进一步优选为2%。所述非必须氨基酸的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。所述谷氨酰胺的添加浓度为0.2-10%,更优选为0.5-2%,更进一步优选为1%。上述百分比均为质量体积比,所述维生素C的添加浓度为10-100ug/ml,更优选为20-50ug/ml,更进一步优选为50ug/ml。所述N-乙酰基-L-半胱氨酸的添加浓度为5-100uM,更优选为10-50uM,更进一步优选为30uM。所述盐酸米诺环素的添加浓度为0.1-20xx,更优选为1-5uM,更进一步优选为2uM。所述胰岛素-转铁蛋白-硒的添加的体积百分比为0.2-10%,更优选为0.5-2%,更进一步优选为1%。
本发明的第三个方面,提供本发明第一方面所述制备方法,或采用本发明第二方面所述 试剂盒制备得到的造血组细胞。
优选的,所述造血组细胞具有CD34+表型。
优选的,所述造血组细胞具有CD43+表型。
优选的,所述造血组细胞具有CD45+表型。
优选的,所述造血组细胞具有CD34+CD45+表型。
优选的,所述造血组细胞具有CD34+CD43+表型。
优选的,所述造血组细胞具有CD43+CD45+表型。
优选的,所述造血组细胞具有CD34+CD117+表型。
优选的,所述造血组细胞具有CD45+CD117+表型。
优选的,所述造血组细胞具有CD43+CD117+表型
优选的,所述造血祖细胞为人的造血祖细胞。
优选的,所述造血祖细胞为造血祖细胞群。
优选的,所述造血祖细胞具有选自下组(A)的1种、2种、3种、4种、5种、6种、7种、8种或9种特征:
(i)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原CD34+;
(ii)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原组合CD43+;
(iii)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原组合CD45+;
(iv)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原组合CD34+CD45+;
(v)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原组合CD34+CD43+;
(vi)80%以上,85%以上,90%以上,91%以上,92%以上,93%以上,94%以上,95%以上,96%以上,97%以上,98%以上,99%以上的细胞具有造血祖细胞表面抗原组合CD43+CD45+;
(vii)30%以上,35%以上,40%以上,45%以上,50%以上,或55%以上的细胞具有造血祖细胞表面抗原组合CD45+CD117+;
(viii)30%以上,35%以上,40%以上,45%以上,50%以上,或55%以上的细胞具有造血祖细胞表面抗原组合CD34+CD117+;
(ix)30%以上,35%以上,40%以上,45%以上,50%以上,或55%以上的细胞具有造血祖细胞表面抗原组合CD43+CD117+。
优选的,本发明第一方面所述方法不需经过纯化和/或富集步骤,就可以获得具有上述(i)-(ix)中1种、2种、3种、4种、5种、6种、7种、8种或9种特征的造血祖细胞。
优选的,所述造血祖细胞具有分化为CD34+CD45+血液前体细胞的能力。
优选的,所述造血祖细胞具有分化为红系血细胞的能力。
优选的,所述的造血祖细胞具有分化为髓系血细胞的能力。
优选的,所述的造血祖细胞还具有分化为淋巴细胞的能力。
本发明的第四个方面,提供包含本发明第三方面所述造血祖细胞的产品。
优选的,所述产品为药物组合物,所述的药物组合物包括:本发明第三方面所述的造血祖细胞,以及药学上可接受的载体。
优选的,所述的药物组合物为液体制剂。优选的,所述的药物组合物为细胞制剂。优选的,所述的药物组合物为静脉注射试剂。优选的,所述药学上可接受的载体包括但并不限于:盐水、缓冲液、葡萄糖、水、DMSO、及其组合。优选的,所述药物组合物中造血祖细胞的浓度为1×10 3个/ml-1×10 7个/ml,较佳地为1×10 4-1×10 6个/ml,更佳地为1×10 5个/ml-9.9×10 5个/ml。
本发明的第五个方面,提供本发明第三方面所述细胞的应用。
优选的,所述应用为在制备治疗和/或预防血液病的药物中的应用。
优选的,提供一种治疗血液病的方法,包括步骤:给需要的对象施用本发明第三方面所述的造血祖细胞、或施用本发明第四方面所述药物组合物。
优选的,所述的对象为哺乳动物,更优选为灵长类,更进一步优先为人。
优选的,施用部位为所述对象的静脉或骨髓内。
所述的血液病指与血液中的细胞病变有关的疾病,包括但不限于:贫血、血小板减少症、白血病、淋巴瘤、重型再障、多发性骨髓瘤或其组合。
本发明的有益效果是:
1本发明的分化方法可用于造血祖细胞的制备、体外扩增、维持;不仅可以快速、高效地制备造血祖细胞,而且所制备的造血祖细胞具有稳定的分化为多种不同血液细胞(包括同时具有红系、髓系及淋巴系细胞)的分化能力。
2本发明的分化方法通过对培养体系的优化,显著提高了分化效率以及获得造血祖细胞的数量;各标志物不仅表达比例高且稳定;而且各标志物在目标细胞中表达均一。
附图说明
图1是实施例1起始干细胞的显微镜下观察图。
图2是实施例1起始干细胞干性标记的流式细胞分析图;图2a是表面干细标记检测;图2b是膜内干系标记检测。
图3是实施例1中D0细胞和分化2天后的中胚层阶段的细胞显微镜下观察图;图3上方是D0开始分化时的细胞形态;图3下方是分化两天后中胚层阶段的细胞形态。
图4是实施例1中细胞分化两天的中胚层细胞的流式细胞分析图。
图5是实施例1中细胞在生血内皮阶段继续分化4天的细胞显微镜下观察图。
图6是实施例1中细胞在生血内皮阶段继续分化4天的流式细胞分析图。
图7是实施例1中细胞在造血祖细胞阶段继续分化6天的细胞显微镜下观察图。
图8是实施例1中细胞在造血祖细胞阶段继续分化6天的流式细胞分析图。
图9是实施例1中各阶段细胞相关基因的QPCR检测结果图。
图10是实施例1中造血组细胞CFU细胞形态图(左上图为CFU-E,右上图为CFU-GEMM,左下图为BFU-E,右下图为CFU-GM)。
图11是实施例1和实施例2的CFU检测实验的结果图。
图12是实施例2起始干细胞的显微镜下观察图。
图13是实施例2起始干细胞干性标记的流式细胞分析图;图13a是表面干细标记检测;图10b是膜内干系标记检测。
图14是实施例2中分化2天后的中胚层阶段的细胞显微镜下观察图。
图15是实施例2中细胞分化两天的中胚层细胞的流式细胞分析图。
图16是实施例2中细胞在生血内皮阶段继续分化4天的细胞显微镜下观察图。
图17是实施例2中细胞在生血内皮阶段继续分化4天的流式细胞分析图。
图18是实施例2中细胞在造血祖细胞阶段继续分化6天的细胞显微镜下观察图。
图19是实施例2中细胞在造血祖细胞阶段继续分化6天的流式细胞分析图。
图20是实施例2中各阶段细胞相关基因的QPCR检测结果图。
图21是实施例3中细胞分化12天得到的造血组细胞的显微镜下观察图。
图22是实施例3中细胞分化12天得到的造血组细胞的流式细胞分析图。
图23是实施例4中细胞分化12天得到的造血组细胞的流式细胞分析图。
图24是实施例5中细胞分化12天得到的造血组细胞的流式细胞分析图。
图25是实施例6中H1-P6细胞分化12天得到的造血组细胞的流式细胞分析图。
图26是实施例6中H1-P7细胞分化12天得到的造血组细胞的流式细胞分析图。
图27是实施例6中H1-P8细胞分化12天得到的造血组细胞的流式细胞分析图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
定义
术语“多能性”是指具有分化成一种或多种组织或器官的所有细胞的潜力的干细胞,例如,三种胚层中的任何一种;内胚层(内部胃壁,胃肠道,肺),中胚层(肌肉,骨骼,血液,泌尿生殖器)或外胚层(表皮组织和神经系统)。
“多能干细胞”是指能够产生所有三个生发层细胞的细胞,即内胚层,中胚层和外胚层。虽然理论上多能干细胞可以分化成身体的任何细胞,但多能性实验测定通常基于多能细胞分化成每个生发层的几种细胞类型。优选的,所述多能干细胞来源于哺乳动物,更优选来源于灵长类,更进一步优选来源于人。所述多能干细胞包括但不限于胚胎干细胞和/或诱导型多能干细胞。优选的,所述人多能干细胞(PSC)为人胚胎干细胞(hESCs)(例如H1、H9)和/或人诱导型多能干细胞(hiPSCs)(例如WC50、IMR90)。优选的,所述人胚胎干细胞为商业化人胚胎干细胞系。在一些实施方式中,所述人胚胎干细胞为未经过体内发育的受精14天以内的人类胚胎分离或者获取的干细胞。
术语“诱导的多能干细胞”,通常缩写为iPS细胞或iPSC,是指通过引入或接触重编程 因子,将非多能细胞(通常是成体体细胞)或终末分化细胞(例如成纤维细胞,造血细胞)人工制备的一种多能干细胞,如肌细胞,神经元,表皮细胞等。
术语“胚胎干细胞”通常缩写为ES细胞或ESC,是源自早期胚胎的多能干细胞。
术语“分化”是一种过程,通过该过程,较不特化的细胞形成至少一种更特化的新细胞类型的后代。
术语“拟胚体”,即胚状体或聚集体,是指包含分化细胞,部分分化细胞和/或悬浮培养的多能干细胞的均质或异质细胞簇。为了概括体内分化固有的一些线索,本发明的某些方面可以使用三维拟胚体作为中间步骤。在细胞聚集开始时,可以启动分化并且细胞可以在有限程度上开始以重现胚胎发育。虽然它们不能形成滋养外胚层组织,但是生物体中存在的几乎所有其他类型的细胞都可以发育。本发明可以进一步促进拟胚体形成后的造血祖细胞分化。
术语“造血祖细胞”,指从多能干细胞定向诱导分化形成的,如本发明第一方面中所述的造血祖细胞。本发明的造血祖细胞是同时具有红系、髓系及淋巴系的分化能力的造血祖细胞。
术语“基础培养基”属于化学成分确定的培养基,包括但不限于如Iscove's modified Dulbecco's medium(IMDM),Eagle's Basal Medium(BME),Eagle MEM,DMEM,Ham,RPMI1640和Fischer培养基等基础细胞培养基。
术语“维生素C”包括维生素C或其各种形式的盐或其多种形式的衍生物。
术语“谷氨酰胺”是指L-谷氨酰胺(L-Glutamine),是蛋白质合成中的编码氨基酸,哺乳动物非必需氨基酸,本发明中作为细胞培养的必需添加物。
术语“SB431542”还包括SB431542及其盐,尤其是药学上可接受的盐。
术语“CHIR99021”包括CHIR99021及其盐,尤其是药学上可接受的盐。
术语“Y-27632”还包括Y-27632及其盐,尤其是药学上可接受的盐。优选的药学上可接受的盐是Y-27632 2HCL。
术语“硫代甘油”即Monothioglycerol,MTG,是培养干细胞所必需的还原剂,与β-巯基乙醇等效。
术语“骨形态发生蛋白4”即bone morphogenetic protein-4,BMP4,在胚胎发育时期对多种细胞的增殖分化具有调控作用。
术语“不含维生素A的B27添加剂”是定制的
Figure PCTCN2022144143-appb-000001
添加剂,不含维生素A。维生素A(视黄醇)能被转化成视黄酸,后者可以诱导干细胞向神经细胞的分化。不含维生素A的配方是干细胞培养的理想选择。
本领域的普通技术人员可以使用常规方法对造血祖细胞进行使用、处理、施用等操作。如:每批造血祖细胞发放或使用之前,必须通过无菌、内毒素和支原体检查,以及DNA同一认定。每批发放的细胞都要符合细胞活力≥95%、细胞纯度(阳性指标≥95%,阴性指标<2%)。造血祖细胞急毒、过敏检测结果均呈阴性。
细胞的检测标记
本发明干细胞的表面干系标记为SSEA4,TRA-1-81,TRA-1-60;膜内干系标记为Nanog,Oct4,Sox2。中胚层阶段细胞检测标记为KDR。生血内皮阶段细胞检测标记为CD31,CD34, CD43,CD309。用本发明方法制备的造血祖细胞,可通过细胞表面抗原CD34,CD43,CD45,CD90,CD117的检测加以验证。
CD34抗原是一种高度糖基化的单次跨膜蛋白,它选择性地表达于人造血干细胞(HSC)造血祖细胞(HPC)和血管内皮细胞(EC)表面。在本发明中,无需纯化和/或富集步骤,步骤(4)分化得到的总细胞群体中,表达CD34的造血祖细胞在总细胞群的比例优选为≥90%。
KDR抗原,即CD309,是一种血管内皮生长因子(VEGF)受体,在发育过程中于多种中胚层组织广泛表达,并表达于胚胎阶段的血管内皮细胞中。体外和体内数据显示KDR对于血管内皮细胞和造血细胞的发育形成至关重要。在本发明中,无需纯化和/或富集步骤,步骤(4)分化得到的总细胞群体中,表达KDR的造血祖细胞在总细胞群的比例优选为≥90%。
CD43抗原是一种由SNP基因编码的糖蛋白,又名白细胞唾液酸糖蛋白或唾液酸蛋白,表达于大多数血液白细胞如B细胞、T细胞、NK细胞、粒细胞表面。在本发明中使用CD43阳性这一特征标记从iPSC分化得到的造血祖细胞。在本发明中,无需纯化和/或富集步骤,步骤(4)分化得到的总细胞群体中,表达CD43的造血祖细胞在总细胞群的比例优选为≥90%。
CD45抗原由一类结构相似,分子量较大的跨膜蛋白组成,广泛存在于白细胞表面,其胞浆区段具有蛋白质酪氨酸磷酸酶的作用,能使底物P56lck和P59fyn上酪氨酸脱磷酸而激活,在细胞的信息传导中发挥重要作用。CD45是成熟的血液前体细胞的表面标志物。在本发明中,无需纯化和/或富集步骤,步骤(4)分化得到的总细胞群体中,表达CD45的造血祖细胞在总细胞群的比例优选为≥90%。
可以使用通用的方法检测本发明造血祖细胞的纯度和分化程度,如流式细胞仪法。检测时,加入不同的与相应细胞表面抗原有针对性的特异抗体,抗体可以是完整的单克隆或多克隆抗体,也可以是具有免疫活性的抗体片段,如Fab'或(Fab)2片段;单链Fv分子(scFV);或嵌合抗体。加入抗体与细胞表面的抗原结合一定时间,可用流式细胞仪对细胞进行自动分析和/或分选。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的试剂和材料。
以下为本发明所采用的试剂的来源如表1所示:
表1试剂来源
试剂名称 公司名称
hiPSCs(XS-iPS) 士泽生物
H1 cell/H9 cell 中乔新舟
DMEM/F12 medium Gibco
E8 medium Gibco
RPM1640 medium Gibco
IMDM medium Gibco
Accutase 四正柏
DPBS 源培
BSA Sigma
B27-supplement without vtaminA Gibco
Y27632 Selleck
RelesR Stem cell
NEAA Gibco
Glutamax Gibco
维生素C Sigma
BMP4 Peprotech
CHIR-99021 Selleck
VEGF-165 Sino Biological
FGF2 Origene
SB431542 Selleck
SCF Peprotech
N-乙酰基-L-半胱氨酸 Sigma
Minocycline hydrochloride Selleck
青霉素-链霉素 Gibco
硫代甘油 Sigma
实施例1造血祖细胞的制备
采用的多能干细胞为ESC,具体为H1 cell细胞系。
1.造血祖细胞的制备
1.1多能干细胞传代分化(H1 cell的培养条件为:六孔板无滋养层贴壁培养;H1 cell的分化为:低贴附六孔板悬浮培养)
D-1培养基:E8培养基+10μM Y27632
将融合度达80%左右的多能干细胞从培养箱中拿出,弃掉培养基,用DPBS清洗一次,弃掉DPBS;每孔加入0.5ml Accutase,37℃二氧化碳培养箱孵育2min,2min后;每孔加入DMEM/F12培养基中止酶作用,吹打细胞,将细胞收集到离心管中,离心1200r,5min,弃上清,加入1ml的E8培养基重悬细胞;将细胞进行AO/PI计数,细胞计数取所需细胞量到D-1培养基中,按照8×10 4cell/well的细胞量接种到低贴附六孔板,记为分化D-1。上下左右(十字交叉法)混匀细胞,放入37℃,5%CO 2培养箱中培养20-24小时。
1.2 Stage Ⅰ:中胚层诱导分化(D0-D1)
Stage Ⅰ培养基:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+20ng/ml BMP4+3uM CHIR-99021。
将D-1接种的细胞从培养箱中拿出,显微镜下观察细胞(拍照记录),细胞呈球状;将低贴附六孔板倾斜放置,吸掉部分上清,留大约500μl左右上清,加入Stage Ⅰ培养基,每 孔2ml,轻轻混匀细胞,放回培养箱,分化两天后,收集细胞将进行流式检测内皮细胞KDR+的表达比例。培养时间为42-48小时。
1.3 Stage Ⅱ:生血内皮诱导分化(D2-D5)
Stage Ⅱ:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+5ng/ml BMP4+50ng/ml VEGF-165+50ng/ml FGF2+10uM SB431542
从培养箱中拿出将分化到D2的细胞,显微镜下观察细胞(拍照记录)。将低贴附六孔板倾斜静置,吸掉部分上清,每孔留大约500μl上清,加入Stage Ⅱ培养基,每孔2ml,轻轻混匀细胞,放回培养箱。分化培养至D4时,从培养箱中拿出细胞,将低贴附六孔板倾斜静置,吸去上清,加入Stage Ⅱ培养基,每孔2ml,混匀细胞,放回培养箱。分化至D6时,收集细胞进行流式检测,检测生血内皮表型的表达比例。
1.4 StageⅢ:造血祖细胞诱导分化(D6-D12)
StageⅢ培养基:IMDM培养基+2%不含维生素A的B27添加剂+1%NEAA+1%GlutaMax+50ug/ml维生素C+5ng/ml BMP4+10ng/ml VEGF-165+20ng/ml SCF+30μM NAC(N-乙酰基-L-半胱氨酸)+2μM Minocycline hydrochloride
将分化到D6的细胞从培养箱中拿出,显微镜下观察细胞(拍照记录)。将低贴附六孔板中的细胞倾斜放置,吸掉上清,每孔留大约500μl左右上清,加入StageⅢ培养基,每孔2ml,轻轻混匀细胞,放回培养箱。分化至D8时,从培养箱中拿出细胞,低贴附六孔板倾斜静置,吸去培养基,加入StageⅢ培养基,每孔2ml,轻轻混匀,放回培养箱。分化至D10时,按照D8同样操作换半液。分化到D12时,将分泌出的细胞离心(1500rpm,5min)收集,弃上清,用5ml的StageⅢ培养基重悬,然后将细胞悬液用40μm的筛网过滤,过滤后的细胞离心(1500rpm,5min),弃上清,用1ml的StageⅢ培养基重悬进行细胞计数,以及造血祖细胞表面CD34+CD45+等标记比例的流式检测。
2.不同阶段细胞的细胞检测
流式细胞分析检测方法如下:
1)将细胞收集,1500转/分钟,离心3分钟;
2)弃上清,用PBS重悬细胞,过40目筛网,细胞AO/PI计数,离心5分钟,1500转/分钟;
3)弃上清后,加入PBS,1500转/分钟,离心3分钟,弃去PBS;
4)加入200μl含0.5%BSA的PBS,重悬细胞,根据需要加入各流式抗体,取用抗体时注意避光,混匀后在4度冰箱中放置30分钟;
5)30分钟后,从4度冰箱中取出细胞,加入1ml的0.5%BSA离心细胞1500转/分钟,离心5分钟;
6)弃上清,用500μl PBS洗细胞,每次离心细胞1500转/分钟,离心5分钟;
7)弃上清,用200μl PBS重悬细胞,上机进行流式(NovoCyte 3000,进样器:NS200,厂家为:Agilent Technologies)分析。
2.1起始干细胞
取实施例1中起始干细胞,在显微镜下观察,形态分别如图1所示。图1左是4x显微镜下细胞形态,图1右是10x显微镜下细胞形态。
对起始干细胞进行流式细胞分析,检测其干性标记。干细胞表面干系标记(SSEA4,TRA-1-81,TRA-1-60)的检测结果如图2a所示,干细胞膜内干系标记(Nanog,Oct4,Sox2)检测结果如图2b所示。如图2a所示,SSEA4的表达比例为99.85%;TRA-1-81的表达比例为99.61%;TRA-1-60的表达比例为99.83%;如图2b所示,Nanog表达比例为79.33%,Oct4的表达比例为93.09%;Sox2的表达比例为94.33%。表明初始的胚胎干细胞没有分化趋势,是正常合格的ESC。
2.2 Stage Ⅰ中胚层阶段(D0-D1)
取实施例1中分化前的D0细胞,以及分化两天后还未进入生血内皮阶段的D2细胞,在显微镜下观察,形态分别如图3所示。图3上方左图是ES细胞D0开始分化时细胞形态(4x),图3上方右图是ES细胞D0开始分化时细胞形态(10x)。图3下方左图是ES细胞分化两天时的中胚层细胞形态图(4x),图3下方右图是ES细胞分化两天时的中胚层细胞形态图(10x)。如图3所示,分化两天的中胚层阶段细胞,其直径在100-200μm左右。
对分化两天的中胚层阶段细胞进行流式细胞分析,检测其-KDR+(CD309)的表达,如图4所示,对于在Stage Ⅰ中胚层阶段分化两天后的细胞,KDR+细胞的比例高达96.23%,表明分化的细胞处于中胚层-内皮细胞阶段。
2.3 Stage Ⅱ生血内皮诱导分化阶段(D2-D5)
取实施例1中在Stage Ⅱ生血内皮诱导分化阶段继续分化4天的还未进入造血祖细胞诱导分化阶段的D6细胞,在显微镜下观察,形态分别如图5所示。图5左图是4x显微镜下细胞形态,右图是10x显微镜下细胞形态。如图5所示,分化6天的生血内皮阶段细胞,其直径在150-300μm左右。
对Stage Ⅱ继续分化4天的生血内皮诱导分化阶段的细胞进行流式细胞分析,检测其标志物CD31、CD34、CD43和KDR(CD309)的表达,如图6所示,FITC-CD31阳性的表达比例为48.08%;PE-Cy7-CD34阳性的表达比例为62.68%;APC-CD43阳性的表达比例为43.22%;PE-CD309阳性的表达比例为53.05%。
2.4 StageⅢ造血祖细胞诱导分化(D6-D12)
取实施例1中在StageⅢ生血内皮诱导分化阶段继续分化6天的D12细胞,在显微镜下观察,形态分别如图7所示。图7是4x显微镜下细胞形态,分化12天的造细胞祖细胞,大小约为8μm,是呈圆形且胞核较大的独核单细胞。
对StageⅢ继续分化6天的造血祖细胞进行流式细胞分析,检测其标志物的表达,如图8所示,分化D12的流式结果:CD34+CD45+比例:96.09%,CD34+CD43+比例:90.09%,CD45+CD43+比例:91.08%。
3.造血祖细胞的数量检测
(1)将分化到D12的细胞从培养箱中取出,显微镜下观察细胞,拍照记录;
(2)将细胞放到超净台中,轻轻摇晃细胞板,然后将细胞收集到离心管中;
(3)将离心管中的细胞离心1500rpm,5min;
(4)弃上清,将细胞用3ml的stageⅢ培养基重悬,之后将细胞悬液用40um的筛网过滤(过滤掉未分化的EB球);
(5)将过滤后的细胞悬液,重新离心1500rpm,5min;
(6)弃上清,用1ml的stageⅢ培养基重悬,之后取15ul的细胞悬液与15ul的AO/PI染料混匀,取20ul的液体到细胞计数板中,然后上机计数。
H1 cell分化D12的细胞计数结果为:AO/PI细胞计数:2.08×10 6(六孔板一个孔分泌的细胞量);细胞活率:90.25%。
4.QPCR检测各阶段分化造血细胞相关基因表达
试剂:FastPure Cell/Tissue Total RNA Isolation Kit V2(诺唯赞)、HiScript III RT SuperMix for qPCR(+gDNA wiper)(诺唯赞)、ChamQ Universal SYBR qPCR Master Mix(诺唯赞);荧光定量PCR仪Light Cycle 480 II 96/384罗氏。
(1)NCBI上查找基因FLK1、SOX17、GATA1、CDX2、CXCL12、GATA2、RUNX1、KITLG、GFI1B、GFI1、MYB、HOXA10、HOXA5、HOXA6、HOXA9,由软件primer5设计引物后,由吉凯基因公司构建;
(2)使用FastPure Cell/Tissue Total RNA Isolation Kit V2进行RNA提取;
(3)使用HiScript III RT SuperMix for qPCR(+gDNA wiper),对提取的Total RNA进行反转录实验:37℃15min;85℃5sec;
(4)使用ChamQ Universal SYBR qPCR Master Mix进行qPCR检测:向逆转录20μL体系的cDNA中加入180μL DNase/RNase Free超纯水作为qPCR模板;配置反应体系,每10μL反应体系中含有2×ChamQ Universal SYBR qPCR Master Mix 5μl,Primer1(10μM)0.2μl,Primer2(10μM)0.2μl,Template cDNA Xμl,ddH2O To 10μl;Roche QPCR仪器检测。引物序列如下所示(第2列为上游引物,第3列为下游引物,最后1列是扩增长度的bp数)。
Figure PCTCN2022144143-appb-000002
对初始细胞、D6细胞以及最终分化完成得到的造血组细胞进行相关基因的QPCR检测,结果如图9所示。图9显示了以下基因的检测效果:FLK1、SOX17、GATA1、CDX2、CXCL12、 GATA2、RUNX1、KITLG、GFI1B、GFI1、MYB、HOXA10、HOXA5、HOXA6、HOXA9。可见,造血细胞相关基因均高表达。
5.集落形成单位(colony-forming unit,CFU)检测实验
集落形成单位(colony-forming unit,CFU)检测实验是体外检测造血干/祖细胞功能的标准,CFU集落一般分为:红系细胞集落形成单位(CFU-E)、爆式红细胞集落形成单位(BFU-E)、粒细胞/巨噬细胞(CFU-GM)和混合细胞系集落形成单位(CFU-GEMM)。具体实验方案如下:
1.将所需要的
Figure PCTCN2022144143-appb-000003
培养基(购买自gibco)在4℃过夜或室温融化
2.用16号钝针头的注射器将培养基分装到15mL离心管中(3.3mL/管)
3.准备细胞:将分化结束收集的细胞,使用细胞计数仪计数后,用0.3mL的IMDM+2%FBS重悬细胞,细胞数为10000个,把该细胞悬液接到
Figure PCTCN2022144143-appb-000004
培养基中(比例为1:10)
4.将该离心管后立刻剧烈涡悬振荡,然后静置5min,使气泡消散。
5.准备一块6孔板,同时将无菌的16号钝端针头接到无菌的10mL注射器上,用注射器吸取
Figure PCTCN2022144143-appb-000005
培养基和细胞的混合物,接种到6孔板的一个孔中,每孔3ml,重复3个孔,每接种一支试管的混合物,用一套新的钝端针头和注射器,轻轻倾斜并旋转培养皿,使培养液在培养皿内铺满,并分布均匀。将其余未接种的孔以及六孔板的空隙中补上PBS。
6.将6孔板置于CO2培养箱中,培养14天。
7.培养14天后,进行克隆计数。检测结果如图10(4x)以及图11所示。结果表明,实施例1分化得到的造血组细胞具有形成CFU-E、BFU-E、CFU-GM和CFU-GEMM的能力,表明实施例1得到的造血祖细胞干性强,质量好,具有分化为红系、髓系等多种血液细胞的能力。
实施例2造血祖细胞的制备
实施例2造血祖细胞的制备与实施例1大致相同,不同之处仅在于采用的胚胎干细胞是H9cell细胞系。
2.不同阶段细胞的细胞检测
按照实施例1方法对实施例2的各阶段细胞进行流式细胞分析。
2.1起始干细胞
取实施例2中起始干细胞,在显微镜下观察,形态分别如图12所示。图12左是4x显微镜下细胞形态,图12右是10x显微镜下细胞形态。
按照实施例1方法对实施例2起始干细胞进行流式细胞分析,检测其干性标记。干细胞表面干系标记(SSEA4,TRA-1-81,TRA-1-60)的检测结果如图13a所示,干细胞膜内干系标记(Nanog,Oct4,Sox2)检测结果如图13b所示。图13表明初始的胚胎干细胞没有分化趋势,是正常合格的ESC。
2.2 Stage Ⅰ中胚层阶段(D0-D1)
取实施例2中分化两天后还未进入生血内皮阶段的D2(中胚层)细胞,在显微镜下观察,形态分别如图14所示。图14左右两图均是H9细胞分化两天时的中胚层细胞形态图(10x)。
对分化两天的中胚层阶段细胞进行流式细胞分析,检测其-KDR+(CD309)的表达,如 图15所示,对于在Stage Ⅰ中胚层阶段分化两天后的细胞,KDR+细胞的比例高达95.96%,表明分化的细胞处于中胚层-内皮细胞阶段。
2.3 Stage Ⅱ生血内皮诱导分化阶段(D2-D5)
取实施例2中在Stage Ⅱ生血内皮诱导分化阶段继续分化4天的还未进入造血祖细胞诱导分化阶段的D6细胞,在显微镜下观察,形态分别如图16所示。图16左图是4x显微镜下细胞形态,右图是10x显微镜下细胞形态。如图16所示,分化6天的生血内皮阶段细胞,其直径在150-300μm左右。
对Stage Ⅱ继续分化4天的生血内皮诱导分化阶段的细胞进行流式细胞分析,检测其标志物CD31、CD34、CD43和KDR(CD309)的表达,如图17所示。
2.4 StageⅢ造血祖细胞诱导分化(D6-D12)
取实施例2中在StageⅢ生血内皮诱导分化阶段继续分化6天的D12细胞,在显微镜下观察,形态如图18所示。图18是4x显微镜下细胞形态,分化12天的造细胞祖细胞,大小约为8μm,是呈圆形且胞核较大的独核单细胞。
对StageⅢ继续分化6天的造血祖细胞进行流式细胞分析,检测其标志物的表达,如图19所示,分化D12的流式结果:CD34+CD45+比例:98.08%,CD34+CD43+比例:88.44%,CD45+CD43+比例:87.55%。
3.造血祖细胞的数量检测
按照实施例1方法对实施例2最终获得的目标细胞进行数量检测。
H9cell分化D12的细胞计数结果为:AO/PI细胞计数:1.8×10 6(六孔板一个孔分泌的细胞量);细胞活率:91.38%。
4.QPCR检测各阶段分化造血细胞相关基因表达
按照实施例1方法,对实施例2的初始细胞、D6细胞以及最终分化完成得到的造血组细胞进行相关基因的QPCR检测,结果如图20所示。图20显示了以下基因的检测效果:FLK1、SOX17、GATA1、CDX2、CXCL12、GATA2、RUNX1、KITLG、GFI1B、GFI1、MYB、HOXA10、HOXA5、HOXA6、HOXA9。可见,造血细胞相关基因均高表达。
5.集落形成单位(colony-forming unit,CFU)检测实验
按照实施例1方法,对实施例2最终分化得到的造血祖细胞进行集落形成单位检测实验。
针对红系细胞集落形成单位(CFU-E)、爆式红细胞集落形成单位(BFU-E)、粒细胞/巨噬细胞(CFU-GM)和混合细胞系集落形成单位(CFU-GEMM)的检测结果如图11所示。结果表明,实施例2分化得到的造血组细胞具有形成CFU-E、BFU-E、CFU-GM和CFU-GEMM的能力,表明实施例2得到的造血祖细胞干性强,质量好,具有分化为红系、髓系等多种血液细胞的能力。实施例1和2的CFU检测实验的结果如表2所示。
表2实施例1和2的CFU检测实验的结果
CFU H1细胞 H9细胞
CFU-GEMM 6 4
CFU-GM 112 98
BFU-E 8 10
CFU-E 23 25
实施例3造血祖细胞的制备
实施例的操作与实施例1大致相同,不同之处仅在于采用的是hiPSCs(购自士泽生物,货号XS-iPS)。
在显微镜下观察最终分化得到的造血祖细胞,形态如图21(4x)所示。对最终分化得到的造血祖细胞进行流式细胞分析,其最终分化得到的造血祖细胞的检测其标志物的表达,如图22所示,在造血祖细胞阶段继续分化6天,即D12的流式结果为:CD34+CD45+比例为:97.30%;CD34+CD43+比例为:91.55%;CD45+CD43+比例为:89.20%;CD34+CD117+比例为:62.18%;CD45+CD117+比例为:53.84%;CD43+CD117+比例为:57.05%。
实施例4造血祖细胞的制备
实施例的操作与实施例1大致相同,不同之处仅在于各阶段培养基如下:
1.1 D-1培养基:E8 medium培养基+10μM Y27632
1.2 Stage Ⅰ:中胚层诱导分化(D0-D1)
Stage Ⅰ培养基:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+10ng/ml BMP4+5uM CHIR-99021。
1.3 Stage Ⅱ:生血内皮诱导分化(D2-D5)
Stage Ⅱ:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+10ng/ml BMP4+20ng/ml VEGFA+20ng/ml FGF2+5uM SB431542
1.4 StageⅢ:造血祖细胞诱导分化(D6-D12)
StageⅢ培养基:IMDM培养基+2%不含维生素A的B27添加剂+1%NEAA+1%GlutaMax+50ug/ml维生素C+10ng/ml BMP4+10ng/ml VEGFA+50ng/ml SCF+30μM NAC(N-乙酰基-L-半胱氨酸)+2μM Minocycline hydrochloride
对最终分化得到的造血祖细胞进行流式细胞分析,检测其标志物的表达,如图23所示,在造血祖细胞阶段继续分化6天,即D12的流式结果为:CD34+CD45+比例为:92.45%;CD34+CD43+比例为:87.73%;CD45+CD43+比例为:91.23%;CD34+CD117+比例为:42.10%;CD45+CD117+比例为:43%;CD43+CD117+比例为:41.87%。
实施例5造血祖细胞的制备
实施例的操作与实施例1大致相同,不同之处仅在于各阶段培养基以及培养时间如下:
1.1 D-1培养基:mTESR培养基+10μM Y27632
1.2 Stage Ⅰ:中胚层诱导分化(D0)
Stage Ⅰ培养基:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+5uM CHIR-99021。
1.3 Stage Ⅱ:生血内皮诱导分化(D1-D4)
Stage Ⅱ:RPM1640培养基+2%不含维生素A的B27添加剂+1%非必须氨基酸+1%谷氨酰胺+50ug/ml维生素C+5ng/ml BMP4+50ng/ml VEGFA+50ng/ml FGF2+10uM SB431542
1.4 StageⅢ:造血祖细胞诱导分化(D5-D12)
StageⅢ培养基:α-MEM培养基+2%不含维生素A的B27添加剂+1%NEAA+1%GlutaMax+胰岛素-转铁蛋白-硒(ITS-G)(100X)+50ug/ml维生素C+5ng/ml BMP4+10ng/ml VEGFA+50ng/ml SCF+30μM NAC(N-乙酰基-L-半胱氨酸)+2μM Minocycline hydrochloride。
对最终分化得到的造血祖细胞进行流式细胞分析,检测其标志物的表达,如图24所示,在造血祖细胞阶段继续分化6天,即D12的流式结果:CD34+CD45+比例为:96.99%;CD34+CD43+比例为:94.02%;CD45+CD43+比例为:94.43%;CD34+CD117+比例为:50.94%;CD45+CD117+比例为:51.47%;CD43+CD117+比例为:50.92%。
实施例6
1.造血祖细胞分化、流式细胞分析、以及数量检测的相关操作与实施例1相同,采用不同批次的H1细胞进行重复。
最终结果如表1所示。对最终分化得到的造血祖细胞其流式细胞分析图如图25-27所示。根据结果可知,本发明所述分化方法重复性好,分化效果十分稳定,分化效率可以维持在90%以上,且能获得大量的造血祖细胞。
表3造血祖细胞分化结果
  起始细胞数量 AO/PI细胞计数 CD34+CD45+ 造血祖细胞数量
H1-P6 8*10^4 2.48*10^6 95.17% 2.36*10^6
H1-P7 8*10^4 3.28*10^6 97.07% 3.18*10^6
H1-P8 8*10^4 3.15*10^6 97.29% 3.06*10^6
对比例1
与实施例1中制备造血组细胞的方法相同,不同之处在于:在Stage Ⅰ、对Stage Ⅱ和StageⅢ培养基中均添加0.1mM的硫代甘油以及1%含量的青霉素-链霉素。
按照实施例1记载的方法检测对比例1得到造血祖细胞的数量和标志物表达。结果显示对比例1造血祖细胞的分化效率以及细胞数量均显著低于实施例;表明本发明通过在各阶段培养基中不添加青霉素-链霉素和硫代甘油,能够显著提高造血祖细胞的数量,以及分化效率。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。

Claims (10)

  1. 造血祖细胞的制备方法,包括以下步骤:
    (1)将多能干细胞进行培养,获得拟胚体;
    (2)将所述拟胚体进行中胚层分化培养,获得中胚层细胞;
    (3)将所述中胚层细胞进行生血内皮分化培养,获得生血内皮细胞;
    (4)将所述生血内皮细胞进行造血祖细胞分化培养,获得造血祖细胞;
    所述制备方法在无抗生素条件下和/或无硫代甘油条件下制备造血祖细胞。
  2. 根据权利要求1所述的制备方法,其特征在于:所述步骤(1)的培养体系包含ROCK抑制剂。
  3. 根据权利要求1所述的制备方法,其特征在于:所述步骤(2)的培养体系包含BMP4和/或GSK-3β抑制剂。
  4. 根据权利要求1所述的制备方法,其特征在于:所述步骤(3)的培养体系包含选自以下的至少一种:BMP4、血管内皮生长因子、成纤维细胞生长因子、和TGFβ/ALK抑制剂。
  5. 根据权利要求1所述的制备方法,其特征在于:所述步骤(4)的培养体系包含选自以下的至少一种:BMP4、血管内皮生长因子和干细胞因子。
  6. 一种制备造血祖细胞的试剂盒,包括选自以下的至少一种:拟胚体培养体系,中胚层分化培养体系,生血内皮分化培养体系,和造血祖细胞分化培养体系;所述各培养体系不含有抗生素和/或硫代甘油。
  7. 权利要求1-5任一项所述制备方法或权利要求6所述试剂盒制备得到的造血组细胞。
  8. 根据权利要求7所述造血祖细胞,其特征在于:所述造血祖细胞具有选自下组(A)的至少一种特征:
    (i)90%以上的细胞具有造血祖细胞表面抗原CD34+;
    (ii)90%以上的细胞具有造血祖细胞表面抗原组合CD43+;
    (iii)90%以上的细胞具有造血祖细胞表面抗原组合CD45+;
    (iv)90%以上的细胞具有造血祖细胞表面抗原组合CD34+CD45+;
    (v)90%以上的细胞具有造血祖细胞表面抗原组合CD34+CD43+;
    (vi)90%以上的细胞具有造血祖细胞表面抗原组合CD43+CD45+;
    (vii)30%以上的细胞具有造血祖细胞表面抗原组合CD45+CD117+;
    (viii)40%以上的细胞具有造血祖细胞表面抗原组合CD34+CD117+;和
    (ix)30%以上的细胞具有造血祖细胞表面抗原组合CD43+CD117+。
  9. 药物组合物,包含权利要求7或8所述的造血祖细胞,以及药学上可接受的载体。
  10. 权利要求7或8所述造血祖细胞在制备治疗和/或预防血液病的药物中的应用。
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