WO2022217955A1 - 一种诱导多能干细胞向造血前体细胞分化的培养基以及方法 - Google Patents

一种诱导多能干细胞向造血前体细胞分化的培养基以及方法 Download PDF

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WO2022217955A1
WO2022217955A1 PCT/CN2021/138097 CN2021138097W WO2022217955A1 WO 2022217955 A1 WO2022217955 A1 WO 2022217955A1 CN 2021138097 W CN2021138097 W CN 2021138097W WO 2022217955 A1 WO2022217955 A1 WO 2022217955A1
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hematopoietic
cells
precursor cells
differentiation
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French (fr)
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雷晓华
张键
马驰原
赵华山
汪宝蓓
李梦霞
李荣荣
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中国科学院深圳先进技术研究院
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Definitions

  • the invention belongs to the field of biotechnology, and relates to a method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells.
  • Pluripotent stem cells including embryonic-derived embryonic stem cells and in vitro reprogramming-derived induced pluripotent stem cells.
  • Pluripotent stem cells can maintain self-renewal ability in long-term culture in vitro, and have the potential of multi-directional differentiation, including differentiation into almost all functional blood cells.
  • Hematopoietic precursor cells are precursor cells that have the ability to self-renew and differentiate into various blood cells, which ultimately generate various blood cell components, including red blood cells, white blood cells, and platelets, which can also differentiate into various other cells.
  • hematopoietic stem/progenitor cells as well as various mature blood cells can serve the major strategic needs of clinical hematopoietic stem cell transplantation and blood cell transfusion therapy.
  • the purpose of the present invention is to provide a simple and efficient pluripotent stem cell to hematopoietic precursor differentiation system, which includes a pluripotent stem cell to hematopoietic precursor differentiation system, and hematopoietic endothelial precursor cells to hematopoietic precursor cells. differentiation system.
  • the present invention provides a medium for the differentiation of pluripotent stem cells into hematopoietic precursor cells with clear chemical components, and uses the medium to explore a method for efficiently inducing pluripotent stem cells to differentiate into hematopoietic cells by using a random gyroscope to simulate the microgravity effect.
  • a method for the differentiation of human hematopoietic precursor cells compared with the existing methods, the method has higher operability and reproducibility, and lays a technical foundation for the large-scale production of hematopoietic precursor cells for biomedicine and clinical treatment in the future.
  • the present invention provides a medium for inducing differentiation of mesoderm cells, which comprises a basal differentiation medium and bone morphogenetic protein 4 (BMP4), Activin A (Activin A) and a GSK-3 ⁇ / ⁇ inhibitor (CHIR99021) ;
  • BMP4 bone morphogenetic protein 4
  • Activin A Activin A
  • CHOK-3 ⁇ / ⁇ inhibitor CHOK-3 ⁇ / ⁇ inhibitor
  • the basal differentiation medium is IF9S complete medium, including IMDM medium and F12 medium mixed in a volume ratio of 1:0.8-1.2, and polyvinyl alcohol (PVA) with a final concentration of 8-12 mg/L, Lipids 100X additive with a volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with a volume percentage of 1.8%-2.2% of the IF9S complete medium, and a monothiol with a final concentration of 35-45 ⁇ l/L Glycerol ( ⁇ MTG), sodium ascorbyl phosphate (AA2P) at a final concentration of 55-70 mg/L, glutalanine dipeptide (GlutaMax TM ) 100X at a volume percentage of 0.8%-1.2% of the IF9S complete medium Supplements and non-essential amino acid supplements 100X (NEAA) in volume percentages of 0.8%-1.2% of the IF9S complete medium.
  • PVA polyvinyl alcohol
  • the IF9S complete medium includes IMDM medium and F12 medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L, and the volume percentage is 0.1% Lipids100X supplement based on IF9S, 2% ITS-X 100X supplement of the IF9S complete medium by volume, monothioglycerol ( ⁇ MTG) at a final concentration of 40 ⁇ l/L, and a final concentration of 64 mg/L of sodium ascorbyl phosphate (AA2P), GlutaMax TM 100X supplement at 1% by volume of the IF9S complete medium, and nonessential amino acid supplement 100X (NEAA) at 1% by volume of the IF9S complete medium.
  • PVA polyvinyl alcohol
  • the mesodermal cell differentiation medium is IF9S complete medium supplemented with 40-60 ng/mL BMP4, 15-25 ⁇ g/mL Activin A, and 0.8-1.2 ⁇ M CHIR99021.
  • the mesodermal cell differentiation medium is IF9S complete medium supplemented with 50 ng/mL of BMP4 and 20 ⁇ g/mL of Activin A, 1 ⁇ M CHIR99021.
  • the present invention provides a medium for inducing differentiation of hematopoietic endothelial precursor cells, which comprises a basal differentiation medium, SB431542, VEGF, bFGF and SCF;
  • the basal differentiation medium is IF9S complete medium, including IMDM medium and F12 medium mixed in a volume ratio of 1:0.8-1.2, and polyvinyl alcohol (PVA) with a final concentration of 8-12 mg/L, Lipids 100X additive with a volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with a volume percentage of 1.8%-2.2% of the IF9S complete medium, and a monothiol with a final concentration of 35-45 ⁇ l/L Glycerol ( ⁇ MTG), Sodium Ascorbyl Phosphate (AA2P) at a final concentration of 55-70 mg/L, GlutaMax TM 100X Supplement at 0.8%-1.2% by volume of the IF9S complete medium, and 100X Supplement by volume of the IF9S 0.8%-1.2% non-essential amino acid supplement 100X (NEAA) in complete medium.
  • PVA polyvinyl alcohol
  • Lipids 100X additive with a volume percentage of 0.08-0.12% of the medium
  • the IF9S complete medium includes IMDM medium and F12 medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L, and the volume percentage is 0.1% Lipids100X supplement based on the base, 2% ITS-X100X supplement of the IF9S complete medium by volume, monothioglycerol ( ⁇ MTG) with a final concentration of 40 ⁇ l/L, and a final concentration of 64 mg/L Sodium Ascorbyl Phosphate (AA2P), GlutaMax TM 100X Supplement at 1% by volume of the IF9S Complete Medium and Nonessential Amino Acids Supplement 100X (NEAA) at 1% by volume of the IF9S Complete Medium.
  • PVA polyvinyl alcohol
  • the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 8-12 ⁇ M SB431542, 40-60 ng/mL VEGF, 40-60 ng/mL bFGF and 40-60 ng/mL SCF ;
  • the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 10 ⁇ M SB431542, 50 ng/mL VEGF, 50 ng/mL bFGF and 50 ng/mL SCF.
  • polyvinyl alcohol PVA
  • deionized water a 5% stock solution before preparing the IF9S complete medium.
  • IMDM medium to dilute monothioglycerol by 2% to prepare a 500X stock solution.
  • F12 medium is used to dissolve AA2P and prepare a 5 mg/mL stock solution.
  • the IMDM medium and the F12 basic base are conventional medium, and commercial medium can be purchased or prepared by themselves.
  • the IMDM medium and the F12 medium are both Available from Gibco.
  • the Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media.
  • the Lipids additive is purchased from Gibco Company, the item number is 11905031.
  • the ITS-X (100X) supplement is a basal medium supplement of insulin-transferrin-seleno-ethanolamine for reducing or replacing fetal bovine serum in cell culture medium.
  • the ITS-X additive is purchased from Gibico Company, the item number is 51500056.
  • the GlutaMax TM (100X) additive is glutalanine dipeptide, which is a cell culture additive, which can directly replace L-glutamine in the cell culture medium.
  • the GlutaMax additive is purchased from Gibico Company, the item number is 35050-061.
  • the NEAA (100X) additive is a non-essential amino acid and is a cell culture additive.
  • the NEAA additive is purchased from Gibico Company, the item number is 35050-061.
  • the present invention provides a combined medium for inducing differentiation of hematopoietic endothelial precursor cells, comprising the above-mentioned medium for inducing differentiation of mesoderm cells and the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells.
  • the medium for inducing differentiation of mesoderm cells and the medium for inducing differentiation of hematopoietic endothelial precursor cells are placed or formulated separately.
  • the present invention provides a medium for inducing hematopoietic endothelial precursor cells to differentiate into hematopoietic precursor cells, comprising a basal differentiation medium, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) , stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6) and thrombopoietin (TPO);
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • IL-3 interleukin 3
  • IL-6 interleukin 6
  • TPO thrombopoietin
  • Described basal differentiation medium is IF9S complete medium, including IMDM basal medium and F12 basal medium mixed by the volume ratio of 1:0.8-1.2, and the final concentration is: polyvinyl alcohol (PVA) of 8-12mg/L. ), the Lipids (100X) additive whose volume percentage is 0.08-0.12% of the medium, the ITS-X (100X) additive whose volume percentage is 1.8%-2.2% of the medium, and the final concentration is 35-45 ⁇ l/ L of monothioglycerol ( ⁇ MTG), AA2P at a final concentration of 55-70 mg/L, 0.8-1.2% GlutaMax TM (100X) supplement by volume of the medium and 0.8-1.2% by volume of the medium 0.8-1.2% of non-essential amino acid additives (NEAA, 100X).
  • PVA polyvinyl alcohol
  • ITS-X 100X
  • NEAA non-essential amino acid additives
  • the basal differentiation medium is IF9S complete medium, including IMDM basal medium and F12 basal medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L , the Lipids (100X) additive whose volume percentage is 0.1% of the medium, the ITS-X (100X) additive whose volume percentage is 2% of the medium, and the monothioglycerol with a final concentration of 40 ⁇ l/L ( ⁇ MTG), AA2P at a final concentration of 64 mg/L, GlutaMax TM (100X) supplement at 1% by volume of the medium, and nonessential amino acid supplement (NEAA, 100X) at 1% by volume of the medium ).
  • PVA polyvinyl alcohol
  • the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 40-60 ng/mL VEGF, 15-25 ng/mL bFGF, 40-60 ng/mL ng/mL SCF, 8-12 ng/mL IL-3, 40-60 ng/mL IL-6, and 15-25 ng/mL TPO.
  • the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 50 ng/mL VEGF, 20 ng/mL bFGF, 50 ng/mL SCF, 10 ng/mL IL-3, 50 ng IL-6/mL and 20 ng/mL TPO.
  • deionized water is used to dissolve polyvinyl alcohol (PVA), and a stock solution with a concentration of 5% is prepared.
  • IMDM medium to dilute monothioglycerol by 2% to prepare a 500X stock solution.
  • F12 medium is used to dissolve AA2P and prepare a 5 mg/mL stock solution.
  • the IMDM basal medium and the F12 basal medium are conventional medium, which can be purchased from commercialized medium or prepared by themselves.
  • the IMDM and F12 medium are both Available from Gibco.
  • the Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media.
  • the Lipids additive is purchased from Gibco Company, the item number is 11905031.
  • the ITS-X (100X) supplement is a basal medium supplement of insulin-transferrin-seleno-ethanolamine for reducing or replacing fetal bovine serum in cell culture medium.
  • the ITS-X additive is purchased from Gibico Company, the item number is 51500056.
  • the GlutaMax TM (100X) additive is glutalanine dipeptide, which is a cell culture additive, which can directly replace L-glutamine in the cell culture medium.
  • the GlutaMax additive is purchased from Gibico Company, the item number is 35050-061.
  • the NEAA (100X) additive is a non-essential amino acid and is an advanced cell culture additive.
  • the NEAA additive is purchased from Gibico Company, the item number is 35050-061.
  • the present invention provides a medium composition for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells, the medium composition comprising:
  • the medium for inducing differentiation of mesoderm cells, the medium for inducing differentiation of hematopoietic endothelial precursor cells and the medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells are placed or formulated separately.
  • the present invention provides a method for promoting the transformation of mesoderm cells into hematopoietic endothelial precursor cells, the method comprising: using a random three-dimensional gyroscope to rotate the cell culture vessel under rotating conditions during the process of culturing the mesodermal cells. Cultivation steps.
  • the culturing under the rotating condition is to randomly rotate the cell culture vessel with a random three-dimensional gyroscope, more preferably, the random rotation is 0.1-10 rpm/min; more preferably, the rotation speed is 5 rpm/ minute.
  • the culture time for culturing under the rotating condition is 48-96 hours, preferably 60-72 hours.
  • the medium for culturing under the rotating condition adopts the above-mentioned hematopoietic endothelial precursor inducing differentiation medium.
  • the present invention also provides a method for differentiating and producing hematopoietic endothelial precursor cells from human pluripotent stem cells, the method comprising the steps of:
  • the cell container is pre-coated with growth factor-reduced basement membrane matrix (Matrigel matrix);
  • the induced mesoderm cells are further differentiated and cultured into hematopoietic endothelial precursor cells:
  • the random three-dimensional gyroscope was placed in a 37°C, 5% CO 2 incubator for 60-96 hours of rotational culture.
  • the growth factor-reduced Matirgel matrix is Growth Factor Reduced produced by Corning Company Matrigel, Catalog No. 354230.
  • the density of seeding in S13) is 50-200 clonal clumps/cm 2 .
  • the density of the inoculation described in S13) is 100 clone clumps/cm 2 ; .
  • the hematopoietic endothelial precursor cell inducing differentiation medium described in S21 is added to 95%-100% of the culture vessel;
  • the rotation speed in S22) is 5 rpm/min;
  • the human pluripotent stem cell clone is amplified and cultured before digestion:
  • the human pluripotent stem cells are seeded in the coated cell container and cultured in TeSR TM -E8 TM medium.
  • the fresh medium is replaced every 24 hours, and when the cell clone grows to a density of 70-80%, the cells are digested and passaged or induced to differentiate and cultured afterwards; the cell passage period is 3-4 days.
  • the extracellular matrix of the embryonic stem cell grade is Matrigel produced by BioCoat Company, the product number is 354277.
  • the present invention also provides a method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, the method comprising, during the culturing process of hematopoietic endothelial precursor cells, using a random gyroscope to place the cell culture vessel in the hematopoietic precursor cell. Steps for culturing under rotational conditions.
  • the culturing under the rotating condition is to randomly rotate the cell culture vessel with a random gyroscope, more preferably, the inner and outer diameters of the random rotation are 0.1-10 rpm/min; more preferably, the rotation speed is an inner and outer diameter of 5rpm/min.
  • the culturing time under the rotating condition is 48-72 hours, preferably 72 hours.
  • the medium for culturing under the rotating condition is the above-mentioned medium for inducing differentiation of vascular/hematopoietic endothelial precursor cells into hematopoietic precursor cells.
  • the medium for inducing hematopoietic endothelial precursor cells to differentiate into hematopoietic precursor cells described in S3) is added to 95%-100% of the culture vessel;
  • the cells are cultured for 48-72 hours, preferably the cells are cultured for 72 hours.
  • the cell culture conditions are 37° C., 5% CO 2 .
  • the present invention also provides a method for generating hematopoietic precursor cells from human pluripotent stem cells, the method comprising the following steps:
  • the present invention provides the use of a random three-dimensional gyroscope for promoting the transformation of mesoderm cells to hematopoietic endothelial precursor cells, or the use of promoting the transformation of hematopoietic endothelial precursor cells to hematopoietic precursor cells.
  • the present invention selects a medium with clear chemical composition for the culture and differentiation of human pluripotent stem cells, and the present invention also describes the use of a random gyrator to simulate the microgravity effect to develop pluripotent stem cells into hematopoietic endothelial precursor cells and pre-hematopoietic cells. Somatic cell differentiation techniques. In a word, compared with the prior art, the present invention has the following advantages:
  • This culture system starts from the culture and expansion of pluripotent stem cells to mesendoderm differentiation, hematopoietic endothelial precursor differentiation and hematopoietic precursor cells are cultured under serum-free, feeder-free and chemically defined conditions.
  • the currently reported methods for in vitro differentiation of human pluripotent stem cells into hematopoietic precursors/stem cells mainly include co-culture with mouse bone marrow stromal cells (OP9 cell line) or mouse aortic gonadal mesonephric stromal cells (mAGM cell line). system, as well as a system using Embryoid Body (EB) differentiation.
  • OP9 cell line mouse bone marrow stromal cells
  • mAGM cell line mouse aortic gonadal mesonephric stromal cells
  • the co-culture system contains animal-derived substances, which increases the instability and safety of the experiment, and increases the complexity of the experimental steps; while the EB differentiation system has low differentiation efficiency.
  • the culture system with clear chemical composition and the random three-dimensional rotation simulating microgravity culture method used in the invention can obtain a large number of hematopoietic endothelial precursor cells and hematopoietic precursor cells derived from pluripotent stem cells in a short time, and the differentiation efficiency is very high , the differentiation efficiency is more than 2 times higher than that of static culture.
  • This low-cost, safe and controllable serum-free and feeder-free random 3D rotational culture strategy can lay the foundation for the large-scale production of hematopoietic endothelial or hematopoietic precursor cells for clinical therapy in the future.
  • FIG. 1 is a bright field picture of hematopoietic endothelial precursor cells induced from human pluripotent stem cells by the method of the present invention.
  • FIG. 2 is a picture of fluorescent staining of hematopoietic endothelial precursor cell marker molecules CD31 and CD34 from hematopoietic endothelial precursor cells induced from human pluripotent stem cells using the method of the present invention.
  • Figure 3 is a flow cytometric analysis of the differentiation of hematopoietic endothelial precursor cells under the random three-dimensional rotary culture of the present invention and a traditional static culture, and compares the efficiency of the cells expressing CD31+CD34+ after differentiation.
  • picture A is the control group
  • picture B is the random three-dimensional rotation group.
  • FIG. 4 is a bright field picture of hematopoietic precursor cells induced from human pluripotent stem cells using the method of the present invention.
  • Fig. 5 is a picture of fluorescent staining of hematopoietic precursor cell marker molecules CD34 and CD43 from hematopoietic precursor cells induced from human pluripotent stem cells using the method of the present invention.
  • Figure 6 is a flow cytometric analysis of the differentiation of hematopoietic precursor cells using the random rotary culture of the present invention and a traditional static culture, and compares the efficiency of the cells expressing CD34+CD43+ after differentiation.
  • Picture A is the control group
  • picture B is the random rotation group.
  • the reagents used in the following examples are all analytical grade reagents and can be obtained commercially from regular channels.
  • the human pluripotent stem cells used in the present invention are the human embryonic stem cell line H1.
  • the reagents used in the following examples are all analytical grade reagents and can be obtained commercially from regular channels.
  • Example 1 Preparation of medium for serum-free culture system
  • IMDM medium F12 medium, polyvinyl alcohol (PVA), Lipids (100X), ITS-X (100X), monothioglycerol ( ⁇ MTG), AA2P, GlutaMax TM (100X), non-essential amino acids ( NEAA, 100X) to prepare IF9S medium.
  • PVA polyvinyl alcohol
  • ITS-X 100X
  • ⁇ MTG monothioglycerol
  • AA2P GlutaMax TM (100X)
  • NEAA, 100X non-essential amino acids
  • each component of the culture medium was purchased from:
  • IMDM medium was purchased from Gibco Company, the article number is 21056-023;
  • F12 medium was purchased from Gibco Company, the product number is 31765-027;
  • Polyvinyl alcohol (PVA) was purchased from Sigma Company, the item number is P8136;
  • Lipids (100X) were purchased from Gibco, Cat. No. 11905031;
  • ITS-X (100X) was purchased from Gibco, Item No. 51500-056;
  • Monothioglycerol ( ⁇ MTG) was purchased from Sigma, the product number is M6145;
  • AA2P was purchased from Sigma, the item number is A8960;
  • GlutaMax (TM) (100X) was purchased from Gibco, Item No. 35050-061;
  • Non-essential amino acids were purchased from Gibco, catalog number 11140-035;
  • the medium is formulated.
  • polyvinyl alcohol (PVA) is powder, which needs to be dissolved in deionized water and made into a 5% concentration stock solution before use.
  • the IF9S medium is prepared by the following method:
  • each reagent is: 10 mg/L polyvinyl alcohol (PVA) ), 0.1% by volume Lipids (100X) additive, 2% by volume ITS-X (100X) additive, 40 ⁇ l/L monothioglycerol ( ⁇ MTG), 64 mg/L AA2P, volume percentage 1% GlutaMax TM (100X) additive and 1% volume percent non-essential amino acid additive (NEAA, 100X).
  • PVA polyvinyl alcohol
  • ITS-X 100X
  • ⁇ MTG monothioglycerol
  • NEAA 1% volume percent non-essential amino acid additive
  • Described mesoderm cell differentiation medium is IF9S medium supplemented with 50 ng/mL of BMP4, 20 ⁇ g/mL of Activin A, 1 ⁇ M CHIR99021.
  • the hematopoietic endothelial precursor differentiation medium is IF9S medium supplemented with 10 ⁇ M SB431542, 50 ng/mL VEGF, 50 ng/mL bFGF, 50 ng/mL of SCF.
  • the hematopoietic precursor/stem cell differentiation medium is IF9S complete medium supplemented with 50 ng/mL VEGF, 20 ng/mL bFGF, 50 ng/mL SCF, 10 ng/mL of IL-3, 50 ng/mL of IL-6 and 20 ng/mL TPO.
  • Example 2 Preparation of hematopoietic endothelial precursor cells derived from human pluripotent stem cells
  • the human pluripotent stem cells used in this example are human embryonic stem cell line (H1) (originated from the University of Wisconsin, USA).
  • the cultured dishes were coated with the extracellular matrix of embryonic stem cell grade, Matrigel, and then the human embryonic stem cells were seeded in the coated dishes and treated with a commercial medium TeSR TM -E8 TM with a clear chemical composition. Incubate with fresh medium every 24 hours. Human embryonic stem cells grow in the form of clones. When the cell clones grow to a density of 70-80%, the cells need to be digested and passaged or induced to differentiate. The cell passaging cycle is generally 3-4 days.
  • the medium in the culture flask is aspirated and replaced with a fresh hematopoietic endothelial precursor cell induction medium, and the culture flask is filled with the culture medium;
  • Example 3 Detection of hematopoietic endothelial precursor cells
  • the vascular/hematopoietic endothelial precursor cells produced in Example 2 were collected for identification by immunofluorescence staining.
  • CD31 and CD34 are important markers of vascular/hematopoietic endothelial precursor cells, and the fluorescence color results can confirm that the method of the present invention can further induce mesoderm cells into CD31 and CD34 positive cells, that is, hematopoietic endothelial precursor cells.
  • Example 4 Efficiency analysis of hematopoietic endothelial precursor cells
  • the resulting hematopoietic endothelial precursor cells produced in Example 2 were collected for identification by flow cytometry analysis.
  • Example 2 The cells described in Example 2 were digested into single cells using Accutase enzyme (Cat. No. A1110501, Gibco), and the digestion was terminated with IF9S medium.
  • Accutase enzyme Cat. No. A1110501, Gibco
  • Cell collection transfer the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes to enrich the cells.
  • Antibody incubation labeling after centrifugation, aspirate the supernatant, resuspend the cells with 0.2% BSA, and set the cell grouping and antibody labeling according to the specific conditions of the experiment.
  • - CD31 antibody Cat. No. 560983, BD Company
  • mouse anti-human FITC-CD34 antibody Cat. No. 560942, BD Company
  • the marker molecule antibodies for hematopoietic precursor/stem cell identification are mouse anti-human FITC-CD34 antibody (Cat. No.: 560942, BD Company) and mouse anti-human APC-CD43 antibody (Cat. No. 560198, BD Corporation).
  • the dilution ratio of the antibody is generally 1:200, and the antibody is labeled with a horizontal shaker at room temperature for 30 minutes.
  • Cell washing add an appropriate amount of PBS, centrifuge at 1000 rpm for 5 minutes to enrich the cells, and aspirate the supernatant.
  • Cell fixation and on-machine detection Resuspend cells in PBS containing 1% paraformaldehyde. Use a cell sieve (40um) for screening before testing on the machine. Pipette the cell suspension into a flow tube in preparation for flow analysis. Flow Cytometry for FACS Calibur analytical flow cytometer (BD company), after the test, use FlowJo 10.0.7 Software for data analysis.
  • Figure 3 shows the results of flow cytometric analysis of hematopoietic endothelial precursor cells in the control group and the random three-dimensional rotation group.
  • the random three-dimensional rotation group cell preparation method is the method of embodiment 2, and the preparation method of control group cells is basically the same as that of embodiment 2, the difference only lies in that after step e, it is directly carried out at 37 ° C, 5% CO 2 in the incubator After culturing for 72 hours, control cells were obtained.
  • Example 5 Preparation of human pluripotent stem cell-derived hematopoietic precursor cells
  • Example 6 Detection of hematopoietic precursor cells
  • the hematopoietic precursor cells produced in Example 5 were collected for identification by immunofluorescence staining.
  • CD34 and CD43 are important markers of hematopoietic precursor cells, and the results of fluorescence color can confirm that hematopoietic endothelial precursor cells can be induced into CD34 and CD43 positive cells, namely hematopoietic precursor cells, by the method of the present invention.
  • Figure 2 shows the results of the identification of hematopoietic precursor cells by staining.
  • Example 7 Efficiency analysis of hematopoietic precursor cells
  • the hematopoietic precursor cells produced in Example 2 were collected for identification by flow cytometry analysis.
  • Example 2 The cells described in Example 2 were digested into single cells using Accutase enzyme (Cat. No. A1110501, Gibco), and the digestion was terminated with IF9S medium respectively.
  • Accutase enzyme Cat. No. A1110501, Gibco
  • Cell collection transfer the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes to enrich the cells.
  • the marker molecule antibodies identified by hematopoietic precursor cells were mouse antibody Antibody to Human FITC-CD34 (Cat. No. 560942, BD Company) and mouse anti-human APC-CD43 antibody (Cat. No. 560198, BD Corporation).
  • the dilution ratio of the antibody is generally 1:200, and the antibody is labeled with a horizontal shaker at room temperature for 30 minutes.
  • Cell washing add an appropriate amount of PBS, centrifuge at 1000 rpm for 5 minutes to enrich the cells, and aspirate the supernatant.
  • Cell fixation and on-board detection Resuspend cells in PBS containing 1% paraformaldehyde. The cells were sieved with a cell sieve (40 ⁇ m) before being tested on the machine. Pipette the cell suspension into a flow tube in preparation for flow analysis. Flow Cytometry for FACS Calibur analytical flow cytometer (BD company), after the test, use FlowJo 10.0.7 Software for data analysis.
  • Figure 6 shows the results of flow cytometric analysis of hematopoietic precursor cells in the control group and the random rotation group.
  • the random rotation group cell preparation method is the method of embodiment 2
  • the preparation method of control group cells is basically the same as that of embodiment 2, the difference only lies in that after step a, directly carry out static culture in 37 ° C, 5% CO 2 incubator After 72 hours, control cells were obtained.
  • Example 8 Induction test of human pluripotent stem cell-derived hematopoietic endothelial precursor cells obtained by other methods to hematopoietic precursor cells
  • the culture from hematopoietic endothelial precursor cells to hematopoietic precursor cells of the present invention can also be induced by the medium and the spin culture method of the present invention.

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Abstract

提供一种诱导多能干细胞向造血前体细胞分化的培养基以及方法。所述方法包括1)从人多能干细胞分化产生生血内皮前体细胞的方法，制备获得生血内皮前体细胞；2)以促进生血内皮前体细胞向造血前体细胞分化的方法，制备造血前体细胞。在制备过程中采用所述培养基并采用随机旋转的方式培养，获得了优异的有效效率，且培养基成分明确。

Description

一种诱导多能干细胞向造血前体细胞分化的培养基以及方法 技术领域
本发明属于生物技术领域，涉及一种诱导多能干细胞向造血前体细胞分化的方法。
 背景技术
多能性干细胞，包括胚胎来源的胚胎干细胞和体外重编程诱导来源的诱导型多能干细胞。多能性干细胞能够在体外具有长期培养保持自我更新能力，并且具有多向分化的潜能，包括分化为几乎所有功能性的血液细胞。造血前体细胞是具有自我更新能力并能分化为各种血细胞的前体细胞，最终生成各种血细胞成分，包括红细胞、白细胞和血小板，它们也可以分化成各种其他细胞。在临床中，造血干/祖细胞以及各种成熟的血液细胞可服务于临床上的造血干细胞移植和血细胞输注治疗的重大战略需求。而如何获得足够多的造血干细胞一直以来是困扰研究人员和医务人员的难题。自2001年，Kaufman等首次实现将人胚胎干细胞分化为CD34+的造血干/祖细胞后[Kaufman D, Hanson E, Lewis et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad USA. 2001; 98(19):10716-21]，人们相继开展了人类多能干细胞体外分化为造血干细胞及相关的研究。虽然在这个领域已经有了一定的进展，从目前临床应用的角度来看，人多能干细胞体外造血分化的研究仍然面临着非常大挑战，例如人多能干细胞诱导分化而来的生血内皮前体细胞和造血干细胞和功能细胞数量不足以满足一次输入的需求；人多能干细胞诱导的分化而来的造血干细胞不具备体内移植的能力；体外培养的条件包含了血清、饲养层细胞等外源性物质，这些问题会大大的限制了目前采用人多能干细胞体外造血分化的临床应用。
由于目前的这些方法培养条件较为复杂，分化周期相对较长，分化效率和产量较低，可操作性较差。基于以上现有分化技术中存在的问题，亟需寻找一种诱导血管/生血内皮前体细胞生成造血前体细胞的方法。
发明内容
本发明的目的是提供一种操作简单、高效的多能干细胞向造血前体诱导分化体系，其包括多能干细胞向生血内皮前体诱导分化体系，以及生血内皮前体细胞向造血前体细胞诱导分化体系。具体地，本发明提供了一种化学成分明确的多能干细胞向造血前体细胞分化的培养基，以及使用该培养基探索了一种利用随机回转仪模拟微重力效应来高效诱导多能干细胞向人造血前体细胞分化的方法；该方法与现有方法相比，具有较高的可操作性与可重复性，为将来大规模生产造血前体细胞用于生物医学和临床治疗奠定技术基础。
第一方面，本发明提供一种中胚层细胞诱导分化培养基，其包括基础分化培养基和骨形成蛋白4（BMP4）、活化素A（Activin A）以及GSK-3α/β抑制剂（CHIR99021）；
所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.08-0.12%的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8%-2.2%的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的0.8%-1.2%的谷丙氨酸二肽（GlutaMax TM）100X添加剂以及体积百分比为所述IF9S完全培养基的0.8%-1.2%的非必需氨基酸添加剂100X（NEAA）。
优选地，所述IF9S完全培养基，包括按1:1的体积比混合的IMDM培养基和F12培养基，以及终浓度为10 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.1%的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的2%的ITS-X 100X添加剂、终浓度为40 μl/L的一硫代甘油（αMTG）、终浓度为64 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的1%的GlutaMax TM100X添加剂以及体积百分比为所述IF9S完全培养基的1%的非必需氨基酸添加剂100X（NEAA）。
优选地，所述中胚层细胞分化培养基为IF9S完全培养基添加40-60 ng/mL的BMP4，15-25μg/mL的Activin A，0.8-1.2μM CHIR99021。
更优选地，所述中胚层细胞分化培养基为IF9S完全培养基添加50 ng/mL的BMP4，20μg/mL的Activin A，1 μM CHIR99021。
第二方面，本发明提供了一种生血内皮前体细胞诱导分化培养基，其包括基础分化培养基、SB431542、VEGF、bFGF和SCF；
所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.08-0.12%的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8%-2.2%的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的0.8%-1.2%的GlutaMax TM100X添加剂以及体积百分比为所述IF9S完全培养基的0.8%-1.2%的非必需氨基酸添加剂100X（NEAA）。
优选地，所述IF9S完全培养基，包括按1:1的体积比混合的IMDM培养基和F12培养基，以及终浓度为10 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.1%的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的2%的ITS-X100X添加剂、终浓度为40 μl/L的一硫代甘油（αMTG）、终浓度为64 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的1%的GlutaMax TM100X添加剂以及体积百分比为所述IF9S完全培养基的1%的非必需氨基酸添加剂100X（NEAA）。
优选地，所述生血内皮前体分化培养基为IF9S完全培养基添加8-12 μM SB431542、40-60 ng/mL的VEGF、40-60 ng/mL的bFGF和40-60 ng/mL的SCF；
更优选地，所述生血内皮前体分化培养基为IF9S完全培养基添加10 μM SB431542、50 ng/mL的VEGF、50 ng/mL的bFGF和50 ng/mL的SCF。
优选地，在配制IF9S完全培养基前，使用去离子水溶解聚乙烯醇（PVA），并配成5%浓度的储液。
优选地，在配制IF9S完全培养基前，使用IMDM培养基将一硫代甘油按2%进行稀释，配成了500 X的储液。
优选地，在配制IF9S完全培养基前，使用F12培养基先将AA2P溶解，并配成5 mg/mL的储液。
其中，所述IMDM培养基和F12基础基为常规培养基，可以购买商业化的培养基，也可以自行配制，在本发明的一个优选的实施例中，所述IMDM培养基和F12培养基均购自Gibco公司。
所述Lipids（100X）为化学成分确定的脂质浓缩物，可用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述Lipids添加剂购自Gibco公司，货号为11905031。
所述ITS-X（100X）添加剂为胰岛素-转铁蛋白-硒-乙醇胺的基础培养基补充物，用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述ITS-X添加剂购自Gibico公司，货号为51500056。
所述GlutaMax TM（100X）添加剂为谷丙氨酸二肽，是一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。在本发明的一个优选的实施例中，所述GlutaMax添加剂购自Gibico公司，货号为35050-061。
所述NEAA（100X）添加剂为非必需氨基酸，是一种细胞培养添加剂。在本发明的一个优选的实施例中，所述NEAA添加剂购自Gibico公司，货号为35050-061。
第三方面，本发明提供了一种生血内皮前体细胞诱导分化组合培养基，其包括上述中胚层细胞诱导分化培养基和上述生血内皮前体细胞诱导分化培养基。
优选地，中胚层细胞诱导分化培养基和生血内皮前体细胞诱导分化培养基分开放置或配制。
第四方面，本发明提供一种诱导生血内皮前体细胞向造血前体细胞分化的培养基，其包括基础分化培养基和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、干细胞因子（SCF）、白介素3（IL-3）、白介素6（IL-6）以及血小板生成素（TPO）；
所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为:8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12%的Lipids(100X)添加剂、体积百分比为所述培养基的1.8%-2.2%的ITS-X (100X)添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的AA2P、体积百分比为所述培养基的:0.8-1.2%的GlutaMax TM (100X)添加剂以及体积百分比为所述培养基的0.8-1.2%的非必需氨基酸添加剂（NEAA，100X）。
优选地，所述基础分化培养基为IF9S完全培养基，包括按1:1的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为10 mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.1%的Lipids(100X)添加剂、体积百分比为所述培养基的2%的ITS-X (100X)添加剂、终浓度为40 μl/L的一硫代甘油（αMTG）、终浓度为64 mg/L的AA2P、体积百分比为所述培养基的1%的GlutaMax TM (100X)添加剂以及体积百分比为所述培养基的1%的非必需氨基酸添加剂（NEAA，100X）。
优选地，所述造血前体细胞分化培养基为IF9S完全培养基添加40-60 ng/mL的VEGF，15-25 ng/mL的bFGF，40-60 ng/mL的SCF，8-12ng/mL的 IL-3，40-60 ng/mL的IL-6以及15-25 ng/mL TPO。
优选地，所述造血前体细胞分化培养基为IF9S完全培养基添加50 ng/mL的VEGF，20 ng/mL的bFGF，50 ng/mL的SCF，10 ng/mL的 IL-3，50 ng/mL的IL-6以及20 ng/mL TPO。
优选地，在配制IF9S完全培养基前，使用去离子水溶解聚乙烯醇（PVA）,并配成5%浓度的储液。
优选地，在配制IF9S完全培养基前，使用IMDM培养基将一硫代甘油按2%进行稀释，配成了500 X的储液。
优选地，在配制IF9S完全培养基前，使用F12培养基先将AA2P溶解，并配成5 mg/mL的储液。
其中，所述IMDM基础培养基和F12基础培养基为常规培养基，可以购买商业化的培养基，也可以自行配制，在本发明的一个优选的实施例中，所述IMDM和F12培养基均购自Gibco公司。
所述Lipids（100X）为化学成分确定的脂质浓缩物，可用于减少或替代细胞培养基中的胎牛血清。在本申请的一个优选的实施例中，所述Lipids添加剂购自Gibco公司，货号为11905031。
所述ITS-X（100X）添加剂为胰岛素-转铁蛋白-硒-乙醇胺的基础培养基补充物，用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述ITS-X添加剂购自Gibico公司，货号为51500056。
所述GlutaMax TM（100X）添加剂为谷丙氨酸二肽，是一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。在本发明的一个优选的实施例中，所述GlutaMax添加剂购自Gibico公司，货号为35050-061。
所述NEAA（100X）添加剂为非必需氨基酸，是一种高级细胞培养添加剂。在本发明的一个优选的实施例中，所述NEAA添加剂购自Gibico公司，货号为35050-061。
第五方面，本发明提供一种诱导多能干细胞向造血前体细胞分化的培养基组合物，所述培养基组合物中包含：
1）上述中胚层细胞诱导分化培养基；
2）上述生血内皮前体细胞诱导分化培养基；
3）上述诱导生血内皮前体细胞向造血前体细胞分化的培养基。
优选地，中胚层细胞诱导分化培养基、生血内皮前体细胞诱导分化培养基分开和诱导生血内皮前体细胞向造血前体细胞分化的培养基放置或配制。
第六方面，本发明提供一种促进中胚层细胞向生血内皮前体细胞转化的方法，所述方法包括在中胚层细胞培养过程中，采用随机三维回转仪将细胞培养容器在旋转条件下进行细胞培养的步骤。
优选地，所述旋转条件下进行培养为以随机三维回转仪将细胞培养容器进行随机旋转，更优选地，随机旋转为0.1-10 rpm/分钟的转速；更优选地，旋转速度为5 rpm/分钟。
优选地，所述旋转条件下进行培养的培养时间为48-96小时，优选为60-72小时。
优选地，所述旋转条件下进行培养的培养基采用上述生血内皮前体诱导分化培养基。
第七方面，本发明还提供了一种从人多能干细胞分化产生生血内皮前体细胞的方法，所述的方法包括如下步骤：
S1)人多能干细胞向中胚层细胞诱导分化培养:
S11).细胞容器预包被生长因子减少的基底膜基质（Matrigel基质）；
S12).采用消化酶将人多能干细胞克隆进行消化、离心处理；
S13).将消化、离心处理后的人多能干细胞克隆小团块接种于S11)获得预包被生长因子减少Matrigel的细胞容器中，添加TeSR TM-E8 TM培养基，静止培养24～48小时；
S14)将培养基更换成上述中胚层细胞诱导分化培养基，静止培养24～48小时，获得诱导的中胚层细胞；
S2)诱导的中胚层细胞进一步向生血内皮前体细胞分化培养:
S21).将中胚层细胞的细胞培养基更换为上述生血内皮前体细胞诱导分化培养基；
S22).将细胞培养容器安装到随机三维回转仪上，设定旋转的方式为随机旋转，转速调整为0.1-10 rpm/分钟的转速；
S23).将随机三维回转仪放入37°C、5% CO 2孵箱中旋转培养60-96小时。
优选的，S11)中其中所述的生长因子减少的Matirgel基质为Corning公司生产的Growth Factor Reduced Matrigel，货号为354230。
优选地，S13)中接种的密度为50-200个克隆团块/cm 2。
再优选地，S13)中所述的接种的密度为100个克隆团块/cm 2 ；。
优选地，S21)中所述的生血内皮前体细胞诱导分化培养基加至培养容器的95%-100%；
优选地，S22)中旋转速度为5 rpm/分钟；
优选地，S22)中培养72小时。
优选地，S12)中，人多能干细胞克隆进行消化前进行扩增培养：
S121)采用胚胎干细胞级别的细胞外基质Matrigel对培养细胞容器进行包被；
S122)将人多能干细胞接种于包被后的细胞容器中并使用TeSR TM-E8 TM培养基进行培养。
优选地，S122)中每24小时更换一次新鲜培养基，当细胞克隆生长至70～80%密度时，对细胞进行消化传代或之后的诱导分化培养；细胞传代的周期为3-4天。
优选地，所述胚胎干细胞级别的细胞外基质为BioCoat公司生产Matrigel，货号为354277。
第八方面，本发明还提供了一种促进生血内皮前体细胞向造血前体细胞分化的方法，所述的方法包括在生血内皮前体细胞培养过程中，采用随机回转仪将细胞培养容器在旋转条件下进行培养的步骤。
优选地，所述旋转条件下进行培养为以随机回转仪将细胞培养容器随机旋转，更优选地，随机旋转内外径均为0.1-10rpm/分钟的转速；更优选地，旋转速度为内外径为5rpm/分钟。
优选地，所述旋转条件下进行培养的培养时间为48-72小时，优选为72小时。
优选地，所述旋转条件下进行培养的培养基采用上述的诱导血管/生血内皮前体细胞向造血前体细胞分化的培养基。
优选地，其包含如下步骤：
S3）在原始培养基中培养72小时后，更换细胞培养基为上述的诱导生血内皮前体细胞向造血前体细胞分化的培养基；
S4）将细胞培养瓶重新安装到随机回转仪上于继续进行回转培养；
优选地，S3）中所述诱导生血内皮前体细胞向造血前体细胞分化的培养基加至培养容器的95%-100%；
优选地，S4)中，细胞培养48-72小时，优选为细胞培养72小时。
优选地，S4)中，细胞培养条件为37°C、5% CO 2。
第九方面，本发明还提供了一种从人多能干细胞分化产生造血前体细胞方法，所述方法包括以下步骤：
1)采用上述从人多能干细胞分化产生生血内皮前体细胞的方法，制备获得生血内皮前体细胞；
2)采用上述促进生血内皮前体细胞向造血前体细胞分化的方法。
第十方面，本发明提供随机三维回转仪在用于促进中胚层细胞向生血内皮前体细胞转化中的用途，或者促进生血内皮前体细胞向造血前体细胞转化的用途。
本发明选择了化学成分明确的培养基用于人多能干细胞的培养和分化，本发明还描述了利用一种随机回转器来模拟微重力效应开展多能干细胞向生血内皮前体细胞和造血前体细胞分化技术。总之与现有技术相比，本发明具有以下优点：
本培养体系从多能干细胞培养扩增开始到中内胚层分化，生血内皮前体分化以及造血前体细胞都是在无血清、无饲养层且化学成分明确的条件下培养。而目前报道的人多能干细胞向造血前体/干细胞体外分化的方法主要包括需要和小鼠骨髓基质细胞（OP9细胞系）或者小鼠主动脉性腺中肾基质细胞（mAGM细胞系）共培养的体系，以及采用拟胚体（Embryoid Body，EB）分化的体系。共培养体系由于含有动物源性物质，增加了实验的不稳定性和安全性，加大了实验步骤的繁琐；而EB分化体系，其分化效率低。本发明使用的化学成分明确的培养体系和随机三维回转模拟微重力的培养方法，可在短时间内获得大量的多能干细胞来源的生血内皮前体细胞以及造血前体细胞，并且分化效率很高，分化效率较静止培养提高2倍以上。这种低成本、安全可控的无血清、无饲养层细胞的随机三维回转培养策略可为将来大规模生产用于临床治疗的生血内皮前体细胞或造血前体细胞奠定了基础。
 附图说明
图1是采用本发明方法从人多能干细胞诱导来源的生血内皮前体细胞明场图片。
图2是采用本发明方法从人多能干细胞诱导来源的生血内皮前体细胞进行生血内皮前体细胞标志分子CD31和CD34的荧光染色图片。
图3是采用本发明随机三维回转培养与传统静止培养下生血内皮前体细胞分化进行流式细胞分析，比较分化后细胞表达CD31+CD34+的效率。其中A图为对照组，B图为随机三维回转组。
图4是采用本发明方法从人多能干细胞诱导来源的造血前体细胞明场图片。
图5是采用本发明方法从人多能干细胞诱导来源的造血前体细胞进行造血前体细胞标志分子CD34和CD43的荧光染色图片。
图6是采用本发明随机回转培养与传统静止培养下造血前体细胞分化进行流式细胞分析，比较分化后细胞表达CD34+CD43+的效率。其中A图为对照组，B图为随机回转组。
具体实施方式
现结合实施例，对本发明做进一步阐述，但本发明的实施并不仅限于此。
除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。
除非特别说明，本发明使用的人多能干细胞为人胚胎干细胞系H1。
除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。
实施例 1 ：无血清培养体系培养基的制备
（1）IF9S培养基的配制：
本申请使用IMDM培养基、F12培养基、聚乙烯醇(PVA)、Lipids(100X)、ITS-X (100X)、一硫代甘油（αMTG）、AA2P、GlutaMax TM (100X)、非必需氨基酸（NEAA，100X）来配制IF9S培养基。
其中，培养基的各组分分别购自：
IMDM培养基购自Gibco公司，货号为21056-023；
F12培养基购自Gibco公司，货号为31765-027；
聚乙烯醇(PVA)购自Sigma公司，货号为P8136；
Lipids (100X) 购自Gibco公司，货号为11905031；
ITS-X (100X)购自Gibco公司，货号为51500-056；
一硫代甘油（αMTG）购自Sigma公司，货号为M6145；
AA2P购自Sigma公司，货号为A8960；
GlutaMax TM (100X) 购自Gibco公司，货号为35050-061；
非必需氨基酸（NEAA，100X）购自Gibco公司，货号为11140-035；
配制所述的培养基。其中聚乙烯醇(PVA)为粉末，使用前需用去离子水溶解并配成5%浓度的储液。
具体地，所述IF9S培养基通过以下方法配制：
首先将向IMDM培养基和F12培养基培养基按1:1混合，搅拌均匀后，在混合后的培养基中添加以下试剂，各试剂的最终浓度为：10 mg/L的聚乙烯醇(PVA)、体积百分比为0.1%的Lipids (100X)添加剂、体积百分比为2%的ITS-X (100X)添加剂、40 μl/L的一硫代甘油（αMTG）、64 mg/L的AA2P、体积百分比为1%的GlutaMax TM (100X)添加剂以及体积百分比为1%的非必需氨基酸添加剂（NEAA，100X）。
各种试剂在添加无严格的顺序，在常温操作即可，配制的过程无需加热，但培养基配制后需用0.22μm的滤器进行无菌过滤，放置4°C冰箱保存。
（2）中胚层细胞分化培养基配制
所述中胚层细胞分化培养基为IF9S培养基添加50 ng/mL的BMP4，20μg/mL的Activin A，1 μM CHIR99021。
各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。
（3）生血内皮前体分化培养基配制
所述生血内皮前体分化培养基为IF9S培养基添加10 μM的SB431542，50 ng/mL的VEGF，50 ng/mL的bFGF，50 ng/mL的SCF。
各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。
（4）造血前体细胞分化培养基配制
所述造血前体/干细胞分化培养基为IF9S完全培养基添加50 ng/mL的VEGF，20 ng/mL的bFGF，50 ng/mL的SCF，10 ng/mL的 IL-3，50 ng/mL的IL-6以及20 ng/mL TPO。
各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。
实施例 2 ：人多能干细胞来源的生血内皮前体细胞的制备
本实施例采用的人多能干细胞是人胚胎干细胞系（H1）（来源于美国威斯康辛大学）。
a.首先采用胚胎干细胞级别的细胞外基质Matrigel对培养细胞的皿进行包被，再将人胚胎干细胞接种于包被后的皿中并使用化学成分明确的商品化培养基TeSR TM-E8 TM进行培养，每24小时更换一次新鲜培养基。人胚胎干细胞以克隆的形式生长，当细胞克隆生长至70～80%密度时，需要对细胞进行消化传代或之后的诱导分化培养。细胞传代的周期一般为3-4天。
b.采用ReLeSRTM消化酶将上述a.中生长后的人胚胎干细胞克隆进行消化处理，37°C消化2～3分钟。消化2分钟后镜下观察细胞消化情况，待细胞克隆边缘出现“卷边”时用TeSR TM-E8 TM培养基终止消化，避免消化过度；
c.将消化、离心处理后的人胚胎干细胞克隆小团块接种于上述预包被生长因子减少Matrigel的培养瓶中，接种的密度为50-200个克隆团块/cm 2。添加适量的TeSR TM-E8 TM培养基，在37°C、5% CO 2孵箱中静置培养24～48小时；培养24～48小时后将培养基更换成中胚层细胞分化培养基；
d.添加中胚层细胞分化培养基后，在37°C、5% CO 2孵箱中静止培养48小时；
e.细胞在中胚层细胞诱导培养基中培养48小时后，将培养瓶中的培养基吸弃更换为新鲜的生血内皮前体细胞诱导培养基，将培养瓶灌满培养基；
f.将细胞培养瓶封口，然后安装到随机三维回转仪上，设定旋转的方式为随机旋转模式，转速调整为0.1-10 rpm/分钟的转速；
g.将随机三维回转移放入37°C、5% CO 2孵箱中旋转培养72小时，培养72小时候后可见细胞组成的血管内皮样网格状形态，此时即为血管/生血内皮前体细胞；生血内皮前体细胞的形态见图1。
实施例 3 ：生血内皮前体细胞的检测
收集实施例2中产生的血管/生血内皮前体细胞进行细胞免疫荧光染色鉴定。
具体地：
a.吸弃培养瓶中的生血内皮前体细胞诱导培养基，用PBS洗涤细胞2遍，以去除死细胞；
b.加入4%的多聚甲醛进行固定，室温静置固定20分钟；
c.吸弃4%多聚甲醛，加入PBS洗涤2-3次；
d.加入5%的驴血清进行封闭，室温封闭1小时；
e.加入荧光标记小鼠抗人PE-CD31抗体（货号：560983, BD公司），荧光标记小鼠抗人FITC-CD34的抗体（货号：560942, BD公司）以及0.1μg/mL的Hoechst33342，抗体稀释倍数按照说明数建议用封闭液进行稀释，室温孵育1小时或4°C孵育过夜；
f.吸弃含抗体的染色液体，加入适量的PBS进行洗涤细胞，洗涤3次，每次5分钟。
g.采用共聚焦荧光显微镜对细胞进行CD31和CD34蛋白的检测，Hoechst33342为标记细胞核，注意操作的时候避光，防止荧光淬灭。图2结果显示的为染色鉴定的结果。
CD31和CD34是血管/生血内皮前体细胞重要的标志物，荧光颜色结果可以证实通过本发明的方法可以将中胚层细胞进一步诱导为CD31和CD34阳性的细胞，即生血内皮前体细胞。
实施例 4 ：生血内皮前体细胞的效率分析
收集实施例2中产生的产生的生血内皮前体细胞进行流式细胞术分析鉴定。
具体地：
a.细胞消化：采用Accutase酶（货号：A1110501，Gibco公司）将实施例2中所述细胞消化为单细胞，用IF9S培养基终止消化。
b.细胞收集：将细胞移入离心管中，1000转/分钟，离心5分钟富集细胞。
c.抗体孵育标记：离心后吸弃上清，用0.2% BSA 重悬细胞，根据实验的具体情况设置细胞的分组及抗体的标记，其中生血内皮前体细胞鉴定标记抗体为小鼠抗人PE-CD31抗体（货号：560983, BD公司）和小鼠抗人FITC-CD34的抗体（货号：560942, BD公司）；造血前体/干细胞鉴定的标志分子抗体分别为小鼠抗人FITC-CD34的抗体（货号：560942, BD公司）以及小鼠抗人APC-CD43抗体（货号：560198, BD公司）。抗体的稀释比例一般为1:200，水平摇床慢摇室温标记抗体30分钟。
d.细胞洗涤：加入适量PBS，1000转/分钟，离心5分钟富集细胞，吸弃上清。
e.细胞固定及上机检测：用含1%多聚甲醛PBS重悬细胞。上机检测前用细胞筛（40um）进行过筛处理。将细胞悬液移入流式管准备进行流式分析。流式细胞仪为FACS Calibur分析型流式细胞仪（BD公司），测试结束后，用FlowJo 10.0.7 软件进行数据分析。
图3显示为对照组和随机三维回转组中生血内皮前体细胞流式细胞分析结果。
其中随机三维回转组细胞制备方法为实施例2的方法，对照组细胞的制备方法与实施例2基本相同，区别仅在于步骤e后直接进行在37°C、5% CO 2孵箱中静置培养72小时，得到对照组细胞。
通过图3结果可知，对照组仅采用本发明的培养基实现了将37.6%的中胚层细胞诱导为生血内皮前体细胞。而通过结合本发明的培养基以及随机三维回转，实现了71.6%的的中胚层细胞诱导为生血内皮前体细胞。本发明的方法证实了随机旋转可以增加向生血内皮前体细胞的诱导效率，而进一步配合本发明的培养基实现了高效诱导生血内皮前体细胞。
实施例 5 ：人多能干细胞来源的造血前体细胞的制备
具体地：
a.弃去培养瓶中生血内皮前体细胞原培养基，更换为实施例1获得的造血前体细胞诱导培养基；培养瓶中应将培养基灌满；
b.将细胞培养瓶安装到随机回转仪上，设定回转的方式为随机回转，转速调整为内外径均为5 rpm/分钟的转速；
c.将随机回转仪置于37°C、5% CO 2孵箱中继续进行回转培养48-72小时；
d.细胞培养48小时候后即可见培养瓶中出现大量的“卵石样”造血前体细胞，部分造血前体细胞从皿底脱离成为悬浮的造血前体细胞。生成的造血前体细胞见图4，其中A为“卵石样”造血前体细胞，B为部分造血前体细胞从皿底脱离的悬浮造血前体细胞。
实施例 6 ：造血前体细胞的检测
收集实施例5中产生的造血前体细胞进行细胞免疫荧光染色鉴定。
h.吸弃培养瓶中的造血前体细胞诱导培养基，用PBS洗涤细胞2遍，以去除死细胞；
i.加入4%的多聚甲醛进行固定，室温静置固定20分钟；
j.吸弃4%多聚甲醛，加入PBS洗涤2-3次；
k.加入5%的驴血清进行封闭，室温封闭1小时；
l.加入荧光标记小鼠抗人FITC-CD34的抗体（货号：560942, BD公司），荧光标记小鼠抗人APC-CD43抗体（货号：560198, BD公司）以及0.1μg/mL的Hoechst33342，抗体稀释倍数按照说明数建议用封闭液进行稀释，室温孵育1小时或4°C孵育过夜；
m.吸弃含抗体的染色液体，加入适量的PBS进行洗涤细胞，洗涤3次，每次5分钟。
n.采用共聚焦荧光显微镜对细胞进行CD34和CD43蛋白的检测，Hoechst33342为标记细胞核，注意操作的时候避光，防止荧光淬灭。图5结果显示的为染色鉴定的结果。
CD34和CD43是造血前体细胞的重要的标志物，荧光颜色结果可以证实通过本发明的方法可以将生血内皮前体细胞诱导为CD34和CD43阳性的细胞，即造血前体细胞。
图2结果显示的为造血前体细胞染色鉴定的结果。
实施例 7 ：造血前体细胞的效率分析
收集实施例2中产生的造血前体细胞进行流式细胞术分析鉴定。
具体地：
f.细胞消化：采用Accutase酶（货号：A1110501，Gibco公司）将实施例2中所述细胞消化为单细胞，分别用IF9S培养基终止消化。
g.细胞收集：将细胞移入离心管中，1000转/分钟，离心5分钟富集细胞。
h.抗体孵育标记：离心后吸弃上清，用0.2% BSA 重悬细胞，根据实验的具体情况设置细胞的分组及抗体的标记，其中造血前体细胞鉴定的标志分子抗体分别为小鼠抗人FITC-CD34的抗体（货号：560942, BD公司）以及小鼠抗人APC-CD43抗体（货号：560198, BD公司）。抗体的稀释比例一般为1:200，水平摇床慢摇室温标记抗体30分钟。
i.细胞洗涤：加入适量PBS，1000转/分钟，离心5分钟富集细胞，吸弃上清。
j.细胞固定及上机检测：用含1%多聚甲醛PBS重悬细胞。上机检测前用细胞筛（40μm）进行过筛处理。将细胞悬液移入流式管准备进行流式分析。流式细胞仪为FACS Calibur分析型流式细胞仪（BD公司），测试结束后，用FlowJo 10.0.7 软件进行数据分析。
图6显示的为对照组和随机回转组中造血前体细胞流式细胞分析结果。其中随机回转组细胞制备方法为实施例2的方法，对照组细胞的制备方法与实施例2基本相同，区别仅在于步骤a后直接进行在37°C、5% CO 2孵箱中静置培养72小时，得到对照组细胞。
通过图3结果可知，对照组仅采用本发明的培养基实现了将28.9%的生血内皮前体细胞诱导为造血前体细胞。而通过结合本发明的培养基以及随机回转，实现了45.9%的生血内皮前体细胞诱导为造血前体细胞。本发明的方法证实了随机旋转可以增加向造血前体细胞的诱导效率，而进一步配合本发明的培养基实现了高效诱导。
实施例 8 ：其他方法获得的人多能干细胞来源生血内皮前体细胞向造血前体细胞诱导试验
采用已知方法制备（例如参照文献Xu Cao, Gopala K. Yakala, Francijna E. van den Hil, Amy Cochrane, Christine L. Mummery, Valeria V. Orlova. Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives. Stem Cell Reports. 2019 June 11; 12(6):1282–1297中描述的）生血内皮前体细胞，并通过以下方法向造血前体细胞诱导培养。
具体地：
e.弃去培养瓶中生血内皮前体细胞原培养基，更换为实施例1获得的造血前体细胞诱导培养基；培养瓶中应将培养基灌满；
f.将细胞培养瓶安装到随机回转仪上，设定回转的方式为随机回转，转速调整为内外径均为5 rpm/分钟的转速；
g.将随机回转仪置于37°C、5% CO 2孵箱中继续进行回转培养48-72小时；
h.细胞培养48小时候后即可见培养瓶中出现大量的“卵石样”造血前体细胞，部分造血前体细胞从皿底脱离成为悬浮的造血前体细胞。
说明本发明的从生血内皮前体细胞向造血前体细胞的培养也可以采用本发明的培养基以及旋转培养方法诱导。

Claims (10)

  1. 一种中胚层细胞诱导分化培养基，其特征在于，其包括基础分化培养基和骨形成蛋白4（BMP4）、活化素A（Activin A）以及GSK-3α/β抑制剂（CHIR99021）；
    所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.08-0.12%的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8%-2.2%的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的0.8%-1.2%的谷丙氨酸二肽（GlutaMax TM）100X添加剂以及体积百分比为所述IF9S完全培养基的0.8%-1.2%的非必需氨基酸添加剂100X（NEAA）；
    优选地，所述中胚层细胞分化培养基为IF9S完全培养基添加40-60 ng/mL的BMP4，15-25μg/mL的Activin A，0.8-1.2μM CHIR99021。
  2. 一种生血内皮前体细胞诱导分化培养基，其特征在于，其包括基础分化培养基、SB431542、VEGF、bFGF和SCF；
    所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12 mg/L的聚乙烯醇（PVA）、体积百分比为所述培养基的0.08-0.12%的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8%-2.2%的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的抗坏血酸磷酸酯钠（AA2P）、体积百分比为所述IF9S完全培养基的0.8%-1.2%的GlutaMax TM100X添加剂以及体积百分比为所述IF9S完全培养基的0.8%-1.2%的非必需氨基酸添加剂100X（NEAA）；
    优选地，所述生血内皮前体分化培养基为IF9S完全培养基添加8-12 μM SB431542、40-60 ng/mL的VEGF、40-60 ng/mL的bFGF和40-60 ng/mL的SCF。
  3. 一种诱导生血内皮前体细胞向造血前体细胞分化的培养基，其特征在于，其包括基础分化培养基和血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、干细胞因子（SCF）、白介素3（IL-3）、白介素6（IL-6）以及血小板生成素（TPO）；
    所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为:8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12%的Lipids(100X)添加剂、体积百分比为所述培养基的1.8%-2.2%的ITS-X (100X)添加剂、终浓度为35-45μl/L的一硫代甘油（αMTG）、终浓度为55-70 mg/L的AA2P、体积百分比为所述培养基的:0.8-1.2%的GlutaMax TM (100X)添加剂以及体积百分比为所述培养基的0.8-1.2%的非必需氨基酸添加剂（NEAA，100X）；
    优选地，所述造血前体细胞分化培养基为IF9S完全培养基添加40-60 ng/mL的VEGF，15-25 ng/mL的bFGF，40-60 ng/mL的SCF，8-12ng/mL的 IL-3，40-60 ng/mL的IL-6以及15-25 ng/mL TPO。
  4. 一种从人多能干细胞分化产生生血内皮前体细胞的方法，其特征在于，所述的方法包括如下步骤：
    S1)人多能干细胞向中胚层细胞诱导分化培养:
    S11).细胞容器预包被生长因子减少的基底膜基质；
    S12).采用消化酶将人多能干细胞克隆进行消化、离心处理；
    S13).将消化、离心处理后的人多能干细胞克隆小团块接种于S11)获得预包被生长因子减少Matrigel的细胞容器中，添加TeSR TM-E8 TM培养基，静止培养24～48小时；
    S14)将培养基更换成权利要求1所述的中胚层细胞诱导分化培养基，静止培养24～48小时，获得诱导的中胚层细胞；
    S2)诱导的中胚层细胞进一步向生血内皮前体细胞分化培养:
    S21).将中胚层细胞的细胞培养基更换为权利要求2所述的生血内皮前体细胞诱导分化培养基；
    S22).将细胞培养容器安装到随机三维回转仪上，设定旋转的方式为随机旋转，转速调整为0.1-10 rpm/分钟的转速；
    S23).将随机三维回转仪放入37°C、5% CO 2孵箱中旋转培养60-96小时，获得生血内皮前体细胞。
  5. 根据权利要求4所述的方法，其特征在于，S22)中旋转速度为5 rpm/分钟；
    优选地，S22)中培养72小时。
  6. 一种促进生血内皮前体细胞向造血前体细胞分化的方法，其特征在于，所述的方法包括在生血内皮前体细胞培养过程中，采用随机回转仪将细胞培养容器在旋转条件下进行培养的步骤；
    所述旋转条件下进行培养的培养基采用权利要求3所述的诱导生血内皮前体细胞向造血前体细胞分化的培养基；
    优选地，其包含如下步骤：
    S3）在原始培养基中培养72小时后，更换细胞培养基为权利要求3所述的诱导生血内皮前体细胞向造血前体细胞分化的培养基；
    S4）将细胞培养瓶重新安装到随机回转仪上于继续进行回转培养。
  7. 根据权利要求6所述的方法，其特征在于，S3中随机旋转内外径均为0.1-10rpm/分钟的转速；
    优选地，S4)中所述旋转条件下进行培养的培养时间为48-72小时；
    优选地，S3）中所述诱导生血内皮前体细胞向造血前体细胞分化的培养基加至培养容器的95%-100%；
    优选地，S4)中，细胞培养48-72小时。
    优选地，S4)中，细胞培养条件为37°C、5% CO 2。
  8. 一种从人多能干细胞分化产生造血前体细胞方法，其特征在于，其所述方法包括以下步骤：
    1)采用权利要求4或5所述的从人多能干细胞分化产生生血内皮前体细胞的方法，制备获得生血内皮前体细胞；
    2)采用权利要求6或7所述促进生血内皮前体细胞向造血前体细胞分化的方法，制备造血前体细胞。
  9. 一种诱导细胞分化的培养基组合物，其特征在于，其包括权利要求1和2所述的中胚层细胞诱导分化培养基以及生血内皮前体细胞诱导分化培养基；或者包括权利要求1、2和3所述的中胚层细胞诱导分化培养基、生血内皮前体细胞诱导分化培养基以及导生血内皮前体细胞向造血前体细胞分化的培养基。
  10. 随机三维回转仪在用于促进中胚层细胞向生血内皮前体细胞转化中的用途，或者促进生血内皮前体细胞向造血前体细胞转化的用途。
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CN116445408A (zh) * 2023-05-22 2023-07-18 呈诺再生医学科技(北京)有限公司 LSD1抑制剂在促进iPSC向HSC分化和HSC干性维持中的应用
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