CN113046318B - Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells - Google Patents

Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells Download PDF

Info

Publication number
CN113046318B
CN113046318B CN202110396567.5A CN202110396567A CN113046318B CN 113046318 B CN113046318 B CN 113046318B CN 202110396567 A CN202110396567 A CN 202110396567A CN 113046318 B CN113046318 B CN 113046318B
Authority
CN
China
Prior art keywords
medium
hematopoietic
cells
precursor cells
culture medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110396567.5A
Other languages
Chinese (zh)
Other versions
CN113046318A (en
Inventor
雷晓华
张键
马驰原
赵华山
汪宝蓓
李梦霞
李荣荣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to CN202110396567.5A priority Critical patent/CN113046318B/en
Publication of CN113046318A publication Critical patent/CN113046318A/en
Priority to PCT/CN2021/138097 priority patent/WO2022217955A1/en
Application granted granted Critical
Publication of CN113046318B publication Critical patent/CN113046318B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a culture medium and a method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells. Specifically, the method comprises 1) differentiating human pluripotent stem cells to generate hematopoietic endothelial precursor cells, and preparing to obtain the hematopoietic endothelial precursor cells; 2) Hematopoietic progenitor cells are prepared by a method of promoting differentiation of hematopoietic endothelial progenitor cells into hematopoietic progenitor cells. The culture medium is adopted in the preparation process and cultured in a random rotation mode, so that excellent effective efficiency is obtained, and the components of the culture medium are clear.

Description

Culture medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells
Technical Field
The invention belongs to the technical field of biology, and relates to a method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells.
Background
Pluripotent stem cells including embryonic stem cells of embryonic origin and induced pluripotent stem cells of in vitro reprogramming-inducing origin. Pluripotent stem cells are capable of long-term culture in vitro to retain self-renewal capacity and have the potential to differentiate in multiple ways, including into almost all functional blood cells. Hematopoietic precursor cells are precursor cells that have self-renewal ability and can differentiate into various blood cells, ultimately producing various blood cell components, including red blood cells, white blood cells, and platelets, which can also differentiate into various other cells. In the clinic, hematopoietic stem/progenitor cells and various mature blood cells can serve as the major strategic demands of clinical hematopoietic stem cell transplantation and blood cell transfusion therapy. How to obtain enough hematopoietic stem cells has been a problem for researchers and medical staff. Since 2001, kaufman et al first achieved differentiation of human embryonic stem cells into CD34+ hematopoietic stem/progenitor cells [ Kaufman D, hanson E, lewis et al, hematopoetic color-forming cells derived from human embryonic stem cells, proc Natl Acad USA.2001;98 10716-21], the differentiation of human pluripotent stem cells into hematopoietic stem cells in vitro and related studies have been carried out. Although there has been some progress in this field, from the perspective of current clinical application, the research of in vitro hematopoietic differentiation of human pluripotent stem cells still faces a great challenge, for example, the number of hematopoietic endothelial precursor cells and hematopoietic stem cells and functional cells induced by human pluripotent stem cells is not enough to meet the requirement of one-time transfusion; hematopoietic stem cells induced by human pluripotent stem cells and differentiated therefrom do not have the capacity of in vivo transplantation; the conditions of in vitro culture comprise exogenous substances such as serum, feeder cells and the like, and the problems can greatly limit the clinical application of in vitro hematopoietic differentiation by adopting human pluripotent stem cells at present.
Because the existing methods have complicated culture conditions, relatively long differentiation period, low differentiation efficiency and yield and poor operability. Based on the above problems in the prior art, there is a need to find a method for inducing the generation of hematopoietic progenitor cells from vascular/hematopoietic endothelial progenitor cells.
Disclosure of Invention
The invention aims to provide a simply-operated and high-efficiency system for inducing differentiation from pluripotent stem cells to hematopoietic precursors, which comprises a system for inducing differentiation from pluripotent stem cells to hematopoietic endothelial precursors and a system for inducing differentiation from hematopoietic endothelial precursor cells to hematopoietic precursor cells. Specifically, the invention provides a culture medium for differentiating pluripotent stem cells into hematopoietic precursor cells with definite chemical components, and a method for efficiently inducing the differentiation of the pluripotent stem cells into human hematopoietic precursor cells by simulating the microgravity effect by using a random gyroscope by using the culture medium; compared with the existing method, the method has higher operability and repeatability, and lays a technical foundation for large-scale production of hematopoietic precursor cells for biomedicine and clinical treatment in the future.
In a first aspect, the present invention provides a mesoderm cell-inducing differentiation medium comprising a basal differentiation medium and bone morphogenetic protein 4 (BMP 4), activin a (Activin a) and a GSK-3 α/β inhibitor (CHIR 99021);
the basic differentiation medium is an IF9S complete medium, which comprises an IMDM medium and an F12 medium mixed according to a volume ratio of 1.8-1.2, polyvinyl alcohol (PVA) with a final concentration of 8-12mg/L, lipid 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, monothioglycerol (alpha MTG) with a final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA 2P) with a final concentration of 55-70mg/L, and glutamine dipeptide (GlutaMax) with a volume percentage of 0.8-1.2% of the IF9S complete medium TM ) 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8% to 1.2% of the IF9S complete medium.
Preferably, the IF9S complete medium comprises IMDM medium and F12 medium mixed in a volume ratio of 1 TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 1% of the IF9S complete medium.
Preferably, the mesodermal cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL BMP4, 15-25. Mu.g/mL Activin A, 0.8-1.2. Mu.M CHIR99021.
More preferably, the mesodermal cell differentiation medium is IF9S complete medium supplemented with 50ng/mL BMP4, 20. Mu.g/mL Activin A, 1. Mu.M CHIR99021.
In a second aspect, the invention provides a hematopoietic endothelial precursor cell-induced differentiation medium comprising basal differentiation medium, SB431542, VEGF, bFGF and SCF;
the basic differentiation medium is an IF9S complete medium, and comprises an IMDM medium and an F12 medium which are mixed according to a volume ratio of 1.8-1.2, polyvinyl alcohol (PVA) with a final concentration of 8-12mg/L, lipid 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, monothioglycerol (alpha MTG) with a final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA 2P) with a final concentration of 55-70mg/L, and GlutaMax with a volume percentage of 0.8-1.2% of the IF9S complete medium TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8% to 1.2% of the IF9S complete medium.
Preferably, the IF9S complete medium comprises IMDM medium and F12 medium mixed in a volume ratio of 1 TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 1% of the IF9S complete medium.
Preferably, the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 8-12 μ M SB431542, 40-60ng/mL VEGF, 40-60ng/mL bFGF and 40-60ng/mL SCF;
more preferably, the hematopoietic progenitor differentiation medium is IF9S complete medium supplemented with 10. Mu.M SB431542, 50ng/mL VEGF,50ng/mL bFGF, and 50ng/mL SCF.
Preferably, polyvinyl alcohol (PVA) is dissolved using deionized water and a stock solution of 5% concentration is prepared before the IF9S complete medium is prepared.
Preferably, a 500 Xstock solution is prepared by diluting monothioglycerol at 2% using IMDM medium before preparing the IF9S complete medium.
Preferably, before preparing the IF9S complete medium, AA2P is dissolved in F12 medium and 5mg/mL stock solution is prepared.
Wherein the IMDM medium and the F12 base medium are conventional media, and commercial media can be purchased, or can be prepared by oneself, and in a preferred embodiment of the present invention, the IMDM medium and the F12 medium are purchased from Gibco company.
The Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the present invention, the Lipids additive is available from Gibco under item number 11905031.
The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine for the reduction or replacement of fetal bovine serum in cell culture medium. In a preferred embodiment of the invention, the ITS-X additive is available from Gibico under the designation 51500056.
The GlutaMax TM The (100X) additive is glutamine-alanine dipeptide, is a cell culture additive, and can directly substitute for L-glutamine in cell culture medium. In a preferred embodiment of the invention, the GlutaMax additive is available from Gibico, cat. 35050-061.
The NEAA (100X) additive is a non-essential amino acid and is a cell culture additive. In a preferred embodiment of the invention, the NEAA additive is available from Gibico corporation under the Cat No. 35050-061.
In a third aspect, the present invention provides a combined culture medium for inducing differentiation of hematopoietic endothelial precursor cells, which comprises the above-mentioned mesodermal cell differentiation-inducing culture medium and the above-mentioned hematopoietic endothelial precursor cell differentiation-inducing culture medium.
Preferably, the mesodermal cell differentiation-inducing medium and the hematopoietic endothelial precursor cell differentiation-inducing medium are placed or formulated separately.
In a fourth aspect, the present invention provides a medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, comprising a basal differentiation medium and 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);
the basic differentiation medium is an IF9S complete medium and comprises an IMDM basic medium and an F12 basic medium which are mixed according to the volume ratio of 1.8-1.2, and polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, lipids (100X) additive with the volume percentage of 0.08-0.12% of the medium, ITS-X (100X) additive with the volume percentage of 1.8-2.2% of the medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, AA2P with the final concentration of 55-70mg/L, glutaMax with the volume percentage of 0.8-1.2% of the medium TM (100X) additives and non-essential amino acid additives (NEAA, 100X) in a volume percentage of 0.8-1.2% of the culture medium.
Preferably, the basal differentiation medium is an IF9S complete medium comprising IMDM basal medium and F12 basal medium mixed in a volume ratio of 1 TM (100X) additives and non-essential amino acid additives (NEAA, 100X) in a volume percentage of 1% of the culture medium.
Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 40-60ng/mL VEGF,15-25ng/mL bFGF,40-60ng/mL SCF,8-12ng/mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO.
Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 50ng/mL VEGF,20ng/mL bFGF,50ng/mL SCF,10ng/mL IL-3, 50ng/mL IL-6 and 20ng/mL TPO.
Preferably, the polyvinyl alcohol (PVA) solution is dissolved using deionized water and a stock solution of 5% concentration is prepared before the IF9S complete medium is prepared.
Preferably, a 500 Xstock solution is prepared by 2% dilution of monothioglycerol in IMDM medium prior to the preparation of IF9S complete medium.
Preferably, before preparing the IF9S complete medium, AA2P is dissolved in F12 medium and 5mg/mL stock solution is prepared.
In a preferred embodiment of the present invention, the IMDM basal medium and the F12 basal medium are both from Gibco, and are conventional media, and commercial media can be purchased or prepared by oneself.
The Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the present application, the Lipids additive is available from Gibco under the serial number 11905031.
The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine for reducing or replacing fetal bovine serum in cell culture medium. In a preferred embodiment of the invention, the ITS-X additive is available from Gibico under the designation 51500056.
The GlutaMax TM The (100X) additive is glutamine dipeptide, is a cell culture additive, and can directly replace L-glutamine in a cell culture medium. In a preferred embodiment of the invention, the GlutaMax additive is available from Gibico, cat. 35050-061.
The NEAA (100X) additive is a non-essential amino acid and is a high-grade cell culture additive. In a preferred embodiment of the invention, the NEAA additive is available from Gibico corporation under the Cat No. 35050-061.
In a fifth aspect, the present invention provides a medium composition for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells, the medium composition comprising:
1) The mesoderm cell induction differentiation medium;
2) The hematopoietic endothelial precursor cell differentiation-inducing medium;
3) The above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.
Preferably, the mesodermal cell differentiation induction medium, the hematopoietic endothelial precursor cell differentiation induction medium are separately placed or formulated and the medium for inducing differentiation of the hematopoietic endothelial precursor cells into hematopoietic precursor cells.
In a sixth aspect, the invention provides a method of promoting transformation of mesodermal cells into hematopoietic endothelial precursor cells, the method comprising the step of culturing the cells in a cell culture vessel under spinning conditions using a random three-dimensional gyroscope during mesodermal cell culture.
Preferably, the culturing under spinning conditions is performed by randomly spinning the cell culture vessel with a random three-dimensional gyroscope, more preferably, the random spinning is at a speed of 0.1-10 rpm/min; more preferably, the rotational speed is 5 rpm/min.
Preferably, the culturing time under the spinning condition is 48 to 96 hours, preferably 60 to 72 hours.
Preferably, the culture medium for culturing under the rotating condition adopts the hematogenous endothelial precursor induced differentiation culture medium.
In a seventh aspect, 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:
s1) inducing differentiation culture of human pluripotent stem cells to mesoderm cells:
s11) cell container pre-coating with growth factor-reduced basement membrane matrix (Matrigel matrix);
s12), adopting digestive enzyme to clone the human pluripotent stem cells, digesting and centrifuging;
s13) inoculating the digested and centrifuged small human pluripotent stem cell clone blocks into the cell container obtained in S11) to obtain the pre-coated growth factor-reduced Matrigel, and adding TeSR TM -E8 TM The culture medium is statically cultured for 24 to 48 hours;
s14) replacing the culture medium with the mesoderm cell induction differentiation culture medium, and statically culturing for 24-48 hours to obtain induced mesoderm cells;
s2) the induced mesodermal cells are further differentiated and cultured to hematogenic endothelial precursor cells:
s21), replacing a cell culture medium of mesoderm cells with the hematopoietic endothelial precursor cell induced differentiation culture medium;
s22), mounting the cell culture container on a random three-dimensional gyroscope, setting the rotation mode to be random rotation, and adjusting the rotation speed to be 0.1-10 rpm/min;
s23) putting the random three-dimensional gyroscope into a chamber with a temperature of 37 ℃ and a content of 5% CO 2 Rotating and culturing in incubator for 60-96 hr.
Preferably, the Growth Factor-Reduced Matirgel matrix described in S11) is a grown Factor Reduced Matrigel manufactured by Corning Corp, cat 354230.
Preferably, the density of the inoculation in S13) is 50-200 clonal clumps/cm 2 。
Still preferably, the density of inoculation described in S13) is 100 clonal clumps/cm 2 ；。
Preferably, the hematopoietic endothelial precursor cell-inducing differentiation medium described in S21) is added to 95% -100% of the culture vessel;
preferably, the rotation speed in S22) is 5 rpm/min;
preferably, S22) for 72 hours.
Preferably, in S12), the human pluripotent stem cell clone is subjected to amplification culture before digestion:
s121) coating a culture cell container with extracellular matrix Matrigel of embryonic stem cell grade;
s122) inoculating the human pluripotent stem cells into the coated cell container and using TeSR TM -E8 TM The culture medium is used for culturing.
Preferably, the fresh culture medium is replaced every 24 hours in S122), and when the cell clone grows to 70-80% of the density, the cell is subjected to digestion passage or induced differentiation culture after the cell clone grows; the cell passage cycle was 3-4 days.
Preferably, the extracellular matrix of embryonic stem cell grade is Matrigel manufactured by BioCoat, cat No. 354277.
In an eighth aspect, the present invention further provides a method for promoting differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, said method comprising the step of culturing the hematopoietic endothelial precursor cells in a rotating cell culture vessel by using a random gyroscope during the culturing of the hematopoietic endothelial precursor cells.
Preferably, the cultivation under the rotation condition is performed by randomly rotating the cell culture vessel with a random gyroscope, and more preferably, the inner and outer diameters of the random rotation are all rotation speeds of 0.1-10 rpm/min; more preferably, the rotation speed is 5 rpm/min for the inner and outer diameters.
Preferably, the culturing time under the rotation condition is 48 to 72 hours, preferably 72 hours.
Preferably, the culture medium for culturing under the rotation condition is the culture medium for inducing the differentiation of the vascular/hematopoietic endothelial precursor cells into the hematopoietic precursor cells.
Preferably, it comprises the following steps:
s3) after culturing for 72 hours in the original culture medium, replacing the cell culture medium with the culture medium for inducing the differentiation of the hematogenic endothelial precursor cells to the hematopoietic precursor cells;
s4) reinstalling the cell culture bottle on a random gyroscope for continuous rotary culture;
preferably, the culture medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells in S3) is added to 95% -100% of the culture vessel;
preferably, in S4), the cells are cultured for 48 to 72 hours, preferably for 72 hours.
Preferably, in S4), the cell culture conditions are 37 ℃ and 5% CO 2 。
In a ninth aspect, the present invention also provides a method of differentiating to produce hematopoietic precursor cells from human pluripotent stem cells, the method comprising the steps of:
1) The method for generating the hematopoietic endothelial precursor cells by differentiating the human pluripotent stem cells is adopted to prepare and obtain the hematopoietic endothelial precursor cells;
2) The method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is used.
In a tenth aspect, the invention provides the use of a random three-dimensional gyroscope for promoting the transformation of mesodermal cells into haematopoietic endothelial precursor cells, or alternatively, for promoting the transformation of haematopoietic endothelial precursor cells into haematopoietic precursor cells.
The invention selects a culture medium with definite chemical components for culturing and differentiating the human pluripotent stem cells, and also describes a technology for carrying out differentiation of the pluripotent stem cells into hematopoietic endothelial precursor cells and hematopoietic precursor cells by simulating the microgravity effect by using a random rotator. In summary, compared with the prior art, the invention has the following advantages:
the culture system is characterized in that the culture system is cultured under the conditions of no serum, no feeding layer and clear chemical composition from the culture and amplification of the pluripotent stem cells to the mesendoderm differentiation, the hematopoietic endothelial precursor differentiation and the hematopoietic precursor cells. The currently reported methods for in vitro differentiation of human pluripotent stem cells into hematopoietic precursors/stem cells mainly include systems requiring co-culture with mouse bone marrow stromal cells (OP 9 cell line) or mouse aortic gonadal mesonephroma stromal cells (mAGM cell line), and systems using Embryoid Bodies (EB) for differentiation. The co-culture system contains animal-derived substances, so that the instability and safety of the experiment are improved, and the complexity of the experiment steps is increased; in the EB differentiation system, the differentiation efficiency was low. The culture system with definite chemical components and the culture method of random three-dimensional rotary simulated microgravity can obtain a large amount of hematopoietic endothelial precursor cells and hematopoietic precursor cells from pluripotent stem cells in a short time, and has high differentiation efficiency which is improved by more than 2 times compared with static culture. The low-cost, safe and controllable random three-dimensional rotary culture strategy of the serum-free and feeder layer-free cells can lay a foundation for the future large-scale production of hematogenic endothelial precursor cells or hematogenic precursor cells for clinical treatment.
Drawings
FIG. 1 is a photograph of a bright field of hematopoietic endothelial precursor cells derived from human pluripotent stem cells induced by the method of the present invention.
FIG. 2 is a photograph of fluorescent staining of hematopoietic endothelial precursor cell marker molecules CD31 and CD34 from hematopoietic endothelial precursor cells derived from human pluripotent stem cell induction using the method of the present invention.
FIG. 3 is a flow cytometric analysis of the differentiation of hematopoietic endothelial precursor cells using the random three-dimensional rotating culture of the present invention and conventional static culture to compare the efficiency of CD31+ CD34+ expression of the differentiated cells. Wherein, the A picture is a control group, and the B picture is a random three-dimensional rotation group.
FIG. 4 is a brightfield image of hematopoietic precursor cells derived from human pluripotent stem cell induction using the method of the invention.
FIG. 5 is a photograph showing fluorescent staining of hematopoietic precursor cell marker molecules CD34 and CD43 from hematopoietic precursor cells derived from human pluripotent stem cells induced by the method of the present invention.
FIG. 6 is a flow cytometric analysis of the differentiation of hematopoietic progenitor cells using the random round culture of the present invention and conventional static culture to compare the efficiency of CD34+ CD43+ expression of the differentiated cells. Wherein, the A picture is a control group, and the B picture is a random rotation group.
Detailed Description
The present invention will now be further illustrated with reference to examples, but the practice of the invention is not limited thereto.
Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from normal sources.
Unless otherwise specified, the human pluripotent stem cells used in the present invention are human embryonic stem cell line H1.
Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from normal sources.
Example 1: preparation of serum-free culture medium
(1) Preparation of IF9S medium:
the present application uses IMDM medium, F12 medium, polyvinyl alcohol (PVA), lipids (100X), ITS-X (100X), monothioglycerol (α MTG), AA2P, glutaMax TM (100X), non-essential amino acids (NEAA, 100X) to prepare IF9S medium.
Wherein, each component of the culture medium is purchased from:
IMDM medium was purchased from Gibco, inc. under the catalog number 21056-023;
the F12 medium was purchased from Gibco, inc. under the product number of 31765-027;
polyvinyl alcohol (PVA) was purchased from Sigma under the trade designation P8136;
lipids (100X) available from Gibco, inc. under the catalog number 11905031;
ITS-X (100X) is available from Gibco, having a Cat number of 51500-056;
monothioglycerol (. Alpha.MTG) was purchased from Sigma under the designation M6145;
AA2P was purchased from Sigma under the accession number A8960;
GlutaMax TM (100X) is available from Gibco corporation under the Cat No. 35050-061;
nonessential amino acids (NEAA, 100X) were purchased from Gibco under the accession number 11140-035;
preparing the culture medium. Wherein, the polyvinyl alcohol (PVA) is powder, and is dissolved by deionized water before use to prepare a stock solution with 5 percent of concentration.
Specifically, the IF9S medium is prepared by the following method:
firstly, mixing an IMDM culture medium and an F12 culture medium according to the proportion of 1, uniformly stirring, and adding the following reagents into the mixed culture medium, wherein the final concentrations of the reagents are as follows: 10mg/L polyvinyl alcohol (PVA), 0.1% by volume additives of Lipids (100X), 2% by volume additives of ITS-X (100X), 40. Mu.l/L monothioglycerol (alpha MTG), 64mg/L AA2P, 1% by volume GlutaMax TM (100X) additives and 1% by volume of non-essential amino acid additives (NEAA, 100X).
The addition of the reagents is not in strict order, and the preparation process is carried out at normal temperature, and the preparation process does not need heating, but the preparation of the culture medium needs sterile filtration by using a filter of 0.22 mu m, and the culture medium is stored in a refrigerator at 4 ℃.
(2) Preparation of mesoderm cell differentiation medium
The mesoderm cell differentiation culture medium is an IF9S culture medium added with 50ng/mL BMP4, 20 mu g/mL Activin A and 1 mu M CHIR99021.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
(3) Preparation of differentiating culture medium for hematogenesis endothelial precursor
The hematopoietic endothelial precursor differentiation medium is IF9S medium supplemented with 10. Mu.M SB431542, 50ng/mL VEGF,50ng/mL bFGF, and 50ng/mL SCF.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
(4) Preparation of hematopoietic precursor cell differentiation medium
The hematopoietic precursor/stem cell differentiation medium was prepared by adding 50ng/mL VEGF,20ng/mL bFGF,50ng/mL SCF,10ng/mL IL-3, 50ng/mL IL-6 and 20ng/mL TPO to IF9S complete medium.
The addition of various factors is not in strict sequence, and the whole process is carried out under the aseptic condition in an ultra-clean workbench.
Example 2: preparation of hematopoietic endothelial precursor cells derived from human pluripotent stem cells
The human pluripotent stem cells used in this example were human embryonic stem cell line (H1) (from university of Wisconsin, USA).
a. Firstly, coating a dish for culturing cells by adopting extracellular matrix Matrigel of embryonic stem cell grade, then inoculating human embryonic stem cells into the coated dish and using a commercial culture medium TeSR with clear chemical components TM -E8 TM The culture was performed with replacement of fresh medium every 24 hours. Human embryonic stem cells grow in a clonal form, and when the cell clones grow to 70-80% of the density, the cells need to be subjected to digestion passage or induced differentiation culture after the cell clones grow. The cell passage cycle is typically 3-4 days.
b. And (3) cloning the human embryonic stem cells grown in the step a by adopting ReLeSRTM digestive enzyme for digestion treatment, and digesting for 2-3 minutes at 37 ℃. Observing the cell digestion condition under the mirror after digesting for 2 minutes, and using the cell after the cell clone edge is curledTeSR TM -E8 TM The digestion of the culture medium is stopped, so that the over digestion is avoided;
c. inoculating the small digested and centrifuged human embryonic stem cell clone blocks into the culture bottle pre-coated with growth factor reduction Matrigel, wherein the inoculation density is 50-200 clone blocks/cm 2 . Adding a proper amount of TeSR TM -E8 TM Medium, at 37 ℃ and 5% CO 2 Standing and culturing in an incubator for 24-48 hours; after culturing for 24-48 hours, replacing the culture medium with a mesoderm cell differentiation culture medium;
d. after addition of the mesoderm cell differentiation medium, the cell differentiation medium was assayed at 37 ℃ and 5% CO 2 Carrying out static culture in an incubator for 48 hours;
e. culturing the cells in a mesoderm cell induction culture medium for 48 hours, then, sucking and replacing the culture medium in a culture bottle with a fresh hematogenous endothelial precursor cell induction culture medium, and filling the culture bottle with the culture medium;
f. sealing the cell culture bottle, then installing the cell culture bottle on a random three-dimensional gyroscope, setting a rotation mode as a random rotation mode, and adjusting the rotation speed to be 0.1-10 rpm/min;
g. placing the random three-dimensional retransfer at 37 ℃ and 5% CO 2 Rotationally culturing for 72 hours in an incubator, and observing a vascular endothelial-like grid shape formed by cells after culturing for 72 hours, wherein the vascular endothelial-like grid shape is blood vessel/hematogenic endothelial precursor cells; the morphology of the hematopoietic endothelial precursor cells is shown in FIG. 1.
Example 3: detection of hematopoietic endothelial precursor cells
The vascular/hematopoietic endothelial precursor cells generated in example 2 were collected for identification by immunofluorescence staining.
Specifically, the method comprises the following steps:
a. removing the hematopoietic endothelial precursor cell induction medium in the culture flask, and washing the cells for 2 times by using PBS (phosphate buffer solution) to remove dead cells;
b. adding 4% paraformaldehyde for fixation, and standing and fixing for 20 minutes at room temperature;
c. removing 4% paraformaldehyde by suction, and washing with PBS for 2-3 times;
d. adding donkey serum with the concentration of 5% for sealing, and sealing at room temperature for 1 hour;
e. adding a fluorescence-labeled mouse anti-human PE-CD31 antibody (cat No. 560983, BD company), a fluorescence-labeled mouse anti-human FITC-CD34 antibody (cat No. 560942, BD company) and 0.1. Mu.g/mL Hoechst33342, wherein the dilution times of the antibodies are recommended to be diluted by using a blocking solution according to the instruction number, and incubating at room temperature for 1 hour or at 4 ℃ overnight;
f. the staining solution containing the antibody was aspirated, and cells were washed 3 times for 5 minutes each by adding an appropriate amount of PBS.
g. CD31 and CD34 proteins are detected by using a confocal fluorescence microscope for cells, hoechst33342 is used for marking cell nuclei, and light is avoided when operation is carried out to prevent fluorescence quenching. The results of FIG. 2 show the results of the staining identification.
CD31 and CD34 are important markers of blood vessel/hematogenic endothelial precursor cells, and the fluorescence color result can confirm that the mesodermal cells can be further induced into CD31 and CD34 positive cells, namely the hematogenic endothelial precursor cells, by the method.
Example 4: efficiency analysis of hematopoietic endothelial precursor cells
The resultant hematogenic endothelial precursor cells produced in example 2 were collected for characterization by flow cytometry analysis.
Specifically, the method comprises the following steps:
a. cell digestion: the cells described in example 2 were digested into single cells using Accutase enzyme (cat. No.: A1110501, gibco Co., ltd.), and the digestion was terminated with IF9S medium.
b. Cell collection: the cells were transferred to a centrifuge tube at 1000 rpm and centrifuged for 5 minutes to enrich the cells.
c. Antibody incubation labeling: after centrifugation, the supernatant was aspirated and the cells were resuspended with 0.2% BSA, and the grouping of the cells and the labeling of the antibody were carried out according to the details of the experiment, wherein the labeled antibody for identifying hematopoietic endothelial precursor cells was mouse anti-human PE-CD31 antibody (cat # 560983, BD Co.) and mouse anti-human FITC-CD34 antibody (cat # 560942, BD Co.); the marker molecule antibodies identified by hematopoietic precursor/stem cells were mouse anti-human FITC-CD34 antibody (cat # 560942, BD Co.) and mouse anti-human APC-CD43 antibody (cat # 560198, BD Co.). The dilution ratio of the antibody is typically 1.
d. And (3) washing cells: adding a proper amount of PBS, rotating at 1000 rpm, centrifuging for 5 minutes to enrich cells, and sucking and removing supernatant.
e. Cell fixation and detection on a machine: resuspend cells with 1% paraformaldehyde in PBS. Sieving with cell sieve (40 um) before detection. The cell suspension was transferred to a flow tube ready for flow analysis. The flow cytometer was a FACS Calibur analysis type flow cytometer (BD), and after the test was completed, data analysis was performed using FlowJo 10.0.7 software.
FIG. 3 shows the flow cytometry analysis results of the hematopoietic endothelial precursor cells in the control group and the random three-dimensional rotation group.
Wherein the method for preparing the cells of the random three-dimensional rotation group was the same as in example 2, and the method for preparing the cells of the control group was substantially the same as in example 2, except that the method was performed directly after step e by removing CO at 37 ℃ and 5% 2 And (5) performing static culture in an incubator for 72 hours to obtain control cells.
As can be seen from the results of fig. 3, the control group achieved induction of 37.6% of mesodermal cells into hematopoietic endothelial precursor cells using only the culture medium of the present invention. By combining the culture medium of the invention and random three-dimensional rotation, 71.6% of mesodermal cells are induced to be blood endothelial precursor cells. The method of the invention proves that the random rotation can increase the induction efficiency to the hematopoietic endothelial precursor cells, and further realizes the high-efficiency induction of the hematopoietic endothelial precursor cells by matching with the culture medium of the invention.
Example 5: preparation of hematopoietic precursor cells derived from human pluripotent stem cells
Specifically, the method comprises the following steps:
a. removing the hematopoietic endothelial precursor cell pro-culture medium in the culture flask, and replacing with the hematopoietic precursor cell induction culture medium obtained in example 1; filling the culture bottle with culture medium;
b. installing a cell culture bottle on a random gyroscope, setting a rotation mode to be random rotation, and adjusting the rotation speed to be the rotation speed with the inner diameter and the outer diameter of 5 rpm/min;
c. subjecting the random gyroscope to 37 deg.C, 5% CO 2 Continuously carrying out rotary culture in the incubator for 48-72 hours;
d. after 48 hours of cell culture, a large amount of "pebble-like" hematopoietic precursor cells appeared in the culture flask, and some of the hematopoietic precursor cells separated from the bottom of the dish to become suspended hematopoietic precursor cells. The resultant hematopoietic precursor cells are shown in FIG. 4, in which A is "pebble-like" hematopoietic precursor cells and B is suspended hematopoietic precursor cells with some of the hematopoietic precursor cells detached from the bottom of the dish.
Example 6: detection of hematopoietic precursor cells
Hematopoietic precursor cells produced in example 5 were collected for identification by immunofluorescence staining.
h. Removing the hematopoietic precursor cell induction medium in the culture flask by aspiration, and washing the cells 2 times with PBS to remove dead cells;
i. adding 4% paraformaldehyde for fixation, and standing and fixing for 20 minutes at room temperature;
j. removing 4% paraformaldehyde by suction, and washing for 2-3 times by adding PBS;
k. adding donkey serum with the concentration of 5% for sealing, and sealing at room temperature for 1 hour;
adding a fluorescence-labeled mouse anti-human FITC-CD34 antibody (cat No. 560942, BD company), a fluorescence-labeled mouse anti-human APC-CD43 antibody (cat No. 560198, BD company) and 0.1. Mu.g/mL Hoechst33342, wherein the dilution times of the antibodies are recommended to be diluted by a blocking solution according to the instruction number, and incubating at room temperature for 1 hour or at 4 ℃ overnight;
m. the staining liquid containing the antibody was aspirated, and the cells were washed 3 times for 5 minutes each by adding an appropriate amount of PBS.
And n, detecting CD34 and CD43 proteins of cells by using a confocal fluorescence microscope, wherein Hoechst33342 is used for marking cell nuclei, and is protected from light during operation to prevent fluorescence quenching. The results of FIG. 5 show the results of the staining identification.
CD34 and CD43 are important markers of hematopoietic precursor cells, and the fluorescence color result can confirm that the hematopoietic endothelial precursor cells can be induced into CD34 and CD43 positive cells, namely hematopoietic precursor cells, by the method of the invention.
The results in FIG. 2 show the results of the identification of the staining of hematopoietic progenitor cells.
Example 7: efficiency assay for hematopoietic precursor cells
The hematopoietic precursor cells produced in example 2 were collected and characterized by flow cytometry analysis.
Specifically, the method comprises the following steps:
f. cell digestion: the cells described in example 2 were digested into single cells using Accutase enzyme (cat. No.: A1110501, gibco Co., ltd.), and the digestion was stopped with IF9S medium, respectively.
g. Cell collection: the cells were transferred to a centrifuge tube at 1000 rpm and centrifuged for 5 minutes to enrich the cells.
h. Antibody incubation labeling: after centrifugation, the supernatant was aspirated and the cells were resuspended in 0.2% BSA, and the cells were grouped and labeled with antibodies according to the specific conditions of the experiment, wherein the antibodies for the marker molecules identified for hematopoietic progenitor cells were mouse anti-human FITC-CD34 antibody (cat # 560942, BD Co.) and mouse anti-human APC-CD43 antibody (cat # 560198, BD Co.). The dilution ratio of the antibody is typically 1.
i. And (3) washing cells: adding a proper amount of PBS, rotating at 1000 rpm, centrifuging for 5 minutes to enrich cells, and sucking and removing supernatant.
j. Cell fixation and detection on a machine: cells were resuspended in 1% paraformaldehyde in PBS. Sieving with cell sieve (40 μm) before testing. The cell suspension was transferred to a flow tube ready for flow analysis. The flow cytometer was a FACS Calibur analysis type flow cytometer (BD), and after the test was completed, data analysis was performed using FlowJo 10.0.7 software.
FIG. 6 shows the results of flow cytometry analysis of hematopoietic precursors in the control and random rotation groups. Wherein the method for preparing the cells of the random rotation group was the method of example 2, and the method for preparing the cells of the control group was substantially the same as that of example 2, except that the method was performed at 37 ℃ and 5% CO directly after step a 2 Standing in incubatorAfter 72 hours of culture, control cells were obtained.
As can be seen from the results of fig. 3, the control group induced 28.9% of hematopoietic endothelial precursor cells into hematopoietic precursor cells using the medium of the present invention alone. By combining the culture medium of the invention and random rotation, 45.9% of the hematopoietic endothelial precursor cells are induced into hematopoietic precursor cells. The method of the invention proves that random rotation can increase the induction efficiency to hematopoietic precursor cells, and further realizes high-efficiency induction by matching with the culture medium of the invention.
Example 8: other methods for obtaining hematopoietic progenitor cells from hematopoietic endothelial progenitor cells to hematopoietic progenitor cells Induction test
Hematopoietic endothelial precursor cells (described, for example, in Xu Cao, gopala K. Yakala, francijna E. Van den Hil, amy Cochrane, christine L. Mummery, valeria V. Orova. Difference and Functional company of Monocytes and Macrophages from PSCs with Peripheral Blood differentiation. Stem Cell reports.2019June 11 (6)) were prepared by a known method and cultured by induction to hematopoietic precursor cells by the following method.
Specifically, the method comprises the following steps:
e. removing the hematopoietic endothelial precursor cell pro-culture medium in the culture flask, and replacing with the hematopoietic precursor cell induction culture medium obtained in example 1; filling the culture bottle with culture medium;
f. installing the cell culture bottle on a random gyroscope, setting a rotation mode to be random rotation, and adjusting the rotation speed to the rotation speed with the inner diameter and the outer diameter of 5 rpm/min;
g. subjecting the random gyroscope to 37 deg.C, 5% CO 2 Continuously carrying out rotary culture in the incubator for 48-72 hours;
h. after 48 hours of cell culture, a large amount of "pebble-like" hematopoietic precursor cells appeared in the culture flask, and some of the hematopoietic precursor cells separated from the bottom of the dish to become suspended hematopoietic precursor cells.
The culture of hematopoietic progenitor cells from hematopoietic endothelial progenitor cells of the invention may also be induced by the culture medium and the spinner culture method of the invention.

Claims (11)

1. A method of differentiating from human pluripotent stem cells to hematopoietic precursor cells, comprising the steps of:
s1) inducing differentiation culture of human pluripotent stem cells to mesoderm cells:
s11) pre-coating the cell container with a growth factor-reduced basement membrane matrix;
s12), adopting digestive enzyme to clone the human pluripotent stem cells, digesting and centrifuging;
s13) inoculating the digested and centrifuged small human pluripotent stem cell clone mass into a cell container obtained in S11) and adding TeSR into the cell container TM -E8 TM The culture medium is statically cultured for 24 to 48 hours;
s14) replacing the culture medium with a mesoderm cell induction differentiation culture medium, and statically culturing for 24-48 hours to obtain induced mesoderm cells;
s2) the induced mesodermal cells are further differentiated and cultured to hematogenic endothelial precursor cells:
s21), replacing a cell culture medium of the mesoderm cells with a hematopoietic endothelial precursor cell induced differentiation culture medium;
s22), mounting the cell culture container on a random three-dimensional gyroscope, setting the rotation mode to be random rotation, and adjusting the rotation speed to be 0.1-10 rpm/min;
s23) placing a random three-dimensional gyroscope at 37 ℃ 5% 2 Rotationally culturing in an incubator for 60-96 hours to obtain hematogenesis endothelial precursor cells;
s3) after culturing for 72 hours in the original culture medium, replacing the cell culture medium with a culture medium for inducing the differentiation of the hematopoietic endothelial precursor cells to the hematopoietic precursor cells;
s4) reinstalling the cell culture bottle on a random three-dimensional gyroscope to continue three-dimensional rotary culture, wherein the three-dimensional rotary culture rotation mode is random rotation, and the rotation speed is 0.1-10 rpm/min;
the mesoderm cell induction differentiation culture medium is an IF9S complete culture medium added with 40-60ng/mL BMP4, 15-25 μ g/mL Activin A and 0.8-1.2 μ M CHIR99021;
the hematopoietic endothelial precursor cell induced differentiation medium is an IF9S complete medium added with 8-12 mu M SB431542, 40-60ng/mL VEGF, 40-60ng/mL bFGF and 40-60ng/mL SCF;
the culture medium for inducing the hematopoietic endothelial precursor cells to differentiate into the hematopoietic precursor cells is an IF9S complete culture medium added with 40-60ng/mL VEGF,15-25ng/mL bFGF,40-60ng/mL SCF,8-12ng/mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO;
the IF9S complete medium comprises an IMDM medium and an F12 medium which are mixed according to a volume ratio of 1.8-1.2, polyvinyl alcohol (PVA) with a final concentration of 8-12mg/L, lipid 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, monothioglycerol (alpha MTG) with a final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA 2P) with a final concentration of 55-70mg/L, and GlutaMax with a volume percentage of 0.8-1.2% of the IF9S complete medium TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8% -1.2% of the IF9S complete medium.
2. The method according to claim 1, wherein the rotation speed in S22) is 5 rpm/min.
3. The method according to claim 2, wherein the culture in S22) is carried out for 72 hours.
4. A method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, which comprises the following steps, in the process of differentiating and culturing the hematopoietic endothelial precursor cells into the hematopoietic precursor cells, adopting a random three-dimensional gyroscope to culture a cell culture container under the rotation condition;
the culture medium for culturing under the rotation condition adopts a culture medium for inducing the differentiation of the hematopoietic endothelial precursor cells to the hematopoietic precursor cells;
the rotation mode of the three-dimensional gyroscope is random rotation, and the rotating speed is 0.1-10 rpm/min;
the induced hematopoietic endothelial precursor cells are added with 40-60ng/mL VEGF,15-25ng/mL bFGF,40-60ng/mL SCF,8-12ng/mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO into an IF9S complete medium;
the IF9S complete culture medium comprises an IMDM basic culture medium and an F12 basic culture medium which are mixed according to the volume ratio of 1.8-1.2, and polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, lipid 100X additive with the volume percentage of 0.08-0.12% of the culture medium, ITS-X100X additive with the volume percentage of 1.8-2.2% of the culture medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, AA2P with the final concentration of 55-70mg/L, glutaMax with the volume percentage of 0.8-1.2% of the culture medium TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8-1.2% of the medium.
5. The method of claim 4, wherein the hematopoietic endothelial precursor cells are cultured for 72 hours prior to their differentiation into hematopoietic precursor cells.
6. The method of claim 4, wherein the random rotation inner and outer diameters are each at a speed of 5 rpm/minute.
7. The method of claim 4, wherein the culturing under spinning conditions is carried out for a period of 48 to 72 hours.
8. The method of claim 4, wherein the medium for inducing differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is added to 95% -100% of the culture vessel.
9. The method of claim 4, wherein the cell culture conditions are 37 ℃, 5% 2 。
10. Use of a random three-dimensional gyroscope for promoting the transformation of mesodermal cells into hematogenic endothelial precursor cells;
culturing mesoderm cells in a random three-dimensional gyroscope, wherein a culture medium is a blood endothelial precursor cell induced differentiation culture medium;
the hematopoietic endothelial precursor cell induced differentiation medium is an IF9S complete medium added with 8-12 mu M SB431542, 40-60ng/mL VEGF, 40-60ng/mL bFGF and 40-60ng/mL SCF;
the IF9S complete medium comprises IMDM medium and F12 medium which are mixed according to the volume ratio of 1.8-1.2, polyvinyl alcohol (PVA) with the final concentration of 8-12mg/L, lipids100X additive with the volume percentage of 0.08-0.12% of the medium, ITS-X100X additive with the volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) with the final concentration of 35-45 mu L/L, sodium ascorbyl phosphate (AA 2P) with the final concentration of 55-70mg/L, and GlutaMax with the volume percentage of 0.8-1.2% of the IF9S complete medium, wherein the IMDM medium and the F12 medium are mixed according to the volume ratio of 1 TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8% -1.2% of the IF9S complete medium;
the random three-dimensional gyroscope rotates randomly with the rotation speed of 0.1-10 rpm/min.
11. Use of a stochastic three-dimensional gyroscope for promoting the transformation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;
culturing the hematopoietic endothelial precursor cells in a random three-dimensional gyroscope, wherein the culture medium is used for inducing the differentiation of the hematopoietic endothelial precursor cells to the hematopoietic precursor cells,
the culture medium for inducing the hematopoietic endothelial precursor cells to differentiate into the hematopoietic precursor cells is an IF9S complete culture medium added with 40-60ng/mL VEGF,15-25ng/mL bFGF,40-60ng/mL SCF,8-12ng/mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO;
the IF9S complete medium comprises IMDM medium and F12 medium which are mixed according to the volume ratio of 1.8-1.2And polyvinyl alcohol (PVA) at a final concentration of 8-12mg/L, lipids100X additive at a volume percentage of 0.08-0.12% of the medium, ITS-X100X additive at a volume percentage of 1.8-2.2% of the IF9S complete medium, monothioglycerol (alpha MTG) at a final concentration of 35-45. Mu.l/L, sodium ascorbyl phosphate (AA 2P) at a final concentration of 55-70mg/L, glutaMax at a volume percentage of 0.8-1.2% of the IF9S complete medium TM 100X additive and non-essential amino acid additive 100X (NEAA) in a volume percentage of 0.8% -1.2% of the IF9S complete medium;
the random three-dimensional gyroscope rotates randomly with the rotation speed of 0.1-10 rpm/min.
CN202110396567.5A 2021-04-13 2021-04-13 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells Active CN113046318B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110396567.5A CN113046318B (en) 2021-04-13 2021-04-13 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells
PCT/CN2021/138097 WO2022217955A1 (en) 2021-04-13 2021-12-14 Culture medium and method for inducing differentiation of pluripotent stem cell into hematopoietic precursor cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110396567.5A CN113046318B (en) 2021-04-13 2021-04-13 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

Publications (2)

Publication Number Publication Date
CN113046318A CN113046318A (en) 2021-06-29
CN113046318B true CN113046318B (en) 2023-04-18

Family

ID=76519330

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110396567.5A Active CN113046318B (en) 2021-04-13 2021-04-13 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

Country Status (2)

Country Link
CN (1) CN113046318B (en)
WO (1) WO2022217955A1 (en)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112760281B (en) * 2019-11-04 2022-05-20 北京基石生命科技有限公司 Culture medium for culturing primary cells of brain tumor solid tumors
CN113046318B (en) * 2021-04-13 2023-04-18 中国科学院深圳先进技术研究院 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells
CN114672455A (en) * 2022-03-25 2022-06-28 中山大学 Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells
CN114958718A (en) * 2022-05-20 2022-08-30 呈诺再生医学科技(北京)有限公司 Method for inducing cells with high FOXA2 expression
CN116064372A (en) * 2022-11-15 2023-05-05 上海交通大学 Method for preparing human ovary somatic cell-like cells
CN115975925B (en) * 2023-03-21 2023-09-08 天九再生医学(天津)科技有限公司 Method for inducing human pluripotent stem cells to differentiate into natural killer cells by variable-speed suspension
CN116445408B (en) * 2023-05-22 2024-02-02 呈诺再生医学科技(北京)有限公司 Use of LSD1 inhibitors to promote iPSC differentiation to HSCs and maintenance of HSC dryness
CN116574672B (en) * 2023-07-11 2023-10-20 北京北启生物医药有限公司 Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells
CN117305241B (en) * 2023-11-28 2024-03-19 上海兴瑞一达生物科技有限公司 Method for inducing and differentiating hiPSCs into NK cells

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8669106B2 (en) * 2006-10-18 2014-03-11 Arteriocyte Inc. Erythrocytes differentiated in vitro from nanofiber expanded CD133+ cells
JP2012080874A (en) * 2010-09-15 2012-04-26 National Institute Of Advanced Industrial Science & Technology Three-dimensional tissue-constructing method in pseudo-microgravity environment
CA2905786C (en) * 2013-03-13 2022-01-25 Wisconsin Alumni Research Foundation Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions
CN104293730B (en) * 2014-09-23 2017-07-28 云南中科灵长类生物医学重点实验室 Multipotential stem cell vitro directed differentiation is the method for cardiac muscle cell
CN105062957B (en) * 2015-06-05 2018-08-21 张竞方 The cultural method of vascular endothelial cell progenitor cells
JP6646311B2 (en) * 2015-07-17 2020-02-14 国立大学法人京都大学 Differentiation induction method from pluripotent stem cells to mesoderm progenitor cells and blood vascular progenitor cells
WO2017164257A1 (en) * 2016-03-23 2017-09-28 国立大学法人京都大学 Mesoderm induction method having high blood cell differentiation capacity
US20200399602A1 (en) * 2018-01-18 2020-12-24 Agency For Science, Technology And Research Method for Differentiation of Human Pluripotent Stem Cell Lines in Suspension Culture
GB201911957D0 (en) * 2019-08-20 2019-10-02 Adaptimmune Ltd Methods of producing haemogenic progenitor cells from pluripotent stem cells
CN111575280B (en) * 2019-08-23 2022-12-23 西北工业大学 miR-194-5p sequence for promoting MEF2C gene expression in mouse myoblast and application thereof
CN111808812B (en) * 2020-08-31 2020-12-15 首都医科大学附属北京友谊医院 Material and method for differentiation of pluripotent stem cells into hematopoietic stem cells
CN113046318B (en) * 2021-04-13 2023-04-18 中国科学院深圳先进技术研究院 Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

Also Published As

Publication number Publication date
CN113046318A (en) 2021-06-29
WO2022217955A1 (en) 2022-10-20

Similar Documents

Publication Publication Date Title
CN113046318B (en) Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells
CN102307991B (en) Multipotential stem cell culture on microcarrier
JP5902092B2 (en) Cardiomyocyte generation
CN111117946B (en) Nasal mucosa organoid culture medium and culture method
CN102388130A (en) Differentiation of pluripotent cells
CN112226409B (en) Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells
CN112048470B (en) Method for preparing clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells
US20060281136A1 (en) Preparation of endodermal stem cells
US20220135947A1 (en) Methods for culturing mesenchymal stem cells, products thereof, and applications thereof
WO2023016029A1 (en) Method for separating fibroblasts derived from human induced pluripotent stem cells, and use thereof
CN114752565A (en) Retina organoid with immune cells and construction method thereof
CN110872574B (en) Efficient and reliable hESC-MSC preparation method
CN109722411B (en) Application method of micromolecules for promoting self-renewal state of embryonic stem cells
CN110951686A (en) Hematopoietic stem cell in-vitro amplification culture system and method
CN112680410B (en) Method for inducing pluripotent stem cells to culture heart fibroblasts in differentiation mode and culture solution thereof
CN115537387A (en) Stentless 3D micro-tissue and construction method thereof
Lu et al. Directed differentiation of red blood cells from human embryonic stem cells
CN114672455A (en) Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells
CN117187174B (en) Muse cell culture medium and extraction method of fat Muse cells
CN114990064B (en) Hematopoietic stem cells, preparation method and application thereof
CN114540282B (en) Method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells
CN117384830A (en) Method for efficiently preparing iPSCs without heterogeneous components
CN116731961A (en) Culture method and application of third pharyngeal cyst endoderm
CN115975930A (en) Method for inducing and differentiating human nasal mucosa mesenchymal stem cells into neural stem cells in batch based on 3D culture
EP3564363A1 (en) Production of megakaryocytes in bioreactors

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant