WO2021088294A1 - Composition, culture medium, method and kit for amplifying hematopoietic stem cell - Google Patents

Composition, culture medium, method and kit for amplifying hematopoietic stem cell Download PDF

Info

Publication number
WO2021088294A1
WO2021088294A1 PCT/CN2020/080030 CN2020080030W WO2021088294A1 WO 2021088294 A1 WO2021088294 A1 WO 2021088294A1 CN 2020080030 W CN2020080030 W CN 2020080030W WO 2021088294 A1 WO2021088294 A1 WO 2021088294A1
Authority
WO
WIPO (PCT)
Prior art keywords
hematopoietic stem
stem cells
cells
jnk
signaling pathway
Prior art date
Application number
PCT/CN2020/080030
Other languages
French (fr)
Chinese (zh)
Inventor
陈立功
孙忠杰
肖雄
刘英全
刘德芳
齐海龙
郭潇
王晓芳
Original Assignee
诺未科技（北京）有限公司
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 诺未科技（北京）有限公司 filed Critical 诺未科技（北京）有限公司
Publication of WO2021088294A1 publication Critical patent/WO2021088294A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • 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
    • C12N2500/40—Nucleotides, nucleosides, bases
    • 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
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to compositions, media, methods, and kits for expanding hematopoietic stem cells.
  • Hematopoietic stem cells are a type of adult stem cells that have both pluripotency and self-renewal ability. There are more than 10 mature cell types in the human blood system. Among them, hematopoietic stem cells are the most representative In addition to being able to differentiate into all cell types in the blood system, cells also retain the ability to proliferate themselves.
  • hematopoietic stem cell can re-establish the entire blood system, and its clinical application prospects are very broad, and it is highly concerned by the scientific and medical circles.
  • Hematopoietic stem cells mainly exist in human bone marrow (BM), mobilized peripheral blood (PB), umbilical cord blood (UCB) and other tissues.
  • BM bone marrow
  • PB mobilized peripheral blood
  • UOB umbilical cord blood
  • the specific cell surface markers and their combinations are usually used to isolate and obtain the required types of hematopoietic stem cells.
  • John E. Dick was able to isolate hematopoietic stem cells at the single-cell level, with a purity of 28%, that is, 5 hematopoietic stem cells with long-term hematopoietic reconstitution ability can be isolated from 18 cells (Notta et al. , 2011).
  • Hematopoietic stem cells are the earliest type of stem cells used in clinical treatment. Since they were first used in the treatment of severe immunodeficiency in 1968, their effectiveness has been repeatedly proven by clinical trials. Dr. Donnall Thomas of the Fred Hutchinson Medical Center of the University of Washington in Seattle, USA, won the Nobel Prize in Physiology in 1990 (Park et al., 2015) for his outstanding contributions to the treatment of leukemia and other blood diseases in hematopoietic stem cell transplantation.
  • hematopoietic stem cells are mainly used clinically to treat malignant hematological diseases such as leukemia and lymphoma, related metabolic diseases, autoimmune diseases, and acquired immunodeficiency.
  • malignant hematological diseases such as leukemia and lymphoma, related metabolic diseases, autoimmune diseases, and acquired immunodeficiency.
  • hematopoietic stem cells have great prospects for medical applications, because the number of hematopoietic stem cells is limited and it is difficult to obtain them in vitro, about 40% of patients in the clinic can only adopt conservative treatment methods due to the failure of HLA matching.
  • Umbilical cord blood is an important source of HSCs. Because of its advantages such as convenient collection, non-invasiveness, and low immunogenicity, it has received high clinical attention in recent years. Nearly 40,000 cord blood is used in clinical research and treatment every year. However, due to the limited number of hematopoietic stem cells contained in a single cord blood, which is not enough to provide the number of cells required by an adult, about 50% of patients cannot receive transplantation, which greatly limits the clinical application of cord blood.
  • Expansion of hematopoietic stem cells in vitro has many advantages, which are mainly summarized as the following three characteristics: (1) The expanded cells have high homogeneity and low risk of cancer; (2) The operation time for expansion of HSC is relatively short, avoiding the introduction of more Exogenous unfavorable factors; (3) The starting cells are hematopoietic stem/progenitor cells, which generally do not have the risk of tumorigenesis, and the clinical application is safer.
  • the present invention aims to solve the technical problems existing in the prior art at least to a certain extent.
  • the present invention finds some chemical small molecule compound combinations through a high-throughput screening platform, which can not only enable efficient expansion of hematopoietic stem cells in vitro, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, but also improve hematopoietic stem cells
  • the reconstruction function and reconstruction efficiency of the body have important scientific research, clinical research and application value.
  • the present invention provides a composition for expanding hematopoietic stem cells.
  • the composition consists of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632.
  • JNK-IN-8 is an inhibitor of the JNK signaling pathway, which regulates the JNK signaling pathway by inhibiting c-Jun phosphorylation and gene transcription; nicotinamide adenine dinucleotide (NAD), which can regulate basic metabolism, is a cell The basic reaction substrate for growth; Rapamycin is an mTOR signal pathway inhibitor, which can be used for anti-aging and inhibiting tumor growth; Y27632 is a ROCK1 inhibitor, which can be used for anti-aging, and has a significant effect on maintaining the vitality of stem cells. .
  • the inventors used high-throughput screening in order to obtain compounds that efficiently expanded hematopoietic stem cells and found that the four small molecules of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 have mutual coordination. , Synergistic effect, can efficiently expand hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve the in vivo reconstruction function and efficiency of hematopoietic stem cells, which has significant scientific research, clinical research and application value .
  • the present invention provides a medium for expanding hematopoietic stem cells.
  • the medium includes: a basal medium; and the aforementioned composition for expanding hematopoietic stem cells. Therefore, the use of the medium according to the embodiments of the present invention can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and improve the in vivo reconstruction function and efficiency of hematopoietic stem cells. It has important scientific research and clinical research. Research and application value.
  • the above-mentioned culture medium may also have the following additional technical features:
  • the concentration of the JNK-IN-8 is 1 ⁇ 5 ⁇ M
  • the concentration of the nicotinamide adenine dinucleotide is 0.5 ⁇ 5 ⁇ M
  • the concentration of the rapamycin is 1 ⁇ 20 nM
  • the concentration of Y27632 is 1-20 ⁇ M.
  • the concentrations of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin, and Y27632 are 2 ⁇ M, 1 ⁇ M, 10 nM, 10 ⁇ M or 3.5 ⁇ M, 0.8 ⁇ M, 15 nM, 7 ⁇ M, respectively Or 1.5 ⁇ M, 3 ⁇ M, 8nM, 15 ⁇ M.
  • the concentration of the JNK-IN-8 is 1 ⁇ 3 ⁇ M
  • the concentration of the nicotinamide adenine dinucleotide is 0.5 ⁇ 2 ⁇ M
  • the concentration of the rapamycin is 6 ⁇ 15nM
  • the concentration of Y27632 is 5-15 ⁇ M.
  • the basic medium is selected from StemSpan SFEM medium containing Flt3 ligand, thrombopoietin (TPO), stem cell growth factor (SCF) and low density lipoprotein (LDL). Therefore, adding the above factors to StemSpan SFEM medium can further improve the amplification efficiency.
  • the concentration of the Flt3 ligand is 60-100 ng/mL
  • the concentration of the thrombopoietin is 20-50 ng/mL
  • the concentration of the stem cell factor is 60-100 ng/mL
  • the concentration of low-density lipoprotein is 5-20 ⁇ g/mL.
  • the concentrations of Flt3 ligand, thrombopoietin, stem cell factor and low-density lipoprotein are respectively 100ng/ml, 50ng/ml, 100ng/ml, 10 ⁇ g/ml or 80ng/ml, 30ng/ml , 80ng/ml, 15 ⁇ g/ml or 90ng/ml, 40ng/ml, 90ng/ml, 6 ⁇ g/ml.
  • the concentration of the Flt3 ligand is 80-100 ng/mL
  • the concentration of thrombopoietin is 40-50 ng/mL
  • the concentration of the stem cell factor is 80-100 ng/mL.
  • the concentration of the low-density lipoprotein is 5-15 ⁇ g/mL.
  • the present invention proposes the use of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 in the preparation of a composition or culture medium. Used to expand hematopoietic stem cells.
  • the present invention provides a method for expanding hematopoietic stem cells.
  • the method includes: using the aforementioned composition to inhibit the following metabolic pathways of hematopoietic stem cells: JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway.
  • the inventors performed high-throughput screening and analysis of many small molecules that can inhibit the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway, and found that JNK-IN-8 (JNK signaling pathway inhibitor), nicotinamide adenine dinucleoside
  • JNK-IN-8 JNK signaling pathway inhibitor
  • Y27632 ROCK signaling pathway inhibitor
  • the method includes: culturing CD34+ cells in the aforementioned medium for expanding hematopoietic stem cells.
  • amplification efficiency can be further improved, the protein phenotype and gene expression profile of hematopoietic stem cells can be effectively maintained, and the in vivo reconstruction function and reconstruction efficiency of hematopoietic stem cells can be improved.
  • the operation is simple and has broad application prospects.
  • the CD34 + cells are derived from bone marrow, liver, spleen, peripheral blood or cord blood.
  • the present invention provides a kit for expanding hematopoietic stem cells.
  • the kit includes: the aforementioned composition for expanding hematopoietic stem cells or the aforementioned culture medium.
  • the use of the kit according to the embodiments of the present invention can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve the in vivo reconstruction function and efficiency of hematopoietic stem cells, which has important scientific research , Clinical research and application value.
  • the present invention proposes the use of the aforementioned composition in the preparation of inhibitors.
  • the inhibitor is used to expand hematopoietic stem cells and inhibit the following metabolic pathways of hematopoietic stem cells: JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway.
  • JNK-IN-8, NAD, rapamycin and Y27632 can effectively inhibit the JNK signaling pathway, mTOR signaling pathway and/or ROCK signaling pathway, thereby efficiently expanding hematopoietic stem cells and effectively maintaining the protein of hematopoietic stem cells Phenotype and gene expression profile, and can improve the in vivo reconstruction function and reconstruction efficiency of hematopoietic stem cells, which has significant scientific research, clinical research and application value.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes: the aforementioned composition or the aforementioned culture medium.
  • the pharmaceutical composition according to the embodiments of the present invention can efficiently expand hematopoietic stem cells, and directly or indirectly administer the composition or the medium as a pharmaceutical composition into the body (animal or cell) to expand
  • hematopoietic stem cells obtained by the method of expanding hematopoietic stem cells described above can also be applied to the body. They have good reconstitution function and efficiency in vivo, and can be widely used in blood system diseases and In the treatment of autoimmune diseases, it has important scientific research, clinical research and application value.
  • the pharmaceutical composition of the present invention may be used in combination with conventional treatment methods and/or therapies, or may be used separately from conventional treatment methods and/or therapies.
  • the pharmaceutical compositions of the present invention are administered in combination therapy with other drugs, they can be administered to the individual sequentially or simultaneously.
  • the pharmaceutical composition of the present invention may also include a combination of a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient and other therapeutic or preventive drugs known in the art.
  • administration refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner.
  • the mesenchymal stem cell of the present invention can be administered by any common route, as long as it can reach the desired tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the present invention is not limited to these exemplified modes of administration.
  • the present invention provides a method for screening drugs.
  • the method includes: culturing the candidate drug with CD34+ cells; determining whether the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway in the cells before and after the culture are inhibited and/or whether the cells expand after the culture And/or whether the surface proteins representing the functions of hematopoietic stem cells in the cells before and after culture are consistent; when the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway are inhibited after culturing and/or cell expansion after culturing and/or in cells before and after culturing The surface proteins representing the functions of hematopoietic stem cells are consistent, which is an indication that the candidate drug is a target drug, and the target drug is the aforementioned composition or the aforementioned culture medium.
  • the composition according to the embodiment of the present invention can inhibit the above three metabolic pathways, thereby achieving the effect of expanding hematopoietic stem cells. At the same time, it can effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells after amplification. Therefore, by adopting the method for screening drugs according to the embodiments of the present invention, the composition of the present invention or the medium or drugs containing it can be effectively screened, which has great scientific research, clinical research and application value.
  • the present invention proposes the use of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 in the expansion of hematopoietic stem cells.
  • JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve hematopoiesis
  • the in vivo reconstruction function and reconstruction efficiency of stem cells have important scientific research, clinical research and application value.
  • 4F contains JNK-IN-8, nicotinamide adenine dinucleotide Culture conditions of four small chemical molecules, rapamycin and Y27632, among which:
  • Fig. 1 shows a schematic diagram of a process flow for amplifying hematopoietic stem cells according to an embodiment of the present invention
  • Figure 2 shows a flow cytometric analysis diagram of CD34+ cells after 7 days of culture according to an embodiment of the present invention
  • Figure 3 shows a flow cytometric analysis diagram of CD34+ cells after 28 days of culture according to an embodiment of the present invention
  • Figure 4 shows a flow cytometric analysis diagram of hematopoietic stem cells cultured in a medium containing different small molecule compounds according to an embodiment of the present invention (the control group is DMSO culture conditions, **P ⁇ 0.01, ***P ⁇ 0.001 );
  • Figures 5-9 show schematic diagrams of flow cytometry analysis of hematopoietic stem cells after hematopoietic stem cell reconstruction according to an embodiment of the present invention, wherein Figure 5 is 4 weeks after transplantation, Figure 6 is 8 weeks after transplantation, Figure 7 is 12 weeks after transplantation, and Figure 8 And 9 is 16 weeks after transplantation;
  • Fig. 10 shows a schematic diagram of the expansion factor analysis of hematopoietic stem cells 16 weeks after hematopoietic stem cell reconstruction according to an embodiment of the present invention.
  • hematopoietic stem cells were expanded according to the following method:
  • the blood from the fetal umbilical cord is collected in a sterile environment in the operating room, stored in a blood bag with anticoagulant, temporarily stored in a 4°C microenvironment, and delivered to the laboratory for use within 24 hours.
  • the liquid level is divided into three layers, the uppermost layer is the plasma/tissue homogenate layer, the lowermost layer is the red blood cells, the middle layer is the separation liquid, and there is a thin and dense layer between the plasma layer and the separation liquid layer.
  • the albuginea is the layer of mononuclear cells (including lymphocytes and monocytes). Carefully pipette the albuginea cells into another 50ml centrifuge tube;
  • the experimental group was hematopoietic stem cells cultured after adding 2 ⁇ M JNK-IN-8, 1 ⁇ M NAD, 10nM Rapamycin, and 10 ⁇ M Y27632 to the above medium (4F group for short), and the control group was replaced by an equal volume of DMSO in the experimental group.
  • Hematopoietic stem cells (DMSO group for short) obtained by culturing the 4 factors of, Fresh group is CD34 + cells freshly isolated from cord blood that have not been cultured in vitro;
  • Figure 4 compares the effects of adding different factors to the medium on the expansion efficiency of hematopoietic stem cells.
  • J is JNK-IN-8
  • Y is Y27632
  • N is NAD
  • V is valproic acid
  • R Rapamycin
  • JY is the simultaneous addition of JNK-IN-8 and Y27632
  • RU is the simultaneous addition of Rapamycin and UM171
  • YR is Add Y27632 and Rapamycin at the same time
  • YU is to add Y27632 and UM171 at the same time
  • JRY is to add JNK-IN-8
  • YRU is to add Y27632, Rapamycin and UM171 at the same time
  • 4F is to add JNK-IN-8, Y27632, NAD and Rapamycin
  • JRU is to add JNK-IN-8, Rapamycin and UM171 at the same time.
  • the expanded cells were injected into NPG severe immunodeficiency mice through the tail vein. After 16 weeks of transplantation, flow cytometry was used to detect the reconstitution ratio of human-derived cells in the peripheral blood of NPG mice to determine the reconstitution function and expansion of hematopoietic stem cells after expansion. multiple.
  • the results are shown in Figures 5-9, where the reconstitution ratio of primary transplanted cells is 3%, the reconstitution ratio of the DMSO culture control group is 5%, and the reconstitution ratio of the 4-factor combination culture experimental group is 28%. This indicates that the use of JNK-IN-8, NAD, Rapamycin and Y27632 can effectively improve the in vivo reconstitution function and efficiency of hematopoietic stem cells.
  • the limiting dilution method was used to calculate the expansion factor of hematopoietic stem cells cultured with 4 factors. It can be seen from the left picture of Figure 10 that the expansion effect of 4 factors is obvious. After 16 weeks of in vivo reconstitution, the proportion of human-derived cells is compared with The control group expanded >10 times. The graph on the right shows the proportion of hematopoietic stem cells in cultured and expanded cells, that is, on average, there is 1 hematopoietic stem cell for every 902 cells. It can be seen from the figure that the proportion of hematopoietic stem cells in the cells expanded under 4F culture conditions is higher than that of the DMSO and Fresh groups.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Provided are a composition for amplifying a hematopoietic stem cell and a culture medium, kit, and pharmaceutical composition comprising the composition, the use thereof and a method for amplifying a hematopoietic stem cell. The composition is composed of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632.

Description

用于扩增造血干细胞的组合物、培养基、方法和试剂盒Composition, culture medium, method and kit for expanding hematopoietic stem cells 技术领域Technical field
本发明涉及生物医药领域。具体地，本发明涉及用于扩增造血干细胞的组合物、培养基、方法和试剂盒。The invention relates to the field of biomedicine. Specifically, the present invention relates to compositions, media, methods, and kits for expanding hematopoietic stem cells.
背景技术Background technique
造血干细胞(hematopoietic stem cells，HSCs)是一类兼具多能性和自我更新能力的成体干细胞，在人体血液系统中，有超过10种成熟的细胞类型，其中，造血干细胞就是最具有代表性的细胞，除了能够分化为血液系统中所有的细胞类型之外，同时还保留了自我增殖的能力。Hematopoietic stem cells (HSCs) are a type of adult stem cells that have both pluripotency and self-renewal ability. There are more than 10 mature cell types in the human blood system. Among them, hematopoietic stem cells are the most representative In addition to being able to differentiate into all cell types in the blood system, cells also retain the ability to proliferate themselves.
据报道，单颗真正的造血干细胞能够重新建立起整个血液系统，其临床应用前景非常广阔，受到科学界和医学界的高度关注。According to reports, a single true hematopoietic stem cell can re-establish the entire blood system, and its clinical application prospects are very broad, and it is highly concerned by the scientific and medical circles.
造血干细胞主要存在于人体骨髓(bone marrow，BM)、动员的外周血(peripheral blood，PB)、脐带血(umbilical cord blood，UCB)等组织。研究过程当中，通常以细胞特殊的表面标记物及其组合来分离获得所需要的造血干细胞类型。2011年，John E.Dick已经能够从单细胞水平上分离获得造血干细胞，其分离纯度可以达到28％，即18颗细胞当中可以分离获得5颗具备长期造血重建能力的造血干细胞(Notta et al.,2011)。Hematopoietic stem cells mainly exist in human bone marrow (BM), mobilized peripheral blood (PB), umbilical cord blood (UCB) and other tissues. During the research process, the specific cell surface markers and their combinations are usually used to isolate and obtain the required types of hematopoietic stem cells. In 2011, John E. Dick was able to isolate hematopoietic stem cells at the single-cell level, with a purity of 28%, that is, 5 hematopoietic stem cells with long-term hematopoietic reconstitution ability can be isolated from 18 cells (Notta et al. , 2011).
随着干细胞科学技术的不断发展，人们对造血干细胞的认识不断加深，同时临床研究对造血干细胞的需求也与日俱增。造血干细胞是最早应用于临床治疗的干细胞类型，自从1968年首次应用于治疗重度免疫缺陷症之后，已被临床试验反复证明其有效性。美国西雅图华盛顿大学佛雷德-哈金森医学中心的Donnall Thomas医师因在造血干细胞移植治疗白血病和其他血液疾病方面作出杰出贡献而获得了1990年的诺贝尔生理学奖(Park et al.,2015)。With the continuous development of stem cell science and technology, people's understanding of hematopoietic stem cells continues to deepen, and the demand for hematopoietic stem cells in clinical research is also increasing day by day. Hematopoietic stem cells are the earliest type of stem cells used in clinical treatment. Since they were first used in the treatment of severe immunodeficiency in 1968, their effectiveness has been repeatedly proven by clinical trials. Dr. Donnall Thomas of the Fred Hutchinson Medical Center of the University of Washington in Seattle, USA, won the Nobel Prize in Physiology in 1990 (Park et al., 2015) for his outstanding contributions to the treatment of leukemia and other blood diseases in hematopoietic stem cell transplantation.
当前，临床上主要应用造血干细胞治疗白血病和淋巴瘤等恶性血液疾病、相关代谢病、自身免疫性疾病和获得性免疫缺陷症等。虽然造血干细胞具备极大的医学应用前景，但是由于造血干细胞数量有限，而且很难在体外获得，导致临床上有40％左右的病人由于HLA配型失败，只能采用保守治疗的方法。Currently, hematopoietic stem cells are mainly used clinically to treat malignant hematological diseases such as leukemia and lymphoma, related metabolic diseases, autoimmune diseases, and acquired immunodeficiency. Although hematopoietic stem cells have great prospects for medical applications, because the number of hematopoietic stem cells is limited and it is difficult to obtain them in vitro, about 40% of patients in the clinic can only adopt conservative treatment methods due to the failure of HLA matching.
脐带血是一种重要的HSCs来源，因为其具备采集方便、无创伤、免疫原性低等诸多优势，近些年被临床高度关注，每年有近4万份脐带血应用于临床研究和治疗。但由于单份脐带血中所含造血干细胞数量有限，不足以提供一名成年人所需细胞数量，大概有50％ 的病人无法接受移植治疗，这极大地限制了脐带血的临床应用。Umbilical cord blood is an important source of HSCs. Because of its advantages such as convenient collection, non-invasiveness, and low immunogenicity, it has received high clinical attention in recent years. Nearly 40,000 cord blood is used in clinical research and treatment every year. However, due to the limited number of hematopoietic stem cells contained in a single cord blood, which is not enough to provide the number of cells required by an adult, about 50% of patients cannot receive transplantation, which greatly limits the clinical application of cord blood.
因此，如何在体外有效获得足量的造血干细胞成为了一大科学难题，未来若能突破这个技术瓶颈，将会书写干细胞临床应用的新篇章。Therefore, how to effectively obtain sufficient amounts of hematopoietic stem cells in vitro has become a major scientific problem. If this technical bottleneck can be broken in the future, a new chapter in the clinical application of stem cells will be written.
当前，限制造血干细胞临床应用的最重要的因素是HSC数量不足。解决这个难题大致有三种策略：(1)通过iPSC或ESC直接分化获得具有功能的HSCs；(2)通过细胞重编程获得HSCs；(3)体外扩增已有的HSCs。由于前面两种方法涉及到的技术难度过大，而且对临床应用造成额外的风险，导致迟迟无法进行临床研究。体外扩增造血干细胞成为了解决这一难题的重要方法。Currently, the most important factor limiting the clinical application of blood stem cells is the insufficient number of HSCs. There are roughly three strategies to solve this problem: (1) Direct differentiation of iPSC or ESC to obtain functional HSCs; (2) Cell reprogramming to obtain HSCs; (3) In vitro expansion of existing HSCs. Due to the technical difficulty involved in the first two methods and additional risks to clinical applications, clinical research is delayed. Expansion of hematopoietic stem cells in vitro has become an important method to solve this problem.
体外扩增造血干细胞具有诸多优势，主要归纳为以下三个特点：(1)扩增的细胞同质性高，癌变风险低；(2)扩增HSC的操作时间相对较短，避免引入更多外源不利因素；(3)起始细胞是造血干/祖细胞，一般不存在致瘤风险，临床应用更加安全。Expansion of hematopoietic stem cells in vitro has many advantages, which are mainly summarized as the following three characteristics: (1) The expanded cells have high homogeneity and low risk of cancer; (2) The operation time for expansion of HSC is relatively short, avoiding the introduction of more Exogenous unfavorable factors; (3) The starting cells are hematopoietic stem/progenitor cells, which generally do not have the risk of tumorigenesis, and the clinical application is safer.
扩增HSCs的研究总结起来大致可以分为两类：(1)利用基因操作手段，比如引入外源转录因子或微小RNA，扩增HSCs；(2)采用化学小分子扩增HSCs。近些年，随着化学小分子技术的不断进步，小分子化合物已经广泛应用于干细胞研究领域。利用小分子药物扩增HSCs具有很多明显的优势：低毒性、易洗脱、临床使用更加安全。但是也存在明显的不足，其中最令人担忧的就是小分子化合物作用靶点不够明确，可能会存在安全隐患，例如小分子的脱靶效应，因此临床上仍然缺乏安全有效且靶点明确的扩增造血干细胞的化学小分子组合方案。The research on the amplification of HSCs can be summarized into two categories: (1) the use of genetic manipulation methods, such as the introduction of exogenous transcription factors or microRNA, to amplify HSCs; (2) the use of chemical small molecules to amplify HSCs. In recent years, with the continuous advancement of chemical small molecule technology, small molecule compounds have been widely used in the field of stem cell research. The use of small molecule drugs to amplify HSCs has many obvious advantages: low toxicity, easy elution, and safer clinical use. However, there are also obvious shortcomings. The most worrying one is that the target of small molecule compounds is not clear enough, and there may be safety risks, such as off-target effects of small molecules. Therefore, there is still a lack of safe, effective and targeted amplification in clinical practice. Chemical small molecule combination scheme for hematopoietic stem cells.
综上，目前利用化学小分子化合物组合以体外扩增造血干细胞的技术方案仍有待研究。In summary, the current technical solution for the use of chemical small molecule compound combinations to expand hematopoietic stem cells in vitro remains to be studied.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决现有技术中存在的技术问题。为此，本发明通过高通量筛选平台，找到一些化学小分子化合物组合，其不仅可以使造血干细胞在体外高效扩增，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention finds some chemical small molecule compound combinations through a high-throughput screening platform, which can not only enable efficient expansion of hematopoietic stem cells in vitro, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, but also improve hematopoietic stem cells The reconstruction function and reconstruction efficiency of the body have important scientific research, clinical research and application value.
在本发明的一个方面，本发明提出了一种用于扩增造血干细胞的组合物。根据本发明的实施例，所述组合物由JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632组成。In one aspect of the present invention, the present invention provides a composition for expanding hematopoietic stem cells. According to an embodiment of the present invention, the composition consists of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632.
JNK-IN-8为JNK信号通路抑制剂，通过抑制c-Jun磷酸化和基因转录来实现调控JNK信号通路的作用；烟酰胺腺嘌呤二核苷酸(NAD)，可以调控基础代谢，是细胞生长的基础反应底物；雷帕霉素(Rapamycin)为mTOR信号通路抑制剂，可用于抗衰老和抑制肿瘤生长；Y27632为ROCK1抑制剂，可用于抗衰老，对维持干细胞的活力有明显提升作用。JNK-IN-8 is an inhibitor of the JNK signaling pathway, which regulates the JNK signaling pathway by inhibiting c-Jun phosphorylation and gene transcription; nicotinamide adenine dinucleotide (NAD), which can regulate basic metabolism, is a cell The basic reaction substrate for growth; Rapamycin is an mTOR signal pathway inhibitor, which can be used for anti-aging and inhibiting tumor growth; Y27632 is a ROCK1 inhibitor, which can be used for anti-aging, and has a significant effect on maintaining the vitality of stem cells. .
发明人采用高通量筛选以便获得高效扩增造血干细胞的化合物，发现JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632这4个小分子化合物之间具有相互配合、增效的作用，可以高效扩增造血干细胞，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。The inventors used high-throughput screening in order to obtain compounds that efficiently expanded hematopoietic stem cells and found that the four small molecules of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 have mutual coordination. , Synergistic effect, can efficiently expand hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve the in vivo reconstruction function and efficiency of hematopoietic stem cells, which has significant scientific research, clinical research and application value .
Figure PCTCN2020080030-appb-000001
Figure PCTCN2020080030-appb-000001
在本发明的另一方面，本发明提出了一种用于扩增造血干细胞的培养基。根据本发明的实施例，所述培养基包括：基础培养基；以及前面所述的用于扩增造血干细胞的组合物。由此，利用根据本发明实施例的培养基可以高效扩增造血干细胞，有效维持造血干细胞的蛋白表型和基因表达谱，提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。In another aspect of the present invention, the present invention provides a medium for expanding hematopoietic stem cells. According to an embodiment of the present invention, the medium includes: a basal medium; and the aforementioned composition for expanding hematopoietic stem cells. Therefore, the use of the medium according to the embodiments of the present invention can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and improve the in vivo reconstruction function and efficiency of hematopoietic stem cells. It has important scientific research and clinical research. Research and application value.
根据本发明的实施例，上述培养基还可以具有下列附加技术特征：According to an embodiment of the present invention, the above-mentioned culture medium may also have the following additional technical features:
根据本发明的实施例，所述JNK-IN-8的浓度为1～5μM，所述烟酰胺腺嘌呤二核苷酸的浓度为0.5～5μM，所述雷帕霉素的浓度为1～20nM，所述Y27632的浓度为1～20μM。根据本发明的具体实施例，JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632的浓度分别为2μM、1μM、10nM、10μM或3.5μM、0.8μM、15nM、7μM或1.5μM、3μM、8nM、 15μM。根据本发明的具体实施例，所述JNK-IN-8的浓度为1～3μM，所述烟酰胺腺嘌呤二核苷酸的浓度为0.5～2μM，所述雷帕霉素的浓度为6～15nM，所述Y27632的浓度为5～15μM。发明人经过大量实验获得上述较优浓度，由此，均可以进一步提高扩增效率。According to an embodiment of the present invention, the concentration of the JNK-IN-8 is 1˜5 μM, the concentration of the nicotinamide adenine dinucleotide is 0.5˜5 μM, and the concentration of the rapamycin is 1˜20 nM The concentration of Y27632 is 1-20 μM. According to specific embodiments of the present invention, the concentrations of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin, and Y27632 are 2 μM, 1 μM, 10 nM, 10 μM or 3.5 μM, 0.8 μM, 15 nM, 7 μM, respectively Or 1.5μM, 3μM, 8nM, 15μM. According to a specific embodiment of the present invention, the concentration of the JNK-IN-8 is 1 ~ 3 μM, the concentration of the nicotinamide adenine dinucleotide is 0.5 ~ 2 μM, and the concentration of the rapamycin is 6 ~ 15nM, the concentration of Y27632 is 5-15μM. The inventors obtained the above-mentioned optimal concentration through a large number of experiments, and thus, the amplification efficiency can be further improved.
根据本发明的实施例，所述基础培养基选自含有Flt3配体、血小板生成素(TPO)、干细胞生长因子(SCF)以及低密度脂蛋白(LDL)的StemSpan SFEM培养基。由此，向StemSpan SFEM培养基中加入上述因子，可以进一步提高扩增效率。According to an embodiment of the present invention, the basic medium is selected from StemSpan SFEM medium containing Flt3 ligand, thrombopoietin (TPO), stem cell growth factor (SCF) and low density lipoprotein (LDL). Therefore, adding the above factors to StemSpan SFEM medium can further improve the amplification efficiency.
根据本发明的实施例，所述Flt3配体的浓度为60～100ng/mL，所述血小板生成素的浓度为20～50ng/mL，所述干细胞因子的浓度为60～100ng/mL，所述低密度脂蛋白的浓度为5～20μg/mL。根据本发明的具体实施例，Flt3配体、血小板生成素、干细胞因子和低密度脂蛋白的浓度分别为100ng/ml、50ng/ml、100ng/ml、10μg/ml或者80ng/ml、30ng/ml、80ng/ml、15μg/ml或者90ng/ml、40ng/ml、90ng/ml、6μg/ml。根据本发明的具体实施例，所述Flt3配体的浓度为80～100ng/mL，所述血小板生成素的浓度为40～50ng/mL，所述干细胞因子的浓度为80～100ng/mL，所述低密度脂蛋白的浓度为5～15μg/mL。发明人经过大量实验优化各组分的浓度，由此，均可以进一步提高扩增效率。According to an embodiment of the present invention, the concentration of the Flt3 ligand is 60-100 ng/mL, the concentration of the thrombopoietin is 20-50 ng/mL, and the concentration of the stem cell factor is 60-100 ng/mL. The concentration of low-density lipoprotein is 5-20μg/mL. According to specific embodiments of the present invention, the concentrations of Flt3 ligand, thrombopoietin, stem cell factor and low-density lipoprotein are respectively 100ng/ml, 50ng/ml, 100ng/ml, 10μg/ml or 80ng/ml, 30ng/ml , 80ng/ml, 15μg/ml or 90ng/ml, 40ng/ml, 90ng/ml, 6μg/ml. According to a specific embodiment of the present invention, the concentration of the Flt3 ligand is 80-100 ng/mL, the concentration of thrombopoietin is 40-50 ng/mL, and the concentration of the stem cell factor is 80-100 ng/mL. The concentration of the low-density lipoprotein is 5-15 μg/mL. The inventor optimized the concentration of each component through a large number of experiments, thus, the amplification efficiency can be further improved.
根据本发明的实施例，本发明提出了JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632在制备组合物或培养基中的用途，所述组合物或培养基用于扩增造血干细胞。According to an embodiment of the present invention, the present invention proposes the use of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 in the preparation of a composition or culture medium. Used to expand hematopoietic stem cells.
在本发明的又一方面，本发明提出了一种扩增造血干细胞的方法。根据本发明的实施例，所述方法包括：采用前面所述的组合物抑制造血干细胞的下列代谢通路：JNK信号通路、mTOR信号通路和ROCK信号通路。发明人分别对能够抑制JNK信号通路、mTOR信号通路和ROCK信号通路的众多小分子物质进行高通量筛选分析，发现JNK-IN-8(JNK信号通路抑制剂)、烟酰胺腺嘌呤二核苷酸、雷帕霉素(mTOR信号通路抑制剂)和Y27632(ROCK信号通路抑制剂)四因子联合使用可以高效扩增造血干细胞，，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，操作简便，具有广泛的应用前景。In another aspect of the present invention, the present invention provides a method for expanding hematopoietic stem cells. According to an embodiment of the present invention, the method includes: using the aforementioned composition to inhibit the following metabolic pathways of hematopoietic stem cells: JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway. The inventors performed high-throughput screening and analysis of many small molecules that can inhibit the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway, and found that JNK-IN-8 (JNK signaling pathway inhibitor), nicotinamide adenine dinucleoside The combination of acid, rapamycin (mTOR signaling pathway inhibitor) and Y27632 (ROCK signaling pathway inhibitor) four factors can efficiently expand hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve The in vivo reconstruction function and reconstruction efficiency of hematopoietic stem cells are easy to operate and have a wide range of application prospects.
根据本发明的实施例，所述方法包括：将CD34 +细胞培养于前面所述用于扩增造血干细胞的培养基中。由此，可以进一步提高扩增效率，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，操作简便，具有广泛的应用前景。 According to an embodiment of the present invention, the method includes: culturing CD34+ cells in the aforementioned medium for expanding hematopoietic stem cells. As a result, amplification efficiency can be further improved, the protein phenotype and gene expression profile of hematopoietic stem cells can be effectively maintained, and the in vivo reconstruction function and reconstruction efficiency of hematopoietic stem cells can be improved. The operation is simple and has broad application prospects.
根据本发明的实施例，所述CD34 +细胞来源于骨髓、肝脏、脾脏、外周血或脐带血。 According to an embodiment of the present invention, the CD34 + cells are derived from bone marrow, liver, spleen, peripheral blood or cord blood.
在本发明的又一方面，本发明提出了一种用于扩增造血干细胞的试剂盒。根据本发明的实施例，所述试剂盒包括：前面所述的用于扩增造血干细胞的组合物或前面所述的培养 基。由此，利用根据本发明实施例的试剂盒可以高效扩增造血干细胞，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。In another aspect of the present invention, the present invention provides a kit for expanding hematopoietic stem cells. According to an embodiment of the present invention, the kit includes: the aforementioned composition for expanding hematopoietic stem cells or the aforementioned culture medium. Thus, the use of the kit according to the embodiments of the present invention can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve the in vivo reconstruction function and efficiency of hematopoietic stem cells, which has important scientific research , Clinical research and application value.
在本发明的又一方面，本发明提出了前面所述的组合物在制备抑制剂中的用途。根据本发明的实施例，所述抑制剂用于扩增造血干细胞，抑制造血干细胞的下列代谢通路：JNK信号通路、mTOR信号通路和ROCK信号通路。如前所述，JNK-IN-8、NAD、雷帕霉素和Y27632可以有效地抑制JNK信号通路、mTOR信号通路和/或ROCK信号通路，从而高效扩增造血干细胞，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。In another aspect of the present invention, the present invention proposes the use of the aforementioned composition in the preparation of inhibitors. According to an embodiment of the present invention, the inhibitor is used to expand hematopoietic stem cells and inhibit the following metabolic pathways of hematopoietic stem cells: JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway. As mentioned earlier, JNK-IN-8, NAD, rapamycin and Y27632 can effectively inhibit the JNK signaling pathway, mTOR signaling pathway and/or ROCK signaling pathway, thereby efficiently expanding hematopoietic stem cells and effectively maintaining the protein of hematopoietic stem cells Phenotype and gene expression profile, and can improve the in vivo reconstruction function and reconstruction efficiency of hematopoietic stem cells, which has significant scientific research, clinical research and application value.
在本发明的又一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的组合物或者前面所述的培养基。如前所述，根据本发明实施例的药物组合物可以高效扩增造血干细胞，直接或者间接将该组合物或者该培养基作为药物组合物给药至机体内(动物或细胞)，起到扩增造血干细胞的目的，也可以将利用前面所述扩增造血干细胞的方法所获得的造血干细胞施加于机体内，其具有较好的体内重建功能和重建效率，可以广泛地用于血液系统疾病以及自身免疫疾病的治疗中，具有重大的科学研究、临床研究和应用价值。In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the aforementioned composition or the aforementioned culture medium. As mentioned above, the pharmaceutical composition according to the embodiments of the present invention can efficiently expand hematopoietic stem cells, and directly or indirectly administer the composition or the medium as a pharmaceutical composition into the body (animal or cell) to expand For the purpose of increasing hematopoietic stem cells, hematopoietic stem cells obtained by the method of expanding hematopoietic stem cells described above can also be applied to the body. They have good reconstitution function and efficiency in vivo, and can be widely used in blood system diseases and In the treatment of autoimmune diseases, it has important scientific research, clinical research and application value.
根据本发明的实施例，本发明的药物组合物可与常规治疗方法和/或疗法相结合使用，或者可与常规治疗方法和/或疗法分开使用。当本发明的药物组合物在采用与其它药物的联合疗法中给药时，它们可序贯地或同时地给予个体。或者，本发明的药物组合物还可以包含药学上可接受的载体或药学上可接受的赋形剂以及本领域已知的其它治疗药或预防药的组合。According to the embodiments of the present invention, the pharmaceutical composition of the present invention may be used in combination with conventional treatment methods and/or therapies, or may be used separately from conventional treatment methods and/or therapies. When the pharmaceutical compositions of the present invention are administered in combination therapy with other drugs, they can be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention may also include a combination of a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient and other therapeutic or preventive drugs known in the art.
在本文中所使用的术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的间充质干细胞可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。The term "administration" as used herein refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The mesenchymal stem cell of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the present invention is not limited to these exemplified modes of administration.
在本发明的又一方面，本发明提出了一种筛选药物的方法。根据本发明的实施例，所述方法包括：将候选药物与CD34 +细胞进行培养；测定培养前后细胞内JNK信号通路、mTOR信号通路和ROCK信号通路是否被抑制和/或培养后细胞是否扩增和/或培养前后细胞内代表造血干细胞功能的表面蛋白是否一致；当培养后所述JNK信号通路、mTOR信号通路和ROCK信号通路被抑制和/或培养后细胞扩增和/或培养前后细胞内代表造血干细胞功能的表面蛋白一致，是所述候选药物为目标药物的指示，所述目标药物为前面所述的组 合物或者前面所述的培养基。如前所述，根据本发明实施例的组合物能够抑制上述三条代谢通路，从而起到扩增造血干细胞的效果。同时，在扩增后可以有效维持造血干细胞的蛋白表型和基因表达谱。因此，通过采用根据本发明实施例的筛选药物的方法，可以有效地筛选出本发明的组合物或者含有其的培养基或者药物，具有重大的科学研究、临床研究和应用价值。 In another aspect of the present invention, the present invention provides a method for screening drugs. According to an embodiment of the present invention, the method includes: culturing the candidate drug with CD34+ cells; determining whether the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway in the cells before and after the culture are inhibited and/or whether the cells expand after the culture And/or whether the surface proteins representing the functions of hematopoietic stem cells in the cells before and after culture are consistent; when the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway are inhibited after culturing and/or cell expansion after culturing and/or in cells before and after culturing The surface proteins representing the functions of hematopoietic stem cells are consistent, which is an indication that the candidate drug is a target drug, and the target drug is the aforementioned composition or the aforementioned culture medium. As mentioned above, the composition according to the embodiment of the present invention can inhibit the above three metabolic pathways, thereby achieving the effect of expanding hematopoietic stem cells. At the same time, it can effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells after amplification. Therefore, by adopting the method for screening drugs according to the embodiments of the present invention, the composition of the present invention or the medium or drugs containing it can be effectively screened, which has great scientific research, clinical research and application value.
需要说明的是，“培养前后细胞内代表造血干细胞功能的表面蛋白是否一致”里面所描述的“一致”应作广义理解，由于代表造血干细胞功能的表面蛋白较多，所有代表造血干细胞功能的表面蛋白在培养前后有至少80％、85％、90％、95％、99％或100％相同均可认定为“一致”。It should be noted that the “consistent” described in “whether the surface proteins representing the functions of hematopoietic stem cells in cells before and after culture are consistent” should be understood in a broad sense. Since there are more surface proteins representing the functions of hematopoietic stem cells, all surfaces representing the functions of hematopoietic stem cells If the protein is at least 80%, 85%, 90%, 95%, 99% or 100% identical before and after the culture, it can be regarded as "identical".
在本发明的又一方面，本发明提出了JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632在扩增造血干细胞中的用途。如前所述，JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632可以高效扩增造血干细胞，有效维持造血干细胞的蛋白表型和基因表达谱，而且能够提高造血干细胞的体内重建功能和重建效率，具有重大的科学研究、临床研究和应用价值。In another aspect of the present invention, the present invention proposes the use of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 in the expansion of hematopoietic stem cells. As mentioned earlier, JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 can efficiently amplify hematopoietic stem cells, effectively maintain the protein phenotype and gene expression profile of hematopoietic stem cells, and can improve hematopoiesis The in vivo reconstruction function and reconstruction efficiency of stem cells have important scientific research, clinical research and application value.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。The additional aspects and advantages of the present invention will be partly given in the following description, and partly will become obvious from the following description, or be understood through the practice of the present invention.
附图说明Description of the drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，图中4F为包含JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632四个化学小分子的培养条件，其中：The above and/or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings. In the figure, 4F contains JNK-IN-8, nicotinamide adenine dinucleotide Culture conditions of four small chemical molecules, rapamycin and Y27632, among which:
图1显示了根据本发明实施例的扩增造血干细胞的工艺流程示意图；Fig. 1 shows a schematic diagram of a process flow for amplifying hematopoietic stem cells according to an embodiment of the present invention;
图2显示了根据本发明实施例的CD34 +细胞培养第7天后的流式细胞分析图； Figure 2 shows a flow cytometric analysis diagram of CD34+ cells after 7 days of culture according to an embodiment of the present invention;
图3显示了根据本发明实施例的CD34 +细胞培养第28天后的流式细胞分析图； Figure 3 shows a flow cytometric analysis diagram of CD34+ cells after 28 days of culture according to an embodiment of the present invention;
图4显示了根据本发明实施例的造血干细胞在含有不同小分子化合物的培养基中培养后的流式细胞分析图(对照组为DMSO培养条件，**P<0.01,***P<0.001)；Figure 4 shows a flow cytometric analysis diagram of hematopoietic stem cells cultured in a medium containing different small molecule compounds according to an embodiment of the present invention (the control group is DMSO culture conditions, **P<0.01, ***P<0.001 );
图5～图9显示了根据本发明实施例的造血干细胞重建后造血干细胞流式细胞分析示意图，其中，图5为移植4周，图6为移植8周，图7为移植12周，图8和9为移植16周；Figures 5-9 show schematic diagrams of flow cytometry analysis of hematopoietic stem cells after hematopoietic stem cell reconstruction according to an embodiment of the present invention, wherein Figure 5 is 4 weeks after transplantation, Figure 6 is 8 weeks after transplantation, Figure 7 is 12 weeks after transplantation, and Figure 8 And 9 is 16 weeks after transplantation;
图10显示了根据本发明实施例的造血干细胞重建16周后造血干细胞扩增倍数分析示意图。Fig. 10 shows a schematic diagram of the expansion factor analysis of hematopoietic stem cells 16 weeks after hematopoietic stem cell reconstruction according to an embodiment of the present invention.
具体实施方式Detailed ways
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, it shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased on the market.
实施例1Example 1
在该实施例中，按照下列方法扩增造血干细胞：In this example, hematopoietic stem cells were expanded according to the following method:
操作流程参见图1，具体步骤如下：Refer to Figure 1 for the operation process, and the specific steps are as follows:
1、脐带血分离CD34 +细胞 1. Isolation of CD34 + cells from cord blood
1)脐带血的采集1) Collection of cord blood
在手术室无菌环境中采集胎儿脐带中血液，保存于加有抗凝剂的血袋，暂时存放于4℃微环境中，于24小时内送达实验室使用。The blood from the fetal umbilical cord is collected in a sterile environment in the operating room, stored in a blood bag with anticoagulant, temporarily stored in a 4°C microenvironment, and delivered to the laboratory for use within 24 hours.
2)分离脐带血中的单核细胞2) Separate monocytes from cord blood
a)在无菌实验台中，将脐带血转移到提前准备好的无菌培养瓶里，按照血液：磷酸盐缓冲溶液＝1.2的体积加入磷酸盐缓冲液，混匀；a) In a sterile laboratory platform, transfer the cord blood to a sterile culture bottle prepared in advance, add phosphate buffer solution according to the volume of blood: phosphate buffer solution = 1.2, and mix;
b)将稀释的脐带血贴壁缓慢加入盛有15ml人淋巴分离液的50ml离心管中，注意缓慢加入，保持两液面界面清晰，不能打破血液和淋巴分离液之间的液面平衡；b) Slowly add the diluted umbilical cord blood adherent to the wall into a 50ml centrifuge tube containing 15ml of human lymphatic separation fluid. Pay attention to adding slowly to keep the interface between the two liquid levels clear and not to break the level balance between blood and lymphatic separation fluid;
c)在室温下，1500转/分钟离心20分钟；c) Centrifuge at 1500 rpm for 20 minutes at room temperature;
d)离心结束后，液面分为三层，最上层是血浆/组织匀浆层，最下面是红细胞，中间层是分离液，在血浆层与分离液层之间是一层薄而致密的白膜，即单核细胞层(包括了淋巴细胞和单核细胞)。小心吸取白膜层细胞到另一个50ml离心管中；d) After centrifugation, the liquid level is divided into three layers, the uppermost layer is the plasma/tissue homogenate layer, the lowermost layer is the red blood cells, the middle layer is the separation liquid, and there is a thin and dense layer between the plasma layer and the separation liquid layer. The albuginea is the layer of mononuclear cells (including lymphocytes and monocytes). Carefully pipette the albuginea cells into another 50ml centrifuge tube;
e)用PBS等比稀释到50ml体积，颠倒混匀；e) Dilute with PBS to a volume of 50ml in an equal proportion, and mix upside down;
f)在室温下，以1600转/分钟离心10分钟；f) Centrifuge at 1600 rpm for 10 minutes at room temperature;
g)弃上清，用PBS重悬备用。g) Discard the supernatant and resuspend in PBS for use.
3)磁珠分选法分离CD34 +细胞 3) Separate CD34 + cells by magnetic bead sorting
a)按照一定比例，将人CD34磁珠与脐带血中分离的单核细胞混合，吹匀，放到4℃冰箱中静置30分钟，同时将磁珠分选所需要的设备放到超净台中，用紫外光照射除菌；a) According to a certain ratio, mix human CD34 magnetic beads with monocytes isolated from cord blood, blow them well, and put them in a refrigerator at 4°C for 30 minutes. At the same time, put the equipment required for magnetic bead sorting in the ultra-clean Taichung, sterilized with ultraviolet light;
b)加入10ml PBS混匀，1600转/分钟离心5分钟；b) Add 10ml PBS and mix well, centrifuge at 1600 rpm for 5 minutes;
c)弃上清，用含有0.5％BSA的PBS重悬，准备过吸附柱；c) Discard the supernatant, resuspend it in PBS containing 0.5% BSA, and prepare for the adsorption column;
d)用含有0.5％BSA的PBS润洗吸附柱，然后加入单核细胞悬液，等待其完全通过吸附柱；d) Rinse the adsorption column with PBS containing 0.5% BSA, then add the mononuclear cell suspension, and wait for it to pass through the adsorption column completely;
e)用1ml含有0.5％BSA的PBS清洗吸附柱，重复3遍；e) Wash the adsorption column with 1ml of PBS containing 0.5% BSA, repeat 3 times;
f)将吸附柱转移到15ml离心管中，再往吸附柱滤膜上加入1ml含有0.5％BSA的PBS，将吸附在滤膜上的携带有CD34磁珠的细胞冲洗到离心管中；f) Transfer the adsorption column to a 15ml centrifuge tube, and then add 1ml of PBS containing 0.5% BSA to the adsorption column filter membrane, and wash the cells with CD34 magnetic beads adsorbed on the filter membrane into the centrifuge tube;
g)离心，弃上清，加入培养基重悬CD34 +细胞。 g) Centrifuge, discard the supernatant, add medium to resuspend the CD34 + cells.
4)流式分析所得单核细胞中CD34 +所占比例 4) The proportion of CD34+ in monocytes obtained by flow cytometry
a)将所得携带有CD34磁珠的细胞取出一小部分到1.5ml离心管中；a) Take out a small part of the cells carrying CD34 magnetic beads into a 1.5ml centrifuge tube;
b)加入相应的表面蛋白抗体，静置于4℃冰箱中；b) Add the corresponding surface protein antibody and place it in a refrigerator at 4°C;
c)30分钟后取出，加入1ml PBS；c) Take it out after 30 minutes and add 1ml PBS;
d)1600转/分钟，离心3分钟；d) 1600 rpm, centrifugation for 3 minutes;
e)弃上清，用200μl预冷的PBS重悬，即可用流式细胞分析仪检测分析所得细胞的表型。e) Discard the supernatant, resuspend it in 200 μl of pre-cooled PBS, and then use a flow cytometer to detect and analyze the phenotype of the obtained cells.
2、CD34 +细胞培养方法 2. CD34 + cell culture method
1)用含有SCF(100ng/ml)、Flt-3L(100ng/ml)、TPO(50ng/ml)、LDL(10μg/ml)的StemSpan SFEM培养基(Stemcell品牌)重悬CD34 +细胞，加入6孔低贴附板中，控制细胞密度为1×10 6颗/ml以下，置于37℃培养箱中培养。 1) Resuspend CD34+ cells in StemSpan SFEM medium (Stemcell brand) containing SCF (100ng/ml), Flt-3L (100ng/ml), TPO (50ng/ml), LDL (10μg/ml), and add 6 In the well-attached plate, control the cell density below 1×10 6 cells/ml and place it in a 37°C incubator for culture.
其中，实验组为在上述培养基中加入2μM JNK-IN-8、1μM NAD、10nM Rapamycin、10μM Y27632后培养所得的造血干细胞(简称4F组)，对照组为用等体积的DMSO代替实验组中的4个因子后培养所得的造血干细胞(简称DMSO组)，Fresh组为未经过体外培养的从脐带血中新鲜分离的CD34 +细胞； Among them, the experimental group was hematopoietic stem cells cultured after adding 2μM JNK-IN-8, 1μM NAD, 10nM Rapamycin, and 10μM Y27632 to the above medium (4F group for short), and the control group was replaced by an equal volume of DMSO in the experimental group. Hematopoietic stem cells (DMSO group for short) obtained by culturing the 4 factors of, Fresh group is CD34 + cells freshly isolated from cord blood that have not been cultured in vitro;
2)将CD34 +细胞放到37℃，5％CO 2的细胞培养箱中培养； 2) Place the CD34 + cells in a 37°C, 5% CO 2 cell incubator for culture;
3)每2天，进行半量更换培养基，保证细胞密度为1×10 6颗/ml以下； 3) Every 2 days, replace the medium in half to ensure that the cell density is less than 1×10 6 cells/ml;
4)培养一段时间后，检测细胞表型变化，并计算细胞数量。4) After culturing for a period of time, detect the changes in cell phenotype and calculate the number of cells.
细胞培养第7天的流式检测图参见图2。可以看出，采用JNK-IN-8、NAD、Rapamycin和Y27632四个因子可以有效地扩增造血干细胞，获得的造血干细胞生存状态良好且代表造血干细胞功能的表面蛋白一直可以维持。相比于DMSO培养基，CD34 +CD45RA -细胞可以在4因子培养基中扩增高达19倍(图3)。 Refer to Figure 2 for the flow cytometry diagram on the 7th day of cell culture. It can be seen that the four factors of JNK-IN-8, NAD, Rapamycin and Y27632 can effectively amplify hematopoietic stem cells, and the hematopoietic stem cells obtained are in a good living condition and the surface protein representing the function of hematopoietic stem cells can always be maintained. Compared to DMSO medium, CD34 + CD45RA - cells can be expanded up to 19 times in 4-factor medium (Figure 3).
实施例2Example 2
在该实施例中，研究不同小分子化合物对于造血干细胞扩增的影响，具体培养方式按照实施例1进行，区别在于培养基中加入不同类型的小分子化合物。In this example, the effect of different small molecule compounds on the expansion of hematopoietic stem cells was studied. The specific culture method was carried out according to Example 1, with the difference that different types of small molecule compounds were added to the medium.
图4比较了培养基中加入不同因子对于造血干细胞扩增效率的影响。其中，J为 JNK-IN-8，Y为Y27632，N为NAD，V为丙戊酸，R为Rapamycin，JY为同时添加JNK-IN-8和Y27632，RU为同时添加Rapamycin和UM171，YR为同时添加Y27632和Rapamycin，YU为同时添加Y27632和UM171，JRY为同时添加JNK-IN-8、Rapamycin和Y27632，YRU为同时添加Y27632、Rapamycin和UM171，4F为同时添加JNK-IN-8、Y27632、NAD和Rapamycin，JRU为同时添加JNK-IN-8、Rapamycin和UM171。Figure 4 compares the effects of adding different factors to the medium on the expansion efficiency of hematopoietic stem cells. Among them, J is JNK-IN-8, Y is Y27632, N is NAD, V is valproic acid, R is Rapamycin, JY is the simultaneous addition of JNK-IN-8 and Y27632, RU is the simultaneous addition of Rapamycin and UM171, and YR is Add Y27632 and Rapamycin at the same time, YU is to add Y27632 and UM171 at the same time, JRY is to add JNK-IN-8, Rapamycin and Y27632 at the same time, YRU is to add Y27632, Rapamycin and UM171 at the same time, 4F is to add JNK-IN-8, Y27632, NAD and Rapamycin, JRU is to add JNK-IN-8, Rapamycin and UM171 at the same time.
结果可以看出，相比于单独添加JNK-IN-8、Y27632、NAD和Rapamycin或者是其他因子而言，同时添加JNK-IN-8、Y27632、NAD和Rapamycin这四个因子，CD34 +CD45RA -标记的造血干祖细胞的扩增效率最高。 The results can be seen that, as compared to adding a separate JNK-IN-8, Y27632, NAD and Rapamycin or other factors, the simultaneous addition of JNK-IN-8, Y27632, NAD and Rapamycin these four factors, CD34 + CD45RA - The labeled hematopoietic stem and progenitor cells have the highest expansion efficiency.
将扩增后细胞通过尾静脉注射到NPG重度免疫缺陷鼠体内，移植16周后，流式检测NPG小鼠外周血中人源细胞重建比例，以判定扩增后造血干细胞的重建功能和扩增倍数。结果如图5～9所示，其中，原代移植细胞的重建比例为3％，DMSO培养对照组的重建比例为5％，4因子组合培养实验组的重建比例为28％。由此，表明采用JNK-IN-8、NAD、Rapamycin和Y27632可以有效地提高造血干细胞的体内重建功能和重建效率。The expanded cells were injected into NPG severe immunodeficiency mice through the tail vein. After 16 weeks of transplantation, flow cytometry was used to detect the reconstitution ratio of human-derived cells in the peripheral blood of NPG mice to determine the reconstitution function and expansion of hematopoietic stem cells after expansion. multiple. The results are shown in Figures 5-9, where the reconstitution ratio of primary transplanted cells is 3%, the reconstitution ratio of the DMSO culture control group is 5%, and the reconstitution ratio of the 4-factor combination culture experimental group is 28%. This indicates that the use of JNK-IN-8, NAD, Rapamycin and Y27632 can effectively improve the in vivo reconstitution function and efficiency of hematopoietic stem cells.
采用极限稀释法计算经过4因子培养的造血干细胞的扩增倍数，由图10的左图可以看出，4因子的扩增效果明显，体内重建16周后，人源细胞嵌合比例相比于对照组扩增>10倍。右图表示培养扩增的细胞中造血干细胞的比例，也即平均每902颗细胞中，就有1颗造血干细胞。由图可以看出，4F培养条件扩增的细胞中造血干细胞的比例相比于DMSO和Fresh组都要高。The limiting dilution method was used to calculate the expansion factor of hematopoietic stem cells cultured with 4 factors. It can be seen from the left picture of Figure 10 that the expansion effect of 4 factors is obvious. After 16 weeks of in vivo reconstitution, the proportion of human-derived cells is compared with The control group expanded >10 times. The graph on the right shows the proportion of hematopoietic stem cells in cultured and expanded cells, that is, on average, there is 1 hematopoietic stem cell for every 902 cells. It can be seen from the figure that the proportion of hematopoietic stem cells in the cells expanded under 4F culture conditions is higher than that of the DMSO and Fresh groups.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (10)

  1. 一种用于扩增造血干细胞的组合物，其特征在于，由JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632组成。A composition for amplifying hematopoietic stem cells, which is characterized by being composed of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632.
  2. 一种用于扩增造血干细胞的培养基，其特征在于，包括：A medium for expanding hematopoietic stem cells, which is characterized in that it comprises:
    基础培养基；以及Basal medium; and
    权利要求1所述的组合物。The composition of claim 1.
  3. 根据权利要求2所述的培养基，其特征在于，所述JNK-IN-8的浓度为1～5μM，所述烟酰胺腺嘌呤二核苷酸的浓度为0.5～5μM，所述雷帕霉素的浓度为1～20nM，所述Y27632的浓度为1～20μM；The medium according to claim 2, wherein the concentration of the JNK-IN-8 is 1-5 μM, the concentration of the nicotinamide adenine dinucleotide is 0.5-5 μM, and the rapamycin The concentration of the element is 1-20 nM, and the concentration of the Y27632 is 1-20 μM;
    所述基础培养基选自含有Flt3配体、血小板生成素、干细胞生长因子以及低密度脂蛋白的StemSpan SFEM培养基；The basic medium is selected from StemSpan SFEM medium containing Flt3 ligand, thrombopoietin, stem cell growth factor and low-density lipoprotein;
    所述Flt3配体的浓度为60～100ng/mL，所述血小板生成素的浓度为20～50ng/mL，所述干细胞因子的浓度为60～100ng/mL，所述低密度脂蛋白的浓度为5～20μg/mL。The concentration of the Flt3 ligand is 60-100 ng/mL, the concentration of the thrombopoietin is 20-50 ng/mL, the concentration of the stem cell factor is 60-100 ng/mL, and the concentration of the low-density lipoprotein is 5～20μg/mL.
  4. 一种扩增造血干细胞的方法，其特征在于，包括：采用权利要求1所述的组合物抑制造血干细胞的下列代谢通路：JNK信号通路、mTOR信号通路和ROCK信号通路。A method for amplifying hematopoietic stem cells, characterized by comprising: using the composition of claim 1 to inhibit the following metabolic pathways of hematopoietic stem cells: JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway.
  5. 根据权利要求4所述的方法，其特征在于，所述方法包括：将CD34 +细胞培养于权利要求2或3所述的培养基中； The method according to claim 4, wherein the method comprises: culturing CD34 + cells in the medium according to claim 2 or 3;
    所述CD34 +细胞来源于骨髓、肝脏、脾脏、外周血或脐带血。 The CD34 + cells are derived from bone marrow, liver, spleen, peripheral blood or cord blood.
  6. 一种用于扩增造血干细胞的试剂盒，其特征在于，包括：权利要求1所述的组合物或权利要求2或3所述的培养基。A kit for expanding hematopoietic stem cells, which is characterized by comprising: the composition according to claim 1 or the medium according to claim 2 or 3.
  7. 权利要求1所述的组合物在制备抑制剂中的用途，其特征在于，所述抑制剂用于扩增造血干细胞，抑制造血干细胞的下列代谢通路：The use of the composition of claim 1 in the preparation of an inhibitor, wherein the inhibitor is used to expand hematopoietic stem cells and inhibit the following metabolic pathways of hematopoietic stem cells:
    JNK信号通路、mTOR信号通路和ROCK信号通路。JNK signal pathway, mTOR signal pathway and ROCK signal pathway.
  8. 一种药物组合物，其特征在于，包括：权利要求1所述的组合物或者权利要求2或3所述的培养基。A pharmaceutical composition, characterized by comprising: the composition according to claim 1 or the medium according to claim 2 or 3.
  9. 一种筛选药物的方法，其特征在于，包括：A method for screening drugs, characterized in that it comprises:
    将候选药物与CD34 +细胞进行培养； Cultivating candidate drugs with CD34 + cells;
    测定培养前后细胞内JNK信号通路、mTOR信号通路和ROCK信号通路是否被抑制和/或培养后细胞是否扩增和/或培养前后细胞内代表造血干细胞功能的表面蛋白是否一致；Determine whether the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway in the cells before and after culture are inhibited and/or whether the cells are expanded after culture and/or whether the surface proteins representing the functions of hematopoietic stem cells in the cells before and after culture are consistent;
    当培养后所述JNK信号通路、mTOR信号通路和ROCK信号通路被抑制和/或培养后细胞扩增和/或培养前后细胞内代表造血干细胞功能的表面蛋白一致，是所述候选药物为目 标药物的指示，When the JNK signaling pathway, mTOR signaling pathway, and ROCK signaling pathway are inhibited after culture and/or cell expansion after culture and/or the surface proteins representing the function of hematopoietic stem cells in the cells are the same before and after culture, the candidate drug is the target drug Instructions,
    所述目标药物为权利要求1所述的组合物或者权利要求2或3所述的培养基。The target drug is the composition of claim 1 or the culture medium of claim 2 or 3.
  10. JNK-IN-8、烟酰胺腺嘌呤二核苷酸、雷帕霉素和Y27632在扩增造血干细胞中的用途。The use of JNK-IN-8, nicotinamide adenine dinucleotide, rapamycin and Y27632 in the expansion of hematopoietic stem cells.
PCT/CN2020/080030 2019-11-05 2020-03-18 Composition, culture medium, method and kit for amplifying hematopoietic stem cell WO2021088294A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911068766.2 2019-11-05
CN201911068766.2A CN110564687B (en) 2019-11-05 2019-11-05 Compositions, media, methods and kits for expanding hematopoietic stem cells

Publications (1)

Publication Number Publication Date
WO2021088294A1 true WO2021088294A1 (en) 2021-05-14

Family

ID=68786082

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/080030 WO2021088294A1 (en) 2019-11-05 2020-03-18 Composition, culture medium, method and kit for amplifying hematopoietic stem cell

Country Status (2)

Country Link
CN (1) CN110564687B (en)
WO (1) WO2021088294A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110564686B (en) * 2019-11-05 2020-04-03 诺未科技（北京）有限公司 Composition for expanding hematopoietic stem cells, expansion method, pharmaceutical composition and use
CN110564687B (en) * 2019-11-05 2020-04-03 诺未科技（北京）有限公司 Compositions, media, methods and kits for expanding hematopoietic stem cells
CN112725275A (en) * 2021-01-09 2021-04-30 海南苏生生物科技有限公司 Signal pathway inhibitor and drug for up-regulating MLLT3 gene expression, and application of signal pathway inhibitor

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030821A (en) * 2010-01-12 2011-04-27 葛龙海 Human placental anti-aging active protein as well as separation method and application thereof
CN103814126A (en) * 2011-05-13 2014-05-21 国立大学法人东京大学 Polynucleated megakaryocytic cell and method for manufacturing platelets
CN108235708A (en) * 2017-12-29 2018-06-29 罗小霞 Cultivating system and its method, the candidate stem cell and hematopoietic progenitor cells of amplifying candidate stem cell and/or hematopoietic progenitor cells
CN108473949A (en) * 2015-12-04 2018-08-31 弗莱德哈钦森癌症研究中心 The purposes of candidate stem cell/progenitor cell populations of amplification
CN109370988A (en) * 2018-11-03 2019-02-22 章毅 Ex vivo expansion of stem cell cultivating system and its method
CN110564687A (en) * 2019-11-05 2019-12-13 诺未科技（北京）有限公司 Compositions, media, methods and kits for expanding hematopoietic stem cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102030821A (en) * 2010-01-12 2011-04-27 葛龙海 Human placental anti-aging active protein as well as separation method and application thereof
CN103814126A (en) * 2011-05-13 2014-05-21 国立大学法人东京大学 Polynucleated megakaryocytic cell and method for manufacturing platelets
CN108473949A (en) * 2015-12-04 2018-08-31 弗莱德哈钦森癌症研究中心 The purposes of candidate stem cell/progenitor cell populations of amplification
CN108235708A (en) * 2017-12-29 2018-06-29 罗小霞 Cultivating system and its method, the candidate stem cell and hematopoietic progenitor cells of amplifying candidate stem cell and/or hematopoietic progenitor cells
CN109370988A (en) * 2018-11-03 2019-02-22 章毅 Ex vivo expansion of stem cell cultivating system and its method
CN110564687A (en) * 2019-11-05 2019-12-13 诺未科技（北京）有限公司 Compositions, media, methods and kits for expanding hematopoietic stem cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAUTHAMAN KALAMEGAM, FONG CHUI-YEE, SUBRAMANIAN ARJUNAN, BISWAS ARIJIT, BONGSO ARIFF: "ROCK Inhibitor Y-27632 Increases Thaw-Survival Rates and Preserves Stemness and Differentiation Potential of Human Wharton’s Jelly Stem Cells After Cryopreservation", STEM CELL REVIEWS AND REPORTS, HUMANA PRESS INC., US, vol. 6, no. 4, 1 December 2010 (2010-12-01), US, pages 665 - 676, XP055810462, ISSN: 1550-8943, DOI: 10.1007/s12015-010-9184-8 *
XIE YINLIANG: "Regulation of Erythroid Differentiation from Human Pluripotent Stem Cells by TGF-β Signaling", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE AND HEALTH SCIENCES, 1 May 2012 (2012-05-01), XP055810461 *
XU GUOSHUN, MENG AI-MIN: "Research progress of regulative effects of mTOR signaling pathways on hematopoietic stem cells", ZHONGGUO YAOLIXUE TONGBAO - CHINESE PHARMACOLOGICAL BULLETIN, LINCHUANG YAOLI YANJIUSUO, HEFEI, CN, vol. 29, no. 9, 1 September 2013 (2013-09-01), CN, pages 1185 - 1188, XP055810459, ISSN: 1001-1978 *

Also Published As

Publication number Publication date
CN110564687B (en) 2020-04-03
CN110564687A (en) 2019-12-13

Similar Documents

Publication Publication Date Title
WO2021088293A1 (en) Composition for expanding hematopoietic stem cells, expansion method, pharmaceutical compositions and uses
WO2021088294A1 (en) Composition, culture medium, method and kit for amplifying hematopoietic stem cell
Li et al. Hematopoietic stem cell repopulating ability can be maintained in vitro by some primary endothelial cells
EP3590520B1 (en) Compositions for providing hematopoietic function without hla matching
CN111575237B (en) Special culture medium and culture method for breast cancer stentless organoid
JP2008518603A (en) Stem cell-derived platelets
US8278101B2 (en) Stem cell bioprocessing and cell expansion
DK2624847T3 (en) HUMAN LUNG STEM CELLS AND APPLICATIONS THEREOF
TW201130977A (en) Mesenchymal stem cells (MSCs) isolated from mobilized peripheral blood
Kurtzberg et al. Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases
WO2008056963A1 (en) Method for proliferating stem cells with leptin
Araki et al. NOTCH-mediated ex vivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia
CN110669732B (en) Use of compositions for reprogramming hematopoietic progenitor cells to hematopoietic stem cells
CN102559579A (en) Novel multi-cell three-dimensional co-culture system for in-vitro detection of newly born blood vessel and kit thereof
EP4012021A1 (en) Method for culturing primary cells of lung cancer solid tumor and primary tumor cells of lung cancer pleural effusion, and supporting reagent
Ghebes et al. Extracellular vesicles derived from adult and fetal bone marrow mesenchymal stromal cells differentially promote ex vivo expansion of hematopoietic stem and progenitor cells
CN113583947B (en) Mesenchymal stem cell and hematopoietic stem cell in vitro culture method and system
JP6348881B2 (en) Method for producing hematopoietic stem cells and progenitor cells
WO2020151033A1 (en) Use of memory lymphocyte population in liver cancer treatment
JP2022511607A (en) Colon Rectal Cancer Solid Tumor Primary Cell and Colon Rectal Adenocarcinoma Ascites Primary Tumor Cell Culture Method and Kit
CN112048474B (en) Method for enhancing hematopoietic stem cell transplantation capability
CN113073078B (en) Universal platelet preparation prepared from umbilical cord-derived mesenchymal stem cells and method
Ogawa et al. In vitro expansion of hematopoietic stem cells
US20090298045A1 (en) Method For Selectively Expanding, Selecting And Enriching Stem/Progenitor Cell Populations
Zeng et al. An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20885737

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20885737

Country of ref document: EP

Kind code of ref document: A1