WO2020057107A1 - 一种组合物及其用途 - Google Patents

一种组合物及其用途 Download PDF

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WO2020057107A1
WO2020057107A1 PCT/CN2019/081855 CN2019081855W WO2020057107A1 WO 2020057107 A1 WO2020057107 A1 WO 2020057107A1 CN 2019081855 W CN2019081855 W CN 2019081855W WO 2020057107 A1 WO2020057107 A1 WO 2020057107A1
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hematopoietic stem
cells
stem cells
hematopoietic
cell
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PCT/CN2019/081855
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English (en)
French (fr)
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孙忠杰
陈立功
刘德芳
肖雄
郭潇
严小娥
齐海龙
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诺未科技（北京）有限公司
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Priority claimed from CN201811084022.5A external-priority patent/CN109207426B/zh
Priority claimed from CN201811625923.0A external-priority patent/CN109593714A/zh
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
Priority to CN201980001857.6A priority Critical patent/CN110972481B/zh
Publication of WO2020057107A1 publication Critical patent/WO2020057107A1/zh

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    • 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
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere

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  • the invention relates to the technical field of hematopoietic stem progenitor cells, in particular to a composition and use thereof.
  • Hematopoietic stem cells are an extremely important type of stem cells in the adult body. Although they only account for less than one ten-thousandth of the human blood cells, they have a strong ability to self-renew and differentiate, and can rebuild the entire blood system and immune system of the body for a long time. Differentiation potential of blood cells and immune cells of various lineages. Therefore, hematopoietic stem cells are widely used in the clinical treatment of malignant blood diseases such as leukemia and lymphoma. Not only that, hematopoietic stem cell transplantation can also help treat metabolic diseases, innate immune deficiency, and diabetes. According to statistics, there are more than 40,000 hematopoietic stem cell transplants worldwide each year.
  • donors of hematopoietic stem cells are mainly derived from bone marrow and mobilized peripheral blood hematopoietic stem cells from donors who are autologous or matched with patients' HLA.
  • the transplantation technique is very effective, due to the strict matching requirements of HLA matching, about 70% of patients still cannot obtain a suitable donor and cannot be treated; even if they receive treatment, most patients will experience different degrees Torment of xenograft versus host disease (GVHD).
  • Umbilical cord blood hematopoietic stem cells have relatively low requirements for HLA matching and low immunogenicity. In addition, they are easy to obtain and rich in sources, and have gradually become a major source of hematopoietic stem cell transplantation donors.
  • Cell reprogramming refers to the process by which differentiated somatic cells differentiate to reverse their fate and return to a totipotent or pluripotent state under specific conditions, or transdifferentiate one type of differentiated cell into another cell type.
  • the transition from hematopoietic progenitor cells to hematopoietic stem cells is a type of cellular reprogramming.
  • Hematopoietic progenitor cells are a type of cell that has lower self-renewal ability and differentiation potential than hematopoietic stem cells. Although it expresses CD34 surface antigen, it does not express CD90 surface molecules specific to hematopoietic stem cells and is positive for CD45RA.
  • CD90 and CD45RA can be used to distinguish hematopoietic stem cells from hematopoietic progenitor cells.
  • Hematopoietic progenitor cells have a short-term (less than one month) ability to be transplanted in vivo and can differentiate into a variety of blood cells such as red blood cells, lymphocytes, and myeloid cells. Because it does not have the ability to rebuild the recipient's blood system for long-term transplantation, it is often excluded from the operation of blood cell transplantation to treat hematological malignancies. However, the content of hematopoietic progenitor cells is much higher than that of hematopoietic stem cells (0.03% vs 0.0001%). If hematopoietic progenitor cells can be reprogrammed into hematopoietic stem cells, the donor source of hematopoietic stem cells will be greatly expanded.
  • hematopoietic stem cell culture system can appropriately expand hematopoietic stem cells by activating Notch signals.
  • PTN secreted by bone marrow endothelial stromal cells can also slightly expand hematopoietic stem cells.
  • Hematopoietic stem cells are under hypoxic conditions under physiological conditions, and oxygen stress generated in vitro can damage hematopoietic stem cells' self-renewal and transplantation functions by increasing ROS levels; it has been found that the addition of antioxidants and the inhibition of mTOR can offset these damages.
  • the above techniques have not been able to significantly expand cord blood hematopoietic stem cells.
  • Apparent modification plays an important role in regulating cell fate.
  • the methylation and acetylation of loci, and the modification of various groups of histones directly affect the degree of openness of genes in adjacent regions and the difficulty of binding transcription factors, thereby regulating the expression of genes and completing the regulation of cell status and destiny. Therefore, apparent modification affects cell function at a higher level.
  • Many epigenetic modifications have been developed to alter cell fate. In recent years, studies have found that the apparent modification can also significantly promote the expansion of artificial blood stem cells in vitro.
  • the small molecule compound MS275 has also attracted great attention in the field of cell reprogramming, because it can promote cell fate change and is used as a catalyst for IPS preparation.
  • the small molecule compound MS275 also known as SNDX-275 or Entinostat, has the molecular formula C 21 H 20 N 4 O 3 and CAS number 209783-80-2. It is a specific inhibitor of type I histone deacetylases HDAC1 and HDAC3. Entinostat is often used in the medical field to refer to MS275.
  • the drug has entered phase I and phase II clinical trials in leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, breast cancer, ovarian cancer, lung cancer, and kidney cancer .
  • MS275 can promote the production of human induced pluripotent stem cells (iPSC), and the maintenance and expansion effects of MS275 on hematopoietic stem cells and the reprogramming of hematopoietic progenitor cells to hematopoietic stem cells have not been reported.
  • iPSC human induced pluripotent stem cells
  • the present invention provides a composition and use thereof in hematopoietic stem progenitor cells.
  • the research of the present invention shows that the small molecule compound MS275 can promote the transformation of hematopoietic progenitor cells to hematopoietic stem cells, and can significantly increase the total number of cells obtained by in vitro expansion of hematopoietic stem cells.
  • the invention provides the application of MS275 in promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells.
  • the invention provides the application of MS275 in the preparation of hematopoietic stem cells.
  • the invention provides the application of MS275 and hematopoietic progenitor cells in the preparation of hematopoietic stem cells.
  • the invention also provides a composition consisting of MS275, TPO, SCF and FLT3L.
  • a composition for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells which consists of MS275, TPO, SCF and FLT3L.
  • a composition for promoting in vitro expansion of hematopoietic stem cells which consists of MS275, TPO, SCF and FLT3L.
  • the composition provided by the present invention is used as an additive of a culture medium to promote the transformation of hematopoietic progenitor cells to hematopoietic stem cells and the expansion of hematopoietic stem cells.
  • the solution of MS275 was configured with DMSO, and the mother liquor concentration was 100 mmol / L.
  • the mass ratio of the MS275, TPO, SCF and FLT3L is (38-3800): (30-70): (80-120): (90-110).
  • the mass ratio of MS275, TPO, SCF and FLT3L in the composition is 380: 30: 80: 90.
  • the mass ratio of MS275, TPO, SCF and FLT3L in the composition is 380: 50: 100: 100.
  • the mass ratio of MS275, TPO, SCF and FLT3L in the composition is 380: 70: 120: 110.
  • composition of the present invention in promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells.
  • composition of the present invention in the preparation of hematopoietic stem cells.
  • compositions and hematopoietic progenitor cells of the present invention in the preparation of hematopoietic stem cells.
  • composition of the invention in promoting the expansion of hematopoietic stem cells.
  • the invention provides a culture system comprising a basal medium and a composition according to the invention.
  • the invention also provides a culture system for promoting the expansion of hematopoietic stem cells, which comprises a basal medium and a composition according to the invention.
  • the invention also provides a culture system for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells, which comprises a basic culture medium and the composition according to the invention.
  • the concentration of the MS275 is 0.1 ⁇ mol / L to 10 ⁇ mol / L. In some embodiments, the concentration of MS275 is 1 ⁇ mol / L.
  • the concentration of the TPO is 30ng / mL to 70ng / mL;
  • the concentration of the SCF is 80 ng / mL to 120 ng / mL;
  • the concentration of the FLT3L is 90 ng / mL to 110 ng / mL.
  • the concentration of the MS275 is 0.1 ⁇ mol / L to 10 ⁇ mol / L;
  • the concentration of the TPO is 30ng / mL to 70ng / mL;
  • the concentration of the SCF is 80 ng / mL to 120 ng / mL;
  • the concentration of the FLT3L is 90 ng / mL to 110 ng / mL.
  • the culture system of the present invention is prepared by adding the composition provided by the present invention to the StemSpan SFEM II medium until the concentration of each component is the concentration according to the present invention.
  • the composition according to the present invention may be a dry powder, which may be a mixture of components or each component may exist separately.
  • the composition may also be a solution or referred to as a mother liquor.
  • the mother liquor includes all or part of the components of the composition.
  • the solvent of MS275 mother liquor is DMSO, and the solvent of TPO, SCF and FTL3L mother liquor is 0.1% BSA.
  • the concentration of the TPO is 30 ng / mL; the concentration of the SCF is 80 ng / mL; and the concentration of the FLT3L is 90 ng / mL.
  • the concentration of the TPO is 50 ng / mL; the concentration of the SCF is 100 ng / mL; and the concentration of the FLT3L is 100 ng / mL.
  • the concentration of the TPO is 70 ng / mL; the concentration of the SCF is 120 ng / mL; and the concentration of the FLT3L is 110 ng / mL.
  • the basal medium is StemPro, RPMI1640, IMDM, ⁇ -MEM or StemSpan SFEM II. In some embodiments, the basal medium is StemSpan SFEM II.
  • the culture system provided by the present invention includes StemSpan and SFEM II medium, 1 ⁇ mol / L MS275, 50ng / mL TPO, 100ng / mL SCF, and 100ng / mL FLT3L.
  • the culture system provided by the present invention includes StemSpan and SFEM II medium, 1 ⁇ mol / L MS275, 30ng / mL TPO, 80ng / mL SCF, and 90ng / mL FLT3L.
  • the culture system provided by the present invention includes StemSpan and SFEM II medium, 1 ⁇ mol / L MS275, 70ng / mL TPO, 120ng / mL SCF, and 110ng / mL FLT3L.
  • the invention also provides a method for promoting the transformation of hematopoietic progenitor cells to hematopoietic stem cells, and the hematopoietic progenitor cells are cultured by the culture system of the invention.
  • the hematopoietic progenitor cells are CD34 + CD90- and CD34 + CD45RA + umbilical cord blood hematopoietic progenitor cells.
  • the density of the cultured and seeded CD34 + CD90- cells is 0.1 to 10 ⁇ 10 4 cells / mL
  • the density of CD34 + CD45RA + cells is 0.1 to 10 ⁇ 10 4 cells / mL.
  • the density of the CD34 + CD90- cells inoculated in the culture is 0.55 ⁇ 10 4 cells / mL, and the density of CD34 + CD45RA + cells is 0.13 ⁇ 10 4 cells / mL.
  • the culture conditions are 37 ° C, 5% CO 2 , and the culture time is 5-10 days.
  • the invention also provides a method for expanding hematopoietic stem cells, and the hematopoietic stem cells are cultured in the culture system of the invention.
  • the hematopoietic stem cells are umbilical cord blood hematopoietic stem cells; the seeding density is 0.1 to 10 ⁇ 10 4 cells / mL.
  • the culture conditions are 37 ° C, 5% CO 2 .
  • Fresh culture medium and composition provided by the present invention are supplemented every 2 days. After 5 to 10 days of culture, the amplification factor is 4 to 20 times.
  • the invention provides the application of MS275 in promoting the expansion of hematopoietic stem cells.
  • Studies have shown that during the expansion and culture of umbilical cord blood hematopoietic stem cells, the addition of cytokines and the addition of MS275 achieves the effect of both increasing the number of hematopoietic stem cells and improving the ability of CFU colonies to form hematopoietic stem cells, allowing hematopoietic stem cells to proliferate and differentiate. Status, which in turn meets the needs of clinical transplantation.
  • the method has the advantages of simple operation, low cost, more hematopoietic stem cells obtained, and solves the defects of low expansion rate and easy differentiation of hematopoietic stem cells in the prior art.
  • MS275 is expected to induce hematopoietic progenitor cells into hematopoietic stem cells through epigenetic modification, and can expand hematopoietic stem cells in vitro. It can be applied to cord blood, placental blood, peripheral blood, bone marrow-derived hematopoietic stem cells; MS275 and artificial blood progenitors are used.
  • the number of hematopoietic stem cells prepared by cells is large, and they have the potential for differentiation of various lineages, which can provide hematopoietic stem cell donors for clinical applications.
  • FIG. 1a is a flow cytometry test of CD34 + CD90-hematopoietic progenitor cells after 5 days of culture (DMSO is the control group);
  • Fig. 1b is a flow cytometry test of CD34 + CD45RA + hematopoietic progenitor cells after 5 days of culture (DMSO is the control group);
  • FIG. 2a is a statistical chart of the proportion and number of hematopoietic stem cells after CD34 + CD90-hematopoietic progenitor cells are cultured for 5 days (DMSO is a control group);
  • Figure 2b is a statistical chart of the proportion and number of hematopoietic stem cells after 5 days of CD34 + CD45RA + hematopoietic progenitor cells culture (DMSO is the control group);
  • Figure 3 shows the analysis of the surface antigen expression of hematopoietic stem cells in groups 1 and 4 on the 5th day; of which, Figure 3a shows the expression of SSC and CD34, Figure 3b shows the expression of CD34 and CD45RA, and Figure 3c shows CD34, CD90 expression;
  • Figure 4 shows representative colony formation of each lineage under an inverted microscope, where (a) shows CFU-E, (b) shows BFU-E, (c) shows CFU-G, (d) shows CFU-M, and (e) shows CFU-GM, (f) shows CFU-GEMM.
  • the invention discloses a composition and a preparation method and preparation thereof for hematopoietic stem cells. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can modify or appropriately modify and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention, to achieve and Apply the technology of the present invention.
  • a method for preparing artificial blood stem cells using artificial blood progenitor cells includes the following steps:
  • S1 obtains CD34 + CD90- and CD34 + CD45RA + umbilical cord blood hematopoietic progenitor cells from human umbilical cord blood;
  • S2 suspend and inoculate the CD34 + CD90- and CD34 + CD45RA + umbilical cord blood hematopoietic progenitor cells in a umbilical cord blood hematopoietic stem cell-specific transformation medium
  • the umbilical cord blood hematopoietic stem cell-specific transformation medium is StemSpan SFEM II serum-free medium
  • MS275 at a concentration of 1 ⁇ M; place at 37 ° C and incubate in a 5% CO 2 incubator.
  • the method for preparing artificial blood stem cells using artificial blood progenitor cells according to the present invention further includes:
  • S3 is supplemented with 500 ⁇ l of the umbilical cord blood hematopoietic stem cell-specific conversion medium every 2 days according to the state of cell culture. A large number of cells are obtained in 5 to 10 days, and the expansion factor is 4 to 20 times.
  • obtaining CD34 + CD90- and CD34 + CD45RA + umbilical cord blood hematopoietic progenitor cells from human umbilical cord blood as described in step S1 further includes the following steps:
  • S12 uses magnetic bead sorting MACS to obtain CD34 + umbilical cord blood hematopoietic stem and progenitor cells from the above PBMC;
  • CD34 + cells are stained with flow antibodies CD34, CD90, and CD45RA for half an hour, and CD34 + CD90- and CD34 + CD45RA + hematopoietic progenitor cells are sorted by a flow sorter.
  • obtaining the peripheral blood mononuclear cell PBMC described in step S11 further includes the following steps:
  • S111 Use a disposable blood bag containing heparin sodium and other anticoagulants to collect 80 to 120 ml of umbilical cord blood, transfer the umbilical cord blood from the blood bag to a 500 ml culture flask, dilute it by 2 to 3 times with normal saline, add it dropwise after mixing 0.4 times the volume of lymphocyte separation fluid;
  • the centrifuge tube was divided into four layers from top to bottom: the first layer was the plasma layer, and the second layer was the annular milky white mononuclear cell layer PBMC. , The third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer;
  • S113 use a pipette to suck the second layer of circular milky white mononuclear cell layer PBMC into another 50ml centrifuge tube, add physiological saline, and centrifuge at 1500 to 2000 rpm / min for 5 to 10 minutes; and
  • step S12 further includes:
  • each umbilical cord blood PBMC was resuspended with a mixture of 50ul human CD34 + magnetic beads, 50ul FcR blocker and 150ul 0.5% BSA, and incubated at 4 ° C for 30 minutes;
  • step S125 resuspend the PBMC agglomerates in step S123 with 500ul 0.5% BSA, mix and transfer to the MACS special adsorption column, and wait for the liquid to completely flow out;
  • step S127 the obtained liquid is frozen and stored in liquid nitrogen with a cryoprotectant dimethyl sulfoxide DMSO.
  • the invention provides the application of MS275 in promoting the expansion of hematopoietic stem cells.
  • the invention also provides a composition for promoting the expansion of hematopoietic stem cells, which consists of MS275, TPO, SCF and FLT3L.
  • the mass ratio of the MS275, TPO, SCF, and FLT3L is (38 to 3800): (30 to 70): (80 to 120): (90 to 110).
  • the invention also provides an expanded hematopoietic stem cell culture system, which includes a basal medium and MS275.
  • the concentration of the MS275 is 0.1 ⁇ mol / L to 10 ⁇ mol / L.
  • TPO, SCF and FLT3L are also included in the culture system of the present invention.
  • the concentration of the TPO is 30 ng / mL to 70 ng / mL; the concentration of the SCF is 80 ng / mL to 120 ng / mL; and the concentration of the FLT3L is 90 ng / mL to 110 ng / mL.
  • the basic medium is StemPro, RPMI1640, IMDM, ⁇ -MEM, or StemSpan SFEM II.
  • the invention also provides a method for expanding hematopoietic stem cells, and the hematopoietic stem cells are cultured in the culture system of the invention.
  • the hematopoietic stem cells are umbilical cord blood hematopoietic stem cells; the initial seeding density is 2 ⁇ 10 4 cells / mL.
  • MS275 is expected to induce hematopoietic progenitor cells into hematopoietic stem cells through the regulation of epigenetic modification, and then to expand hematopoietic stem cells in vitro. It can be applied to hematopoietic stem cells derived from umbilical cord blood, placental blood, peripheral blood, and bone marrow. Hematopoietic stem cells are high in number and have the potential for differentiation of various lineages, which can provide hematopoietic stem cell donors for clinical applications.
  • the hematopoietic stem cells are a type of cells that have self-renewal ability and differentiation ability, can differentiate into various types of blood cells, and can rebuild the entire blood system and immune system of the recipient in the body for a long time. They express CD34 and CD90 surface antigens. CD45RA is not expressed, that is, CD34 + CD90 + CD45RA-. In the present invention, CD34 + CD90 + and CD34 + CD45RA- both represent hematopoietic stem cells.
  • the hematopoietic progenitor cell is a type of cell that has lower self-renewal ability and differentiation ability than hematopoietic stem cells, and can differentiate into a variety of blood cells, but cannot rebuild the entire blood system and immune system of the recipient in the body for a long time.
  • CD34 surface antigen but does not express CD90 surface molecules specific for hematopoietic stem cells, and CD45RA is positive, that is, CD34 + CD90-CD45RA +.
  • CD34 + CD90- and CD34 + CD45RA + both represent hematopoietic progenitor cells.
  • the transformation may also be referred to as transformation, that is, the transformation of hematopoietic progenitor cells into hematopoietic stem cells; it may also be referred to as reprogramming, that is, hematopoietic progenitor cells are reprogrammed into hematopoietic stem cells.
  • the reprogramming refers to the reversion of hematopoietic progenitor cells to the state of hematopoietic stem cells.
  • the expansion refers to a process in which hematopoietic stem cells are cultured to increase their number.
  • the number of cells can be expanded by more than 17 times.
  • the composition refers to the combination of the small molecule compound MS275 and the growth factors TPO, SCF and FLT3L.
  • Each component in the composition of the present invention may exist independently or be mixed with each other, which is not limited in the present invention.
  • Each component can be a solution or a powder. In the present invention, each component exists as a solution, and each component is independent of each other.
  • the culture system refers to nutrients for culturing cells under in vitro conditions, and may also be referred to as a culture medium.
  • the culture system according to the present invention can be used immediately before use, or can be made into a finished product for long-term storage.
  • the basal medium refers to a medium capable of realizing the expansion or growth of hematopoietic stem cells, which is a required basic nutrient substance.
  • the basal medium according to the present invention may be a powder or a culture solution.
  • test materials, reagents or experimental equipment used in the present invention are common commercial products, and are all available on the market. Specifically, the raw material manufacturers involved in the following examples are as follows (Table 1):
  • StemSpanSFEM II is a serum-free medium, the manufacturer is StemCell Technologies, the article number is 09655;
  • Recombined human stem cell factor rhSCF (recombined human stem cell factor), the manufacturer is Stemimune LLC, the article number is HHM-SF-1000;
  • Recombined human thrombopoietin rhTPO (recombined human thrombopoietin), the manufacturer is Stemimune LLC, the article number is HHM-TP-0100;
  • Recombinant human FMS-like tyrosine kinase 3 ligand rhFLT3L (recombined human FMS-like tyrosinekinase 3ligand), referred to as FLT3 or FLT3L, the manufacturer is Stemimune LLC, the article number is HHM-FT-1000;
  • PBMCs Peripheral blood mononuclear cells
  • MACS magnetic bead sorting
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffered saline
  • MethoCult TM GF H4435 is a semi-solid medium
  • CFU-E Colony Forming Unit of Erythrocyte
  • Chinese name is erythrocyte colony forming unit
  • BFU-E Burst Forming Unit of Erythrocyte
  • Chinese name Burst Erythrocyte Colony Forming Unit
  • CFU-G Colony Forming Unit of Granulocyte
  • Chinese name Granulocyte Colony Forming Unit
  • CFU-M Colony Forming Unit of Macrophage
  • Chinese name is Macrophage Colony Forming Unit
  • CFU-GM Colony Forming Unit of Granulocyte-Macrophage
  • Chinese name is granulocyte-macrophage colony forming unit
  • CFU-GEMM The full name of CFU-GEMM is Colony, Forming, Granululocyte, erythrocyte, macrophage / monocyte, megakaryocyte, mixed colony. Its Chinese name is granulocyte, red blood cell, macrophage / monocyte, megakaryocyte colony forming unit;
  • the preparation of the umbilical cord blood hematopoietic stem cells includes: diluting umbilical cord blood with physiological saline 2 to 3 times, adding lymphocyte separation solution, centrifuging at 1500 to 2000 rpm / min for 20 minutes to take a mononuclear cell layer (PBMC), washing with saline and resuspending PBMC cell masses were obtained; CD34 + cells were then isolated by magnetic bead method.
  • PBMC mononuclear cell layer
  • the composition or culture system provided by the present invention can be suitable for in vitro expansion of hematopoietic stem cells, and the hematopoietic stem cells can be derived from experimental animals (such as mice) or humans.
  • Human hematopoietic stem cells can be derived from bone marrow, peripheral blood, umbilical cord blood, and placental blood.
  • umbilical cord blood hematopoietic stem cells are used as examples.
  • umbilical cord blood has been tested for hepatitis B, hepatitis C, syphilis, AIDS, The cytomegalovirus, TORCH test, mycoplasma, chlamydia, G-6PD, and thalassemia were negative.
  • the isolated human umbilical cord blood hematopoietic stem cells expressed the following membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation antigen CD34, and leukocyte differentiation Antigen CD90, leukocyte differentiation antigen CD49f.
  • PBMC peripheral blood mononuclear cells
  • Centrifuge tubes are divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, and the second layer is the circular milky white mononuclear cell layer (PBMC). Three layers are transparent separation liquid layer, and the fourth layer is red blood cell layer.
  • PBMC circular milky white mononuclear cell layer
  • PBMC circular milky white mononuclear cell layer
  • MCS magnetic bead sorting
  • Each umbilical cord blood PBMC was resuspended with 50ul of human CD34 + magnetic beads, 50ul of FcR blocker and 150ul of 0.5% BSA, and incubated at 4 ° C for 30 minutes.
  • DMSO dimethyl sulfoxide
  • CD34 + cells were stained with flow antibodies CD34, CD90, and CD45RA for half an hour, and CD34 + CD90- and CD34 + CD45RA + hematopoietic progenitor cells were sorted by a flow sorter.
  • the CD34 + CD90- and CD34 + CD45RA + umbilical cord blood hematopoietic progenitor cells are suspended and inoculated in a special conversion medium for umbilical cord blood hematopoietic stem cells and cultured.
  • Transformation medium StemSpan, SFEM, II serum-free medium + 100ng / ml, SCF + 100ng / ml, FLT3 + 50ng / ml, TPO + 1 ⁇ M MS275;
  • Control medium StemSpan, SFEM, II serum-free medium + 100ng / ml, SCF + 100ng / ml, FLT3 + 50ng / ml, TPO + 0.1% DMSO;
  • Phenotypic identification, viability and purity testing, and functional identification of the isolated human placental blood hematopoietic stem cells include the following steps:
  • CD34 + CD90 + and CD34 + CD45RA-hematopoietic stem cells were counted after culture.
  • BD company FACS Verse flow cytometer was used to take 20 ⁇ l of cell suspension and add 0.2 ⁇ l each of the following four antibodies dissolved in 0.5% BSA: FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, APC Tagged CD90. After vortexing each tube, incubate for 15 minutes at room temperature in the dark, add an appropriate amount of PBS, centrifuge at 1600 rpm for 5 minutes at room temperature, discard the supernatant, add 200 ⁇ l PBS, and analyze on the machine.
  • MethoCult TM GF H4534 semi-solid medium was added to a six-well plate with 1 ml / well of medium, CD34 + cells were seeded at a density of 1000 cells / well, and cultured in a 37 ° C 5% CO 2 incubator for 14 days, and each lineage was calculated. Number of colonies and take photos.
  • Centrifuge tubes are divided into four layers from top to bottom due to different densities: the first layer is the plasma layer, the second layer is the annular milky white mononuclear cell layer (PBMC), the first Three layers are transparent separation liquid layer, and the fourth layer is red blood cell layer;
  • PBMC annular milky white mononuclear cell layer
  • Each umbilical cord blood PBMC was resuspended with 50 ⁇ L of human CD34 + magnetic beads, 50 ⁇ L of FcR blocker reagent and 150 ⁇ L of 0.5% BSA, and incubated at 4 ° C for 30 minutes;
  • the CD34 + umbilical cord blood hematopoietic stem cells prepared in Example 1 were suspended and inoculated in the cell culture medium of each group for culture.
  • the cell seeding density in the 24-well plate was 1 ⁇ 10 4 cells / well, the volume was 0.5 mL, and the cells were cultured in a 5% CO 2 incubator at 37 ° C. According to the state of cell culture, 500 ⁇ L of fresh cell culture medium of each group was added every 2 days. A large number of hematopoietic stem cells could be obtained in 5 to 10 days, and the expansion factor was about 4 to 20 times.
  • the umbilical cord blood hematopoietic stem cells cultured in each group of Example 4 were subjected to cell counting, phenotypic identification, and colony forming unit analysis.
  • CD34 + cells cultured on MS275 or DMSO on day 0 and 5 were analyzed by flow cytometry.
  • a BD company FACS Verse flow cytometer was used to take 20 ⁇ L of the cell suspension and add FITC-labeled CD34, PE-labeled CD38, APC-Cy7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA. After vortexing each tube, incubate at room temperature for 15 minutes in the dark, add an appropriate amount of PBS, centrifuge at 1600 rpm for 5 minutes at room temperature, discard the supernatant, add 200 ⁇ L of PBS, and analyze on the machine. The test results of Group 2 and Group 4 are shown in Figure 3.
  • Colony forming unit analysis was performed on CD34 + cells cultured on MS275 or DMSO on day 0 and day 5, respectively.
  • MethoCult TM GF H4435 semi-solid medium was added to a six-well plate with 1 ml / well of medium, and CD34 + cells were seeded at a density of 500 cells / well. After being cultured in a 37 ° C 5% CO 2 incubator for 14 days, colonies of each lineage were calculated Number and take photos. The representative figure of colony formation of each lineage under an inverted microscope is shown in Figure 4, and the statistics of colony formation number is shown in Table 6:

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Abstract

提供了一种包含MS275的组合物及其促使人造血祖细胞向人造血干细胞转变的用途。在脐带血造血干细胞扩增培养过程中，加入细胞因子的同时加入MS275，能够既增加造血干细胞数量，又提高造血干细胞CFU集落形成能力，使造血干细胞处于增殖不分化的状态，达到临床移植需求。

Description

一种组合物及其用途
本申请要求于2018年09月17日提交中国专利局、申请号为201811084022.5、发明名称为“一种将人造血祖细胞转变为造血干细胞的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。并且要求于2018年12月28日提交中国专利局、申请号为201811625923.0、发明名称为“扩增造血干细胞的培养体系、方法及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及造血干祖细胞技术领域，特别涉及一种组合物及其用途。
背景技术
造血干细胞是成体内极其重要的一类干细胞，尽管其仅占人体血细胞比例不到万分之一，但具备极强的自我更新能力和分化能力，能长期重建机体整个血液系统和免疫系统，具备各世系血细胞和免疫细胞的分化潜能。因此，造血干细胞被广泛应用于白血病、淋巴瘤等恶性血液疾病的临床治疗。不仅如此，造血干细胞移植还能帮助治疗代谢性疾病、先天性免疫缺陷、糖尿病等病症。据统计，每年全世界有超过40,000例造血干细胞移植手术。目前，造血干细胞的供体主要来源于患者自体或者与患者HLA匹配的捐献者的骨髓和动员的外周血造血干细胞。尽管该移植技术疗效很好，但由于其存在HLA配型的严格配对要求，仍有大约70％的患者不能获得合适的供体而无法接受治疗；即使接受了治疗，多数患者也会经受程度各异的移植物抗宿主病(GVHD)的折磨。脐带血造血干细胞对于HLA配型的要求相对较低，且免疫原性低，再加上其获取方便、来源丰富，逐渐成为造血干细胞移植供体的一大来源。然而由于单份脐带血所含造血干细胞数量少，不足以短时间内重建成人患者的免疫系统，从而增高机会性感染致死率，急需一种增加脐带血造血干细胞数量的方法。
细胞重编程是指分化的体细胞在特定的条件下去分化逆转命运回到全能性或多能性状态，或者将一种类型的分化细胞转分化变成另外一种细 胞类型的过程。从造血祖细胞到造血干细胞的转变，是一种细胞重编程。造血祖细胞是一类自我更新能力和分化潜能低于造血干细胞的细胞类型，虽然表达CD34表面抗原，但不表达造血干细胞特异的CD90表面分子，而为CD45RA阳性。因此，可以利用CD90和CD45RA区分造血干细胞和造血祖细胞。造血祖细胞具备短期(不到一个月)的体内移植能力，能分化为红细胞、淋巴细胞、髓系细胞等多种血液细胞。由于其不具备长期移植重建受体血液系统的能力，常常被排除在血液细胞移植治疗恶性血液病的手术之外。但造血祖细胞体内含量远远高于造血干细胞(0.03％vs0.0001％)，若能够将造血祖细胞重编程为造血干细胞，则将大大拓宽造血干细胞的供体来源。
近年来，人们对于脐带血造血干细胞的体外扩增进行了大量尝试，但都没有取得理想效果。早期人们利用血液中的细胞因子来培养造血干细胞，结果导致细胞分化，而移植功能减弱。后来，人们发现骨髓造血干细胞微环境中的Wnt信号分子、Notch配体、视黄酸拮抗因子等能够有效扩增CD34+造血干/祖细胞。利用CHIR99021或者BIO激活Wnt信号通路维持体外培养的造血干细胞的移植能力；而在造血干细胞的培养体系中添加DLL1，DSL1等，能够通过激活Notch信号而适度扩增造血干细胞。另有研究发现，骨髓内皮基质细胞分泌的PTN也能够轻微扩增造血干细胞。生理状态下造血干细胞处在低氧条件下，而体外培养产生的氧胁迫会通过增高ROS水平损害造血干细胞的自我更新和移植功能；人们发现，抗氧化剂的添加以及mTOR的抑制能够抵消这些损害。然而，上述技术并没能够显著扩增脐带血造血干细胞。偶然的发现，铜离子螯合剂TEPA，SIRT抑制剂Nicotinamide能够显著提高造血干细胞移植水平，且在临床实验中显示初步疗效，但扩增后的细胞体内存活时间不够长，且分化谱系不够完整。近几年的高通量筛选化学小分子发现一类氮杂环化合物SR1和吲哚类似物UM171能够更有效的扩增具备长期移植能力的造血干细胞。临床实验表明，SR1扩增的造血干细胞具备重建患者免疫系统的能力，但其依然没有摆脱对双份脐带血移植的依赖。总的来说，HSC最佳的体外扩增条件至今尚没有明确的共识。
表观修饰在调控细胞命运方面具有重要作用。基因座的甲基化、乙酰化，组蛋白的各类基团修饰直接影响邻近区域基因的开放程度和转录因子结合难度，进而调控基因的表达，完成对细胞状态及命运的调节。因此，表观修饰在更高的层面上影响细胞功能。人们研发了许多表观修饰物来改变细胞命运。近年研究发现表观修饰物对人造血干细胞的体外扩增也有明显促进作用。小分子化合物MS275在细胞重编程领域也引起高度重视，因其能够促进细胞命运改变，被用作IPS制备的催化剂。
小分子化合物MS275，又名SNDX-275或Entinostat，分子式为C 21H 20N 4O 3，CAS号为209783-80-2，是I型组蛋白去乙酰化酶HDAC1和HDAC3的特异抑制剂。医疗领域多用Entinostat来指称MS275，该药物在白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、乳腺癌、卵巢癌、肺癌、肾癌等多种恶性肿瘤已经进入I期和II期临床试验。据报道，MS275能促进人诱导型多潜能干细胞(iPSC)的产生，而MS275对造血干细胞的维持和扩增作用，以及对造血祖细胞向造血干细胞重编程的作用尚无报道。
发明内容
针对相关技术中的上述技术问题，本发明提供了一种组合物及其在造血干祖细胞中的用途。本发明研究表明，小分子化合物MS275能够促进造血祖细胞向造血干细胞转变，并且能够显著提高造血干细胞体外扩增所得的细胞总量。
本发明提供了MS275在促进造血祖细胞向造血干细胞转变中的应用。
本发明提供了，MS275在制备造血干细胞中的应用。
本发明提供了，MS275和造血祖细胞在制备造血干细胞中的应用
本发明还提供了一种组合物，其由MS275、TPO、SCF和FLT3L组成。
一种促进造血祖细胞向造血干细胞转变的组合物，其由MS275、TPO、SCF和FLT3L组成。
一种促进造血干细胞体外扩增的组合物，其由MS275、TPO、SCF 和FLT3L组成。
本发明实施例中，本发明提供的组合物作为培养基的添加剂，用于促进造血祖细胞向造血干细胞转变和促进造血干细胞扩增。其中，MS275的溶液以DMSO配置，母液浓度为100mmol/L。
本发明提供的组合物中，所述MS275、TPO、SCF和FLT3L的质量比为(38～3800)：(30～70)：(80～120)：(90～110)。
一些实施例中，所述组合物中MS275、TPO、SCF和FLT3L的质量比为380：30：80：90。
一些实施例中，所述组合物中MS275、TPO、SCF和FLT3L的质量比为380：50：100：100。
一些实施例中，所述组合物中MS275、TPO、SCF和FLT3L的质量比为380：70：120：110。
本发明所述的组合物在促进造血祖细胞向造血干细胞转变中的应用。
本发明所述的组合物在制备造血干细胞中的应用。
本发明所述的组合物和造血祖细胞在制备造血干细胞中的应用。
本发明所述的组合物在促进造血干细胞扩增中的应用。
本发明提供了一种培养体系，其包括基础培养基和本发明所述的组合物。
本发明还提供了一种促进造血干细胞扩增的培养体系，其包括基础培养基和本发明所述的组合物。
本发明还提供了一种促进造血祖细胞向造血干细胞转变的培养体系，其包括基础培养基和本发明所述的组合物。
本发明提供的培养体系中，所述MS275的浓度为0.1μmol/L～10μmol/L。一些实施例中，所述MS275的浓度为1μmol/L。
本发明所述的培养体系中：
所述TPO的浓度为30ng/mL～70ng/mL；
所述SCF的浓度为80ng/mL～120ng/mL；
所述FLT3L的浓度为90ng/mL～110ng/mL。
实施例中，
本发明所述的培养体系中：
所述MS275的浓度为0.1μmol/L～10μmol/L；
所述TPO的浓度为30ng/mL～70ng/mL；
所述SCF的浓度为80ng/mL～120ng/mL；
所述FLT3L的浓度为90ng/mL～110ng/mL。
本发明所述的培养体系其制备方法为，在StemSpan SFEM II培养基中，加入本发明提供的组合物直至各组分的浓度为本发明所述的浓度。本发明所述组合物可为干粉，其可为各组分的混合物或各组分分别单独存在。所述组合物亦可为溶液，或称为母液。所述母液中包括组合物的全部或部分组分。MS275母液的溶剂为DMSO，TPO、SCF和FTL3L母液的溶剂为0.1％BSA。
一些实施例中，所述TPO的浓度为30ng/mL；所述SCF的浓度为80ng/mL；所述FLT3L的浓度为90ng/mL。
一些实施例中，所述TPO的浓度为50ng/mL；所述SCF的浓度为100ng/mL；所述FLT3L的浓度为100ng/mL。
一些实施例中，所述TPO的浓度为70ng/mL；所述SCF的浓度为120ng/mL；所述FLT3L的浓度为110ng/mL。
本发明中，所述基础培养基为StemPro、RPMI1640、IMDM、α-MEM或StemSpan SFEM II。一些实施例中，所述基础培养基为StemSpan SFEM II。
一些具体实施例中，本发明提供的培养体系包括StemSpan SFEM II培养基、1μmol/L MS275、50ng/mL TPO、100ng/mL SCF和100ng/mL FLT3L。
一些具体实施例中，本发明提供的培养体系包括StemSpan SFEM II培养基、1μmol/L MS275、30ng/mL TPO、80ng/mL SCF和90ng/mL FLT3L。
一些具体实施例中，本发明提供的培养体系包括StemSpan SFEM II培养基、1μmol/L MS275、70ng/mL TPO、120ng/mL SCF和110ng/mL FLT3L。
本发明所述的培养体系在促进造血祖细胞向造血干细胞转变中的应 用。
本发明所述的培养体系在制备造血干细胞中的应用。
本发明所述的培养体系和造血祖细胞在制备造血干细胞中的应用。
本发明所述的培养体系在促进造血干细胞扩增中的应用。
本发明还提供了一种促进造血祖细胞向造血干细胞转变的方法，其以本发明所述的培养体系对造血祖细胞进行培养。
本发明提供的方法中，所述造血祖细胞为CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞。
本发明提供的方法中，所述培养接种的CD34+CD90-细胞密度为0.1～10×10 4cells/mL，CD34+CD45RA+细胞密度为0.1～10×10 4cells/mL。
一些具体实施例中，所述培养接种的CD34+CD90-细胞密度为0.55×10 4cells/mL，CD34+CD45RA+细胞密度为0.13×10 4cells/mL。
所述培养的条件为37℃,5％CO 2，培养时长为5～10天。
本发明还提供了一种扩增造血干细胞的方法，其以本发明所述的培养体系对造血干细胞进行培养。
本发明所述的方法中，所述造血干细胞为脐带血造血干细胞；接种的密度为0.1～10×10 4cells/mL。
所述培养的条件为37℃,5％CO 2。每隔2天补加新鲜的本发明提供的培养基及组合物。培养5～10天扩增倍数为4～20倍。
本发明提供了MS275在促进造血干细胞扩增中的应用。研究表明，在脐带血造血干细胞扩增培养过程中，加入细胞因子的同时加入MS275，达到了既增加造血干细胞数量同时又提高造血干细胞CFU集落形成能力的效果，使得造血干细胞能够处于增殖不分化的状态，进而达到临床移植需求。本发明的操作简便，成本低廉，得到的造血干细胞数量更多，解决了现有技术中造血干细胞扩增率低、易分化等缺陷。
MS275通过表观修饰的调节有望将造血祖细胞诱导为造血干细胞，并且能实现体外扩增造血干细胞，可应用于脐带血、胎盘血、外周血、骨髓来源的造血干细胞；利用MS275和人造血祖细胞制备的造血干细胞数量多，且具备各谱系分化潜能，能够为临床应用提供造血干细胞供体。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1a是CD34+CD90-造血祖细胞培养5天后的造血干细胞流式检测图(DMSO为对照组)；
图1b是CD34+CD45RA+造血祖细胞培养5天后的造血干细胞流式检测图(DMSO为对照组)；
图2a是CD34+CD90-造血祖细胞培养5天后的造血干细胞比例和数目统计图(DMSO为对照组)；
图2b是CD34+CD45RA+造血祖细胞培养5天后的造血干细胞比例和数目统计图(DMSO为对照组)；
图3示组1和组4的造血干细胞在第5天的表面抗原表达情况分析图；其中，图3a示SSC、CD34的表达情况，图3b示CD34、CD45RA的表达情况，图3c示CD34、CD90的表达情况；
图4示倒置显微镜下各谱系集落形成代表图，其中(a)示CFU-E，(b)示BFU-E，(c)示CFU-G，(d)示CFU-M，(e)示CFU-GM，(f)示CFU-GEMM。
具体实施方式
本发明公开了一种组合物及其用于造血干细胞的制备方法、制剂，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
一种利用人造血祖细胞制备人造血干细胞的方法，包括以下步骤：
S1从人脐带血获取CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞；
S2将所述CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞悬浮接种于脐带血造血干细胞专用转变培养基中培养，所述脐带血造血干细胞专用转变培养基采用StemSpan SFEM II无血清培养基，添加100ng/ml SCF，100ng/ml FLT3，50ng/ml TPO；初始细胞接种密度为：CD34+CD90-细胞为0.1～10×10 4/ml，CD34+CD45RA+细胞为0.1～10×10 4/mL；添加MS275浓度为1μM；置于37℃,5％CO 2培养箱培养。
本发明所述的利用人造血祖细胞制备人造血干细胞的方法，还进一步包括：
S3根据细胞培养状态，每隔2天补加所述脐带血造血干细胞专用转变培养基500μl，5～10天获得数量较多的细胞，扩增倍数为4～20倍。
本发明所述方法中，步骤S1所述的从人脐带血获取CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞进一步包括以下步骤：
S11获取外周血单个核细胞PBMC；
S12利用磁珠分选MACS从上述PBMC中获得CD34 +脐带血造血干、祖细胞；和
S13将CD34 +细胞利用流式抗体CD34、CD90、CD45RA染色半小时，通过流式分选仪分选得到CD34+CD90-和CD34+CD45RA+造血祖细胞。
本发明所述方法中，步骤S11所述的获取外周血单个核细胞PBMC进一步包括以下步骤：
S111用含肝素钠等抗凝剂的一次性血袋采集脐带血80～120ml，将脐带血由血袋转移至500ml培养瓶中，加生理盐水稀释2～3倍，混匀后逐滴加入到0.4倍体积淋巴细胞分离液中；
S112置于离心管中以1500～2000rpm/分钟离心20分钟，因密度不同离心管中由上到下分为四层：第一层为血浆层，第二层为环状乳白色单个核细胞层PBMC，第三层为透明分离液层，第四层为红细胞层；
S113用吸管吸取第二层环状乳白色单个核细胞层PBMC到另一50ml离心管中，补加生理盐水，1500～2000rpm/分钟离心5～10分钟；和
S114弃上清，加生理盐水重悬，1500～2000rpm/分钟离心5～10分钟，弃上清，得到PBMC细胞团块。
本发明所述方法中，所述的步骤S12进一步包括：
S121每份脐带血PBMC采用50ul人CD34+磁珠和50ul FcR阻滞剂及150ul 0.5％BSA的混合液重悬，4℃孵育30分钟；
S122同时将磁铁和磁力架至于超净台中紫外线照射30分钟；
S123加入10ml无菌PBS,混匀后，1500～2000rpm/分钟离心5～10分钟，弃上清；
S124将MACS专用吸附柱放进磁铁中，加入500ul 0.5％BSA润洗，流出的液体用15ml管接住；
S125用500ul 0.5％BSA重悬步骤S123中PBMC团块，混匀，转移到MACS专用吸附柱中，待液体完全流出；
S126用500ul 0.5％BSA洗涤3次，取下吸附柱，置于15ml管中；和
S127加入1ml 0.5％BSA，用活塞把液体推入15ml管中，所得液体即含CD34 +脐带血造血干、祖细胞；和
S128稀释，计数。
本发明所述方法中，所述的步骤S127中，用冻存保护剂二甲亚砜DMSO将所得的液体冻存于液氮中。
本发明提供了MS275在促进造血干细胞扩增中的应用。
本发明还提供了一种促进造血干细胞扩增的组合物，其由MS275、TPO、SCF和FLT3L组成。
本发明所述组合物中，所述MS275、TPO、SCF和FLT3L的质量比为(38～3800)：(30～70)：(80～120)：(90～110)。
本发明还提供了扩增造血干细胞培养体系，其包括基础培养基和MS275。
本发明所述培养体系中，所述MS275的浓度为0.1μmol/L～10μmol/L。
本发明所述培养体系中还包括TPO、SCF和FLT3L。
本发明所述培养体系中，所述TPO的浓度为30ng/mL～70ng/mL；所 述SCF的浓度为80ng/mL～120ng/mL；所述FLT3L的浓度为90ng/mL～110ng/mL。
本发明所述培养体系中，所述基础培养基为StemPro、RPMI1640、IMDM、α-MEM或StemSpan SFEM II。
本发明还提供了一种扩增造血干细胞的方法，其以本发明所述的培养体系对造血干细胞进行培养。
本发明实施例中，所述造血干细胞为脐带血造血干细胞；初始接种密度为2×10 4cells/mL。
MS275通过表观修饰的调节有望将造血祖细胞诱导为造血干细胞，进而实现体外扩增造血干细胞，可应用于脐带血、胎盘血、外周血、骨髓来源的造血干细胞；利用人造血祖细胞制备的造血干细胞数量高，且具备各谱系分化潜能，能够为临床应用提供造血干细胞供体。
本发明中，所述造血干细胞是一类具有自我更新能力和分化能力，能向各类血细胞分化，在体内能长期重建受体整个血液系统和免疫系统的细胞，其表达CD34和CD90表面抗原，不表达CD45RA，即为CD34+CD90+CD45RA-，本发明中CD34+CD90+和CD34+CD45RA-均代表造血干细胞。
本发明中，所述造血祖细胞是一类自我更新能力和分化能力低于造血干细胞的细胞类型，能向多种血细胞分化，但在体内不能长期重建受体整个血液系统和免疫系统，虽然表达CD34表面抗原，但不表达造血干细胞特异的CD90表面分子，而CD45RA阳性，即为CD34+CD90-CD45RA+，本发明中CD34+CD90-和CD34+CD45RA+均代表造血祖细胞。
本发明中，所述转变，亦可称为转化，即造血祖细胞向造血干细胞转化；也可以称为是重编程，即造血祖细胞重编程为造血干细胞。所述重编程是指使造血祖细胞逆转恢复到造血干细胞的状态。
本发明中，所述扩增是指将造血干细胞进行培养，使其数量得到增加的过程。本发明中，经过本发明提供的培养体系对造血干细胞进行扩增培养后，细胞数量可以得到17倍以上的扩增。本发明中，所述组合物是指小分子化合物MS275，以及生长因子TPO、SCF和FLT3L的组合。本发 明所述组合物中的各组分可以各自独立存在，也可相互混合，本发明对此不做限定。各组分可以为溶液也可为粉末。在本发明中，各组分以溶液形式存在，各组分相互独立。
本发明中，所述培养体系是指体外条件下培养细胞的营养物质，亦可称为培养基。本发明所述的培养体系可于使用前现配现用，也可制成成品长期储存。
本发明中，所述基础培养基是指能够实现造血干细胞扩增或生长的所需基本营养物质的培养基。本发明所述的基础培养基可为粉末状也可为培养液。
本发明采用的试材、试剂或实验器材皆为普通市售品，皆可于市场购得。具体地，关于下面实施例中涉及的原料生产厂家如下表(表1)：
表1原料信息
试剂名称 品牌
青链霉素 GIBCO
淋巴细胞分离液(ficoll) GE
生理盐水 石家庄四药
胶原酶Ⅰ gibco
CD34+磁珠 美天旎
MS275 Sigma-Aldrich
StemSpanSFEM II是无血清培养基，生产厂商为StemCell Technologies，货号为09655；
重组人干细胞因子rhSCF(recombined human stem cell factor)，生产厂商为Stemimmune LLC，货号为HHM-SF-1000；
重组人血小板生成素rhTPO(recombined human thrombopoietin)，生产厂商为Stemimmune LLC，货号为HHM-TP-0100；
重组人FMS样酪氨酸激酶3配体rhFLT3L(recombined human FMS-like tyrosine kinase 3ligand)，简称FLT3或FLT3L，生产厂商为Stemimmune LLC，货号为HHM-FT-1000；
外周血单个核细胞PBMC(peripheral blood mononuclear cell)
MACS：磁珠分选；
DMSO：二甲亚砜；
PBS:磷酸盐缓冲液；
MethoCult TMGF H4435，是半固体培养基；
CFU-E全称Colony Forming Unit of Erythrocyte，中文名为红细胞集落形成单位；
BFU-E全称Burst Forming Unit of Erythrocyte，中文名为爆发式红细胞集落形成单位；
CFU-G全称ColonyForming Unit of Granulocyte，中文名为粒细胞集落形成单位；
CFU-M全称Colony Forming Unit of Macrophage，中文名为巨噬细胞集落形成单位；
CFU-GM全称Colony Forming Unit of Granulocyte-Macrophage，中文名为粒细胞-巨噬细胞集落形成单位；
CFU-GEMM全称Colony Forming Unit of granulocyte,erythrocyte,macrophage/monocyte,megakaryocyte，混合集落，其中文名为粒细胞，红细胞，巨噬/单核细胞，巨核细胞集落形成单位；
所述脐血造血干细胞的制备包括：将脐带血以生理盐水稀释2～3倍后加入淋巴细胞分离液，经1500～2000rpm/min离心20min取单个核细胞层(PBMC)，生理盐水洗涤并重悬得到PBMC细胞团块；然后以磁珠法分离CD34+细胞。
本发明提供的组合物或培养体系能够适用于造血干细胞的体外扩增，所述造血干细胞可来源于实验动物(例如小鼠等)或人类。人类造血干细胞可来源于骨髓、外周血、脐带血和胎盘血，在本发明实施例中，以脐带血造血干细胞为例，其中，脐带血经检测乙型肝炎、丙型肝炎、梅毒、艾滋病、巨细胞病毒、TORCH检测、支原体、衣原体、G-6PD和地贫均为阴性，经检测，分离的人脐带血造血干细胞表达如下几种膜分子：白细胞分化抗原CD45、白细胞分化抗原CD34、白细胞分化抗原CD90、白细胞分化抗原CD49f。
下面结合附图和具体实施方式进一步详细说明本发明。
实施例1：
利用人脐带血造血干细胞专用转变培养基由造血祖细胞获得大量的脐带血造血干细胞，包括如下步骤：
1.获取外周血单个核细胞(PBMC)。
(1)用一次性血袋(含肝素钠等抗凝剂)采集脐带血80～120ml，将脐带血由血袋转移至500ml培养瓶中，加生理盐水稀释2～3倍，混匀后逐滴加入0.4倍体积淋巴细胞分离液中，注意不要破坏界面。
(2)1500～2000rpm/分钟离心20分钟，因密度不同离心管中由上到下分为四层：第一层为血浆层，第二层为环状乳白色单个核细胞层(PBMC)，第三层为透明分离液层，第四层为红细胞层。
(3)用吸管小心吸取第二层环状乳白色单个核细胞层(PBMC)到另一50ml离心管中，补加生理盐水，1500～2000rpm/分钟离心5～10分钟。
(4)弃上清，加生理盐水重悬，1500～2000rpm/分钟离心5～10分钟，弃上清，得到PBMC细胞团块。
2.利用磁珠分选(MACS)从上述PBMC中获得CD34 +脐带血造血干、祖细胞。
(1)每份脐带血PBMC采用50ul人CD34+磁珠和50ul FcR阻滞剂(blocker reagent)及150ul 0.5％BSA的混合液重悬，4℃孵育30分钟。
(2)与此同时，将磁铁和磁力架至于超净台中紫外线照射30分钟。
(3)加入10ml无菌PBS,混匀后，1500～2000rpm/分钟离心5～10分钟，弃上清。
(4)将MACS专用吸附柱放进磁铁中，加入500ul 0.5％BSA润洗，流出的液体用15ml管接住。
(5)500ul 0.5％BSA重悬步骤(3)中PBMC团块，混匀，转移到MACS专用吸附柱中，待液体完全流出。
(6)500ul 0.5％BSA洗涤3次，取下吸附柱，置于15ml管中。
(7)加入1ml 0.5％BSA,用活塞把液体推入15ml管中，所得液体即含CD34 +脐带血造血干、祖细胞。
(8)稀释，计数，如要必要，用冻存保护剂二甲亚砜(DMSO)将上述液体冻存于液氮中。
3.将CD34 +细胞利用流式抗体CD34、CD90、CD45RA染色半小时，通过流式分选仪分选得到CD34+CD90-和CD34+CD45RA+造血祖细胞。
4.所述CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞悬浮接种于脐带血造血干细胞专用转变培养基中培养。采用StemSpan SFEM II无血清培养基，添加100ng/ml SCF，100ng/ml FLT3，50ng/ml TPO；24孔板中细胞接种密度为：CD34+CD90-细胞为0.55×10 4/孔，CD34+CD45RA+细胞为0.13×10 4/孔，体积均为0.5mL；化合物M(MS275)添加至1μM，对照组添加DMSO(0.1％)；置于37℃,5％CO 2培养箱培养。
转变培养基：StemSpan SFEM II无血清培养基+100ng/ml SCF+100ng/ml FLT3+50ng/ml TPO+1μM MS275；
对照组培养基：StemSpan SFEM II无血清培养基+100ng/ml SCF+100ng/ml FLT3+50ng/ml TPO+0.1％DMSO；
5.根据细胞培养状态，每隔2天补加脐带血造血干细胞专用转变培养基500μl，5～10天可获得数量较多的细胞，扩增倍数约为4～20倍。
实施例2：
将上述分离的人胎盘血造血干细胞进行表型鉴定、活率和纯度检测及功能鉴定，包括如下步骤：
1.细胞计数
分别对培养后的CD34+CD90+和CD34+CD45RA-造血干细胞进行计数。
表2 CD34+CD90-造血祖细胞培养后的细胞数目统计
Figure PCTCN2019081855-appb-000001
表3 CD34+CD45RA+造血祖细胞培养后的细胞数目统计
Figure PCTCN2019081855-appb-000002
2.细胞流式分析
采用BD公司FACS Verse流式检测仪，取细胞悬液20μl，加入溶解于0.5％BSA中的以下四种抗体各0.2μl：FITC标记的CD34，PE标记的CD38，APC-Cy7标记的CD45RA，APC标记的CD90。各管涡旋后室温下避光孵育15分钟，加入适量PBS，1600rpm室温离心5分钟，弃上清液，加入PBS 200μl,然后上机分析。
3.集落形成单位分析
采用MethoCult TMGF H4534半固体培养基，在六孔板中加入培养基1ml/孔，CD34 +细胞接种密度为1000细胞/孔，置于37℃5％CO 2培养箱培养14天后，计算各谱系集落数目，并拍摄照片。
由图1a、图1b及图2a、图2b可以得知，含MS275的专用转变培养基能诱导产生CD34+CD90+和CD34+CD45RA-表型的造血干细胞，而普通培养基不能。
实施例3
1、获取脐带血单个核细胞；
(1)将脐带血加生理盐水稀释2～3倍，混匀后逐滴加入到0.4倍体积淋巴细胞分离液中，注意不要破坏界面；
(2)使用1500～2000rpm/min离心20min，因密度不同离心管中由上到下分为四层：第一层为血浆层、第二层为环状乳白色单个核细胞层(PBMC)、第三层为透明分离液层、第四层为红细胞层；
(3)用吸管小心吸取第二层环状乳白色单个核细胞层(PBMC)到另一50ml离心管中，补加生理盐水，再次使用1500～2000rpm/min离心5～10min；
(4)弃上清加生理盐水重悬，最后使用1500～2000rpm/min离心5～10min，再次弃上清，得到PBMC细胞团块。
2、利用MACS从上述PBMC中获得CD34+脐带血造血干细胞；
(1)每份脐带血PBMC采用50μL人CD34+磁珠和50μL FcR blocker reagent及150μL 0.5％BSA的混合液重悬，4℃孵育30min；
(2)与此同时，将磁铁和磁力架至于超净台中紫外线照射30min；
(3)加入10ml无菌PBS,混匀后，使用1500～2000rpm/min离心5～10min后弃上清；
(4)将MACS专用吸附柱放进磁铁中，加入500ul 0.5％BSA润洗，流出的液体用15ml tube接住；
(5)500μL 0.5％BSA重悬获取脐带血单个核细胞的步骤3)中PBMC团块，混匀后转移到MACS专用吸附柱中，待液体完全流出；
(6)500μL 0.5％BSA洗涤3次，取下吸附柱，置于15ml tube中；
(7)加入1ml 0.5％BSA,用活塞把液体推入15ml tube中，所得液体即含CD34+脐带血造血干细胞。
实施例4
各组培养基中因子的含量如表4：
表4各组培养基中因子的含量
  MS275 SCF TPO FLT3
组1 1μM 80ng/ml 30ng/ml 90ng/ml
组2 1μM 100ng/ml 50ng/ml 100ng/ml
组3 1μM 120ng/ml 70ng/ml 110ng/ml
组4 0 80ng/ml 30ng/ml 90ng/ml
将各物质以表1浓度添加入StemSpan SFEM II无血清培养基。
将实施例1制得的CD34+脐带血造血干细胞悬浮接种于各组细胞培养基中进行培养。24孔板中细胞接种密度为1×10 4cells/孔，体积为0.5mL，置于37℃，5％CO 2培养箱培养。根据细胞培养状态，每隔2天补加各组新鲜的细胞培养基500μL，5～10天可获得数量较多的造血干细胞，扩增 倍数约为4～20倍。
效果检测
对实施例4各组培养的脐带血造血干细胞进行细胞计数、表型鉴定及集落形成单位分析。
1、细胞计数
分别对第5天的MS275或DMSO培养的细胞进行计数，并计算相比第0天的细胞数目扩增倍数。各组培养基培养结果如表5：
表5:各组条件细胞数目扩增倍数统计表
Figure PCTCN2019081855-appb-000003
结果表明：添加MS275的组相对于组4，获得的CD34+CD90+细胞数量更多，扩增倍数更大，经统计学分析，组1～3的扩增效果与组4存在显著性差异，p<0.05。组1～3中，组2的扩增效果更佳。
2、细胞流式分析
分别对第0天、第5天的MS275或DMSO培养的CD34+细胞进行流式分析。采用BD公司FACS Verse流式检测仪，取细胞悬液20μL，加入溶解于0.5％BSA中的FITC标记的CD34，PE标记的CD38，APC-Cy7标记的CD45RA，APC标记的CD90。各管涡旋后室温下避光孵育15min，加入适量PBS，1600rpm室温水平离心5min，弃上清液，加入PBS 200μL,然后上机分析。组2和组4的检测结果如图3。结果表明，相对于未添加MS275的组4，组2扩增获得的CD34+CD90+细胞比例更高，这说明表明组2扩增获得的这些造血干细胞更加原始，具有更强的重建血液系统的分化潜能，可以更有效地支持临床治疗需要。组1、3扩增获得的细胞中，CD34+CD90+细胞比例与组2相似。
3、集落形成单位分析
分别对第0天、第5天的MS275或DMSO培养的CD34+细胞进行集落形成单位分析。采用MethoCult TMGF H4435半固体培养基，在六孔板中加入培养基1ml/孔，CD34+细胞接种密度为500细胞/孔，置于37℃5％CO 2培养箱培养14天后，计算各谱系集落数目，并拍摄照片。倒置显微镜下各谱系集落形成代表图如4，集落形成数目统计如表6：
表6各组培养后集落形成数目
Figure PCTCN2019081855-appb-000004
如表6所示，添加MS275的组相对于组4，获得的细胞集落数目更多，经统计学分析，组1～3的集落数目与组4存在显著性差异，p<0.05。组1～3中，组2的群落数目最多。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. MS275在促进造血祖细胞向造血干细胞转变和/或促进造血干细胞扩增中的应用。
  2. 一种组合物，其特征在于，由MS275、TPO、SCF和FLT3L组成。
  3. 根据权利要求2所述的组合物，其特征在于，所述MS275、TPO、SCF和FLT3L的质量比(38～3800)：(30～70)：(80～120)：(90～110)。
  4. 权利要求2或3所述的组合物在促进造血祖细胞向造血干细胞转变和/或促进造血干细胞扩增中的应用。
  5. 一种培养体系，其特征在于，包括基础培养基和权利要求2或3所述的组合物。
  6. 根据权利要求5所述的培养体系，其特征在于，
    所述MS275浓度为0.1μmol/L～10μmol/L；
    所述TPO的浓度为30ng/mL～70ng/mL；
    所述SCF的浓度为80ng/mL～120ng/mL；
    所述FLT3L的浓度为90ng/mL～110ng/mL。
  7. 根据权利要求5～6任一项所述的培养体系，其特征在于，所述基础培养基为StemPro、RPMI1640、IMDM、α-MEM或StemSpan SFEM II。
  8. 一种促进造血祖细胞向造血干细胞转变的方法，其特征在于，以权利要求5～7任一项所述的培养体系对造血祖细胞进行培养。
  9. 根据权利要求8所述的方法，其特征在于，所述造血祖细胞为CD34+CD90-和CD34+CD45RA+脐带血造血祖细胞。
  10. 根据权利要求9所述的方法，其特征在于，所述培养接种的CD34+CD90-细胞密度为0.1～10×10 4cells/mL，CD34+CD45RA+细胞密度为0.1～10×10 4cells/mL。
  11. 一种扩增造血干细胞的方法，其特征在于，以权利要求5～7任一项所述的培养体系对造血干细胞进行培养。
  12. 根据权利要求11所述的方法，其特征在于，所述造血干细胞为脐带血造血干细胞，或权利要求8～10任一项所述方法培养获得的造血干细胞；初始细胞接种的密度为0.1～10×10 4cells/mL。
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