CN110972481B - Composition and application thereof - Google Patents
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Abstract
A composition comprising MS275 and its use to promote the conversion of human hematopoietic progenitor cells to human hematopoietic stem cells is provided. In the process of expansion culture of umbilical cord blood hematopoietic stem cells, the addition of the MS275 together with the addition of the cell factors can increase the number of the hematopoietic stem cells and improve the CFU colony forming capability of the hematopoietic stem cells, so that the hematopoietic stem cells are in a state of proliferation and no differentiation, and the clinical transplantation requirement is met.
Description
The present application claims priority from a chinese patent application entitled "a method for converting human hematopoietic progenitor cells into hematopoietic stem cells" filed by the chinese patent office on 2018, 09, month 17, application No. 201811084022.5, the entire contents of which are incorporated herein by reference. And claims priority of chinese patent application filed in 2018, 12, 28, under the name of "culture system for expanding hematopoietic stem cells, method and use thereof" with chinese patent office, application No. 201811625923.0, the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the technical field of hematopoietic stem and progenitor cells, in particular to a composition and application thereof.
Background
The hematopoietic stem cells are the most important stem cells in adults, and although the proportion of the hematopoietic stem cells is less than one ten thousandth of that of human blood cells, the hematopoietic stem cells have extremely strong self-renewal capacity and differentiation capacity, can reconstruct the whole blood system and immune system of an organism for a long time, and have the differentiation potential of blood cells and immune cells of various lineages. Therefore, hematopoietic stem cells are widely used for clinical treatment of malignant blood diseases such as leukemia and lymphoma. Moreover, hematopoietic stem cell transplantation can also help in the treatment of metabolic diseases, congenital immunodeficiency, diabetes and other conditions. Statistically, there are over 40,000 hematopoietic stem cell transplantation surgeries worldwide each year. Currently, donors of hematopoietic stem cells are derived primarily from bone marrow and mobilized peripheral blood hematopoietic stem cells of donors autologous to the patient or HLA matched to the patient. Although this transplantation technique works well, due to its strict match requirements for HLA matching, approximately 70% of patients do not receive a suitable donor and are not treated; even with treatment, most patients suffer from varying degrees of Graft Versus Host Disease (GVHD). The cord blood hematopoietic stem cells have relatively low requirement on HLA matching, low immunogenicity, convenient acquisition and abundant sources, and gradually become a large source of hematopoietic stem cell transplantation donors. However, since a single cord blood contains a small number of hematopoietic stem cells and is not enough to reconstitute the immune system of an adult patient in a short time, thereby increasing the mortality rate of opportunistic infections, a method for increasing the number of umbilical cord blood hematopoietic stem cells is urgently needed.
Cell reprogramming refers to the process by which differentiated somatic cells differentiate under specific conditions to reverse the fate back to a totipotent or pluripotent state, or to transdifferentiate one type of differentiated cell into another. The transition from hematopoietic progenitor cells to hematopoietic stem cells is a type of cell reprogramming. Hematopoietic progenitor cells are a cell type with lower self-renewal capacity and differentiation potential than hematopoietic stem cells, and although expressing the CD34 surface antigen, do not express the hematopoietic stem cell-specific CD90 surface molecule and are CD45RA positive. Thus, 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) in vivo transplantation ability and can differentiate into various blood cells such as erythrocytes, lymphocytes and myeloid cells. Because of their inability to transplant long-term, reconstitute the recipient's blood system, they are often excluded from blood cell transplantation surgery for the treatment of hematological malignancies. However, the in vivo content of hematopoietic progenitor cells is much higher than that of hematopoietic stem cells (0.03% vs 0.0001%), which would greatly broaden the donor sources of hematopoietic stem cells if they could be reprogrammed into hematopoietic stem cells.
In recent years, many attempts have been made to expand umbilical cord blood hematopoietic stem cells in vitro, but none of them have achieved the desired effect. In the early days, hematopoietic stem cells were cultured using cytokines in blood, and as a result, the cells were differentiated and the transplantation function was weakened. Subsequently, it was discovered that Wnt signaling molecules, Notch ligands, retinoic acid antagonists, etc., in the myeloid hematopoietic stem cell microenvironment were able to efficiently expand CD34+ hematopoietic stem/progenitor cells. Using CHIR99021 or BIO to activate Wnt signal path to maintain the transplantation ability of the in vitro cultured hematopoietic stem cells; on the other hand, by adding DLL1, DSL1, or the like to a hematopoietic stem cell culture system, hematopoietic stem cells can be appropriately expanded by activating a Notch signal. In addition, PTN secreted by the endothelial stromal cells of the bone marrow can slightly expand the hematopoietic stem cells. Under the physiological condition, the hematopoietic stem cells are under the condition of hypoxia, and the oxygen stress generated by in vitro culture damages the self-renewal and transplantation functions of the hematopoietic stem cells by increasing the ROS level; it was found that the addition of antioxidants and the inhibition of mTOR could counteract these lesions. However, the above-mentioned techniques have not been able to significantly expand umbilical cord blood hematopoietic stem cells. Incidentally, the copper ion chelating agent TEPA and the SIRT inhibitor Nicotinamide can obviously improve the hematopoietic stem cell transplantation level and show primary curative effect in clinical experiments, but the survival time of the amplified cells in vivo is not long enough, and the differentiation lineage is not complete enough. In recent years, chemical small molecules are screened in a high-throughput manner, and a class of nitrogen heterocyclic compounds SR1 and indole analogues UM171 are found to be capable of more effectively amplifying hematopoietic stem cells with long-term transplantation capability. Clinical experiments show that SR1 expanded hematopoietic stem cells have the capability of reconstructing the immune system of patients, but the hematopoietic stem cells still do not get rid of the dependence on double cord blood transplantation. Overall, there is no clear consensus on optimal in vitro expansion conditions for HSCs to date.
Apparent modification plays an important role in regulating cell fate. Methylation and acetylation of a gene locus, and various group modifications of histone directly influence the openness degree of genes in the adjacent region and the combination difficulty of transcription factors, so that the expression of the genes is regulated, and the regulation of the cell state and the fate is completed. Thus, the apparent modification affects cell function at a higher level. Many appearance modifiers have been developed to alter cell fate. In recent years, the apparent modifier is found to have obvious promotion effect on the in vitro amplification of the human hematopoietic stem cells. The small molecule compound MS275 also attracts high attention in the field of cell reprogramming, and is used as a catalyst for IPS production because it can promote cell fate change.
A small molecule compound MS275, also known as SNDX-275 or Entinostat, with molecular formula C21H20N4O3CAS number 209783-80-2, is a specific inhibitor of class I histone deacetylase HDAC1 and HDAC 3. The Entinostat is generally named as MS275 in the medical field, and the medicine enters clinical tests of stages I and II in leukemia, non-Hodgkin lymphoma, breast cancer, ovarian cancer, lung cancer, kidney cancer and other malignant tumors. MS275 reportedly promotes human induced multi-potentialsThe production of stem cells (ipscs) is possible, but the maintenance and expansion of hematopoietic stem cells by MS275, and the reprogramming of hematopoietic progenitor cells to hematopoietic stem cells, have not been reported.
Disclosure of Invention
In response to the above-mentioned problems of the related art, the present invention provides a composition and its use in hematopoietic stem and progenitor cells. The research of the invention shows that the small molecular compound MS275 can promote the conversion of hematopoietic progenitor cells to hematopoietic stem cells and can obviously improve the total amount of cells obtained by the in vitro expansion of the hematopoietic stem cells.
The present invention provides the use of MS275 in promoting the conversion of hematopoietic progenitor cells to hematopoietic stem cells.
The invention provides application of MS275 in preparing hematopoietic stem cells.
The invention provides the use of MS275 and hematopoietic progenitor cells in the preparation of hematopoietic stem cells
The present invention also provides a composition consisting of MS275, TPO, SCF and FLT 3L.
A composition for promoting the conversion of hematopoietic progenitor cells to hematopoietic stem cells, consisting of MS275, TPO, SCF, and FLT 3L.
A composition for promoting expansion of hematopoietic stem cells in vitro, consisting of MS275, TPO, SCF, and FLT 3L.
In the embodiment of the invention, the composition provided by the invention is used as an additive of a culture medium and is used for promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells and promoting the expansion of the hematopoietic stem cells. Wherein the solution of MS275 is prepared by DMSO, and the concentration of the mother liquor is 100 mmol/L.
In the composition provided by the invention, the mass ratio of the MS275, the TPO, the SCF and the FLT3L is (38-3800): (30-70): (80-120): (90-110).
In some embodiments, the mass ratio of MS275, TPO, SCF, and FLT3L in the composition is 380: 30: 80: 90.
in some embodiments, the mass ratio of MS275, TPO, SCF, and FLT3L in the composition is 380: 50: 100: 100.
in some embodiments, the mass ratio of MS275, TPO, SCF, and FLT3L in the composition is 380: 70: 120: 110.
the composition of the invention is applied to promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells.
The composition of the invention is applied to the preparation of hematopoietic stem cells.
The composition and the application of the hematopoietic progenitor cells in preparing hematopoietic stem cells are disclosed.
The composition disclosed by the invention is applied to promoting the expansion of hematopoietic stem cells.
The invention provides a culture system, which comprises a basic culture medium and the composition.
The invention also provides a culture system for promoting the expansion of hematopoietic stem cells, which comprises a basal culture medium and the composition.
The invention also provides a culture system for promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells, which comprises a basal medium and the composition of the invention.
In the culture system provided by the invention, the concentration of the MS275 is 0.1-10 mu mol/L. In some embodiments, the concentration of the MS275 is 1. mu. mol/L.
In the culture system of the invention:
the concentration of the TPO is 30 ng/mL-70 ng/mL;
the concentration of the SCF is 80 ng/mL-120 ng/mL;
the concentration of the FLT3L is 90 ng/mL-110 ng/mL.
In an embodiment of the present invention,
in the culture system of the invention:
the concentration of the MS275 is 0.1 to 10 mu mol/L;
the concentration of the TPO is 30 ng/mL-70 ng/mL;
the concentration of the SCF is 80 ng/mL-120 ng/mL;
the concentration of the FLT3L is 90 ng/mL-110 ng/mL.
The preparation method of the culture system comprises the step of adding the composition provided by the invention into a StemBan SFEM II culture medium until the concentration of each component is the concentration of the invention. The compositions of the present invention may be dry powders which may be mixtures of the components or the components may be present separately. The composition may also be a solution, or referred to as a mother liquor. All or a portion of the components of the composition are included in the mother liquor. The solvent for the MS275 mother liquor was DMSO and the solvents for the TPO, SCF and FTL3L mother liquors were 0.1% BSA.
In some embodiments, the concentration of said TPO is 30 ng/mL; the concentration of the SCF is 80 ng/mL; the concentration of the FLT3L is 90 ng/mL.
In some embodiments, the concentration of said TPO is 50 ng/mL; the concentration of the SCF is 100 ng/mL; the concentration of the FLT3L is 100 ng/mL.
In some embodiments, the concentration of said TPO is 70 ng/mL; the concentration of the SCF is 120 ng/mL; the concentration of the FLT3L is 110 ng/mL.
In the invention, the basic culture medium is StemPro, RPMI1640, IMDM, alpha-MEM or StemBan SFEMII. In some embodiments, the basal medium is StemSpan SFEM II.
In some embodiments, the culture system provided herein comprises StemBan SFEM II medium, 1. mu. mol/L MS275, 50ng/mL TPO, 100ng/mL SCF, and 100ng/mL FLT 3L.
In some embodiments, the present invention provides a culture system comprising StemBan SFEM II medium, 1. mu. mol/L MS275, 30ng/mL TPO, 80ng/mL SCF, and 90ng/mL FLT 3L.
In some embodiments, the culture system provided herein comprises StemBan SFEM II medium, 1. mu. mol/L MS275, 70ng/mL TPO, 120ng/mL SCF, and 110ng/mL FLT 3L.
The culture system disclosed by the invention is applied to promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells.
The culture system of the invention is applied to the preparation of hematopoietic stem cells.
The culture system and the application of the hematopoietic progenitor cells in the preparation of hematopoietic stem cells are disclosed.
The culture system of the invention is applied to the promotion of the expansion of hematopoietic stem cells.
The invention also provides a method for promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells, which cultures the hematopoietic progenitor cells by using the culture system.
In the method provided by the invention, the hematopoietic progenitor cells are CD34+ CD 90-and CD34+ CD45RA + cord blood hematopoietic progenitor cells.
In the method provided by the invention, the density of the cultured and inoculated CD34+ CD 90-cells is 0.1-10 multiplied by 104cell/mL, CD34+ CD45RA + cell density of 0.1-10 × 104cells/mL。
In some embodiments, the culture inoculated CD34+ CD 90-cells have a density of 0.55X 104cell/mL, CD34+ CD45RA + cell density of 0.13X 104cells/mL。
The culture conditions were 37 ℃ and 5% CO2The culture time is 5-10 days.
The invention also provides a method for expanding hematopoietic stem cells, which cultures the hematopoietic stem cells by using the culture system.
In the method of the present invention, the hematopoietic stem cells are cord blood hematopoietic stem cells; the density of the inoculation is 0.1-10 multiplied by 104cells/mL。
The culture conditions were 37 ℃ and 5% CO2. Every 2 days, fresh culture medium and composition provided by the invention are supplemented. The amplification times of the cells are 4-20 times after 5-10 days of culture.
The invention provides an application of MS275 in promoting hematopoietic stem cell expansion. Research shows that in the process of expansion culture of umbilical cord blood hematopoietic stem cells, the addition of the MS275 is performed while adding the cell factors, so that the effects of increasing the number of the hematopoietic stem cells and improving the formation capacity of CFU colonies of the hematopoietic stem cells are achieved, the hematopoietic stem cells can be in a state of proliferation and no differentiation, and the clinical transplantation requirement is further met. The method has the advantages of simple operation and low cost, and the obtained hematopoietic stem cells have more quantity, thereby overcoming the defects of low expansion rate, easy differentiation and the like of the hematopoietic stem cells in the prior art.
MS275 is expected to induce hematopoietic progenitor cells into hematopoietic stem cells through regulation of apparent modification, can realize in vitro expansion of the hematopoietic stem cells, and can be applied to umbilical cord blood, placental blood, peripheral blood and bone marrow-derived hematopoietic stem cells; the hematopoietic stem cells prepared by using the MS275 and the human hematopoietic progenitor cells have a large number, have differentiation potential of various lineages and can provide hematopoietic stem cell donors for clinical application.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1a is a flowgram of hematopoietic stem cells after 5 days of culture of CD34+ CD 90-hematopoietic progenitor cells (DMSO is control);
FIG. 1b is a flowgram of hematopoietic stem cells 5 days after culture of CD34+ CD45RA + hematopoietic progenitor cells (DMSO is control);
FIG. 2a is a statistical plot of the proportion and number of hematopoietic stem cells after 5 days of culture of CD34+ CD 90-hematopoietic progenitor cells (DMSO is control);
FIG. 2b is a statistical plot of the proportion and number of hematopoietic stem cells after 5 days of culture of CD34+ CD45RA + hematopoietic progenitor cells (DMSO is control);
FIG. 3 is a graph showing an analysis of surface antigen expression profiles on day 5 of hematopoietic stem cells of groups 1 and 4; wherein, FIG. 3a shows the expression of SSC and CD34, FIG. 3b shows the expression of CD34 and CD45RA, and FIG. 3c shows the expression of CD34 and CD 90;
FIG. 4 shows representative graphs of colony formation of each lineage under an inverted microscope, in which (a) shows CFU-E, (b) shows BFU-E, (c) shows CFU-G, (d) shows CFU-M, (E) shows CFU-GM, and (f) shows CFU-GEMM.
Detailed Description
The invention discloses a composition, a preparation method and a preparation thereof for hematopoietic stem cells, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
A method for preparing human hematopoietic stem cells from human hematopoietic progenitor cells, comprising the steps of:
s1 obtaining CD34+ CD 90-and CD34+ CD45RA + cord blood hematopoietic progenitor cells from human cord blood;
s2, the CD34+ CD 90-and CD34+ CD45RA + cord blood hematopoietic progenitor cells are inoculated in a cord blood hematopoietic stem cell special conversion culture medium in suspension, the cord blood hematopoietic stem cell special conversion culture medium adopts StemBanSFEM II serum-free culture medium, 100ng/ml SCF, 100ng/ml FLT3 and 50ng/ml TPO are added; initial cell seeding density was: CD34+ CD 90-cells are 0.1-10 multiplied by 104The concentration of CD34+ CD45RA + cells is 0.1-10 multiplied by 104Per mL; MS275 was added at a concentration of 1. mu.M; standing at 37 deg.C for 5% CO2Culturing in an incubator.
The method for preparing human hematopoietic stem cells using human hematopoietic progenitor cells according to the present invention further comprises:
s3, according to the cell culture state, 500 mu l of the special conversion culture medium for the umbilical cord blood hematopoietic stem cells is supplemented every 2 days, a large number of cells are obtained in 5-10 days, and the amplification multiple is 4-20 times.
In the method of the present invention, the step of obtaining hematopoietic progenitor cells from human umbilical cord blood in step S1, which are CD34+ CD 90-and CD34+ CD45RA + umbilical cord blood, further comprises the following steps:
s11 obtaining Peripheral Blood Mononuclear Cells (PBMCs);
s12 obtaining CD34 from the PBMCs by magnetic bead sorting MACS+Hematopoietic stem and progenitor cells from umbilical cord blood; and
s13 CD34+The cells were stained with flow antibodies CD34, CD90, CD45RA for half an hour and sorted by flow sorter to give CD34+ CD 90-and CD34+ CD45RA + hematopoietic progenitor cells.
In the method of the present invention, the step of obtaining PBMCs of peripheral blood mononuclear cells in step S11 further comprises the steps of:
s111, collecting 80-120 ml of umbilical cord blood by using a disposable blood bag containing anticoagulants such as heparin sodium and the like, transferring the umbilical cord blood from the blood bag to a 500ml culture bottle, adding physiological saline for diluting by 2-3 times, uniformly mixing, and dropwise adding the mixture into 0.4-fold volume of lymphocyte separation liquid;
s112, placing the mixture into a centrifugal tube, centrifuging the mixture at 1500-2000 rpm/min for 20min, and dividing the centrifugal tube into four layers from top to bottom due to different densities: the first layer is a plasma layer, the second layer is annular milky mononuclear cell layer PBMC, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer;
s113 sucking the second annular milky white mononuclear cell layer PBMC layer into another 50ml centrifuge tube by using a suction tube, supplementing physiological saline, and centrifuging at 1500-2000 rpm/min for 5-10 min; and
s114, abandoning the supernatant, adding physiological saline for resuspension, centrifuging at 1500-2000 rpm/min for 5-10 min, abandoning the supernatant, and obtaining PBMC cell pellets.
In the method of the present invention, the step S12 further includes:
s121 resuspending each cord blood PBMC with a mixture of 50ul human CD34+ magnetic beads, 50ul FcR blocker and 150ul 0.5% BSA, and incubating at 4 ℃ for 30 min;
s122, simultaneously placing the magnet and the magnetic frame in a super clean bench to irradiate ultraviolet rays for 30 minutes;
s123, adding 10ml of sterile PBS, uniformly mixing, centrifuging at 1500-2000 rpm/min for 5-10 min, and discarding the supernatant;
s124, putting the MACS special adsorption column into a magnet, adding 500ul of 0.5% BSA for rinsing, and catching the effluent liquid by using a 15ml tube;
s125 resuspending the PBMC pellet obtained in the step S123 with 500ul 0.5% BSA, mixing uniformly, transferring to a MACS special adsorption column, and allowing the liquid to flow out completely;
s126, washing the column with 500ul of 0.5% BSA for 3 times, taking down the adsorption column, and placing the column in a 15ml tube; and
s127 Add 1ml 0.5% BSA and push the liquid into a 15ml tube with a piston, the resulting liquid i.e.Containing CD34+Hematopoietic stem and progenitor cells from umbilical cord blood; and
and S128, diluting and counting.
In the method of the present invention, in the step S127, the obtained liquid is cryopreserved in liquid nitrogen using a cryopreservation agent, namely dimethylsulfoxide DMSO.
The invention provides an application of MS275 in promoting hematopoietic stem cell expansion.
The invention also provides a composition for promoting expansion of hematopoietic stem cells, which consists of MS275, TPO, SCF and FLT 3L.
In the composition, the mass ratio of the MS275, the TPO, the SCF and the FLT3L is (38-3800): (30-70): (80-120): (90-110).
The invention also provides an expanded hematopoietic stem cell culture system, which comprises a basal medium and MS 275.
In the culture system, the concentration of the MS275 is 0.1-10 mu mol/L.
The culture system of the invention also comprises TPO, SCF and FLT 3L.
In the culture system, the concentration of TPO is 30 ng/mL-70 ng/mL; the concentration of the SCF is 80 ng/mL-120 ng/mL; the concentration of the FLT3L is 90 ng/mL-110 ng/mL.
In the culture system, the basic culture medium is StemPro, RPMI1640, IMDM, alpha-MEM or StemSpan SFEM II.
The invention also provides a method for expanding hematopoietic stem cells, which cultures the hematopoietic stem cells by using the culture system.
In the embodiment of the invention, the hematopoietic stem cells are cord blood hematopoietic stem cells; initial inoculum density was 2X 104cells/mL。
MS275 hopefully induces hematopoietic progenitor cells into hematopoietic stem cells through the regulation of apparent modification, further realizes the in vitro expansion of the hematopoietic stem cells, and can be applied to the hematopoietic stem cells from umbilical cord blood, placental blood, peripheral blood and bone marrow; the hematopoietic stem cells prepared by the human hematopoietic progenitor cells have high number, have differentiation potential of various lineages and can provide hematopoietic stem cell donors for clinical application.
The hematopoietic stem cells are cells which have self-renewal capacity and differentiation capacity, can differentiate into various blood cells, can reconstruct the whole blood system and immune system of a receptor for a long time in vivo, express CD34 and CD90 surface antigens, do not express CD45RA, and are CD34+ CD90+ CD45RA-, and CD34+ CD90+ and CD34+ CD45 RA-represent the hematopoietic stem cells in the invention.
In the invention, the hematopoietic progenitor cells are a cell type with self-renewal capacity and differentiation capacity lower than those of hematopoietic stem cells, can be differentiated into various blood cells, but cannot be used for reconstructing the whole blood system and immune system of a receptor for a long time in vivo, although the CD34 surface antigen is expressed, the CD90 surface molecule specific to the hematopoietic stem cells is not expressed, and the CD45RA is positive, namely CD34+ CD90-CD45RA +, and CD34+ CD 90-and CD34+ CD45RA + both represent the hematopoietic progenitor cells.
In the present invention, the transformation, which may also be referred to as transformation, is the transformation of hematopoietic progenitor cells into hematopoietic stem cells; it may also be referred to as reprogramming, i.e., the reprogramming of hematopoietic progenitor cells into hematopoietic stem cells. The reprogramming refers to the reversion of hematopoietic progenitor cells back to the hematopoietic stem cell state.
In the present invention, the expansion refers to a process of culturing hematopoietic stem cells to increase the number thereof. In the invention, after the hematopoietic stem cells are subjected to amplification culture by the culture system provided by the invention, the number of the cells can be amplified by more than 17 times. In the present invention, the composition refers to the small molecule compound MS275, and the combination of growth factors TPO, SCF and FLT 3L. The components of the composition of the present invention may be present independently or mixed with each other, and the present invention is not limited thereto. The components can be in the form of solution or powder. In the present invention, the components are present in the form of a solution, and the components are independent of each other.
In the present invention, the culture system refers to a nutrient for culturing cells under in vitro conditions, and may be referred to as a culture medium. The culture system can be prepared before use, and can also be prepared into finished products for long-term storage.
In the present invention, the basic medium refers to a medium of essential nutrients required for expansion or growth of hematopoietic stem cells. The basic culture medium can be powder or culture solution.
The test material, reagent or experimental device adopted by the invention is a common commercial product and can be purchased in the market. In particular, the following table (table 1) is referred to with respect to the raw material manufacturers involved in the following examples:
table 1 raw material information
StemScanSFEM II is serum free media, StemShell Technologies, Cat # 09655;
recombinant human stem cell factor rhSCF (recombined human stem cell factor), the manufacturer is Stemimmune LLC, the product number is HHM-SF-1000;
recombinant human thrombopoietin rhTPO (recombinant human thrombopoietin), the manufacturer is Stemimmune LLC, the product number is HHM-TP-0100;
recombinant human FMS-like tyrosine kinase 3ligand rhFLT3L (recombined human FMS-like tyrosine kinase 3ligand), abbreviated as FLT3 or FLT3L, and produced by Stemimmune LLC with the product number of HHM-FT-1000;
peripheral blood mononuclear cells PBMC (peripheral blood monuclear cell)
MACS: sorting magnetic beads;
DMSO, DMSO: dimethyl sulfoxide;
PBS phosphate buffer solution;
MethoCultTMGF H4435, semi-solid medium;
CFU-E is called Colony Forming Unit of erythrocytes in its entirety, and is called erythroid Colony-Forming Unit in Chinese;
BFU-E is called Burst Forming Unit of Erythrocyte in its entirety, and Chinese name is Burst red blood cell colony Forming Unit;
CFU-G is called ColonyForming Unit of Granulocyte, and Chinese name is Granulocyte colony forming Unit;
CFU-M is called Colony Forming Unit of Macrophage, and Chinese name is Macrophage Colony Forming Unit;
CFU-GM is called Colony Forming Unit of Granulocyte-macro, Chinese name is Granulocyte-Macrophage Colony Forming Unit;
CFU-GEMM is called Colony Forming Unit of grandilocyte, erythrocyte, macrocage/monocyte, megakaryocyte, mixed Colony, wherein the literal names are granulocyte, erythrocyte, macrophage/monocyte, megakaryocyte Colony Forming Unit;
the preparation of the cord blood hematopoietic stem cells comprises the following steps: diluting the umbilical cord blood by 2-3 times with physiological saline, adding a lymphocyte separation solution, centrifuging at 1500-2000 rpm/min for 20min, taking a mononuclear cell layer (PBMC), washing with the physiological saline, and resuspending to obtain a PBMC cell pellet; CD34+ cells were then isolated by the magnetic bead method.
The composition or culture system provided by the invention can be suitable for in vitro expansion of hematopoietic stem cells, which can be derived from experimental animals (such as mice) or humans. In the embodiment of the present invention, the cord blood hematopoietic stem cells are taken as an example, wherein the cord blood hematopoietic stem cells are negative in hepatitis b, hepatitis c, syphilis, aids, cytomegalovirus, TORCH detection, mycoplasma, chlamydia, G-6PD and thalassemia detection, and the isolated human cord blood hematopoietic stem cells express the following membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation antigen CD34, leukocyte differentiation antigen CD90, and leukocyte differentiation antigen CD49 f.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Example 1:
the method for obtaining a large number of umbilical cord blood hematopoietic stem cells from hematopoietic progenitor cells by using the special conversion culture medium for the human umbilical cord blood hematopoietic stem cells comprises the following steps:
1. peripheral Blood Mononuclear Cells (PBMCs) were obtained.
(1) Collecting 80-120 ml of umbilical cord blood by using a disposable blood bag (containing anticoagulant such as heparin sodium), transferring the umbilical cord blood from the blood bag to a 500ml culture bottle, adding physiological saline for diluting by 2-3 times, uniformly mixing, dropwise adding into 0.4-fold volume of lymphocyte separation liquid, and paying attention to not damage an interface.
(2) Centrifuging at 1500-2000 rpm/min for 20min, wherein the centrifugal tube is divided into four layers from top to bottom due to different densities: the first layer is a plasma layer, the second layer is a ring-shaped 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.
(3) Carefully sucking the second annular milky white mononuclear cell layer (PBMC) into another 50ml centrifuge tube by using a pipette, supplementing physiological saline, and centrifuging at 1500-2000 rpm/min for 5-10 min.
(4) And discarding the supernatant, adding physiological saline for resuspension, centrifuging at 1500-2000 rpm/min for 5-10 min, and discarding the supernatant to obtain PBMC cell pellets.
2. CD34 was obtained from the PBMC using magnetic bead sorting (MACS)+The cord blood is hematopoietic stem and progenitor cells.
(1) Each cord blood PBMC was resuspended in a mixture of 50ul human CD34+ magnetic beads, 50ul FcR blocker (blocker reagent) and 150ul 0.5% BSA and incubated at 4 ℃ for 30 minutes.
(2) At the same time, the magnet and the magnetic force were set up in an ultraclean bench and the ultraviolet ray was irradiated for 30 minutes.
(3) Adding 10ml of sterile PBS, mixing uniformly, centrifuging at 1500-2000 rpm/min for 5-10 min, and discarding the supernatant.
(4) The adsorption column dedicated for MACS was placed in a magnet, 500ul of 0.5% BSA was added for rinsing, and the effluent was caught in a 15ml tube.
(5) Resuspending the PBMC pellet from step (3) with 500ul 0.5% BSA, mixing well, transferring to a MACS adsorption column until the liquid completely flows out.
(6)500ul of 0.5% BSA was washed 3 times, and the column was removed and placed in a 15ml tube.
(7) 1ml of 0.5% BSA was added and the liquid, i.e. containing CD34, was pushed into a 15ml tube by a plunger+The cord blood is hematopoietic stem and progenitor cells.
(8) The solution was diluted and counted, and if necessary, the solution was frozen in liquid nitrogen using a cryoprotectant, Dimethylsulfoxide (DMSO).
3. Will CD34+The cells were stained with flow antibodies CD34, CD90, CD45RA for half an hour and sorted by flow sorter to give CD34+ CD 90-and CD34+ CD45RA + hematopoietic progenitor cells.
4. The CD34+ CD 90-and CD34+ CD45RA + cord blood hematopoietic progenitor cells are inoculated in a cord blood hematopoietic stem cell special conversion culture medium in suspension for culture. Adopting StemBan SFEM II serum-free culture medium, adding 100ng/ml SCF, 100ng/ml FLT3 and 50ng/ml TPO; the cell seeding density in 24-well plates was: CD34+ CD 90-cells were 0.55X 104Well, CD34+ CD45RA + cells 0.13X 104/The volume of the holes is 0.5 mL; compound M (MS275) was added to 1 μ M, control added DMSO (0.1%); standing at 37 deg.C for 5% CO2Culturing in an incubator.
Transformation medium: StemBan SFEM II serum free medium +100ng/ml SCF +100ng/ml FLT3+50ng/ml TPO + 1. mu.M MS 275;
control group medium: StemBan SFEM II serum free medium +100ng/ml SCF +100ng/ml FLT3+50ng/ml TPO + 0.1% DMSO;
5. according to the cell culture state, 500 mul of special conversion culture medium for cord blood hematopoietic stem cells is supplemented every 2 days, a large number of cells can be obtained in 5-10 days, and the amplification factor is about 4-20 times.
Example 2:
the phenotype identification, the survival rate and the purity detection and the function identification of the separated human placental blood hematopoietic stem cells are carried out, and the method comprises the following steps:
1. cell counting
The number of cultured CD34+ CD90+ and CD34+ CD45 RA-hematopoietic stem cells was counted, respectively.
TABLE 2 statistics of cell numbers after culture of CD34+ CD 90-hematopoietic progenitor cells
TABLE 3 cell count after culture of CD34+ CD45RA + hematopoietic progenitors
2. Cell flow analysis
Using a FACS Verse flow meter manufactured by BD company, 20. mu.l of the cell suspension was taken, and 0.2. mu.l each of the following four antibodies dissolved in 0.5% BSA was added: FITC-labeled CD34, PE-labeled CD38, APC-Cy 7-labeled CD45RA, APC-labeled CD 90. After vortexing, each tube was incubated in the dark at room temperature for 15 minutes, an appropriate amount of PBS was added, the mixture was centrifuged at 1600rpm for 5 minutes at room temperature, the supernatant was discarded, 200. mu.l of PBS was added, and the tube was then loaded onto a machine for analysis.
3. Colony Forming Unit analysis
By using MethoCultTMGF H4534 semisolid culture medium, wherein 1 ml/well of culture medium, CD34, is added into a six-well plate+The cell seeding density is 1000 cells/well, and the cells are placed in 5% CO at 37 DEG C2After 14 days of incubator culture, the number of each lineage colony was counted and a photograph was taken.
As can be seen from FIGS. 1a and 1b and FIGS. 2a and 2b, the dedicated transformation medium containing MS275 induced the generation of hematopoietic stem cells of the phenotypes CD34+ CD90+ and CD34+ CD45RA-, whereas the normal medium did not.
Example 3
1. Obtaining umbilical cord blood mononuclear cells;
(1) diluting the umbilical cord blood by 2-3 times by adding physiological saline, uniformly mixing, and then dropwise adding the mixture into 0.4 volume of lymphocyte separation liquid, wherein the interface is not damaged;
(2) centrifuging at 1500-2000 rpm/min for 20min, wherein the centrifugal tube is divided into four layers from top to bottom due to different densities: the first layer is a plasma layer, the second layer is a ring-shaped 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;
(3) carefully sucking the second annular milky white mononuclear cell layer (PBMC) into another 50ml centrifuge tube by using a suction tube, supplementing physiological saline, and centrifuging for 5-10 min by using 1500-2000 rpm/min again;
(4) and (3) discarding the supernatant, adding physiological saline for resuspension, finally centrifuging at 1500-2000 rpm/min for 5-10 min, and discarding the supernatant again to obtain PBMC cell masses.
2. Obtaining CD34+ cord blood hematopoietic stem cells from the PBMC by using MACS;
(1) resuspend each cord blood PBMC with a mixture of 50. mu.L human CD34+ magnetic beads, 50. mu.L FcR blocker reagent and 150. mu.L 0.5% BSA, incubate for 30min at 4 ℃;
(2) meanwhile, the magnet and the magnetic force are erected in an ultra-clean bench for ultraviolet irradiation for 30 min;
(3) adding 10ml of sterile PBS, uniformly mixing, centrifuging at 1500-2000 rpm/min for 5-10 min, and then removing the supernatant;
(4) putting the adsorption column special for MACS into a magnet, adding 500ul of 0.5% BSA for rinsing, and catching the effluent liquid with 15 mltube;
(5) resuspending 500 mu L of 0.5% BSA to obtain the PBMC pellet in the step 3) of obtaining umbilical cord blood mononuclear cells, uniformly mixing, transferring the PBMC pellet to a MACS special adsorption column, and allowing the liquid to completely flow out;
(6) washing with 500 μ L of 0.5% BSA for 3 times, removing the adsorption column, and placing in 15ml tube;
(7) 1ml of 0.5% BSA was added and the liquid, i.e., cord blood hematopoietic stem cells containing CD34+, was pushed into a 15ml tube by a plunger.
Example 4
The factor content in each group of media is shown in table 4:
TABLE 4 factor content in the culture media of each group
| MS275 | SCF | TPO | FLT3 | |
| Group 1 | 1μM | 80ng/ml | 30ng/ml | 90ng/ml |
| Group 2 | 1μM | 100ng/ml | 50ng/ml | 100ng/ |
| Group | ||||
| 3 | 1μM | 120ng/ml | 70ng/ml | 110ng/ |
| Group | ||||
| 4 | 0 | 80ng/ml | 30ng/ml | 90ng/ml |
The substances were added to the StemBan SFEM II serum-free medium at the concentrations of Table 1.
The CD34+ cord blood hematopoietic stem cells obtained in example 1 were cultured by inoculating them in suspension in each cell culture medium. The density of cell inoculation in 24-well plate is 1X 104cells/well, volume 0.5mL, placed at 37 ℃ in 5% CO2Culturing in an incubator. According to the cell culture state, 500 mu L of fresh cell culture medium of each group is supplemented every 2 days, more hematopoietic stem cells can be obtained in 5-10 days, and the amplification multiple is about 4-20 times.
Effect detection
The hematopoietic stem cells of umbilical cord blood cultured in each group of example 4 were subjected to cell counting, phenotypic identification, and colony forming unit analysis.
1. Cell counting
Cells cultured in MS275 or DMSO at day 5 were counted, respectively, and fold expansion of cell number compared to day 0 was calculated. The culture results of each group of culture media are shown in table 5:
TABLE 5 statistical table of the number expansion fold of cells for each set of conditions
The results show that: compared with the group 4, the group added with MS275 has more CD34+ CD90+ cells and larger amplification fold, and the amplification effects of the groups 1-3 are remarkably different from the group 4 by statistical analysis, wherein p is less than 0.05. In groups 1 to 3, group 2 had a better amplification effect.
2. Cell flow analysis
Flow analysis was performed on MS275 or DMSO cultured CD34+ cells at day 0, day 5, respectively. 20. mu.L of the cell suspension was taken by a FACS Verse flow meter manufactured by BD Co, and FITC-labeled CD34, PE-labeled CD38, APC-Cy 7-labeled CD45RA, and APC-labeled CD90 dissolved in 0.5% BSA were added. After vortexing, each tube was incubated in the dark at room temperature for 15min, an appropriate amount of PBS was added, the mixture was horizontally centrifuged at 1600rpm at room temperature for 5min, the supernatant was discarded, 200. mu.L of PBS was added, and the mixture was then loaded onto a computer for analysis. The results of the tests of groups 2 and 4 are shown in FIG. 3. The results show that the proportion of CD34+ CD90+ cells obtained by expansion of group 2 is higher relative to group 4 without addition of MS275, which indicates that the hematopoietic stem cells obtained by expansion of group 2 are more primitive, have stronger differentiation potential for reconstructing a blood system, and can more effectively support clinical treatment needs. The ratio of CD34+ CD90+ cells in the cells obtained by amplification in groups 1 and 3 was similar to that in group 2.
3. Colony Forming Unit analysis
Colony forming unit analysis was performed on MS275 or DMSO cultured CD34+ cells at day 0, day 5, respectively. By using MethoCultTMGF H4435 semi-solid culture medium, adding 1ml of culture medium into six-hole plate, inoculating CD34+ cells at density of 500 cells/hole, and placing at 37 deg.C with 5% CO2After 14 days of incubator culture, the number of each lineage colony was counted and a photograph was taken. The colony formation of each lineage under an inverted microscope is shown in the graph of 4, and the colony formation number is shown in the graph of 6:
TABLE 6 number of colony formations after cultivation of each group
As shown in Table 6, the group added with MS275 obtained more cell colonies than group 4, and the statistical analysis showed that the colony numbers of groups 1-3 were significantly different from that of group 4, with p < 0.05. In groups 1 to 3, group 2 had the largest number of colonies.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A culture system comprising basal medium, MS275, TPO, SCF, and FLT 3L; wherein,
the concentration of the MS275 is 1 mu mol/L;
the concentration of the TPO is 30 ng/mL-70 ng/mL;
the concentration of the SCF is 80 ng/mL-120 ng/mL;
the concentration of the FLT3L is 90 ng/mL-110 ng/mL.
2. The culture system of claim 1, wherein the basal medium is StemPro, RPMI1640, IMDM, α -MEM, or StemSpan SFEM II.
3. A method for promoting the conversion of hematopoietic progenitor cells into hematopoietic stem cells, comprising culturing the hematopoietic progenitor cells in the culture system according to claim 1 or 2.
4. The method of claim 3, wherein the hematopoietic progenitor cells are CD34+ CD90 "and CD34+ CD45RA + cord blood hematopoietic progenitor cells.
5. The method of claim 4, wherein the culture inoculated CD34+ CD 90-cell density is 0.1-10 x 104cell/mL, CD34+ CD45RA + cell density of 0.1-10 × 104cells/mL。
6. A method for expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the culture system according to any one of claims 1 or 2.
7. The method according to claim 6, wherein the hematopoietic stem cells are cord blood hematopoietic stem cells, or hematopoietic stem cells obtained by culturing according to any one of claims 3 to 5; the density of initial cell inoculation is 0.1-10 multiplied by 104cells/mL。
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| CN2018116259230 | 2018-12-28 | ||
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