CN109593716B

CN109593716B - Culture system and method for expanding hematopoietic stem cells and application thereof

Info

Publication number: CN109593716B
Application number: CN201811628989.5A
Authority: CN
Inventors: 肖雄; 郭潇; 刘德芳; 孙忠杰
Original assignee: Nuowei Technology Beijing Co ltd
Current assignee: Nuowei Technology Beijing Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2021-04-09
Anticipated expiration: 2038-12-28
Also published as: CN109593716A

Abstract

The invention relates to the technical field of cell culture, in particular to a culture system and a method for amplifying hematopoietic stem cells and application thereof. Research shows that in the process of expansion culture of umbilical cord blood hematopoietic stem cells, the cell factor and the EPZ6438 are added, so that the effects of increasing the number of the hematopoietic stem cells and improving the formation capacity of hematopoietic stem cell CFU colonies 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.

Description

Culture system and method for expanding hematopoietic stem cells and application thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture system and a method for amplifying hematopoietic stem cells and application thereof.

Background

The hematopoietic stem cell transplantation technology is a common and effective treatment means for clinically treating various blood diseases and immune system diseases such as leukemia, lymphoma, aplastic anemia, thalassemia and the like. Hematopoietic stem cells are generally derived from three sources: bone marrow, peripheral blood and cord blood. Compared with bone marrow and peripheral blood hematopoietic stem cells, the umbilical cord blood hematopoietic stem cells are convenient to obtain, rich in sources, free of damage and side effects on donors, and therefore become an important source of hematopoietic stem cell transplantation donors.

At present, the bottleneck of cord blood hematopoietic stem cell transplantation technology is that the cell content is low, and the number of hematopoietic stem cells and progenitor cells contained in one cord blood is not enough to quickly restore the immune system of an adult patient, so that the fatality rate of opportunistic infection is increased. The current tentative strategy is transplantation of two umbilical cord blood, i.e. one patient receives transplantation of two umbilical cord blood after marrow removal, but this increases the difficulty of matching HLA of donors, so a method for amplifying umbilical cord blood hematopoietic stem cells is urgently needed to obtain sufficient hematopoietic stem cells for transplantation.

Many attempts have been made to expand umbilical cord blood hematopoietic stem cells in vitro, but none 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.

EPZ6438 (chemical formula C)₃₄H₄₄N₄O₄CAS No. 1403254-99-8) is a specific inhibitor of histone methyltransferase EZH 2. In the field of clinical treatment, EPZ6438, also known as Tazemetostat, is a tentative anticancer drug used to treat lymphoma, non-small cell lung cancer, melanoma, and melanomaEndometrial cancer has entered into phase I and II clinical trials, and the maintenance and expansion of human hematopoietic stem cells by EPZ6438 has not been reported.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide the use of EPZ6438, and the research of the present invention shows that EPZ6438 can significantly increase the total amount of cells obtained by in vitro expansion of hematopoietic stem cells.

The invention provides application of EPZ6438 in promoting hematopoietic stem cell expansion.

The research of the invention shows that the addition of EPZ6438 obviously improves the total amount of cells obtained by in-vitro expansion of umbilical cord blood hematopoietic stem cells, and when EPZ6438 is used together with TPO, SCF and FLT3L, the synergistic effect can be generated, thereby better promoting the in-vitro expansion of the hematopoietic stem cells.

The invention also provides a composition for promoting expansion of hematopoietic stem cells, which consists of EPZ6438, TPO, SCF and FLT 3L.

In the composition for promoting expansion of hematopoietic stem cells, the mass ratio of the EPZ6438 to the TPO to the SCF to the FLT3L is (57.2-5720): (30-70): (80-120): (90-110).

In some embodiments, the composition has a mass ratio of EPZ6438, TPO, SCF, and FLT3L of 114.4:30:80: 90.

in some embodiments, the composition has a mass ratio of EPZ6438, TPO, SCF, and FLT3L of 114.4:50:100: 100.

in some embodiments, the composition has a mass ratio of EPZ6438, TPO, SCF, and FLT3L of 114.4:70: 120: 110.

the components of the composition provided by the present invention may be present independently or mixed with each other, which is not limited in the present invention. 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. Wherein, the solution of EPZ6438 is prepared by DMSO, and the concentration of the mother liquor is 100 mmol/L.

The composition provided by the invention is used as an additive of a culture medium and is used for promoting the in vitro amplification of hematopoietic stem cells.

The invention also provides an expanded hematopoietic stem cell culture medium, which comprises a basal medium and EPZ 6438.

In the culture medium for amplifying hematopoietic stem cells, the concentration of the EPZ6438 is 0.1 to 10 mu mol/L.

In a specific embodiment, the concentration of EPZ6438 is 0.2. mu. mol/L.

The culture medium for expanding hematopoietic stem cells provided by the invention also comprises TPO, SCF and FLT 3L.

In 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 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 present invention, the base medium is StemPro, RPMI1640, IMDM, alpha-MEM or StemBan SFEM II. In some embodiments, the basal medium is StemSpan SFEM II.

In some embodiments, the media provided herein include StemBan SFEM II media, 0.2. mu. mol/L EPZ6438, 50ng/mL TPO, 100ng/mL SCF, and 100ng/mL FLT 3L.

In some embodiments, the media provided herein include StemBan SFEM II media, 0.2. mu. mol/L EPZ6438, 30ng/mL TPO, 80ng/mL SCF, and 90ng/mL FLT 3L.

In some embodiments, the media provided herein include StemBan SFEM II media, 0.2. mu. mol/L EPZ6438, 70ng/mL TPO, 120ng/mL SCF, and 110ng/mL FLT 3L.

The culture medium can be prepared just before use, and can also be prepared into a finished product for long-term storage. The preparation method 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 described by 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 of the mother liquor is DMSO.

The method for expanding hematopoietic stem cells according to the present invention comprises culturing hematopoietic stem cells in the medium according to the present invention.

In the method of the present invention, the hematopoietic stem cells are cord blood hematopoietic stem cells; the density of the inoculation is 2X 10⁴cells/mL。

The culture conditions were 37 ℃ and 5% CO₂. Every 2 days, fresh medium provided by the present invention was supplemented. The amplification times of the cells are 4-20 times after 5-10 days of culture.

Research shows that in the process of expansion culture of umbilical cord blood hematopoietic stem cells, the cell factor and the EPZ6438 are added, so that the effects of increasing the number of the hematopoietic stem cells and improving the formation capacity of hematopoietic stem cell CFU colonies 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.

Drawings

FIG. 1 is a graph showing the analysis of the surface antigen expression profiles on day 5 of hematopoietic stem cells of groups 1 and 4; wherein (a) shows the expression conditions of CD45 and CD34, (b) shows the expression conditions of CD34 and CD45RA, and (c) shows the expression conditions of CD34 and CD 90;

FIG. 2 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 provides the application of EPZ6438, and a person skilled in the art can use the content to review the content and appropriately improve the process parameters to realize the EPZ. 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.

The test material, reagent or experimental device adopted by the invention is a common commercial product and can be purchased in the market.

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.

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), the manufacturer is Stemimmune LLC, the product number is HHM-FT-1000;

EPZ6438, Sigma-Aldrich;

peripheral blood mononuclear cells PBMC (peripheral blood monuclear cell)

MACS: sorting magnetic beads;

DMSO, DMSO: dimethyl sulfoxide;

PBS phosphate buffer solution;

MethoCult^TMGF 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 composition or culture medium 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 invention is further illustrated by the following examples:

example 1

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) resuspending each cord blood PBMC with a mixture of 50. mu.L human CD34+ magnetic beads, 50. mu.L FcR blockeregent and 150. mu.L 0.5% BSA, and incubating at 4 ℃ for 30 min;

(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 15ml tube;

(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 2

The factor content in each group of media is shown in table 1:

TABLE 1 factor content in the culture media of each group

	EPZ6438	SCF	TPO	FLT3
					Group 1	0.2μM	80ng/ml	30ng/ml	90ng/ml
Group
	2	0.2μM	100ng/ml	50ng/ml	100ng/ml
Group 3						0.2μM	120ng/ml	70ng/ml	110ng/ml
	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, 5% CO at 37 ℃₂Culturing 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 2 were subjected to cell counting, phenotypic identification, and colony forming unit analysis.

1. Cell counting

The cells cultured in EPZ6438 or DMSO at day 5 were counted, respectively, and the fold expansion of the cell number compared to day 0 was calculated. The culture results of each group of culture media are shown in Table 2:

TABLE 2 statistical table of the number expansion fold of cells under each set of conditions

The results show that: compared with the group 4, the group added with the EPZ6438 has the advantages that the number of the obtained CD34+ CD90+ cells is more, the amplification multiple is larger, the amplification effect of the groups 1-3 is remarkably different from that of the group 4 through statistical analysis, and 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 day 0, day 5 EPZ6438 or DMSO cultured CD34+ cells, 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. 1. The results show that the proportion of CD34+ CD90+ cells obtained by the expansion of the group 2 is higher compared with the group 4 without adding EPZ6438, which shows that the hematopoietic stem cells obtained by the expansion of the group 2 are more primitive, have stronger differentiation potential for reconstructing a blood system and can more effectively support the clinical treatment requirement. 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 day 0, day 5 EPZ6438 or DMSO cultured CD34+ cells, respectively. By using MethoCult^TMGF H4435 semisolid culture medium, adding 1ml culture medium into six-well plate, inoculating CD34+ cells at density of 500 cells/well, 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 2, and the colony formation number is shown in the graph of 3:

TABLE 3 number of colony formations after cultivation for each group

As shown in Table 3, the group added with EPZ6438 obtained more cell colonies than group 4, and the colonies of groups 1-3 were significantly different from group 4 by statistical analysis, 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 it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

Use of EPZ6438 to promote hematopoietic stem cell expansion in vitro.
2. A composition for promoting hematopoietic stem cell expansion, which consists of EPZ6438, TPO, SCF and FLT3L, wherein the mass ratio of the EPZ6438, the TPO, the SCF and the FLT3L is 114.4:30:80:90 or 114.4:50:100:100 or 114.4:70: 120: 100.
3. an expanded hematopoietic stem cell culture medium comprising basal medium, EPZ6438, TPO, SCF and FLT 3L;

wherein the concentration of EPZ6438 is 0.2 μmol/L, the concentration of SCF is 80ng/mL, the concentration of TPO is 30ng/mL, and the concentration of FLT3L is 90 ng/mL;

or the concentration of EPZ6438 is 0.2 μmol/L, the concentration of SCF is 100ng/mL, the concentration of TPO is 50ng/mL, and the concentration of FLT3L is 100 ng/mL;

or the concentration of EPZ6438 is 0.2. mu. mol/L, the concentration of SCF is 120ng/mL, the concentration of TPO is 70ng/mL, and the concentration of FLT3L is 110 ng/mL.
4. The culture medium according to claim 3, wherein the basal medium is StemPro, RPMI1640, IMDM, α -MEM or StemSpan SFEM II.
5. A method for in vitro expansion of hematopoietic stem cells, comprising culturing hematopoietic stem cells in the medium according to claim 3.
6. The method of claim 5, wherein the hematopoietic stem cells are cord blood hematopoietic stem cells; the density of the inoculation is 2X 10⁴cells/mL。