CN110511909B - Growth factor composition for in vitro expansion of hematopoietic stem cells and application thereof - Google Patents
Growth factor composition for in vitro expansion of hematopoietic stem cells and application thereof Download PDFInfo
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- CN110511909B CN110511909B CN201910690702.XA CN201910690702A CN110511909B CN 110511909 B CN110511909 B CN 110511909B CN 201910690702 A CN201910690702 A CN 201910690702A CN 110511909 B CN110511909 B CN 110511909B
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Abstract
The invention discloses a growth factor composition for in vitro expansion of hematopoietic stem cells, which comprises: interferon alpha, heparin and interleukin 12. The invention also discloses application of the growth factor composition in-vitro expansion of hematopoietic stem cells, and the growth factor composition is added into a culture solution for in-vitro culture of the hematopoietic stem cells, so that the hematopoietic stem cells can be quickly and effectively expanded, the expansion quantity of the in-vitro hematopoietic stem cells can be increased by 7689 times, and the problems of low culture efficiency and insufficient quantity in the prior art are solved.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a growth factor composition for in vitro expansion of hematopoietic stem cells and application thereof.
Background
Bone marrow transplantation, i.e., transplantation of Hematopoietic Stem Cells (HSCs), is currently the most mature method of stem cell therapy in clinical applications, and is also considered to be the only therapeutic approach for treating diseases such as leukemia, aplastic anemia, and lymphoma. So far, HSCs are transplanted according to bone marrow matching, peripheral hematopoietic stem/progenitor cells or umbilical cord blood after donor bone marrow mobilization are collected and directly transplanted without in vitro culture, and simultaneously, the problem of difficulty in finding matched HSCs exists; in addition, matched donor HSCs require donors to provide a large amount of hematopoietic stem cells, which has certain influence on the physiology and psychology of the donors, and is also the main reason why many hematopoietic donors temporarily regress the donation in China in recent years. If the donor is cord blood, since the cord blood HSCs are in small quantities, multiple copies of the cord blood are needed to meet the needs of adult patients, which causes great difficulty in collecting the blood source, and this also leads to many views that are not useful in storing cord blood. Based on the problem that the presence of the above-mentioned various factors causes an insufficient amount of HSCs, it is difficult to satisfy the amount required for transplantation of HSCs. In the in vitro amplification, the environment in vivo is simulated outside the organism, and certain nutrition and stimulating factors are given to promote cell proliferation. The number of the hematopoietic stem cells is increased by an in vitro amplification technology, and the self-renewal capacity of the hematopoietic stem cells is not reduced so as to meet the requirement of bone marrow transplantation, which is a direction of research of domestic and foreign scientists.
Initially, HSCs were cultured in vitro using stromal cells as helper cells, which secrete large amounts of growth factors or cytokines, which are also key factors in promoting the proliferation of HSCs. For many years, the discovery of cytokines that promote the proliferation of HSCs has been the key topic of scientists at home and abroad for studying HSCs, and a plurality of cytokines and active substances have been reported to promote the proliferation and/or self-renewal of HSCs in vitro. From the literature at home and abroad today, stem cell growth factor (SCF), interleukin 3(IL3) and interleukin 6(IL6) are typically the 3 cytokines that must be added to culture mouse and human hematopoietic stem cells in vitro, while different laboratories or researchers may choose fibroblast growth factor (FGF1 or FGF2) [ de Haan, g., E, 2003; yeoh JS, R, 2006), insulin-like growth factor (IGF2) [ Zhang CC, 2004], Thrombopoietin (TPO) [ Sauvaeau G, 2004; ko KH, 2011], Erythropoietin (EPO), Fms-like tyrosine kinase receptor 3 ligand (Fltl3) [ punton LE, 2000; ko KH, 2011), granulocyte-macrophage colony stimulating factor (GM-CSF), and other cytokines, and is added into cell culture medium for proliferation of HSCs in vitro. At present, when hematopoietic stem cells are cultured in vitro by adopting single or multiple cytokines, the proliferation of the hematopoietic stem cells can be increased to a certain extent, but when the hematopoietic stem cells are cultured by multiple cytokines together, different components may have antagonism or have the coexistence of growth factors and cytokines with the same functions, so that the problems of low in vitro culture efficiency or acquired cell defects and the like exist, and the method is difficult to be applied to scientific research or industrial production, such as: after human cord blood CD34+ cells are cultured in vitro with IL-3, IL-6 and SCF or IL-11, SCF and FL for 6 days, the expanded cells interfere bone marrow homing due to the change of expression of VLA-4 and VLA-5, so that the consistency of expanded HSCS is poor [ zhai qiongli, 2004], which causes difficult clinical application and can not solve the problem of insufficient hematopoietic stem cell number. At present, various cytokines exist to play respective roles, and the amplification efficiency is poor. Therefore, it is necessary to design a different cytokine composition to achieve the optimal expansion effect, so as to satisfy the problems of low expansion efficiency and insufficient quantity of hematopoietic stem cells.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to provide a composition for in vitro expansion of hematopoietic stem cells, which can rapidly and efficiently expand hematopoietic stem cells in vitro by using the growth factor and the active substance of the composition, thereby increasing the number of expanded hematopoietic stem cells in vitro and securing the self-renewal ability thereof.
To achieve these objects and other advantages in accordance with the present invention, there is provided a growth factor composition for in vitro expansion of hematopoietic stem cells, comprising: interferon alpha, heparin and interleukin 12.
Preferably, the method further comprises the following steps: stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor.
The invention also provides an application of the growth factor composition in-vitro expansion of hematopoietic stem cells, which is to mix a DMEM culture solution containing 10% serum with the growth factor composition to expand the hematopoietic stem cells in vitro.
Preferably, the growth factor comprises interferon alpha, heparin, interleukin 12, stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor.
Preferably, the interferon alpha, heparin, interleukin 12, stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor 2(IGF2) are added at concentrations of 1-100ng/ml, respectively.
Preferably, the interferon alpha addition concentration is 50ng/mL, the heparin addition concentration is 10ug/mL, the interleukin 12 addition concentration is 10ng/mL, the stem cell growth factor addition concentration is 100ng/mL, the interleukin 3 addition concentration is 100ng/mL, the thrombopoietin addition concentration is 100ng/mL, the Fms-like tyrosine kinase receptor 3 ligand addition concentration is 100ng/mL, and the insulin-like growth factor addition concentration is 10 ng/mL.
Preferably, the in vitro expanded hematopoietic stem cells are obtained from a sample of mouse or human bone marrow or umbilical cord blood hematopoietic stem cells.
Preferably, the conditions for in vitro expansion of hematopoietic stem cells are: culturing at 37 deg.C under 5% carbon dioxide for 14-21 days.
The invention at least comprises the following beneficial effects:
the interleukin 12(IL12) contained in the growth factor composition disclosed by the invention is an important regulatory factor for promoting TH1 response, can promote TH0 cells to differentiate to TH1 cells and inhibit the differentiation to TH2 cells, and the Interferon (IFN) is one of main secretions of TH1 cytokines, can promote the expression of IL12 receptor mRNA in Th0 cells through the simultaneous action of IL12 and IFN alpha, and also increases the binding probability of IL12 and IL12 receptors; in addition, IL12 can reverse the inhibition of IL12 receptor mRNA by allergen and also increase the combination of IL-12 and IL-12R, therefore, IL12 and IFN alpha are added together to culture hematopoietic stem cells in vitro, which is favorable for enhancing the expression of histocompatibility antigen and FC receptor on the surface of peripheral blood mononuclear cells. Heparin (heparin) is a mucopolysaccharide containing sulfate groups, can strengthen antithrombin III (AT-III) to inactivate serine protease, thereby having various effects of preventing thrombin formation, antithrombin and preventing platelet aggregation, and the like, and can prevent the coagulation of hematopoietic stem cells after in vitro amplification by mixing the heparin with IL-12 and IL-12R, thereby greatly improving the amplification efficiency. Furthermore, the hematopoietic stem/progenitor cells can self-secrete or paracrine growth factors such as FGF, and heparin can promote the binding of FGF and FGF receptors on the surfaces of the hematopoietic stem cells and activate signaling pathways such as AKT and ERK to promote cell proliferation. According to the application, through the addition of the growth factor composition IL-12, IFN alpha and heparin and the combination of other cytokines and in-vitro amplification culture media, the mouse/human hematopoietic stem cell in-vitro amplification can be 7689 times amplified, compared with the prior art, a large number of hematopoietic stem cells can be quickly obtained through in-vitro amplification technology, the application to scientific research or industrial production is facilitated, and a foundation is laid for bone marrow transplantation of modern medicine through in-vitro amplification hematopoietic stem cells.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows the results of screening different growth factors according to the present invention;
FIG. 2 shows the number of CD117+ mouse hematopoietic stem cells expanded by adding growth factors at different concentrations according to the present invention;
FIG. 3 shows the expansion efficiency of hematopoietic stem cells of CD117+ mice when different concentrations of growth factors are added;
FIG. 4 is a graph showing the potential of CD117+ mouse hematopoietic stem cells to generate Colonies (CFU) in different growth factor compositions according to the present invention;
FIG. 5 shows the number of hematopoietic stem cells expanded by human cord blood cells of the present invention in various growth factor compositions.
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides a growth factor composition for in vitro expansion of hematopoietic stem cells, which comprises: interferon alpha, heparin and interleukin 12.
In a preferred embodiment, the method further comprises: stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor.
The invention also provides application of the growth factor composition in-vitro expansion of hematopoietic stem cells, which is characterized in that the application is to mix DMEM culture solution containing 10% serum with the growth factor composition to expand the hematopoietic stem cells in vitro.
In a preferred embodiment, the growth factor comprises interferon alpha, heparin, interleukin 12, stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor.
In a preferred embodiment, the interferon alpha, heparin, interleukin 12, stem cell growth factor, interleukin 3, thrombopoietin, ligand for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor are added at concentrations of 1 to 100ng/ml, respectively.
In a preferred embodiment, the interferon alpha is added at a concentration of 50ng/mL, the heparin is added at a concentration of 10ug/mL, the interleukin 12 is added at a concentration of 10ng/mL, the stem cell growth factor is added at a concentration of 100ng/mL, the interleukin 3 is added at a concentration of 100ng/mL, the thrombopoietin is added at a concentration of 100ng/mL, the Fms-like tyrosine kinase receptor 3 ligand is added at a concentration of 100ng/mL, and the insulin-like growth factor is added at a concentration of 10 ng/mL.
In a preferred embodiment, the in vitro expanded hematopoietic stem cells are obtained from human bone marrow or umbilical cord blood hematopoietic stem cell samples and mouse and other mammalian hematopoietic stem cells.
In a preferred embodiment, the conditions for in vitro expansion of hematopoietic stem cells are: culturing at 37 deg.C under 5% carbon dioxide for 14-21 days.
Interferon alpha (IFN alpha), a soluble substance, can enhance the expression of histocompatibility antigen and peripheral blood mononuclear cell surface FC receptor, inhibit the proliferation of lymphocytes, enhance the cytotoxic activity of macrophages, regulate immune homeostasis function, and the like.
Heparin (heparin) is a mucopolysaccharide containing sulfate groups, can promote the binding of FGF and FGFR, and can strengthen the inactivation of serine protease by antithrombin III (AT-III), thereby having various effects of preventing thrombin formation, antithrombin and preventing platelet aggregation.
Interleukin 12(IL12) is a heterodimeric cytokine and is considered to be a key regulator in the cellular immune response.
The stem cell growth factor (SCF) is a combination of protein and enzyme which can stimulate the growth of autologous endogenous stem cells, is a stimulating factor of early hematopoietic stem/progenitor cells, and plays an important role in maintaining the survival of hematopoietic cells, promoting the proliferation and differentiation of the hematopoietic cells and regulating the growth and development of the hematopoietic cells.
Interleukin-3 (IL3) is an important member of the interleukin family, a glycoprotein produced by T lymphocytes and capable of stimulating the proliferation and differentiation and enhancing the function of cells involved in the immune response.
Thrombopoietin (TPO) is a glycosylated polypeptide chain comprising 834 amino acids that physiologically regulates the proliferation and differentiation of hematopoietic progenitor cells evolving into mature megakaryocytes.
Fms-like tyrosine kinase receptor 3 ligand (Fltl3)) is an early hematopoietic growth factor involved in hematopoietic regulation and may act synergistically with a variety of cytokines. The ligand of Flt3 can regulate the proliferation and differentiation of primitive hemopoietic stem cell/progenitor cell after being combined with Flt3, mobilize and stimulate the proliferation and differentiation of myeloid and lymphoid progenitor cell, dendritic cell, natural killer cell and natural killer dendritic cell, and is the most important growth factor of dendritic cell.
Insulin-like growth factors (IGFs), a class of multifunctional cell proliferation regulators, now including both IGF-1 and IGF-2, have been discovered, and IGF2 used herein can promote the proliferation rate of hematopoietic stem cells.
1. Object
Animal-derived cells: (ii) obtained from mouse CD117+ stem/progenitor cells;
human cells: hematopoietic stem cell samples from bone marrow, umbilical cord blood, not affected by hematological disorders (from maternal umbilical cord blood, with maternal informed notes).
2. Test method
2.1 obtaining mouse CD117+ stem/progenitor cells comprising the steps of:
1) mice were sacrificed by CO2 asphyxiation;
2) fixing the mouse on a dissection plate, and directly spraying 75% ethanol on the surface of the mouse for disinfection;
3) transferring the sterilized mice to a biosafety cabinet;
4) subtracting the skin of the rear leg of the mouse to expose the leg of the mouse, and separating the foot of the mouse at the ankle of the mouse;
5) fascia and muscle behind the knee joint of the mouse are cut off, the knee joint is fixed by forceps, and the femur near the knee joint is broken towards the joint direction. Pushing the muscle tissue down to the pelvis, exposing and cutting the whole femur;
6) fixing the knee joint by using a forceps, breaking the position of the tibia close to the knee joint in the opposite direction, and downwards pushing muscle tissues to take down the tibia;
7) the mouse femur and tibia were placed in 6-well plates with 3ml sterile PBS;
8) sucking PBS (phosphate buffer solution) for soaking thighbone and shinbone by using a 1ml syringe, flushing bone marrow from bones with openings at two ends, repeating the steps for several times until the bones are flushed and whitened, and then blowing the flushed flushing liquid to disperse cells by using the syringe;
9) placing a 70-micron filter on a 50ml centrifuge tube, adding the cell suspension obtained in the step 8 into the filter, and centrifuging for 10min at 1200 RPM;
10) the bone marrow cells were transferred to sterile 5mL sterile flow cytometry tubes, concentrated to a volume of 200 μ L by centrifugation, and then PE-labeled anti-CD 117 antibody was added, following procedures detailed in kit instructions (EasySep)TMMouse CD117(cKIT) Positive Selection kit, STEMCELL) and magnetic bead sorting method were used to obtain CD117+ murine hematopoietic stem cells.
2.2 in vitro expansion of CD117+ murine hematopoietic Stem cells
2.2.1 exploring the optimal composition of different growth factors
7000 CD117+ mouse hematopoietic stem cells obtained from 2.1 above were seeded in 96-well plates (round bottom) of 10% serum-containing DMEM (Hyclone-DMEM/HIGH GLUCOSE) at 200. mu.L of DMEM (10% serum-containing) per well, growth factors were added in different combinations, 5 replicate wells were set for each combination, and 5% CO was plated at 37 ℃ in each combination2After culturing in vitro for 14-21d, the cells in the culture product were collected and the total number of cells was calculated.
Analyzing the percentage of each cell group by flow cytometry, which comprises the following steps: adding 4uL of the antibody containing the following flow-down antibody to each sampleMixing the antibodies, dyeing for 30min at room temperature or 4 ℃, adding PBS buffer solution, washing for 1-2 times, adding 200ul PBS buffer solution, and detecting by a flow cytometer (BD Accuri)TM C6Flow Cytometer,BD)。
Wherein, the flow-type antibodies are not specially indicated and are all purchased from eBioscience, wherein Lin comprises FITC labeled CD11B, Gr-1, TER-119, CD3, CD4, CD8, B220 and CD 41; APC-labeled c-Kit and PE-cy 5.5-labeled Sca-1.
Preparation of PBS buffer: weighing NaCl 8g, KCL0.2g and Na2HPO4·12H2PO4Dissolving 0.24g in 900mL double distilled water, adjusting pH to 7.4 with hydrochloric acid, adding water to constant volume of 1L, and storing at normal temperature for use. The sterile PBS was obtained from the prepared PBS buffer at 121 deg.C and autoclaved for 20-30 min.
The method for adding the growth factors with different combinations comprises the following steps: (INF. alpha. is 10ng/mL, heparin concentration is 20ug/mL, and the concentration of other various factors is 50ng/mL)
The growth factor with the highest amplification efficiency was obtained by adding IL3, IL6, INF α, EPO, TPO, heparin, fibroblast growth factor 1(FGF1), fibroblast growth factor 2(FGF2), fibroblast growth factor 9(FGF9), and fibroblast growth factor 18(FGF18) to DMEM culture solution containing 10% serum + SCF as a control for culturing mononuclear CD117+ cells, respectively, and was named a.
1. In addition to the above screening results, Fltl3, IGF2, Hepatocyte Growth Factor (HGF), TPO, IL12, stromal cell derived factor 1(SDF1), Epidermal Growth Factor (EGF), growth inhibitory specific gene product 6(Gas6), EPO, and M-CSF were added to DMEM medium containing 10% serum + SCF + a as a control for culturing mononuclear CD117+ cells, respectively, to obtain a growth factor having the highest amplification efficiency, which was designated as B.
2. Based on the screening results 1, the growth factor having the highest amplification efficiency was obtained by adding INF α, IGF2, EGF, IL12, FGF1, FGF2, FGF9, FGF18, and platelet-derived factor (PDGF) to DMEM culture solution containing 10% serum + SCF + a + B as a control for culturing mononuclear CD117+ cells, and was named C.
3. Based on the screening results of 2, the growth factor having the highest amplification efficiency was obtained by adding IL7, heparin, SDF1, and fibroblast growth factor 21(FGF21) to DMEM medium containing 10% serum + SCF + a + B + C as a control for culturing mononuclear CD117+ cells, and was designated as D.
4. Based on the results of the screening 3, the growth factor having the highest amplification efficiency was obtained by adding IL6, IGF2, IL12, EPO, HGF and Fltl3 to DMEM medium containing 10% serum + SCF + a + B + C + D as a control for culturing mononuclear CD117+ cells, and was designated as E.
5. In addition to the results of the 4 screening, a growth factor having the highest amplification efficiency was obtained by adding osteogenesis protein 2(BMP2), IGF2, Fltl3, Wnt3a, SDF1, and EPO to DMEM medium containing 10% serum + SCF + a + B + C + D + E as a control for culturing mononuclear CD117+ cells, and the growth factor was named F.
6. Based on the results of the 5-step screening, a growth factor having the highest amplification efficiency was obtained by adding IGF2, EPO, Wnt3a, and SDF1 to DMEM medium containing 10% serum + SCF + a + B + C + D + E + F as a control for culturing mononuclear CD117+ cells, respectively, and was designated as G.
7. In addition to the 6 screening results, a composition of growth factors that promote the expansion of mononuclear CD117+ cells (a standard for determining the composition to be obtained when each of the added growth factors inhibits or exerts the same effect as the control) was obtained by adding EPO, Wnt3a, and SDF1 to DMEM medium containing 10% serum + SCF + a + B + C + D + E + F + G as a control for culturing mononuclear CD117+ cells.
2.2.2 exploring the optimal concentration of different growth factors in the optimal composition
On the basis of the optimal growth factor combination obtained by 2.2.1 exploration, 7000 CD117+ mouse hematopoietic stem cells obtained from 2.1 are taken, and in a 96-well plate (round bottom) containing 10% serum DMEM culture solution, the DMEM culture solution (containing 10% serum) dosage in each well is 200 μ L, and according to the experimental requirements, growth factors and growth factor combinations in the optimal composition with different concentrations are added, wherein the addition concentration of the single growth factor is as follows: SCF, IL3, TPO, Fltl3, IGF2, INF alpha and IL12 were added at concentrations of 1ng/mL, 10ng/mL, 50ng/mL and 100ng/mL, respectively, and heparin was added at concentrations of 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL.
The combination of the growth factor compositions and their respective growth factor concentrations are as follows: composition Z1 contained SCF100ng/mL, IL3100ng/mL, and TPO100 ng/mL; composition Z2 contained SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, and INF α 50 ng/mL; composition Z3 included SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, INF α 50ng/mL, heparin10ug/mL and Fltl 3100 ng/mL; composition Z4 included SCF100ng/mL, IL3100ng/mL, F1t3L100ng/mL, heparin10ug/mL and IGF210 ng/mL; composition Z5 included SCF100ng/mL, IL3100ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL and INF α 50 ng/mL; composition Z6 includes SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, INF α 50ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL and IL 1210 ng/mL), each in combination with 5 duplicate wells, cultured in vitro for 14-21d, and then collected and counted for total number of cells, while using CD117+ mouse hematopoietic stem cells cultured with a composition supplemented with SCF100ng/mL and IL3100ng/mL as a control. The flow cytometer analyzed the percentage of each cell population, and the specific procedure is shown in reference to 2.2.1.
2.2.3 testing the potential of CD117+ murine hematopoietic Stem cells to generate Colonies (CFU)
CD117+ mouse hematopoietic stem cells are cultured by a methyl fiber semisolid culture method. 10000 of the CD117+ mouse hematopoietic stem cells obtained from the 2.1 are planted in a methyl fiber semisolid culture medium, different growth factor combinations are added according to the experimental requirements, and a colony formation test is carried out by setting 3 times for each combination.
The method comprises the following specific steps: resuspending the obtained cells, counting by trypan blue staining, mixing 10000 cells with a methyl fiber semisolid culture medium as a control group, and adding the following compositions into the methyl fiber semisolid culture medium respectively: composition F1 included SCF100ng/mL, IL3100ng/mL, and TPO100 ng/mL; composition F2 included SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, and INF α 50 ng/mL; composition F3 comprises SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, INF α 50ng/mL, heparin10 ug/mL; composition F4Comprises 100ng/mL of SCF, 100ng/mL of IL3100ng/mL, 100ng/mL of TPO, 3 t3L100ng/mL of F1, 10ug/mL of heparin and 210ng/mL of IGF; composition F5 includes SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL and INF alpha 50 ng/mL; composition F6 includes SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL, INF α 50ng/mL and IL 1210 ng/mL; then respectively placing the mixture at 37 ℃ and 5% CO2After 14-21 days of culture under the conditions, the number of colonies of CD117+ mouse hematopoietic stem cells in the methyl fiber semisolid culture medium was measured.
2.3 in vitro expansion of human hematopoietic Stem cells in different combinations of growth factors
2.3.1 obtaining human cells, comprising the method steps of:
(1) after a sample of hematopoietic stem cells from bone marrow or umbilical cord blood without hematological disorders is obtained, the sample is pipetted into a 50ml centrifuge tube using a 3ml plastic pipette, added with PBS buffer to 50ml, and centrifuged at 1200RPM for 5 min.
(2) The supernatant was discarded, and the corresponding volume of erythrocyte lysate was added according to the erythrocyte lysate (Solarbio) instructions, lysed at 4 ℃ for 10min, and centrifuged at 1200RPM for 5 min.
(3) Discard the supernatant and repeat step 2 until the red blood cells are lysed completely.
2.3.2 in vitro expansion of human hematopoietic Stem cells
Obtaining human cells according to the above method, planting 50000 cells in 24-well plate containing 10% serum DMEM culture solution, adding 2.2.1 optimal growth factor and its composition according to experimental requirements, and culturing at 37 deg.C and 5% CO2The cells were cultured in vitro for 14-21 days under the conditions, and the CD34+ cell rate was analyzed every 2d flow cytometer. The detection method is carried out by adopting a flow cytometer, and the specific steps are shown as reference 2.2.1.
3. Statistical analysis
All experimental data were tested using Student's t test, GraphPad Prism Version 5.04, and data were considered statistically significant using Means ± SEM, P < 0.05.
Results
1. Optimal composition of different growth factors
1.1 CD117+ hematopoietic Stem cell culture results with different combinations of growth factors
1. DMEM medium + SCF with 10% serum was used as a control for culturing mononuclear CD117+ cells, and the results showed that: compared with the control, the total cell number was 17.5-fold, 9-fold, 8.5-fold, 7.5-fold and 10.5-fold after adding the growth factors IL3, IL6, INF alpha, TPO and heparin, and the rest of the growth factors were the same as the control effect, as shown in FIG. 1 a.
2. DMEM medium with 10% serum + SCF + IL3 was used as a control for culturing mononuclear CD117+ cells, and the results showed: in comparison with the control, the total number of cells was amplified 10.5-fold, 11.5-fold, 13.5-fold, 17-fold, 16-fold, 11-fold, and 9.5-fold after adding Fltl3, IGF2, HGF, TPO, IL12, SDF1, and EPO, respectively, and the remaining growth factors were the same as the control effect, as shown in fig. 1 b.
3. When DMEM culture solution containing 10% serum + SCF + IL3+ TPO was used as a control for culturing mononuclear CD117+ cells, INF α, IGF2, EGF, FGF1, FGF2, FGF9, FGF18, and PDGF were added, respectively, and the results showed that: compared to the control, with the addition of INF α, EGF, FGF2, FGF9 and PDGF, the total number of cells expanded 6.5-fold, 5.25-fold, 5.75-fold, 6-fold and 5.35-fold, respectively, and the remaining growth factors inhibited the expansion or the same effect as the control, as shown in fig. 1 c.
4. As a result of adding IL7, heparin, SDF1 and FGF21 to a control containing 10% serum in DMEM culture solution + SCF + IL3+ TPO + INF α for culturing mononuclear CD117+ cells, the total number of cells was 6-fold and 5.4-fold, respectively, compared to the control, and the remaining growth factors were the same as the control, as shown in FIG. 1 d.
5. As a result of adding IL6, IGF2, IL12, EPO, HGF and Fltl3 to a control containing 10% serum in DMEM culture solution + SCF + IL3+ TPO + INF α + heparin, respectively, cultured mononuclear CD117+ cells, the total number of cells was 14.5-fold and 9-fold, respectively, compared to the control, and the remaining growth factors were the same as the control, as shown in FIG. 1 e.
6. The results of using DMEM culture solution containing 10% serum + SCF + IL3+ TPO + INF α + heparin + IL12 as a control for culturing mononuclear CD117+ cells and adding BMP2, IGF2, Fltl3, Wnt3a, SDF1, and EPO, respectively, showed that the total number of cells added with IGF2, Fltl3, Wnt3a, SDF1, and EPO was 6.5-fold, 10.1-fold, 7.5-fold, 6.1-fold, and 6-fold, respectively, compared to the control, and that the effect of adding BMP2 was similar to the control, as shown in fig. 1 f.
7. The results of adding IGF2, EPO, Wnt3a and SDF1 to DMEM culture solution + SCF + IL3+ TPO + INF α + heparin + IL12+ Fltl3 containing 10% serum as a control for culturing mononuclear CD117+ cells, respectively, showed that the total number of cells added with IGF2, Wnt3a and SDF1 was 7-fold, 5.9-fold and 5.9-fold, respectively, compared to the control, and that the addition of EPO was similar to the control, as shown in fig. 1 g.
8. The results of adding EPO, Wnt3a and SDF1 to DMEM culture medium + SCF + IL3+ TPO + INF α + heparin + IL12+ Fltl3+ IGF2 containing 10% serum as controls for culturing mononuclear CD117+ cells showed similar effects of adding SDF1 and inhibition of amplification of the other two cells, as shown in fig. 1 h.
Growth factors are interactive when cultured in vitro on mononuclear CD117+ cells, as analyzed by a screening process for over 20 cytokines, such as: in the above 2 or 6, when the culture medium is DMEM culture solution containing 10% serum + SCF + IL3 or DMEM culture solution containing 10% serum + SCF + IL3+ INF α + heparin + IL12+ Fltl3, the addition of EPO increases the efficiency of cell total expansion, whereas the addition of EPO to the above 4, 5, 7, and 8 inhibits cell expansion, indicating that IL12 and EPO have a synergistic effect in promoting expansion, while Fltl3 or Fltl3+ IGF2 combination inhibits the activity of EPO in regulating erythropoiesis. For another example: the amplification is inhibited by adding IL12 to the medium used in the above 3, while the amplification of the total number of cells is enhanced by adding IL12 to the medium used in 2 or 5, respectively, which shows that the addition of IL3 alone promotes the activity of IL12 to enhance the amplification efficiency based on the medium used in1, but the addition of IL3+ TPO simultaneously to the medium used in1 inhibits the activity of IL12 to inhibit the amplification of cells; in addition, on the basis of the culture medium used in1, after IL3+ TPO, INF alpha + heparin is added to stimulate the activity of IL12 to improve the cell amplification efficiency, so that each growth factor does not play a separate role in the in vitro amplification process, but the amplification efficiency of the in vitro amplified hematopoietic stem cells is improved through the synergistic effect of a plurality of growth factors, namely, the type of the growth factor added into the in vitro amplification culture solution is very critical. The present inventors have screened various growth factors, and the growth factors capable of promoting CD117+ mouse hematopoietic stem cell expansion include: SCF, IL3, TPO, FLTL3, IGF2, heparin, INF alpha and IL 12.
When 8 growth factors are added simultaneously, the expansion efficiency of the hematopoietic stem cells of the CD117+ mouse is the highest (as shown in figure 2), and the total mononuclear cells can reach 21358300, the proportion of the hematopoietic stem cells is 1.26 percent, and the number of the total hematopoietic stem cells reaches 269115 by detection of a flow cytometer, so that the expansion of 7689 times is obtained. In the prior art, after different growth factor compositions are added, the growth factor composition can be improved by 3139 times after being cultured for 14 days, the hematopoietic stem cells can reach 109881, and the growth factor composition can be expanded by 7689 times after being cultured for 14-21 days.
1. Optimal concentration of optimal composition of different growth factors
When the addition concentrations of SCF, IL3, TPO, Fltl3, IGF2, INF alpha and IL12 were 1ng/mL, 10ng/mL, 50ng/mL and 100ng/mL, respectively, and the addition amounts of heparin were 1. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL, the results showed that: when only SCF is added, the in vitro amplification is not obvious at four adding concentrations, but the total number of cells is more when the addition is relative to 100 ng/mL; when SCF100ng/mL was added, the amplification ratio of IL3 at a concentration of 100ng/mL was 1-fold, and for reference, the amplification ratio of SCF + IL3 at 100ng/mL was selected as a control.
As shown in FIG. 3, the optimal concentrations of TPO, Fltl3, IGF2, heparin, IL12 and INF alpha were determined to be 100ng/mL, 10. mu.g/mL, 10ng/mL and 50ng/mL, respectively, for the control of CD117+ mouse hematopoietic stem cells cultured in DMEM containing 10% serum in which SCF and IL3 were added.
On the basis of the preferred optimum concentrations of the different growth factors in the separate addition of the optimum composition, different combinations are made, namely: composition Z1-composition Z6, the addition of the composition was found to increase the efficiency of amplification compared to the addition of either growth factor alone in the optimal composition; as analyzed from Table 1, the amplification ratios of the different combinations of composition Z1-composition Z6 were different, and Z6 showed the best amplification ratio, 7689 times that of the control. Namely: when SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, INF alpha 50ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL and IL 1210 ng/mL are added simultaneously, the highest amplification rate can be obtained, and the invention can effectively improve the in vitro proliferation rate of hematopoietic stem cells under the optimal growth factor composition and corresponding concentration.
TABLE 1 in vitro expansion of hematopoietic stem cells with different growth factor compositions
2. Testing the potential of CD117+ mouse hematopoietic Stem cells to generate Colonies (CFU)
As can be seen from the analysis of fig. 4, different growth factor compositions exhibited different increases of hematopoietic cell colonies compared to the control, and the greatest number of hematopoietic colonies formed in the methyl fiber semisolid medium containing the optimal combinations and concentrations of growth factors shown in the results of fig. 2 and 3, i.e., SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL, INF α 50ng/mL, and IL 1210 ng/mL), again demonstrating that the selected growth factors all increased the expansion efficiency of hematopoietic stem cells in vitro, but synergistically exhibited the optimal colony potential only when 8 growth factors were simultaneously added and the hematopoietic stem cells were cultured at optimal concentrations; the number of hematopoietic colonies formed was the highest with F6, 6.5 times that of the control.
3. Proliferation number of human cells in different growth factor combination
As can be seen from the analysis of FIG. 5, the different growth factor compositions exhibited different increases in hematopoietic cell numbers compared to the control, and the highest number of hematopoietic stem cells was obtained under the culture conditions with the optimal combinations and concentrations of growth factors (i.e., SCF100ng/mL, IL3100ng/mL, TPO100ng/mL, INF α 50ng/mL, F1t3L100ng/mL, heparin10ug/mL, IGF210ng/mL, and IL 1210 ng/mL) as shown in the results of FIGS. 2 and 3, again demonstrating that the selected growth factors all enhanced the expansion efficiency of hematopoietic stem cells in vitro, but only the hematopoietic stem cells were cultured at optimal concentrations with the simultaneous addition of 8 growth factors, which synergistically exhibited the optimal expansion efficiency.
While the embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made thereto by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein but is not limited to the particular arrangements shown and described without departing from the general concept defined by the appended claims and their equivalents.
Claims (4)
1. A growth factor composition for expanding hematopoietic stem cells in vitro comprising: interferon alpha, heparin, interleukin 12, stem cell growth factor, interleukin 3, thrombopoietin, ligands for Fms-like tyrosine kinase receptor 3, and insulin-like growth factor;
the interferon alpha addition concentration is 50ng/mL, the heparin addition concentration is 10 mug/mL, the interleukin 12 addition concentration is 10ng/mL, the stem cell growth factor addition concentration is 100ng/mL, the interleukin 3 addition concentration is 100ng/mL, the thrombopoietin addition concentration is 100ng/mL, the Fms-like tyrosine kinase receptor 3 ligand addition concentration is 100ng/mL, and the insulin-like growth factor addition concentration is 10 ng/mL.
2. The use of the growth factor composition according to claim 1 for the in vitro expansion of hematopoietic stem cells by mixing a DMEM culture solution containing 10% serum with the growth factor composition.
3. The use of a growth factor composition according to claim 2 for the in vitro expansion of hematopoietic stem cells obtained from human bone marrow, cord blood hematopoietic stem cell samples, or other mammalian sources of hematopoietic stem cells.
4. Use of a growth factor composition according to claim 2 for the in vitro expansion of hematopoietic stem cells under conditions such that: culturing at 37 deg.C under 5% carbon dioxide for 14-21 days.
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