Erythroid progenitor cell expansion culture medium, expansion culture method and application
By using N6-methyl-2’-deoxyadenosine (6mdA) and other growth factors in the amplification medium of erythrocyte progenitor cells, the activation of c-Kit was solved, and the problem of limited proliferation of erythrocyte progenitor cells in the prior art was achieved efficient proliferation and normal differentiation of erythrocyte progenitor cells was achieved.
Patent Information
- Application Number
- CN202310511638.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-09
- Publication Date
- 2025-04-25
- Estimated Expiration
- 2043-05-09
AI Technical Summary
In the prior art, the growth factor used for in vitro culture of erythrocyte progenitor cells has limited effect and it is difficult to effectively stimulate the proliferation of erythrocyte progenitor cells.
A erythrocyte progenitor cell expansion medium including N6-methyl-2’-deoxyadenosine (6mdA) was used to promote the proliferation of erythrocyte progenitor cells by maintaining c-Kit activation. The medium also contains basal medium, L-glutamine, thioglycerol, glucocorticoid, erythropoietin (EPO), and stem cell growth factor (SCF).
By using 6mdA and other growth factors, the proliferation of erythrocytes was significantly promoted. The proliferation ability of erythrocytes was increased by about 10 times on 10 days of expansion. After 6mdA was removed, erythrocytes could still differentiate normally into erythrocytes.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of in vitro culture of erythroid progenitor cells and therapeutic drugs for erythroid-related diseases, and in particular to an expansion culture medium, an expansion culture method and an application of erythroid progenitor cells. Background Art
[0002] Red blood cells are the most abundant type of cells in the blood, and they provide the oxygen needed for all tissues and cells in the human body. Erythropoiesis is the process of gradual development and differentiation from hematopoietic stem cells to form red blood cells. The culture of erythroid progenitor cells in vitro is an indispensable part of studying the regulation of erythropoiesis, erythroid-related diseases, and in vitro red blood cell preparation. In vitro erythroid progenitor cell culture requires the participation of multiple growth factors. However, the effects of some commonly used growth factors are limited, and other chemical molecules that can stimulate the proliferation of erythroid progenitor cells are still being sought. Summary of the invention
[0003] The main purpose of the present invention is to provide an expansion culture medium, an expansion culture method and an application of erythroid progenitor cells, in order to solve the above technical problems at least to a certain extent.
[0004] To achieve the above object, the technical solution of the present invention is as follows:
[0005] As one aspect of the present invention, there is provided an expansion medium for erythroid progenitor cells, comprising N6-methyl-2'-deoxyadenosine (6mdA, CAS: 2002-35-9), having a structure as shown in the following formula:
[0006]
[0007] According to an embodiment of the present invention, by adding 6mdA, proliferation can be promoted by maintaining the activation of c-Kit (ie, CD117, type III receptor tyrosine kinase) in erythroid progenitor cells.
[0008] According to an embodiment of the present invention, in the expansion medium, the concentration of 6mdA is in the range of 5-200 mM. Within a suitable concentration range, 6mdA can more effectively stimulate the proliferation of erythroid progenitor cells derived from fetal liver or adult bone marrow.
[0009] According to an embodiment of the present invention, the expansion medium further comprises a basal medium, L-glutamine, monothioglycerol, glucocorticoids, erythropoietin (Erythropoietin, EPO) and a stem cell growth factor (Stem Cell Factor, SCF).
[0010] According to an embodiment of the present invention, L-glutamine can provide the required nutrition for erythroid progenitor cells and promote the proliferation of erythroid progenitor cells; preferably, the concentration of L-glutamine is 2~4 mM, for example, it can be 2 mM, 2.5 mM, 3.5 mM, 4 mM, etc.
[0011] According to an embodiment of the present invention, thioglycerol is used as a reducing agent to maintain cell growth through antioxidant effect during cell proliferation; preferably, the concentration of thioglycerol is 50~100 μM, for example, it can be 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM.
[0012] According to an embodiment of the present invention, glucocorticoids, EPO and SCF are respectively in vitro erythroid progenitor cell proliferation stimulating factors, which can stimulate the proliferation of erythroid progenitor cells. Glucocorticoids include but are not limited to paramethasone, dexamethasone, etc., preferably dexamethasone (dex), which can enhance the proliferation effect of SCF.
[0013] According to an embodiment of the present invention, in the expansion medium, the concentration of glucocorticoid is 0.1-1 μM, for example, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, etc. Within the appropriate concentration range, the proliferation of erythroid progenitor cells can be effectively stimulated.
[0014] According to an embodiment of the present invention, in the expansion medium, the concentration of EPO is 0.5-1 U / mL, for example, 0.5 U / mL, 0.6 U / mL, 0.7 U / mL, 0.8 U / mL, 0.9 U / mL, 1.0 U / mL, etc. Within a suitable concentration range, the proliferation of erythroid progenitor cells can be effectively stimulated.
[0015] According to an embodiment of the present invention, in the expansion medium, the concentration of SCF is 10-100 ng / mL, for example, 10 ng / mL, 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, etc. Within a suitable concentration range, the proliferation of erythroid progenitor cells can be effectively stimulated.
[0016] According to an embodiment of the present invention, the basal culture medium is a StemPro-34 culture medium supplemented with 1x nutrient supplement, but is not limited thereto, and may also be an IMDM culture medium, for example.
[0017] As another aspect of the present invention, a method for expanding and culturing erythroid progenitor cells is provided, comprising: culturing erythroid progenitor cells using the expansion medium as described above to promote the proliferation of erythroid progenitor cells.
[0018] According to an embodiment of the present invention, the erythroid progenitor cells may be cultured under conventional culture conditions, for example, the erythroid progenitor cells may be cultured under culture conditions of 37° C. and 5% CO 2 .
[0019] According to an embodiment of the present invention, the erythroid progenitor cells are derived from mouse embryos or bone marrow, but are not limited thereto. The erythroid progenitor cells may also be human erythroid progenitor cells, etc.
[0020] According to an embodiment of the present invention, the erythroid progenitor cells are early erythroid progenitor cells BFU-E (erythroid burst colony), and 6mdA can promote the proliferation of early erythroid progenitor cells BFU-E. In vitro expansion for 10 days can increase its expansion capacity by about 10 times.
[0021] As another aspect of the present invention, there is provided a use of N6-methyl-2'-deoxyadenosine in the expansion culture of erythroid progenitor cells.
[0022] As another aspect of the present invention, there is provided a use of N6-methyl-2'-deoxyadenosine in the preparation of a drug for treating erythroid-related diseases.
[0023] According to an embodiment of the present invention, when 6mdA is added during in vitro culture of erythroid progenitor cells, proliferation can be promoted by maintaining the activation of c-Kit, and the effect of 6mdA on c-Kit does not produce an irreversible effect, and the erythroid progenitor cells proliferated by 6mdA can differentiate normally and produce mature red blood cells. After removing 6mdA before inducing differentiation of these erythroid progenitor cells induced to proliferate by 6mdA, these erythroid progenitor cells can normally enter terminal differentiation and produce hemoglobin.
[0024] In general, the chemical molecule 6mdA, which stimulates the proliferation of erythroid progenitor cells, can be used for long-term culture of erythroid progenitor cells in vitro, thereby obtaining more erythroid progenitor cells with normal differentiation ability. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The MTS method used in Example 1 of the present invention is used to detect the proliferation of fetal liver-derived erythroid progenitor cells promoted by 6mdA, wherein A is the cell proliferation effect of 6mdA at a concentration of 0-100 μM, and B is the cell proliferation effect of 6mdA at a concentration of 100-200 μM.
[0026] Figure 2 This is the sorting and proliferation detection of fetal liver-derived early erythroid progenitor cells BFU-E in Example 2 of the present invention, wherein A is a flow sorting diagram of BFU-E cells, and B is a MTS detection diagram of BFU-E cell proliferation.
[0027] Figure 3It is the effect of 6mdA on the proliferation of erythroid progenitor cells derived from the bone marrow of adult mice in Example 3 of the present invention, wherein A is a schematic diagram of the BFU-E clone formation experiment of adult mouse bone marrow cells, B is a graphic display of the size and number of BFU-E clones after 6mdA treatment, C is the statistical result of the number of BFU-E clones, D is the statistical result of the size of BFU-E clones, E is a schematic diagram of the separation of erythroid progenitor cells derived from the bone marrow of adult mice, and F is the MTS detection of the proliferation of erythroid progenitor cells derived from the bone marrow of adult mice.
[0028] Figure 4 It is the effect of the erythroid progenitor cell proliferation stimulating factor on the proliferation of embryonic-derived erythroid progenitor cells in Example 4 of the present invention, wherein A and D are the cell proliferation effects detected by MTS under the adjustment of SCF and 6mdA concentrations, B is the cell proliferation effect detected by MTS under the adjustment of EPO and 6mdA concentrations, and C is the cell proliferation effect detected by MTS under the adjustment of dex and 6mdA concentrations.
[0029] Figure 5 It is the effect of 6mdA on the expression of c-Kit in erythroid progenitor cells in Example 5 of the present invention, wherein A is the immunoglobulin detection result of c-Kit and pc-Kit; B is the immunoglobulin statistical result of c-Kit and pc-Kit.
[0030] Figure 6 This is Example 6 of the present invention, inducing differentiation of erythroid progenitor cells whose proliferation is promoted by 6mdA, and detecting their nuclear extrusion and hemoglobin production, wherein A is a schematic diagram of 6mdA treatment of erythroid progenitor cells and induced differentiation, B is the differentiation result of erythroid progenitor cells, C is the differentiation result of early erythroid progenitor cells BFU-E, D is the nuclear extrusion of BFU-E cells, E is the hemoglobin production after erythroid progenitor cell differentiation, and F is the hemoglobin production after BFU-E cell differentiation. DETAILED DESCRIPTION
[0031] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be noted in advance that, unless otherwise defined, the technical terms used in the following embodiments have the same meanings as those generally understood by technicians in the field to which the present invention belongs. The experimental reagents used in the following embodiments, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.
[0032] In the process of realizing the present invention, it was unexpectedly discovered that 6mdA can stimulate the proliferation and growth of erythroid progenitor cells and can be used for long-term in vitro culture of erythroid progenitor cells. Further studies have found that it promotes their proliferation by maintaining the activation of c-Kit, and that 6mdA does not have an irreversible effect on the effect of c-Kit. After the removal of 6mdA, erythroid progenitor cells can still undergo normal differentiation and produce mature red blood cells by expelling the nucleus.
[0033] Example 1: Isolation and expansion of fetal liver-derived erythroid progenitor cells
[0034] Isolation of embryonic-derived erythroid progenitor cells
[0035] Erythroid progenitor cell isolation was performed according to the description of McIver et al. (Methods Mol, Biol, 2018, 1698(1698):67-89). Mouse erythroid progenitor cells were isolated from mouse fetal liver cells using magnetic bead negative selection. First, fetal liver cells from C57 / 6J mouse embryos at 13.5 days of gestation were obtained and diluted to 100 x 10 cells with PBS containing 5% BSA. 6 cells / mL, according to every 10 6 2 μL of antibody was added to the cells. Biotin anti-mouse Lineage Panel was incubated on ice for 15 min, and then Streptavidin Microbeads magnetic beads were added and incubated at 4 °C for 30 min. After the incubation, the cells were passed through the LS column to collect the cells that were not bound to the antibody and obtain Lin - Cell. - After the second round of magnetic bead sorting, the cells were incubated with Biotin anti-mouseCD19 and Biotin anti-mouse CD71 antibodies to obtain Lin - CD71 - CD19 - Erythroid progenitor cells.
[0036] Proliferation and culture of embryonic-derived erythroid progenitor cells
[0037] The Lin isolated above - CD71 - CD19 -Erythroid progenitor cells were cultured in a specific expansion medium. The expansion medium was StemPro-34 supplemented with 1x nutrient supplement (Thermo), 2 mM L-glutamine, 1% penicillin / streptomycin solution, 100 μM monothioglycerol, 1 μM dexamethasone dex, 0.5 U / mL erythropoietin EPO, and 100 ng / mL stem cell growth factor SCF, and then 6mdA was added at concentrations of 0, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM, respectively.
[0038] According to the MTS proliferation assay, Figure 1 As shown in the data, 6mdA can stimulate the proliferation of erythroid progenitor cells in the range of 5-200 μM, but when the concentration of 6mdA is greater than 50 μM, the effect of promoting proliferation is relatively small. There is no significant difference in the effect of 6mdA at a concentration of 100 μM or 200 μM in promoting proliferation. Therefore, 100 μM 6mdA was selected when preparing the culture medium.
[0039] Example 2: Isolation and expansion of fetal liver-derived erythroid progenitor cells
[0040] Isolation of embryonic-derived erythroid progenitor cells
[0041] For the sorting of BFU-E cells, magnetic bead negative selection combined with flow cytometry was used (Blood, 2011, 117(12): 3435-3444). First, fetal liver cells from C57 / 6J mouse embryos at 13.5 days of gestation were obtained and diluted to 100 x 10 cells with PBS containing 5% BSA. 6 cells / mL, incubated with magnetic beads with Biotin anti-mouse Lineage Panel, biotin anti-mouse CD34, biotin anti-mouse CD41, biotin anti-mouse Sca-1 and biotin anti-mouse CD16 / 32 antibodies, and Lin was removed + CD34 + CD41 + Sca + CD16 / 32 + The remaining cells were separated into BFU-E cells by flow cytometry based on the expression of cell surface markers c-Kit and CD71. Figure 2 Figure A shows the flow cytometry sorting of BFU-E cells. The sorted BFU-E cells are c-Kit + CD71 10%low .
[0042] Proliferation and culture of embryonic-derived erythroid progenitor cells
[0043] The BFU-E cells obtained by the above sorting were cultured in the same amplification medium containing 100 μM 6mdA as in Example 1, and the proliferation of BFU-E cells under 100 μM 6mdA was detected by MTS. Figure 2 As shown in Figure B, the results show that 6mdA has a stronger promoting effect on the proliferation of BFU-E cells, and its proliferation can be increased by about 10 times after 10 days of expansion.
[0044] Example 3: Isolation and expansion of bone marrow-derived erythroid progenitor cells
[0045] Isolation of bone marrow-derived erythroid progenitor cells
[0046] Bone marrow cells of mice at about 10 weeks old were taken and the BFU-E cells were isolated according to the method in Example 2, except that only magnetic beads were used for separation and Lin was removed. + CD34 + CD41 + Sca + CD16 / 32 + cells, and obtain a mixture of erythroid progenitor cells, such as Figure 3 As shown in Figure E.
[0047] Expansion and culture of bone marrow-derived erythroid progenitor cells
[0048] like Figure 3 As shown in Figure A, 1.2 mL of MethoCult medium (STEMCELL) containing 0 and 100 μM 6mdA was added to mouse bone marrow cells (3×10 4 ) were mixed to conduct BFU-E clone formation experiment, and the size and number of BFU-E clones were recorded after 14 days. As shown in the results of Figures B to D, 6mdA can promote bone marrow cells to produce more BFU-E cell clones, and after 6mdA treatment, BFU-E clones are larger, indicating that 6mdA promotes the proliferation of BFU-E cells in the bone marrow.
[0049] like Figure 3 Figure E shows that the erythroid progenitor cell mixture was separated from the bone marrow cells and its proliferation was detected using the same expansion medium containing 100 μM 6mdA as in Example 1. Figure 3 As shown in Figure F, MTS proliferation assay found that 6mdA significantly promoted the expansion of bone marrow-derived erythroid progenitor cells. After 6 days of expansion culture, the cells in the 6mdA-treated group expanded more than 10 times, while the cells in the untreated group only expanded about 2 times.
[0050] Example 4: Evaluation of the proliferation effect of erythroid progenitor cell proliferation stimulating factor
[0051] The same operation as in Example 1 was used, except that the dosage of EPO, SCF, dex and 6mdA was adjusted. Figure 4 As shown in Figures A to D, EPO, SCF, dex and 6mdA all showed a cell proliferation-promoting effect, especially there was a synergistic effect between 6mdA and EPO and SCF, that is, in the presence of SCF and EPO, 6mdA could more significantly promote the proliferation of erythroid progenitor cells.
[0052] Example 5: 6mdA can maintain the activation of c-Kit
[0053] The erythroid progenitor cells were cultured using the same expansion medium as in Example 1 supplemented with 20 μM and 100 μM 6mdA, respectively, and then the expression of c-Kit and pc-Kit was detected by Western blotting on days 2, 4, and 6 of the expansion culture.
[0054] Tyrosine kinase receptor c-Kit is an essential gene in the proliferation of erythroid progenitor cells. The results of investigating the effect of 6mdA on c-Kit expression and phosphorylation showed that Figure 5 As shown in Figures A and B, 6mdA can maintain the expression and phosphorylation of c-Kit, indicating that 6mdA promotes the proliferation of erythroid progenitor cells by maintaining the activation of c-Kit.
[0055] Example 6: Erythroid progenitor cells proliferated by 6mdA can differentiate normally
[0056] 6mdA can increase the ability of erythroid progenitor cells to expand in vitro, thereby obtaining more erythroid progenitor cells. In order to explore whether these erythroid progenitor cells can undergo normal differentiation to produce more mature red blood cells, such as Figure 6 In the induced differentiation experiment shown in Figure A, the erythroid progenitor cells isolated from Examples 1 and 2 were expanded and cultured for 3 days in the presence of 6mdA, then 6mdA was removed and replaced with a differentiation medium containing high concentrations of EPO for 3 days, and then cell differentiation, nuclear exocytosis, and hemoglobin production were detected. When erythroid progenitor cells differentiated into the proerythroid stage, Ter119 on the cell surface began to gradually express, so we used the Ter119+ cell population to indicate the differentiation of erythroid progenitor cells. Figure 6 Flow cytometry analysis shown in Figures B to D of FIG. 5 shows that the Ter119+ cell population slightly increases after differentiation of erythroid progenitor cells treated with 6mdA. Similarly, the Ter119+ cell population slightly increases after differentiation of BFU-E cells treated with 6mdA. Figure 6As shown in Figures E and F, the hemoglobin detected after differentiation of erythroid progenitor cells treated with 6mdA increased. These results indicate that 6mdA treatment during the expansion period did not affect the subsequent terminal differentiation of erythroid progenitor cells.
[0057] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. An expansion medium for erythroid progenitor cells, comprising N6-methyl-2'-deoxyadenosine.
2. The expansion medium according to claim 1, characterized in that The concentration of N6-methyl-2'-deoxyadenosine was 5~200 μM.
3. The expansion medium according to claim 1, characterized in that The expansion culture medium also includes: basic culture medium, L-glutamine, thioglycerol, glucocorticoid, erythropoietin and stem cell growth factor.
4. The expansion medium according to claim 3, characterized in that In the expansion medium, the concentration of L-glutamine is 2-4 mM; the concentration of thioglycerol is 50-100 μM; the concentration of glucocorticoid is 0.1-1 μM; the concentration of erythropoietin is 0.5-1 U / mL; and the concentration of stem cell growth factor is 10-100 ng / mL.
5. The expansion medium according to claim 3, characterized in that The basic culture medium is StemPro-34 supplemented with 1xnutrient supplement; the glucocorticoid is dexamethasone.
6. A method for expanding and culturing erythroid progenitor cells, comprising: The erythroid progenitor cells are cultured using the expansion medium according to any one of claims 1 to 5 to promote the proliferation of the erythroid progenitor cells.
7. The method for amplification and culture according to claim 6, characterized in that: The erythroid progenitor cells are derived from mouse fetal liver or bone marrow.
8. The method for amplification and culture according to claim 6, characterized in that: The erythroid progenitor cells are human erythroid progenitor cells.
9. Use of N6-methyl-2'-deoxyadenosine in the expansion culture of erythroid progenitor cells.