CN116445402A - Erythroid progenitor cell expansion culture medium, expansion culture method and application - Google Patents
Erythroid progenitor cell expansion culture medium, expansion culture method and application Download PDFInfo
- Publication number
- CN116445402A CN116445402A CN202310511638.0A CN202310511638A CN116445402A CN 116445402 A CN116445402 A CN 116445402A CN 202310511638 A CN202310511638 A CN 202310511638A CN 116445402 A CN116445402 A CN 116445402A
- Authority
- CN
- China
- Prior art keywords
- erythroid progenitor
- progenitor cells
- erythroid
- 6mda
- expansion culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000003013 erythroid precursor cell Anatomy 0.000 title claims abstract description 68
- 238000012136 culture method Methods 0.000 title claims abstract description 7
- 230000010261 cell growth Effects 0.000 title claims description 4
- 239000001963 growth medium Substances 0.000 title abstract description 7
- 230000035755 proliferation Effects 0.000 claims abstract description 31
- 230000000925 erythroid effect Effects 0.000 claims abstract description 30
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 11
- DYSDOYRQWBDGQQ-XLPZGREQSA-N N6-Methyl-2'-deoxyadenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 DYSDOYRQWBDGQQ-XLPZGREQSA-N 0.000 claims abstract description 9
- 230000001605 fetal effect Effects 0.000 claims abstract description 9
- DYSDOYRQWBDGQQ-UHFFFAOYSA-N N6-Methyldeoxyadenosine Natural products C1=NC=2C(NC)=NC=NC=2N1C1CC(O)C(CO)O1 DYSDOYRQWBDGQQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 210000004185 liver Anatomy 0.000 claims abstract description 6
- 201000010099 disease Diseases 0.000 claims abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 24
- 230000003321 amplification Effects 0.000 claims description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 16
- 102000003951 Erythropoietin Human genes 0.000 claims description 13
- 108090000394 Erythropoietin Proteins 0.000 claims description 13
- 229940105423 erythropoietin Drugs 0.000 claims description 12
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 9
- 239000003862 glucocorticoid Substances 0.000 claims description 7
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 6
- 229930182816 L-glutamine Natural products 0.000 claims description 6
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 claims description 6
- 229940035024 thioglycerol Drugs 0.000 claims description 6
- 239000007640 basal medium Substances 0.000 claims description 4
- 229960003957 dexamethasone Drugs 0.000 claims description 4
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 4
- 210000000130 stem cell Anatomy 0.000 claims description 4
- 235000015872 dietary supplement Nutrition 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 229940037128 systemic glucocorticoids Drugs 0.000 claims description 3
- 102100032528 C-type lectin domain family 11 member A Human genes 0.000 claims description 2
- 101710167766 C-type lectin domain family 11 member A Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 229960002743 glutamine Drugs 0.000 claims 1
- 210000002960 bfu-e Anatomy 0.000 abstract description 27
- 238000000338 in vitro Methods 0.000 abstract description 12
- 210000003743 erythrocyte Anatomy 0.000 abstract description 8
- 230000007812 deficiency Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 19
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 16
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 description 16
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 230000004663 cell proliferation Effects 0.000 description 13
- 230000004069 differentiation Effects 0.000 description 11
- 229960002685 biotin Drugs 0.000 description 8
- 235000020958 biotin Nutrition 0.000 description 8
- 239000011616 biotin Substances 0.000 description 8
- 238000002955 isolation Methods 0.000 description 8
- 102000001554 Hemoglobins Human genes 0.000 description 6
- 108010054147 Hemoglobins Proteins 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 4
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 4
- 210000002798 bone marrow cell Anatomy 0.000 description 4
- 230000005757 colony formation Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 101001078143 Homo sapiens Integrin alpha-IIb Proteins 0.000 description 3
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 3
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 3
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 3
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 3
- 238000000719 MTS assay Methods 0.000 description 3
- 231100000070 MTS assay Toxicity 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 238000001516 cell proliferation assay Methods 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- -1 SCF Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000011712 cell development Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000010437 erythropoiesis Effects 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000009044 synergistic interaction Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/18—Erythrocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/14—Erythropoietin [EPO]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
An expansion culture medium, an expansion culture method and application of erythroid progenitor cells, wherein the expansion culture medium comprises N6-methyl-2' -deoxyadenosine, can stimulate the proliferation of erythroid progenitor cells (including fetal liver and bone marrow sources) from multiple sources, and especially can improve the proliferation of early erythroid progenitor cells BFU-E by about 10 times. And these proliferated erythroid progenitors can differentiate normally to produce mature erythrocytes. The molecule can be used for in vitro culture of erythroid progenitor cells and treatment of diseases related to erythroid progenitor cell deficiency.
Description
Technical Field
The invention relates to the technical field of erythroid progenitor cell in-vitro culture and erythroid related disease treatment medicines, in particular to an expansion culture medium, an expansion culture method and application of erythroid progenitor cells.
Background
Erythrocytes are the most abundant type of cells in blood and provide all tissues and cells of the human body with the necessary oxygen. Erythropoiesis is the process by which hematopoietic stem cells develop and differentiate to form erythrocytes. The culture of erythroid progenitors in vitro is an essential part of research into regulation of erythropoiesis, erythrocyte-related diseases, and in vitro erythrocyte production. In vitro erythroid progenitor cell culture requires participation of various growth factors, however, some of the growth factors commonly used at present have limited effects, and other chemical molecules capable of stimulating erythroid progenitor cell proliferation are still sought.
Disclosure of Invention
The invention mainly aims at an expansion culture medium, an expansion culture method and application of erythroid progenitor cells, and aims at solving the technical problems at least to a certain extent.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
as one aspect of the present invention, there is provided an expansion medium for erythroid progenitor cells, comprising N6-methyl-2'-deoxyadenosine (N6-methyl-2' -deoxydenosine, 6mdA, CAS: 2002-35-9), having a structure represented by the following formula:
proliferation may be promoted by maintaining activation of the c-Kit (i.e., CD117, type iii receptor tyrosine kinase) in erythroid progenitors by addition of 6mdA according to embodiments of the invention.
In accordance with embodiments of the present invention, 6mdA may be more effective in stimulating proliferation of erythroid progenitors, including fetal liver-derived or adult bone marrow-derived, in an expansion medium at a concentration in the range of 5-200 mM, in a suitable concentration range.
According to an embodiment of the invention, the expansion medium further comprises basal medium, L-glutamine, thioglycerol, glucocorticoid, erythropoietin (EPO), and Stem Cell Factor (SCF).
According to the embodiment of the invention, the L-glutamine can provide needed nutrition for erythroid progenitor cells and promote the proliferation of the erythroid progenitor cells; preferably, the concentration of L-glutamine is 2 to 4 mM, and may be, for example, 2 mM, 2.5 mM, 3.5 mM, 4 mM, or the like.
According to embodiments of the present invention, thioglycerol acts as a reducing agent to maintain cell growth during cell proliferation by antioxidant action; the concentration of thioglycerol is preferably 50 to 100. Mu.M, and may be, for example, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, or 100. Mu.M.
According to an embodiment of the present invention, glucocorticoids, EPO, and SCF are in vitro erythroid progenitor cell proliferation stimulators, respectively, capable of stimulating proliferation of erythroid progenitor cells. Glucocorticoids including, but not limited to, praethasone, dexamethasone, and the like, preferably Dexamethasone (dex), enhance the proliferative effects of SCF.
According to an embodiment of the present invention, the glucocorticoid concentration in the amplification medium is 0.1 to 1. Mu.M, for example, 0.1. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1. Mu.M, etc. Within a suitable concentration range, proliferation of erythroid progenitor cells can be effectively stimulated.
According to the embodiment of the invention, the concentration of EPO in the amplification medium is 0.5-1U/mL, for example, 0.5U/mL, 0.6U/mL, 0.7U/mL, 0.8U/mL, 0.9U/mL, 1.0U/mL, etc. Within a suitable concentration range, proliferation of erythroid progenitor cells can be effectively stimulated.
According to the embodiment of the invention, the concentration of SCF in the amplification medium 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, proliferation of erythroid progenitor cells can be effectively stimulated.
According to an embodiment of the invention, the basal medium is Stempro-34 medium supplemented with 1x nutrient supplement. However, the present invention is not limited thereto, and for example, IMDM medium and the like may be used.
As another aspect of the present invention, there is provided a method for expansion culture of erythroid progenitor cells, comprising: erythroid progenitor cells are cultured using an expansion medium as described above to promote proliferation of erythroid progenitor cells.
According to embodiments of the invention, erythroid progenitor cells may be cultured under conventional conditions, e.g., 37℃and 5% CO 2 Is cultured under culture conditions of erythroid progenitor cells.
According to an embodiment of the present invention, erythroid progenitor cells are derived from mouse embryos or bone marrow, but are not limited thereto, and erythroid progenitor cells may also be human erythroid progenitor cells or the like.
According to an embodiment of the invention, erythroid progenitor cells are early erythroid progenitor cells BFU-E (erythroid blast colonies), and 6mdA can promote proliferation of early erythroid progenitor cells BFU-E. The amplification capacity of the in vitro amplification can be improved by about 10 times after 10 days of in vitro amplification.
As a further aspect of the invention, there is provided the use of N6-methyl-2' -deoxyadenosine for the expansion culture of erythroid progenitors.
As a further aspect of the present invention, there is provided the use of N6-methyl-2' -deoxyadenosine in the manufacture of a medicament for the treatment of red-related diseases.
According to embodiments of the invention, the addition of 6mdA to culture erythroid progenitors in vitro promotes proliferation by maintaining activation of the c-Kit, and the effect of 6mdA on the c-Kit does not have an irreversible effect, and erythroid progenitors that proliferate upon stimulation of 6mdA can differentiate normally to produce mature erythrocytes. After 6mdA was removed before induced differentiation of these erythroid progenitors that proliferated by 6mdA, these erythroid progenitors can normally enter terminal differentiation and produce hemoglobin.
In general, chemical molecule 6mdA, which stimulates the proliferation of erythroid progenitors, can be used for long-term in vitro culture of erythroid progenitors, thereby allowing more erythroid progenitors with normal differentiation capacity to be obtained.
Drawings
FIG. 1 shows how 6mdA promotes proliferation of fetal liver-derived erythroid progenitors according to example 1 of the present invention, wherein A is the cell proliferation effect of 6mdA at a concentration of 0 to 100. Mu.M and B is the cell proliferation effect of 6mdA at a concentration of 100 to 200. Mu.M.
FIG. 2 is a flow chart showing the separation and proliferation of BFU-E cells, wherein A is the flow chart of BFU-E cells, and B is the chart of MTS detection BFU-E cells, in example 2 of the present invention.
FIG. 3 is a graph showing the effect of 6mdA on the proliferation of adult mouse bone marrow-derived erythroid progenitors in example 3 of the present invention, wherein A is a schematic representation of the colony formation experiments of adult mouse bone marrow cells BFU-E, B is an image display of the colony formation sizes and numbers of BFU-E after 6mdA treatment, C is a statistical result of the colony formation numbers of BFU-E, D is a statistical result of the colony formation sizes of BFU-E, E is a schematic representation of the isolation of adult mouse bone marrow-derived erythroid progenitors, and F is MTS for detecting the proliferation of adult mouse bone marrow-derived erythroid progenitors.
FIG. 4 shows the effect of erythroid progenitor cell proliferation stimulators on proliferation of embryo-derived erythroid progenitor cells in example 4 of the invention, wherein A and D are the effects of MTS assay cell proliferation at SCF and adjusted concentration of 6mdA, B is the effects of MTS assay cell proliferation at EPO and adjusted concentration of 6mdA, and C is the effects of MTS assay cell proliferation at dex and adjusted concentration of 6mdA.
FIG. 5 is the effect of 6mdA on the expression of erythroid progenitor c-Kit in example 5 of the invention, wherein A is the result of immunoWestern blot detection of c-Kit and p-c-Kit; b is the western blot statistics of c-Kit and p-c-Kit.
FIG. 6 shows the erythroid progenitor cells induced to differentiate and examined for nuclear and hemoglobin production by promoting proliferation of 6mdA in example 6 of the present invention, wherein A is a schematic diagram of treatment of erythroid progenitor cells with 6mdA 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 ejection of BFU-E cells, E is the hemoglobin production after erythroid progenitor cells differentiation, and F is the hemoglobin production after BFU-E cell differentiation.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that, unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.
In the process of realizing the invention, the 6mdA can stimulate the proliferation and growth of erythroid progenitor cells, can be used for long-term culture of erythroid progenitor cells in vitro, and further research shows that the proliferation of the erythroid progenitor cells is promoted by maintaining the activation of c-Kit, the effect of 6mdA on the c-Kit does not generate irreversible influence, and after the 6mdA is removed, the erythroid progenitor cells can still be normally differentiated, and the erythroid nuclei are generated to mature erythrocytes.
Example 1: isolation and expansion culture of fetal liver-derived erythroid progenitor cells
Isolation of erythroid progenitor cells of embryonic origin
Erythroid progenitor cell isolation the mouse erythroid progenitor cells were isolated from mouse fetal liver cells using the method of magnetic bead anion selection, as described by McIver et al (Methods Mol, biol, 2018, 1698 (1698): 67-89). First, fetal liver cells from C57/6J mouse embryos pregnant for 13.5 days were taken and diluted to 100X 10 with PBS containing 5% BSA 6 cells/mL per 10 6 2 mu L of the cell antibody is added into Biotin anti-mouse Lineage Panel, and after incubation on ice for 15 min, streptavidin Microbeads magnetic beads are added and incubated at 4 ℃ for 30 min. Passing the cells after incubation through LS column, collecting cells not bound with antibody to obtain Lin - And (3) cells. Lin (line) - The cells are subjected to a second round of magnetic bead separation, and are incubated with Biotin anti-mouse CD19 and Biotin anti-mouse CD71 antibodies, so that the Lin can be obtained - CD71 - CD19 - Erythroid progenitor cells.
Expansion culture of erythroid progenitor cells of embryonic origin
Lin obtained by separating the above - CD71 - CD19 - Erythroid progenitor cells are cultured in a specific expansion medium. The amplification medium was StemPro-34 and 1x nutrient supplement (Thermo), 2 mM L-glutamine, 1% penicillin/streptomycin solution, 100. Mu.M thioglycerol, 1. Mu.M dexamethasone dex, 0.5U/mL erythropoietin EPO, and 100 ng/mL stem cell growth factor SCF were added followed by 6mdA at concentrations of 0, 5. Mu.M, 10. Mu.M, 20. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, respectively.
According to MTS proliferation assay, as shown in FIG. 1, 6mdA can stimulate proliferation of erythroid progenitor cells in the range of 5-200. Mu.M, but after the concentration of 6mdA is more than 50. Mu.M, the difference of proliferation promoting effect is small, and the proliferation promoting effect of 6mdA in the concentration of 100. Mu.M or 200. Mu.M is not significantly different, so that 100. Mu.M 6mdA is selected for preparing the culture medium.
Example 2: isolation and expansion culture of fetal liver-derived erythroid progenitor cells
Isolation of erythroid progenitor cells of embryonic origin
For BFU-E cell sorting, magnetic bead negative sorting is adopted and combined with flow sortingMethods (Blood, 2011, 117 (12): 3435-3444). First, fetal liver cells from C57/6J mouse embryos pregnant for 13.5 days were also taken and diluted to 100X 10 with PBS containing 5% BSA 6 cell/mL, bead incubation Biotin anti-mouse Lineage Panel, biotin anti-mouse CD34, biotin anti-mouse CD41, biotin anti-mouse Sca-1, biotin anti-mouse CD16/32 antibody, lin removal + CD34 + CD41 + Sca + CD16/32 + And (3) cells. The remaining cells were isolated from BFU-E cells by flow cell sorting based on the expression of cell surface markers c-Kit and CD 71. Wherein, FIG. 2A shows that the BFU-E cells sorted by the flow sort of BFU-E cells are c-Kit + CD71 10%low 。
Expansion culture of erythroid progenitor cells of embryonic origin
The BFU-E cells obtained by the sorting are cultured in the same amplification medium containing 100 mu M6 mdA as in example 1, MTS is used for detecting the proliferation of the BFU-E cells under the condition of 100 mu M6 mdA, and as shown in a B diagram of FIG. 2, the result shows that 6mdA has stronger promotion effect on the proliferation of the BFU-E cells, and the proliferation can be improved by about 10 times after being amplified for 10 days.
Example 3: isolation and expansion culture of bone marrow-derived erythroid progenitor cells
Isolation of bone marrow-derived erythroid progenitor cells
About 10 weeks of mouse bone marrow cells were taken and BFU-E cells were isolated according to the method of example 2, except that only magnetic beads were used to isolate Lin + CD34 + CD41 + Sca + CD16/32 + Cells, a mixture of erythroid progenitors was obtained, as shown in FIG. 3, panel E.
Expansion culture of bone marrow-derived erythroid progenitor cells
As shown in FIG. 3A, 1.2. 1.2 mL MethoCurt Medium (STEMCELL) containing 0 and 100. Mu.M 6mdA, respectively, was prepared from mouse bone marrow cells (number 3X 10) 4 ) The BFU-E clone formation experiments were performed by mixing and recording the BFU-E clone formation size and number after 14 days. As shown in the results of the B-D graphs, 6mdA can promote bone marrow cells to generate more BFU-E cell clones, and after 6mdA treatment, BFU-E clones are larger, which indicates that 6mdA promotes BFU-E cells in bone marrowProliferation.
As shown in figure 3, panel E, the erythroid progenitor mixture was isolated from bone marrow cells and examined for proliferation using the same 100 μm 6 mdA-containing expansion medium as in example 1, and as shown in figure 3, panel F, MTS proliferation assay found that 6mdA significantly promoted expansion of bone marrow-derived erythroid progenitor cells. After 6 days of expansion culture, cells from the 6mdA treated group were expanded more than 10-fold, and untreated groups were expanded only about 2-fold.
Example 4: proliferation effect evaluation of erythroid progenitor cell proliferation stimulating factor
A similar procedure to that of example 1 was employed, except that the amounts of EPO, SCF, dex and 6mdA were adjusted. According to the MTS proliferation assay, as shown in figures A to D of FIG. 4, both EPO, SCF, dex and 6mdA show a pro-cell proliferation effect, especially the synergistic interaction between 6mdA and EPO, SCF, i.e., 6mdA is able to exert a pro-proliferation effect on erythroid progenitors more significantly in the presence of SCF, EPO.
Example 5:6mdA can maintain activation of c-Kit
Erythroid progenitor cells were cultured using the same amplification medium added with 20. Mu.M and 100. Mu.M 6mdA, respectively, as in example 1, and then the expression of c-Kit and p-c-Kit was examined by immunoWestern blot experiments on days 2, 4, and 6 of the amplification culture.
The tyrosine kinase receptor c-Kit is an essential gene in the erythroid progenitor cell proliferation process, and the results of exploring the c-Kit expression and phosphorylation of 6mdA show that 6mdA can maintain the c-Kit expression and phosphorylation, as shown in the A diagram and the B diagram of fig. 5, and indicate that 6mdA promotes erythroid progenitor cell proliferation by maintaining the c-Kit activation.
Example 6: erythroid progenitor cells proliferated by 6mdA undergo normal differentiation
6mdA increases the ability of erythroid progenitors to expand in vitro to give more erythroid progenitors, and to investigate whether these erythroid progenitors can differentiate normally to give more mature erythrocytes, the erythroid progenitors isolated from examples 1 and 2 were expanded in the presence of 6mdA for 3 days in an induced differentiation experiment as shown in panel A of FIG. 6, followed by removal of 6mdA, and replacement with a differentiation medium containing high concentrations of EPO for 3 days, and then examined for cell differentiation, nuclear and hemoglobin production. When erythroid progenitor cells differentiate into the pro-erythroid stage, cell surface Ter119 begins to express gradually, and we therefore represent erythroid progenitor cell differentiation as a population of Ter119+ cells. Flow cytometric analysis as shown in panels B-D of fig. 6 showed a slight increase in ter119+ cell populations after differentiation of erythroid progenitor cells after 6mdA treatment. Similarly, the Ter119+ cell population was slightly increased after differentiation of BFU-E cells treated with 6mdA. As shown in fig. 6E and F, the red blood progenitor cells after 6mdA treatment differentiated and the detected hemoglobin increased. These results indicate that treatment at stage 6mdA of expansion did not affect the subsequent terminal differentiation of erythroid progenitors.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. An expansion medium for erythroid progenitor cells comprising N6-methyl-2' -deoxyadenosine.
2. The amplification medium of claim 1, wherein the concentration of N6-methyl-2' -deoxyadenosine is 5-200 μm.
3. The amplification medium of claim 1, wherein the amplification medium further comprises: basal medium, L-glutamine, thioglycerol, glucocorticoids, erythropoietin and stem cell growth factors.
4. The amplification medium according to claim 3, wherein the concentration of L-glutamine in the amplification medium is 2 to 4 mM; the concentration of the thioglycerol is 50-100 mu M; the concentration of the glucocorticoid is 0.1-1 mu M; the concentration of erythropoietin is 0.5-1U/mL; the concentration of the stem cell growth factor is 10-100 ng/mL.
5. The amplification medium of claim 1, wherein the basal medium is StemPro-34 supplemented with 1x nutrient supplement; the glucocorticoid is dexamethasone.
6. An expansion culture method of erythroid progenitor cells, comprising: culturing erythroid progenitor cells using an expansion medium according to any one of claims 1 to 5, to promote proliferation of the erythroid progenitor cells.
7. The method according to claim 6, wherein the erythroid progenitor cells are derived from mouse fetal liver or bone marrow.
8. The expansion culture method according to claim 6, wherein the erythroid progenitor cells are human erythroid progenitor cells.
9. Use of N6-methyl-2' -deoxyadenosine in the expansion culture of erythroid progenitors.
10. An application of N6-methyl-2' -deoxyadenosine in preparing medicine for treating red system related diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310511638.0A CN116445402A (en) | 2023-05-09 | 2023-05-09 | Erythroid progenitor cell expansion culture medium, expansion culture method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310511638.0A CN116445402A (en) | 2023-05-09 | 2023-05-09 | Erythroid progenitor cell expansion culture medium, expansion culture method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116445402A true CN116445402A (en) | 2023-07-18 |
Family
ID=87128612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310511638.0A Pending CN116445402A (en) | 2023-05-09 | 2023-05-09 | Erythroid progenitor cell expansion culture medium, expansion culture method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116445402A (en) |
-
2023
- 2023-05-09 CN CN202310511638.0A patent/CN116445402A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3702459B1 (en) | Method for improving fetal hemoglobin expression | |
| EP0455482B1 (en) | A subset of human progenitor cells | |
| US7919316B2 (en) | Hematopoietic stem cell identification and isolation | |
| US20080118477A1 (en) | Umbilical cord mesenchymal stem cells support cord blood hematopoiesis | |
| CN109652369B (en) | Method for preparing mature red blood cells in vitro by using peripheral blood and preparation | |
| WO2018082316A1 (en) | Application of cepharanthine and culture medium and method for expanding hematopoietic stem cells | |
| CN113136362A (en) | Vesicle and application thereof | |
| Freed | Continuous cultivation of cells derived from haploid Rana pipiens embryos | |
| CN112080469B (en) | Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro | |
| WO2018172420A1 (en) | Methods of improving hematopoietic grafts | |
| CN116445402A (en) | Erythroid progenitor cell expansion culture medium, expansion culture method and application | |
| Kannourakis et al. | Proliferative properties of unfractionated, purified, and single cell human progenitor populations stimulated by recombinant human interleukin-3 | |
| CN103540566A (en) | Methods and compositions for long term hematopoietic repopulation | |
| Janoshwitz et al. | Density gradient segregation of bone marrow cells with the capacity to form granulocytic and macrophage colonies in vitro | |
| CN114507642B (en) | Method for separating single cells of pericytes of animal nervous system | |
| CN115197909B (en) | NK cell in-vitro culture method | |
| CN112779220B (en) | Culture medium for neural stem cell expansion | |
| CN113005082B (en) | T cell serum-free culture medium and application thereof | |
| CN110055217B (en) | Method for obtaining megakaryocyte and platelet | |
| Lu et al. | The correlation of growth with incorporation of radioactive metabolites into nucleic acids in embryonic tissue culture | |
| CN116162659B (en) | In vitro gene expression intervention method of primary MDSC | |
| WO2022145144A1 (en) | Therapeutic agent for x-linked sideroblastic anemia | |
| CN115197907A (en) | Preparation method of stimulated cells for autologous or universal NK cell in-vitro culture | |
| CN115975920A (en) | Method for removing aged mesenchymal stem cells | |
| CN116121187A (en) | Method for artificially inducing and differentiating umbilical cord blood hematopoietic stem cells into erythrocytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |