WO2021121266A1 - 扩增造血干细胞的小分子化合物及其组合 - Google Patents
扩增造血干细胞的小分子化合物及其组合 Download PDFInfo
- Publication number
- WO2021121266A1 WO2021121266A1 PCT/CN2020/136790 CN2020136790W WO2021121266A1 WO 2021121266 A1 WO2021121266 A1 WO 2021121266A1 CN 2020136790 W CN2020136790 W CN 2020136790W WO 2021121266 A1 WO2021121266 A1 WO 2021121266A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- small molecule
- cells
- saha
- hscs
- inhibitors
- Prior art date
Links
- 210000003958 hematopoietic stem cell Anatomy 0.000 title abstract description 174
- -1 Small molecule compounds Chemical class 0.000 title description 5
- 239000003112 inhibitor Substances 0.000 claims abstract description 249
- 150000003384 small molecules Chemical class 0.000 claims abstract description 199
- 238000000338 in vitro Methods 0.000 claims abstract description 30
- 230000001737 promoting effect Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 281
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 152
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 152
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 108
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 108
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 claims description 83
- 239000002067 L01XE06 - Dasatinib Substances 0.000 claims description 82
- 229960002448 dasatinib Drugs 0.000 claims description 82
- 239000000203 mixture Substances 0.000 claims description 59
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 57
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 claims description 57
- 239000003102 growth factor Substances 0.000 claims description 44
- 238000000034 method Methods 0.000 claims description 36
- 230000037361 pathway Effects 0.000 claims description 33
- 230000008685 targeting Effects 0.000 claims description 32
- WXRGFPHDRFQODR-ICLZECGLSA-N 1-[3-[[(2R,3S,4R,5R)-5-(4-amino-7-pyrrolo[2,3-d]pyrimidinyl)-3,4-dihydroxy-2-oxolanyl]methyl-propan-2-ylamino]propyl]-3-(4-tert-butylphenyl)urea Chemical compound C([C@@H]1[C@H]([C@@H](O)[C@@H](O1)N1C2=NC=NC(N)=C2C=C1)O)N(C(C)C)CCCNC(=O)NC1=CC=C(C(C)(C)C)C=C1 WXRGFPHDRFQODR-ICLZECGLSA-N 0.000 claims description 31
- 230000005754 cellular signaling Effects 0.000 claims description 31
- GJFCSAPFHAXMSF-UXBLZVDNSA-N 3-[[(e)-4-(dimethylamino)but-2-enoyl]amino]-n-[3-methyl-4-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide Chemical compound CN(C)C\C=C\C(=O)NC1=CC=CC(C(=O)NC=2C=C(C)C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)=CC=2)=C1 GJFCSAPFHAXMSF-UXBLZVDNSA-N 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 25
- 102000003964 Histone deacetylase Human genes 0.000 claims description 21
- 108090000353 Histone deacetylase Proteins 0.000 claims description 21
- 230000019491 signal transduction Effects 0.000 claims description 15
- ANEBQUSWQAQFQB-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-[2-methyl-5-[[3-(trifluoromethyl)benzoyl]amino]phenyl]-1,3-thiazole-5-carboxamide Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC(C(=CC=1)C)=CC=1NC(=O)C1=CC=CC(C(F)(F)F)=C1 ANEBQUSWQAQFQB-UHFFFAOYSA-N 0.000 claims description 14
- 108090001005 Interleukin-6 Proteins 0.000 claims description 13
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 12
- AXRCEOKUDYDWLF-UHFFFAOYSA-N 3-(1-methyl-3-indolyl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-3-indolyl]pyrrole-2,5-dione Chemical compound C12=CC=CC=C2N(C)C=C1C(C(NC1=O)=O)=C1C(C1=CC=CC=C11)=CN1C(CC1)CCN1CC1=CC=CC=N1 AXRCEOKUDYDWLF-UHFFFAOYSA-N 0.000 claims description 11
- 230000035755 proliferation Effects 0.000 claims description 10
- 229950002189 enzastaurin Drugs 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 7
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 6
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 6
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 6
- 229960001285 quercetin Drugs 0.000 claims description 6
- 235000005875 quercetin Nutrition 0.000 claims description 6
- 210000003995 blood forming stem cell Anatomy 0.000 claims 10
- 150000004922 Dasatinib derivatives Chemical group 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 5
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 5
- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 description 94
- 229960000604 valproic acid Drugs 0.000 description 47
- 210000004700 fetal blood Anatomy 0.000 description 41
- BGFHMYJZJZLMHW-UHFFFAOYSA-N 4-[2-[[2-(1-benzothiophen-3-yl)-9-propan-2-ylpurin-6-yl]amino]ethyl]phenol Chemical compound N1=C(C=2C3=CC=CC=C3SC=2)N=C2N(C(C)C)C=NC2=C1NCCC1=CC=C(O)C=C1 BGFHMYJZJZLMHW-UHFFFAOYSA-N 0.000 description 38
- 238000012216 screening Methods 0.000 description 31
- AZXXGVPWWKWGAE-UHFFFAOYSA-N 4-n-[2-benzyl-7-(2-methyltetrazol-5-yl)-9h-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine Chemical compound CN1N=NC(C=2C=C3C(C4=C(NC5CCC(N)CC5)N=C(CC=5C=CC=CC=5)N=C4N3)=CC=2)=N1 AZXXGVPWWKWGAE-UHFFFAOYSA-N 0.000 description 29
- 239000006285 cell suspension Substances 0.000 description 28
- 238000001514 detection method Methods 0.000 description 23
- 238000012360 testing method Methods 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 19
- 210000003743 erythrocyte Anatomy 0.000 description 19
- 230000006698 induction Effects 0.000 description 19
- 238000002054 transplantation Methods 0.000 description 18
- 210000001185 bone marrow Anatomy 0.000 description 17
- 210000005259 peripheral blood Anatomy 0.000 description 17
- 239000011886 peripheral blood Substances 0.000 description 17
- 239000002953 phosphate buffered saline Substances 0.000 description 17
- 238000010195 expression analysis Methods 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 14
- 239000002458 cell surface marker Substances 0.000 description 13
- 238000000684 flow cytometry Methods 0.000 description 13
- 210000000952 spleen Anatomy 0.000 description 11
- 230000004069 differentiation Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 210000000130 stem cell Anatomy 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 9
- 238000012423 maintenance Methods 0.000 description 9
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 8
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 8
- 239000002504 physiological saline solution Substances 0.000 description 8
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 7
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 7
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 7
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 7
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 102000003951 Erythropoietin Human genes 0.000 description 6
- 108090000394 Erythropoietin Proteins 0.000 description 6
- 210000002798 bone marrow cell Anatomy 0.000 description 6
- 229940105423 erythropoietin Drugs 0.000 description 6
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 6
- 210000000822 natural killer cell Anatomy 0.000 description 6
- 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 description 6
- 238000012546 transfer Methods 0.000 description 6
- DNTSIBUQMRRYIU-UHFFFAOYSA-N GW 9662 Chemical compound [O-][N+](=O)C1=CC=C(Cl)C(C(=O)NC=2C=CC=CC=2)=C1 DNTSIBUQMRRYIU-UHFFFAOYSA-N 0.000 description 5
- 101100383038 Homo sapiens CD19 gene Proteins 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 210000002960 bfu-e Anatomy 0.000 description 5
- 230000001332 colony forming effect Effects 0.000 description 5
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 5
- 229960002986 dinoprostone Drugs 0.000 description 5
- 210000003013 erythroid precursor cell Anatomy 0.000 description 5
- 208000024908 graft versus host disease Diseases 0.000 description 5
- 102000056982 human CD33 Human genes 0.000 description 5
- 102000049018 human NCAM1 Human genes 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 210000000066 myeloid cell Anatomy 0.000 description 5
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000013592 cell lysate Substances 0.000 description 4
- 210000004748 cultured cell Anatomy 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000003394 haemopoietic effect Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- INAICWLVUAKEPB-QSTFCLMHSA-N PFI-3 Chemical compound OC1=CC=CC=C1C(=O)\C=C\N1[C@@H](CN2C=3N=CC=CC=3)C[C@@H]2C1 INAICWLVUAKEPB-QSTFCLMHSA-N 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 238000011773 genetically engineered mouse model Methods 0.000 description 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000004989 spleen cell Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 210000002303 tibia Anatomy 0.000 description 3
- 210000000689 upper leg Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 108010002386 Interleukin-3 Proteins 0.000 description 2
- 102000000646 Interleukin-3 Human genes 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000002473 artificial blood Substances 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 230000000925 erythroid effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- SIXVRXARNAVBTC-UHFFFAOYSA-N gsk2606414 Chemical compound C12=C(N)N=CN=C2N(C)C=C1C(C=C1CC2)=CC=C1N2C(=O)CC1=CC=CC(C(F)(F)F)=C1 SIXVRXARNAVBTC-UHFFFAOYSA-N 0.000 description 2
- 230000006195 histone acetylation Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 229940076264 interleukin-3 Drugs 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000006148 magnetic separator Substances 0.000 description 2
- 210000003593 megakaryocyte Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- HTOYBIILVCHURC-UHFFFAOYSA-N santacruzamate A Chemical compound CCOC(=O)NCCCC(=O)NCCC1=CC=CC=C1 HTOYBIILVCHURC-UHFFFAOYSA-N 0.000 description 2
- LUPNKHXLFSSUGS-UHFFFAOYSA-M sodium;2,2-dichloroacetate Chemical compound [Na+].[O-]C(=O)C(Cl)Cl LUPNKHXLFSSUGS-UHFFFAOYSA-M 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 1
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 1
- SGYJGGKDGBXCNY-QXUYBEEESA-N (3s,9s,12r)-3-benzyl-6,6-dimethyl-9-[6-[(2s)-oxiran-2-yl]-6-oxohexyl]-1,4,7,10-tetrazabicyclo[10.3.0]pentadecane-2,5,8,11-tetrone Chemical compound C([C@H]1C(=O)NC(C(N[C@@H](CC=2C=CC=CC=2)C(=O)N2CCC[C@@H]2C(=O)N1)=O)(C)C)CCCCC(=O)[C@@H]1CO1 SGYJGGKDGBXCNY-QXUYBEEESA-N 0.000 description 1
- FMSSKCBYHCEFQE-UHFFFAOYSA-N 1h-indole;pyrimidine Chemical class C1=CN=CN=C1.C1=CC=C2NC=CC2=C1 FMSSKCBYHCEFQE-UHFFFAOYSA-N 0.000 description 1
- HJGMCDHQPXTGAV-UHFFFAOYSA-N 2-(4-chlorophenoxy)-n-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide Chemical compound C1=CC(Cl)=CC=C1OCC(=O)NC1CCC(NC(=O)COC=2C=CC(Cl)=CC=2)CC1 HJGMCDHQPXTGAV-UHFFFAOYSA-N 0.000 description 1
- WJRRGYBTGDJBFX-UHFFFAOYSA-N 4-(2-methyl-3-propan-2-yl-4-imidazolyl)-N-(4-methylsulfonylphenyl)-2-pyrimidinamine Chemical compound CC(C)N1C(C)=NC=C1C1=CC=NC(NC=2C=CC(=CC=2)S(C)(=O)=O)=N1 WJRRGYBTGDJBFX-UHFFFAOYSA-N 0.000 description 1
- 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
- 101710164309 56 kDa type-specific antigen Proteins 0.000 description 1
- ZYRLHJIMTROTBO-UHFFFAOYSA-N 6,8-bis(benzylsulfanyl)octanoic acid Chemical compound C=1C=CC=CC=1CSC(CCCCC(=O)O)CCSCC1=CC=CC=C1 ZYRLHJIMTROTBO-UHFFFAOYSA-N 0.000 description 1
- VGXRQCOVGLGFIM-UHFFFAOYSA-N 7-oxo-5-phenyl-6-propan-2-yl-1H-pyrazolo[1,5-a]pyrimidine-3-carbonitrile Chemical compound N1=C2C(C#N)=CNN2C(=O)C(C(C)C)=C1C1=CC=CC=C1 VGXRQCOVGLGFIM-UHFFFAOYSA-N 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- FVVDKUPCWXUVNP-UHFFFAOYSA-M Aminosalicylate sodium anhydrous Chemical compound [Na+].NC1=CC=C(C([O-])=O)C(O)=C1 FVVDKUPCWXUVNP-UHFFFAOYSA-M 0.000 description 1
- 208000031872 Body Remains Diseases 0.000 description 1
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- SGYJGGKDGBXCNY-UHFFFAOYSA-N Chlamydocin Natural products N1C(=O)C2CCCN2C(=O)C(CC=2C=CC=CC=2)NC(=O)C(C)(C)NC(=O)C1CCCCCC(=O)C1CO1 SGYJGGKDGBXCNY-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 1
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- ZGSXEXBYLJIOGF-ALFLXDJESA-N IWR-1-endo Chemical compound C=1C=CC2=CC=CN=C2C=1NC(=O)C(C=C1)=CC=C1N1C(=O)[C@@H]2[C@H](C=C3)C[C@H]3[C@@H]2C1=O ZGSXEXBYLJIOGF-ALFLXDJESA-N 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 239000012825 JNK inhibitor Substances 0.000 description 1
- 229940118135 JNK inhibitor Drugs 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- WRKPZSMRWPJJDH-UHFFFAOYSA-N N-(6-methyl-1,3-benzothiazol-2-yl)-2-[(4-oxo-3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]acetamide Chemical compound S1C2=CC(C)=CC=C2N=C1NC(=O)CSC1=NC=2CCSC=2C(=O)N1C1=CC=CC=C1 WRKPZSMRWPJJDH-UHFFFAOYSA-N 0.000 description 1
- KYRVNWMVYQXFEU-UHFFFAOYSA-N Nocodazole Chemical compound C1=C2NC(NC(=O)OC)=NC2=CC=C1C(=O)C1=CC=CS1 KYRVNWMVYQXFEU-UHFFFAOYSA-N 0.000 description 1
- 101710185562 Peroxiredoxin 2 Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- VSWDORGPIHIGNW-UHFFFAOYSA-N Pyrrolidine dithiocarbamic acid Chemical compound SC(=S)N1CCCC1 VSWDORGPIHIGNW-UHFFFAOYSA-N 0.000 description 1
- 229940100514 Syk tyrosine kinase inhibitor Drugs 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 229930189037 Trapoxin Natural products 0.000 description 1
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 230000009045 body homeostasis Effects 0.000 description 1
- CJGYSWNGNKCJSB-YVLZZHOMSA-N bucladesine Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](OC(=O)CCC)[C@@H]2N1C(N=CN=C2NC(=O)CCC)=C2N=C1 CJGYSWNGNKCJSB-YVLZZHOMSA-N 0.000 description 1
- 229960005263 bucladesine Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 108700023145 chlamydocin Proteins 0.000 description 1
- 229960001338 colchicine Drugs 0.000 description 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 229960001425 deferoxamine mesylate Drugs 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- IDDIJAWJANBQLJ-UHFFFAOYSA-N desferrioxamine B mesylate Chemical compound [H+].CS([O-])(=O)=O.CC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCN IDDIJAWJANBQLJ-UHFFFAOYSA-N 0.000 description 1
- 229940121548 devimistat Drugs 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- BNJOZDZCRHCODO-UHFFFAOYSA-N dimethyloxalylglycine Chemical compound COC(=O)CNC(=O)C(=O)OC BNJOZDZCRHCODO-UHFFFAOYSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000009109 downstream regulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940030275 epigallocatechin gallate Drugs 0.000 description 1
- QHPJWPQRZMBKTG-KPKJPENVSA-N ethyl 2-[2-methoxy-4-[(e)-(4-oxo-2-sulfanylidene-1,3-thiazolidin-5-ylidene)methyl]phenoxy]acetate Chemical compound C1=C(OC)C(OCC(=O)OCC)=CC=C1\C=C\1C(=O)NC(=S)S/1 QHPJWPQRZMBKTG-KPKJPENVSA-N 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 229960000282 metronidazole Drugs 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- DOBKQCZBPPCLEG-UHFFFAOYSA-N n-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide Chemical compound C=1SC(NC=2N=CN=CC=2)=NC=1C(=O)NCC1=CC=CC=C1 DOBKQCZBPPCLEG-UHFFFAOYSA-N 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229950006344 nocodazole Drugs 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009703 regulation of cell differentiation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- LCOIAYJMPKXARU-VAWYXSNFSA-N salubrinal Chemical compound C=1C=CC2=CC=CN=C2C=1NC(=S)NC(C(Cl)(Cl)Cl)NC(=O)\C=C\C1=CC=CC=C1 LCOIAYJMPKXARU-VAWYXSNFSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940074404 sodium succinate Drugs 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000011222 transcriptome analysis Methods 0.000 description 1
- 108010060597 trapoxin A Proteins 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- OMOMUFTZPTXCHP-UHFFFAOYSA-N valpromide Chemical compound CCCC(C(N)=O)CCC OMOMUFTZPTXCHP-UHFFFAOYSA-N 0.000 description 1
- 229960001930 valpromide Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 229960004982 vinblastine sulfate Drugs 0.000 description 1
- KDQAABAKXDWYSZ-PNYVAJAMSA-N vinblastine sulfate Chemical compound OS(O)(=O)=O.C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 KDQAABAKXDWYSZ-PNYVAJAMSA-N 0.000 description 1
- 238000004260 weight control Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- 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
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
- A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- C12N2501/145—Thrombopoietin [TPO]
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2306—Interleukin-6 (IL-6)
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
Definitions
- This application relates to the field of biomedicine, in particular, to a small molecule compound used to amplify hematopoietic stem cells (HSCs), specifically a small molecule inhibitor of cell signaling pathway, its composition, and the small molecule inhibitor and the same Use of the composition in the expansion of hematopoietic stem cells.
- HSCs hematopoietic stem cells
- Hematopoietic stem cells are a group of heterogeneous primitive hematopoietic cells in the blood system, which have two important characteristics: self-renewal and multi-lineage differentiation.
- HSCs When the body is in a healthy state, HSCs in the body remain in a resting state for a long time.
- HSCs When the body undergoes disease or severe blood loss, HSCs are activated and enter a state of self-renewal and multi-line differentiation, maintaining the stability of the blood system and the body's homeostasis.
- HSCs help the offspring HSCs to maintain stemness, and the multi-lineage differentiation properties of HSCs can make them differentiate into a variety of mature blood cells, such as myeloid cells (granulocytes, monocytes, red blood cells and platelets), lymphocytes ( T cells and B cells).
- myeloid cells granulocytes, monocytes, red blood cells and platelets
- lymphocytes T cells and B cells.
- the characteristics of HSCs are conducive to the differentiation of HSCs when the body needs it.
- HSCs include long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs).
- LT-HSCs long-term HSCs
- ST-HSCs short-term HSCs
- the former has a high degree of self-renewal ability and can carry out hematopoietic reconstruction throughout the life cycle of the body; while the latter can only maintain hematopoietic reconstruction function for a limited time.
- Thomas and others used bone marrow hematopoietic stem cells to perform the first hematopoietic stem cell transplantation in human history to treat leukemia clinically to restore normal hematopoietic function in the patient.
- hematopoietic stem cell transplantation is not only used to treat a variety of blood system diseases, but also used to treat immunodeficiency diseases and neurodegenerative diseases.
- HSCs mainly come from three sources, bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (CB).
- BM bone marrow
- mPB mobilized peripheral blood
- CB umbilical cord blood
- G-CSF granulocyte colony stimulating factor
- HSCs collected by these two methods need to be matched with the human leukocyte antigen (HLA) between the donor and the patient. HLA matching is difficult. Once a mismatch occurs, a graft-versus-host reaction (GVHD) will occur. Patients with GVHD will die of immune system disorders.
- HLA human leukocyte antigen
- umbilical cord blood As a new source of HSCs, umbilical cord blood has many advantages. First, umbilical cord blood HSCs have low requirements for HLA matching, allowing partial HLA mismatches, low incidence of GVHD after transplantation, and alleviating the traditional HSCT matching difficulties; secondly, umbilical cord Blood collection is convenient, harmless to the donor, and there is no ethical problem. HSCs have strong hematopoietic ability. These advantages make cord blood the preferred source of HSCs for the treatment of diseases in the future.
- the safety and effectiveness of hematopoietic stem cell transplantation depend on the amount of HSCs transplanted. If the number of HSCs can be expanded in vitro, the success rate of hematopoietic stem cell transplantation can be improved.
- HSCs small-molecule compounds
- SMCs small-molecule compounds
- SMCs are easy to obtain, easy to mass produce, stable in nature, clear in structure, and convenient to control their concentration. They have been widely used in medical research.
- SMCs can significantly increase the amplification factor of HSCs.
- the AhR aromatic hydrocarbon receptor inhibitor StemRegenin1 (SR1) is the first SMC screened to expand HSCs in vitro.
- the pyrimidine indole derivative UM171 can also amplify HSCs in vitro, but it does not function through the AhR cell signaling pathway.
- Transcriptome analysis results showed that UM171 did not down-regulate the AhR cell signaling pathway, but inhibited genes related to the differentiation of red blood cells and megakaryocytes. The combination of the two increases the amplification factor of HSCs.
- Histone deacetylases are known cell signal transduction pathways. Histones can regulate the transcription process of specific genes, cell proliferation and differentiation through acetylation or deacetylation.
- HDAC inhibitors TSA, trapoxin, and chlamydocin can regulate histone acetylation in vitro to promote the self-renewal and proliferation of HSCs.
- Src is encoded by the Src proto-oncogene and is a non-receptor protein kinase with tyrosine protein kinase activity. It exists in the cytoplasm and can be activated by a variety of cell surface receptors to mediate multiple cell signal transduction pathways, thereby regulating cell proliferation, differentiation and other processes. It is a key molecule of multiple cell signal transduction pathways. For example, after Src is activated, it cooperates with p52Shc to activate the mitogen-activated protein kinase (MAPK) cell signal transduction pathway and participate in the downstream regulation of cell growth and differentiation. Src can also activate the STAT cell signal transduction pathway and promote the transcription of related genes.
- MAPK mitogen-activated protein kinase
- the problem to be solved in this application is to screen out the best small molecule compounds and their compositions that promote the in vitro expansion of HSCs while maintaining a high proportion of dryness of HSCs by studying the key factors that regulate cell signal transduction pathways, thereby solving the prior art
- the number of HSCs amplified in vitro is still insufficient.
- the applicant’s research found that multiple small molecule inhibitors that act on the Src target of the STAT cell signal transduction pathway can maintain the dryness of HSCs in vitro, and the Src target can effectively expand the STAT cell signal transduction pathway.
- the increase of hematopoietic stem cells and the maintenance of the stemness of hematopoietic stem cells play an important role, which has not been reported in previous studies.
- HDAC inhibitors such as SAHA, Valproic acid (VPA)
- SAHA Valproic acid
- VPA Valproic acid
- this application discovered that small molecule inhibitors of signal transduction and activator of transcription (STAT) cell signaling pathways, such as small molecule inhibitors targeting Src targets, can promote the proliferation of HSCs and maintain the dryness characteristics of HSCs.
- this application provides a method for promoting the proliferation of HSCs and maintaining the dryness of HSCs, including in vitro exposure of HSCs to small molecule inhibitors containing STAT cell signaling pathways or the cultivation of small molecule inhibitors of other cell signaling pathways liquid.
- the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor of the Src target.
- the small molecule inhibitor of the Src target is selected from one or more of the following: Dasatinib, Quercetin, UM-164, KX2-391, and KX1-004. In some embodiments, the small molecule inhibitor of the Src target is selected from one or more of the following: Dasatinib, UM-164, and KX1-004.
- the small molecule inhibitor of the Src target is used in combination with other small molecule inhibitors of cell signaling pathways.
- the small molecule inhibitors of other cell signaling pathways are selected from small molecule inhibitors targeting HDAC, small molecule inhibitors targeting PKC, and small molecule inhibitors targeting JNK One or two or more of them.
- the small molecule inhibitor of the Src target is combined with a small molecule inhibitor targeting HDAC, a small molecule inhibitor targeting PKC, or a small molecule inhibitor targeting JNK use.
- the small molecule inhibitor of the Src target and the small molecule inhibitor VPA targeting HDAC, the small molecule inhibitor SAHA targeting HDAC, and the small molecule inhibitor targeting PKC Enzastaurin or the small molecule inhibitor JNK-IN-8 targeting JNK can be used in combination.
- the small molecule inhibitor Dasatinib of the Src target is used in combination with VPA or SAHA.
- the small molecule inhibitor of the signal transduction and activator of transcription (STAT) cell signaling pathway such as a small molecule inhibitor of the Src target, such as Dasatinib, UM-164, or KX1-004, maintains CD34 +CD45+CD90+CD45RA-CD38- phenotype HSCs accounted for more than 8%, 10%, 15%, 20%, 25%, 30% of all cells, such as maintaining CD34+CD45+CD90+CD45RA -CD38-phenotypic HSCs cells account for 5%-35%, 10%-35%, 15%-35%, 20%-35%, 25%-35%, 5%-30% of all cells %, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%; maintaining the proportion of CD34+ cells in all cells exceeds 65%, 70%, 75%, 80%, 85%.
- STAT signal transduction and activator of transcription
- the present application provides a composition for maintaining the dryness of HSCs, which includes a small molecule inhibitor containing a STAT cell signaling pathway.
- the small molecule inhibitor containing the STAT cell signaling pathway is a small molecule inhibitor of the Src target.
- the small molecule inhibitor of the Src target is selected from one or more of the following: Dasatinib, Quercetin, UM-164, KX2-391, and KX1-004.
- the composition also includes other small molecule inhibitors of cell signaling pathways.
- the other small molecule inhibitors of cell signaling pathways include small molecule inhibitors that target HDAC, small molecule inhibitors that target PKC, and small molecule inhibitors that target JNK.
- the small molecule inhibitors of other cell signaling pathways include the small molecule inhibitor VPA that targets HDAC, the small molecule inhibitor SAHA that targets HDAC, and the small molecule inhibitor that targets PKC. Enzastaurin and the small molecule inhibitor JNK-IN-8 targeting JNK.
- the composition further includes SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6.
- the composition is composed of a small molecule inhibitor containing STAT cell signaling pathway and/or other small molecule inhibitors of cell signaling pathway, SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor Composition of TPO and growth factor IL-6.
- the composition maintains the proportion of HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in more than 8%, 10%, 15%, 20%, 25% or 30%, for example, the proportion of cells maintaining HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in all cells is 5%-35%, 10%-35%, 15%-35%, 20%- 35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25% , 5%-20%, 10%-20%, 15%-20%. In some embodiments, the composition maintains a ratio of CD34+ cells to more than 65%, 70%, 75%, 80%, or 85% of all cells.
- the small molecule inhibitor of the signal transduction and activator of transcription (STAT) cell signaling pathway such as a small molecule inhibitor of the Src target, such as Dasatinib, UM-164 or KX1-004 and other cells
- Small molecule inhibitors of signaling pathways such as VPA or SAHA, used in combination to maintain HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype accounted for more than 8%, 10%, 15%, 20% of all cells %, 25%, 30%, for example, the proportion of cells maintaining HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in all cells is 5%-35%, 10%-35%, 15%-35% %, 20%-35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%; maintaining the proportion of CD34+ cells in all cells exceeds 65%, 70%, 7
- Dasatinib 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, more preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- SAHA 10nM-20 ⁇ M, preferably 20nM-15 ⁇ M, more preferably 30nM-10 ⁇ M, most preferably 0.1 ⁇ M-10 ⁇ M;
- VPA 10 ⁇ M-2000 ⁇ M, preferably 10 ⁇ M-1500 ⁇ M, more preferably 10 ⁇ M-1000 ⁇ M, most preferably 100 ⁇ M-1000 ⁇ M;
- JNK-IN-8 0.1 ⁇ M-20 ⁇ M, preferably 0.5 ⁇ M-15 ⁇ M, more preferably 0.5 ⁇ M-10 ⁇ M, most preferably 1 ⁇ M-10 ⁇ M;
- EPZ004777 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, more preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- DZNeP 1nM-500nM, preferably 5nM-400nM, more preferably 10nM-300nM, most preferably 10nM-250nM;
- UM-164 0.1 ⁇ M-1000 ⁇ M, preferably 0.5 ⁇ M-500 ⁇ M, more preferably 1 ⁇ M-100 ⁇ M, most preferably 1 ⁇ M-10 ⁇ M;
- KX2-391 0.1nM-1000nM, preferably 1nM-1000nM, more preferably 10nM-500nM, most preferably 10nM-100nM;
- KX1-004 0.1 ⁇ M-1000 ⁇ M, preferably 1 ⁇ M-1000 ⁇ M, more preferably 10 ⁇ M-500 ⁇ M, most preferably 10 ⁇ M-100 ⁇ M.
- this application provides a method for promoting the proliferation of HSCs and maintaining the dryness of HSCs, which includes contacting HSCs in vitro with a culture medium containing one or more of the following small molecule inhibitors: 1) Small molecules targeting HDACs Molecular inhibitor VPA; 2) small molecule inhibitor SAHA targeting HDAC; 3) small molecule inhibitor Enzastaurin targeting PKC; and 4) small molecule inhibitor JNK-IN- targeting JNK 8.
- the present application provides a composition for maintaining the dryness of HSCs, comprising any combination selected from: SAHA+EPZ004777, SAHA+DZNeP, SAHA+Dasatinib, VPA+Dasatinib, SAHA+JNK-IN- 8 or SAHA+VPA.
- the composition further includes SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6.
- the composition consists of any combination selected from SAHA+EPZ004777, SAHA+DZNeP, SAHA+Dasatinib, VPA+Dasatinib, SAHA+JNK-IN-8 or SAHA+VPA and SFEMII medium, growth It is composed of factor Flt-3L, growth factor SCF, growth factor TPO and growth factor IL-6.
- the composition for maintaining the dryness of HSCs also helps to maintain the ratio of CD34+ cells.
- the composition maintains the proportion of HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in more than 8%, 10%, 15%, 20%, 25%, 30%, for example, the proportion of cells maintaining HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in all cells is 5%-35%, 10%-35%, 15%-35%, 20%- 35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25% , 5%-20%, 10%-20%, 15%-20%; maintaining the proportion of CD34+ cells in all cells exceeds 65%, 70%, 75%, 80%, 85%.
- the concentration of each inhibitor in the culture medium is:
- Dasatinib 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, more preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- SAHA 10nM-20 ⁇ M, preferably 20nM-15 ⁇ M, more preferably 30nM-10 ⁇ M, most preferably 0.1 ⁇ M-10 ⁇ M;
- VPA 10 ⁇ M-2000 ⁇ M, preferably 10 ⁇ M-1500 ⁇ M, more preferably 10 ⁇ M-1000 ⁇ M, most preferably 100 ⁇ M-1000 ⁇ M;
- JNK-IN-8 0.1 ⁇ M-20 ⁇ M, preferably 0.5 ⁇ M-15 ⁇ M, more preferably 0.5 ⁇ M-10 ⁇ M, most preferably 1 ⁇ M-10 ⁇ M;
- EPZ004777 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, more preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- DZNeP 1nM-500nM, preferably 5nM-400nM, more preferably 10nM-300nM, most preferably 10nM-250nM.
- the present application provides a composition for maintaining the dryness of HSCs, comprising any combination selected from: SAHA+EPZ004777+DZNeP or SAHA+VPA+Dasatinib.
- the composition further includes SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6.
- the composition consists of SAHA+EPZ004777+DZNeP or SAHA+VPA+Dasatinib and SFEMII medium, growth factor Flt-3L, growth factor SCF, growth factor TPO, and growth factor IL-6.
- the composition maintains the proportion of HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in more than 8%, 10%, 15%, 20%, 25%, 30%, for example, the proportion of cells maintaining HSCs with CD34+CD45+CD90+CD45RA-CD38- phenotype in all cells is 5%-35%, 10%-35%, 15%-35%, 20%- 35%, 25%-35%, 5%-30%, 10%-30%, 20%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-25% , 5%-20%, 10%-20%, 15%-20%; maintaining the proportion of CD34+ cells in all cells exceeds 65%, 70%, 75%, 80%, 85%.
- the concentration of each inhibitor in the culture medium is: Dasatinib: 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, further preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- SAHA 10nM-20 ⁇ M, preferably 20nM-15 ⁇ M, more preferably 30nM-10 ⁇ M, most preferably 0.1 ⁇ M-10 ⁇ M;
- VPA 10 ⁇ M-2000 ⁇ M, preferably 10 ⁇ M-1500 ⁇ M, more preferably 10 ⁇ M-1000 ⁇ M, most preferably 100 ⁇ M-1000 ⁇ M;
- EPZ004777 0.1 ⁇ M-50 ⁇ M, preferably 0.5 ⁇ M-40 ⁇ M, more preferably 0.5 ⁇ M-30 ⁇ M, most preferably 0.5 ⁇ M-10 ⁇ M;
- DZNeP 1nM-500nM, preferably 5nM-400nM, more preferably 10nM-300nM, most preferably 10nM-250nM.
- the small molecule inhibitor of the STAT cell signaling pathway is a small molecule inhibitor of the Src target.
- the small molecule inhibitor of the Src target is selected from one or more of Dasatinib, Quercetin, UM-164, KX2-391 and KX1-004 as follows.
- the small molecule inhibitor of the Src target is selected from one or more of the following: Dasatinib, UM-164, and KX1-004.
- the above-mentioned small molecule inhibitors of cell signaling pathways can well maintain the dryness of HSCs and the proportion of CD34+ cells during in vitro expansion, and the combination of these small molecule inhibitors is effective for HSCs. Renewal, dryness maintenance, etc. are superior to the combination with small molecules that have been reported in the prior art.
- the applicant’s research results have found an inhibitor of Src, which can maintain the dryness of HSCs in terms of in vitro expansion and culture of HSCs; and found an HDAC inhibitor, which can be combined with Src inhibitors.
- an inhibitor of Src which can maintain the dryness of HSCs in terms of in vitro expansion and culture of HSCs
- HDAC inhibitor which can be combined with Src inhibitors.
- the effect is better than that of the reported small molecule inhibitors and the effect of these small molecule inhibitors alone.
- the applicant's research results can achieve the in vitro expansion of HSCs while maintaining a high proportion of HSCs dryness, laying the foundation for the realization of the clinical application of HSCs.
- the “stemness” of the above-mentioned hematopoietic stem cells is the abbreviation for the characteristics of hematopoietic stem cells.
- Hematopoietic stem cells mainly present two major cell biological characteristics: self-renewal capacity and pluripotency. These properties of hematopoietic stem cells are referred to as “stemness” for short.
- the molecular phenotype expressed on the cell surface of hematopoietic stem cells can reflect to some extent whether they maintain "stemness".
- hematopoietic stem cells are CD34+CD45+CD90+CD45RA-CD38-, it means that they are LT-HSCs and maintain their "dryness".
- hematopoietic stem cells with CD34+CD45+CD90+CD45RA-CD38- phenotype are defined as LT-HSCs; hematopoietic stem cells with CD34+CD45+CD90+CD45RA-CD38- phenotype are defined as maintenance or maintenance The characteristics of hematopoietic stem cells, that is, "dryness".
- the all cells refer to all the progeny cells after the initial CD34+ cells have been cultured.
- hematopoietic stem cells are exposed in vitro to small molecule inhibitors containing STAT cell signaling pathways, such as small molecule inhibitors of the Src target, or other cell signaling pathway small molecule inhibitors, such as small molecule inhibitors targeting HDAC, and PKC.
- small molecule inhibitors and JNK targeted small molecule inhibitors can well maintain the dryness of HSCs during in vitro expansion and the proportion of CD34+ cells in all HSCs, and the combination of these small molecule inhibitors
- the self-renewal and dryness maintenance of HSCs are better than the combination with small molecules that have been reported in the prior art.
- the cell transplantation efficiency after treatment with the above small molecule inhibitors is significantly higher than that reported in the prior art Cell transplantation efficiency after treatment with small molecule inhibitors.
- Figure 1 shows the logic gate of the target cell population CD34+CD45+CD45RA-CD90+CD38- cell population and the determination of the gate position.
- Figure 2 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the first round of screening that can maintain the dryness of HSCs.
- Table 4 shows the induction of small molecule inhibitors (4 concentrations for each small molecule test) 6-7 days later, flow cytometric detection of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis chart, where the abscissa represents the name and concentration of the inhibitor, and the ordinate is CD34+CD45+CD90 +CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 3 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the second round of screening that can maintain the dryness of HSCs.
- Table 5 shows the flow cytometric detection of LT-HSCs 6-7 days after induction of small molecule inhibitors.
- Figure 4 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the third round of screening that can maintain the dryness of HSCs.
- Table 6 shows small molecule inhibitors (except SAHA-1 ⁇ M, each inhibitor Test 3 concentrations) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the name and concentration of the inhibitor, and the vertical axis
- the coordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 5 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the fourth round of screening that can maintain the dryness of HSCs.
- Table 7 shows small molecule inhibitors (except SAHA-1 ⁇ M, each inhibitor Test 3 concentrations) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the name and concentration of the inhibitor, and the vertical axis
- the coordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 6 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the fifth round of screening that can maintain the dryness of HSCs.
- Table 8 shows small molecule inhibitors (except SAHA-1 ⁇ M, each inhibitor Test 3 concentrations) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the name and concentration of the inhibitor, and the vertical axis
- the coordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 7 shows the first round of screening of the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- the coordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 8 shows the second round of screening of the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- the coordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 9 shows the third round of screening of the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood, the small molecule combination SAHA+Butyrate, SAHA+EPZ004777, SAHA+DZNeP, SAHA+ Vitamin C (except SAHA, each inhibitor tested at 3 concentrations) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38-) expression analysis chart, in which, horizontal The coordinates represent the name and concentration of the inhibitor, the ordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs. S stands for SAHA (1 ⁇ M).
- Figure 10 shows the fourth round of screening of the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- SAHA+Enzastaurin (LY317615) (except SAHA, 3 concentrations are tested for each inhibitor) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38-) expression Analysis chart, where the abscissa represents the name and concentration of the inhibitor, the ordinate CD34+CD45+CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs . S stands for SAHA (1 ⁇ M).
- Figure 11 shows the fifth round of screening of the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- Figure 12 shows the first round of screening of the best tri-molecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- the small molecule combination SAHA+EPZ004777+DZNeP is induced for 6-7 days after induction by flow cytometry LT -HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the name and concentration of the inhibitor, and the ordinate is CD34+CD45+CD90+CD45RA-CD38- (%) Represents the proportion of LT-HSCs in all cells, and CD34+CD45+ (%) represents the purity of HSCs. There are 3 replicates in each group, and * represents a significant difference.
- Figure 13 shows the second round of screening of the best tri-molecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood, the small molecule combination SAHA+Dasatinib+EPZ004777, SAHA+JNK-IN-8+ EPZ004777, SAHA+JNK-IN-8+DZNeP, SAHA+JNK-IN-8+Dasatinib, SAHA+JNK-IN-8+EPZ004777+DZNeP induced 6-7 days after the flow cytometric detection of LT-HSCs cell surface markers (CD34 +CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the combination name and concentration of the inhibitor, and the ordinate CD34+CD45+CD90+CD45RA-CD38- (%) represents that LT-HSCs account for all cells The ratio of CD34+CD45+(%) represents the purity of HSCs. There are 3 replicates in each group, and *
- Figure 14 shows the third round of screening of the best tri-molecular combination of small molecule inhibitors to maintain the dryness of HSCs on CD34+ cells derived from cord blood.
- the small molecule combination SAHA+VPA+Dasatinib induced 6-7 days after the flow cytometric detection of LT -HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the combination name and concentration of the inhibitor, the ordinate is CD34+CD45+CD90+CD45RA-CD38-(% ) Represents the proportion of LT-HSCs in all cells, and CD34+CD45+ (%) represents the purity of HSCs. There are 3 replicates in each group, and * represents a significant difference.
- Figure 15 shows the comparison of the small molecule inhibitors screened on CD34+ cells derived from cord blood and the small molecule inhibitors SR1 and UM171 reported in the literature.
- Figure 16 shows the analysis of in vitro cloning ability of screened small molecule inhibitors and small molecule inhibitors SR1 and UM171 that have been screened on CD34+ cells derived from cord blood.
- BFU-E, CFU-E, CFU-GM, CFU-GEMM represent clones of different lineages of the blood system such as erythroid, myeloid, and lymphoid lines.
- the abscissa represents the name of the inhibitor combination and the concentration used
- the ordinate colonies number represents the total number of clones
- the colonies number of GEMM represents the number of CFU-GEMM clones. There are 3 replicates in each group, and * represents a significant difference.
- S stands for SAHA (1 ⁇ M).
- Figure 17 shows the optimal concentration of small molecule inhibitors on CD34+ cells derived from cord blood and the screening that can maintain the dryness of HSCs.
- Table 9 shows small molecule inhibitors (except SAHA-1 ⁇ M, each inhibitor tested 3 Concentration) 6-7 days after induction, flow cytometric detection of LT-HSCs cell surface markers (CD34+/CD45+/CD90+/CD45RA-/CD38-) expression analysis graph, the abscissa represents the name and concentration of the inhibitor, the ordinate is CD34+CD45 +CD90+CD45RA-CD38-(%) represents the proportion of LT-HSCs in all cells, and CD34+CD45+(%) represents the purity of HSCs.
- Figure 18 18A and 18C show the flow cytometric detection of LT-HSCs cell surface markers after 8 days of treatment with small molecule Mock (DMSO), SR1 (5 ⁇ M), UM171 (350nM), and Dasatinib (50nM) on CD34+ cells derived from cord blood (CD34+CD45+CD90+CD45RA-CD38-) expression analysis graph, where the abscissa represents the name of the inhibitor, and the ordinate CD34+CD45+CD90+CD45RA-CD38- (%) represents the proportion of LT-HSCs in all cells , CD34+CD45+(%) represents the purity of HSCs.
- DMSO small molecule Mock
- SR1 5 ⁇ M
- UM171 350nM
- Dasatinib 50nM
- 18B and 18D show the absolute number of LT-HSCs and CD34+ cell proliferation after 8 days of treatment with small molecule Mock (DMSO), SR1 (5 ⁇ M), UM171 (350nM), and Dasatinib (50nM) on CD34+ cells derived from cord blood.
- DMSO small molecule Mock
- SR1 5 ⁇ M
- UM171 350nM
- Dasatinib 50nM
- Figure 19 shows the determination of logic gates and gate positions of hCD45+ and mCD45+ cell populations.
- NC represents the control without antibody
- 33 represents sample No. 33 with antibody
- hCD45 represents human CD45+ cells
- mCD45 represents mouse CD45+ cells.
- Figure 20 shows the determination of logic gates and gate positions of hCD45+, hCD3+, hCD33+, hCD56+, hCD19+ and mCD45+ cell populations.
- hCD3+ stands for human-derived CD3+ cells and is a surface marker of T lymphocytes.
- hCD33+ stands for CD33+ cells of human origin and is a surface marker of myeloid cells.
- hCD56+ stands for human-derived CD56+ cells and is a surface marker of natural killer cells (NK cells).
- hCD19+ stands for human-derived CD19+ cells and is a surface marker of B lymphocytes.
- mCD45 stands for CD45+ cells of murine origin.
- Figure 21 shows the small molecule Mock (DMSO), SR1 (5 ⁇ M), and Dasatinib (50nM) treatment on CD34+ cells derived from cord blood for 7 days. All cells were collected and transplanted into mice. At the 4th week and 8th week after transplantation The proportion of human-derived CD45 cells in the peripheral blood of mice was measured by flow cytometry at weeks, 12 and 16 weeks. The proportion of human-derived CD45 cells in the bone marrow and spleen of mice was measured by flow cytometry at the 16th week after transplantation. In Fig.
- DMSO small molecule Mock
- SR1 5 ⁇ M
- 50nM Dasatinib
- the abscissa represents the name of the inhibitor and the transplantation time
- the ordinate hCD45-PB (%) represents the proportion of human-derived CD45+ cells detected in the peripheral blood of the mouse.
- PB stands for Peripheral Blood
- hCD45 stands for CD45+ cells of human origin.
- the abscissa represents the name of the inhibitor
- the ordinate hCD45-BM (%) represents the proportion of human-derived CD45+ cells detected in the bone marrow of mice.
- BM stands for Bone Marrow
- hCD45 stands for CD45+ cells of human origin.
- the abscissa represents the name of the inhibitor
- the ordinate hCD45-SP (%) represents the proportion of human-derived CD45+ cells detected in the mouse spleen.
- SP stands for Spleen
- hCD45 stands for CD45+ cells of human origin.
- Figure 22 shows the small molecule Mock (DMSO), SR1 (5 ⁇ M), Dasatinib (50nM) treatment on CD34+ cells derived from cord blood for 7 days. All cells were collected and transplanted into mice. Flow cytometry was detected 16 weeks after transplantation. The proportion of human-derived hCD3+, hCD33+, hCD56+, and hCD19+ cells in mouse peripheral blood, bone marrow, and spleen represents human-derived T lymphocytes (T), myeloid cells (My), natural killer cells (NK), and B lymphocytes, respectively Cell (B).
- T lymphocytes T
- My myeloid cells
- NK natural killer cells
- B B lymphocytes
- the abscissa represents the name of the inhibitor
- the ordinate in Figure 22A represents the proportion of hCD45+ cells of different lineages in the peripheral blood.
- the ordinate in Fig. 22B represents the proportion of hCD45+ cells of different lineages in bone marrow.
- the ordinate in Fig. 22C represents the proportion of hCD45+ cells of different lineages in the spleen.
- Example 1 Cord blood sorting CD34+HSCs for subsequent small molecule screening
- the operation is as follows: first add FcR Blocking Reagent (Miltenyi Biotec, item number: 130- 100-453, the amount of reagent is determined according to the result of cell count) Resuspend the cells, then add pre-mixed CD34MicroBeads (CD34MicroBead Kit UltraPure, human: MiltenyiBiotec, catalog number: 130-100-453), mix well, and incubate in a refrigerator at 4°C for 30min . Add physiological saline (1% HSA) to the centrifuge tube to a final volume of 50ml, transfer to a high-speed centrifuge, and centrifuge at 500g for 10 minutes.
- FcR Blocking Reagent Miltenyi Biotec, item number: 130- 100-453
- CD34MicroBead Kit UltraPure human: MiltenyiBiotec, catalog number: 130-100-453
- Magnetic separator (MiltenyiBiotec, model: 130-042-102) and a magnetic stand (MiltenyiBiotec, model: 130-042-303), adjust the magnetic separator to an appropriate height, and put it in the MS Column (MiltenyiBiotec, item number: 130 -042-201) or LS Column (MiltenyiBiotec, Item No. 130-042-401) (The type of column is determined according to the cell mass, please refer to the relevant product instructions for details), and a 15ml centrifuge tube (Corning, Item No.
- the storage solution of the small molecule inhibitor configures the storage solution of the small molecule inhibitor. Then proceed to the preparation of the basic medium: SFEMII medium (stem cell, article number: 09655) + 50ng/ml growth factor Flt-3L (PeProtech, article number: 300-100UG) + 50ng/ml growth factor SCF (PeProtech, article number: 300 -07-100UG)+50ng/ml growth factor TPO (PeProtech, article number: 300-18-100UG)+10ng/ml growth factor IL-6 (PeProtech, article number: 200-06-20UG) + 1% double antibody (HyClone , Item No.: sv30010). According to the set concentration gradient of small molecule inhibitors, the storage solution and basal medium are used to prepare medium containing different concentrations of small molecule inhibitors.
- the final volume of the cell culture medium per well is 1ml
- the total number of cells per well is calculated based on the cell density of each well as 2*10 ⁇ 5 cells
- the density of 50 ⁇ l cell suspension per well is 4*10 ⁇ 6/ml
- Example 3 Flow cytometric detection of dryness of HSCs and maintenance of CD34+
- FITC anti-human CD45RA Biolegend 304106 APC Mouse IgG2a, ⁇ Isotype Ctrl Biolegend 400220 APC/Cyanine7Mouse IgG1, ⁇ Isotype Ctrl Biolegend 400128 PE Mouse IgG2a, ⁇ Isotype Ctrl Biolegend 400212 FITC Mouse IgG2b, ⁇ Isotype Ctrl Biolegend 402208 Brilliant Violet 510™ Mouse IgG2a, ⁇ Isotype Ctrl Biolegend 400268
- control is set to NC group and ISO group respectively, and the cell selection is any sample or mixed cells of the samples to be tested in this batch of experiments, depending on the number of cells. In the same batch of experiments, each control group does not have duplicate testing. See Table 3 for group settings.
- the test results are analyzed according to the following methods: 1) The target cell population is CD34+CD45+CD45RA-CD90+CD38- cell population; 2) The logic gate and gate position determination are shown in Figure 1: First, circle the cell population, P1 gate; source The cell population in the P1 gate removes the adherent cells and becomes the P2 gate; the cell population derived from the P2 gate uses NC or ISO to delimit the CD34, CD45, and CD45RA negative cell population, which is the Q3-LL gate (CD34-/CD45-), Q5- UL+Q5-LL gate (CD45RA-); FMO90 delineates the CD90-negative cell population, which is the Q5-LL+Q5-LR gate; FMO38 delineates the CD38-negative cell population, which is the Q6-LR gate; the use of NC, ISO, FMO delineated Gate, confirm that the cells delineated by Q3-UR-Q5-UL-Q6-LR gate are CD34+CD45+CD45RA-CD90+CD
- Example 2 On the cord blood-derived CD34+ cells sorted in Example 1, the optimal concentration of small molecule inhibitors and the screening of the ability to maintain the dryness of HSCs were performed in the same way as in Example 2. After 6-7 days of small molecule induction, The same method as in Example 3 was used to detect the expression of long-term hematopoietic stem cells (LT-HSCs) cell surface markers (CD34+CD45+CD90+CD45RA-CD38-) by flow cytometry.
- LT-HSCs long-term hematopoietic stem cells
- Valproic acid (VPA) in Table 8 is significantly higher than other small molecules about 30 times, and about 1 times higher than SAHA.
- SAHA is 15 times that of other small molecules (except VPA). In maintaining the ratio of CD34+ cells, SAHA and VPA are superior to other small molecules.
- Small molecule inhibitor name Test concentration VE821 0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M AZ20 0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M PFI-3 0.2 ⁇ M, 2 ⁇ M, 5 ⁇ M, 10 ⁇ M Sodium 4-Aminosalicylate(S-4-A) 0.1mM, 1mM, 5mM, 10mM PDTC 1nM, 5nM, 10nM, 50nM SAHA 0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M Santacruzamate A(SIS3HCL) 0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M SR1 0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M
- Example 3 On the cord blood-derived CD34+ cells sorted in Example 1, the best bimolecular combination of small molecule inhibitors to maintain the dryness of HSCs was screened in the same way as in Example 2. After 6-7 days of induction of the small molecule combination, The expression of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) was detected by flow cytometry using the same method as in Example 3.
- Figure 7 shows that in terms of HSCs stemness maintenance and CD34+ cell ratio, the combination of inhibitors of SAHA and the four types of hematopoietic stem cell expansion related cell signaling pathways in Table 4 SAHA+SR1, SAHA+VE821, SAHA+ The comparison of PFI-3 and SAHA+S-4-A is not significantly better than SAHA alone.
- Fig. 9 show that in terms of maintaining the dryness of HSCs, in the combination of SAHA and Butyrate in Table 6, Butyrate is not as effective as SAHA alone when used at low concentrations. Butyrate is more effective than SAHA alone when used at high concentrations, but under high concentration conditions, Butyrate It is cytotoxic, and the results obtained in Example 4 show that Butyrate alone is not as effective as SAHA alone, and it is the same target inhibitor as SAHA, so Butyrate will not be further studied in the future.
- SAHA is combined with EPZ004777 and DZNeP in Table 6, and the combination of SAHA+EPZ004777 and SAHA+DZNeP is better than SAHA alone.
- Example 3 On the cord blood-derived CD34+ cells sorted in Example 1, the best tri-molecular combination of small molecule inhibitors to maintain the dryness of HSCs was screened in the same way as in Example 2. After 6-7 days of induction of the small molecule combination, The expression of LT-HSCs cell surface marker (CD34+CD45+CD90+CD45RA-CD38-) was detected by flow cytometry using the same method as in Example 3.
- Fig. 14 show that the bimolecular combinations SAHA+Dasatinib and SAHA+VPA that can maintain the dryness of HSCs screened in Example 5 were recombined.
- the tri-molecular combination of SAHA+VPA+Dasatinib is 50 times that of the Mock group and 1.5-2 times that of the bi-molecular combination.
- the tri-molecular combination of SAHA+VPA+Dasatinib was 20% higher than that of the Mock group.
- Example 7 Comparison of the screened inhibitors SAHA, VPA, Dasatinib and the reported inhibitors UM171 and SR1 alone and in combination
- the dual-molecule combination SAHA+Dasatinib and VPA+Dasatinib are more effective than small molecules alone, the dual-molecule combination SAHA+SR1 and SAHA+UM171 and tri-molecules Combination SAHA+DZNeP+EPZ004777.
- Example 8 CD34+ hematopoietic stem cell colony formation and culture
- colony-forming unit was used to detect the in vitro function of cord blood-derived hematopoietic stem cells induced by small molecule inhibitors for qualitative and quantitative detection, and to verify their in vitro differentiation potential.
- Carry out cell seeding Take the small-molecule inhibitor-induced cells (CD34+ hematopoietic stem cells induced by small-molecule inhibitors from cord blood) suspension cell count after 7 days of expansion and culture, and draw 100 times the inoculation density of the cell suspension (e.g. , The inoculation density is 100cells/well/3ml, 10000cells should be collected), add 1ml of 2% FBS (Gibco, article number: 16000-044)-IMDM (Gibco, article number: 12440-053) medium, and mix well for later use.
- FBS Gabco, article number: 16000-044
- IDM Ibco, article number: 12440-053
- 3 cc Syringes (Stem cell, item number: 28240) used in conjunction with Blunt-End Needles 16 Gauge (Stemcell, item number: 28110), draw up the cell suspension to 1 mL, push out the syringe to exhaust the gas in the syringe, and re-absorb all the obtained
- inject 3 mL into a hole of SmsrtDishTM-6 (stem cell, catalog number: 27370, 6-well plate), and slowly tilt the 6-well plate to make the cell suspension evenly spread over the bottom of the well.
- Add 3ml of sterile PBS to the gap of each well of the 6-well plate to prevent the medium from drying up. Cover the 6-well plate and place it in a carbon dioxide incubator (Thermo, model: 3111) at 37°C, 5% CO 2 , and 95% relative humidity for 14 days.
- CFU-GEMM (CFU-G, CFU-E, CFU-MM): granulocyte-erythrocyte-macrophage-megakaryocyte colony forming unit.
- a colony contains red blood cells and 20 or more non-red blood cells (granulocytes, macrophages and/or megakaryocytes). Usually, there are red blood cells in the center of the colony and non-red blood cells around. The non-red blood cells can also be concentrated on one side of the red blood cells.
- the colony of CFU-GEMM is usually larger than the colony of CFU-GM or BFU-E. It is relatively rare in most cell samples (usually 10% of the total number of colonies).
- CFU-GM A colony containing more than 20 granulocytes (CFU-G) and/or macrophages (CFU-M). It does not appear red or brown, and individual cells in the colony can usually be distinguished, especially at the edge of the colony. Large colonies may have one or more dense dark nuclei. Erythropoietin (EPO) is not required for colony growth and differentiation.
- CFU-G granulocytes
- CFU-M macrophages
- BFU-E Burst red blood cell colony forming unit, forming a colony composed of single or multiple cell clusters, each colony contains >200 mature red blood cells. When cells are hemoglobinized, they appear red or brown, and it is difficult to distinguish individual cells in each cluster. BFU-E is a more immature progenitor cell, and its growth requires erythropoietin (EPO) and other cytokines, especially interleukin-3 (IL-3) and Stem Cell Factor (SCF) to promote the optimal growth of its colonies.
- EPO erythropoietin
- IL-3 interleukin-3
- SCF Stem Cell Factor
- CFU-E Red blood cell colony forming unit, which can form 1-2 cell clusters containing 8-200 red blood cells. When the cells are hemoglobinized, they appear red or brown, and it is difficult to distinguish individual cells in the colony.
- CFU-E is a progenitor cell of a mature red blood cell lineage and requires erythropoietin (EPO) to promote its differentiation.
- Example 9 Comparison of in vitro clone formation ability of screened inhibitors SAHA, VPA, Dasatinib and literature reported inhibitors UM171 and SR1 alone and in combination
- the umbilical cord blood-derived CD34+ cells sorted in Example 1 were used to compare the cloning ability of the screened inhibitors SAHA, VPA, and Dasatinib with the inhibitors UM171 and SR1 reported in the literature alone and in combination.
- the in vitro clone (CFU) formation test was performed using the same method as in Example 8. 14 days after cell inoculation, the number of clones was counted and the CFU-GEMM was analyzed. The results are shown in Figure 16. Among them, BFU-E, CFU-E, CFU-GM, CFU-GEMM represent clones of different blood system lineages such as erythroid, myeloid, and lymphoid lines.
- VPA+Dasatinib and SAHA+Dasatinib are better than the small molecule SR1, UM171 used alone and in combination with SAHA reported in the literature.
- Example 2 On the cord blood-derived CD34+ cells sorted in Example 1, the same method as in Example 2 was used to screen other small molecule inhibitors of the Src target. After 6-7 days of induction of the small molecule combination, use the same method as in Example 2. 3 The same method was used to detect the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38-) using the same method. The results are shown in Figure 17. Among them, the small molecule inhibitors and their concentrations screened in this round See Table 9.
- Example 11 Comparison of the ability of the screened inhibitor Dasatinib and the inhibitors UM171 and SR1 reported in the literature on the in vitro expansion and dryness maintenance of hematopoietic stem cells
- Example 3 On CD34+ cells derived from umbilical cord blood sorted in Example 1, the screening inhibitor Dasatinib was compared with the inhibitors UM171 and SR1 reported in the literature for in vitro expansion and dryness maintenance ability. After 6-8 days of induction by small molecule inhibitors, flow cytometry was used to detect the expression of LT-HSCs cell surface markers (CD34+CD45+CD90+CD45RA-CD38-) using the same method as in Example 3.
- Example 12 Verification of the effect of the screened inhibitor Dasatinib and the reported inhibitor SR1 on hematopoietic stem cell transplantation in vivo
- Example 1 On the cord blood-derived CD34+ cells sorted in Example 1, comparisons were made between the small molecule inhibitor Dasatinib that has been screened and the ability of the inhibitor SR1 to be used alone to rebuild the hematopoietic system in vivo.
- the concentration and grouping of small molecule inhibitors used in this example are shown in Table 10.
- the initial amount of cultured cells transplanted to each mouse is 1*10 ⁇ 5/mouse, and the cells expanded in each well of the 24-well plate can be transplanted to one mouse. Counting every other day during the cell culture process, the technical method and cell counter are the same as in Example 1, to ensure that the cell density does not exceed 8*10 ⁇ 5/ml. If the cells are too dense, separate wells in time and add fresh medium.
- mice Prepare mice, each group set 8 mice. Mice were purchased from Beijing Vital Mastery Biotechnology Co., strains of NPG (NOD-Prkdc scid ll2rg null / Vst), 6 weeks old, female, the difference between the weight control mice in grams 3g. The mice were irradiated with a half-lethal dose before cell transplantation, and the irradiation dose was 1.6Gy.
- mice peripheral blood of mice was collected at 4th week, 8th week, and 12th week, and flow cytometry was performed to detect human CD45 ratio; at 16th week, mice were sacrificed, and peripheral blood, bone marrow cells and spleen of mice were collected.
- Flow cytometry detects the proportion of human CD45 in peripheral blood of mice. See Table 12 for the set of cell detection groups.
- Collect mouse peripheral blood (approximately 100 ⁇ l), and add antibodies according to the groups set in Table 12. Vortex to mix, and incubate for 15 min at room temperature in the dark. After the incubation, add 1.2ml of 1X red blood cell lysate to the NC and each sample, vortex to mix, and lyse at room temperature for 15 minutes in the dark, during which time the sample centrifuge tube is turned upside down every 3 minutes. After lysis, centrifuge at 400g for 5min at room temperature. After centrifugation, the supernatant was discarded, and 1ml of PBS containing 1% HSA was added to each experimental sample, mixed well, and centrifuged at 400g for 5min at room temperature.
- the test results are analyzed according to the following methods: 1) The target cell population is human CD45+ cell population; 2) The determination of the logic gate and gate position is shown in Figure 19: first delineate the cell population, which is the gate P1; remove the cell population from the gate P1 Adhesive cells are in gate P2; cell populations derived from P2 gate are delineated by 7-AAD to delineate live cell populations, which are gate P3; cell populations derived from P3 gate are delineated by NC mouse CD45- and human CD45-cell populations (Q2-LL Gate); Use the gate delineated by NC to determine that the cells delineated by the Q2-UL gate are human CD45+ target cells.
- the efficiency of artificial blood stem cell transplantation is expressed by the proportion of human CD45 cells, and the calculation method is human CD45%/(human CD45%+mouse CD45%).
- mice peripheral blood (approximately 100 ⁇ l), and add antibodies according to the groups set in Table 13. Subsequent blood sample processing is consistent with the aforementioned procedure for detecting human CD45 ratios in peripheral blood of mice. After the operation, use a flow cytometer for detection.
- mice were sacrificed by neck dissection, and the tibia and femur of one hind leg of the mouse were taken.
- Use ophthalmic scissors and ophthalmic forceps to cut off both ends of the tibia and femur to expose the bone marrow cavity.
- Use a 1ml syringe to suck up the pre-cooled PBS containing 1% HSA, pierce the needle into one end of the bone marrow cavity, push the PBS strongly, and flush out the bone marrow cells from the other end of the bone marrow cavity.
- the bone marrow cavity of the tibia and femur was rinsed with 2ml PBS respectively.
- mice were sacrificed by neck dissection, and their spleens were taken and placed in pre-chilled PBS containing 1% HSA. Cut the spleen with ophthalmological scissors, pipette repeatedly to suck the spleen tissue suspension, filter with 40 ⁇ m cell mesh, 400g, centrifuge at room temperature for 5min. After the centrifugation, the supernatant was discarded and the spleen cells were used for later use.
- the test results are analyzed according to the following methods: 1) The target cell group is human CD45+ cell group, human CD19+ cell group, human CD3+ cell group, human CD33+ cell group, and human CD56+ cell group; 2) The determination of logic gates and gate positions is shown in the figure Figure 20: First delimit the cell population, P1 gate; the cell population derived from P1 gate removes the adherent cells, and is the P2 gate; the cell population derived from P2 gate uses 7-AAD to delimit the living cell population, which is the P3 gate; derived from P3 The cell population of the gate uses NC to delineate mouse CD45+ (P4 gate) and human CD45+ cell population (P5 gate); the cell population from P5 gate uses NC to delineate human CD33+ (P6 gate) and human CD56+ cell population (P7 gate); source The cell population in gate P5 is delineated by NC to delineate human CD19+ (P8 gate) and human CD3+ cell population (P9 gate).
- the efficiency of artificial blood stem cell transplantation is expressed by the proportion of human CD45 cells, and the calculation method is human CD45%/(human CD45%+mouse CD45%).
- the efficiency of human hematopoietic stem cells differentiated into blood cells of various lineages in mice is human CD19% (representing B cells), human CD3% (representing T cells), human CD33% (representing myeloid cells), and human CD56% (representing NK cells) ) Means.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Pain & Pain Management (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
抗体名称 | 厂家 | 货号 |
APC/Cy7 anti-human CD45 | Biolegend | 304014 |
APC anti-human CD38 | Biolegend | 356606 |
Brilliant Violet 510™;anti-human CD34 | Biolegend | 343528 |
PE anti-human CD90(Thy1) | Biolegend | 328110 |
FITC anti-human CD45RA | Biolegend | 304106 |
APC Mouse IgG2a,κIsotype Ctrl | Biolegend | 400220 |
APC/Cyanine7Mouse IgG1,κIsotype Ctrl | Biolegend | 400128 |
PE Mouse IgG2a,κIsotype Ctrl | Biolegend | 400212 |
FITC Mouse IgG2b,κIsotype Ctrl | Biolegend | 402208 |
Brilliant Violet 510™;Mouse IgG2a,κIsotype Ctrl | Biolegend | 400268 |
小分子抑制剂名称 | 测试浓度 |
VE821 | 0.1μM,1μM,5μM,10μM |
AZ20 | 0.1μM,1μM,5μM,10μM |
PFI-3 | 0.2μM,2μM,5μM,10μM |
Sodium 4-Aminosalicylate(S-4-A) | 0.1mM,1mM,5mM,10mM |
PDTC | 1nM,5nM,10nM,50nM |
SAHA | 0.1μM,1μM,5μM,10μM |
Santacruzamate A(SIS3HCL) | 0.1μM,1μM,5μM,10μM |
SR1 | 0.1μM,1μM,5μM,10μM |
小分子抑制剂名称 | 测试浓度 |
SAHA | 1μM |
SR1 | 5μM |
UM171 | 350nM |
PGE2 | 10μM |
GW9662 | 1μM |
FLU | 1μM |
小分子抑制剂名称 | 测试浓度 |
SAHA | 1μM |
Vitamin C | 5μg/ml,25μg/ml,50μg/ml |
EPZ004777 | 0.5μM,5μM,10μM |
Forskolin | 5μM,10μM,20μM |
CPI-455 | 1μM,5μM,10μM |
DZNeP | 10nM,50nM,250nM |
CHIR-99021 | 1μM,3μM,10μM |
Butyrate | 50μM,250μM,500μM, |
SB203580 | 1μM,5μM,10μM |
IWP-2 | 1μM,5μM,10μM |
IWR-1-endo | 1μM,5μM,10μM |
JNK-inhibitor iX | 1μM,5μM,10μM |
Dibutyryl-cAMP | 1μM,5μM,10μM |
Thiazovivin | 1μM,5μM,10μM |
IMR-1 | 1μM,5μM,10μM |
SB431542 | 1μM,5μM,10μM |
Quercetin | 0.5μM,1μM,5μM |
小分子抑制剂名称 | 测试浓度 |
SAHA | 1μM |
Dasatinib | 0.5μM 5μM 10μM |
SGC0496 | 1μM 5μM 10μM |
JNK-IN-8 | 1μM 2μM 10μM |
Enzastaurin(LY317615) | 1μM 5μM 10μM |
小分子抑制剂名称 | 测试浓度 |
SAHA | 1μM |
Valproic acid | 100μM 500μM 1000μM |
Valpromide | 100μM 500μM 1000μM |
AZD5438 | 1μM 5μM 10μM |
Go 6983 | 1μM 5μM 10μM |
Sodium succinate | 5μM 10μM 50μM |
Sodium dichloroacetate(DCA) | 0.1mM 1mM 5mM |
Salubrinal | 1μM 5μM 10μM |
CPI-613 | 5μM 10μM 50μM |
ISRIB(trans-isomer) | 1μM 5μM 10μM |
Acetylcysteine | 1μM 5μM 10μM |
GSK2606414 | 10nM 50nM 100nM |
DMOG | 1μM 5μM 10μM |
Metronidazole | 1μM 5μM 10μM |
DHT | 1nM 5nM 50nM |
Nocodazole | 1μM 5μM 10μM |
Vinblastine sulfate | 1μM 5μM 10μM |
Epigallocatechin Gallate | 1μM 5μM 10μM |
Deferoxamine mesylate: | 10nM 50nM 500nM |
Epicatechin gallate | 10nM 50nM 500nM |
Colchicine | 50nM 500nM 1000nM |
Y27632 | 5μM 10μM 20μM |
小分子抑制剂名称 | 测试浓度 |
SAHA | 1μM |
UM-164 | 1μM 5μM 10μM |
KX2-391 | 10nM 50nM 100nM |
KX1-004 | 10μM 50μM 100μM |
组别 | 小分子抑制剂使用浓度 |
Mock | —— |
SR1 | 5μM |
Dasatinib | 50nM |
抗体名称 | 厂家 | 货号 |
FITC anti-mouse CD45 | Biolegend | 103108 |
APC/Cy7 anti-human CD45 | Biolegend | 304014 |
Brilliant Violet 510 TM anti-human CD3 | Biolegend | 300448 |
PE anti-human CD19 | Biolegend | 363004 |
Brilliant Violet 421 TM anti-human CD33 | Biolegend | 303416 |
APC anti-human CD56 | Biolegend | 304610 |
7-AAD Viability Staining Solution | Biolegend | 420404 |
Claims (31)
- 一种促进HSCs增殖并维持HSCs干性的方法,包括使HSCs体外接触含有STAT细胞信号传导通路的小分子抑制剂或者其它细胞信号通路小分子抑制剂的培养液。
- 根据权利要求1的方法,所述含有STAT细胞信号传导通路的小分子抑制剂为Src靶点的小分子抑制剂。
- 根据权利要求2的方法,所述Src靶点的小分子抑制剂选自如下的一种或多种:Dasatinib、Quercetin、UM-164、KX2-391和KX1-004。
- 根据权利要求2-3任一项的方法,其中所述Src靶点的小分子抑制剂与所述其它细胞信号通路小分子抑制剂联合使用。
- 根据权利要求4的方法,其中,所述其它细胞信号通路小分子抑制剂选自以HDAC为靶点的小分子抑制剂、以PKC为靶点的小分子抑制剂和以JNK为靶点的小分子抑制剂中的一种或两种以上。
- 根据权利要求5的方法,其中,所述Src靶点的小分子抑制剂与以HDAC为靶点的小分子抑制剂、以PKC为靶点的小分子抑制剂或以JNK为靶点的小分子抑制剂联合使用。
- 根据权利要求6的方法,其中,所述Src靶点的小分子抑制剂与以HDAC为靶点的小分子抑制剂VPA、以HDAC为靶点的小分子抑制剂SAHA、以PKC为靶点的小分子抑制剂Enzastaurin或以JNK为靶点的小分子抑制剂JNK-IN-8联合使用。
- 根据权利要求7的方法,其中,所述Src靶点的小分子抑制剂为Dasatinib。
- 根据权利要求8的方法,其中,Dasatinib与VPA或SAHA联合使用。
- 根据权利要求2-8任一项的方法,其中所述Src靶点的小分子抑制剂单独或与其它抑制剂的联合使用维持具有CD34+CD45+CD90+CD45RA-CD38-表型的HSCs的细胞占全部细胞中的比例超过8%、10%、15%、20%、25%或30%。
- 根据权利要求2-9任一项的方法,其中所述Src靶点的小分子抑制剂单独或与其它抑制剂的联合使用维持CD34+细胞占全部细胞中的比例超 过65%、70%、75%、80%或85%。
- 一种用于维持HSCs干性的组合物,其包括含有STAT细胞信号传导通路的小分子抑制剂。
- 根据权利要求12的组合物,其中,所述含有STAT细胞信号传导通路的小分子抑制剂为Src靶点的小分子抑制剂。
- 根据权利要求13的组合物,其中,所述Src靶点的小分子抑制剂选自如下的一种或多种:Dasatinib、Quercetin、UM-164、KX2-391和KX1-004。
- 根据权利要求13-14任一项的组合物,所述组合物还包括其它细胞信号通路小分子抑制剂。
- 根据权利要求15的组合物,其中,所述其它细胞信号通路小分子抑制剂包括以HDAC为靶点的小分子抑制剂、以PKC为靶点的小分子抑制剂和以JNK为靶点的小分子抑制剂。
- 根据权利要求16所述的组合物,其中,所述其它细胞信号通路小分子抑制剂包括以HDAC为靶点的小分子抑制剂VPA、以HDAC为靶点的小分子抑制剂SAHA、以PKC为靶点的小分子抑制剂Enzastaurin和以JNK为靶点的小分子抑制剂JNK-IN-8。
- 根据权利要求12-17任一项的组合物,其中,所述组合物还包括SFEMII培养基、生长因子Flt-3L、生长因子SCF、生长因子TPO和生长因子IL-6。
- 根据权利要求12-18任一项的组合物,其中,所述组合物维持具有CD34+CD45+CD90+CD45RA-CD38-表型的HSCs的细胞占全部细胞中的比例超过8%、10%、15%、20%、25%或30%。
- 根据权利要求12-19任一项的组合物,其中,所述组合物维持CD34+细胞占全部细胞中的比例超过65%、70%、75%、80%或85%。
- 一种用于维持HSCs干性的组合物,包含选自如下的任一组合:SAHA+EPZ004777、SAHA+DZNeP、SAHA+Dasatinib、VPA+Dasatinib、SAHA+JNK-IN-8或SAHA+VPA。
- 权利要求21的组合物,其中所述组合物维持具有CD34+CD45+CD90+CD45RA-CD38-表型的HSCs的细胞占全部细胞中的比例超过8%、10%、15%、20%、25%或30%。
- 权利要求21或22的组合物,其中所述组合物维持CD34+细胞占全部细胞中的比例超过65%、70%、75%、80%或85%。
- 根据权利要求21-23任一项的组合物,其中,所述组合物还包括SFEMII培养基、生长因子Flt-3L、生长因子SCF、生长因子TPO和生长因子IL-6。
- 一种用于维持HSCs干性的组合物,包含选自如下的任一组合:SAHA+EPZ004777+DZNeP或SAHA+VPA+Dasatinib。
- 根据权利要求25的组合物,其中,所述组合物维持具有CD34+CD45+CD90+CD45RA-CD38-表型的HSCs的细胞占全部细胞中的比例超过8%、10%、15%、20%、25%或30%。
- 根据权利要求25或26的组合物,其中所述组合物维持CD34+细胞占全部细胞中的比例超过65%、70%、75%、80%或85%。
- 根据权利要求25-27任一项的组合物,其中,所述组合物还包括SFEMII培养基、生长因子Flt-3L、生长因子SCF、生长因子TPO和生长因子IL-6。
- 根据权利要求17-19任一项的组合物,其中每种抑制剂在培养基中的浓度为:Dasatinib:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;SAHA:10nM-20μM,优选为20nM-15μM,进一步优选为30nM-10μM,最优选为0.1μM-10μM;VPA:10μM-2000μM,优选为10μM-1500μM,进一步优选为10μM-1000μM,最优选为100μM-1000μM;JNK-IN-8:0.1μM-20μM,优选为0.5μM-15μM,进一步优选为0.5μM-10μM,最优选为1μM-10μM;EPZ004777:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;DZNeP:1nM-500nM,优选为5nM-400nM,进一步优选为10nM-300nM,最优选为10nM-250nM;UM-164:0.1μM-1000μM,优选为0.5μM-500μM,进一步优选为 1μM-100μM,最优选为1μM-10μM;KX2-391:0.1nM-1000nM,优选为1nM-1000nM,进一步优选为10nM-500nM,最优选为10nM-100nM;KX1-004:0.1μM-1000μM,优选为1μM-1000μM,进一步优选为10μM-500μM,最优选为10μM-100μM。
- 根据权利要求21-24任一项的组合物,其中每种抑制剂在培养基中的浓度为:Dasatinib:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;SAHA:10nM-20μM,优选为20nM-15μM,进一步优选为30nM-10μM,最优选为0.1μM-10μM;VPA:10μM-2000μM,优选为10μM-1500μM,进一步优选为10μM-1000μM,最优选为100μM-1000μM;JNK-IN-8:0.1μM-20μM,优选为0.5μM-15μM,进一步优选为0.5μM-10μM,最优选为1μM-10μM;EPZ004777:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;DZNeP:1nM-500nM,优选为5nM-400nM,进一步优选为10nM-300nM,最优选为10nM-250nM。
- 根据权利要求25-28任一项的组合物,其中每种抑制剂在培养基中的浓度为:Dasatinib:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;SAHA:10nM-20μM,优选为20nM-15μM,进一步优选为30nM-10μM,最优选为0.1μM-10μM;VPA:10μM-2000μM,优选为10μM-1500μM,进一步优选为10μM-1000μM,最优选为100μM-1000μM;EPZ004777:0.1μM-50μM,优选为0.5μM-40μM,进一步优选为0.5μM-30μM,最优选为0.5μM-10μM;DZNeP:1nM-500nM,优选为5nM-400nM,进一步优选为10nM-300nM, 最优选为10nM-250nM。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202080086704.9A CN114787342A (zh) | 2019-12-16 | 2020-12-16 | 扩增造血干细胞的小分子化合物及其组合 |
KR1020227024470A KR20220116508A (ko) | 2019-12-16 | 2020-12-16 | 조혈 줄기 세포의 증폭을 위한 저분자 화합물 및 이의 조합 |
EP20904181.3A EP4079842A4 (en) | 2019-12-16 | 2020-12-16 | SMALL MOLECULE COMPOUNDS FOR AMPLIFYING HEMATOPOIETIC STEM CELLS, AND THEIR COMBINATION |
US17/786,433 US20230027247A1 (en) | 2019-12-16 | 2020-12-16 | Small molecule compounds for amplifying hematopoietic stem cells, and combination thereof |
CA3162030A CA3162030A1 (en) | 2019-12-16 | 2020-12-16 | Small molecule compounds for amplifying hematopoietic stem cells, and combination thereof |
JP2022537871A JP2023507486A (ja) | 2019-12-16 | 2020-12-16 | 造血幹細胞を増幅するための小分子化合物、及びそれらの組み合わせ |
AU2020404285A AU2020404285A1 (en) | 2019-12-16 | 2020-12-16 | Small molecule compounds for amplifying hematopoietic stem cells, and combination thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2019125687 | 2019-12-16 | ||
CNPCT/CN2019/125687 | 2019-12-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021121266A1 true WO2021121266A1 (zh) | 2021-06-24 |
Family
ID=76478496
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/136790 WO2021121266A1 (zh) | 2019-12-16 | 2020-12-16 | 扩增造血干细胞的小分子化合物及其组合 |
Country Status (9)
Country | Link |
---|---|
US (1) | US20230027247A1 (zh) |
EP (1) | EP4079842A4 (zh) |
JP (1) | JP2023507486A (zh) |
KR (1) | KR20220116508A (zh) |
CN (1) | CN114787342A (zh) |
AU (1) | AU2020404285A1 (zh) |
CA (1) | CA3162030A1 (zh) |
TW (1) | TW202136503A (zh) |
WO (1) | WO2021121266A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11661596B2 (en) | 2019-07-12 | 2023-05-30 | Peking University | Targeted RNA editing by leveraging endogenous ADAR using engineered RNAs |
US11702658B2 (en) | 2019-04-15 | 2023-07-18 | Edigene Therapeutics (Beijing) Inc. | Methods and compositions for editing RNAs |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20230050123A (ko) * | 2021-10-07 | 2023-04-14 | 삼성전자주식회사 | 전자 장치 및 그 제어 방법 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101287706A (zh) * | 2005-09-21 | 2008-10-15 | 尼科梅德有限责任公司 | 作为组蛋白脱乙酰基酶抑制剂的磺酰基吡咯盐酸盐 |
CN104278008A (zh) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | 一种通过小分子化合物处理来制备多潜能干细胞的方法、试剂盒和用途 |
CN105705021A (zh) * | 2013-05-20 | 2016-06-22 | 位于西奈山的伊坎医学院 | 用于治疗血液病症的富集和扩增的人脐带血干细胞 |
CN108060120A (zh) * | 2016-11-07 | 2018-05-22 | 云南济慈再生医学研究院有限公司 | 用于分化的细胞重编程的小分子化合物组合、试剂盒及应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2018354418A1 (en) * | 2017-10-27 | 2020-05-14 | Immunebridge Inc. | Compositions and methods of making expanded hematopoietic stem cells using derivatives of fluorene |
-
2020
- 2020-12-16 KR KR1020227024470A patent/KR20220116508A/ko not_active Application Discontinuation
- 2020-12-16 US US17/786,433 patent/US20230027247A1/en active Pending
- 2020-12-16 AU AU2020404285A patent/AU2020404285A1/en active Pending
- 2020-12-16 EP EP20904181.3A patent/EP4079842A4/en active Pending
- 2020-12-16 WO PCT/CN2020/136790 patent/WO2021121266A1/zh unknown
- 2020-12-16 TW TW109144495A patent/TW202136503A/zh unknown
- 2020-12-16 CA CA3162030A patent/CA3162030A1/en active Pending
- 2020-12-16 JP JP2022537871A patent/JP2023507486A/ja active Pending
- 2020-12-16 CN CN202080086704.9A patent/CN114787342A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101287706A (zh) * | 2005-09-21 | 2008-10-15 | 尼科梅德有限责任公司 | 作为组蛋白脱乙酰基酶抑制剂的磺酰基吡咯盐酸盐 |
CN105705021A (zh) * | 2013-05-20 | 2016-06-22 | 位于西奈山的伊坎医学院 | 用于治疗血液病症的富集和扩增的人脐带血干细胞 |
CN104278008A (zh) * | 2013-07-12 | 2015-01-14 | 北京大学科技开发部 | 一种通过小分子化合物处理来制备多潜能干细胞的方法、试剂盒和用途 |
CN108060120A (zh) * | 2016-11-07 | 2018-05-22 | 云南济慈再生医学研究院有限公司 | 用于分化的细胞重编程的小分子化合物组合、试剂盒及应用 |
Non-Patent Citations (9)
Title |
---|
BOITANO A E ET AL., SCIENCE, 2010 |
EVANS T., CELL STEM CELL., 2009 |
FARES I ET AL., SCIENCE, 2014 |
GUO B ET AL., NATURE MEDICINE, 2018 |
GUO B ET AL., NATURE MEDICINE., 2017 |
S HIBINO ET AL: "Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells", 《ONCOGENESIS》, vol. 3, no. 5, 26 May 2014 (2014-05-26), XP055649533, ISSN: 2157-9024, DOI: 10.1038/oncsis.2014.17 * |
See also references of EP4079842A4 |
WARREN FISKUS ET AL: "Cotreatment with vorinostat (suberoylanilide hydroxamic acid) enhances activity of dasatinib (BMS-354825) against imatinib mesylate-sensitive or imatinib mesylate-resistant chronic myelogenous leukemia cells", 《CLIN CANCER RES》, vol. 12, no. 19, 1 October 2006 (2006-10-01), XP002672182, ISSN: 1078-0432, DOI: 10.1158/1078-0432.ccr-06-0980 * |
XIONG ZHANG ET AL: "Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells", STEM CELL RES, vol. 13, no. 3, 30 November 2014 (2014-11-30), XP055483780, ISSN: 1873-5061, DOI: 10.1016/j.scr.2014.09.007 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11702658B2 (en) | 2019-04-15 | 2023-07-18 | Edigene Therapeutics (Beijing) Inc. | Methods and compositions for editing RNAs |
US11661596B2 (en) | 2019-07-12 | 2023-05-30 | Peking University | Targeted RNA editing by leveraging endogenous ADAR using engineered RNAs |
Also Published As
Publication number | Publication date |
---|---|
US20230027247A1 (en) | 2023-01-26 |
EP4079842A1 (en) | 2022-10-26 |
TW202136503A (zh) | 2021-10-01 |
AU2020404285A1 (en) | 2022-07-07 |
EP4079842A4 (en) | 2024-01-24 |
CA3162030A1 (en) | 2021-06-24 |
KR20220116508A (ko) | 2022-08-23 |
JP2023507486A (ja) | 2023-02-22 |
CN114787342A (zh) | 2022-07-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2021121266A1 (zh) | 扩增造血干细胞的小分子化合物及其组合 | |
Peled et al. | Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment | |
Vukovic et al. | Microglia modulate hippocampal neural precursor activity in response to exercise and aging | |
AU770896B2 (en) | Methods of controlling proliferation and differentiation of stem and progenitor cells | |
CN112980789A (zh) | 扩增造血干细胞的小分子化合物及其组合 | |
BR112014020119A2 (pt) | cultura de células-tronco mesenquimais | |
US20120093782A1 (en) | Enhanced Hematopoietic Stem Cell Engraftment | |
CN108478599A (zh) | 间充质基质细胞及其相关用途 | |
EP4095240A1 (en) | Serum-free medium and culturing method suited for culturing blood cells such as human hematopoietic stem cells | |
Kurtzberg et al. | Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases | |
Toya et al. | Interaction of a Specific Population of Human Embryonic Stem Cell–Derived Progenitor Cells with CD11b+ Cells Ameliorates Sepsis-Induced Lung Inflammatory Injury | |
JP2002502617A (ja) | 幹細胞および前駆細胞の増殖と分化を制御する方法 | |
BRPI0620523A2 (pt) | método de aumentar o potencial de implante e de atração celular, método de preparar células para transplante em um sujeito, população de células e composição farmacêutica | |
CN114075547A (zh) | 扩增造血干细胞的方法及其组合物 | |
Gupta et al. | Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase | |
CN112805015A (zh) | 造血干细胞和造血祖细胞扩增系统 | |
EP1807509A1 (en) | Multipotent stem cells isolated from umbilical cord blood and the cellular therapeutic agent comprisin the same for treating ischemic disease | |
CN110551688B (zh) | 一种诱导体细胞重编程为造血干/祖细胞且促进造血干/祖细胞体外扩增的组合物及其应用 | |
WO2022143675A1 (zh) | 用于扩增并维持HSCs自我更新能力和分化潜能的培养基组合物及其应用 | |
WO2022063226A1 (zh) | 化合物用于提高人造血干细胞移植效率的用途 | |
KR101132858B1 (ko) | 내피전구세포의 체외 증폭방법 | |
Chivu et al. | Ex vivo differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium | |
KR20100061794A (ko) | 경동맥체로부터 유래된 줄기세포 및 이의 용도 | |
Arlat et al. | Generation of functionally active resident macrophages from adipose tissue by 3-D cultures | |
Almalki | Investigation of Haematopoietic Stem Cell Homing Post-transplantation Using an in Vitro Model of the Bone Marrow/vasculature Interface |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20904181 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3162030 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2022537871 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020404285 Country of ref document: AU Date of ref document: 20201216 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20227024470 Country of ref document: KR Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2020904181 Country of ref document: EP Effective date: 20220718 |