US20210238548A9 - Pluripotent stem cell and t cell differentiated therefrom and application thereof - Google Patents
Pluripotent stem cell and t cell differentiated therefrom and application thereof Download PDFInfo
- Publication number
- US20210238548A9 US20210238548A9 US16/312,794 US201816312794A US2021238548A9 US 20210238548 A9 US20210238548 A9 US 20210238548A9 US 201816312794 A US201816312794 A US 201816312794A US 2021238548 A9 US2021238548 A9 US 2021238548A9
- Authority
- US
- United States
- Prior art keywords
- cells
- medium
- pluripotent stem
- stem cell
- recombinant mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000001778 pluripotent stem cell Anatomy 0.000 title claims abstract description 80
- 210000004027 cell Anatomy 0.000 title claims description 51
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 31
- 210000000130 stem cell Anatomy 0.000 claims abstract description 28
- 108010043471 Core Binding Factor Alpha 2 Subunit Proteins 0.000 claims abstract description 25
- 108010027263 homeobox protein HOXA9 Proteins 0.000 claims abstract description 24
- 102000002664 Core Binding Factor Alpha 2 Subunit Human genes 0.000 claims abstract description 23
- 239000013604 expression vector Substances 0.000 claims abstract description 7
- 230000004069 differentiation Effects 0.000 claims description 51
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 34
- 239000002243 precursor Substances 0.000 claims description 33
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 claims description 30
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 claims description 30
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 21
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 21
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 21
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 claims description 18
- 101001033276 Mus musculus Interleukin-3 Proteins 0.000 claims description 18
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 claims description 18
- 101000716728 Mus musculus Kit ligand Proteins 0.000 claims description 18
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 17
- 229960003722 doxycycline Drugs 0.000 claims description 16
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 claims description 16
- 230000001939 inductive effect Effects 0.000 claims description 16
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 12
- 108010023082 activin A Proteins 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 238000003501 co-culture Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 206010028980 Neoplasm Diseases 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 8
- 101001052849 Homo sapiens Tyrosine-protein kinase Fer Proteins 0.000 claims description 7
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 claims description 7
- 102100024537 Tyrosine-protein kinase Fer Human genes 0.000 claims description 7
- 239000000654 additive Substances 0.000 claims description 7
- 230000000996 additive effect Effects 0.000 claims description 7
- 230000002708 enhancing effect Effects 0.000 claims description 7
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 claims description 7
- 229940097277 hygromycin b Drugs 0.000 claims description 7
- 239000003446 ligand Substances 0.000 claims description 7
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 6
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 6
- 102000004338 Transferrin Human genes 0.000 claims description 6
- 108090000901 Transferrin Proteins 0.000 claims description 6
- 229960005070 ascorbic acid Drugs 0.000 claims description 6
- 235000010323 ascorbic acid Nutrition 0.000 claims description 6
- 239000011668 ascorbic acid Substances 0.000 claims description 6
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 claims description 6
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- 210000002536 stromal cell Anatomy 0.000 claims description 6
- 229940035024 thioglycerol Drugs 0.000 claims description 6
- 239000012581 transferrin Substances 0.000 claims description 6
- 108010049976 Bone Morphogenetic Protein 5 Proteins 0.000 claims description 5
- 102100022526 Bone morphogenetic protein 5 Human genes 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 claims description 5
- 230000028993 immune response Effects 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 210000002325 somatostatin-secreting cell Anatomy 0.000 claims description 5
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical group C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims description 4
- 108091033409 CRISPR Proteins 0.000 claims description 4
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims description 4
- 230000006801 homologous recombination Effects 0.000 claims description 4
- 238000002744 homologous recombination Methods 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 4
- 201000001322 T cell deficiency Diseases 0.000 claims description 3
- 208000027912 T-cell immunodeficiency Diseases 0.000 claims description 3
- 239000012894 fetal calf serum Substances 0.000 claims description 3
- 238000009169 immunotherapy Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 238000010354 CRISPR gene editing Methods 0.000 claims description 2
- 238000010459 TALEN Methods 0.000 claims description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 2
- 208000036142 Viral infection Diseases 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 230000009385 viral infection Effects 0.000 claims description 2
- 108010049951 Bone Morphogenetic Protein 3 Proteins 0.000 claims 2
- 102100024504 Bone morphogenetic protein 3 Human genes 0.000 claims 2
- 239000013598 vector Substances 0.000 abstract description 8
- 231100000588 tumorigenic Toxicity 0.000 abstract description 5
- 230000000381 tumorigenic effect Effects 0.000 abstract description 5
- 230000004186 co-expression Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 63
- 230000003394 haemopoietic effect Effects 0.000 description 20
- 210000002242 embryoid body Anatomy 0.000 description 11
- 239000003814 drug Substances 0.000 description 10
- 238000002054 transplantation Methods 0.000 description 10
- 210000000601 blood cell Anatomy 0.000 description 9
- 238000000684 flow cytometry Methods 0.000 description 9
- 210000000952 spleen Anatomy 0.000 description 9
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 8
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 8
- 206010061598 Immunodeficiency Diseases 0.000 description 7
- 208000029462 Immunodeficiency disease Diseases 0.000 description 7
- 230000007813 immunodeficiency Effects 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 210000005259 peripheral blood Anatomy 0.000 description 6
- 239000011886 peripheral blood Substances 0.000 description 6
- 210000001541 thymus gland Anatomy 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 108010010803 Gelatin Proteins 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 239000003636 conditioned culture medium Substances 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 229940076144 interleukin-10 Drugs 0.000 description 3
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 101000823089 Equus caballus Alpha-1-antiproteinase 1 Proteins 0.000 description 2
- 101000651211 Homo sapiens Transcription factor PU.1 Proteins 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 102000003814 Interleukin-10 Human genes 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 description 2
- 238000011579 SCID mouse model Methods 0.000 description 2
- 102100027654 Transcription factor PU.1 Human genes 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000011132 hemopoiesis Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000004264 monolayer culture Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 210000003751 DN2 alpha-beta immature T lymphocyte Anatomy 0.000 description 1
- 210000001086 DN3 alpha-beta immature T lymphocyte Anatomy 0.000 description 1
- 210000001570 DN4 alpha-beta immature T lymphocyte Anatomy 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 102100030339 Homeobox protein Hox-A10 Human genes 0.000 description 1
- 102100025110 Homeobox protein Hox-A5 Human genes 0.000 description 1
- 102100021090 Homeobox protein Hox-A9 Human genes 0.000 description 1
- 101001083164 Homo sapiens Homeobox protein Hox-A10 Proteins 0.000 description 1
- 101001077568 Homo sapiens Homeobox protein Hox-A5 Proteins 0.000 description 1
- 101001078143 Homo sapiens Integrin alpha-IIb Proteins 0.000 description 1
- 101100454393 Homo sapiens LCOR gene Proteins 0.000 description 1
- 101000931462 Homo sapiens Protein FosB Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101001010792 Homo sapiens Transcriptional regulator ERG Proteins 0.000 description 1
- 101001059220 Homo sapiens Zinc finger protein Gfi-1 Proteins 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 102100038260 Ligand-dependent corepressor Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000001190 Q-PCR Methods 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 102100029004 Zinc finger protein Gfi-1 Human genes 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 210000004524 haematopoietic cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011536 re-plating Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
-
- 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
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
-
- 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
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- 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
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- 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]
- C12N2501/2303—Interleukin-3 (IL-3)
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1394—Bone marrow stromal cells; whole marrow
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- 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
- C12N2510/00—Genetically modified cells
Definitions
- the invention belongs to the technical field of medical bio-engineering and relates to a pluripotent stem cell and a T cell differentiated therefrom and application thereof.
- Pluripotent stem cells which are currently the focus in stem cell research, are a class of cells with unlimited proliferative potential, having the ability to differentiate into different lineages of cellular tissues, and are easily genetically modified. Inducing autologous pluripotent stem cells to differentiate into different tissues is an application hotspot in the field of regenerative medicine, which can not only avoid ethical controversy, but also reduce the risk of immune rejection. As an emerging immune cell therapy, CAR-T has received extensive attentions due to the characteristics of high specificity and high cancer cell removal efficiency. At present, the immune cells for the CAR-T therapy are mainly derived from the patient's own T cells.
- hematopoietic stem and progenitor cells with multi-lineage hematopoietic reconstitution ability by expressing transcription factors ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1 in the human pluripotent stem cell-derived hematopoietic endothelium, followed by transplantation to produce multiple hematopoietic lineage cells (including T cells)
- T cells multiple hematopoietic lineage cells
- human hematopoietic multipotent progenitors having the ability to reconstitute partial lineage haematopoiesis (without the ability to reconstitute T cell lineage haematopoiesis) and mouse hematopoietic stem cells having the ability to reconstitute all lineage haematopoiesis were obtained by expressing transcription factors FOSB, GFI1, RUNX1 and SPI1 in endothelial cells (V. M. Sandler et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 213-318 (2014); R. Lis et al.
- the present invention provides a pluripotent stem cell and a T cell differentiated therefrom and application thereof.
- the obtained pluripotent stem cell-derived T cells not only function normally, but also has no tumorigenic risk.
- the present invention provides a vector comprising Runx1 and Hoxa9 in which Runx1 and Hoxa9 are co-expressed in tandem.
- the cDNA sequences of Runx1 and Hoxa9 are expressed in tandem in the same vector for infecting host cells, resulting in host cells stably expressing Runx1 and Hoxa9, which are easy to operate and efficient, and the obtained host cells have the ability to differentiate into T cells.
- the present invention provides a nucleic acid expressing the vector as described in the first aspect.
- the present invention provides a host cell comprising the vector as described in the first aspect
- the host cell is a pluripotent stem cell.
- the present invention provides a method for directed differentiation of T cells using pluripotent stem cells, comprising the steps of:
- step (1) directionally differentiating the pluripotent stem cells of step (1) into hematopoietic stem cell precursors
- step (3) co-culturing the hematopoietic stem cell precursors of step (2) with mouse bone marrow stromal cells to obtain T-lineage progenitor cells;
- step (3) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells.
- functionally normal T cells with no tumorigenic risk are obtained by subjecting the pluripotent stem cell line co-expressing Runx1 and Hoxa9 to directional differentiation condition to obtain hematopoietic stem cell precursors, which are then co-cultured with the OP9-DL1 cell line to generate T-lineage progenitor cells, followed by further differentiation.
- the expression vector in which Runx1 and Hoxa9 are in a tandem arrangement in step (1) is integrated into Rosa26 site of the pluripotent stem cells.
- the pluripotent stem cells in step (1) are genetically-edited inducible pluripotent stem cells and/or embryonic pluripotent stem cell lines.
- the method for integrating in step (1) comprises any one of homologous recombination, CRISPR/Cas9, TALEN, transfection or viral infection, or combination thereof, preferably homologous recombination.
- the resistance screening in step (1) employs Hygromycin B.
- the method for directed differentiation in step (2) is to culture the pluripotent stem cells with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially to obtain the hematopoietic stem cell precursors.
- the D0 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), wherein the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL.
- BMP4 bone morphogenetic protein 4
- the D2.5 medium is a basic differentiation medium containing 3-8 ng/mL activin A and 3-8 ng/mL basic fibroblast growth factor (bFGF), wherein the concentration of activin A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the basic fibroblast growth factor may be, for example, 3 ng/mL, 5 Ng/mL or 8 ng/mL, preferably 5 ng/mL.
- bFGF basic fibroblast growth factor
- the D3 medium is a basic differentiation medium containing 3-8 ng/mL Activin A, 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor, wherein the concentration of activin A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
- the D4 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor (VEGF), wherein the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL
- BMP4 bone morphogenetic protein 4
- VEGF vascular endothelial growth factor
- the D5 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO) and 10-30 ng/mL human Fins-associated tyrosine kinase 3 ligand (hFlt3L), wherein the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL
- the D6 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO), and 10-30 ng/mL hFlt3L and 1-2 ⁇ g/mL Doxycycline (Dox), wherein the concentration of the bone morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL
- the D7 medium is a basic differentiation medium containing 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO), and 10-30 ng/mL hFlt3L and 1-2 ⁇ g/mL Doxycycline (Dox), wherein the concentration of the recombinant mouse interleukin 3 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant mouse interleukin 6 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant recomb
- the basic differentiation medium is IMDM medium comprising 10-20% fetal calf serum, 180-220 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 ⁇ 4 M thioglycerol, 1-3 mM GlutaMAXTM-I additive and 0.4-0.6 mM ascorbic acid, wherein the concentration of the fetal bovine serum may be, for example, 10%, 15% or 20%, preferably 15%, the concentration of the iron-saturated transferrin may be, for example, 180 ⁇ g/mL, 200 ⁇ g/mL or 220 ⁇ g/mL, preferably 200 ⁇ g/mL, the concentration of the thioglycerol may be, for example, 4 ⁇ 10 ⁇ 4 M, 4.5 ⁇ 10 ⁇ 4 M or 5 ⁇ 10 ⁇ 4 M, preferably 4.5 ⁇ 10 ⁇ 4 M, the concentration of the GlutaMAXTM-I additive may be, for example, 1 mM, 2 mM or 3 mM, preferably
- the inventors designed and optimized the directed hematopoietic differentiation system by changing the additive substances in the medium and induced the hematopoietic differentiation of the pluripotent stem cells into hematopoietic stem cell precursors, which were further co-cultured with mouse bone marrow stromal cells to obtain T-lineage progenitor cells.
- the stromal cells in step (3) are OP9-DL1 cells.
- Doxycycline is used for inducing during the co-culture in step (3).
- the T cells in step (4) are mainly CD3 + T cells.
- the T cells are TCR ⁇ cells and/or TCR ⁇ / ⁇ cells.
- the present invention provides a method for the directional differentiation of pluripotent stem cells into T cells, comprising the steps of:
- step (1) (2) culturing the pluripotent stem cells of step (1) with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially, and directionally differentiating the same into hematopoietic stem cell precursors on day 11;
- step (3) co-culturing the hematopoietic stem cell precursors of step (2) with OP9-DL1 cells and inducing with Doxycycline to obtain T-lineage progenitor cells;
- step (3) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells which are TCR ⁇ cells and/or TCR ⁇ / ⁇ cells.
- the present invention provides a T-lineage progenitor cell and/or a T cell prepared by the method of the first aspect.
- the present invention provides a pharmaceutical composition comprising any one of the vector as described in the first aspect, the host cell as described in the third aspect, and the T-lineage progenitor cell or the T cell as described in the fifth aspect or combination thereof.
- the pharmaceutical composition further comprises any one of a pharmaceutically acceptable carrier, excipient or diluent, or combination thereof.
- the present invention provides the pharmaceutical composition according to the fourth aspect for use in the preparation of a medicament for enhancing an immune response, preferably for the preparation of a medicament for enhancing an immune response of a T cell.
- the pharmaceutical composition can be used to enhance an immune response, in particular, to enhance the immune response of a T cell.
- the present invention provides the pharmaceutical composition according to the fourth aspect for use in preparation of a medicament for preventing and/or treating immunodeficiency, preferably for preparation of a medicament for preventing and/or treating T cell immunodeficiency.
- the pharmaceutical composition can be used for preventing and/or treating immunodeficiency, in particular, for preventing and/or treating T cell immunodeficiency.
- the present invention provides the pharmaceutical composition according to the fourth aspect for use in preparation of a medicament used for treating a tumor with T cell immunotherapy.
- the pharmaceutical composition can be used in a T cell immunotherapy.
- the present invention has the following beneficial effects:
- Pluripotent stem cells which inducibly co-express exogenous Runx1 and Hoxa9 are successfully constructed in the present invention by introducing an exogenous vector co-expressing Runx1 and Hoxa9 into pluripotent stem cells.
- the pluripotent stem cells have the ability to differentiate into T cells, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors;
- a directed differentiation system and a co-culture method are adopted in the present invention to directionally differentiate the pluripotent stem cells into T-lineage progenitor cells which can be induced to differentiate into T cells, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors;
- the pluripotent stem cell-derived T cells obtained by the method of the present invention function normally without tumorigenic risk, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors.
- FIG. 1(A) is a schematic diagram showing an inducible expression system to site-specifically knock-in at the Rosa26 site of pluripotent stem cells.
- the expression system employed a p2a sequence to link the cDNA sequences of Runx1 and Hoxa9 in tandem, and Doxycycline was used to induce gene expression;
- FIG. 1(B) is a light field diagram of the iRunx1-p2a-Hoxa9 pluripotent stem cells which were obtained by resistance screening with Hygromycin; and
- FIG. 1(C) shows the relative expression levels of Runx1 and Hoxa9 after 24 hours of treatment with Doxycycline;
- FIG. 2(A) is a schematic diagram showing the embryoid body-monolayer culture system for inducing iRunx1-p2a-Hoxa9 pluripotent stem cells to directionally differentiate into hematopoietic precursors, hematopoietic stem cell precursors and blood cells;
- FIG. 2 (B) is a diagram showing the cell morphology on day 11 during the induction of the directional differentiation of iRunx1-p2a-Hoxa9 pluripotent stem cells;
- FIG. 2(C) shows the composition and proportion of hematopoietic-related cells on day 11 of the directed differentiation which were analyzed by flow cytometry;
- FIG. 3(A) shows the flow cytometry sorting strategy for hematopoietic stem cell precursors
- FIG. 3(B) is a schematic diagram showing co-culture of the sorted hematopoietic stem cell precursor population (CD31 + CD41 low CD45 ⁇ c-Kit + CD201 high ) and OP9-DL1 cell line
- FIG. 3(C) shows the number of cobblestone-like formation areas observed under the microscope after 10 days of the co-culture of hematopoietic stem cell precursor population with OP9-DL1 cell line
- FIG. 3(D) shows the light field diagram of the cobblestone-like-like formation areas observed under the microscope after 10 days of the co-culture of hematopoietic stem cell precursor population with OP9-DL1 cell line.
- FIG. 4(A) shows the transplantation of T-lineage progenitor cells which were harvested after the co-culture of hematopoietic stem cell precursors with OP9-DL1 cell line into a CD45.1 + NOD/SCID immunodeficient mouse
- FIG. 4(B) shows the identification of pluripotent stem cell-derived blood cells by flow cytometry 4 weeks after the transplantation, wherein hematopoietic chimera were detected in the iRunx1-p2a-Hoxa9 group
- FIG. 4(B) shows the transplantation of T-lineage progenitor cells which were harvested after the co-culture of hematopoietic stem cell precursors with OP9-DL1 cell line into a CD45.1 + NOD/SCID immunodeficient mouse
- FIG. 4(B) shows the identification of pluripotent stem cell-derived blood cells by flow cytometry 4 weeks after the transplantation, wherein hematopoietic chimera were detected in the iRunx1
- FIG. 4(C) shows the lineage distribution of pluripotent stem cell-derived hematopoietic cells and the phenotype of CD3 + T lymphocytes in peripheral blood, bone marrow, spleen and thymus of the recipient mouse which was sacrificed 5 weeks after the transplantation; and
- FIG. 4(D) shows the PCR and sequencing identification of the genome of the pluripotent stem cell-derived blood cells.
- FIG. 5(A) shows the analysis for the pluripotent stem cell-derived DN cell population (DN1/DN2/DN3/DN4) in the thymus of the recipient mouse which was sacrificed 4 weeks after the transplantation
- FIG. 5(B) shows the analysis for the TCR- ⁇ and TCR- ⁇ / ⁇ populations in the pluripotent stem cell-derived CD3 + T cells in the peripheral blood, spleen and lymph nodes of the recipient mouse which was sacrificed 4 weeks after the transplantation
- FIG. 5(B) shows the analysis for the TCR- ⁇ and TCR- ⁇ / ⁇ populations in the pluripotent stem cell-derived CD3 + T cells in the peripheral blood, spleen and lymph nodes of the recipient mouse which was sacrificed 4 weeks after the transplantation
- 5(C) shows a mixed lymphocyte reaction (MLR) experiment of the recipient mouse which was sacrificed 4 weeks after the transplantation, wherein the PSC-T is CD3 + T cells enriched in the spleen by magnetic beads 6 weeks after the transplantation of T-lineage progenitor cells which were obtained by inducing the differentiation of pluripotent stem cell line with Runx1-p2a-Hoxa9 into a NOD-SCID recipient mouse.
- MLR mixed lymphocyte reaction
- FIG. 6 shows the representative cytokines secreted by T cells after in vitro stimulation which are detected by ELISA, wherein IL10-interleukin 10, IFN- ⁇ - ⁇ interferon, IL-2-interleukin 2, TNF- ⁇ -tumor necrosis factor ⁇ .
- an inducible expression sequence was site-specifically knocked-in at the Rosa26 site of pluripotent stem cells by electro-transformation in combination with gene recombination, as shown in FIG. 1(A) , wherein the knocked-in sequence comprised Runx1-p2a-Hoxa9 tandem sequence and a Hygromycin B resistance gene sequence for resistance screening.
- a pluripotent stem cell medium containing Hygromycin B 150 ⁇ g/mL was added 20 hours after the electro-transformation and the medium was replaced every day.
- the medium was replaced every day when the clone mass was adhered in the MEF cell layer. After 3 days, the clone mass was digested with 0.25% trypsin and passaged into a 12-well plate. The cell morphology was shown in FIG. 1(B) , and the clone mass was in logarithmic growth phase, the edge was neat and transparent, there was a clear boundary with the MEF cell layer and there was no differentiation. The cells were passaged, amplified and frozen according to their state and growth density.
- the total mRNA of the iRunx1-p2a-Hoxa9 pluripotent stem cells was extracted after 24 hours of Dox treatment (a Dox-free group was used as a control group), and the expression levels of Runx1 and Hoxa9 mRNA were obtained by Q-PCR. It was shown in FIG. 1(C) that the addition of Dox could induce the expression of Runx1 and Hoxa9.
- the directed hematopoietic differentiation system as shown in FIG. 2(A) was used to induce the hematopoietic differentiation of pluripotent stem cells.
- the formulation of each medium in the directional hematopoietic differentiation system was:
- Basic differentiation medium BDM IMDM medium containing 15% fetal bovine serum, 200 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 ⁇ 4 M thioglycerol, 2 mM GlutaMAXTM-I additive and 0.5 mM ascorbic acid;
- D0 medium a basal differentiation medium containing 5 ng/mL bone morphogenetic protein 4;
- D2.5 medium a basic differentiation medium containing 5 ng/mL activin A and 5 ng/mL basic fibroblast growth factor;
- D3 medium a basal differentiation medium containing 5 ng/mL activin A, 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor;
- D4 medium a basal differentiation medium containing 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelial growth factor;
- D5 medium a basal differentiation medium containing 5 ng/mL bone morphogenetic protein 4 and 5 ng/mL vascular endothelium growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin and 20 ng/mL hFlt3L;
- D6 medium a basal differentiation medium containing 5 ng/mL bone morphogenetic protein 4, 5 ng/mL intravascular growth factor, 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin, 20 ng/mL hFlt3L and 1 ⁇ g/mL Doxycycline;
- D7 medium a basal differentiation medium containing 20 ng/mL recombinant mouse interleukin 3, 20 ng/mL recombinant mouse interleukin 6, 20 ng/mL recombinant mouse stem cell factor, 20 ng/mL recombinant human thrombopoietin, 20 ng/mL hFlt3L and 1 ⁇ g/mL Doxycycline.
- the suspended cells were collected, centrifuged at 250 g for 5 min, and washed once with DPBS. The cells were resuspended with D0 medium and counted, and the cell concentration was adjusted to 1 ⁇ 10 5 /mL. Added 5-10 mL of cell suspension into a tilted 10 cm dish, pipetted 20 ⁇ L of cell suspension and added the same into a 15 cm culture dish to suspend the embryoid body (EB) with 20 ⁇ L (about 2000 cells) per single EB. The culture dish was then inverted and a 10 cm culture dish lid was placed at the bottom of the culture dish and 5-6 mL of cell culture water was added into the lid. Incubated in an incubator at 37° C. for 2.5 days.
- EB embryoid body
- the EB was collected into a centrifuge tube with a Pasteur pipette, and the bottom of the dish was washed with DPBS. The supernatant was carefully aspirated when the EB has settled naturally. Alternatively, the supernatant was removed by centrifugation at a low speed of 90 g for 5 min. DPBS was added to rinse once. The supernatant was removed again by sediment or centrifugation. The EB was resuspended with D2.5 medium, transferred into a low-adherence 24-well plate and cultured for 12 hours to observe whether the EB was contaminated.
- the EB was collected into a 15 mL centrifuge tube, and the supernatant was carefully aspirated when the EB has settled naturally.
- DPBS was added to rinse once. 400 ⁇ L of 0.05% trypsin was added, transferred into a 24-well low-adhesive culture dish and digested at 37° C. for 3 min, followed by repeated gentle blistering of the EB, and D3 medium was added to terminate the digestion when the EB exhibited a single cell state, centrifuged at 350 g for 5 min.
- the viable cells were resuspended with D3 medium and counted, and inoculated into a 12-well plate which was pre-coated with 0.1% gelatin at a density of 2 ⁇ 10 5 cells/well.
- the medium was then replaced every other day with D7 medium.
- FIG. 2(B) obvious hematopoietic clusters were observed in the iRunx1-p2a-Hoxa9 differentiation group on day 11; the flow cytometry analysis as shown in FIG. 2(C) showed that the hematopoiesis-related cell populations were CD41 + hematopoietic precursor cells and CD45 + blood cells on day 11 of the directed differentiation.
- the inventors co-cultured hematopoietic stem cell precursors with mouse bone marrow stromal cells to verify that the hematopoietic precursor cells which were differentiated from pluripotent stem cells have the proliferative ability as an embryonic hematopoietic stem cell precursor population, that is, the ability to form cobblestone-like areas with high expansion potential on stromal cells.
- the co-culture medium was alpha-MEM medium containing 15% DFBS, 200 ⁇ g/mL iron-saturated transferrin, 4.5 ⁇ 10 ⁇ 4 M thioglycerol, 2 mM GlutaMAXTM-I additive, 0.5 mM ascorbic acid, 2% AFT024-mSCF conditioned medium, 2% AFT024-mIL3 conditioned medium, 2% AFT024-hFlt3L conditioned medium and 1 ⁇ g/mL Dox.
- hematopoietic stem cell precursors (CD31 + CD41 low CD45 ⁇ c-Kit + CD201 high ) were sorted by flow cytometry by using the sorting strategy as shown in FIG. 3(A) .
- a cobblestone-like area forming experiment (CAFC) was used to examine whether the hematopoietic stem cell precursors which were differentiated from pluripotent stem cells have the same proliferative ability as the embryonic-derived hematopoietic stem cell precursors. As shown in FIG.
- the sorted hematopoietic stem cell precursor population was re-plated onto OP9-DL1 stromal cells, and the number of the cobblestone-like areas formed per 100 hematopoietic stem cell precursors was counted 10 days later.
- the inventors further designed a post-co-culture transplantation strategy to obtain T cells by utilizing the in vivo microenvironment.
- the hematopoietic stem cell precursors were placed onto the OP9-DL1 stromal cells and Dox was added for inducing for 10 days to obtain T-lineage progenitor cells.
- the OP9-DL1 cell line was resuscitated 4 days in advance, and the cells were passaged in time according to their growth state to prevent the cells from aging due to excessive growth. A passage was carried out one day before use by re-plating 50,000 cells per well (a 12-well plate) for use the next day.
- T-lineage progenitor cells obtained by co-culture of the pluripotent stem cell-derived hematopoietic stem cell precursors were transplanted into a 6-8 weeks old CD45.1 NOD/SCID mouse via ocular vein, and the hematopoietic chimera of peripheral blood were detected by flow cytometry 4 weeks after the transplantation.
- the T-lineage progenitor cells obtained by co-culturing the iRunx1-p2a-Hoxa9 pluripotent stem cell-derived hematopoietic stem cell precursor population formed hematopoietic chimera in the peripheral blood of the recipient NOD/SCID mouse, which were mainly CD3 + T cells (97.7%), achieving effective reconstruction of the T lymphatic system.
- the mouse was sacrificed and the blood cell lineages in its peripheral blood, bone marrow, spleen and thymus were analyzed by flow cytometry to further clarify the distribution of iRunx1-p2a-Hoxa9 pluripotent stem cell-derived blood cells in other hematopoietic and lymphoid tissues. It was found in the flow cytometry analysis that, as shown in FIG. 4(C) , in the bone marrow, thymus and spleen, the pluripotent stem cell-derived blood cells were also mainly T-lineage hematopoiesis.
- This group of CD3 + T cells including both CD4 + single positive cells and CD8 + single positive cells, contained a small amount of both CD4 + CD8 + double positive cells and CD4 ⁇ CD8 ⁇ double negative cells in the spleen, bone marrow and thymus.
- the thymus DN cell population was analyzed to further identify the type of the pluripotent stem cell-derived immune cells in the mouse. It was shown in FIG. 5(A) that the T cells in the recipient mouse were normally developed and DN1, DN2, DN3 and DN4 cell populations were detectable. The TCR receptors of pluripotent stem cell-derived T cells in the peripheral blood, spleen and lymphatic vessels were subjected to a detection. As shown in FIG. 5(B) , a certain proportion of TCR ⁇ / ⁇ cells (0.37-1.71%) were found in the T cells, while most of them were TCR ⁇ cells.
- a mixed lymphocyte reaction was carried out with spleen cells of a Balb/C mouse and T cells obtained from the spleen of the recipient mouse by CD3 magnetic bead enrichment, and a detection was performed on days 3 and 6, respectively.
- the pluripotent stem cell-derived T cells were able to proliferate after activation, confirming that these T cells have proliferative ability after stimulation.
- the culture supernatant was analyzed by ELISA. As shown in FIG. 6 , the regenerated T cells after stimulation and proliferation were able to secrete a large amount of interleukin 10 (IL10), interferon gamma (IFN- ⁇ ), interleukin 2 (IL-2) and tumor necrosis factor ⁇ (TNF- ⁇ ).
- IL10 interleukin 10
- IFN- ⁇ interferon gamma
- IL-2 interleukin 2
- TNF- ⁇ tumor necrosis factor ⁇
- pluripotent stem cells which inducibly co-express exogenous Runx1 and Hoxa9 are successfully constructed in the present invention by introducing an exogenous vector co-expressing Runx1 and Hoxa9 into pluripotent stem cells.
- the pluripotent stem cells were directionally differentiated into T-lineage progenitor cells which will be developed into T cells.
- the pluripotent stem cell-derived T cells obtained by the method of the present invention are not only functionally normal but also have no tumorigenic risk, and can be used for preparing a medicine for enhancing an immune effect, preventing and/or treating immunodeficiency and treating a tumor.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Transplantation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
- The invention belongs to the technical field of medical bio-engineering and relates to a pluripotent stem cell and a T cell differentiated therefrom and application thereof.
- Pluripotent stem cells (PSCs), which are currently the focus in stem cell research, are a class of cells with unlimited proliferative potential, having the ability to differentiate into different lineages of cellular tissues, and are easily genetically modified. Inducing autologous pluripotent stem cells to differentiate into different tissues is an application hotspot in the field of regenerative medicine, which can not only avoid ethical controversy, but also reduce the risk of immune rejection. As an emerging immune cell therapy, CAR-T has received extensive attentions due to the characteristics of high specificity and high cancer cell removal efficiency. At present, the immune cells for the CAR-T therapy are mainly derived from the patient's own T cells. However, some patients (such as infants, immunodeficiency patients with advanced tumor and patients received extensive chemotherapy) are unable to provide effective doses of T cells and the CAR-T therapy is expensive, greatly limiting the application of this therapy. The above problems can be solved by obtaining functional T cells by pluripotent stem cells.
- A basic research has been carried out to obtain hematopoietic stem and progenitor cells (HSPCs) with multi-lineage hematopoietic reconstitution ability by expressing transcription factors ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1 in the human pluripotent stem cell-derived hematopoietic endothelium, followed by transplantation to produce multiple hematopoietic lineage cells (including T cells) (R. Sugimura et al. Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature, 545, 432-438 (2017)). However, the above study requires up to seven transcription factors for stem cell induction, having the disadvantages of complex operation, poor stability and low efficiency.
- It has also been reported that human hematopoietic multipotent progenitors having the ability to reconstitute partial lineage haematopoiesis (without the ability to reconstitute T cell lineage haematopoiesis) and mouse hematopoietic stem cells having the ability to reconstitute all lineage haematopoiesis were obtained by expressing transcription factors FOSB, GFI1, RUNX1 and SPI1 in endothelial cells (V. M. Sandler et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 213-318 (2014); R. Lis et al. Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature 545, 439-445 (2017)). However, the above studies have problems such as inconvenient access to endothelial cells, difficulty in gene editing, cumbersome technical methods and low efficiency of T-lineage generation. Therefore, there is a need for a simple method for inducing pluripotent stem cells to solely obtain T lineage cell.
- In view of the deficiencies of the prior art, the present invention provides a pluripotent stem cell and a T cell differentiated therefrom and application thereof. The obtained pluripotent stem cell-derived T cells not only function normally, but also has no tumorigenic risk.
- In a first aspect, the present invention provides a vector comprising Runx1 and Hoxa9 in which Runx1 and Hoxa9 are co-expressed in tandem.
- In the present invention, the cDNA sequences of Runx1 and Hoxa9 are expressed in tandem in the same vector for infecting host cells, resulting in host cells stably expressing Runx1 and Hoxa9, which are easy to operate and efficient, and the obtained host cells have the ability to differentiate into T cells.
- In a second aspect, the present invention provides a nucleic acid expressing the vector as described in the first aspect.
- In a third aspect, the present invention provides a host cell comprising the vector as described in the first aspect;
- Preferably, the host cell is a pluripotent stem cell.
- In a fourth aspect, the present invention provides a method for directed differentiation of T cells using pluripotent stem cells, comprising the steps of:
- (1) integrating an expression vector in which Runx1 and Hoxa9 are of in tandem into pluripotent stem cells and performing resistance screening;
- (2) directionally differentiating the pluripotent stem cells of step (1) into hematopoietic stem cell precursors;
- (3) co-culturing the hematopoietic stem cell precursors of step (2) with mouse bone marrow stromal cells to obtain T-lineage progenitor cells; and
- (4) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells.
- In the present invention, functionally normal T cells with no tumorigenic risk are obtained by subjecting the pluripotent stem cell line co-expressing Runx1 and Hoxa9 to directional differentiation condition to obtain hematopoietic stem cell precursors, which are then co-cultured with the OP9-DL1 cell line to generate T-lineage progenitor cells, followed by further differentiation.
- Preferably, the expression vector in which Runx1 and Hoxa9 are in a tandem arrangement in step (1) is integrated into Rosa26 site of the pluripotent stem cells.
- Preferably, the pluripotent stem cells in step (1) are genetically-edited inducible pluripotent stem cells and/or embryonic pluripotent stem cell lines.
- Preferably, the method for integrating in step (1) comprises any one of homologous recombination, CRISPR/Cas9, TALEN, transfection or viral infection, or combination thereof, preferably homologous recombination.
- Preferably, the resistance screening in step (1) employs Hygromycin B.
- Preferably, the method for directed differentiation in step (2) is to culture the pluripotent stem cells with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially to obtain the hematopoietic stem cell precursors.
- Preferably, the D0 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), wherein the concentration of the bone
morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL. - Preferably, the D2.5 medium is a basic differentiation medium containing 3-8 ng/mL activin A and 3-8 ng/mL basic fibroblast growth factor (bFGF), wherein the concentration of activin A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the basic fibroblast growth factor may be, for example, 3 ng/mL, 5 Ng/mL or 8 ng/mL, preferably 5 ng/mL.
- Preferably, the D3 medium is a basic differentiation medium containing 3-8 ng/mL Activin A, 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor, wherein the concentration of activin A may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the bone
morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL - Preferably, the D4 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4) and 3-8 ng/mL vascular endothelial growth factor (VEGF), wherein the concentration of the bone
morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, and the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL - Preferably, the D5 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO) and 10-30 ng/mL human Fins-associated
tyrosine kinase 3 ligand (hFlt3L), wherein the concentration of the bonemorphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of therecombinant mouse interleukin 3 may be, for example, ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of therecombinant mouse interleukin 6 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant mouse stem cell factor may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/mL or30 ng/mL, preferably 20 ng/mL, and the concentration of the hFlt3L may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL - Preferably, the D6 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 (BMP4), 3-8 ng/mL vascular endothelial growth factor (VEGF), 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO), and 10-30 ng/mL hFlt3L and 1-2 μg/mL Doxycycline (Dox), wherein the concentration of the bone
morphogenetic protein 4 may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of the vascular endothelial growth factor may be, for example, 3 ng/mL, 5 ng/mL or 8 ng/mL, preferably 5 ng/mL, the concentration of therecombinant mouse interleukin 3 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of therecombinant mouse interleukin 6 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant mouse stem cell factor may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the hFlt3L may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, and the concentration of the Doxycycline may be, for example, 1 μg/mL or 2 μg/mL, preferably 1 μg/mL - Preferably, the D7 medium is a basic differentiation medium containing 10-30 ng/mL recombinant mouse interleukin 3 (mIL3), 10-30 ng/mL recombinant mouse interleukin 6 (mIL6), 10-30 ng/mL recombinant mouse stem cell factor (mSCF), 10-30 ng/mL recombinant human thrombopoietin (hTPO), and 10-30 ng/mL hFlt3L and 1-2 μg/mL Doxycycline (Dox), wherein the concentration of the
recombinant mouse interleukin 3 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of therecombinant mouse interleukin 6 may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant mouse stem cell factor may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the recombinant human thrombopoietin may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, the concentration of the hFlt3L may be, for example, 10 ng/mL, 20 ng/mL or 30 ng/mL, preferably 20 ng/mL, and the concentration of the Doxycycline may be, for example, 1 μg/mL or 2 μg/mL, preferably 1 μg/mL - Preferably, the basic differentiation medium is IMDM medium comprising 10-20% fetal calf serum, 180-220 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 1-3 mM GlutaMAX™-I additive and 0.4-0.6 mM ascorbic acid, wherein the concentration of the fetal bovine serum may be, for example, 10%, 15% or 20%, preferably 15%, the concentration of the iron-saturated transferrin may be, for example, 180 μg/mL, 200 μg/mL or 220 μg/mL, preferably 200 μg/mL, the concentration of the thioglycerol may be, for example, 4×10−4 M, 4.5×10−4 M or 5×10−4 M, preferably 4.5×10−4 M, the concentration of the GlutaMAX™-I additive may be, for example, 1 mM, 2 mM or 3 mM, preferably 2 mM, and the concentration of the ascorbic acid may be, for example, 0.4 mM, 0.5 mM or 0.6 mM, preferably 0.5 mM.
- In the present invention, the inventors designed and optimized the directed hematopoietic differentiation system by changing the additive substances in the medium and induced the hematopoietic differentiation of the pluripotent stem cells into hematopoietic stem cell precursors, which were further co-cultured with mouse bone marrow stromal cells to obtain T-lineage progenitor cells.
- Preferably, the stromal cells in step (3) are OP9-DL1 cells.
- Preferably, Doxycycline is used for inducing during the co-culture in step (3).
- Preferably, the T cells in step (4) are mainly CD3+ T cells.
- Preferably, the T cells are TCR β cells and/or TCR γ/δ cells.
- As a preferred technical solution, the present invention provides a method for the directional differentiation of pluripotent stem cells into T cells, comprising the steps of:
- (1) integrating an expression vector wherein Runx1 and Hoxa9 are linked in tandem into pluripotent stem cells at the Rosa26 site by gene recombination and performing resistance screening with Hygromycin B;
- (2) culturing the pluripotent stem cells of step (1) with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially, and directionally differentiating the same into hematopoietic stem cell precursors on
day 11; - (3) co-culturing the hematopoietic stem cell precursors of step (2) with OP9-DL1 cells and inducing with Doxycycline to obtain T-lineage progenitor cells; and
- (4) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells which are TCR β cells and/or TCR γ/δ cells.
- In a fifth aspect, the present invention provides a T-lineage progenitor cell and/or a T cell prepared by the method of the first aspect.
- In a sixth aspect, the present invention provides a pharmaceutical composition comprising any one of the vector as described in the first aspect, the host cell as described in the third aspect, and the T-lineage progenitor cell or the T cell as described in the fifth aspect or combination thereof.
- Preferably, the pharmaceutical composition further comprises any one of a pharmaceutically acceptable carrier, excipient or diluent, or combination thereof.
- In a seventh aspect, the present invention provides the pharmaceutical composition according to the fourth aspect for use in the preparation of a medicament for enhancing an immune response, preferably for the preparation of a medicament for enhancing an immune response of a T cell.
- In the present invention, the pharmaceutical composition can be used to enhance an immune response, in particular, to enhance the immune response of a T cell.
- In an eighth aspect, the present invention provides the pharmaceutical composition according to the fourth aspect for use in preparation of a medicament for preventing and/or treating immunodeficiency, preferably for preparation of a medicament for preventing and/or treating T cell immunodeficiency.
- In the present invention, the pharmaceutical composition can be used for preventing and/or treating immunodeficiency, in particular, for preventing and/or treating T cell immunodeficiency.
- In a ninth aspect, the present invention provides the pharmaceutical composition according to the fourth aspect for use in preparation of a medicament used for treating a tumor with T cell immunotherapy.
- In the present invention, the pharmaceutical composition can be used in a T cell immunotherapy.
- Compared with the prior art, the present invention has the following beneficial effects:
- (1) Pluripotent stem cells which inducibly co-express exogenous Runx1 and Hoxa9 are successfully constructed in the present invention by introducing an exogenous vector co-expressing Runx1 and Hoxa9 into pluripotent stem cells. The pluripotent stem cells have the ability to differentiate into T cells, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors;
- (2) A directed differentiation system and a co-culture method are adopted in the present invention to directionally differentiate the pluripotent stem cells into T-lineage progenitor cells which can be induced to differentiate into T cells, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors;
- (3) The pluripotent stem cell-derived T cells obtained by the method of the present invention function normally without tumorigenic risk, and can be used for preparing a medicine for enhancing immune effects, preventing and/or treating immunodeficiency and treating tumors.
-
FIG. 1(A) is a schematic diagram showing an inducible expression system to site-specifically knock-in at the Rosa26 site of pluripotent stem cells. The expression system employed a p2a sequence to link the cDNA sequences of Runx1 and Hoxa9 in tandem, and Doxycycline was used to induce gene expression;FIG. 1(B) is a light field diagram of the iRunx1-p2a-Hoxa9 pluripotent stem cells which were obtained by resistance screening with Hygromycin; andFIG. 1(C) shows the relative expression levels of Runx1 and Hoxa9 after 24 hours of treatment with Doxycycline; -
FIG. 2(A) is a schematic diagram showing the embryoid body-monolayer culture system for inducing iRunx1-p2a-Hoxa9 pluripotent stem cells to directionally differentiate into hematopoietic precursors, hematopoietic stem cell precursors and blood cells;FIG. 2 (B) is a diagram showing the cell morphology onday 11 during the induction of the directional differentiation of iRunx1-p2a-Hoxa9 pluripotent stem cells; andFIG. 2(C) shows the composition and proportion of hematopoietic-related cells onday 11 of the directed differentiation which were analyzed by flow cytometry; -
FIG. 3(A) shows the flow cytometry sorting strategy for hematopoietic stem cell precursors;FIG. 3(B) is a schematic diagram showing co-culture of the sorted hematopoietic stem cell precursor population (CD31+CD41lowCD45−c-Kit+CD201high) and OP9-DL1 cell line;FIG. 3(C) shows the number of cobblestone-like formation areas observed under the microscope after 10 days of the co-culture of hematopoietic stem cell precursor population with OP9-DL1 cell line; andFIG. 3(D) shows the light field diagram of the cobblestone-like-like formation areas observed under the microscope after 10 days of the co-culture of hematopoietic stem cell precursor population with OP9-DL1 cell line. -
FIG. 4(A) shows the transplantation of T-lineage progenitor cells which were harvested after the co-culture of hematopoietic stem cell precursors with OP9-DL1 cell line into a CD45.1+ NOD/SCID immunodeficient mouse;FIG. 4(B) shows the identification of pluripotent stem cell-derived blood cells byflow cytometry 4 weeks after the transplantation, wherein hematopoietic chimera were detected in the iRunx1-p2a-Hoxa9 group;FIG. 4(C) shows the lineage distribution of pluripotent stem cell-derived hematopoietic cells and the phenotype of CD3+ T lymphocytes in peripheral blood, bone marrow, spleen and thymus of the recipient mouse which was sacrificed 5 weeks after the transplantation; andFIG. 4(D) shows the PCR and sequencing identification of the genome of the pluripotent stem cell-derived blood cells. -
FIG. 5(A) shows the analysis for the pluripotent stem cell-derived DN cell population (DN1/DN2/DN3/DN4) in the thymus of the recipient mouse which was sacrificed 4 weeks after the transplantation;FIG. 5(B) shows the analysis for the TCR-β and TCR-γ/δ populations in the pluripotent stem cell-derived CD3+ T cells in the peripheral blood, spleen and lymph nodes of the recipient mouse which was sacrificed 4 weeks after the transplantation; andFIG. 5(C) shows a mixed lymphocyte reaction (MLR) experiment of the recipient mouse which was sacrificed 4 weeks after the transplantation, wherein the PSC-T is CD3+ T cells enriched in the spleen bymagnetic beads 6 weeks after the transplantation of T-lineage progenitor cells which were obtained by inducing the differentiation of pluripotent stem cell line with Runx1-p2a-Hoxa9 into a NOD-SCID recipient mouse. -
FIG. 6 shows the representative cytokines secreted by T cells after in vitro stimulation which are detected by ELISA, wherein IL10-interleukin 10, IFN-γ-γ interferon, IL-2-interleukin 2, TNF-α-tumor necrosis factor α. - In order to further illustrate the technical measures adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and accompanying drawings. It can be understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the present invention.
- In the examples, techniques or conditions, which are not specifically indicated, are performed according to techniques or conditions described in the literature of the art, or according to product instructions. The reagents or instruments for use, which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.
- In this example, an inducible expression sequence was site-specifically knocked-in at the Rosa26 site of pluripotent stem cells by electro-transformation in combination with gene recombination, as shown in
FIG. 1(A) , wherein the knocked-in sequence comprised Runx1-p2a-Hoxa9 tandem sequence and a Hygromycin B resistance gene sequence for resistance screening. In order to obtain homologous recombined pluripotent stem cells successfully, a pluripotent stem cell medium containing Hygromycin B (150 μg/mL) was added 20 hours after the electro-transformation and the medium was replaced every day. After screening with Hygromycin B for 10 days, individual clones were selected under a microscope into a 12-well plate which was pre-incubated with MEF cells, with one pluripotent stem cell clone per well, and cultured in a Hygromycin-free medium. - The medium was replaced every day when the clone mass was adhered in the MEF cell layer. After 3 days, the clone mass was digested with 0.25% trypsin and passaged into a 12-well plate. The cell morphology was shown in
FIG. 1(B) , and the clone mass was in logarithmic growth phase, the edge was neat and transparent, there was a clear boundary with the MEF cell layer and there was no differentiation. The cells were passaged, amplified and frozen according to their state and growth density. - The total mRNA of the iRunx1-p2a-Hoxa9 pluripotent stem cells was extracted after 24 hours of Dox treatment (a Dox-free group was used as a control group), and the expression levels of Runx1 and Hoxa9 mRNA were obtained by Q-PCR. It was shown in
FIG. 1(C) that the addition of Dox could induce the expression of Runx1 and Hoxa9. - The directed hematopoietic differentiation system as shown in
FIG. 2(A) was used to induce the hematopoietic differentiation of pluripotent stem cells. The formulation of each medium in the directional hematopoietic differentiation system was: - Basic differentiation medium BDM: IMDM medium containing 15% fetal bovine serum, 200 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 2 mM GlutaMAX™-I additive and 0.5 mM ascorbic acid;
- D0 medium: a basal differentiation medium containing 5 ng/mL bone
morphogenetic protein 4; - D2.5 medium: a basic differentiation medium containing 5 ng/mL activin A and 5 ng/mL basic fibroblast growth factor;
- D3 medium: a basal differentiation medium containing 5 ng/mL activin A, 5 ng/mL bone
morphogenetic protein - D4 medium: a basal differentiation medium containing 5 ng/mL bone
morphogenetic protein - D5 medium: a basal differentiation medium containing 5 ng/mL bone
morphogenetic protein recombinant mouse interleukin recombinant mouse interleukin - D6 medium: a basal differentiation medium containing 5 ng/mL bone
morphogenetic protein recombinant mouse interleukin recombinant mouse interleukin - D7 medium: a basal differentiation medium containing 20 ng/mL
recombinant mouse interleukin recombinant mouse interleukin - The specific steps were as follows:
- 1 mL of 0.1% gelatin was placed in a 6-
well plate 40 min before use. The pluripotent stem cells were digested into single cells with 0.05% trypsin, centrifuged and resuspended. The 0.1% gelatin was aspirated, and the pluripotent stem cell suspension was transferred into a gelatin-coated well and placed in an incubator for 40 min to remove MEF cells. - The suspended cells were collected, centrifuged at 250 g for 5 min, and washed once with DPBS. The cells were resuspended with D0 medium and counted, and the cell concentration was adjusted to 1×105/mL. Added 5-10 mL of cell suspension into a tilted 10 cm dish, pipetted 20 μL of cell suspension and added the same into a 15 cm culture dish to suspend the embryoid body (EB) with 20 μL (about 2000 cells) per single EB. The culture dish was then inverted and a 10 cm culture dish lid was placed at the bottom of the culture dish and 5-6 mL of cell culture water was added into the lid. Incubated in an incubator at 37° C. for 2.5 days.
- The EB was collected into a centrifuge tube with a Pasteur pipette, and the bottom of the dish was washed with DPBS. The supernatant was carefully aspirated when the EB has settled naturally. Alternatively, the supernatant was removed by centrifugation at a low speed of 90 g for 5 min. DPBS was added to rinse once. The supernatant was removed again by sediment or centrifugation. The EB was resuspended with D2.5 medium, transferred into a low-adherence 24-well plate and cultured for 12 hours to observe whether the EB was contaminated.
- The EB was collected into a 15 mL centrifuge tube, and the supernatant was carefully aspirated when the EB has settled naturally. DPBS was added to rinse once. 400 μL of 0.05% trypsin was added, transferred into a 24-well low-adhesive culture dish and digested at 37° C. for 3 min, followed by repeated gentle blistering of the EB, and D3 medium was added to terminate the digestion when the EB exhibited a single cell state, centrifuged at 350 g for 5 min. The viable cells were resuspended with D3 medium and counted, and inoculated into a 12-well plate which was pre-coated with 0.1% gelatin at a density of 2×105 cells/well.
- Rinsed with DPBS once, replaced with D4 medium and cultured for one day.
- Rinsed with DPBS once, replaced with D5 medium and cultured for one day.
- Rinsed with DPBS once, replaced with D6 medium and cultured for one day.
- Rinsed with DPBS once, replaced with D7 medium and cultured for one day.
- The medium was then replaced every other day with D7 medium. As shown in
FIG. 2(B) , obvious hematopoietic clusters were observed in the iRunx1-p2a-Hoxa9 differentiation group onday 11; the flow cytometry analysis as shown inFIG. 2(C) showed that the hematopoiesis-related cell populations were CD41+ hematopoietic precursor cells and CD45+ blood cells onday 11 of the directed differentiation. - The inventors co-cultured hematopoietic stem cell precursors with mouse bone marrow stromal cells to verify that the hematopoietic precursor cells which were differentiated from pluripotent stem cells have the proliferative ability as an embryonic hematopoietic stem cell precursor population, that is, the ability to form cobblestone-like areas with high expansion potential on stromal cells. The co-culture medium was alpha-MEM medium containing 15% DFBS, 200 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 2 mM GlutaMAX™-I additive, 0.5 mM ascorbic acid, 2% AFT024-mSCF conditioned medium, 2% AFT024-mIL3 conditioned medium, 2% AFT024-hFlt3L conditioned medium and 1 μg/mL Dox.
- On
day 11 of the embryoid body-monolayer culture, hematopoietic stem cell precursors (CD31+CD41lowCD45−c-Kit+CD201high) were sorted by flow cytometry by using the sorting strategy as shown inFIG. 3(A) . Subsequently, a cobblestone-like area forming experiment (CAFC) was used to examine whether the hematopoietic stem cell precursors which were differentiated from pluripotent stem cells have the same proliferative ability as the embryonic-derived hematopoietic stem cell precursors. As shown inFIG. 3(B) , the sorted hematopoietic stem cell precursor population was re-plated onto OP9-DL1 stromal cells, and the number of the cobblestone-like areas formed per 100 hematopoietic stem cell precursors was counted 10 days later. The results inFIG. 3(C) andFIG. 3(D) indicated that the iRunx1-p2a-Hoxa9 pluripotent stem cell-derived hematopoietic stem cell precursors had a strong ability to form cobblestone-like areas, and the pluripotent stem cell-derived hematopoietic stem cell precursors formed highly uniform small, round and bright blood cells on stromal cells OP9-DL1. - The inventors further designed a post-co-culture transplantation strategy to obtain T cells by utilizing the in vivo microenvironment. As shown in
FIG. 4(A) , the hematopoietic stem cell precursors were placed onto the OP9-DL1 stromal cells and Dox was added for inducing for 10 days to obtain T-lineage progenitor cells. The OP9-DL1 cell line was resuscitated 4 days in advance, and the cells were passaged in time according to their growth state to prevent the cells from aging due to excessive growth. A passage was carried out one day before use by re-plating 50,000 cells per well (a 12-well plate) for use the next day. The T-lineage progenitor cells obtained by co-culture of the pluripotent stem cell-derived hematopoietic stem cell precursors were transplanted into a 6-8 weeks old CD45.1 NOD/SCID mouse via ocular vein, and the hematopoietic chimera of peripheral blood were detected byflow cytometry 4 weeks after the transplantation. - It was shown in
FIG. 4(B) that the T-lineage progenitor cells obtained by co-culturing the iRunx1-p2a-Hoxa9 pluripotent stem cell-derived hematopoietic stem cell precursor population formed hematopoietic chimera in the peripheral blood of the recipient NOD/SCID mouse, which were mainly CD3+ T cells (97.7%), achieving effective reconstruction of the T lymphatic system. - After 5 weeks, the mouse was sacrificed and the blood cell lineages in its peripheral blood, bone marrow, spleen and thymus were analyzed by flow cytometry to further clarify the distribution of iRunx1-p2a-Hoxa9 pluripotent stem cell-derived blood cells in other hematopoietic and lymphoid tissues. It was found in the flow cytometry analysis that, as shown in
FIG. 4(C) , in the bone marrow, thymus and spleen, the pluripotent stem cell-derived blood cells were also mainly T-lineage hematopoiesis. This group of CD3+ T cells, including both CD4+ single positive cells and CD8+ single positive cells, contained a small amount of both CD4+CD8+ double positive cells and CD4−CD8−double negative cells in the spleen, bone marrow and thymus. - Primers were designed for PCR amplification and sequencing identification to confirm, from the genome level, that CD45.2+ hematopoietic cells (mainly T cells) in the recipient mouse were derived from iRunx1-p2a-Hoxa9 pluripotent stem cells. First, bone marrow- and spleen-derived CD45.2+ cells were sorted by flow cytometry, the genome thereof was extracted, and the specific primers of the knocked-in gene sequence were used for PCR identification.
FIG. 4(D) showed that iRunx1-p2a-Hoxa9 plasmid-derived sequences were found in the genome of these cells, confirming that the CD45.2+ blood cells (primarily T cells) were derived from the iRunx1-p2a-Hoxa9 pluripotent stem cells. - The thymus DN cell population was analyzed to further identify the type of the pluripotent stem cell-derived immune cells in the mouse. It was shown in
FIG. 5(A) that the T cells in the recipient mouse were normally developed and DN1, DN2, DN3 and DN4 cell populations were detectable. The TCR receptors of pluripotent stem cell-derived T cells in the peripheral blood, spleen and lymphatic vessels were subjected to a detection. As shown inFIG. 5(B) , a certain proportion of TCR γ/δ cells (0.37-1.71%) were found in the T cells, while most of them were TCR β cells. A mixed lymphocyte reaction was carried out with spleen cells of a Balb/C mouse and T cells obtained from the spleen of the recipient mouse by CD3 magnetic bead enrichment, and a detection was performed ondays FIG. 5(C) , the pluripotent stem cell-derived T cells were able to proliferate after activation, confirming that these T cells have proliferative ability after stimulation. - The culture supernatant was analyzed by ELISA. As shown in
FIG. 6 , the regenerated T cells after stimulation and proliferation were able to secrete a large amount of interleukin 10 (IL10), interferon gamma (IFN-γ), interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α). - In summary, pluripotent stem cells which inducibly co-express exogenous Runx1 and Hoxa9 are successfully constructed in the present invention by introducing an exogenous vector co-expressing Runx1 and Hoxa9 into pluripotent stem cells. The pluripotent stem cells were directionally differentiated into T-lineage progenitor cells which will be developed into T cells. The pluripotent stem cell-derived T cells obtained by the method of the present invention are not only functionally normal but also have no tumorigenic risk, and can be used for preparing a medicine for enhancing an immune effect, preventing and/or treating immunodeficiency and treating a tumor.
- The applicant states that detailed methods of the present invention are demonstrate in the present invention through the above embodiments, however, the present invention is not limited to the above detailed methods, and does not mean that the present invention must rely on the above detailed methods to implement. It should be apparent to those skilled in the art that, for any improvement of the present invention, the equivalent replacement of the raw materials of the present invention, the addition of auxiliary components, and the selection of specific modes, etc., will all fall within the protection scope and the disclosure scope of the present invention.
Claims (15)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711490483.8 | 2017-12-30 | ||
CN201711490483.8A CN108220243B (en) | 2017-12-30 | 2017-12-30 | A kind of multipotential stem cell and its T cell and application of differentiation |
PCT/CN2018/072254 WO2019127664A1 (en) | 2017-12-30 | 2018-01-11 | Multipotent stem cells and differentiated t-cells and use thereof |
Publications (3)
Publication Number | Publication Date |
---|---|
US20200208107A1 US20200208107A1 (en) | 2020-07-02 |
US20210238548A9 true US20210238548A9 (en) | 2021-08-05 |
US11299709B2 US11299709B2 (en) | 2022-04-12 |
Family
ID=62642102
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/312,794 Active 2039-08-08 US11299709B2 (en) | 2017-12-30 | 2018-01-11 | Pluripotent stem cell and T cell differentiated therefrom and application thereof |
Country Status (4)
Country | Link |
---|---|
US (1) | US11299709B2 (en) |
JP (1) | JP7098187B2 (en) |
CN (1) | CN108220243B (en) |
WO (1) | WO2019127664A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113046311B (en) * | 2021-03-03 | 2022-09-30 | 中山大学孙逸仙纪念医院 | Method for inducing pluripotent stem cells to directionally differentiate into lymphoid tissue inducing cells |
CN115247151B (en) * | 2022-09-21 | 2022-12-27 | 呈诺再生医学科技(北京)有限公司 | Method for preparing hematopoietic endothelial cells and method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells |
CN115948472B (en) * | 2022-12-06 | 2024-02-06 | 广州市天河诺亚生物工程有限公司 | Application of RUNX1 for inducing over-expression in construction of depletion T cell model |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2970881A4 (en) | 2013-03-14 | 2017-01-25 | Children's Medical Center Corporation | Compositions and methods for reprogramming hematopoietic stem cell lineages |
CN104789529A (en) * | 2015-04-28 | 2015-07-22 | 济南劲牛生物科技有限公司 | Method for promoting mouse bone marrow hematopoietic stem cell in vitro clone formation and differentiation ability |
WO2017070337A1 (en) * | 2015-10-20 | 2017-04-27 | Cellular Dynamics International, Inc. | Methods for directed differentiation of pluripotent stem cells to immune cells |
WO2017192708A1 (en) * | 2016-05-03 | 2017-11-09 | The Children's Medical Center Corporation | Hematopoietic stem and progenitor cells derived from hemogenic endothelial cells |
-
2017
- 2017-12-30 CN CN201711490483.8A patent/CN108220243B/en active Active
-
2018
- 2018-01-11 US US16/312,794 patent/US11299709B2/en active Active
- 2018-01-11 JP JP2020535615A patent/JP7098187B2/en active Active
- 2018-01-11 WO PCT/CN2018/072254 patent/WO2019127664A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
CN108220243B (en) | 2019-05-28 |
US11299709B2 (en) | 2022-04-12 |
US20200208107A1 (en) | 2020-07-02 |
JP2021509272A (en) | 2021-03-25 |
CN108220243A (en) | 2018-06-29 |
WO2019127664A1 (en) | 2019-07-04 |
JP7098187B2 (en) | 2022-07-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102575976B1 (en) | Proliferation method of natural killer cells | |
EP2841563B1 (en) | Method for developing natural killer cells from stem cells | |
KR101443478B1 (en) | Cell Preparation Containing Mesenchymal Stem Cell, and Method for Producing Same | |
EP2669368B1 (en) | Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates | |
CN114774365B (en) | Method for obtaining CD34+ cells and NK cells by inducing iPSC differentiation and application thereof | |
US11299709B2 (en) | Pluripotent stem cell and T cell differentiated therefrom and application thereof | |
Guo et al. | Generation and clinical potential of functional T lymphocytes from gene-edited pluripotent stem cells | |
CN114402065A (en) | Low density cell culture | |
US20230071538A1 (en) | Cytotoxic t cells derived from human t cell-derived ips cells | |
CN113939302A (en) | Pharmaceutical composition | |
AU4419396A (en) | System for the maintenance, growth and differentiation of human and non-human primate pluripotent stem, progenitor and mature bone marrow cells | |
WO2022242359A1 (en) | Method for regenerating humoral immunity system and use thereof | |
CN116479041A (en) | Gene construct and method for producing multi-lineage hematopoietic stem/progenitor cells | |
AU2005285222A1 (en) | Liver stromal cells for prevention and treatment of immune responses in transplantation | |
CN115418373A (en) | Method for regenerating humoral immune system and application thereof | |
US20100209396A1 (en) | Method of Enhancing Proliferation and/or Hematopoietic Differentiation of Stem Cells | |
WO2006085482A1 (en) | Self-replication factor and amplification method of hematopoietic stem cell | |
Lisini et al. | Stem Cell Production: Processes, Practices, and Regulation | |
Romee et al. | Development of Diverse Hematopoietic Cell Populations from Human Embryonic Stem Cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
AS | Assignment |
Owner name: GUANGZHOU INSTITUTES OF BIOMEDICINE AND HEALTH CHINESE ACADEMY OF SCIENCES, CHINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WANG, JINYONG;GUO, RONGQUN;ZHANG, MENGYUN;AND OTHERS;REEL/FRAME:047868/0176 Effective date: 20181212 |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED |
|
FEPP | Fee payment procedure |
Free format text: PETITION RELATED TO MAINTENANCE FEES GRANTED (ORIGINAL EVENT CODE: PTGR); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |