US20150140657A1 - Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts - Google Patents
Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts Download PDFInfo
- Publication number
- US20150140657A1 US20150140657A1 US14/465,533 US201414465533A US2015140657A1 US 20150140657 A1 US20150140657 A1 US 20150140657A1 US 201414465533 A US201414465533 A US 201414465533A US 2015140657 A1 US2015140657 A1 US 2015140657A1
- Authority
- US
- United States
- Prior art keywords
- cells
- hemangioblasts
- dcs
- culturing
- cell
- 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.)
- Abandoned
Links
- 210000004443 dendritic cell Anatomy 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 82
- 210000003566 hemangioblast Anatomy 0.000 title claims abstract description 78
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 50
- 210000001671 embryonic stem cell Anatomy 0.000 title claims description 19
- 210000004027 cell Anatomy 0.000 claims abstract description 159
- 102000004127 Cytokines Human genes 0.000 claims description 50
- 108090000695 Cytokines Proteins 0.000 claims description 50
- 229920000609 methyl cellulose Polymers 0.000 claims description 34
- 239000001923 methylcellulose Substances 0.000 claims description 34
- 238000012258 culturing Methods 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 25
- 102000003812 Interleukin-15 Human genes 0.000 claims description 24
- 108090000172 Interleukin-15 Proteins 0.000 claims description 24
- 210000002966 serum Anatomy 0.000 claims description 22
- 230000014509 gene expression Effects 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 230000035800 maturation Effects 0.000 claims description 12
- 102000006354 HLA-DR Antigens Human genes 0.000 claims description 10
- 108010058597 HLA-DR Antigens Proteins 0.000 claims description 10
- 238000003306 harvesting Methods 0.000 claims description 9
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 8
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 8
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 8
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 8
- 108091036414 Polyinosinic:polycytidylic acid Proteins 0.000 claims description 8
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 claims description 8
- 229960002986 dinoprostone Drugs 0.000 claims description 8
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 claims description 8
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 claims description 8
- 210000001939 mature NK cell Anatomy 0.000 claims description 7
- 230000003442 weekly effect Effects 0.000 claims description 7
- 102100022297 Integrin alpha-X Human genes 0.000 claims description 6
- 210000003509 immature nk cell Anatomy 0.000 claims description 6
- 102100021992 CD209 antigen Human genes 0.000 claims description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 5
- 102100035793 CD83 antigen Human genes 0.000 claims description 4
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 claims description 4
- 210000004748 cultured cell Anatomy 0.000 claims description 4
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims 4
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims 4
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims 4
- 230000004069 differentiation Effects 0.000 description 23
- 210000001082 somatic cell Anatomy 0.000 description 23
- 210000002242 embryoid body Anatomy 0.000 description 16
- 230000008672 reprogramming Effects 0.000 description 16
- 241000894007 species Species 0.000 description 15
- 238000003556 assay Methods 0.000 description 13
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 210000001778 pluripotent stem cell Anatomy 0.000 description 11
- 210000001744 T-lymphocyte Anatomy 0.000 description 10
- 230000024245 cell differentiation Effects 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 210000001185 bone marrow Anatomy 0.000 description 7
- 230000003013 cytotoxicity Effects 0.000 description 7
- 231100000135 cytotoxicity Toxicity 0.000 description 7
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 6
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 6
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 6
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 6
- 238000003501 co-culture Methods 0.000 description 6
- 230000001024 immunotherapeutic effect Effects 0.000 description 6
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 239000000546 pharmaceutical excipient Substances 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 208000021841 acute erythroid leukemia Diseases 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 238000002784 cytotoxicity assay Methods 0.000 description 5
- 231100000263 cytotoxicity test Toxicity 0.000 description 5
- 238000002825 functional assay Methods 0.000 description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 241000282472 Canis lupus familiaris Species 0.000 description 4
- 241000283153 Cetacea Species 0.000 description 4
- 241000282326 Felis catus Species 0.000 description 4
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 4
- -1 Nanog Proteins 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 3
- 208000031637 Erythroblastic Acute Leukemia Diseases 0.000 description 3
- 208000036566 Erythroleukaemia Diseases 0.000 description 3
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 3
- 101000971513 Homo sapiens Natural killer cells antigen CD94 Proteins 0.000 description 3
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 3
- 102100021462 Natural killer cells antigen CD94 Human genes 0.000 description 3
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000002458 cell surface marker Substances 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000003394 haemopoietic effect Effects 0.000 description 3
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229940092253 ovalbumin Drugs 0.000 description 3
- 230000008186 parthenogenesis Effects 0.000 description 3
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 241001455214 Acinonyx jubatus Species 0.000 description 2
- 241000282452 Ailuropoda melanoleuca Species 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 241001481833 Coryphaena hippurus Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 241000283070 Equus zebra Species 0.000 description 2
- 102000003951 Erythropoietin Human genes 0.000 description 2
- 108090000394 Erythropoietin Proteins 0.000 description 2
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 101000973177 Homo sapiens Nuclear factor interleukin-3-regulated protein Proteins 0.000 description 2
- 101000914496 Homo sapiens T-cell antigen CD7 Proteins 0.000 description 2
- 102000013462 Interleukin-12 Human genes 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- 102000003810 Interleukin-18 Human genes 0.000 description 2
- 108090000171 Interleukin-18 Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000014736 Notch Human genes 0.000 description 2
- 108010070047 Notch Receptors Proteins 0.000 description 2
- 102100022163 Nuclear factor interleukin-3-regulated protein Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 208000025174 PANDAS Diseases 0.000 description 2
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 2
- 240000004718 Panda Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- 241000282320 Panthera leo Species 0.000 description 2
- 241000282376 Panthera tigris Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108010056995 Perforin Proteins 0.000 description 2
- 102000004503 Perforin Human genes 0.000 description 2
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 2
- 241000283080 Proboscidea <mammal> Species 0.000 description 2
- 241000282330 Procyon lotor Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 101100247004 Rattus norvegicus Qsox1 gene Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 102100027208 T-cell antigen CD7 Human genes 0.000 description 2
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 2
- 206010043276 Teratoma Diseases 0.000 description 2
- 238000010817 Wright-Giemsa staining Methods 0.000 description 2
- 210000005006 adaptive immune system Anatomy 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 238000011319 anticancer therapy Methods 0.000 description 2
- 210000003969 blast cell Anatomy 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000001109 blastomere Anatomy 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940105423 erythropoietin Drugs 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 210000001654 germ layer Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000001401 hemangioblastic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 210000005007 innate immune system Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000003071 memory t lymphocyte Anatomy 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 229930192851 perforin Natural products 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010374 somatic cell nuclear transfer Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108010037897 DC-specific ICAM-3 grabbing nonintegrin Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 102100035716 Glycophorin-A Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 1
- 101001074244 Homo sapiens Glycophorin-A Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 1
- 101000642523 Homo sapiens Transcription factor SOX-7 Proteins 0.000 description 1
- 101000976622 Homo sapiens Zinc finger protein 42 homolog Proteins 0.000 description 1
- 102000002698 KIR Receptors Human genes 0.000 description 1
- 108010043610 KIR Receptors Proteins 0.000 description 1
- 108700021430 Kruppel-Like Factor 4 Proteins 0.000 description 1
- 108010013709 Leukocyte Common Antigens Proteins 0.000 description 1
- 102000017095 Leukocyte Common Antigens Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 102100040120 Prominin-1 Human genes 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000011579 SCID mouse model Methods 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- 102100023550 Zinc finger protein 42 homolog Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005889 cellular cytotoxicity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 108091008034 costimulatory receptors Proteins 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000000925 erythroid effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 210000003953 foreskin Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000002064 heart cell Anatomy 0.000 description 1
- 102000052241 human SOX7 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 1
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 101150111214 lin-28 gene Proteins 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011536 re-plating Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000005222 synovial tissue Anatomy 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012447 xenograft mouse model Methods 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
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- 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
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
- A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/48—Blood cells, e.g. leukemia or lymphoma
-
- 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
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- 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]
-
- 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]
- C12N2501/2304—Interleukin-4 (IL-4)
-
- 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
- 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
Definitions
- This invention relates to the generation of natural killer (NK) cells and dendritic cells (DCs) from hemangioblasts.
- NK natural killer
- DCs dendritic cells
- hemangioblast a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture.
- the inventors' group has developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which is important for their productive use in regenerative medicine.
- Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown.
- Natural killer (NK) cells which arise through the lymphoid lineage and are part of the innate immune system, may be used in anti-cancer therapy as they have been found to detect and kill certain types of tumor cells.
- Dendritic cells which mostly arise through the myeloid lineage (from monocytes) and are part of the adaptive immune system, may be used to enhance antigen-specific immune responses through their ability to present antigen to and stimulate both naive and memory T cells (e.g., DC-based vaccine therapy).
- NK natural killer
- DCs dendritic cells
- Various embodiments of the present invention provide for a method of generating natural killer (NK) cells comprising: providing hemangioblasts; culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, SCF and FL; harvesting the cultured cells; and culturing the harvested cells in liquid media comprising human serum, and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate NK cells.
- NK natural killer
- the methylcellulose can be H4236 methylcellulose. In other embodiments, the methylcellulose can be H4536 methylcellulose.
- the concentration of IL2 can be about 5-10 ng/ml
- IL3 can be about 1-10 ng/ml
- IL6 can be about 1-10 ng/ml
- IL7 can be about 5-20 ng/ml
- IL15 can be about 5-10 ng/ml
- SCF can be about 10-50 ng/ml
- FL can be about 10-50 ng/ml.
- culturing the hemangioblasts can be for about 6 to 8 days. In various embodiments, cluturing the harvested cells can be for about 14 to 21 days. In various embodiments, the method can further comprise weekly media changes to refresh the second cytokine mixture.
- the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.
- hESCs human embryonic stem cells
- iPS induced pluripotent
- the NK cells can be immature NK cells and can be CD56+ and CD16 ⁇ . In other embodiments, the NK cells can be mature NK cells and can be CD56 ⁇ and CD 16+, or CD56lo and CD16+.
- Various embodiments of the present invention provide for a method of generating natural killer (NK) cells comprising: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and SCF; harvesting the cultured cells; and culturing the harvested cells in liquid media comprising human serum and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate the NKs.
- NK natural killer
- the concentration of IL2 can be about 5-10 ng/ml
- IL3 can be about 1-10 ng/ml
- IL6 can be about 1-10 ng/ml
- IL7 can be about 5-20 ng/ml
- IL15 can be about 5-10 ng/ml
- SCF can be about 10-50 ng/ml
- FL can be about 10-50 ng/ml.
- culturing the hemangioblasts can be for about 6 to 8 days. In various embodiments, culturing the harvested cells can be for about 14 to 21 days.
- the method can further comprise weekly media changes to refresh the second cytokine mixture.
- the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.
- hESCs human embryonic stem cells
- iPS induced pluripotent
- the NK cells can be immature NK cells and can be CD56+ and CD16 ⁇ . In other embodiments, the NK cells can be mature NK cells and can be CD56 ⁇ and CD16+, or CD56lo and CD16+.
- NK natural killer
- Other embodiments of the present invention provide for a pharmaceutically acceptable composition comprising a quantity of the NK cells generated by any of the method of the present invention.
- DCs dendritic cells
- a method to generate dendritic cells comprising: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF; adding IL4 to the liquid media; and further culturing the hemangioblasts to generate the DCs.
- culturing the hemangioblasts can be for about 7 to 11 days. In various embodiments, the culturing of the hemangioblast after the addition of IL4 can be for about 8 to 10 days.
- the method can further comprise adding a cytokine mixture comprising IL1b, TNF ⁇ and IL6 to induce maturation of the DCs.
- the cytokine mixture can be added for about 48 hours.
- the cytokine mixture can further comprise a cytokine selected from the group consisting of PGE2, IFN ⁇ 2b, poly I:C, IFN ⁇ and combinations thereof.
- the method can further comprise adding LPS, IFN ⁇ and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs.
- the concentration of SCF can be about 20-100 ng/ml
- FL can be about 10-50 ng/ml
- IL3 can be about 5-50 ng/ml
- GM-CSF can be about 50-100 ng/ml
- IL4 can be about 50-100 ng/ml.
- the concentration of IL1b can be about 10 ng/ml
- TNF ⁇ can be about 10 ng/ml
- IL6 can be about 150 ng/ml.
- the concentration of PGE2 can be about 1 ⁇ g/ml
- IFN ⁇ 2b can be about 3000 units/ml
- poly I:C can be about 20 ⁇ g/ml
- IFN ⁇ can be about 20 ng/ml.
- the DCs can be mature DCs and express CD83. In other embodiments, the DCs can be mature DCs and the expression of CD209, HLA DR and/or CD11c is increased.
- Various embodiments of the present invention provide for a dendritic cell (DC) generated by any of the methods of the present invention.
- Other embodiments of the present invention provide for a pharmaceutically acceptable composition comprising a quantity of the DCs generated by any of the method of the present invention.
- FIG. 1 shows that hemangioblasts are bipotential precursor cells that can give rise to both hematopoietic (A-C) and vascular (D-F) lineages in accordance with various embodiments of the present invention.
- FIG. 2 shows that cells with immunotherapeutic potential, such as dendritic cells can be differentiated from hemangioblasts in accordance with various embodiments of the present invention.
- FIG. 3 shows that upon exposure to lymphoid-inducing cytokines, hESC-derived hemangioblasts can give rise to CD56low/CD16+ natural killer cells in a feeder-free culture system in accordance with various embodiments of the present invention.
- C Emergence of CD56low/CD16+ NK cells after total of 28-40 days differentiation.
- FIG. 4 shows that similar to the classic 51Cr release assay, intracellular flow cytometry can be used to assess NK-mediated cellular cytotoxicity in accordance with various embodiments of the present invention.
- FIG. 5 shows that hemangioblast-derived NK effector cells can induce apoptosis in K562 erythroleukemia target cells after standard 4 hour co-culture in accordance with an embodiment of the present invention.
- FIG. 6 shows a process of generating the NK cells in accordance with an embodiment of the present invention.
- ES cells embryonic stem cells
- This term includes cells derived from the inner cell mass of human blastocysts or morulae, including those that have been serially passaged as cell lines.
- the ES cells may be derived from fertilization of an egg cell with sperm, as well as using DNA, nuclear transfer, parthenogenesis, or by means to generate ES cells with homozygosity in the HLA region.
- ES cells are also cells derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, androgenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce a cell.
- Embryonic stem cells regardless of their source or the particular method use to produce them, can be identified based on (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) ability to produce teratomas when transplanted into immunodeficient animals.
- pluripotent stem cells includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived.
- Pluripotent stem cells are defined functionally as stem cells that are: (a) capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc).
- SCID immunodeficient
- Exemplary pluripotent stem cells can be generated using, for example, methods known in the art.
- Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo).
- embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis.
- SCNT somatic cell nuclear transfer
- pluripotent stem cells include induced pluripotent stem cells (iPS cells) generated by reprogramming a somatic cell by expressing a combination of factors (herein referred to as reprogramming factors).
- iPS cells can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells.
- factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4.
- factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct 4, Sox2, Nanog, and Lin28.
- somatic cells are reprogrammed by expressing at least 2 reprogramming factors, at least three reprogramming factors, or four reprogramming factors.
- Induced pluripotent stem cells can be produced by expressing a combination of reprogramming factors in a somatic cell.
- at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.
- at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.
- at least four reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell.
- Induced pluripotent stem cells can be produced by protein transduction of reprogramming factors in a somatic cell.
- at least two reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell.
- at least three reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell.
- at least four reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell.
- additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram a somatic cell to a pluripotent stem cell.
- Induced pluripotent stem cells are defined functionally and include cells that are reprogrammed using any of a variety of methods (integrative vectors, non-integrative vectors, chemical means, etc).
- the pluripotent stem cells can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.
- iPS cells can be from any species. iPS cells have been successfully generated using mouse and human cells. iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species.
- the species is an endangered species.
- the species is a currently extinct species.
- Induced pluripotent stem cells can be generated using, as a starting point, virtually any somatic cell of any developmental stage.
- the cell can be from an embryo, fetus, neonate, juvenile, or adult donor.
- Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by a skin sample or biopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. Although skin and cheek provide a readily available and easily attainable source of appropriate cells, virtually any cell can be used.
- the somatic cell is not a fibroblast.
- hemangioblast and “hemangio-colony forming cell” will be used interchangeably throughout this application.
- the cells have numerous structural and functional characteristics. Amongst the characteristics of these cells is the ability to engraft into the bone marrow when administered to a host. These cells can be described based on numerous structural and functional properties including, but not limited to, expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers.
- Hemangio-colony forming cells are capable of differentiating to give rise to at least hematopoietic cell types or endothelial cell types.
- Hemangio-colony forming cells are preferably bi-potential and capable of differentiating to give rise to at least hematopoietic cell types and endothelial cell types.
- hemangio-colony forming cells of the present invention are at least uni-potential, and preferably bi-potential. Additionally however, hemangio-colony forming cells may have a greater degree of developmental potential and can, in certain embodiments, differentiate to give rise to cell types of other lineages.
- the hemangio-colony forming cells are capable of differentiating to give rise to other mesodermal derivatives such as cardiac cells (for example, cardiomyocytes) and/or smooth muscle cells.
- non-engrafting hemangioblasts or “non-engrafting hemangio cells” are used throughout this application to refer to a population of cells that share some of the characteristics of hemangio-colony forming cells. However, the non-engrafting hemangio cells are distinguishable in that they do not engraft into the bone marrow when administered to an immunodeficient host. Despite this difference, non-engrafting hemangio cells may share one or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional or structural characteristics and properties of hemangio-colony forming cells. For example, in certain embodiments, the non-engrafting hemangio cells are loosely adherent to each other.
- non-engrafting hemangio cells do not express one or more than one (2, 3, 4) of the following proteins: CD34, KDR, CD133, CD31.
- non-engrafting hemangio cells may provide a distinct stem cell population that is somewhat more committed than hemangio-colony forming cells, and yet still capable of producing a range of hematopoietic cell types.
- the inventors have developed an in vitro culture system to produce cells with immunotherapeutic potential from human ESCs. This strategy differs from prior art in that it involves the use of hESC-derived hemangioblasts as an intermediate cell source.
- the inventors have been able to direct the differentiation of hemangioblasts into both natural killer (NK) and dendritic cells (DCs) using feeder-free culture conditions.
- NK cells which arise through the lymphoid lineage and are part of the innate immune system, may be used in anti-cancer therapy as they have been found to detect and kill certain types of tumor cells.
- DCs which mostly arise through the myeloid lineage (from monocytes) and are part of the adaptive immune system, may be used to enhance antigen-specific immune responses through their ability to present antigen to and stimulate both na ⁇ ve and memory T cells (e.g. DC-based vaccine therapy).
- NK cells which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity.
- hemangioblasts generated from both H7 and HuES-3 hESC lines the inventors have been able to produce mature CD56low/ ⁇ , CD16+ NK cells and found that their production does not require the use of stromal feeder layers.
- the differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 10-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population.
- NK cells An additional 14-21 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry.
- a non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture.
- Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro, and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential.
- Embodiments of the present invention provide a method of generating lymphoid lineage cells.
- the present invention provides a method of generating natural killer (NK) cells.
- the method of generating NK cells comprises: providing hemangioblasts; plating and culturing the hemangioblasts on methylcellulose and IL2, IL3, IL6, IL7, IL15, SCF and FL; harvesting the cells; and replating/culturing the harvested cells in liquid media comprising human serum, IL7, IL15, SCF and FL.
- the liquid media is changed weekly.
- the methylcelllulose is H4236 methylcellulose.
- the methylcellulose is H4536 methylcellulose.
- the concentrations of the cytokines are IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml).
- harvesting the cells is done after 6-8 days of culturing the cells.
- the cells are cultured for an additional 14-21 days after the cells are replaced.
- the liquid media is ⁇ MEM or DMEM:F12.
- the method of generating natural killer (NK) cells comprises: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising IL2, IL3, IL6, IL7, IL15, SCF, and human serum; harvesting the cells; and culturing the harvested cells in liquid media comprising human serum, IL7, IL15, SCF and FL.
- the liquid media is changed weekly. Similar to the foregoing, in various embodiments the concentrations of the cytokines are IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml). In various embodiments, harvesting the cells is done after 6-8 days of culturing the cells. In various embodiments, the cells are cultured for an additional 14-21 days after the cells are replated. In various embodiments, the liquid media is ⁇ MEM or DMEM:F12.
- NK cells are provided in a pharmaceutically acceptable composition comprising a quantity of the NK cells generated by the methods of the present invention.
- the method to generate DCs comprises: providing hemangioblasts; plating and culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF; adding IL4 to the liquid media; and further culturing the cells.
- the method further comprises adding a cytokine cocktail comprising IL1b, TNF ⁇ and IL6 to induce maturation of the DCs.
- the cytokine cocktail further comprises a cytokine selected from the group consisting of PGE2, IFN ⁇ 2b, poly I:C, IFN ⁇ and combinations thereof.
- the method further comprising adding LPS, IFN ⁇ and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs.
- the liquid media is changed every 6 to 7 days. In various embodiments the liquid media is ⁇ MEM or DMEM:F12. In various embodiments, the cells are cultured for 7-11 days. In various embodiments, the IL4 is added after the cells are cultured for 7-11 days. In various embodiments, the cells are cultured for an additional 8-10 days after adding the IL4.
- the concentrations of the human serum, SCF, FL, IL3 and GM-CSF are human serum (10-20%), SCF (20-100 ng/ml), FL (10-50 ng/ml), IL3 (5-50 ng/ml) and GM-CSF (50-100 ng/ml).
- the concentration of IL4 is 50-100 ng/ml.
- the concentrations of IL1b, TNF ⁇ and IL6 are IL1b (10 ng/ml), TNF ⁇ (10 ng/ml) and IL6 (150 ng/ml).
- the concentrations of PGE2, IFN ⁇ 2b, poly I:C, and IFN ⁇ are PGE2 (1 ⁇ g/ml), IFN ⁇ 2b (3000 units/ml), poly I:C (20 ⁇ /ml), and IFN ⁇ (20 ng/ml).
- DC maturation cocktail may improve the IL12p70 secretion assay.
- LPS and/or IFN ⁇ may be required in order to stimulate IL12p70 production from blast-derived DCs.
- synthetic compound, S-28463 may help increase both IL12p70 production and HLA-DR expression.
- the DCs are provided in a pharmaceutically acceptable composition comprising a quantity of the DCs generated by the methods of the present invention.
- the hemangioblast may be obtained by a method comprising: providing hESCs; culturing the hESCs in media comprising cytokines to generate embryoid bodies (EBs); disaggregating the EBs: filtering individual cells; seeding the individual cells into methylcellulose comprising TPO, VEGF, FL, and bFGF; and harvesting the blast-like cells from methylcellulose.
- EBs embryoid bodies
- the hESCs are first cultured for 4 days before disaggregating the EBs.
- the cytokines to generate embryoid bodies comprise VEGF and BMP4.
- VEGF and BMP4 are used throughout the EB formation.
- the cytokines to generate embryoid bodies further comprise bFGF.
- the bFGF is added after the first 2 days of culturing the hESCs.
- disaggregation of the EBs comprises disaggregating the EBs with trypsin and then inactivating the trypsin with serum-containing media. In one embodiment, the trypsin is 0.05%.
- filtering the individual cells comprise filtering the individual cells through a 40 ⁇ M cell strainer.
- the methylcellulose is H4436 or H4536 methylcellulose.
- the concentration of cytokines are TPO (50 ⁇ g/ml), VEGF (50 ⁇ g/ml), FL (50 ⁇ g/ml), and bFGF (20-50 ⁇ g/ml).
- the blast-like cells are harvested from methylcellulose between day 6 and day 10.
- the methylcellulose may comprise additional cytokines.
- additional cytokines are selected from the group consisting of IL2, IL7, IL15 and combinations thereof.
- the concentrations of these cytokines are IL2 (1-10 ⁇ g/ml), IL7 (1-20 ⁇ g/ml), and IL15 (1-10 ⁇ g/ml).
- methylcellulose cultures are plated at a concentration of 50,000 to 150,000 cells/ml.
- the hemangioblasts are generated in an erythropoietin free methylcellulose.
- the erythropoietin free methylcellulose is H4536 methylcellulose.
- pluripotent stem cells including iPS cells and human iPS cells
- the hemangioblasts may be non-engrafting hemangioblasts.
- the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the natural kill cells or dendritic cells of the present invention.
- “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
- compositions according to the invention may be formulated for delivery via any route of administration.
- Route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.
- Parenteral refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
- the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
- compositions according to the invention can also contain any pharmaceutically acceptable carrier.
- “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
- the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
- Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
- the initial differentiation procedure for both cell types is the same and involves a 4 day culture of hESCs in Stemline II (Sigma) plus cytokines in order to generate embryoid bodies (EBs).
- the cytokines, VEGF and BMP4 are used throughout the EB culture while bFGF is added after the first 2 days. After 4 days total, the resulting EBs are disaggregated with 0.05% trypsin and then the trypsin is inactivated with serum-containing media.
- H4436 or H4536 methylcellulose Stem Cell Technologies
- additional cytokines such as TPO (50 ⁇ g/ml), VEGF (50 ⁇ g/ml), FL (50 ⁇ g/ml) and bFGF (20-50 ⁇ g/ml).
- TPO 50 ⁇ g/ml
- VEGF 50 ⁇ g/ml
- FL 50 ⁇ g/ml
- bFGF 20-50 ⁇ g/ml
- the cytokines IL2 (1-10 ⁇ g/ml
- IL7 1-20 ⁇ g/ml
- IL15 IL15
- Methylcellulose cultures are plated at a concentration of 50,000 to 150,000 cells per ml for the production and expansion of a hemangioblastic population.
- Blast-like cells are harvested from methylcellulose between day 6 and 10 and further differentiated by one of the following procedures.
- Blast cells may be replated in H4236 methylcellulose plus IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml) or in liquid culture containing the same cytokines and 10-20% human serum.
- cytokine cocktail After 6-8 days culture, cells are harvested and replated in liquid media ( ⁇ MEM or DMEM:F12) plus 10-20% human serum and the cytokines IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml) for an additional 14-21 days. Weekly media changes are used to refresh the cytokine cocktail.
- Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of cells and the acquisition of NK cell surface markers.
- Cell surface markers include CD34, CD45, CD56, CD16, CD94, NKG2D, CD3, CD7, CD4, CD8a, and CD45RA.
- Tests to examine the function of hemangioblast-derived NK cells include (1) natural cytotoxicity assay using K562 erythroleukemia target cells, (2) IFN ⁇ production in response to IL12/IL18 or phorbol myristate acetate treatment, (3) intracelluar flow cytometry for presence of perforin and granzyme B enzymes, and (4) antibody-dependent cellular cytotoxicity assay using Raji cells and anti-CD20 antibodies.
- NK cells from both H7 and HuES3 hESCs.
- a non-radioactive cytotoxicity assay similar to the 51 Cr release assay shows that our hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture.
- Blast cells are plated in liquid media ( ⁇ MEM or DMEM:F12) plus 10-20% human serum and the cytokines, SCF (20-100 ng/ml), FL (10-50 ng/ml), IL3 (5-50 ng/ml), and GM-CSF (50-100 ng/ml). After 7-11 days culture. IL4 (50-100 ng/ml) is also added to the culture and cells are allowed to grow for as additional 8-10 days. Media changes are performed every 6-7 days. An additional cytokine cocktail (10 ng/ml IL1b, 10 ng/ml TNF ⁇ , 150 ng/ml IL6) can be added to the culture for 48 hours in order to induce maturation of DCs.
- cytokine cocktail (10 ng/ml IL1b, 10 ng/ml TNF ⁇ , 150 ng/ml IL6) can be added to the culture for 48 hours in order to induce maturation of DCs.
- Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of cells and the acquisition of DC surface markers.
- Cell surface markers include CD11c, CD209 (DC-SIGN), HLA ABC (MHC class I), HLA DR (MHC class II), CD1a, and CD14. Maturation is assessed by the acquisition of T-cell co-stimulatory receptor, CD83 while the expression of CD209, HLA DR, and CD11c may also increase upon maturation.
- Additional cytokines such as 1 ⁇ g/ml PGE2, 3000 units/ml IFN ⁇ 2b, 20 ⁇ g/ml poly I:C, or IFN ⁇ 20 ng/ml may be added to the maturation cocktail to enhance DC functional response in certain assays.
- Assays to address the functionality of hemangioblast-derived DCs include (1) antigen uptake via DQ-ovalbumin (BD Biosciences) processing, (2) transwell migration in response to the chemoattractant MIP-3b, (3) allogenic mixed lymphocyte reaction assay to determine the ability of DCs to increase proliferation of HLA-mismatched T cells, (4) IL12-p70 secretion upon DC stimulation, and (5) antigen presenting assay to determine if antigen-loaded DCs can induce IFN ⁇ production in previously antigen-primed peripheral blood mononuclear cells.
- DQ-ovalbumin BD Biosciences
- the inventors have been able to generate DCs from both HuES3 and MA01 hESCs. Upregulation of CD83, HLA-DR, CD11c, and CD209 in response to the 48 hour maturation cytokine cocktail was observed. These DCs are found to be able to uptake and proteolyse DQ-ovalbumin in a 30 minute assay.
- H4536 an erythropoietin-free methylcellulose from Stem Cell Technologies can also be used to efficiently generate hemangioblasts.
- epo minus hemangioblasts are quite similar to the original “epo plus” blasts; they are capable of differentiating into a variety of hematopoietic and vascular cell types.
- Preliminary results suggest that the use of H4536 may provide a significant advantage over H4436 methylcellulose for the differentiation of hemangioblasts into various hematopoietic lineages, including NK cells and DCs.
- epo-minus growth conditions may enhance differentiation down myeloid and/or lymphoid lineages.
- MKs can be generated from iPS cells.
- a few thousand CD41a+ MKs were generated from a few hundred thousand iPS cells.
- H7 ESCs were differentiated into embryoid bodies (EBs) for 4 days.
- EBs were harvested and transferred to cytokine-rich methylcellulose for 10-15 days for hemangioblast production and expansion.
- Hemangioblasts were harvested and placed into feeder-free liquid culture medium plus 10-20% human AB serum, with a panel of cytokines for an additional 14-17 days with media half changes every 3-4 days.
- Immature NK cells were CD56bright, CD16lo, KIRlo, CD117+, CD94 ⁇ , NKG2D+. By using a variation of the above procedure, 20-30% of the viable cells after 32 days of differentiation were CD56+ CD16 ⁇ . Mature NK cells were CD56dim, CD16hi, KIRhi, CD117lo/ ⁇ , CD94+, NKG2D+. By using the above procedure, 20% of the viable cells after 31 days of differentiation were CB56 ⁇ CD16+ and 5% of them were CD56loCD16+.
- NK cells can elicit apoptosis of target cells such as human K562 erythroleukemia, MCF7, U87, PC3, NTERA2 cells.
- a “3FC” assay is used to assess efficiency of cytotoxicity. It is similar to 51Cr release assay but does not require radioactivity. See Derby et al., Immunol. Letters 78: 35-39 (2001).
- the heterogeneous population of mature NK cells described above (item B2-a) was found to elicit apoptosis in 65-70% of K562 cells in a standard 4 hour experiment.
- ADCC Antibody-dependent cell-mediated cytotoxicity: The FcyRIII (CD16) on the NK cell surface binds to the fc region of anti-CD20 antibodies attached to target cells and induces ADCC. Raji cells (derived from Burkett's lymphoma) are preincubated with anti-CD20 antibody and used as targets in ADCC assay. (Tsirigotis et al., J of Steroid Biochem and Mol Bio 108: 267-271 (2008)).
- IFN ⁇ cytokine production Immature NK cells produce large amounts of IFN ⁇ in response to overnight treatment with PMA (phorbol myristate acetate) plus ionomycin or IL12 plus IL18. IFN ⁇ secretion is blocked with brefeldin a, cells are stained for cell surface markers and IFN ⁇ using intracellular flow cytometry. See Woll et al. J. of Immunol 175: 5095-5103 (2005).
- NK cells In vivo immunotherapy potential of NK cells using xenograft mouse model: Bioluminescent (luciferase-containing) K562 cells are injected into NOD/SCID mice for engraftment of tumors, followed by bolus of NK cells and daily IP injections of IL2 and IL15. Bioluminescence imaging is used to monitor in vivo NK immunotherapeutic potential over time. See: Wollet al. Blood 113 (24): 6094-6101 (2009).
- hemangioblasts as bone-marrow-repopulating cells or by differentiating them into dendritic, natural killer, T cells, and/or mesenchymal stem cells (MSCs)
- MSCs mesenchymal stem cells
- the inventors were able to achieve differentiation of dendritic cells (DCs) from both hESCs and iPS cells with 40-55% efficiency.
- DCs dendritic cells
- a side by side comparison to DCs derived from human bone marrow in addition to two new functional assays, has now confirmed that the hESC-derived DCs share many comparable features with human BM-derived DCs and also identified areas that need further optimization.
- NK cell differentiation For natural killer cell differentiation, a side by side comparison to human bone marrow-derived NK cells has confirmed that in vitro NK cell differentiation is not very efficient, even when using a bone marrow cell source.
- the inventors have found that blast-derived NK cells display natural cytotoxicity capabilities. Antibody-dependent cellular cytotoxicity assays are performed.
- T cell differentiation the inventors have successfully created a human delta-ligand expressing OP9 stroma cell line to stimulate Notch signaling and are using this stroma cell line to stimulate T cell differentiation of hemangioblasts.
- CD11c, 45, 209 show comparable expression on blastderived DCs and human bone marrow-derived DCs, while HLA-DR is expressed at much lower levels on blast DCs than on human BM DCs.
- IL12-p70 secretion IL12p70 is secreted by mature DCs in order to elicit a Th1-directed response from CD4+ T cells.
- Human BM-derived DCs can produce >500 pg/ml of IL12p70 yet blast-derived DCs did not produce any detectable IL12p70 upon maturation.
- Mixed lymphocyte reaction (MLR) assay The MLR assay determines the ability of DCs to stimulate proliferation of allogenic T cells.
- CBMCs Cord blood mononuclear cells
- T cells cord blood mononuclear cells
- Hemangioblast-derived natural killer cells Cell surface markers CD45, CD7, CD94, CD56, CD16, and NKG2D were evaluated in blast-derived and human bone marrow-derived NK cells.
- NK differentiation efficiency Recombinant human Sox7 protein was being investigated for its ability to increase the pool of CD34+ starting progenitors for NK differentiation. Results suggest that rhSox7 does not dramatically affect % of CD34+ cells.
- Murine AFT024 was investigated as a stroma feeder layer that may provide critical cell-cell contacts and secreted factors for NK cell differentiation. As indicated by cell surface marker expression, AFT024 stroma co-culture did not enhance NK differentiation of either human BM or blasts.
- Hemangioblast-derived T cells Notch signaling is crucial for T cell differentiation.
- the cDNA for human delta-like ligand 1 (hDLL1) was cloned into an MSCV-ires-GFP based retroviral vector and used the resulting viral supernatant to infect OP9 stroma cells.
- OP9 cells Upon viral integration, OP9 cells will express hDLL1 on their cell surface and be gfp positive.
- FACS-based sorting was used to purify the highest gfp-positive cells from the heterogeneous pool of infected OP9 cells.
- S OP9-hDL1S
- Both cord blood and peripheral blood mononuclear cells are used as responders and the inventors use human bone marrow-derived DCs as positive control effectors.
- E4BP4 has been shown to be critical for NK lineage development (see Gascoyne et al. Nature Immunology 10(10): 1118-1125, 2009) and may provide the transcriptional program necessary for more efficient in vitro.
- NK cell differentiation The inventors clone E4BP4 cDNA into a retroviral vector for its overexpression in hemangioblasts and evaluate its ability to increase NK differentiation.
- RT-PCR is used to monitor the expression of various KIR receptor isoforms and the enzymes, perforin and granzyme B, which are critical for NK cell functionality.
- the inventors have shown that blast-derived NK cells display natural cytotoxicity, so their antibody-dependent cellular cytotoxicity (ADCC) capabilities are assessed.
- Required reagents for the ADCC assay include the Burkitt's lymphoma-derived Raji cells and anti-CD20 antibodies. Co-culture of CD20-marked Raji cells with NK cells should elicit a specific ADCC response, which will be monitored through flow cytometric means.
Abstract
Description
- This invention relates to the generation of natural killer (NK) cells and dendritic cells (DCs) from hemangioblasts.
- All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
- Studies with human and mouse embryonic stem cells (ESCs) have shown that a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture. The inventors' group has developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which is important for their productive use in regenerative medicine. Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown.
- Natural killer (NK) cells, which arise through the lymphoid lineage and are part of the innate immune system, may be used in anti-cancer therapy as they have been found to detect and kill certain types of tumor cells. Dendritic cells (DCs), which mostly arise through the myeloid lineage (from monocytes) and are part of the adaptive immune system, may be used to enhance antigen-specific immune responses through their ability to present antigen to and stimulate both naive and memory T cells (e.g., DC-based vaccine therapy).
- Given the immunotherapeutic potential, there exists a need in the art for a method of generating natural killer (NK) cells and dendritic cells (DCs).
- The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.
- Various embodiments of the present invention provide for a method of generating natural killer (NK) cells comprising: providing hemangioblasts; culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, SCF and FL; harvesting the cultured cells; and culturing the harvested cells in liquid media comprising human serum, and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate NK cells.
- In various embodiments, the methylcellulose can be H4236 methylcellulose. In other embodiments, the methylcellulose can be H4536 methylcellulose.
- In various embodiments, the concentration of IL2 can be about 5-10 ng/ml, IL3 can be about 1-10 ng/ml, IL6 can be about 1-10 ng/ml, IL7 can be about 5-20 ng/ml, IL15 can be about 5-10 ng/ml, SCF can be about 10-50 ng/ml, and FL can be about 10-50 ng/ml.
- In various embodiments, culturing the hemangioblasts can be for about 6 to 8 days. In various embodiments, cluturing the harvested cells can be for about 14 to 21 days. In various embodiments, the method can further comprise weekly media changes to refresh the second cytokine mixture.
- In various embodiments, the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.
- In various embodiments, the NK cells can be immature NK cells and can be CD56+ and CD16−. In other embodiments, the NK cells can be mature NK cells and can be CD56− and
CD 16+, or CD56lo and CD16+. - Various embodiments of the present invention provide for a method of generating natural killer (NK) cells comprising: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and SCF; harvesting the cultured cells; and culturing the harvested cells in liquid media comprising human serum and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate the NKs.
- In various embodiments, the concentration of IL2 can be about 5-10 ng/ml, IL3 can be about 1-10 ng/ml, IL6 can be about 1-10 ng/ml, IL7 can be about 5-20 ng/ml, IL15 can be about 5-10 ng/ml, SCF can be about 10-50 ng/ml, and FL can be about 10-50 ng/ml.
- In various embodiments, culturing the hemangioblasts can be for about 6 to 8 days. In various embodiments, culturing the harvested cells can be for about 14 to 21 days.
- In various embodiments, the method can further comprise weekly media changes to refresh the second cytokine mixture.
- In various embodiments,, the hemangioblasts can be differentiated from human embryonic stem cells (hESCs). In other embodiments, the hemangioblasts can be differentiated from induced pluripotent (iPS) cells.
- In various embodiments, the NK cells can be immature NK cells and can be CD56+ and CD16−. In other embodiments, the NK cells can be mature NK cells and can be CD56− and CD16+, or CD56lo and CD16+.
- Various embodiments of the present invention provide for a natural killer (NK) cell generated by any of the methods of the present invention. Other embodiments of the present invention provide for a pharmaceutically acceptable composition comprising a quantity of the NK cells generated by any of the method of the present invention.
- Various embodiments of the present invention provide for a method to generate dendritic cells (DCs), comprising: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF; adding IL4 to the liquid media; and further culturing the hemangioblasts to generate the DCs.
- In various embodiments, culturing the hemangioblasts can be for about 7 to 11 days. In various embodiments, the culturing of the hemangioblast after the addition of IL4 can be for about 8 to 10 days.
- In various embodiments, the method can further comprise adding a cytokine mixture comprising IL1b, TNFα and IL6 to induce maturation of the DCs. In various embodiments, the cytokine mixture can be added for about 48 hours. In various embodiments, the cytokine mixture can further comprise a cytokine selected from the group consisting of PGE2, IFNα2b, poly I:C, IFNγ and combinations thereof.
- In various embodiments, the method can further comprise adding LPS, IFNγ and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs.
- In various embodiments, the concentration of SCF can be about 20-100 ng/ml, FL can be about 10-50 ng/ml, IL3 can be about 5-50 ng/ml, GM-CSF can be about 50-100 ng/ml, and IL4 can be about 50-100 ng/ml. In various embodiments, the concentration of IL1b can be about 10 ng/ml, TNFγ can be about 10 ng/ml, and IL6 can be about 150 ng/ml. In various embodiments, the concentration of PGE2 can be about 1 μg/ml, IFNα2b can be about 3000 units/ml, poly I:C can be about 20 μg/ml, and IFNγ can be about 20 ng/ml.
- In various embodiments, the DCs can be mature DCs and express CD83. In other embodiments, the DCs can be mature DCs and the expression of CD209, HLA DR and/or CD11c is increased.
- Various embodiments of the present invention provide for a dendritic cell (DC) generated by any of the methods of the present invention. Other embodiments of the present invention provide for a pharmaceutically acceptable composition comprising a quantity of the DCs generated by any of the method of the present invention.
- Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.
- Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
-
FIG. 1 shows that hemangioblasts are bipotential precursor cells that can give rise to both hematopoietic (A-C) and vascular (D-F) lineages in accordance with various embodiments of the present invention. -
FIG. 2 shows that cells with immunotherapeutic potential, such as dendritic cells can be differentiated from hemangioblasts in accordance with various embodiments of the present invention. A. Dendritic cell surface marker expression at day 28 of differentiation. B. Bar graph showing 48 hours exposure to maturation cytokines can increase DC surface marker expression. C. Histogram showing DCs can take up and process DQ-ovalbumin antigen in a 30 minute assay. D. Wright-Giemsa stain of DCs, 20×. E. Wright-Giemsa stain of DCs, 100 ×. -
FIG. 3 shows that upon exposure to lymphoid-inducing cytokines, hESC-derived hemangioblasts can give rise to CD56low/CD16+ natural killer cells in a feeder-free culture system in accordance with various embodiments of the present invention. A. During methylcellulose culture, a subset of hemangioblasts acquires the common leukocyte antigen, CD45. B. Transfer of hemangioblasts to liquid culture (containing human serum and a cocktail of cytokines) allows acquisition of NK cell marker, CD56. C. Emergence of CD56low/CD16+ NK cells after total of 28-40 days differentiation. -
FIG. 4 shows that similar to the classic 51Cr release assay, intracellular flow cytometry can be used to assess NK-mediated cellular cytotoxicity in accordance with various embodiments of the present invention. -
FIG. 5 shows that hemangioblast-derived NK effector cells can induce apoptosis in K562 erythroleukemia target cells after standard 4 hour co-culture in accordance with an embodiment of the present invention. -
FIG. 6 shows a process of generating the NK cells in accordance with an embodiment of the present invention. - All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and
Molecular Biology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York. N.Y. 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with a general guide to many of the terms used in the present application. - One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
- As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
- The term “embryonic stem cells” (ES cells) is used herein as it is used in the art. This term includes cells derived from the inner cell mass of human blastocysts or morulae, including those that have been serially passaged as cell lines. The ES cells may be derived from fertilization of an egg cell with sperm, as well as using DNA, nuclear transfer, parthenogenesis, or by means to generate ES cells with homozygosity in the HLA region. ES cells are also cells derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, androgenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce a cell. Embryonic stem cells, regardless of their source or the particular method use to produce them, can be identified based on (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) ability to produce teratomas when transplanted into immunodeficient animals.
- As used herein, the term “pluripotent stem cells” includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are defined functionally as stem cells that are: (a) capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g.,
express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc). Exemplary pluripotent stem cells can be generated using, for example, methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst stage embryos, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying the remainder of the embryo). Such embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. Further exemplary pluripotent stem cells include induced pluripotent stem cells (iPS cells) generated by reprogramming a somatic cell by expressing a combination of factors (herein referred to as reprogramming factors). iPS cells can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to asOct 3/4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination ofOct 4, Sox2, Nanog, and Lin28. In other embodiments, somatic cells are reprogrammed by expressing at least 2 reprogramming factors, at least three reprogramming factors, or four reprogramming factors. Induced pluripotent stem cells can be produced by expressing a combination of reprogramming factors in a somatic cell. In certain embodiments, at least two reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least three reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least four reprogramming factors are expressed in a somatic cell to successfully reprogram the somatic cell. Induced pluripotent stem cells can be produced by protein transduction of reprogramming factors in a somatic cell. In certain embodiments, at least two reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least three reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell. In other embodiments, at least four reprogramming proteins are transduced into a somatic cell to successfully reprogram the somatic cell. - In other embodiments, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram a somatic cell to a pluripotent stem cell. Induced pluripotent stem cells are defined functionally and include cells that are reprogrammed using any of a variety of methods (integrative vectors, non-integrative vectors, chemical means, etc).
- The pluripotent stem cells can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.
- Similarly, iPS cells can be from any species. iPS cells have been successfully generated using mouse and human cells. iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species. Thus, one can generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like. In certain embodiments, the species is an endangered species. In certain embodiments, the species is a currently extinct species.
- Induced pluripotent stem cells can be generated using, as a starting point, virtually any somatic cell of any developmental stage. For example, the cell can be from an embryo, fetus, neonate, juvenile, or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by a skin sample or biopsy, synoviocytes from synovial tissue, foreskin cells, cheek cells, or lung fibroblasts. Although skin and cheek provide a readily available and easily attainable source of appropriate cells, virtually any cell can be used. In certain embodiments, the somatic cell is not a fibroblast.
- The terms “hemangioblast” and “hemangio-colony forming cell” will be used interchangeably throughout this application. The cells have numerous structural and functional characteristics. Amongst the characteristics of these cells is the ability to engraft into the bone marrow when administered to a host. These cells can be described based on numerous structural and functional properties including, but not limited to, expression (RNA or protein) or lack of expression (RNA or protein) of one or more markers. Hemangio-colony forming cells are capable of differentiating to give rise to at least hematopoietic cell types or endothelial cell types. Hemangio-colony forming cells are preferably bi-potential and capable of differentiating to give rise to at least hematopoietic cell types and endothelial cell types. As such, hemangio-colony forming cells of the present invention are at least uni-potential, and preferably bi-potential. Additionally however, hemangio-colony forming cells may have a greater degree of developmental potential and can, in certain embodiments, differentiate to give rise to cell types of other lineages. In certain embodiments, the hemangio-colony forming cells are capable of differentiating to give rise to other mesodermal derivatives such as cardiac cells (for example, cardiomyocytes) and/or smooth muscle cells.
- The terms “non-engrafting hemangioblasts” or “non-engrafting hemangio cells” are used throughout this application to refer to a population of cells that share some of the characteristics of hemangio-colony forming cells. However, the non-engrafting hemangio cells are distinguishable in that they do not engraft into the bone marrow when administered to an immunodeficient host. Despite this difference, non-engrafting hemangio cells may share one or more than one (2, 3, 4, 5, 6, 7, 8, 9, 10) of the functional or structural characteristics and properties of hemangio-colony forming cells. For example, in certain embodiments, the non-engrafting hemangio cells are loosely adherent to each other. In other embodiments, the non-engrafting hemangio cells do not express one or more than one (2, 3, 4) of the following proteins: CD34, KDR, CD133, CD31. Without being bound by theory, non-engrafting hemangio cells may provide a distinct stem cell population that is somewhat more committed than hemangio-colony forming cells, and yet still capable of producing a range of hematopoietic cell types.
- The inventors have developed an in vitro culture system to produce cells with immunotherapeutic potential from human ESCs. This strategy differs from prior art in that it involves the use of hESC-derived hemangioblasts as an intermediate cell source. The inventors have been able to direct the differentiation of hemangioblasts into both natural killer (NK) and dendritic cells (DCs) using feeder-free culture conditions. NK cells, which arise through the lymphoid lineage and are part of the innate immune system, may be used in anti-cancer therapy as they have been found to detect and kill certain types of tumor cells. DCs, which mostly arise through the myeloid lineage (from monocytes) and are part of the adaptive immune system, may be used to enhance antigen-specific immune responses through their ability to present antigen to and stimulate both naïve and memory T cells (e.g. DC-based vaccine therapy).
- Interplay between various activating and inhibitory signals control the three main functions of NK cells, which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, the inventors have been able to produce mature CD56low/−, CD16+ NK cells and found that their production does not require the use of stromal feeder layers. The differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 10-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population. An additional 14-21 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture. Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro, and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential.
- Embodiments of the present invention provide a method of generating lymphoid lineage cells. In various embodiments, the present invention provides a method of generating natural killer (NK) cells.
- In various embodiments, the method of generating NK cells comprises: providing hemangioblasts; plating and culturing the hemangioblasts on methylcellulose and IL2, IL3, IL6, IL7, IL15, SCF and FL; harvesting the cells; and replating/culturing the harvested cells in liquid media comprising human serum, IL7, IL15, SCF and FL.
- In various embodiments, the liquid media is changed weekly. In various embodiments, the methylcelllulose is H4236 methylcellulose. In other embodiments, the methylcellulose is H4536 methylcellulose. In various embodiments, the concentrations of the cytokines are IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml). In various embodiments, harvesting the cells is done after 6-8 days of culturing the cells. In various embodiments, the cells are cultured for an additional 14-21 days after the cells are replaced. In various embodiments, the liquid media is αMEM or DMEM:F12.
- In another embodiment, the method of generating natural killer (NK) cells comprises: providing hemangioblasts; culturing the hemangioblasts in liquid media comprising IL2, IL3, IL6, IL7, IL15, SCF, and human serum; harvesting the cells; and culturing the harvested cells in liquid media comprising human serum, IL7, IL15, SCF and FL.
- In various embodiments, the liquid media is changed weekly. Similar to the foregoing, in various embodiments the concentrations of the cytokines are IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml). In various embodiments, harvesting the cells is done after 6-8 days of culturing the cells. In various embodiments, the cells are cultured for an additional 14-21 days after the cells are replated. In various embodiments, the liquid media is αMEM or DMEM:F12.
- Various embodiments of the present invention provide for natural killer cells generated by the methods of the present invention. In various embodiments, the NK cells are provided in a pharmaceutically acceptable composition comprising a quantity of the NK cells generated by the methods of the present invention.
- Other embodiments of the present invention provide for a method to generate dendritic cells (DCs). In one embodiment, the method to generate DCs comprises: providing hemangioblasts; plating and culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF; adding IL4 to the liquid media; and further culturing the cells.
- In other embodiments, the method further comprises adding a cytokine cocktail comprising IL1b, TNFα and IL6 to induce maturation of the DCs. In further embodiments, the cytokine cocktail further comprises a cytokine selected from the group consisting of PGE2, IFNα2b, poly I:C, IFNγ and combinations thereof. In other embodiments, the method further comprising adding LPS, IFNγ and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs.
- In various embodiments, the liquid media is changed every 6 to 7 days. In various embodiments the liquid media is αMEM or DMEM:F12. In various embodiments, the cells are cultured for 7-11 days. In various embodiments, the IL4 is added after the cells are cultured for 7-11 days. In various embodiments, the cells are cultured for an additional 8-10 days after adding the IL4.
- In various embodiments, the concentrations of the human serum, SCF, FL, IL3 and GM-CSF are human serum (10-20%), SCF (20-100 ng/ml), FL (10-50 ng/ml), IL3 (5-50 ng/ml) and GM-CSF (50-100 ng/ml). In another embodiment, the concentration of IL4 is 50-100 ng/ml. In various embodiments, the concentrations of IL1b, TNFα and IL6 are IL1b (10 ng/ml), TNFα (10 ng/ml) and IL6 (150 ng/ml). In various embodiments, the concentrations of PGE2, IFNα2b, poly I:C, and IFNγ are PGE2 (1 μg/ml), IFNα2b (3000 units/ml), poly I:C (20 μ/ml), and IFNγ (20 ng/ml).
- Since the efficiency of producing dendritic cells from hemangioblasts is relatively good, future work with DCs can involve optimizing conditions for and developing various new functional assays. For example, altering the components that comprise the DC maturation cocktail may improve the IL12p70 secretion assay. While the inventors currently use a cocktail of 6 different cytokines for maturation, the addition of LPS and/or IFNγ may be required in order to stimulate IL12p70 production from blast-derived DCs. Similarly, the synthetic compound, S-28463 may help increase both IL12p70 production and HLA-DR expression.
- Various embodiments of the present invention provide for dendritic cells generated by the methods of the present invention. In various embodiments, the DCs are provided in a pharmaceutically acceptable composition comprising a quantity of the DCs generated by the methods of the present invention.
- In various embodiments of the present invention, the hemangioblast may be obtained by a method comprising: providing hESCs; culturing the hESCs in media comprising cytokines to generate embryoid bodies (EBs); disaggregating the EBs: filtering individual cells; seeding the individual cells into methylcellulose comprising TPO, VEGF, FL, and bFGF; and harvesting the blast-like cells from methylcellulose.
- In various embodiments the hESCs are first cultured for 4 days before disaggregating the EBs. In various embodiments, the cytokines to generate embryoid bodies comprise VEGF and BMP4. In various embodiments, VEGF and BMP4 are used throughout the EB formation. In another embodiment, the cytokines to generate embryoid bodies further comprise bFGF. In one embodiment, the bFGF is added after the first 2 days of culturing the hESCs. In various embodiments, disaggregation of the EBs comprises disaggregating the EBs with trypsin and then inactivating the trypsin with serum-containing media. In one embodiment, the trypsin is 0.05%. In various embodiments, filtering the individual cells comprise filtering the individual cells through a 40 μM cell strainer. In various embodiments, the methylcellulose is H4436 or H4536 methylcellulose. In various embodiments the concentration of cytokines are TPO (50 μg/ml), VEGF (50 μg/ml), FL (50 μg/ml), and bFGF (20-50 μg/ml). In various embodiments, the blast-like cells are harvested from methylcellulose between day 6 and
day 10. - In particular embodiments, such as preparing hemangioblasts for use in generating NK cells, the methylcellulose may comprise additional cytokines. These additional cytokines are selected from the group consisting of IL2, IL7, IL15 and combinations thereof. In various embodiments, the concentrations of these cytokines are IL2 (1-10 μg/ml), IL7 (1-20 μg/ml), and IL15 (1-10 μg/ml). In various embodiments, methylcellulose cultures are plated at a concentration of 50,000 to 150,000 cells/ml.
- In a particular embodiment, the hemangioblasts are generated in an erythropoietin free methylcellulose. In one embodiment the erythropoietin free methylcellulose is H4536 methylcellulose.
- In alternate embodiments, pluripotent stem cells (including iPS cells and human iPS cells) are used in place of hESCs. In other embodiments, the hemangioblasts may be non-engrafting hemangioblasts.
- International Application Nos. PCT/US09/43050 and PCT/US09/43043 both filed May 6, 2009 and herein incorporated by reference as though fully set forth in their entirety, provide additional guidance on the generation of hemangioblasts and non-engrafting hemangioblasts.
- In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of the natural kill cells or dendritic cells of the present invention. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
- In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.
- “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
- The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
- The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
- The initial differentiation procedure for both cell types is the same and involves a 4 day culture of hESCs in Stemline II (Sigma) plus cytokines in order to generate embryoid bodies (EBs). The cytokines, VEGF and BMP4 are used throughout the EB culture while bFGF is added after the first 2 days. After 4 days total, the resulting EBs are disaggregated with 0.05% trypsin and then the trypsin is inactivated with serum-containing media. Individual cells are subsequently filtered through a 40 μM cell strainer, counted, and seeded into H4436 or H4536 methylcellulose (Stem Cell Technologies) containing additional cytokines, such as TPO (50 μg/ml), VEGF (50 μg/ml), FL (50 μg/ml) and bFGF (20-50 μg/ml). For NK differentiation, the cytokines IL2 (1-10 μg/ml), IL7 (1-20 μg/ml), and/or IL15 (1-10 μg/ml) may also be added at this stage. Methylcellulose cultures are plated at a concentration of 50,000 to 150,000 cells per ml for the production and expansion of a hemangioblastic population. Blast-like cells are harvested from methylcellulose between
day 6 and 10 and further differentiated by one of the following procedures. - Blast cells may be replated in H4236 methylcellulose plus IL2 (5-10 ng/ml), IL3 (1-10 ng/ml), IL6 (1-10 ng/ml), IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml) or in liquid culture containing the same cytokines and 10-20% human serum. After 6-8 days culture, cells are harvested and replated in liquid media (αMEM or DMEM:F12) plus 10-20% human serum and the cytokines IL7 (5-20 ng/ml), IL15 (5-10 ng/ml), SCF (10-50 ng/ml), and FL (10-50 ng/ml) for an additional 14-21 days. Weekly media changes are used to refresh the cytokine cocktail.
- Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of cells and the acquisition of NK cell surface markers. Cell surface markers include CD34, CD45, CD56, CD16, CD94, NKG2D, CD3, CD7, CD4, CD8a, and CD45RA. Tests to examine the function of hemangioblast-derived NK cells include (1) natural cytotoxicity assay using K562 erythroleukemia target cells, (2) IFNγ production in response to IL12/IL18 or phorbol myristate acetate treatment, (3) intracelluar flow cytometry for presence of perforin and granzyme B enzymes, and (4) antibody-dependent cellular cytotoxicity assay using Raji cells and anti-CD20 antibodies.
- Thus far, the inventors have been able to generate NK cells from both H7 and HuES3 hESCs. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that our hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture.
- Blast cells are plated in liquid media (αMEM or DMEM:F12) plus 10-20% human serum and the cytokines, SCF (20-100 ng/ml), FL (10-50 ng/ml), IL3 (5-50 ng/ml), and GM-CSF (50-100 ng/ml). After 7-11 days culture. IL4 (50-100 ng/ml) is also added to the culture and cells are allowed to grow for as additional 8-10 days. Media changes are performed every 6-7 days. An additional cytokine cocktail (10 ng/ml IL1b, 10 ng/ml TNFγ, 150 ng/ml IL6) can be added to the culture for 48 hours in order to induce maturation of DCs.
- Flow cytometry is used intermittently throughout the differentiation procedure to assess the immunophenotype of cells and the acquisition of DC surface markers. Cell surface markers include CD11c, CD209 (DC-SIGN), HLA ABC (MHC class I), HLA DR (MHC class II), CD1a, and CD14. Maturation is assessed by the acquisition of T-cell co-stimulatory receptor, CD83 while the expression of CD209, HLA DR, and CD11c may also increase upon maturation. Additional cytokines such as 1 μg/ml PGE2, 3000 units/ml IFNα2b, 20 μg/ml poly I:C, or
IFNγ 20 ng/ml may be added to the maturation cocktail to enhance DC functional response in certain assays. Assays to address the functionality of hemangioblast-derived DCs include (1) antigen uptake via DQ-ovalbumin (BD Biosciences) processing, (2) transwell migration in response to the chemoattractant MIP-3b, (3) allogenic mixed lymphocyte reaction assay to determine the ability of DCs to increase proliferation of HLA-mismatched T cells, (4) IL12-p70 secretion upon DC stimulation, and (5) antigen presenting assay to determine if antigen-loaded DCs can induce IFNγ production in previously antigen-primed peripheral blood mononuclear cells. - The inventors have been able to generate DCs from both HuES3 and MA01 hESCs. Upregulation of CD83, HLA-DR, CD11c, and CD209 in response to the 48 hour maturation cytokine cocktail was observed. These DCs are found to be able to uptake and proteolyse DQ-ovalbumin in a 30 minute assay.
- The above NK and DC differentiation procedures were performed using hemangioblasts grown in H4436 methylcellulose. However, H4536, an erythropoietin-free methylcellulose from Stem Cell Technologies can also be used to efficiently generate hemangioblasts. These “epo minus” hemangioblasts are quite similar to the original “epo plus” blasts; they are capable of differentiating into a variety of hematopoietic and vascular cell types. Preliminary results suggest that the use of H4536 may provide a significant advantage over H4436 methylcellulose for the differentiation of hemangioblasts into various hematopoietic lineages, including NK cells and DCs. The absence of epo in the blast growth media has been found to reduce the percentage of cells expressing the erythrocyte marker CD235a and increase the percentage of cells expressing CD34, CD45, and CD41a. Due to this difference in cell surface marker expression, “epo-minus” growth conditions may enhance differentiation down myeloid and/or lymphoid lineages.
- Using OP9 co-culture system, the inventors show that MKs can be generated from iPS cells. A few thousand CD41a+ MKs were generated from a few hundred thousand iPS cells.
- Differentiation procedure was performed as follows:
- H7 ESCs were differentiated into embryoid bodies (EBs) for 4 days. EBs were harvested and transferred to cytokine-rich methylcellulose for 10-15 days for hemangioblast production and expansion. Hemangioblasts were harvested and placed into feeder-free liquid culture medium plus 10-20% human AB serum, with a panel of cytokines for an additional 14-17 days with media half changes every 3-4 days.
- Immunophenotyping (Using Flow Cytometry):
- Immature NK cells were CD56bright, CD16lo, KIRlo, CD117+, CD94−, NKG2D+. By using a variation of the above procedure, 20-30% of the viable cells after 32 days of differentiation were CD56+ CD16−. Mature NK cells were CD56dim, CD16hi, KIRhi, CD117lo/−, CD94+, NKG2D+. By using the above procedure, 20% of the viable cells after 31 days of differentiation were CB56−CD16+ and 5% of them were CD56loCD16+.
- Functional Assays:
- Natural cytotoxicity: mature NK cells can elicit apoptosis of target cells such as human K562 erythroleukemia, MCF7, U87, PC3, NTERA2 cells. A “3FC” assay is used to assess efficiency of cytotoxicity. It is similar to 51Cr release assay but does not require radioactivity. See Derby et al., Immunol. Letters 78: 35-39 (2001). The heterogeneous population of mature NK cells described above (item B2-a) was found to elicit apoptosis in 65-70% of K562 cells in a standard 4 hour experiment.
- Antibody-dependent cell-mediated cytotoxicity (ADCC): The FcyRIII (CD16) on the NK cell surface binds to the fc region of anti-CD20 antibodies attached to target cells and induces ADCC. Raji cells (derived from Burkett's lymphoma) are preincubated with anti-CD20 antibody and used as targets in ADCC assay. (Tsirigotis et al., J of Steroid Biochem and Mol Bio 108: 267-271 (2008)).
- IFNγ cytokine production: Immature NK cells produce large amounts of IFNγ in response to overnight treatment with PMA (phorbol myristate acetate) plus ionomycin or IL12 plus IL18. IFNγ secretion is blocked with brefeldin a, cells are stained for cell surface markers and IFNγ using intracellular flow cytometry. See Woll et al. J. of Immunol 175: 5095-5103 (2005).
- In vivo immunotherapy potential of NK cells using xenograft mouse model: Bioluminescent (luciferase-containing) K562 cells are injected into NOD/SCID mice for engraftment of tumors, followed by bolus of NK cells and daily IP injections of IL2 and IL15. Bioluminescence imaging is used to monitor in vivo NK immunotherapeutic potential over time. See: Wollet al. Blood 113 (24): 6094-6101 (2009).
- By using hemangioblasts as bone-marrow-repopulating cells or by differentiating them into dendritic, natural killer, T cells, and/or mesenchymal stem cells (MSCs), we can produce large-scale, effective cell-based therapies to combat cancer, HIV, and/or automimmune diseases. The inventors were able to achieve differentiation of dendritic cells (DCs) from both hESCs and iPS cells with 40-55% efficiency. A side by side comparison to DCs derived from human bone marrow, in addition to two new functional assays, has now confirmed that the hESC-derived DCs share many comparable features with human BM-derived DCs and also identified areas that need further optimization. For natural killer cell differentiation, a side by side comparison to human bone marrow-derived NK cells has confirmed that in vitro NK cell differentiation is not very efficient, even when using a bone marrow cell source. The inventors have found that blast-derived NK cells display natural cytotoxicity capabilities. Antibody-dependent cellular cytotoxicity assays are performed. For T cell differentiation, the inventors have successfully created a human delta-ligand expressing OP9 stroma cell line to stimulate Notch signaling and are using this stroma cell line to stimulate T cell differentiation of hemangioblasts.
- Hemangioblast-Derived Dendritic Cells (40-55% Efficiency)
- Cell surface markers: CD11c, 45, 209 show comparable expression on blastderived DCs and human bone marrow-derived DCs, while HLA-DR is expressed at much lower levels on blast DCs than on human BM DCs.
- Functional assays (previously reported on antigen, uptake and migration assays). IL12-p70 secretion: IL12p70 is secreted by mature DCs in order to elicit a Th1-directed response from CD4+ T cells. Human BM-derived DCs can produce >500 pg/ml of IL12p70 yet blast-derived DCs did not produce any detectable IL12p70 upon maturation. Mixed lymphocyte reaction (MLR) assay: The MLR assay determines the ability of DCs to stimulate proliferation of allogenic T cells. Cord blood mononuclear cells (CBMCs, which include T cells) were used as responders, fluorescently labeled, and their proliferation was measured after 4-5 days coculture with immature or mature blast-derived DCs. Preliminary results show that the responder cells proliferate in response to mature (m)DCs.
- Hemangioblast-derived natural killer cells: Cell surface markers CD45, CD7, CD94, CD56, CD16, and NKG2D were evaluated in blast-derived and human bone marrow-derived NK cells.
- NK differentiation efficiency: Recombinant human Sox7 protein was being investigated for its ability to increase the pool of CD34+ starting progenitors for NK differentiation. Results suggest that rhSox7 does not dramatically affect % of CD34+ cells. Murine AFT024 was investigated as a stroma feeder layer that may provide critical cell-cell contacts and secreted factors for NK cell differentiation. As indicated by cell surface marker expression, AFT024 stroma co-culture did not enhance NK differentiation of either human BM or blasts.
- Hemangioblast-derived T cells: Notch signaling is crucial for T cell differentiation. As such, the cDNA for human delta-like ligand 1 (hDLL1) was cloned into an MSCV-ires-GFP based retroviral vector and used the resulting viral supernatant to infect OP9 stroma cells. Upon viral integration, OP9 cells will express hDLL1 on their cell surface and be gfp positive. FACS-based sorting was used to purify the highest gfp-positive cells from the heterogeneous pool of infected OP9 cells. These OP9-hDL1S (“S” for sorted) were expanded and characterized. Q-RT-PCR, immunofluorescence, and flow cytometry all confirm expression of the hDLL1 protein in these cells.
- Both cord blood and peripheral blood mononuclear cells are used as responders and the inventors use human bone marrow-derived DCs as positive control effectors.
- E4BP4 has been shown to be critical for NK lineage development (see Gascoyne et al. Nature Immunology 10(10): 1118-1125, 2009) and may provide the transcriptional program necessary for more efficient in vitro.
- NK cell differentiation. The inventors clone E4BP4 cDNA into a retroviral vector for its overexpression in hemangioblasts and evaluate its ability to increase NK differentiation. RT-PCR is used to monitor the expression of various KIR receptor isoforms and the enzymes, perforin and granzyme B, which are critical for NK cell functionality. For functional assays, the inventors have shown that blast-derived NK cells display natural cytotoxicity, so their antibody-dependent cellular cytotoxicity (ADCC) capabilities are assessed. Required reagents for the ADCC assay include the Burkitt's lymphoma-derived Raji cells and anti-CD20 antibodies. Co-culture of CD20-marked Raji cells with NK cells should elicit a specific ADCC response, which will be monitored through flow cytometric means.
-
- 1. Woll, P, et al. J. of Immunology. 175: 5095-5103 (2005).
- 2. Woll, P et al. Blood 113(24): 6094-101 (2009).
- 3. Bordoni et al. Hepatology 39: 1508-1516 (2004).
- 4. Tabatoabaei-Zavareh et al. PLOS One
Issue 2, e232 (2007). - 5. McCullar, V et al. Exp. Hematology 36(5): 598-608 (2008).
- 6. Freud, A G et al. Immunity 22:295-304 (2005).
- 7. Yu, H et al. Blood 92 (10): 3647-3657 (1998).
-
- 1. Su et al. Clinical Cancer Research 14(19): 6207-6217 (2008).
- 2. Tseng et al. Regenerative Medicine 4(4): 513-526 (2009).
- 3. Bandi et a. AIDS Research and Therapy 5:1 (2008). (open-access)
- 4. Slukvin, II et al. J of Immunology 176: 2924-2932 (2006).
- Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
- The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
- While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term, “including” should be interpreted as “including but not limited to,” the term, “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
Claims (38)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/465,533 US20150140657A1 (en) | 2009-12-04 | 2014-08-21 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
US16/669,931 US20200131475A1 (en) | 2009-12-04 | 2019-10-31 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US26666109P | 2009-12-04 | 2009-12-04 | |
PCT/US2010/058593 WO2011068896A1 (en) | 2009-12-04 | 2010-12-01 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
US201213512795A | 2012-07-18 | 2012-07-18 | |
US14/465,533 US20150140657A1 (en) | 2009-12-04 | 2014-08-21 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2010/058593 Continuation WO2011068896A1 (en) | 2009-12-04 | 2010-12-01 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
US13/512,795 Continuation US8822218B2 (en) | 2009-12-04 | 2010-12-01 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/669,931 Continuation US20200131475A1 (en) | 2009-12-04 | 2019-10-31 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Publications (1)
Publication Number | Publication Date |
---|---|
US20150140657A1 true US20150140657A1 (en) | 2015-05-21 |
Family
ID=44115272
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/512,795 Active US8822218B2 (en) | 2009-12-04 | 2010-12-01 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
US14/465,533 Abandoned US20150140657A1 (en) | 2009-12-04 | 2014-08-21 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
US16/669,931 Pending US20200131475A1 (en) | 2009-12-04 | 2019-10-31 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/512,795 Active US8822218B2 (en) | 2009-12-04 | 2010-12-01 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/669,931 Pending US20200131475A1 (en) | 2009-12-04 | 2019-10-31 | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
Country Status (11)
Country | Link |
---|---|
US (3) | US8822218B2 (en) |
EP (1) | EP2507365A4 (en) |
JP (4) | JP6013187B2 (en) |
KR (4) | KR101947588B1 (en) |
CN (1) | CN102822332A (en) |
AU (1) | AU2010326027A1 (en) |
BR (1) | BR112012014104A2 (en) |
CA (3) | CA2781969A1 (en) |
IN (1) | IN2012DN05053A (en) |
RU (1) | RU2012127811A (en) |
WO (1) | WO2011068896A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10894065B2 (en) | 2012-12-21 | 2021-01-19 | Astellas Institute For Regenerative Medicine | Methods for production of platelets from pluripotent stem cells and compositions thereof |
US11566228B2 (en) | 2006-04-14 | 2023-01-31 | Astellas Institute For Regenerative Medicine | Hemangio-colony forming cells |
Families Citing this family (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8822218B2 (en) | 2009-12-04 | 2014-09-02 | Stem Cell & Regenerative Medicine International, Inc. | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
EP2841563B1 (en) * | 2012-04-24 | 2019-06-12 | Dan S. Kaufman | Method for developing natural killer cells from stem cells |
JP6588465B2 (en) * | 2014-01-03 | 2019-10-09 | アカデミア シニカAcademia Sinica | Modified natural killer cells, compositions and uses thereof |
CN105219710B (en) * | 2014-06-05 | 2020-01-10 | 上海厚超生物科技有限公司 | Method for culturing immune cell population with high killing activity |
DE102014014993B4 (en) * | 2014-10-09 | 2017-02-02 | Helga Schmetzer | Use of immunomodulatory effective kits for the immunotherapeutic treatment of patients with myeloid leukemia |
CN104711225B (en) * | 2015-04-09 | 2018-05-22 | 奥思达干细胞有限公司 | The external preparation method of NK cells |
CN108697641A (en) | 2015-08-18 | 2018-10-23 | 安斯泰来再生医药协会 | Clinical preparation |
CN105219722A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating kidney specific immune response |
CN105219727A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating colorectal cancer specific immune response |
CN105219717A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | An a kind of type polarization dendritic cell and induction method thereof and application |
CN105219716A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating incidence cancer specific immune response |
CN105219728A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of for activating the immunoreactive test kit of Breast Cancer-Specific |
CN105219718A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of for activating the immunoreactive test kit of prostatic cancer specific |
CN105219726A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of method of efficient preparation one type polarization dendritic cell and application thereof |
CN105219723A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating cancer of the stomach specific immune response |
CN105219719A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating ovarian cancer specific immune response |
CN105219725A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating sarcoma specific immune response |
GB201810486D0 (en) * | 2018-06-26 | 2018-08-08 | Imperial Innovations Ltd | Natural killer cells |
US11453862B2 (en) | 2019-07-08 | 2022-09-27 | Immunitybio, Inc. | Mononuclear cell derived NK cells |
JP7213976B2 (en) | 2019-07-08 | 2023-01-27 | イミュニティーバイオ、インコーポレイテッド | NK cells derived from mononuclear cells |
US11547726B2 (en) * | 2020-03-26 | 2023-01-10 | Honed Life Sciences, Llc | Enhancement of production of NK cells from stem cells |
US11591381B2 (en) | 2020-11-30 | 2023-02-28 | Crispr Therapeutics Ag | Gene-edited natural killer cells |
KR102292843B1 (en) * | 2020-12-29 | 2021-08-25 | 주식회사 온코인사이트 | Induced pluripotent stem cell(iPSC) derived natural killer cell and its use |
EP4271798A1 (en) | 2020-12-30 | 2023-11-08 | CRISPR Therapeutics AG | Compositions and methods for differentiating stem cells into nk cells |
CN115247149B (en) * | 2022-08-22 | 2023-06-16 | 华域生物科技(天津)有限公司 | Culture medium composition suitable for NK cells and culture method |
WO2024047368A1 (en) * | 2022-09-02 | 2024-03-07 | Imperial College Innovations Limited | Natural killer cells |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6479286B1 (en) * | 1997-05-21 | 2002-11-12 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Methods and compositions for making dendritic cells from expanded populations of monocytes and for activating T cells |
US20050032210A1 (en) * | 2003-03-18 | 2005-02-10 | Kirin Beer Kabushiki Kaisha | Method of preparing immuno-regulatory dendritic cells and the use thereof |
US20050042751A1 (en) * | 2001-10-31 | 2005-02-24 | Michel Goldman | Generation and use of new types of dendritic cells |
US20080014183A1 (en) * | 2004-06-24 | 2008-01-17 | Dnavec Research Inc. | Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto |
Family Cites Families (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69530980T2 (en) * | 1994-06-15 | 2004-05-19 | Systemix, Inc., Palo Alto | POPULATION OF CELLS ENRICHED WITH MYELOID AND / OR LYMPHOID PRECURSORS AND METHOD FOR THEIR RECOVERY AND USE |
CA2278847A1 (en) * | 1997-01-31 | 1998-08-06 | Hemosol Inc. | Method for the production of selected lymphocytes |
EP1034288A4 (en) * | 1997-12-04 | 2002-11-27 | Univ Duke | Methods of isolating and using cd7+cd34-lin-hematopoietic cells |
FR2801056B1 (en) * | 1999-11-12 | 2003-03-28 | Commissariat Energie Atomique | PROTEIN PRESENT ON THE SURFACE OF HEMATOPOIETIC STEM CELLS OF THE LYMPHOID LINE AND NK CELLS, AND ITS APPLICATIONS |
KR20020083634A (en) * | 2001-04-27 | 2002-11-04 | 크레아젠 주식회사 | Pharmaceutical Compositions Comprising Dendritic Cells for Immunotherapy of Autoimmune Disease and Treatment Method Using the Same |
WO2004050855A2 (en) * | 2002-12-04 | 2004-06-17 | Baylor Research Institute | Rapid one-step method for generation of antigen loaded dendritic cell vaccine from precursors |
CA2521217C (en) * | 2003-04-01 | 2013-05-14 | United States Of America Department Of Veteran's Affairs | Stem-cell, precursor cell, or target cell-based treatment of multi-organ failure and renal dysfunction |
AU2004291559B2 (en) * | 2003-11-19 | 2008-10-16 | Australian Stem Cell Centre Limited | Methods for producing blood products from pluripotent cells in cell culture |
WO2005085426A1 (en) * | 2004-03-04 | 2005-09-15 | Tanabe Seiyaku Co., Ltd. | Medium for feeder-free differentiation and feeder-free differentiation method from primate embryonic stem cell |
DK1809737T3 (en) * | 2004-10-07 | 2011-03-07 | Argos Therapeutics Inc | Compositions with mature dendrite cells and methods for growing them |
US20080166751A1 (en) * | 2005-02-23 | 2008-07-10 | Takayuki Asahara | Method of Analyzing Dynamics in the Differentiation of Vascular Endothelial Progenitor Cells |
USRE46152E1 (en) * | 2005-04-08 | 2016-09-20 | Argos Therapeutics, Inc. | Dendritic cell compositions and methods |
US7811821B2 (en) * | 2005-06-01 | 2010-10-12 | Wisconsin Alumni Research Foundation | Method of forming dendritic cells from embryonic stem cells |
DK1941027T3 (en) * | 2005-09-28 | 2014-10-13 | Ipd Therapeutics B V | PROCEDURES AND AGENTS FOR STEM CELL INCREASE AND SUBSEQUENT GENERATION AND EXPANSION OF PROGENITOR CELLS AND PRODUCTION OF EFFECT CELLS AS CLINICAL THERAPEUTICS |
CN101528915A (en) | 2006-04-14 | 2009-09-09 | 先进细胞技术公司 | Hemangio-colony forming cells |
WO2008038279A2 (en) * | 2006-09-25 | 2008-04-03 | Kfir Nissan | Force transducer and method |
JP5067949B2 (en) | 2006-11-09 | 2012-11-07 | 独立行政法人国立国際医療研究センター | Method for culturing and subculture of primate embryonic stem cells and method for inducing differentiation thereof |
DE102006054041B3 (en) * | 2006-11-16 | 2008-05-08 | Pierburg Gmbh | Regulating device for an internal combustion engine |
ES2530995T3 (en) * | 2007-09-28 | 2015-03-09 | Anthrogenesis Corp | Tumor suppression using human placental perfusate and intermediate natural killer cells that come from human placenta |
WO2009120891A2 (en) * | 2008-03-27 | 2009-10-01 | Geron Corporation | Differentiation of primate pluripotent stem cells to hematopoietic lineage cells |
KR20170102048A (en) | 2008-05-06 | 2017-09-06 | 아스텔라스 인스티튜트 포 리제너러티브 메디슨 | Methods for producing enucleated erythroid cells derived from pluripotent stem cells |
US9410123B2 (en) | 2008-05-06 | 2016-08-09 | Ocata Therapeutics, Inc. | Hemangio colony forming cells and non-engrafting hemangio cells |
CA2753679C (en) * | 2009-02-27 | 2021-03-02 | Cellular Dynamics International, Inc. | Differentiation of pluripotent cells |
US8822218B2 (en) | 2009-12-04 | 2014-09-02 | Stem Cell & Regenerative Medicine International, Inc. | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts |
-
2010
- 2010-12-01 US US13/512,795 patent/US8822218B2/en active Active
- 2010-12-01 KR KR1020127017370A patent/KR101947588B1/en active IP Right Grant
- 2010-12-01 RU RU2012127811/10A patent/RU2012127811A/en not_active Application Discontinuation
- 2010-12-01 IN IN5053DEN2012 patent/IN2012DN05053A/en unknown
- 2010-12-01 KR KR1020197003655A patent/KR20190016602A/en not_active Application Discontinuation
- 2010-12-01 EP EP10835074.5A patent/EP2507365A4/en not_active Withdrawn
- 2010-12-01 KR KR1020207018660A patent/KR20200083645A/en not_active IP Right Cessation
- 2010-12-01 CA CA2781969A patent/CA2781969A1/en not_active Abandoned
- 2010-12-01 WO PCT/US2010/058593 patent/WO2011068896A1/en active Application Filing
- 2010-12-01 KR KR1020217030824A patent/KR20210120136A/en not_active Application Discontinuation
- 2010-12-01 CA CA3080368A patent/CA3080368A1/en not_active Abandoned
- 2010-12-01 AU AU2010326027A patent/AU2010326027A1/en not_active Abandoned
- 2010-12-01 CA CA3095576A patent/CA3095576A1/en active Pending
- 2010-12-01 BR BR112012014104A patent/BR112012014104A2/en not_active IP Right Cessation
- 2010-12-01 JP JP2012542163A patent/JP6013187B2/en active Active
- 2010-12-01 CN CN2010800629687A patent/CN102822332A/en active Pending
-
2014
- 2014-08-21 US US14/465,533 patent/US20150140657A1/en not_active Abandoned
-
2016
- 2016-03-24 JP JP2016060101A patent/JP2016119910A/en active Pending
-
2019
- 2019-02-12 JP JP2019022273A patent/JP7343984B2/en active Active
- 2019-10-31 US US16/669,931 patent/US20200131475A1/en active Pending
-
2021
- 2021-11-19 JP JP2021188673A patent/JP2022027790A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6479286B1 (en) * | 1997-05-21 | 2002-11-12 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Methods and compositions for making dendritic cells from expanded populations of monocytes and for activating T cells |
US20050042751A1 (en) * | 2001-10-31 | 2005-02-24 | Michel Goldman | Generation and use of new types of dendritic cells |
US20050032210A1 (en) * | 2003-03-18 | 2005-02-10 | Kirin Beer Kabushiki Kaisha | Method of preparing immuno-regulatory dendritic cells and the use thereof |
US20080014183A1 (en) * | 2004-06-24 | 2008-01-17 | Dnavec Research Inc. | Anticancer Agent Containing Dendritic Cell Having Rna Virus Transferred Thereinto |
Non-Patent Citations (2)
Title |
---|
Fedele et al. (CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction, Eur. J. Immunol. 2004. 34: 1342-1350) * |
Lechman et al. (CD83 on dendritic cells: more than just a marker for maturation. TRENDS in Immunology Vol.23 No.6 June 2002. Pages 1-3) * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11566228B2 (en) | 2006-04-14 | 2023-01-31 | Astellas Institute For Regenerative Medicine | Hemangio-colony forming cells |
US10894065B2 (en) | 2012-12-21 | 2021-01-19 | Astellas Institute For Regenerative Medicine | Methods for production of platelets from pluripotent stem cells and compositions thereof |
US11400118B2 (en) | 2012-12-21 | 2022-08-02 | Astellas Institute For Regenerative Medicine | Methods for production of platelets from pluripotent stem cells and compositions thereof |
Also Published As
Publication number | Publication date |
---|---|
JP2013512673A (en) | 2013-04-18 |
US20120282693A1 (en) | 2012-11-08 |
KR20200083645A (en) | 2020-07-08 |
BR112012014104A2 (en) | 2016-07-05 |
EP2507365A4 (en) | 2014-01-01 |
US20200131475A1 (en) | 2020-04-30 |
KR20190016602A (en) | 2019-02-18 |
WO2011068896A1 (en) | 2011-06-09 |
RU2012127811A (en) | 2014-01-10 |
CA3095576A1 (en) | 2011-06-09 |
JP2019083819A (en) | 2019-06-06 |
JP2016119910A (en) | 2016-07-07 |
CA3080368A1 (en) | 2011-06-09 |
JP2022027790A (en) | 2022-02-14 |
JP7343984B2 (en) | 2023-09-13 |
AU2010326027A1 (en) | 2012-06-21 |
EP2507365A1 (en) | 2012-10-10 |
JP6013187B2 (en) | 2016-10-25 |
IN2012DN05053A (en) | 2015-10-09 |
US8822218B2 (en) | 2014-09-02 |
CA2781969A1 (en) | 2011-06-09 |
CN102822332A (en) | 2012-12-12 |
KR101947588B1 (en) | 2019-02-14 |
KR20210120136A (en) | 2021-10-06 |
KR20120112511A (en) | 2012-10-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200131475A1 (en) | Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts | |
AU2020264375B2 (en) | Method for developing natural killer cells from stem cells | |
US9988602B2 (en) | Methods for producing enucleated erythroid cells derived from pluripotent stem cells | |
US9399758B2 (en) | SSEA3(+) pluripotent stem cell that can be isolated from body tissue | |
US20050221482A1 (en) | Methods and compositions for obtaining hematopoietic stem cells derived from embryonic stem cells and uses thereof | |
JP2024026891A (en) | Methods and systems for producing hematopoietic lineage cells | |
D'Souza et al. | GSK3β inhibition promotes efficient myeloid and lymphoid hematopoiesis from non-human primate-induced pluripotent stem cells | |
O'Brien | Induced Human Pluripotent Stem Cell–derived NK Cells as an Alternative Source of Lymphocytes for Anti-Cancer Immunotherapy | |
Thomson et al. | Saritha S. D’Souza, John Maufort, Akhilesh Kumar, Jiuchun Zhang, 2 Kimberley Smuga-Otto, 2 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
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: FINAL REJECTION MAILED |
|
AS | Assignment |
Owner name: ASTELLAS INSTITUTE FOR REGENERATIVE MEDICINE, MASS Free format text: CHANGE OF NAME;ASSIGNOR:STEM CELL & REGENERATIVE MEDICINE INTERNATIONAL, INC.;REEL/FRAME:049861/0416 Effective date: 20190719 |
|
AS | Assignment |
Owner name: STEM CELL & REGENERATIVE MEDICINE INTERNATIONAL, I Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIMBREL, ERIN;LU, SHI-JIANG;REEL/FRAME:049913/0721 Effective date: 20120703 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: ADVISORY ACTION MAILED |
|
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 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |