CN117018033A - Biological agent containing myocardial precursor-like cells, and preparation method and application thereof - Google Patents
Biological agent containing myocardial precursor-like cells, and preparation method and application thereof Download PDFInfo
- Publication number
- CN117018033A CN117018033A CN202310522477.5A CN202310522477A CN117018033A CN 117018033 A CN117018033 A CN 117018033A CN 202310522477 A CN202310522477 A CN 202310522477A CN 117018033 A CN117018033 A CN 117018033A
- Authority
- CN
- China
- Prior art keywords
- cells
- precursor
- myocardial
- cell
- cardiomyocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000002107 myocardial effect Effects 0.000 title claims abstract description 192
- 239000003124 biologic agent Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 22
- 108010014608 Proto-Oncogene Proteins c-kit Proteins 0.000 claims abstract description 22
- 102000016971 Proto-Oncogene Proteins c-kit Human genes 0.000 claims abstract description 21
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 claims abstract description 15
- 102100038081 Signal transducer CD24 Human genes 0.000 claims abstract description 15
- 108091054438 MHC class II family Proteins 0.000 claims abstract description 13
- 102000043131 MHC class II family Human genes 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 303
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 115
- 239000001963 growth medium Substances 0.000 claims description 43
- 230000008672 reprogramming Effects 0.000 claims description 34
- 239000003112 inhibitor Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 26
- 235000015872 dietary supplement Nutrition 0.000 claims description 20
- 230000001079 digestive effect Effects 0.000 claims description 17
- 239000003102 growth factor Substances 0.000 claims description 16
- 230000029087 digestion Effects 0.000 claims description 15
- 239000000556 agonist Substances 0.000 claims description 14
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 14
- 229940043355 kinase inhibitor Drugs 0.000 claims description 14
- 210000004165 myocardium Anatomy 0.000 claims description 14
- 239000003757 phosphotransferase inhibitor Substances 0.000 claims description 14
- 239000011435 rock Substances 0.000 claims description 14
- 229940124647 MEK inhibitor Drugs 0.000 claims description 13
- 108010019160 Pancreatin Proteins 0.000 claims description 13
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 claims description 13
- 229940055695 pancreatin Drugs 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 229940088597 hormone Drugs 0.000 claims description 12
- 239000005556 hormone Substances 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 230000004156 Wnt signaling pathway Effects 0.000 claims description 10
- 239000007640 basal medium Substances 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 230000011664 signaling Effects 0.000 claims description 10
- 238000010171 animal model Methods 0.000 claims description 8
- 230000032459 dedifferentiation Effects 0.000 claims description 8
- 230000035755 proliferation Effects 0.000 claims description 8
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 7
- 230000003321 amplification Effects 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000001727 in vivo Methods 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 3
- 208000037891 myocardial injury Diseases 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims 5
- 238000011316 allogeneic transplantation Methods 0.000 abstract description 7
- 230000004069 differentiation Effects 0.000 description 34
- 238000003556 assay Methods 0.000 description 24
- 239000000872 buffer Substances 0.000 description 23
- 238000010586 diagram Methods 0.000 description 23
- 239000000203 mixture Substances 0.000 description 23
- 239000002609 medium Substances 0.000 description 17
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 16
- 238000005119 centrifugation Methods 0.000 description 15
- 238000001514 detection method Methods 0.000 description 15
- 238000010186 staining Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 239000003153 chemical reaction reagent Substances 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 9
- 101710165323 Troponin T, cardiac muscle Proteins 0.000 description 9
- 239000008188 pellet Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 239000013049 sediment Substances 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 102100026893 Troponin T, cardiac muscle Human genes 0.000 description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 description 8
- 239000001569 carbon dioxide Substances 0.000 description 8
- 239000002243 precursor Substances 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 7
- 239000012091 fetal bovine serum Substances 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 239000012981 Hank's balanced salt solution Substances 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical group C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 4
- 208000009329 Graft vs Host Disease Diseases 0.000 description 4
- 108010058597 HLA-DR Antigens Proteins 0.000 description 4
- 102000006354 HLA-DR Antigens Human genes 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 102000013814 Wnt Human genes 0.000 description 4
- 108050003627 Wnt Proteins 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 208000024908 graft versus host disease Diseases 0.000 description 4
- 208000019622 heart disease Diseases 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 210000001704 mesoblast Anatomy 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 238000010212 intracellular staining Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 101710197650 Actin-7 Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- QWOJMRHUQHTCJG-UHFFFAOYSA-N CC([CH2-])=O Chemical compound CC([CH2-])=O QWOJMRHUQHTCJG-UHFFFAOYSA-N 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102100032352 Leukemia inhibitory factor Human genes 0.000 description 2
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010010378 HLA-DP Antigens Proteins 0.000 description 1
- 102000015789 HLA-DP Antigens Human genes 0.000 description 1
- 108010062347 HLA-DQ Antigens Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 210000004322 M2 macrophage Anatomy 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000003510 anti-fibrotic effect Effects 0.000 description 1
- 230000003409 anti-rejection Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000003690 classically activated macrophage Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002837 heart atrium Anatomy 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000001023 pro-angiogenic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval 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
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
-
- 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
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
-
- 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
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal 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
- A61K35/37—Digestive system
- A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
-
- 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
- A61K35/37—Digestive system
- A61K35/407—Liver; Hepatocytes
-
- 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
- A61K35/37—Digestive system
- A61K35/413—Gall bladder; Bile
-
- 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
- A61K35/42—Respiratory system, e.g. lungs, bronchi or lung 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
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
-
- 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
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Physiology (AREA)
- Genetics & Genomics (AREA)
- Nutrition Science (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pulmonology (AREA)
- General Engineering & Computer Science (AREA)
- Reproductive Health (AREA)
- Cardiology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Vascular Medicine (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
Abstract
The invention provides a biological agent containing myocardial precursor-like cells, a preparation method and application thereof, wherein the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules. The invention solves the problem that the myocardial precursor-like cells in the prior art have immune rejection after allogeneic transplantation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a biological agent containing myocardial precursor-like cells, and a preparation method and application thereof.
Background
Heart disease is a leading cause of death in humans, and at the time of heart attack, an adult human heart may lose up to 10 billion cardiomyocytes, and only less than 1% of adult cardiomyocytes can regenerate. For heart diseases such as myocardial necrosis, heart failure, etc., the biggest difficulty with conservative treatment is the inability to compensate for the missing functional myocardium.
From a regenerative medicine point of view, the lack of seed cell sources is an important factor limiting the use of cell therapies for the treatment of heart diseases, since mature cardiomyocytes cannot be expanded in vitro. The specific reasons for the lack of seed cells are two aspects, namely, the first aspect is that primary cardiomyocytes are difficult to obtain, even if donated hearts can be obtained, it is a very difficult matter to isolate primary cardiomyocytes, and the primary cardiomyocytes cannot be expanded in vitro and therefore cannot be used as seed cells for cell therapy; the second aspect is that recent studies have revealed that cardiac muscle precursor-like cells and cardiac muscle cells can be differentiated from pluripotent stem cells including embryonic stem cells (Embryonic Stem cell, ES) and induced pluripotent stem cells (Induced pluripotent stem cells, iPSCs), but this technical route is quite complex and has the following problems: the first, undifferentiated ES and undifferentiated ipscs may be tumorigenic during differentiation; secondly, the purity of the myocardial precursor-like cells and the myocardial cells obtained by differentiation of the ES and the iPSC is difficult to ensure, and the application of the myocardial precursor-like cells and the myocardial cells is limited; third, cardiomyocyte precursor-like cells obtained by differentiation of ES and iPSC cannot be expanded in large amounts, and thus, for each batch application, differentiation from scratch is required.
At present, myocardial precursor-like cells in the prior art can generate immune rejection after allogeneic transplantation, and the immune rejection can be solved by oral anti-rejection medicine. Accordingly, there is a need to provide a biological agent comprising myocardial precursor-like cells, and a preparation method and application thereof, to solve the above-mentioned problems in the prior art.
Disclosure of Invention
The invention aims to provide a biological agent containing myocardial precursor-like cells, and a preparation method and application thereof, so as to solve the problem that the myocardial precursor-like cells in the prior art have immune rejection after allogeneic transplantation.
To achieve the above object, the present invention provides a biological agent comprising a cardiac muscle precursor-like cell that positively expresses CD24 and c-Kit, the cardiac muscle precursor-like cell not expressing MHC class ii molecules.
The biological agent comprising myocardial precursor-like cells of the present invention has the beneficial effects that: through positive expression of CD24 and c-Kit by the myocardial precursor-like cells, the myocardial precursor-like cells do not express MHC class II molecules, so that the myocardial precursor-like cells can be well adapted to human bodies during allogeneic transplantation, and the problem that immune rejection occurs after allogeneic transplantation of the myocardial precursor-like cells in the prior art is solved.
The invention also provides a preparation method of the biological agent containing the myocardial precursor-like cells, which comprises the following steps:
s0: providing a cardiomyocyte precursor-like cell;
s1: mixing the myocardial precursor-like cells with a pharmaceutically acceptable carrier to obtain the biological agent containing the myocardial precursor-like cells, wherein the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules.
The preparation method of the biological agent containing myocardial precursor-like cells has the beneficial effects that: the preparation method of the biological agent containing the myocardial precursor-like cells is simple, and the biological agent containing the myocardial precursor-like cells can be well adapted to the human body after being returned to the human body, and the medicine is not needed to be used to avoid immune rejection caused by allograft.
Preferably, the preparation method of the myocardial precursor-like cells comprises the following steps:
s00: providing a mature cardiomyocyte;
s01: placing the mature myocardial cells into a reprogramming culture medium for performing dedifferentiation culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and performing digestion treatment on the myocardial precursor-like cells by using pancreatin digestive juice to obtain the myocardial precursor-like cells, wherein the reprogramming culture medium comprises a basal culture medium, a MEK inhibitor, an inhibitor and a protein hormone, and the reprogramming culture medium is used for inducing the myocardial precursor-like cells and promoting proliferation of the myocardial precursor-like cells. The beneficial effects are that: the MEK inhibitor is used for inducing myocardial precursor-like cells, and the inhibitor and the protein hormone are used for promoting proliferation of myocardial precursor-like cells.
Preferably, the preparation method of the myocardial precursor-like cells comprises the following steps: s02: and placing the myocardial precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and then using the pancreatin digestive juice to digest the myocardial precursor-like cells so as to obtain the passaged myocardial precursor-like cells. The beneficial effects are that: the MEK inhibitor is co-acting with growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements for induction of cardiomyocyte precursor-like cells; the inhibitor and the protein hormone act together with growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors and nutritional supplements to promote proliferation of the cardiomyocyte precursor-like cells, enabling a substantial expansion of the cardiomyocyte precursor-like cells in vitro.
Preferably, the reprogramming media further comprises growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements.
Preferably, the MEK inhibitor is present in an amount of 1-10. Mu.M, the inhibitor is present in an amount of 5-20ng/mL, and the protein hormone is present in an amount of 1-10. Mu.g/mL, based on the volume of the basal medium.
Preferably, the content of the growth factor is 50-80ng/mL, the content of the ROCK kinase inhibitor is 10-50 mu M, the content of the Wnt signal path agonist is 1-10 mu M, the content of the TGF-beta signal inhibitor is 1-10 mu M, and the content of the nutritional supplement is 1% -10% based on the volume of the basal medium.
Preferably, the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.
Preferably, the mature cardiomyocytes are obtained from myocardial tissue by digestion.
The invention also provides the application of the biological agent containing the myocardial precursor-like cells, and the biological agent containing the myocardial precursor-like cells is used for intervening in vivo animal models, wherein the animal models comprise drug-induced myocardial injury animal models.
The beneficial effects of the application of the biological agent containing myocardial precursor-like cells of the present invention are that: because the myocardial precursor-like cells do not express MHC class II molecules, the biological preparation containing the myocardial precursor-like cells does not need to take anti-expulsion drugs after being transplanted, and the injury to human bodies can be reduced.
Drawings
FIG. 1 is a schematic photograph showing the morphology of primary cardiomyocytes according to example 1 of the present invention;
FIG. 2 is a photograph showing the morphology of the third generation of mature cardiomyocytes according to example 1 of the present invention;
FIG. 3 is a schematic photograph showing the morphology of third generation myocardial precursor-like cells obtained by the culture of example 1 of the present invention;
FIG. 4 is a photograph showing the morphology of third generation control myocardial precursor-like cells obtained by the culture of example 1 of the present invention;
FIG. 5 is a graph showing the growth of cardiomyocyte precursor-like cells in reprogramming media and mature cardiomyocyte cells in cardiomyocyte media in accordance with example 1 of the present invention;
FIG. 6 is a schematic diagram showing the results of flow assay for positive expression of c-Kit by third generation cardiomyocyte precursor-like cells in example 1 of the present invention;
FIG. 7 is a schematic diagram showing the results of flow assay for positive expression of CD24 by third generation myocardial precursor-like cells in example 1 of the present invention;
FIG. 8 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by third generation cardiomyocyte precursor-like cells according to example 1 of the present invention;
FIG. 9 is a schematic diagram showing the cell morphology of the third generation cardiomyocyte precursor-like cells of example 1 of the present invention after 7 days of differentiation;
FIG. 10 is a schematic diagram showing the results of flow assay for positive expression of cTnT 7 days after differentiation of third generation cardiomyocyte precursor-like cells according to example 1 of the present invention;
FIG. 11 is a schematic diagram showing the results of flow assay for positive expression of α -Sarcometric actin 7 days after differentiation of third-generation cardiomyocyte precursor-like cells according to example 1 of the present invention;
FIG. 12 is a schematic diagram showing the results of flow assay for positive expression of cTnT by mature cardiomyocytes according to example 2 of the present invention;
FIG. 13 is a schematic diagram showing the results of flow assay for positive expression of c-kit by myocardial precursor-like cells in example 2 of the present invention;
FIG. 14 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by myocardial precursor-like cells in example 2 of the present invention;
FIG. 15 is a schematic diagram showing the results of flow assay for positive expression of c-kit by myocardial precursor-like cells in example 3 of the present invention;
FIG. 16 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by myocardial precursor-like cells in example 3 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.
In the prior art, there are methods for preparing mature cardiomyocytes and cardiomyocyte precursor-like cells, and the methods for preparing the mature cardiomyocytes comprise the following three methods: the first preparation method comprises the following steps: isolated from primary heart organ tissue, preferably new tissue, to obtain mature cardiomyocytes; the second preparation method comprises the following steps: the mature myocardial cells can be obtained by differentiating ES or iPSC, and the stage of the differentiation is that the mesoderm cells are obtained by differentiating ES or iPSC, the myocardial mesoderm cells are obtained by differentiating the mesoderm cells, the myocardial precursor-like cells are obtained by differentiating the myocardial mesoderm cells, and the mature myocardial cells are obtained by differentiating the myocardial precursor-like cells; the third preparation method comprises the following steps: mature cardiomyocytes can be transdifferentiated from other cells, such as fibroblasts. A method for preparing a cardiomyocyte precursor-like cell comprising the steps of: the cardiomyocyte precursor-like cells can be obtained by differentiating ES or iPSC, and the stage of differentiation is that the mesodermal cells are obtained by differentiating ES or iPSC, that the cardiomyocyte mesodermal cells are obtained by differentiating mesodermal cells, and that the cardiomyocyte precursor-like cells are obtained by differentiating cardiomyocyte mesodermal cells.
The above prior art has drawbacks including: the first preparation method of mature cardiomyocytes has the following drawbacks: 1. ethical issues; 2. the harvest rate is extremely low; 3. cannot be amplified and can not meet the treatment requirement; 4. the immunogenicity is high, and the method is not suitable for allograft transplantation; the second method of preparation of mature cardiomyocytes has the following drawbacks: 1. ES or iPSC residues are prone to cause tumors; 2. differentiation purity is difficult to ensure; 3. the differentiation period is long, and the cost is high; 4. the immunogenicity is high, and the method is not suitable for allograft transplantation; the third preparation method of mature cardiomyocytes has the following drawbacks: 1. the efficiency is extremely low; 2. the purity is extremely low, the immunogenicity is high, and the method is not suitable for allograft transplantation; the preparation method of the myocardial precursor-like cells has the following defects: 1. ES or iPSC residues are prone to cause tumors; 2. differentiation purity is difficult to ensure; 3. the differentiation period is long, and the cost is high; 4. failure to maintain the cells in a precursor state can spontaneously mature to give mature cardiomyocytes; 5. in the precursor state, large amounts cannot be amplified; 6. high immunogenicity and is not suitable for allograft.
The invention can culture the mature myocardial cells from different sources in vitro by using a reprogramming culture medium to obtain myocardial precursor-like cells, and the myocardial precursor-like cells obtained by the invention can be greatly expanded in vitro, wherein the mature myocardial cells from different sources comprise the mature myocardial cells obtained from primary cells and the mature myocardial cells obtained from ES or iPSC differentiation. Meanwhile, the invention can greatly expand the myocardial precursor-like cells differentiated from ES or iPSC in vitro, and solves the problem that the myocardial precursor-like cells cannot be greatly expanded in vitro.
Mature cardiomyocytes differentiated from ES or iPSC, or mature cardiomyocytes isolated from heart tissue by digestion, subjecting the mature cardiomyocytes to dedifferentiation treatment in the medium provided by the present invention to obtain cardiomyocyte precursor-like cells, which have the following commonalities: 1. the myocardial precursor-like cells can be greatly amplified in vitro, and have industrialized application prospect; 2. positive expression of CD24 and c-Kit by myocardial precursor cell; 3. myocardial precursor-like cells possess the general properties of precursor cells, such as the ability to proliferate rapidly; 4. the myocardial precursor-like cells have the functions of precursor cells and can be re-differentiated into mature myocardial cells under the action of a myocardial differentiation medium, so that cTnT and alpha-Sarcometric actin are positively expressed; 5. low immunogenicity, no or almost no expression (< 5%) of MHC class II antigens HLA-DP, HLA-DQ and HLA-DR, thus being suitable for allograft and not easy to cause graft-versus-host disease (the English name of graft-versus-host disease is graft-versus-host disease:, the English name of graft-versus-host disease is GvHD); 6. inhibiting inflammation, inhibiting PBMC activation, and simultaneously inhibiting M1 macrophages and promoting their conversion to M2 macrophages; 7. antifibrotic, apoptosis can be induced by paracrine action in vivo or in vitro in astrocytes and other fibrosis-derived cells.
The present invention provides a biological agent comprising a cardiomyocyte precursor-like cell that positively expresses CD24 and c-Kit, the cardiomyocyte precursor-like cell not expressing MHC class ii molecules.
Specifically, CD24 and c-Kit are positively expressed by the myocardial precursor-like cells, and the myocardial precursor-like cells do not express MHC class II molecules, so that the myocardial precursor-like cells can be well adapted to human bodies during allogeneic transplantation, and the problem that immune rejection occurs after allogeneic transplantation of the myocardial precursor-like cells in the prior art is solved.
In some embodiments of the invention, the MHC class II molecules include HLA-DR/DP/DQ, abbreviated HLA-DRPQ.
The invention also provides a preparation method of the biological agent containing the myocardial precursor-like cells, which comprises the following steps:
s0: providing a cardiomyocyte precursor-like cell;
s1: mixing the myocardial precursor-like cells with a pharmaceutically acceptable carrier to obtain the biological agent containing the myocardial precursor-like cells, wherein the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules.
Specifically, the preparation method of the biological agent containing the myocardial precursor-like cells is simple, and the biological agent containing the myocardial precursor-like cells can be well adapted to the human body after being returned to the human body, and the medicine is not needed to be used for avoiding immune rejection caused by allograft.
In some embodiments of the invention, the method for preparing the myocardial precursor-like cells comprises the steps of:
s00: providing a mature cardiomyocyte;
s01: placing the mature myocardial cells into a reprogramming culture medium for performing dedifferentiation culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and performing digestion treatment on the myocardial precursor-like cells by using pancreatin digestive juice to obtain the myocardial precursor-like cells, wherein the reprogramming culture medium comprises a basal culture medium, a MEK inhibitor, an inhibitor and a protein hormone, and the reprogramming culture medium is used for inducing the myocardial precursor-like cells and promoting proliferation of the myocardial precursor-like cells. The MEK inhibitor is used for inducing myocardial precursor-like cells, and the inhibitor and the protein hormone are used for promoting proliferation of myocardial precursor-like cells.
In some embodiments of the invention, the inhibitor is leukemia inhibitor, the protein hormone is insulin, the MEK inhibitor PD0325901 is from Shanghai-associated biological engineering Co., ltd, the leukemia inhibitor LIF is from Shanghai-Kanglan biological technology Co., ltd, and the insulin is from Gan Li pharmaceutical industry.
In some embodiments of the invention, the method for preparing the myocardial precursor-like cells comprises the steps of: s02: and placing the myocardial precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and then using the pancreatin digestive juice to digest the myocardial precursor-like cells so as to obtain the passaged myocardial precursor-like cells. The MEK inhibitor is co-acting with growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements for induction of cardiomyocyte precursor-like cells; the inhibitor and the protein hormone act together with growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors and nutritional supplements to promote proliferation of the cardiomyocyte precursor-like cells, enabling a substantial expansion of the cardiomyocyte precursor-like cells in vitro.
In some embodiments of the invention, the reprogramming media further comprises a growth factor, a ROCK kinase inhibitor, a Wnt signaling pathway agonist, a TGF- β signaling inhibitor, and a nutritional supplement.
In some embodiments of the invention, the growth factors comprise an epithelial cell growth factor EGF and a basic fibroblast growth factor bFGF, the epithelial cell growth factor EGF is derived from Peprotech, the basic fibroblast growth factor bFGF is derived from Peprotech, the ROCK kinase inhibitor Y-27632 is derived from Tao Shu, the Wnt signal pathway agonist CHIR99021 is derived from TargetMol, the TGF-beta signal inhibitor A830 is derived from TargetMol, and the nutritional supplements comprise an N2 nutritional supplement and a B27 nutritional supplement, the N2 nutritional supplement is derived from Invitrogen, and the B27 nutritional supplement is derived from Invitrogen.
According to some embodiments of the invention, the MEK inhibitor is present in an amount of 1-10. Mu.M, the inhibitor is present in an amount of 5-20ng/mL, and the protein hormone is present in an amount of 1-10. Mu.g/mL, based on the volume of the basal medium. In some embodiments, the MEK inhibitor is present in an amount of any of 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, and 9. Mu.M, the inhibitor is present in an amount of any of 6ng/mL, 8ng/mL, 10ng/mL, 12ng/mL, 14ng/mL, 16ng/mL, 18ng/mL, and 19ng/mL, and the protein hormone is present in an amount of any of 2. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, and 9. Mu.g/mL, based on the volume of the basal medium.
According to some embodiments of the invention, the growth factor is present in an amount of 50-80ng/mL, the ROCK kinase inhibitor is present in an amount of 10-50. Mu.M, the Wnt signaling pathway agonist is present in an amount of 1-10. Mu.M, the TGF-beta signaling inhibitor is present in an amount of 1-10. Mu.M, and the nutritional supplement is present in an amount of 1% -10% based on the volume of the basal medium. In some embodiments, the growth factor is present in an amount of any one of 55ng/mL, 60ng/mL, 65ng/mL, 70ng/mL, and 75ng/mL, the ROCK kinase inhibitor is present in an amount of any one of 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, and 45 μM, the Wnt signaling pathway agonist is present in an amount of any one of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, and 9 μM, the TGF- β signaling inhibitor is present in an amount of any one of 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, and 9 μM, and the nutritional supplement is present in an amount of any one of 2%, 3%, 4%, 5%, 6%, 7%, 8%, and 9% based on the volume of the basal medium.
In some embodiments of the invention, the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.
In some embodiments of the invention, the mature cardiomyocytes are obtained from myocardial tissue by digestion.
The invention also provides the application of the biological agent containing the myocardial precursor-like cells, and the biological agent containing the myocardial precursor-like cells is used for intervening in vivo animal models, wherein the animal models comprise drug-induced myocardial injury animal models.
Specifically, since the cardiac muscle precursor-like cells do not express MHC class II molecules, the biological preparation containing the cardiac muscle precursor-like cells does not need to take anti-expulsion drugs after being transplanted, so that the injury to human bodies can be reduced.
The abscissas in fig. 6, 7, 8, 10, 11, 12, 13, 14, 15, and 16 represent the same meaning, wherein the abscissas in fig. 6 represent relative fluorescence intensities, and the abscissas represent cell numbers.
Example 1: and separating and dedifferentiating the mature myocardial cells of the mice to obtain myocardial precursor-like cells, and carrying out characteristic identification on the mature myocardial cells and the myocardial precursor-like cells.
FIG. 1 is a schematic photograph showing the morphology of primary cardiomyocytes according to example 1 of the present invention; FIG. 2 is a photograph showing the morphology of the third generation of mature cardiomyocytes according to example 1 of the present invention; FIG. 3 is a schematic photograph showing the morphology of third generation myocardial precursor-like cells obtained by the culture of example 1 of the present invention; FIG. 4 is a photograph showing the morphology of third generation control myocardial precursor-like cells obtained by the culture of example 1 of the present invention; FIG. 5 is a graph showing the growth of cardiomyocyte precursor-like cells in reprogramming media and mature cardiomyocyte cells in cardiomyocyte media in accordance with example 1 of the present invention.
Isolation of mouse mature cardiomyocytes:
the heart of the mice 1 day after birth was selected.
Reagent:
primary cell culture fluid: to the basic culture solution DMEM/F12 (derived from source culture, model L310 KJ) were added fetal bovine serum (fetal bovine serum English name: fetal calf serum, english name: FBS, derived from Corning) with a volume content of 10% and penicillin-streptomycin double antibody solution (derived from source culture, concentration 100X) with a volume content of 1% based on the volume of the basic culture solution DMEM/F12 to obtain a primary cell culture solution.
The digestive juice comprises the following components: the digestive juice is prepared by taking the volume of pancreatin (pancreatin is derived from source culture, model S330JV, concentration is 0.25%), collagenase II (collagenase II is derived from Thermo Fisher, concentration is 2 mg/ml) with the volume content of 25% and then using a D-Hanks solution (derived from Wuhanplauosai Life technologies Co., ltd.) with the volume content of 50% to obtain the digestive juice, and preserving the digestive juice at minus 20 ℃, wherein the pH value of the D-Hanks solution is 7.2-7.8.
Cardiomyocyte medium: mouse cardiomyocyte medium, derived from procall company; the complete culture medium of the myocardial cells of the mice is derived from the life technology Co.Ltd.
The experimental steps are as follows:
sterilizing the skin of a suckling mouse 1 day after birth, fixing the head and limbs of the suckling mouse with 75% ethanol, cutting off the chest skin, sterilizing subcutaneous tissue of the suckling mouse with 75% ethanol, replacing forceps and scissors, opening chest to take out the heart of the suckling mouse, putting the heart of the suckling mouse into a plate (or penicillin bottle) containing D-Hanks solution (from the Ghancinoma life technologies Co., ltd.), cutting off atrium, cutting off ventricle, flushing with D-Hanks solution three times, removing residual blood, cutting the heart into heart fragments with the size of 1 cubic millimeter, transferring the heart fragments into a first centrifuge tube, adding 5 milliliters of digestive juice, digesting for 5 minutes at 37 ℃, naturally precipitating, discarding supernatant, adding 5 milliliters of digestive juice, digesting for 20 minutes at 37 ℃, shaking a few first centrifuge tube every 2 minutes in the digestion process, and blowing off the first centrifuge tube for 1 minute to obtain cell suspension; sucking out and transferring undigested heart fragments in a first centrifuge tube to a second centrifuge tube, adding 2 ml of digestive juice pre-cooled at 4 ℃ into the second centrifuge tube to stop digestion, putting the second centrifuge tube into a centrifuge, centrifuging the second centrifuge tube at a speed of 1000rpm for 5 minutes, discarding the supernatant, adding 2 ml of D-Hanks solution into the sediment of the second centrifuge tube, putting the second centrifuge tube into the centrifuge again, centrifuging the second centrifuge tube at a speed of 1500rpm for 10 minutes, discarding the supernatant, adding 2 ml of culture solution into the sediment of the second centrifuge tube, and blowing the second centrifuge tube with a suction tube to obtain a cell suspension; and supplementing 5 milliliters of digestive juice of undigested heart fragments in the second centrifuge tube, continuing to digest, repeating the operation, combining the cell suspensions in each centrifuge tube, placing the cell suspensions into a culture flask, placing the culture flask into a carbon dioxide incubator for culture, wherein the temperature of the carbon dioxide incubator is 37 ℃, and the carbon dioxide content of the carbon dioxide incubator is maintained at 5%.
Culture results of cell suspension: the cell suspension was cultured in a carbon dioxide incubator for 24 hours, and then a pulsating monolayer of mature cardiomyocytes was visible.
The mature myocardial cells of the mice are dedifferentiated to obtain myocardial precursor-like cells:
reagent:
the reprogramming media consisted of: basic medium DMEM/F-12 (from the Ghancinolone acetonide life sciences Co., ltd.) contains, based on the volume of the basic medium DMEM/F-12, epithelial cell growth factor EGF in an amount of 20 ng/ml, basic fibroblast growth factor bFGF in an amount of 50 ng/ml, N2 nutritional supplement (1X) in an amount of 1%, B27 nutritional supplement (1X) in an amount of 1%, ROCK kinase inhibitor Y-27632 in an amount of 10uM, wnt signal pathway agonist CHIR99021 in an amount of 3uM, TGF-beta signal inhibitor A830 in an amount of 1uM, MEK inhibitor PD0325901 in an amount of 1uM, leukemia inhibitory factor LIF in an amount of 10 ng/ml, insulin in an amount of 1 microgram/ml;
the control medium consisted of: basic culture medium DMEM/F-12 (from the Ghancinolone acetonide life sciences Co., ltd.) contains, based on the volume of the basic culture medium DMEM/F-12, epithelial cell growth factor EGF in an amount of 20 ng/ml, basic fibroblast growth factor bFGF in an amount of 50 ng/ml, N2 nutritional supplement (1X) in an amount of 1%, B27 nutritional supplement (1X) in an amount of 1%, ROCK kinase inhibitor Y-27632 in an amount of 10uM, wnt signal pathway agonist CHIR99021 in an amount of 3uM, TGF-beta signal inhibitor A830 in an amount of 1 uM;
Cardiomyocyte medium: mouse cardiomyocyte medium, derived from procall company;
the composition of the cardiomyocyte differentiation medium was as follows: basal medium alphSub>A-MEM (from Shanghai Huiyang Biotechnology Co., ltd.) was 1. Mu.M dexamethasone, 50 ng/mL vitamin C, 10mM sodium betSub>A-glycerophosphate, 3% fetal bovine serum FBS, 5ng/mL TGF-betSub>A 1, 10ng/mL VEGF-A based on the volume of basal medium alphSub>A-MEM.
The experimental steps are as follows:
primary cardiomyocytes: inoculating cell suspension obtained by digestion of myocardial tissue into 6-well plate, adding 2 ml DMEM/F-12+10% FBS culture solution into each well, and adding CO 2 The cells were cultured in an incubator at 37 degrees celsius to obtain primary cardiomyocytes, designated P0.
Passaging of cardiomyocyte precursor-like cells: when the confluence of the primary myocardial cells reaches 80%, digesting single-layer mature myocardial cells by trypsin (derived from source culture), inoculating the mature myocardial cells into a culture dish of 10 cm at an inoculation density of 20000 per square cm, adding 10 ml of reprogramming culture medium into each dish for carrying out dedifferentiation culture to obtain first-generation myocardial precursor-like cells (marked as P1), changing the reprogramming culture medium every 2-3 days in the process of amplifying and culturing the first-generation myocardial precursor-like cells, and carrying out passage and passage specific operations when the confluence of the cells reaches 80 percent: mature cardiomyocytes were digested with trypsin (derived from source culture), inoculated into 10 cm dishes at an inoculation density of 20000 cells/cm, dedifferentiated cultured with 10 ml of reprogramming medium per dish, second generation cardiomyocyte precursor-like cells (denoted as P2) were cultured, the process of expansion culture was cycled until fifteenth generation cardiomyocyte precursor-like cells (denoted as P15) were cultured, the passage algebra were photographed, counted and recorded every 3 days, and a growth curve was drawn from the recorded data, see fig. 5. The schematic of the cell morphology of the third generation of cardiomyocyte precursor-like cells is shown in fig. 3.
Passaging control cardiomyocyte precursor-like cells: when the confluence of the primary myocardial cells reaches 80%, digesting single-layer mature myocardial cells by trypsin (derived from source culture), inoculating the mature myocardial cells into 10 cm culture dishes at an inoculation density of 20000 per square cm, adding 10 ml of control culture medium into each culture dish for dedifferentiation culture to obtain first-generation control myocardial precursor-like cells (marked as P1), changing the control culture medium every 2-3 days in the amplification culture process of the control culture medium, and carrying out passage for the cells until the confluence of the cells reaches 80%, wherein the specific operation of the passage is as follows: mature cardiomyocytes were digested with trypsin (from source culture), inoculated into 10 cm dishes at an inoculation density of 20000 cells/cm, each dish was subjected to dedifferentiation culture with 10 ml of control medium, second generation control cardiomyocyte precursor-like cells (denoted as P2) were cultured, and the culture process was cycled through expansion until third generation control cardiomyocyte precursor-like cells (denoted as P3) were cultured, and the photographic cell morphology diagram of the third generation control cardiomyocyte precursor-like cells is shown in fig. 4.
Referring to fig. 3 and 4, when the third-generation control myocardial precursor-like cells are cultured in the control medium, the third-generation control myocardial precursor-like cells become worse, and spread out like a purse string, become larger in diameter, and fail to proliferate.
Passaging mature cardiomyocytes: when the confluence of the primary myocardial cells reaches 80%, digesting single-layer mature myocardial cells with trypsin (source culture), inoculating the mature myocardial cells into culture dishes of 10 cm at an inoculation density of 20000 per square cm, adding 10 ml of myocardial cell culture medium into each culture dish to culture until the confluence of the mature myocardial cells is not lower than 80% and the growth state is good, completing amplification culture to obtain first-generation mature myocardial cells (denoted as P1), changing the myocardial cell culture medium every 2-3 days in the amplification culture process, circularly amplifying the culture process, culturing second-generation mature myocardial cells (denoted as P2) until the second-generation mature myocardial cells are cultured into fifth-generation mature myocardial cells (denoted as P5), photographing, counting every 3 days, recording passage algebra, and drawing a growth curve according to the recorded data, referring to FIG. 5.
Referring to fig. 5, the third generation of mature cardiomyocytes obtained from the cardiomyocyte culture medium almost all die, while the third generation of cardiomyocyte precursor-like cells obtained from the reprogramming culture medium provided by the present invention can proliferate stably, compared to the case where the mature cardiomyocytes are cultured using the cardiomyocyte culture medium, the reprogramming culture medium provided by the present invention can stably culture the cardiomyocyte precursor-like cells up to the fifteenth generation, and the curve of the total cell number is always in an ascending trend, indicating that the reprogramming culture medium provided by the present invention can continue to proliferate the cardiomyocyte precursor-like cells in vitro, and the proliferation number of the total cells increases exponentially.
FIG. 6 is a schematic diagram showing the results of flow assay for positive expression of c-Kit by third generation cardiomyocyte precursor-like cells in example 1 of the present invention; FIG. 7 is a schematic diagram showing the results of flow assay for positive expression of CD24 by third generation myocardial precursor-like cells in example 1 of the present invention; FIG. 8 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by third generation cardiomyocyte precursor-like cells according to example 1 of the present invention; FIG. 9 is a schematic diagram showing the cell morphology of the third generation cardiomyocyte precursor-like cells of example 1 of the present invention after 7 days of differentiation; FIG. 10 is a schematic diagram showing the results of flow assay for positive expression of cTnT 7 days after differentiation of third generation cardiomyocyte precursor-like cells according to example 1 of the present invention; FIG. 11 is a schematic diagram showing the results of flow assay for positive expression of α -Sarcometric actin 7 days after differentiation of third-generation cardiomyocyte precursor-like cells according to example 1 of the present invention.
Each of the third generation (i.e., P3) of myocardial precursor-like cells and the third generation of control myocardial cells was 1.0X10 × 6 The method comprises the following specific steps of:
cell surface staining:
p3 myocardial precursor-like cells were aspirated and reprogrammed in 5ml of sterile PBS buffer, and then digested with 2 ml of pancreatin digest in a petri dish, and the cell mixture was placed in a 15ml centrifuge tube, which was subjected to centrifugation at a speed of 200g for 5 minutes, and the supernatant of the centrifuged cell mixture was discarded to obtain a cell pellet.
220 microliters of staining buffer was added to the cell pellet to resuspend the cell pellet, and the resuspended cell pellet was transferred to 2 1.5 milliliter centrifuge tubes, 100 microliters/tube format. And respectively adding 5 microliters of the flow antibody to be detected, and blowing and uniformly mixing. After placing the centrifuge tubes in a refrigerator at 2-8 degrees celsius for 30 minutes, sterile PBS buffer was added to each centrifuge tube at a dose of 800 μl/tube, and the centrifuge tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400 μl of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of surface markers.
The antibody names used for the surface marker flow detection are as follows: CD24, HLV-DRPQ.
CD24 was purchased from abcam under the accession number ab290730; HLV-DRPQ was purchased from abcam under the trade designation ab7856; staining buffer was purchased from BD Biosciences under the trade designation 554656.
Intracellular staining of cells:
p3 myocardial precursor sample cells are sucked and removed from the reprogramming culture medium, the reprogramming culture medium is rinsed by using 5 milliliters of sterile PBS buffer solution, then 2 milliliters of pancreatin digestion solution is dripped into a culture dish for digestion treatment to obtain a cell mixture, the cell mixture is placed into a 15 milliliter centrifuge tube, the centrifuge tube is placed into a centrifuge, centrifugal treatment is carried out for 5 minutes at the speed of 200g, and supernatant liquid of the cell mixture after the centrifugal treatment is discarded to obtain cell sediment.
Adding 1 ml of fixed membrane penetrating fluid into the cell sediment, placing the centrifuge tube in a refrigerator at 2-8 ℃ for standing for 50 minutes, adding 2 ml of PBS buffer into the centrifuge tube, centrifuging the centrifuge tube at a rotating speed of 300g for 5 minutes, and adding 100 microliters of dyeing buffer into the centrifuge tube for resuspension after centrifugation. 5 microliters of the flow antibody to be detected is added into the 1.5 milliliter centrifuge tube, the mixture is blown and evenly mixed, and the centrifuge tube is placed into an incubator containing 5 percent of carbon dioxide by volume at 37 ℃ for standing for 30 minutes. After incubation, sterile PBS buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400 μl of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of intracellular markers.
The antibody name used for the flow assay of the intracellular marker is c-Kit. c-Kit was purchased from abcam under the accession number ab283653; staining buffer was purchased from BD Biosciences under the trade designation 554656.
The results of the flow detection are shown in fig. 6, 7 and 8.
Referring to FIGS. 6, 7 and 8, third generation cardiomyocyte-like cells expressed CD24, c-Kit positively and HLA-DRPQ negatively.
Taking third generation (namely P3) myocardial precursor-like cells, differentiating the third generation myocardial precursor-like cells by using a myocardial cell differentiation medium for 7 days to obtain mature myocardial cells, changing fresh myocardial cell differentiation medium every 3 days after differentiating the third generation myocardial precursor-like cells for 7 days, and performing flow detection on the obtained mature myocardial cells, wherein the detection results are shown in fig. 10 and 11.
Referring to fig. 10 and 11, the third generation cardiomyocyte precursor-like cells differentiated for 7 days to obtain mature cardiomyocytes, and the obtained mature cardiomyocytes can positively express markers cTnT and α -Sarcometric actin, which prove that the cells are mature cardiomyocytes. cTnT is purchased from abcam, cat No. ab209813; alpha-Sarcometric actin was purchased from Biyun Tian, cat No. AG0101.
Example 2iPSC differentiated to give mature cardiomyocytes, and then dedifferentiated with reprogramming media to give cardiomyocyte precursor-like cells:
FIG. 12 is a schematic diagram showing the results of flow assay for positive expression of cTnT by mature cardiomyocytes according to example 2 of the present invention; FIG. 13 is a schematic diagram showing the results of flow assay for positive expression of c-kit by myocardial precursor-like cells in example 2 of the present invention; FIG. 14 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by myocardial precursor-like cells in example 2 of the present invention.
Reagent:
iPSC culture reagent: mTESR1 culture medium, from Stemcell Tech company;
agent for differentiation of iPSC into mature myocardium: myocardial differentiation reagent, from Stemcell Tech company.
The experimental steps are as follows:
placing iPSC into mTESR1 culture medium for culturing to obtain induced pluripotent stem cells, wherein the specific operation process is carried out according to the specification of the mTESR1 culture medium; the iPSC was put into a cardiomyocyte differentiation reagent and subjected to differentiation culture to obtain mature cardiomyocytes, and the specific steps of the differentiation culture were performed according to the instructions of the cardiomyocyte differentiation reagent. Special precautions are: the iPSC was cultured for 15 days or more after differentiation on day 8, after changing to a maintenance medium (from Stemcell Tech Co.), to obtain mature cardiomyocytes. Markers for mature cardiomyocytes were: cardiomyocytes stopped proliferating and spontaneously beating in the petri dish. The method for detecting the mature myocardial cells in a flow mode comprises the following specific steps of:
intracellular staining of cells:
the iPSC differentiated mature myocardial cells, sucking and removing the reprogramming culture medium, using 5 ml of sterile PBS buffer solution for rinsing, then using 2 ml of pancreatin digestive solution to be dripped into a culture dish for digestion treatment to obtain a cell mixture, placing the cell mixture into a 15 ml centrifuge tube, placing the centrifuge tube into a centrifuge, carrying out centrifugation treatment at the speed of 200g for 5 minutes, and discarding supernatant of the cell mixture after the centrifugation treatment to obtain cell sediment.
Adding 1 ml of fixed membrane penetrating fluid into the cell sediment, placing the centrifuge tube in a refrigerator at 2-8 ℃ for standing for 50 minutes, adding 2 ml of PBS buffer into the centrifuge tube, centrifuging the centrifuge tube at a rotating speed of 300g for 5 minutes, and adding 100 microliters of dyeing buffer into the centrifuge tube for resuspension after centrifugation. 5 microliters of the flow antibody to be detected is added into the 1.5 milliliter centrifuge tube, the mixture is blown and evenly mixed, and the centrifuge tube is placed into an incubator containing 5 percent of carbon dioxide by volume at 37 ℃ for standing for 30 minutes. After incubation, sterile PBS buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400 μl of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of intracellular markers.
The antibody used for the flow assay of intracellular markers was designated cTnT, purchased from abcam under the designation ab209813. The flow detection results are shown in fig. 12.
Referring to fig. 12, ipscs differentiated for 15 days to obtain mature cardiomyocytes, and a flow assay of the mature cardiomyocytes showed positive expression of cTnT genes.
Culturing the obtained mature myocardial cells with a reprogramming culture medium to obtain myocardial precursor-like cells, and carrying out flow detection on the obtained myocardial precursor-like cells, wherein the specific steps of the flow detection comprise:
cell surface staining:
the myocardial precursor cell is sucked and removed, a reprogramming culture medium is used for rinsing by using 5 ml of sterile PBS buffer solution, then 2 ml of pancreatin digestive solution is dripped into a culture dish for digestion treatment to obtain a cell mixture, the cell mixture is placed into a 15 ml centrifuge tube, the centrifuge tube is placed into a centrifuge for centrifugation treatment at the speed of 200g for 5 minutes, and the supernatant of the cell mixture after the centrifugation treatment is discarded to obtain a cell precipitate.
And (3) adding 100 microliters of staining buffer into the cell sediment to re-suspend the cell sediment, adding 5 microliters of antibody to be detected, and blowing and uniformly mixing. After placing the centrifuge tubes in a refrigerator at 2-8 degrees celsius for 30 minutes, sterile PBS buffer was added to each centrifuge tube at a dose of 800 μl/tube, and the centrifuge tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400 μl of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of surface markers.
The antibody names used for the surface marker flow detection are as follows: HLA-DRPQ, available from abcam under the designation ab7856; staining buffer was purchased from BD Biosciences under the trade designation 554656.
Intracellular staining of cells:
the myocardial precursor cell is sucked and removed, a reprogramming culture medium is used for rinsing by using 5 ml of sterile PBS buffer solution, then 2 ml of pancreatin digestive solution is dripped into a culture dish for digestion treatment to obtain a cell mixture, the cell mixture is placed into a 15 ml centrifuge tube, the centrifuge tube is placed into a centrifuge for centrifugation treatment at the speed of 200g for 5 minutes, and the supernatant of the cell mixture after the centrifugation treatment is discarded to obtain a cell precipitate.
Adding 1 ml of fixed membrane penetrating fluid into the cell sediment, placing the centrifuge tube in a refrigerator at 2-8 ℃ for standing for 50 minutes, adding 2 ml of PBS buffer into the centrifuge tube, centrifuging the centrifuge tube at a rotating speed of 300g for 5 minutes, and adding 100 microliters of dyeing buffer into the centrifuge tube for resuspension after centrifugation. 5 mu L of the flow antibody to be detected is added into the 1.5 ml centrifuge tube, the mixture is blown and evenly mixed, and the centrifuge tube is placed into an incubator containing 5% carbon dioxide by volume at 37 ℃ for standing for 30 minutes. After incubation, sterile PBS buffer was added to each centrifuge tube at a dose of 800. Mu.L/tube, and the tubes were placed in a centrifuge and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was discarded, 400 μl of staining buffer was added to each centrifuge tube to resuspend the cell pellet, and then transferred to a flow tube, and the resuspended cell mixture was subjected to flow detection of intracellular markers.
The antibody name used for the flow detection of the intracellular marker is c-Kit, which is purchased from abcam and is sold under the product number ab283653; staining buffer was purchased from BD Biosciences under the trade designation 554656.
The results of the detection are shown in FIGS. 13 and 14, and the results show that the obtained myocardial precursor-like cells positively express c-kit and negatively express HLA-DRPQ.
Example 3 obtaining cardiomyocyte precursor-like cells from ipsc differentiation:
FIG. 15 is a schematic diagram showing the results of flow assay for positive expression of c-kit by myocardial precursor-like cells in example 3 of the present invention; FIG. 16 is a schematic diagram showing the results of flow assay for negative expression of HLA-DRPQ by myocardial precursor-like cells in example 3 of the present invention.
Reagent:
iPSC culture reagent: mTESR1 culture medium, from Stemcell Tech company;
agent for differentiation of iPSC into mature myocardium: myocardial differentiation reagent purchased from Stemcell Tech company.
The experimental steps are as follows:
placing iPSC into mTESR1 culture medium for culturing to obtain induced pluripotent stem cells, wherein the specific operation process is carried out according to the specification of the mTESR1 culture medium; the iPSC was placed in a myocardial differentiation reagent and subjected to differentiation culture to obtain myocardial precursor-like cells, and specific steps of the differentiation culture were performed according to the instructions of the myocardial differentiation reagent. Notice that: on day 8 of differentiation, the cells were changed to maintenance medium (from Stemcell Tech company) to be cardiomyocyte precursor-like cells, and the markers of cardiomyocyte precursor-like cells were: myocardial precursor-like cells can proliferate and beat involuntarily in culture dishes. The flow assay was performed on the obtained myocardial precursor-like cells, and the results are shown in fig. 15 and 16.
Referring to FIGS. 15 and 16, the cardiomyocyte-like cells differentiated by iPSC expressed positively the c-kit gene and HLA-DRPQ gene.
The technical core of the invention is as follows:
1. obtaining of myocardial precursor-like cells: can be amplified in vitro on a large scale.
Mature myocardial cells are isolated and cultured by a reprogramming culture medium to obtain myocardial precursor-like cells.
iPSC is differentiated into mature myocardial cells, and the myocardial precursor-like cells are obtained by performing dedifferentiation culture on the mature myocardial cells by using a reprogramming culture medium.
2. The application of the myocardial precursor-like cells obtained by the invention: the MHC class II molecule HLA-DR/DP/DQ associated with immune rejection is not expressed. The myocardial precursor-like cells can transdifferentiate into new mature myocardial cells to promote myocardial regeneration; and recruits capillaries by secreting pro-angiogenic factors, new endocardial vessels. Because the MHC class II molecule HLA-DR/DP/DQ associated with immune rejection is not expressed, patients do not need to take anti-expulsion drugs after transplantation of myocardial precursor-like cells. The myocardial precursor-like cells obtained by the invention have higher safety in treating heart diseases, wider application range and stronger patient acceptance.
While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.
Claims (10)
1. A biologic comprising a cardiomyocyte precursor-like cell that positively expresses CD24 and c-Kit, wherein the cardiomyocyte precursor-like cell does not express MHC class ii molecules.
2. A method of preparing a biologic comprising cardiomyocyte-like cells according to claim 1, comprising the steps of:
s0: providing a cardiomyocyte precursor-like cell;
s1: mixing the myocardial precursor-like cells with a pharmaceutically acceptable carrier to obtain the biological agent containing the myocardial precursor-like cells, wherein the myocardial precursor-like cells positively express CD24 and c-Kit, and the myocardial precursor-like cells do not express MHC class II molecules.
3. The method for producing a biological agent comprising a cardiac muscle precursor-like cell according to claim 2, wherein the method for producing a cardiac muscle precursor-like cell comprises the steps of:
s00: providing a mature cardiomyocyte;
s01: placing the mature myocardial cells into a reprogramming culture medium for performing dedifferentiation culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and performing digestion treatment on the myocardial precursor-like cells by using pancreatin digestive juice to obtain the myocardial precursor-like cells, wherein the reprogramming culture medium comprises a basal culture medium, a MEK inhibitor, an inhibitor and a protein hormone, and the reprogramming culture medium is used for inducing the myocardial precursor-like cells and promoting proliferation of the myocardial precursor-like cells.
4. The method for producing a biological agent comprising a cardiac muscle precursor-like cell according to claim 3, wherein the method for producing a cardiac muscle precursor-like cell comprises the steps of:
s02: and placing the myocardial precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the myocardial precursor-like cells is not lower than 80%, and then using the pancreatin digestive juice to digest the myocardial precursor-like cells so as to obtain the passaged myocardial precursor-like cells.
5. The method of claim 4, wherein the reprogramming media further comprises growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements.
6. The method according to claim 5, wherein the MEK inhibitor is contained in an amount of 1 to 10. Mu.M, the inhibitor is contained in an amount of 5 to 20ng/mL, and the protein hormone is contained in an amount of 1 to 10. Mu.g/mL, based on the volume of the basal medium.
7. The method of claim 6, wherein the growth factor is present in an amount of 50-80ng/mL, the ROCK kinase inhibitor is present in an amount of 10-50 μm, the Wnt signaling pathway agonist is present in an amount of 1-10 μm, the TGF- β signaling inhibitor is present in an amount of 1-10 μm, and the nutritional supplement is present in an amount of 1% -10% based on the volume of the basal medium.
8. The method of claim 3, wherein the mature cardiomyocytes are differentiated from embryonic stem cells or induced pluripotent stem cells.
9. The method for producing a biological agent comprising a cardiac muscle precursor-like cell according to claim 3, wherein the mature cardiac muscle cell is obtained by digestion treatment of a cardiac muscle tissue.
10. Use of a biologic comprising a cardiomyocyte precursor-like cell according to claim 1 for intervention in an in vivo animal model, including a drug-induced myocardial injury animal model.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210503678 | 2022-05-10 | ||
| CN2022105036786 | 2022-05-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117018033A true CN117018033A (en) | 2023-11-10 |
Family
ID=88621424
Family Applications (9)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310519697.2A Pending CN117025502A (en) | 2022-05-10 | 2023-05-09 | Prostate precursor-like cells, cell preparation, preparation method and application thereof |
| CN202310519744.3A Pending CN117025505A (en) | 2022-05-10 | 2023-05-09 | Gastric mucosal epithelial precursor-like cell, and preparation method and application thereof |
| CN202310522421.XA Pending CN117025506A (en) | 2022-05-10 | 2023-05-09 | Biological agent containing kidney precursor-like cells, preparation method and application thereof |
| CN202310522452.5A Pending CN117018032A (en) | 2022-05-10 | 2023-05-09 | Biological agent containing skeletal muscle precursor-like cells, and preparation method and application thereof |
| CN202310519743.9A Pending CN117025504A (en) | 2022-05-10 | 2023-05-09 | Preparation method and application of gall bladder precursor-like cells |
| CN202310519740.5A Pending CN117025503A (en) | 2022-05-10 | 2023-05-09 | Intrahepatic duct precursor-like cell, cell preparation, preparation method and application |
| CN202310522470.3A Pending CN117025508A (en) | 2022-05-10 | 2023-05-09 | Kidney epithelial precursor-like cell, its preparation method, preparation and application |
| CN202310522477.5A Pending CN117018033A (en) | 2022-05-10 | 2023-05-09 | Biological agent containing myocardial precursor-like cells, and preparation method and application thereof |
| CN202310522433.2A Pending CN117025507A (en) | 2022-05-10 | 2023-05-09 | Preparation method and application of lung precursor-like cells |
Family Applications Before (7)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310519697.2A Pending CN117025502A (en) | 2022-05-10 | 2023-05-09 | Prostate precursor-like cells, cell preparation, preparation method and application thereof |
| CN202310519744.3A Pending CN117025505A (en) | 2022-05-10 | 2023-05-09 | Gastric mucosal epithelial precursor-like cell, and preparation method and application thereof |
| CN202310522421.XA Pending CN117025506A (en) | 2022-05-10 | 2023-05-09 | Biological agent containing kidney precursor-like cells, preparation method and application thereof |
| CN202310522452.5A Pending CN117018032A (en) | 2022-05-10 | 2023-05-09 | Biological agent containing skeletal muscle precursor-like cells, and preparation method and application thereof |
| CN202310519743.9A Pending CN117025504A (en) | 2022-05-10 | 2023-05-09 | Preparation method and application of gall bladder precursor-like cells |
| CN202310519740.5A Pending CN117025503A (en) | 2022-05-10 | 2023-05-09 | Intrahepatic duct precursor-like cell, cell preparation, preparation method and application |
| CN202310522470.3A Pending CN117025508A (en) | 2022-05-10 | 2023-05-09 | Kidney epithelial precursor-like cell, its preparation method, preparation and application |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310522433.2A Pending CN117025507A (en) | 2022-05-10 | 2023-05-09 | Preparation method and application of lung precursor-like cells |
Country Status (2)
| Country | Link |
|---|---|
| CN (9) | CN117025502A (en) |
| WO (9) | WO2023217134A1 (en) |
Family Cites Families (38)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10590391B2 (en) * | 2007-06-08 | 2020-03-17 | Wake Forest University Health Sciences | Selective cell therapy for the treatment of renal failure |
| JP5458112B2 (en) * | 2009-02-03 | 2014-04-02 | コーニンクレッカ ネザーランド アカデミー ヴァン ウェテンシャッペン | Culture medium for epithelial stem cells and organoids containing the stem cells |
| AU2009354350B2 (en) * | 2009-10-19 | 2015-09-03 | Tristem Trading (Cyprus) Limited | Treatment using reprogrammed mature adult cells |
| EP2638149B1 (en) * | 2010-11-12 | 2019-05-15 | Georgetown University | Immortalization of epithelial cells and methods of use |
| WO2013049856A2 (en) * | 2011-09-30 | 2013-04-04 | Hare Joshue M | Renal stem cells isolated from kidney |
| CN103031270A (en) * | 2013-01-05 | 2013-04-10 | 绍兴文理学院 | Efficient amplifying and culturing method for biliary epithelial cells |
| CN104152398A (en) * | 2013-05-13 | 2014-11-19 | 百奥迈科生物技术有限公司 | Method for separating prostate tissues and establishing primary epithelial cells and/or interstitial cells |
| ITFI20130303A1 (en) * | 2013-12-24 | 2015-06-25 | Azienda Ospedaliero Universitaria M Eyer | METHOD FOR THE INSULATION, PURIFICATION AND AMPLIFICATION OF RHYNAL PROJECTS CD133 + CD24 + FROM URINE OF PATIENTS WITH RENAL DISEASES. |
| CN103860597A (en) * | 2014-03-03 | 2014-06-18 | 奥思达干细胞有限公司 | Stem cell preparation for treating ischemic cardiomyopathy and preparation method of stem cell preparation |
| US9687512B2 (en) * | 2014-03-12 | 2017-06-27 | Banner Health | Isolated cardiac stem cells and methods of their use |
| JP5924750B2 (en) * | 2014-05-01 | 2016-05-25 | ｉＨｅａｒｔ Ｊａｐａｎ株式会社 | CD82 positive myocardial progenitor cells |
| US10258652B2 (en) * | 2014-12-18 | 2019-04-16 | Pluristem Ltd. | Methods and compositions for treating and preventing muscle wasting disorders |
| CN107636149A (en) * | 2015-04-03 | 2018-01-26 | 普罗帕格尼克斯公司 | The vitro proliferation of epithelial cell |
| KR101782488B1 (en) * | 2015-05-19 | 2017-09-28 | 주식회사 스템랩 | Method for Inducing Oligodendrocyte Progenitor Cells from Human Somatic Cells Introduced with Oct4 Through Direct Reprogramming |
| CN104928235A (en) * | 2015-07-10 | 2015-09-23 | 奥思达干细胞有限公司 | Composition based on stem cells and application thereof in preparing preparation for coronary heart disease |
| EP3892718A1 (en) * | 2015-09-11 | 2021-10-13 | Propagenix Inc. | Ex vivo proliferation of epithelial cells |
| CN106754636B (en) * | 2015-11-19 | 2019-08-30 | 中国人民解放军第二军医大学 | External evoked primary hepatocyte bile ductization and long-term cultivation, amplification and the method and its application of differentiation |
| JP2017108705A (en) * | 2015-12-18 | 2017-06-22 | 国立大学法人京都大学 | Cardiomyocyte production method |
| GB201603569D0 (en) * | 2016-03-01 | 2016-04-13 | Koninklijke Nederlandse Akademie Van Wetenschappen | Improved differentiation method |
| JP7090544B2 (en) * | 2016-04-27 | 2022-06-24 | 武田薬品工業株式会社 | Method for producing skeletal muscle progenitor cells and skeletal muscle cells |
| JP7089484B2 (en) * | 2016-06-01 | 2022-06-22 | ミルテニイ ビオテック ベー．ファー． ウント コー．カーゲー | Methods for Generating, Identifying and Isolating Cardiomyocytes and Cardiomyocyte Subpopulations Derived from Human Pluripotent Stem Cells |
| CN107142240B (en) * | 2016-06-03 | 2021-01-29 | 中国人民解放军军事医学科学院野战输血研究所 | Method for reprogramming epithelial cells derived from digestive tract into endoderm stem/progenitor cells and application |
| CN107151645A (en) * | 2017-05-16 | 2017-09-12 | 武汉大学深圳研究院 | A kind of method and culture medium that in vitro individuation drug test is provided for lung cancer |
| GB201709704D0 (en) * | 2017-06-19 | 2017-08-02 | Cambridge Entpr Ltd | Methods of expanding cholangiocytes |
| CN108570443A (en) * | 2017-12-05 | 2018-09-25 | 皓昇莱生物制药有限公司 | A kind of culture medium for cultivating urine derived cell |
| CN110218696A (en) * | 2018-03-01 | 2019-09-10 | 中国科学院广州生物医药与健康研究院 | A kind of cultivating system generated for chemical induction multipotent stem cells and the chemical reprogramming method using the cultivating system |
| WO2019210279A1 (en) * | 2018-04-27 | 2019-10-31 | The Regents Of The University Of California | De novo formation of the biliary system by hepatocyte transdifferentiation |
| EP3790957A1 (en) * | 2018-05-08 | 2021-03-17 | Universität Zürich | Method for xeno-free generation of a population of hmpc |
| KR102071302B1 (en) * | 2018-06-28 | 2020-01-30 | 한국과학기술연구원 | A method for inducing transdifferentiation of cardiomyocytes based on exosome |
| CN109576215A (en) * | 2018-12-27 | 2019-04-05 | 广州赛莱拉干细胞科技股份有限公司 | A method of induction dental pulp stem cell Cardiocytes differentiation |
| CA3135189A1 (en) * | 2019-04-03 | 2020-10-08 | The Johns Hopkins University | Methods, compositions, and kits for producing skeletal muscle stem cells and treating disorders |
| KR102150489B1 (en) * | 2019-04-09 | 2020-09-01 | 고려대학교 산학협력단 | Method for Direct Conversion from Human Urine Cells into Nephron Progenitor Cells and Composition for Preventing or Treating Kidney Failure Comprising Induced Nephron Progenitor Cells Thereof |
| CN110184299B (en) * | 2019-04-23 | 2023-11-24 | 中国科学院广州生物医药与健康研究院 | Factor inducing reprogramming of cells and method for inducing reprogramming of cells using the same |
| CN112126618B (en) * | 2019-06-24 | 2023-09-12 | 上海拜羡生物科技有限公司 | Method for obtaining human gallbladder stem cells and long-term in vitro culture |
| CN112481192B (en) * | 2019-09-12 | 2024-05-28 | 海门雨霖细胞科技有限责任公司 | Chemical small molecule composition and method for directly reprogramming in vitro and in vivo chemically induced fibroblast into hepatocyte |
| CN111440761A (en) * | 2020-04-09 | 2020-07-24 | 上海赛尔维医疗科技有限公司 | Method for expanding and differentiating pancreatic cells and uses thereof |
| CN115322947B (en) * | 2022-06-25 | 2024-02-09 | 同济大学 | Method for constructing kidney micro-organ by suspension differentiation culture of adult kidney precursor cells and application |
| CN115992090B (en) * | 2022-11-09 | 2024-09-10 | 同济大学 | Secretion set derived from kidney precursor cells, and preparation method and application thereof |
-
2023
- 2023-05-09 WO PCT/CN2023/092969 patent/WO2023217134A1/en unknown
- 2023-05-09 CN CN202310519697.2A patent/CN117025502A/en active Pending
- 2023-05-09 CN CN202310519744.3A patent/CN117025505A/en active Pending
- 2023-05-09 WO PCT/CN2023/092959 patent/WO2023217126A1/en unknown
- 2023-05-09 WO PCT/CN2023/092967 patent/WO2023217132A1/en unknown
- 2023-05-09 CN CN202310522421.XA patent/CN117025506A/en active Pending
- 2023-05-09 WO PCT/CN2023/092954 patent/WO2023217121A1/en unknown
- 2023-05-09 WO PCT/CN2023/092965 patent/WO2023217130A1/en unknown
- 2023-05-09 WO PCT/CN2023/092963 patent/WO2023217128A1/en unknown
- 2023-05-09 WO PCT/CN2023/092956 patent/WO2023217123A1/en unknown
- 2023-05-09 CN CN202310522452.5A patent/CN117018032A/en active Pending
- 2023-05-09 WO PCT/CN2023/092958 patent/WO2023217125A1/en unknown
- 2023-05-09 CN CN202310519743.9A patent/CN117025504A/en active Pending
- 2023-05-09 CN CN202310519740.5A patent/CN117025503A/en active Pending
- 2023-05-09 CN CN202310522470.3A patent/CN117025508A/en active Pending
- 2023-05-09 WO PCT/CN2023/092964 patent/WO2023217129A1/en unknown
- 2023-05-09 CN CN202310522477.5A patent/CN117018033A/en active Pending
- 2023-05-09 CN CN202310522433.2A patent/CN117025507A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023217128A1 (en) | 2023-11-16 |
| WO2023217123A1 (en) | 2023-11-16 |
| CN117025507A (en) | 2023-11-10 |
| WO2023217134A1 (en) | 2023-11-16 |
| WO2023217126A1 (en) | 2023-11-16 |
| CN117025508A (en) | 2023-11-10 |
| CN117025504A (en) | 2023-11-10 |
| WO2023217121A1 (en) | 2023-11-16 |
| CN117025506A (en) | 2023-11-10 |
| CN117025503A (en) | 2023-11-10 |
| WO2023217130A1 (en) | 2023-11-16 |
| CN117025502A (en) | 2023-11-10 |
| WO2023217132A1 (en) | 2023-11-16 |
| WO2023217125A1 (en) | 2023-11-16 |
| CN117025505A (en) | 2023-11-10 |
| WO2023217129A1 (en) | 2023-11-16 |
| CN117018032A (en) | 2023-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11821006B2 (en) | Method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord | |
| TWI535377B (en) | Storage, culture and application of umbilical cord tissue and its derived cells | |
| WO2010069204A1 (en) | Pluripotent stem cells, method for preparation thereof and uses thereof | |
| CN110179826B (en) | Human umbilical cord mesenchymal stem cell source stem cell factor microvesicle preparation and preparation method thereof | |
| US20050059152A1 (en) | In vitro culture of mesenchymal stem cells (MSC) and a process for the preparation thereof for therapeutic use | |
| JP6711757B2 (en) | Method to differentiate into hepatocytes using pluripotent stem cells derived from mesenchymal stem cells | |
| CN115975914B (en) | Method for inducing pluripotent stem cells by reprogramming chemical small molecule drugs | |
| WO2019199234A1 (en) | A method of inducing or improving wound healing properties of mesenchymal stem cells | |
| CN112852709B (en) | Method for culturing mouse lung organoid | |
| CN114807034A (en) | Preparation method of Muller cells derived from human pluripotent stem cells | |
| US11186828B2 (en) | Method of making human cells expressing OCT4, SOX2, and Nanog using an Ecklonia cava extract | |
| CN116355832A (en) | Method for preparing human pancreatic beta cells by inducing differentiation of iPSCs derived from urine epithelial cells and application of iPSCs | |
| WO2019199230A1 (en) | A method of transporting mesenchymal stem cells by means of a cell culture medium and a method of administering stem cells to wounds | |
| CN117018033A (en) | Biological agent containing myocardial precursor-like cells, and preparation method and application thereof | |
| CN107058225B (en) | Compound induction culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by adopting culture medium | |
| CN115612664A (en) | Scleral plate osteogenesis/cartilage interstitial precursor cells derived from human pluripotent stem cells, and preparation method and application thereof | |
| CN110592007B (en) | Mesenchymal stem cell and preparation method and application thereof | |
| US20200325443A1 (en) | Method of inducing or improving wound healing properties of mesenchymal stem cells | |
| CN111394301A (en) | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity | |
| CN118048296B (en) | Culture system, kit and method for reprogramming cells | |
| CN117264897A (en) | Culture medium for culturing gastric cancer organoids and application thereof | |
| CN116790493A (en) | Menstrual blood source stem cell exosome delivery system and preparation method and application thereof | |
| CN116333971A (en) | Method for improving differentiation efficiency of human induced pluripotent stem cells to insulin-producing cells by co-culturing human mature islet cells and application | |
| CN115386548A (en) | Preparation method of nasal cavity mucosa-derived mesenchymal stem cells | |
| CN118064350A (en) | Kit for constructing endometrium organoid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication |