WO2023049079A2 - Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof - Google Patents
Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof Download PDFInfo
- Publication number
- WO2023049079A2 WO2023049079A2 PCT/US2022/044024 US2022044024W WO2023049079A2 WO 2023049079 A2 WO2023049079 A2 WO 2023049079A2 US 2022044024 W US2022044024 W US 2022044024W WO 2023049079 A2 WO2023049079 A2 WO 2023049079A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- insulin
- cells
- glucagon
- secreting
- cell population
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 91
- 238000000034 method Methods 0.000 title claims abstract description 69
- 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 title claims description 368
- 108090001061 Insulin Proteins 0.000 title claims description 184
- 102000004877 Insulin Human genes 0.000 title claims description 184
- 229940125396 insulin Drugs 0.000 title claims description 184
- 210000002660 insulin-secreting cell Anatomy 0.000 title claims description 84
- 210000001511 glucagon-secreting cell Anatomy 0.000 title claims description 83
- 206010012601 diabetes mellitus Diseases 0.000 title claims description 47
- 210000004923 pancreatic tissue Anatomy 0.000 title description 30
- 230000001225 therapeutic effect Effects 0.000 title description 9
- 208000016222 Pancreatic disease Diseases 0.000 claims abstract description 15
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 187
- 230000003248 secreting effect Effects 0.000 claims description 50
- 102000051325 Glucagon Human genes 0.000 claims description 47
- 108060003199 Glucagon Proteins 0.000 claims description 47
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 47
- 229960004666 glucagon Drugs 0.000 claims description 47
- 210000004153 islets of langerhan Anatomy 0.000 claims description 43
- 239000006143 cell culture medium Substances 0.000 claims description 34
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 33
- 229920001184 polypeptide Polymers 0.000 claims description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 28
- 210000000496 pancreas Anatomy 0.000 claims description 25
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 claims description 24
- 102100040120 Prominin-1 Human genes 0.000 claims description 24
- 238000013188 needle biopsy Methods 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 238000011282 treatment Methods 0.000 claims description 13
- 238000000338 in vitro Methods 0.000 claims description 12
- 206010033645 Pancreatitis Diseases 0.000 claims description 11
- 238000002513 implantation Methods 0.000 claims description 11
- 238000001802 infusion Methods 0.000 claims description 10
- 208000024691 pancreas disease Diseases 0.000 claims description 10
- 230000001902 propagating effect Effects 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 230000037396 body weight Effects 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 4
- 150000001413 amino acids Chemical group 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 5
- 230000001413 cellular effect Effects 0.000 description 38
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 25
- 239000008103 glucose Substances 0.000 description 25
- 238000002054 transplantation Methods 0.000 description 24
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 17
- 239000002953 phosphate buffered saline Substances 0.000 description 17
- 125000003275 alpha amino acid group Chemical group 0.000 description 16
- 241000124008 Mammalia Species 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 210000000130 stem cell Anatomy 0.000 description 13
- 210000004369 blood Anatomy 0.000 description 12
- 239000008280 blood Substances 0.000 description 12
- 230000000644 propagated effect Effects 0.000 description 12
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 210000004748 cultured cell Anatomy 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 230000000638 stimulation Effects 0.000 description 10
- 239000012981 Hank's balanced salt solution Substances 0.000 description 9
- 230000003914 insulin secretion Effects 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 238000001574 biopsy Methods 0.000 description 8
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 239000012894 fetal calf serum Substances 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- 208000000668 Chronic Pancreatitis Diseases 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 5
- 229930182816 L-glutamine Natural products 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 206010033649 Pancreatitis chronic Diseases 0.000 description 5
- -1 SST Proteins 0.000 description 5
- 239000000654 additive Substances 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 201000001421 hyperglycemia Diseases 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 4
- 101150022636 MAFB gene Proteins 0.000 description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 4
- 229960003942 amphotericin b Drugs 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 229960003405 ciprofloxacin Drugs 0.000 description 4
- 238000005138 cryopreservation Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 101000727462 Homo sapiens Reticulon-3 Proteins 0.000 description 3
- 101001132698 Homo sapiens Retinoic acid receptor beta Proteins 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 3
- 102100041030 Pancreas/duodenum homeobox protein 1 Human genes 0.000 description 3
- 101710144033 Pancreas/duodenum homeobox protein 1 Proteins 0.000 description 3
- 101150075928 Pax4 gene Proteins 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- 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 3
- 230000006378 damage Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 238000005538 encapsulation Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 2
- 108010022452 Collagen Type I Proteins 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 2
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 2
- 108010032788 PAX6 Transcription Factor Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 229940096422 collagen type i Drugs 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000002124 endocrine Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000008029 eradication Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012595 freezing medium Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000009707 neogenesis Effects 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229960001052 streptozocin Drugs 0.000 description 2
- 230000009469 supplementation Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 101150109188 CKX3 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102100039511 Chymotrypsin-C Human genes 0.000 description 1
- 102000016736 Cyclin Human genes 0.000 description 1
- 108010058546 Cyclin D1 Proteins 0.000 description 1
- 102000006311 Cyclin D1 Human genes 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 101100129232 Danio rerio mafaa gene Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000018711 Facilitative Glucose Transport Proteins Human genes 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 108091052347 Glucose transporter family Proteins 0.000 description 1
- 101001098029 Homo sapiens 40S ribosomal protein S2 Proteins 0.000 description 1
- 101000889306 Homo sapiens Chymotrypsin-C Proteins 0.000 description 1
- 101001081567 Homo sapiens Insulin-like growth factor-binding protein 1 Proteins 0.000 description 1
- 101001065568 Homo sapiens Lymphocyte antigen 6E Proteins 0.000 description 1
- 101000603702 Homo sapiens Neurogenin-3 Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 102100027636 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 1
- 102000036770 Islet Amyloid Polypeptide Human genes 0.000 description 1
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 description 1
- 101150051019 Klrg1 gene Proteins 0.000 description 1
- 102100032131 Lymphocyte antigen 6E Human genes 0.000 description 1
- 101150084866 MAFA gene Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 102000008730 Nestin Human genes 0.000 description 1
- 108010088225 Nestin Proteins 0.000 description 1
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 description 1
- 108050000588 Neurogenic differentiation factor 1 Proteins 0.000 description 1
- 102100038553 Neurogenin-3 Human genes 0.000 description 1
- 101150081664 PAX6 gene Proteins 0.000 description 1
- 102100036691 Proliferating cell nuclear antigen Human genes 0.000 description 1
- 101710183548 Pyridoxal 5'-phosphate synthase subunit PdxS Proteins 0.000 description 1
- 102100035459 Pyruvate dehydrogenase protein X component, mitochondrial Human genes 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700042768 University of Wisconsin-lactobionate solution Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- PLOPBXQQPZYQFA-AXPWDRQUSA-N amlintide Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)CSSC1)[C@@H](C)O)C(C)C)C1=CC=CC=C1 PLOPBXQQPZYQFA-AXPWDRQUSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 230000006329 citrullination Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 150000001875 compounds Chemical group 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000013118 diabetic mouse model Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008393 encapsulating agent Substances 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 102000044741 human RPS2 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000000774 hypoallergenic effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 210000005055 nestin Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000020925 non fasting Nutrition 0.000 description 1
- 231100001222 nononcogenic Toxicity 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000009996 pancreatic endocrine effect Effects 0.000 description 1
- 230000004203 pancreatic function Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 210000004043 pneumocyte Anatomy 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011536 re-plating Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 210000002325 somatostatin-secreting cell Anatomy 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Chemical group 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012808 vapor phase Substances 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic 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
- A61K35/39—Pancreas; Islets of Langerhans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Definitions
- Cell-based therapies offer the promise of treating and altering the course of pancreatic disorders, such as Type 1 diabetes (T1 D), which cannot be addressed adequately by existing therapies, yet cell-based therapies present myriad issues, mainly related to safety and efficacy and scalability of manufacture. Many of the problems associated with cell-based therapies are described in Engineering the next generation of cell-based therapeutics by Bashor, C.J., et al., Nat Rev Drug Discov (2022) (and available online at https://doi.org/10.1038/s41573-022-00476-6).
- compositions and methods for generating compositions comprising cell-based therapeutics useful for treating pancreatic disorders comprising Type 1 diabetes.
- a composition as disclosed herein comprises an insulin and glucagon secreting cell population generated from non-insulin secreting pancreatic cells collected via needle biopsy from a Type 1 diabetic donor pancreas.
- a composition as disclosed herein comprises an insulin-and glucagon secreting cell population generated from pancreatic cells collected via needle biopsy from a patient or donor suffering from chronic pancreatitis.
- non-insulin secreting Type 1 diabetic pancreatic cells are treated, in vitro, with an islet cell culture medium comprising a base medium and an effective amount of a polypeptide according to an amino acid sequence listed in SEQ ID 1 or 2, wherein treatment causes the treated cells to differentiate and propagate into a population of islet progenitor cells that secrete both insulin and glucagon in response to stimuli and are CD133 positive.
- the resulting insulin and glucagon secreting progenitor cells can be propagated to a desirable cell count for subsequent use in transplantation or injection and as a cell-based therapeutic for Type 1 diabetes or chronic pancreatitis.
- the cellular composition comprising an effective amount of an insulin and glucagon secreting progenitor cell population may be administered to a subject by infusion, injection, transplantation, intra portal delivery, or by other suitable delivery means such as with a medical device, as method for restoring secretion of insulin and glucagon in response to stimuli.
- compositions and methods disclosed herein have implications for producing large volumes of insulin and glucagon secreting pancreatic cells useful for cell-based therapies and cellular transplantations, namely autologous or allogenic transplantation for the treatment of Type 1 diabetes or chronic pancreatitis.
- Also disclosed herein is a method of treating a pancreatic disorder, such as Type 1 diabetes or pancreatitis, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising an insulin and glucagon secreting pancreatic cell population, wherein the insulin and glucagon secreting pancreatic cell population is generated by treating pancreatic cells collected from diseased pancreatic tissue (for example, from a Type 1 diabetic subject or one suffering from chronic pancreatitis), such as via needle biopsy, with an islet cell culture media comprising a base medium and a peptide comprising an amino acid sequence according to SEQ ID 1 or 2.
- the composition when administered to a subject in need thereof, provides delivery of healthy pancreatic progenitor cells to a target site in the subject, wherein the healthy pancreatic progenitor cells are capable of producing insulin and glucagon in response to stimulation.
- composition comprising a therapeutically effective amount of insulin and glucagon secreting progenitor cells generated by the methods disclosed herein may be used as an autologous or allogenic cell based therapeutic to supplement the loss of insulin production or replace insulin production in patients with Type 1 diabetes, or with other diseases characterized by severe insulin deficiency, such as after total or partial pancreatectomy, with and without autologous or allogenic islet transplantation.
- compositions may be prepared for transplantation by supplementing the compositions with human serum albumin and/or human serum from the recipient prior to administration.
- an islet cell culture medium useful for stimulating growth, propagation and differentiation of insulin and glucagon secreting cells from pancreatic cells derived from Type 1 diabetic pancreatic tissue comprises a base medium and an effective amount of a polypeptide, wherein the polypeptide comprising an amino acid sequence according to one or more of SEQ ID NO. 1 - 2 (listed in Table 1), or active fragment thereof.
- the polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO. 01 ; in another embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO.
- the polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2; in another embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2.
- a cellular composition comprises a population of insulin and glucagon secreting cells generated by treatment of isolated Type 1 diabetic pancreas tissue with an islet cell culture medium comprising a base medium and an effective amount of a polypeptide according to SEQ ID NO. 1 or 2 or active fragment thereof; further comprising measuring the response of the cells to glucose; wherein the cellular composition comprises a population of cells capable of secreting insulin and glucagon in response to appropriate stimuli.
- a method of manufacturing a cellular composition comprises applying, in vitro, an islet cell culture medium comprising a base medium and an effective amount of a polypeptide according to SEQ ID NO. 1 or 2, or active fragment thereof, to human pancreatic tissue collected from a Type 1 diabetic; incubating the cells in the islet cell culture medium; screening the incubated cells for one or more cell markers selective for CD133 and insulin; and collecting the cells identified by screening as CD133 and insulin-positive from the cultured cell population; continuing to grow the cultured cells until a desired quantity of cells are propagated.
- cellular compositions comprising insulin and glucagon secreting cells derived from T1 D pancreatic tissue are packaged or encapsulated for administration or implantation into a mammal for in vivo therapy, specifically to restore insulin production and secretion.
- the cellular compositions may be packaged as a delivery solution, or in a delivery vehicle, and administered by implantation, injection or infusion, whether administration is systemic, localized or directed to a target site.
- a method of treating a pancreatic disorder, wherein the pancreatic disorder is characterized by an insufficient production of insulin, in a mammal comprises: culturing, in vitro, a population of insulin and glucagon secreting cells from pancreatic tissue collected from a Type 1 diabetic donor pancreas, in an islet cell culture medium comprising a base medium and an effective amount of a polypeptide according to SEQ ID NO.
- a population of CD133 positive, insulin and glucagon secreting cells are produced; further comprising isolating and expanding the population to generate a predominantly (at least 60% or greater) insulin and glucagon secreting cell population; and further comprising collecting the insulin and glucagon secreting cells and suspending the collected cells in a physiologic buffer, such as phosphate buffered saline (PBS) or Hanks Balanced Salt Solution (HBSS), and implanting or injecting into a mammal a cellular composition comprising the insulin and glucagon secreting cells in suspension with physiologic buffer.
- a physiologic buffer such as phosphate buffered saline (PBS) or Hanks Balanced Salt Solution (HBSS)
- the composition may be delivered as an aqueous solution, a suspension, an encapsulation, a microencapsulation, and/or an encapsulated, or semisolid formulation; wherein the composition may be delivered to the mammal via one or more of an injection, infusion, omental or peritoneal pouch, surgical implantation, or via packaging the composition as part of a device to a target site in the mammal.
- a cellular composition comprises an insulin and glucagon secreting cell population further comprising one or more of a buffer, a pharmaceutically acceptable carrier, a pharmaceutically acceptable additive, an antibiotic or other pharmaceutical agent.
- IPCs insulinproducing cells
- FIG. 1 shows that T1 D-derived insulin and glucagon secreting cells propagated according to methods herein are greater than 50% triple positive for CD 133, insulin and glucagon.
- FIG. 2 shows T1 D pancreatic tissue cultured with islet cell culture medium comprising a peptide according to SEQ ID NO. 1 or 2 produce cells that secrete insulin in response to glucose stimulation, as shown by the stimulation index, which is the ratio between insulin secretion under high glucose conditions vs basal release under unstimulated conditions.
- the stimulation index which is the ratio between insulin secretion under high glucose conditions vs basal release under unstimulated conditions.
- a value above 2 represents glucose responsiveness in the cells.
- Sample 1 is a cell population propagated from normal pancreatic tissue according to methods herein;
- Samples 2 - 4 are cell populations propagated from T1 D pancreatic tissue according to methods herein.
- FIG. 3 shows the down regulation and up regulation of gene (families) associated with pancreatic function in a single T1 D biopsy-derived cell preparation compared to native pancreatic tissue.
- Each family of genes includes 5 to 13 genes.
- cell populations comprising insulin and glucagon secreting cells generated according to the methods herein exhibit upregulation of gene families essential for mature islet cells, beta cell maturation, GSIS, insulin granules and cell cycle.
- FIG. 4 shows serum insulin levels following transplantation in streptozotocin (STZ, a 0- cell-specific toxin that induces irreversible damage to pancreatic islets and induces diabetes) treated mice of cellular compositions comprising insulin and glucagon propagated according to methods herein.
- STZ streptozotocin
- the cellular compositions were shown to promote the secretion of human insulin in vivo, which was present in the serum for up to 100 days of STZ mice treated with cellular compositions disclosed herein.
- FIG. 5 shows that insulin and glucagon secreting cells generated from Type 1 diabetic (T1 D) cells propagated according to methods herein can normalize blood glucose levels upon injection in a STZ diabetic mouse model.
- M1 - 4 refer to STZ mouse sample numbers, ie, Mouse-1 , Mouse-2, Mouse-3, Mouse-4.
- SEQ ID NO 1 or 2 refers to a protein, polypeptide, peptide fragment, or analogue thereof, and including any modification thereto, having an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO 1 or 2 (See Table 1). Also contemplated is a peptide fragment, or analogue thereof, and including any modification thereto, having an amino acid sequence having at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO(s): 1 and 2.
- insulin and glucagon secreting cells or “insulin and glucagon secreting islet cells” or “insulin and glucagon secreting progenitor cells” are used interchangeably herein to refer to the cellular composition comprising insulin and glucagon secreting cells and/or cell population(s), which are generated from non-insulin secreting T1 D pancreatic cells according to the methods described herein, and which are positive for the cell markers: CD133, insulin, glucagon, and that produce insulin and glucagon in response to stimuli, and are further characterized by cell markers PDX-1, SST, HAP, Pax4, Pax6, Nkx2, Nkx6, NeuroDI , MafA, MafB.
- propagation refers to an increase in the number of cells present in a culture as a result of cell division.
- culture refers to removal or isolation of cells from an environment (such as in a host mammal) and their subsequent growth in a favorable artificial environment in vitro.
- “Cultured cells” is intended to include sub- cultured (i.e., passaged) by transferring the cells to a new vessel with fresh growth medium to provide more room for continued growth, differentiation and/or propagation.
- pancreatic cells include those cells normally found in the pancreas of a mammal, and include pancreatic islet cells, e.g., glucagon-synthesizing alpha cells, insulin-producing beta cells, and any combination thereof.
- target site refers to a region in the recipient host (a mammal, preferably human) that requires treatment or supplementation.
- the target site can be a single region within a specific organ or can be multiple regions in the host.
- the supplementation or replacement results in the same physiological response as normal tissue, such as pancreatic tissue, whether or not targeting the pancreas.
- the terms “treat,” “treating” or “treatment,” and other grammatical equivalents as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and prophylaxis.
- the terms further include achieving a therapeutic benefit and/or a prophylactic benefit.
- therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
- a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
- an "effective amount” refers to an amount that is sufficient to achieve the stated effect.
- a therapeutically effective amount to treat a condition is an amount capable of achieving a clinically relevant end-point in a patient or patient population.
- administration of an effective amount of a composition comprising insulin and glucagon secreting cells is an amount of approximately 1 .2 to about 2.5 x 10 6 cells/kg, or greater than 200 x 10 6 cells, in order to produce sufficient insulin to cause a reduction in blood glucose levels to approximately 100 to 125 mg/dl (5.6 to 6.9 mmol/L), or to under 250 mg/dl.
- compositions include approximately 3 x 10 6 cells to about 25 x 10 6 cells per kg of body mass; or approximately 5 x 10 6 to about 10 x 10 6 millions cells/kilo.
- the appropriate dose of the composition may depend on the route of administration, such as injection or infusion or transplantation, and may depend on the subject being treated as well as the severity of the condition to be treated.
- scaling methods such as allometric scaling, it is possible to predict suitable and exemplary dosage ranges for the administration of compositions, as disclosed herein, to adult humans. Dose scaling is an empirical approach, is well characterized and understood in the art.
- the human pancreas based on the literature, has between 6 x 10 5 to about 2 x 10 6 islets; hence there are approximately 600 x 10 6 islet cells in the normal human pancreas, with half of them being beta cells. Considering that an islet mass of 30% is sufficient to maintain normoglycemia, a dose of 1.2 x 10 6 of the insulin and glucagon secreting cells as disclosed herein would be expected to sufficiently replace the insulin producing capacity of a non-diseased human pancreas.
- sequence identity refers to the identity between two or more amino acid sequences expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. The percentage identity is calculated over the entire length of the sequence. Homologs or orthologs of amino acid sequences possess a relatively high degree of sequence identity when aligned using standard methods. This homology is more significant when the orthologous proteins are derived from species which are more closely related (e.g., human and mouse sequences), compared to species more distantly related (e.g., human and C. elegans sequences). Methods of alignment of sequences for comparison are well known in the art.
- NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990), which is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894, US) and on the Internet.
- NCBI NCBI Basic Local Alignment Search Tool
- Representative cultures of the insulin and glucagon secreting cells characterized herein have been deposited with ATCC on September 7, 2022 [Accession Number ] under the terms of the Budapest Treaty. Cultured cells, propagated cells, isolated cells, and the like may be protected from external mutagenic stimuli, such as, for example UV radiation.
- T1 D pancreatic tissue Utilizing the methods disclosed herein, it has been determined that 30 days post isolation of T1 D pancreatic tissue, a single pancreas can product 77 billion insulin and glucagon secreting (islet) cells; 2 trillion cells by day 60, which is enough cells to infuse 100 - 150 patients in need of treatment (depending on the severity of illness or dosage used), or bank (freeze/store) cells for future expansion and re-infusion.
- Amino acid residues of the active agents may be post-translationally modified or conjugated with other functional or non-functional molecular groups. See, e.g., Guo et al. Mol. Biosyst. 7(7): 2286-2295, 2011 , describing generally antagonistic citrullination and methylation of human ribosomal protein S2 (e.g., SEQ ID NO. 1). Naturally, such modified amino acid residues are included in the amino acid sequences and within the scope of the active agents described herein.
- polypeptides and/or polypeptide fragments according to, for example, SEQ ID NO(s): 1 and 2 may be produced under conditions known in the art for protein production, such as production in bacteria, yeast or by synthetic means, or as described in United States Patent Application No.15/811,060.
- the cellular compositions may be packaged as a delivery solution, or in a delivery vehicle comprising a medical device, and may be administered by implantation, injection, or infusion, whether administration is parenteral, systemic, localized, or directed to a target site.
- encapsulation of in vitro- generated insulin and glucagon secreting islet cells and implantation into a mammal have been previously characterized in the art (see, for example, Altman, et al., 1984, Trans. Am. Soc. Art. Organs 30:382-386, and U.S. Pat. No.
- the encapsulant is hypoallergenic, is easily and stably situated in a target tissue, and provides added protection to the implanted cellular composition, to protect and prevent from the destruction of the implanted cells.
- the appropriate implantation dosage in humans can be determined from existing information relating to ex vivo islet transplantation in humans, further in vitro and animal experiments, and from human clinical trials. From data relating to transplantation of ex vivo islets in humans, it is expected that about 8,000-12,000 islets per patient kg may be required. Assuming long-term survival of the implants following transplantation, less than the number of naturally occurring islets (about 2 million in a normal human adult pancreas), or possibly even less than the amount used in ex vivo islet transplantation may be necessary.
- the cellular compositions are of therapeutic benefit for treating a pancreatic disorder, wherein the pancreatic disorder is hyperglycemia, Type 1 diabetes, or chronic pancreatitis, in a mammal, which comprises: administering a therapeutically effective amount of the insulin and glucagon secreting cell population, thereby providing a treatment for the pancreatic disorder.
- the composition comprising a therapeutically effective amount of an insulin and glucagon secreting cell population may be formulated as an aqueous solution, a suspension, an encapsulation, a microencapsulation, and/or an encapsulated, or semi-solid formulation; wherein the composition may be delivered to the patient in need via one or more of an injection, infusion, omental or peritoneal pouch, port, surgical implantation, or via packaging the composition as part of a device to a target site in the mammal.
- a composition comprises an insulin and glucagon secreting progenitor cell population further comprising one or more of a pharmaceutically acceptable excipients, and/or one or more of a pharmaceutically acceptable additive and/or one or more a pharmaceutical agent.
- Suitable excipients and additives include, but are not limited to buffering agents such as PBS, or HBSS, amino acids, stabilizer or bulking agents, surfactants, antimicrobial/preservatives, antifungal agents, metal ions/chelators, polymers, polyanions, salts, sugars, cyclodextrin based excipients, lyoprotectants, solubilizing agents, antioxidants, complexing agents, anti-adhesive agents, dispersing agents, serum additives.
- the disclosure provides a method for treating a mammal, preferably a human, suffering from, or at risk of developing Type 1 diabetes or severe pancreatitis, which comprises: removing pancreatic tissue from the mammal; culturing the excised pancreatic tissue in vitro to propagate a population of insulin and glucagon secreting islet cells; and implanting, transplanting, infusing, injecting, or otherwise inserting the population of insulin and glucagon secreting islet cells, alone or together with a medical or delivery device, into the mammal.
- Cell cultures performed under the Examples were incubated at 37°C under standard CO (5%) conditions; culturing (plating, splitting of cells) was performed using standard aseptic techniques and conditions in a vertical laminar flow hood. Unless stated otherwise, cells (including controls) were cultured in the islet cell culture media described in Table 2 and 3. Cells were split when they reached confluency of approximately 70 - 80% in culture.
- the splitting technique involved removal of supernatant from the culture plates (supernatant was preserved). Plates were then washed with 2 - 5 ml PBS (wash was preserved). Cells were detached using approximately 3 - 5 ml of trypsin (such as available from Sigma-Aldrich) by incubating the cells in the presence of trypsin at 37°C for approximately 3 - 5 minutes until cells detached. Plates were then washed a second time with PBS. The trypsinized cells, as well as the preserved PBS washes and collected cell culture supernatant, were then centrifuged at 300 g for 7 minutes at 4°C.
- trypsin such as available from Sigma-Aldrich
- the resulting supernatant was decanted, and the pellet resuspended in 2 ml PBS, and recentrifuged. The supernatant was then removed, and the pellet resuspended in culture medium containing SEQ ID 1 or 2 and re-plated at a cell density of ⁇ 1000 cells/cm2. While the Examples may refer to cell culture plates, it will be understood that cell culture flasks are an acceptable alternative to plates.
- Type 1 diabetic (T1 D) donor patient 58-year-old female; 53 years diabetic.
- Biopsies of 1x1 mm3 were obtained from the donor pancreas (procured from a center for organ recovery and education) preserved on ice in a commercially available solution (sold under names such as Viaspan, Belzer UW, Bel-Gen or StoreProtec).
- CMRL such as Mediatech #99-663-CV Transplant Medium (CMRL 1066) without phenol red
- L-glutamine 2mmol
- Ciprofloxacin 2mg/L
- Amphotericin B 0.1mg/L
- Penicillin 100,000 units/L
- Streptomycin 100,000 micrograms/L
- FCS fetal calf serum
- Control culture medium is one which comprises CMRL supplemented with L-glutamine (2mmol), Ciprofloxacin (2mg/L), Amphotericin B (0.1mg/L) Penicillin (100,000 units/L) and Streptomycin (100,000 micrograms/L) and fetal calf serum (FCS) (10%) and human serum (10%) (without the addition of a polypeptide according to SEQ ID NO: 1
- Tissue is cultured on plates or in flasks coated with an attachment factor mixture (AFM) comprising Collagen Type I (Collagen from rat tail, Sigma-Aldrich C3867) and Endothelial Cell Attachment Factor (ECAF, Sigma-Aldrich E9765).
- AFM attachment factor mixture
- ECAF Endothelial Cell Attachment Factor
- Various ratios of ECAF and collagen may be used, including but not limited to a 50/50 ratio of collagen to ECAF. Briefly, plates (or flasks) were prepared by applying a thin layer of AFM (between 3 - 10 ml) to the plates, and after setting for 30 minutes the excess AFM was removed. The plates were allowed to dry for 45 minutes in a hood. Prior to use the plates were washed with PBS to remove any potential contaminants. The collected tissue was incubated on the AFM-treated plates in islet cell culture medium supplemented with a polypeptide according to SEQ ID NO. 1 or 2 until cells began to mobiliz
- biopsy-derived cells After 12-15 days in culture the biopsy-derived cells were mobilized and began to adhere to the dish and to proliferate. During a subsequent 4 - 6-week period, the adherent cells continued to proliferate, became confluent and continue to multiply, doubling every 3 days.
- the biopsy-derived cells in culture exhibited morphological similarities to cellular compositions comprising insulin secreting cells generated from non-diabetic donors, previously characterized in US 2021/0205371 , published July 8, 2021 . Similarly, the cells formed islet-like cell clusters with size consistent with that of islets of Langerhans and were shown to secrete insulin in response to stimulation with glucose.
- the resulting cell cultures derived from T1 D pancreatic biopsy tissue were also assayed for the expression of CD133, as well as intracellular insulin and glucagon expression, by fluorescence activated cell sorting (FACS) using a flow cytometry instrument (Becton Dickinson FACS Aria cell sorter).
- FACS fluorescence activated cell sorting
- the cultured cells were initially labeled for CD 133 expression and then fixed and permeabilized with FOXP3 Fixation/Permeabilization Buffer, as per the manufacturer's instructions, and stained with conjugated fluorometric antibodies for glucagon and intra-cellular insulin, respectively.
- FACS analysis followed FOXP3 Fixation Permeabilization and staining with conjugated fluorometric antibodies.
- T1 D pancreas biopsy-derived cells cultured in islet cell culture medium comprising a peptide according to SEQ ID NO. 1 or 2 were found to be positive for CD133, glucagon and insulin (referred to herein as “triple positive”), specifically, 48 - 73% triple positive for insulin, glucagon and CD133, 26 - 42% double positive for glucagon and CD133, 14 - 18% triple negative for insulin, glucagon and CD133, 9 - 23% single positive for glucagon, and 0 - 7% single positive CD133, and negative for insulin and glucagon; negative for insulin and CD133; and negative for insulin. It was determined that, overall, the cultured cell population was greater than 65% positive for insulin, CD133 and glucagon (triple positive). (See FIG. 1)
- the insulin and glucagon secreting cells were subjected to a glucose stimulation insulin secretion assay. Approximately 1 x 10 6 cells/well were plated in a 6-well dish and underwent 2 stimulus conditions to assess insulin secretion. The cells were incubated for 30 minutes with either: (1) islet cell culture medium (See Table 2); or (2) islet cell culture medium supplemented with a higher glucose concentration (final concentration 16.7 mM, as a stimulus for insulin secretion). Following incubation, the supernatant was stored at -20°C until undergoing a standard ELISA assay for insulin quantification.
- the cells cultured in the islet cell culture medium supplemented with a higher glucose concentration were shown to secrete higher insulin amounts than those cells treated with standard islet cell culture medium (unstimulated controls).
- FIG 2 shows the stimulation index (the measure of the ratio between insulin secretion with high glucose versus basal release) of the cells treated with islet cell culture medium supplemented with high glucose, compared to unstimulated controls.
- RNA sequencing methods characteristics of several cell populations were determined, including pancreatic tissue from a deceased donor, insulin and glucagon secreting cells generated by methods described herein, and denovo-pseudo-islets from the same deceased donor. Characteristics of the various cell types were assessed using an Illumina® NovaSeqTM platform. Markers assessed were: insulin, glucagon, PDX-1 , SST, HAP, Pax4, Pax-6, NKx2, Nkx6, NeuroDI , MafA, and MafB.
- compositions comprising insulin and glucagon secreting cells generated from T1 D-derived pancreatic tissue exhibited a substantial decrease in markers of exocrine function (AMY and CTRC), which are expressed in the native pancreas.
- markers of proliferation PCNA and CCND1 cyclin family
- increased in expression in the cellular compositions potentially signifying dedifferentiation to motile, proliferating cells, consistent with the observation of cell expansion in vitro.
- the insulin and glucagon secreting cells generated from pancreatic tissue collected by needle biopsy from a T1D donor underwent morphological re-arrangement and spontaneously generated de novo-pseudo-islets.
- islet-like structures were characterized by a significant increase in expression of an endocrine progenitor and islet signature markers that included insulin, glucagon, PDX-1 , SST, HAP, Pax4, Pax-6, NKx2, Nkx6, NeuroDI , MafA, and MafB, while exhibiting downregulation of the cell proliferation pathways.
- IGFBP1 a marker of p— cell regeneration, was found expressed at higher levels in pseudo-islets when compared to the cellular compositions comprising insulin and glucagon secreting cells, whereas the stem cell markers LY6E and PROM1 were more highly expressed in the cellular compositions, suggesting the maturation to a more differentiated endocrine progenitor cell population committed to generate alpha, beta and delta cells.
- Microarray mRNA profile of the cells confirmed a gene profile compatible with a pancreatic endocrine islet cell population, with expression of insulin, and glucagon.
- T1D-derived cell populations also expressed the pancreatic transcription factors PDX1 , Nkx6, ngn3, NeuroD, and MafA and MafB, as well as the islet neogenesis factor nestin, the glucose transporter Glut-2, the secretory product of 0- cells IAPP, and somatostatin which, is secreted by islet 5-cells.
- pancreatic transcription factors PDX1 , Nkx6, ngn3, NeuroD, and MafA and MafB as well as the islet neogenesis factor nestin, the glucose transporter Glut-2, the secretory product of 0- cells IAPP, and somatostatin which, is secreted by islet 5-cells.
- mice were injected with approximately 2.5 x 10 6 T1 D-derived biopsy-derived insulin and glucagon secreting cells (cells were counted using a Neubauer Chamber) twice, one week apart (total of 2 doses).
- T1 D-derived biopsy-derived insulin and glucagon secreting cells cells were counted using a Neubauer Chamber
- Serum was obtained and stored at -20°C until used to measure human insulin and human C-peptide concentrations by ELISA (Abeam and Alpco, respectively).
- T1D-derived Insulin and glucagon secreting cells were generated using methods described herein, for example by culturing T1D pancreatic tissue in a culture medium comprising a polypeptide according to SEQ ID NO. 1 or 2 at a concentration ranging from 3 to 20 pg/ml.
- a culture medium comprising a polypeptide according to SEQ ID NO. 1 or 2 at a concentration ranging from 3 to 20 pg/ml.
- islet cell culture medium is described as follows (and shown in Table 2 and Example 1): CMRL supplemented with L-glutamine (2mmol), Ciprofloxacin (2mg/L), Amphotericin B (0.1mg/L) Penicillin (100,000 units/L) and Streptomycin (100,000 micrograms/L), and a polypeptide according to SEQ ID NO. 1 or 2 ( at 10 pg/ml,) and Fetal calf serum (FCS) (10%) and human serum (10%).
- Control culture medium
- cells Prior to transplantation, cells were cultured for approximately 50 - 60 days. At the time of transplant, cells were detached from the bottom of the plate/flask using trypsin (available from Gibco). Following filtration via a 40pm sterile mesh, single cells were washed in a phosphate buffer solution (without Calcium and Magnesium), Hanks Balanced Salt Solution may also be used (both available from Sigma Aldrich) by spinning (from 180 g - 300 g, for approximately 10 minutes) counted and resuspended in approximately 200 pl of sterile PBS (or HBSS) at a dose of 1.25 x 10 6 /1 OOpI and injected into anesthetized mice via the tail vein. Injection was carried out over the course of one minute.
- trypsin available from Gibco.
- Human insulin was detected in all mice on day 14 and day 30 (the concentration range was measured as 12.5 to 33 pmol/L) post first injection. Human C-peptide confirmed positive results when measured on day 30 (at a level up to 10 pmol).
- the cells can be resuspended in PBS at a more concentrated volume of 20 - 50 pl and inserted in the sub capsular space (occupying an area of approximately 1cm 2 ) of the kidney by using a PE-50 tubing connected to a syringe (method described by Bertera et al, Journal of Transplantation Volume 2012, Article ID 856386, 9 pages doi:10.1155/2012/856386).
- a PE-50 tubing connected to a syringe method described by Bertera et al, Journal of Transplantation Volume 2012, Article ID 856386, 9 pages doi:10.1155/2012/856386.
- larger doses of cells can be administered at once (for example, from about 5 - 10 x 10 6 cells), in contrast to intravenous injection of human cells, of which doses higher than 2.5 x 10 6 cells may not be as well tolerated.
- compositions comprising insulin and glucagon secreting cells propagated from murine islets, using the islet cell culture medium described herein, can be safely injected and/or transplanted via the kidney capsule into animals, such as mice.
- human insulin secretion following implantation of insulin and glucagon secreting cells under the kidney capsule in mice was detected for a duration of at least 100 days (See FIG. 4).
- the insulin and glucagon secreting cells propagated were collected, then suspended in a solution, such as one comprising Hanks Balanced Salt Solution (HBSS) or Phosphate Buffered Saline (PBS) (available from Sigma Aldrich) to form a cellular composition.
- HBSS Hanks Balanced Salt Solution
- PBS Phosphate Buffered Saline
- the cellular composition comprising insulin and glucagon secreting cells suspended in Hanks Balanced Salt Solution was transplanted under the kidney capsule of streptozotocin-diabetic (STZ) nude mice (5-6 weeks old; Jackson Laboratory, Bar Harbor, ME) using known methods, such as an approach described in Bertera et al 2012.
- STZ streptozotocin-diabetic
- mice Prior to transplantation, mice were injected with streptozotocin (240mg/kg IP) and hyperglycemia (non-fasting blood glucose levels > 350mg/dl on 2 consecutive readings) was confirmed.
- a cellular composition is made by detaching the insulin and glucagon secreting cells from culture, such as with trypsin; centrifuging (at 300 g) and counting the detached cells. Approximately 4 x 10 6 cells; were suspended in a solution comprising HBSS; and loaded into a tubing or catheter, such as PE50 tubing. The cellular composition is then transplanted into an STZ mouse by placing the catheter or tubing containing the cellular composition under the kidney capsule of a fully anesthetized STZ mouse, via a small incision of the left flank, and following exposure of the kidney.
- Cellular compositions comprising insulin and glucagon secreting cells generated by treating non-type 1 diabetic pancreatic tissue (human and murine in origin) with an islet cell culture medium comprising a base medium and a polypeptide according to SQ ID NO: 1 or 2 have been shown to secrete insulin following glucose stimulation at sufficient levels to lower blood glucose.
- These cellular compositions comprising approximately 4 x 10 6 cells, when injected intravenously, not only secrete insulin and glucagon but home and engraft in the pancreas.
- Cellular compositions comprising insulin and glucagon secreting cells may be delivered via cell transplantation for the treatment of pancreatic disorders, including diabetes and hyperglycemia.
- the expression of the stem cell marker CD133 correlates with the capacity of cells to engraft long-term, and these cells have the innate capability to migrate and home to injury sites. It was determined that cellular compositions as disclosed herein, when injected into an STZ-diabetic mouse, migrate to the pancreas and are shown to normalize hyperglycemia by a corresponding lowering of blood glucose levels following treatment. For example, six (6) four STZ-treated mice were injected with a cellular composition comprising (approximately) 20 x 10 6 cells isolated from murine pancreatic tissue and treated with islet cell culture medium comprising a polypeptide according to SEQ ID NO. 1 or 2.
- Results showed that by day 10 all mice had lower blood glucose levels; by day 22 post injection two of the four animals exhibited a fasting blood glucose lower than 200 mg/dl. All recipient animals had a decrease in blood glucose levels, two of the animals-maintained blood glucose levels close to 250 mg/dl, with one animal having levels as low as 180 mg/dl on day 85 of the experiment. See FIG. 5.
- an islet cell culture medium comprises CMRL-1066 (Mediatech, #99-663-CV; Transplant Medium (CMRL 1066) without phenol red) supplemented with: 10% Heat Inactivated Fetal Calf Serum (Gibco, #16140071), 10% Human Serum (Gemini, #100512), L-Glutamine (2mM, Gibco, #25030081), Ciprofloxacin (2ml/L, Bioworld, #403100313), Amphotericin-B (0.1mg/L, Gibco, #15290026), Penicillinstreptomycin (100,000 U/L-100,000 pg/L, Gibco, #10378016), and a peptide according to SEQ ID 1 or 2 (range of 3 - 20 pg/ml, such as, 3 pg/ml, 5 pg/ml, or 10.0 pg/ml).
- a cell culture medium for cryopreservation comprises a cryoprotectant medium (Gibco #12648010).
- viability testing was carried out using methods comprising utilizing fluoroscein diacetate (FDA) (Sigma Aldrich #F7378) and propidium Iodide (PI) (Sigma Aldrich #P4170)
- human pancreatic tissue was collected via needle biopsy from human donors affected by Type 1 diabetes or severe pancreatitis and cultured in vitro in islet cell culture medium comprising CMRL 1066 supplemented with 10% fetal calf serum, 10% human serum, 2 mmol/L-glutamine, antibiotics, and a peptide according to SEQ ID NO 1 or 2 at a concentration range of 3 pg/mL to 20 pg/ml, such as 10.0 pg/mL, on plates (or flasks) coated with an attachment factor mixture (AFM) comprising Collagen Type I and Endothelial Cell Attachment Factor (ECAF).
- AFM attachment factor mixture
- ECAF Endothelial Cell Attachment Factor
- ECAF and collagen were used, including a 50/50 ratio of collagen to ECAF.
- a thin layer of AFM (between 3 - 10 ml) was applied and after setting for 30 minutes the excess AFM was removed; the plates were dried for 45 minutes in a hood. Plates are then washed with Phosphate Buffer Solution (PBS) to remove any potential contaminants.
- PBS Phosphate Buffer Solution
- the collected pancreatic tissue was incubated on AFM-treated flasks/plates in islet cell culture medium for 10 to 20 days until cells began to mobilize and proliferate. Cultured cells are split when they reached confluency of approximately 70 - 80% in culture using standard aseptic techniques in a vertical laminar flow hood.
- the splitting technique involved removal of supernatant from the culture plates (supernatant was preserved). Plates are then washed with 2 - 5 ml PBS (wash was preserved). The cultured cells were detached using approximately 3 - 5 ml of trypsin (25% solution) by incubating the cells in the presence of trypsin at 37°C for approximately 3 - 5 minutes until cells detached. The plate was then washed a second time with PBS. The trypsinized cells, as well as the preserved PBS washes and collected cell culture supernatant, were then centrifuged at 1000 rpm for 7 minutes at 4°C. The resulting supernatant was decanted and the pellet resuspended in 2 ml PBS, and centrifuged.
- the supernatant was then removed, and the pellet resuspended in islet cell culture medium comprising SEQ ID 1 or 2, and re-plated at a cell density of ⁇ 1000 cells/cm2.
- Cultured cells that were not used for replating were resuspended at the maximum concentration of 1x10 6 /ml in a cryopreservation medium.
- Cultured cells were centrifuged (1000 rpm for 7 minutes) at 4°C.
- the supernatant was aspirated and replaced by fresh cryopreservation medium and the cells were transferred into a freezing container Mr. Frosty (Thermo-Fisher #5100-0036) overnight and then transferred and stored in vapor phase liquid nitrogen.
- a composition comprising an insulin and glucagon secreting cell population generated from non-insulin secreting pancreatic cells collected via needle biopsy from a Type 1 diabetic donor pancreas, a pancreatitis donor pancreas, or a combination thereof.
- Aspect 2 The composition of Aspect 1 , wherein at least about 50% of the insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin.
- Aspect 3 The composition of any one of Aspects 1 — 2, wherein about 50% to about 100% of insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin, including all values in between, such as, for example, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%; specify percent as a total of medium cell population).
- Aspect 4 The composition of any one of Aspects 1 - 3, wherein the insulin and glucagon secreting cell population comprises from about 3 x 10 6 cells to about 25 x 10 6 cells per kg of body weight in a human recipient, including all values in between, such as, for example about 4 x 10 6 cells, about 5 x 10 6 cells, about 6 x 10 6 cells, about 7 x 10 6 cells, about 8 x 10 6 cells, about 9 x 10 6 cells, about 10 x 10 6 cells, about 11 x 10 6 cells, about 12 x 10 6 cells, about 13 x 10 6 cells, about 14 x 10 6 cells, about 15 x 10 6 cells, about 16 x 10 6 cells, about 17 x 10 6 cells, about 18 x 10 6 cells, about 19 x 10 6 cells, about 20 x 10 6 cells, about 21 x 10 6 cells, about 22 x 10 6 cells, about 23 x 10 6 cells, and about 24 x 10 6 cells; where the mass (in kg) for typical human recipient depends on, for example,
- Aspect 5 The composition of any one of Aspects 1 - 4, wherein the non-insulin secreting pancreatic cells are collected via needle biopsy are obtained from a Type 1 diabetic donor pancreas.
- Aspect 6 The composition of any one of Aspects 1 - 5, wherein the non-insulin secreting pancreatic cells collected via needle biopsy are obtained from a pancreatitis donor pancreas.
- Aspect 7 The composition of any one of Aspectsl - 6, wherein the non-insulin secreting pancreatic cells are isogenic, allogenic, or a combination thereof.
- Aspect 8 A method of treating a pancreatic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of Aspectsl - 7, wherein the pancreatic disorder comprises Type 1 diabetes, pancreatitis, or a combination thereof.
- Aspect 9 The method of Aspect 8, wherein said administering comprises delivering to the subject the therapeutically effective amount of the composition of any one of Aspectsl - 7 via one or more of an injection, infusion, omental or peritoneal pouch, surgical implantation, or via packaging the composition as part of a device to a target site in the subject.
- a method for preparing a composition comprising an insulin and glucagon secreting cell population which comprises: treating in vitro a population of non-insulin secreting Type 1 diabetic pancreatic cells with an islet cell culture medium comprising a base medium and an effective amount of a polypeptide comprising SEQ ID NO. 1, an active fragment of SEQ ID No. 1 , SEQ ID NO. 2, an active fragment of SEQ ID No. 2, or a combination thereof.
- Aspect 11 The method of Aspect 10, wherein the treating further comprises differentiating the population of non-insulin secreting Type 1 diabetic pancreatic cells and propagating the insulin and glucagon secreting cell population.
- Aspect 12 The method of Aspect 10, wherein the treating further comprises differentiating the population of non-insulin secreting Type 1 diabetic pancreatic cells and propagating the insulin and glucagon secreting cell population, wherein the insulin and glucagon secreting cell population at least about 50% of insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin.
- Aspect 13 The method of Aspect 10, wherein the treating further comprises differentiating the population of non-insulin secreting Type 1 diabetic pancreatic cells and propagating the insulin and glucagon secreting cell population, wherein the insulin and glucagon secreting cell population about 50% to about 100% of insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin.
- Aspect 14 The method of Aspect 10, wherein the treating further comprises differentiating the population of non-insulin secreting Type 1 diabetic pancreatic cells and propagating the insulin and glucagon secreting cell population, wherein the insulin and glucagon secreting cell population comprises from about 3 x 10 6 cells to about 25 x 10 6 cells per kg of body weight in a human recipient.
- Aspect 15 The method of any one of Aspects 10 - 14 further comprising extracting the population of non-insulin secreting Type 1 diabetic pancreatic cells from a donor.
- Aspect 16 The method of any one of Aspects 10 - 14 further comprising extracting the population of non-insulin secreting Type 1 diabetic pancreatic cells from a donor via needle biopsy.
- Aspect 17 The method of any one of Aspects 10 - 16 further comprising extracting the population of non-insulin secreting Type 1 diabetic pancreatic cells from an isogenic donor, and allogenic donor, or a combination thereof.
- Aspect 18 The method of any one of Aspects 10 - 17, wherein the islet cell culture medium comprises the polypeptide in amount that ranges from about 3 pg/mL to about 20 pg/mL and all values in between, such as, for example, about 4 pg/mL, about 5 pg/mL, about 6 pg/mL, about 7 pg/mL, about 8 pg/mL, about 9 pg/mL, about 10 pg/mL, about 11 pg/mL, about 12 pg/mL, about 13 pg/mL, about 14 pg/mL, about 15 pg/mL, about 16 pg/mL, about 17 pg/mL, about 18 pg/mL, and about 19 pg/mL.
- Aspect 19 The method of any one of Aspects 10 - 19, wherein the treating further comprises differentiating the population of non-insulin secreting Type 1 diabetic pancreatic cells and propagating the insulin and glucagon secreting cell population to obtain a therapeutically amount of the insulin and glucagon secreting cell population and the method further comprises administering to a subject in need thereof the therapeutically effective amount of the insulin and glucagon secreting cell population.
- a composition (e.g., any one of Aspects 1 - 7) comprising an insulin and glucagon secreting cell population generated from non-insulin secreting pancreatic cells collected via needle biopsy from a Type 1 diabetic donor pancreas for use in the treatment of Type 1 diabetes, pancreatitis, or a combination thereof.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2022349361A AU2022349361A1 (en) | 2021-09-22 | 2022-09-19 | Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof |
CA3231258A CA3231258A1 (en) | 2021-09-22 | 2022-09-19 | Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202163247252P | 2021-09-22 | 2021-09-22 | |
US63/247,252 | 2021-09-22 | ||
US202263337137P | 2022-05-01 | 2022-05-01 | |
US63/337,137 | 2022-05-01 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023049079A2 true WO2023049079A2 (en) | 2023-03-30 |
WO2023049079A3 WO2023049079A3 (en) | 2023-10-12 |
Family
ID=85721374
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2022/044024 WO2023049079A2 (en) | 2021-09-22 | 2022-09-19 | Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU2022349361A1 (en) |
CA (1) | CA3231258A1 (en) |
WO (1) | WO2023049079A2 (en) |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPR311601A0 (en) * | 2001-02-15 | 2001-03-08 | Adp Pharmaceutical Pty Limited | Matrix gene expression in chondrogenesis |
EP1745154B1 (en) * | 2004-05-07 | 2012-08-01 | Celera Corporation | Genetic polymorphisms associated with liver fibrosis methods of detection and uses thereof |
CN102791851B (en) * | 2010-03-01 | 2017-07-14 | 詹森生物科技公司 | The method of cell of the purifying derived from multipotential stem cell |
US11534466B2 (en) * | 2016-03-09 | 2022-12-27 | Aal Scientifics, Inc. | Pancreatic stem cells and uses thereof |
US20210205371A1 (en) * | 2018-06-25 | 2021-07-08 | Imagine Pharma Llc | Composition and Methods for Producing Insulin Producing Islet Cells |
-
2022
- 2022-09-19 WO PCT/US2022/044024 patent/WO2023049079A2/en active Application Filing
- 2022-09-19 AU AU2022349361A patent/AU2022349361A1/en active Pending
- 2022-09-19 CA CA3231258A patent/CA3231258A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
AU2022349361A1 (en) | 2024-03-07 |
CA3231258A1 (en) | 2023-03-30 |
WO2023049079A3 (en) | 2023-10-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU749675B2 (en) | Uses for human non-autologous mesenchymal stem cells | |
Domouky et al. | Mesenchymal stem cells and differentiated insulin producing cells are new horizons for pancreatic regeneration in type I diabetes mellitus | |
EP2367419B1 (en) | Cellular compositions for use in therapy | |
Yu et al. | Human adipose-derived stem cells enriched with VEGF-modified mRNA promote angiogenesis and long-term graft survival in a fat graft transplantation model | |
US20230033991A1 (en) | Neo-Islets Comprising Stem and Islet Cells and Treatment of Diabetes Mellitus Therewith | |
KR101524079B1 (en) | Method for inducing differentiation of adult cells into insulin producing cells using exosome | |
WO2007143139A1 (en) | Abcb5 positive mesenchymal stem cells as immunomodulators | |
KR20140040696A (en) | Cell therapy composition for preventing or treating immune disease comprising mesenchymal stem cell and regulatory t cell | |
Aizman et al. | Cell injury-induced release of fibroblast growth factor 2: relevance to intracerebral mesenchymal stromal cell transplantations | |
He et al. | MSCs promote the development and improve the function of neonatal porcine islet grafts | |
EP3083941B1 (en) | Pancreatic islet-like cell structures and a method of preparing thereof | |
AU2019295576B2 (en) | Compositions and methods for propagating insulin-producing islet cells and therapeutic uses thereof | |
WO2023049079A2 (en) | Compositions and methods for propogating insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof | |
Dietrich et al. | Trophic effects of adipose derived stem cells on Langerhans islets viability | |
S Reddi et al. | Human umbilical cord blood cells and diabetes mellitus: recent advances | |
CN117917961A (en) | Compositions and methods for proliferation of insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof | |
JP6644702B2 (en) | A therapeutic agent for muscular dystrophy containing pluripotent stem cells derived from dental pulp | |
CN113750110A (en) | Application of mesenchymal stem cell exosome in preparation of medicine for preventing or treating type 1 diabetes and related diseases thereof | |
KR20160064954A (en) | Method for promoting growth of hair | |
KR102216646B1 (en) | A composition comprising mesenchymal stem cell for inhibiting adipogenesis | |
KR102314158B1 (en) | A composition comprising mesenchymal stem cell for inhibiting adipogenesis | |
US20240084261A1 (en) | Cell Clusters Comprising Stem and Islet Cells, Methods of Making, and Treatment of Diabetes Mellitus Therewith | |
Wehbe et al. | Type 2 diabetes mellitus and stem cell therapy: a review | |
Zhang | Optimization of Feline Adipose-Derived Multipotent Stromal Cell Isolation and Canine Cranial Cruciate Ligament Regeneration with Intra-Articular Adipose-Derived Multipotent Stromal Cells | |
Zhang et al. | A novel population of human bone marrow multipotent mesenchymal stem cells regenerates infarcted myocardium in rats |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22873465 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2022349361 Country of ref document: AU Ref document number: AU2022349361 Country of ref document: AU |
|
ENP | Entry into the national phase |
Ref document number: 2022349361 Country of ref document: AU Date of ref document: 20220919 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 3231258 Country of ref document: CA Ref document number: 808806 Country of ref document: NZ |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112024004061 Country of ref document: BR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 311468 Country of ref document: IL |