Differentiering af humane pluripotente stamceller i insulinproducerende øklynger
Summary
Differentieringen af stamceller i øceller giver en alternativ løsning til konventionel diabetesbehandling og sygdomsmodellering. Vi beskriver en detaljeret stamcellekulturprotokol, der kombinerer et kommercielt differentieringssæt med en tidligere valideret metode til at hjælpe med at producere insulinudskillende, stamcelleafledte øer i en skål.
Abstract
Differentiering af humane pluripotente stamceller (hPSC’er) i insulinudskillende betaceller giver materiale til undersøgelse af betacellefunktion og diabetesbehandling. Der er dog stadig udfordringer med at opnå stamcelleafledte betaceller, der i tilstrækkelig grad efterligner indfødte humane betaceller. På baggrund af tidligere undersøgelser er hPSC-afledte øceller blevet genereret for at skabe en protokol med forbedrede differentieringsresultater og konsistens. Protokollen beskrevet her bruger et pancreas stamfadersæt i trin 1-4, efterfulgt af en protokol ændret fra et papir, der tidligere blev offentliggjort i 2014 (kaldet “R-protokol” i det følgende) i trin 5-7. Detaljerede procedurer for anvendelse af pancreas stamfadersæt og mikrobrøndplader med en diameter på 400 μm til generering af pancreas stamfaderklynger, R-protokol for endokrin differentiering i et 96-brønds statisk suspensionsformat og in vitro-karakterisering og funktionel evaluering af hPSC-afledte øer er inkluderet. Den komplette protokol tager 1 uge for indledende hPSC-ekspansion efterfulgt af ~ 5 uger for at opnå insulinproducerende hPSC-øer. Personale med grundlæggende stamcellekulturteknikker og træning i biologiske assays kan reproducere denne protokol.
Introduction
Pancreas beta-celler udskiller insulin, der reagerer på stigninger i blodsukkerniveauet. Patienter, der mangler tilstrækkelig insulinproduktion på grund af autoimmun destruktion af betaceller i type 1-diabetes (T1D)1 eller på grund af betacelledysfunktion i type 2-diabetes (T2D)2, behandles typisk med administration af eksogent insulin. På trods af denne livreddende behandling kan den ikke nøjagtigt matche den udsøgte kontrol af blodsukkeret, som opnås ved dynamisk insulinsekretion fra bona fide betaceller. Som sådan lider patienter ofte konsekvenserne af livstruende hypoglykæmiske episoder og andre komplikationer som følge af kroniske hyperglykæmiske udflugter. Transplantation af humane kadaverøer genopretter med succes stram glykæmisk kontrol hos T1D-patienter, men er begrænset af tilgængeligheden af ødonorer og vanskeligheder med at rense sunde øer til transplantation 3,4. Denne udfordring kan i princippet løses ved at anvende hPSC’er som alternativt udgangsmateriale.
Nuværende strategier til generering af insulinudskillende øer fra hPSC’er in vitro sigter ofte mod at efterligne processen med embryonal udvikling af bugspytkirtlen in vivo 5,6. Dette kræver kendskab til de ansvarlige signalveje og tidsbestemt tilsætning af tilsvarende opløselige faktorer for at efterligne kritiske stadier af den udviklende embryonale bugspytkirtel. Bugspytkirtelprogrammet starter med forpligtelsen til endelig endoderm, som er præget af transkriptionsfaktorer gaffelhovedboks A2 (FOXA2) og kønsbestemmende region Y-boks 17 (SOX17)7. Successiv differentiering af endelig endoderm involverer dannelsen af et primitivt tarmrør, mønster i en bageste fortarm, der udtrykker bugspytkirtlen og duodenal homeobox 1 (PDX1)7,8,9 og epitelekspansion til bugspytkirtelforfædre, der co-udtrykker PDX1 og NK6 homeobox 1 (NKX6.1)10,11.
Yderligere forpligtelse til endokrine øceller ledsages af den forbigående ekspression af pro-endokrin masterregulator neurogenin-3 (NGN3)12 og stabil induktion af nøgletranskriptionsfaktorer neuronal differentiering 1 (NEUROD1) og NK2 homeobox 2 (NKX2.2)13. De vigtigste hormonekspressive celler, såsom insulinproducerende betaceller, glukagonproducerende alfaceller, somatostatinproducerende deltaceller og pancreas polypeptidproducerende PPY-celler, programmeres efterfølgende. Med denne viden samt opdagelser fra omfattende lægemiddelscreeningsundersøgelser med høj kapacitet har nylige fremskridt muliggjort generering af hPSC-øer med celler, der ligner betaceller, der er i stand til insulinsekretion 14,15,16,17,18,19.
Trinvise protokoller er blevet rapporteret til generering af glukose-responsive betaceller 6,14,18,19. Bygget på disse undersøgelser involverer denne protokol brugen af et pancreas stamfadersæt til generering af PDX1+/NKX6.1+ stamceller i bugspytkirtlen i en plan kultur, efterfulgt af mikrobrøndpladeaggregering i klynger af ensartet størrelse og yderligere differentiering mod insulinudskillende hPSC-øer med R-protokollen i en statisk 3D-suspensionskultur. Kvalitetskontrolanalyser, herunder flowcytometri, immunfarvning og funktionel vurdering, udføres til streng karakterisering af de differentierende celler. Dette papir giver en detaljeret beskrivelse af hvert trin i den rettede differentiering og skitserer de vitro-karakteriseringsmetoderne.
Protocol
Representative Results
Discussion
Dette papir beskriver en syv-trins hybridprotokol, der muliggør generering af hPSC-øer, der er i stand til at udskille insulin ved glukoseudfordring inden for 40 dage efter dyrkning in vitro. Blandt disse mange trin menes effektiv induktion af endelig endoderm at sætte et vigtigt udgangspunkt for de endelige differentieringsresultater18,27,28. I producentens protokol anbefales en såtæthed på 2,6 × 105 …
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi anerkender taknemmeligt støtten fra STEMCELL Technologies, Michael Smith Health Research BC, Stem Cell Network, JDRF og Canadian Institutes of Health Research. Jia Zhao og Shenghui Liang er modtagere af Michael Smith Health Research BC Trainee Award. Mitchell J.S. Braam er modtager af Mitacs Accelerate Fellowship. Diepiriye G. Iworima er modtager af Alexander Graham Bell Canada Graduate Scholarship og CFUW 1989 Ecole Polytechnique Commemorative Award. Vi takker oprigtigt Dr. Edouard G. Stanley fra MCRI og Monash University for at dele Mel1 INS GFP / W-linjen og Alberta Diabetes Institute Islet Core for at isolere og distribuere menneskelige øer. Vi anerkender også støtten fra Life Sciences Institute Imaging and Flow Cytometry faciliteter ved University of British Columbia. Figur 1 blev oprettet med BioRender.com.
Materials
| 3,3’,5-Triiodo-L-thyronine (T3) | Sigma | T6397 | Thyroid hormone |
| 4% PFA solution | Santa Cruz Biotechnology | sc-281692 | Should be handled in fume hood |
| 96-Well, Ultralow Attachment, flat bottom | Corning Costar (VWR) | CLS3474 | Flat bottom; for static suspension culture in the last three stages |
| Accutase | STEMCELL Technologies | 07920 | Dissociation reagent for Stage 4 cells |
| Aggrewell400 plates | STEMCELL Technologies | 34415 | 400 µm diameter microwell plates |
| Aggrewell800 plates | STEMCELL Technologies | 34815 | 800 µm diameter microwell plates |
| Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG) | R&D Systems | IC2400G | 1:100 in flow cytometry; used for assaying Stage 1 cells |
| Alexa Fluor 488 Goat IgG Isotype Control | R&D Systems | IC108G | 1:100 in flow cytometry |
| Alexa Fluor 488 Mouse anti-Human SST (mouse IgG2B) | BD Sciences | 566032 | 1:250 in flow cytometry; used for assaying Stage 7 cells |
| Alexa Fluor 488 Mouse IgG2B Isotype Control | R&D Systems | IC0041G | 1:500 in flow cytometry |
| Alexa Fluor 647 Mouse anti-Human C-peptide (mouse IgG1κ) | BD Pharmingen | 565831 | 1:2,000 in flow cytometry; used for assaying Stage 7 cells |
| Alexa Fluor 647 Mouse anti-Human INS (mouse IgG1κ) | BD Sciences | 565689 | 1:2,000 in flow cytometry |
| Alexa Fluor 647 Mouse anti-Human NKX6.1 (mouse IgG1κ) | BD Sciences | 563338 | 1:33 in flow cytometry; used for assaying Stage 4 cells |
| Alexa Fluor 647 Mouse anti-Human SOX17 (mouse IgG1κ) | BD Sciences | 562594 | 1:50 in flow cytometry; used for assaying Stage 1 cells |
| Alexa Fluor 647 Mouse IgG1κ Isotype Control | BD Sciences | 557714 | 1:50 in flow cytometry |
| ALK5i II | Cayman Chemicals | 14794 | TGF-beta signaling inhibitor |
| Anti-Adherence Rinsing Solution | STEMCELL Technologies | 7010 | Microwell Rinsing Solution |
| Assay chamber | Cellvis | D35-10-1-N | For static GSIS and confocal imaging purposes |
| Bovine serum albumin (BSA) | Thermo Fisher Scientific | BP1600-100 | For immunostaining procedure |
| CK19 antibody | DAKO | M0888 | 1:50 in whole mount immunofluorescence |
| D-glucose | Sigma | G8769 | Medium supplement |
| DAPI | Sigma | D9542 | For nuclear counterstaining |
| DMEM/F12, HEPES | Thermo Fisher Scientific | 11330032 | Matrix diluting solution |
| Donkey anti-goat Alexa Fluor 555 | Life technologies | A21432 | 1:500 in whole mount immunofluorescence |
| Donkey anti-goat Alexa Fluor 647 | Life technologies | A21447 | 1:500 in whole mount immunofluorescence |
| Donkey anti-mouse Alexa Fluor 555 | Life technologies | A31570 | 1:500 in whole mount immunofluorescence |
| Donkey anti-mouse Alexa Fluor 647 | Life technologies | A31571 | 1:500 in whole mount immunofluorescence |
| Donkey anti-rabbit Alexa Fluor 555 | Life technologies | A31572 | 1:500 in whole mount immunofluorescence |
| Donkey anti-rabbit Alexa Fluor 647 | Life technologies | A31573 | 1:500 in whole mount immunofluorescence |
| Donkey anti-sheep Alexa Fluor 647 | Life technologies | A21448 | 1:500 in whole mount immunofluorescence |
| DPBS | Sigma | D8537 | Without Ca2+ and Mg2+ |
| ELISA, insulin, human | Alpco | 80-INSHU-E01.1 | For human insulin measurement |
| Fatty acid-free BSA | Proliant | 68700 | Medium supplement |
| Fixation and Permeabilization Solution Kit | BD Sciences | 554714 | Fix/Perm and 10x Perm/Wash solutions included |
| Gentle Cell Dissociation Reagent | STEMCELL Technologies | 7174 | For clump passaging hPSCs during maintenance culture |
| Glucagon antibody | Sigma | G2654 | 1:400 in whole mount immunofluorescence |
| GLUT1 antibody | Thermo Fisher Scientific | PA1-37782 | 1:200 in whole mount immunofluorescence |
| GlutaMAX-I (100x) | Gibco | 35050061 | L-glutamine supplement |
| Glycerol | Thermo Fisher Scientific | G33-4 | For tissue clearing and mounting |
| GSi XX | Sigma Millipore | 565789 | Notch inhibitor |
| Heparin | Sigma | H3149 | Medium supplement |
| ITS-X (100x) | Thermo Fisher Scientific | 51500056 | Insulin-Transferrin-Selenium-Ethanolamine; medium supplement |
| LDN193189 | STEMCELL Technologies | 72147 | BMP antagonist |
| MAFA antibody | Abcam | ab26405 | 1:200 in whole mount immunofluorescence |
| Matrigel, hESC-qualified | Thermo Fisher Scientific | 08-774-552 | Extracellular matrix for vessel surface coating |
| MCDB131 medium | Life technologies | 10372019 | Base medium |
| mTeSR1 Complete Kit | STEMCELL Technologies | 85850 | stem cell medium and 5x supplement included |
| N-Cys (N-acetyl cysteine) | Sigma | A9165 | Antioxidant |
| NaHCO3 | Sigma | S6297 | Medium supplement |
| NEUROD1 antibody | R&D Systems | AF2746 | 1:20 in whole mount immunofluorescence |
| NKX6.1 antibody | DSHB | F55A12-c | 1:50 in whole mount immunofluorescence |
| Pancreatic polypeptide antibody | R&D Systems | AF6297 | 1:200 in whole mount immunofluorescence |
| PBS | Sigma | D8662 | With Ca2+ and Mg2+ |
| PDX1 antibody | Abcam | ab47267 | 1:200 in whole mount immunofluorescence |
| PE Mouse anti-Human GCG (mouse IgG1κ) | BD Sciences | 565860 | 1:2,000 in flow cytometry; used for assaying Stage 7 cells |
| PE Mouse anti-Human NKX6.1 (mouse IgG1k) | BD Sciences | 563023 | 1:250 in flow cytometry |
| PE Mouse anti-Human PDX1 (mouse IgG1k) | BD Sciences | 562161 | 1:200 in flow cytometry; used for assaying Stage 4 cells |
| PE Mouse IgG1κ Isotype Control | BD Sciences | 554680 | 1:2,000 in flow cytometry |
| PE Mouse-Human Chromogranin A (CHGA, mouse IgG1k) | BD Sciences | 564563 | 1:200 in flow cytometry |
| R428 | Cayman Chemicals | 21523 | AXL tyrosine kinase inhibitor |
| Retinoid acid, all-trans | Sigma | R2625 | Light-sensitive |
| RIPA lysis buffer, 10x | Sigma | 20-188 | For hormone extraction |
| SANT-1 | Sigma | S4572 | SHH inhibitor |
| SLC18A1 antibody | Sigma | HPA063797 | 1:200 in whole mount immunofluorescence |
| Somatostatin antibody | Sigma | HPA019472 | 1:100 in whole mount immunofluorescence |
| STEMdiff Pancreatic Progenitor Kit | STEMCELL Technologies | 05120 | Basal media and supplements included |
| Synaptophysin antibody | Novus | NB120-16659 | 1:25 in whole mount immunofluorescence |
| Triton X-100 | Sigma | X100 | For permeabilization |
| Trolox | Sigma Millipore | 648471 | Vitamin E analog |
| TrypLE Enzyme Express | Life technologies | 12604-021 | cell dissociation enzyme reagent for single cell passaging hPSCs |
| Trypsin1/2/3 antibody | R&D Systems | AF3586 | 1:25 in whole mount immunofluorescence |
| Y-27632 | STEMCELL Technologies | 72304 | ROCK inhibitor |
| Zinc sulfate | Sigma | Z0251 | Medium supplement |
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