In Vitro Pancreas Organogenesis fra spredte Mouse Embryonale Progenitorer
Summary
Den tredimensionale kultur beskrevet i denne protokol metode sammenfatter bugspytkirtlen udvikling fra spredte embryonale mus bugspytkirtel stamfædre, herunder deres betydelige ekspansion, differentiering og morfogenese i en forgrenet orgel. Denne metode er modtagelig for billedbehandling, funktionelle indblanding og manipulation af den niche.
Abstract
The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens.
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas.
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.
Introduction
Organ kultur giver en nyttig model, der bygger bro mellem det komplekse, men yderst relevante in vivo undersøgelser og bekvem men omtrentlige simulering af cellelinje modeller. I tilfælde af bugspytkirtlen, der er helt svarer til bugspytkirtel progenitorer selvom der transformeres cellelinjer simulerer endokrine og eksokrine celler ikke cellelinie. Den voksne hele bugspytkirtlen kan ikke dyrkes; isolerede endokrine øer kan opretholdes for få uger uden celleproliferation og vævssnit kan opbevares in vitro i få timer 5. Embryonale bugspytkirtel kultur har været meget anvendt til ikke blot at studere dens udvikling, men også at undersøge epitel-mesenkymale interaktioner 4,6,7, til billede processer 8 eller kemisk forstyrre dem 9.. To orgel dyrkningsmetoder anvendes hovedsageligt: Den første består i dyrkning af pancreas knopper på fibronectincoatede coatede plader 2, som er bekvemfår du let til billeddiagnostiske formål; den anden mulighed er at dyrke organer på filtre på luft-væske grænsefladen 3,4, som bedst bevarer morfogenese. Selvom meget nyttigt, disse metoder fører til en vis grad af udfladning; udvidelse af progenitorer er meget begrænset i forhold til den normale udvikling og starte befolkning er kompleks, der omfatter alle typer af pancreasceller og mesenchymale celler.
Evnen til kultur og udvide spredte primære celler er værdifuldt at studere afstamning relationer og afdække de iboende egenskaber af isolerede celletyper 10. Sugiyama m.fl. 11. Kunne opretholde bugspytkirtel stamfædre og hormonforstyrrende forfædre at tilbageholdte nogle funktionelle tegn i 3-5 dage i kultur på feeder lag. Pancreatospheres, beslægtet med neurosfærer 12 og mammospheres 13 er blevet udvidet fra voksne øer og duktale celler, selv om arten af de stamceller / stamcellerat generere disse kugler er ikke klart. Desuden, i modsætning til fysiologisk udvikling, pancreatospheres indeholdt nogle neuroner 14,15. Sfærer blev også for nylig produceret fra embryonale bugspytkirtel stamfædre 16,17 og regenererende pancreata 18 med god stamfader ekspansion og efterfølgende differentiering, men undlod at rekapitulere morfogenese.
3D-modeller fra spredte og ofte definerede celler, der selv-organisere i miniaturiserede organer har for nylig blomstrede og simulere udviklingen eller voksen omsætning af flere organer, såsom tarmen 19,20, maven 21, lever 22, prostata 23 og luftrøret 24.. I nogle tilfælde har udviklingsmæssige morfogenese og differentiering er sammenfattet i 3D fra ES-celler, som det er tilfældet af optiske kopper 25, tarme 26 eller hjerne 27.
Her har vi desCribe en metode til at udvide dissocierede multipotente bugspytkirtlen forfædre i en 3D Matrigel stillads, hvor de kan differentiere og selv-organisere.
Protocol
Representative Results
Discussion
Storstilet produktion af funktionelle betaceller in vitro er stadig ineffektiv 1. I denne udfordrende sammenhæng kan Developmental Biology undersøgelser hjælpe decifrere de nøjagtige signaler, der kræves for differentieringen af funktionelle betaceller. Denne protokol giver mulighed for vedligeholdelse, udbygning og differentiering af embryonale pancreas stamfædre in vitro. Dette omfatter dannelsen af insulin-producerende beta-celler, der ikke co-udtrykke andre endokrine hormoner…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansieret sekventielt af en NCCR Frontiers in Genetics pilot award, Juvenile Diabetes Research Foundation Grant 41-2009-775 og Grant 12-126875 fra There Frie Forskningsråd / health and Sygdom. Forfatterne takker Spagnoli laboratorium for hosting video optagelse.
Materials
| Penicillin-Streptomycin | Gibco | 15070-063 | Stock keept at -20°C | |
| KnockOut Serum replacement (supplement) | Gibco | 10828-028 | Stock keept at -20°C | |
| 2-mercaptoethanol | Sigma Aldrich | 3148-25ML | Stock keept at 4°C | |
| Phorbol Myristate Acetate (PMA) | Calbiotech | 524400-1MG | Stock keept at -20°C | |
| Y-27632 (ROCK inhibitor) | Sigma Aldrich | ab120129 | Stock keept at -20°C- Attention! Stability/source is a frequent source of problems | |
| EGF | Sigma Aldrich | E9644-2MG | Stock keept at -80°C | |
| Recombinant Human R-spondin 1 | R&D | 4645-RS-025/CF | Stock keept at -80°C | |
| - or - | ||||
| Recombinant Mouse R-spondin 1 | R&D | 3474-RS-050 | Stock keept at -80°C | |
| Recombinant Human FGF1 (aFGF) | R&D | 232-FA-025 | Stock keept at -80°C- do not include to increase beta cell production | |
| Heparin (Liquemin) | Drossapharm | Stock keept at 4°C | ||
| Recombinant Human FGF10 | R&D | 345-FG-025 | Stock keept at -80°C | |
| DMEM/F-12 | Gibco | 21331-020 | ||
| Penicillin-Streptomycin | Gibco | 15070-063 | Stock keept at -20°C | |
| B27 x50 (supplement) | Gibco | 17504-044 | Stock keept at -20°C | |
| Recombinant Human FGF2 (bFGF) | R&D | 233-FB-025 | Stock keept at -80°C | |
| Y-27632 (ROCK inhibitor) | Sigma Aldrich | ab120129 | Stock keept at -20°C- Attention! Stability/source is a frequent source of problems | |
| DMEM/F-12 | Gibco | 21331-020 | ||
| Matrigel | Corning | 356231 | Stock keept at -20°C | |
| Trypsin 0.05% | Gibco | 25300-054 | Stock keept at 4°C | |
| RNAlater – RNA stabilizing reagent | Qiagen | 76104 | Store at room temperature | |
| Dispase | Sigma Aldrich | D4818-2MG | Stock keept at -20°C | |
| BSA for reconstitution | Milipore | 81-068 | For reconstituition of cytokines – Stock keept at -20°C | |
| Fetal calf serum (FCS) | Gibco | 16141079 | Stock keept at -20°C | |
| 60 well MicroWell trays | Sigma Aldrich | M0815-100EA | ||
| 4-well plates | Thermo Scientific | 176740 | ||
| 95-well plates F bottom | Greiner Bio | 6555180 | ||
| Glas bottom plates | Ibidi | 81158 | ||
| Disposal micropittes | Blaubrand | 708745 |
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