Effektiv vaskularisering af nyreorganoider gennem intracelomisk transplantation i kyllingembryoner
Summary
Her præsenterer vi en detaljeret protokol til transplantation af nyreorganoider i det celomiske hulrum af kyllingembryoner. Denne metode inducerer vaskularisering og forbedret modning af organoiderne inden for 8 dage og kan bruges til at studere disse processer på en effektiv måde.
Abstract
Nyreorganoider afledt af humane inducerede pluripotente stamceller indeholder nefronlignende strukturer, der til en vis grad ligner dem i den voksne nyre. Desværre hæmmes deres kliniske anvendelighed af manglen på en funktionel vaskulatur og følgelig begrænset modning in vitro. Transplantationen af nyreorganoider i det celomiske hulrum af kyllingembryoner inducerer vaskularisering ved perfunderede blodkar, herunder dannelsen af glomerulære kapillærer, og forbedrer deres modning. Denne teknik er meget effektiv, hvilket giver mulighed for transplantation og analyse af et stort antal organoider. Dette papir beskriver en detaljeret protokol for intracelomisk transplantation af nyreorganoider i kyllingembryoner efterfulgt af injektion af fluorescerende mærket lektin for at plette den perfunderede vaskulatur og indsamling af transplanterede organoider til billeddannelsesanalyse. Denne metode kan bruges til at inducere og studere organoid vaskularisering og modning for at finde spor til forbedring af disse processer in vitro og forbedre sygdomsmodellering.
Introduction
Human induceret pluripotent stamcelle (hiPSC)-afledte nyreorganoider har vist sig at have potentiale for udviklingsstudier 1,2,3,4, toksicitetsscreening 5,6 og sygdomsmodellering 5,7,8,9,10,11,12,13 . Imidlertid er deres anvendelighed til disse og eventuelle kliniske transplantationsformål begrænset af manglen på et vaskulært netværk. Under embryonal nyreudvikling interagerer podocytter, mesangialceller og vaskulære endotelceller (EC’er) for at danne glomerulus indviklede struktur. Uden denne interaktion kan den glomerulære filtreringsbarriere, der består af podocytter, den glomerulære kældermembran (GBM) og EC’er, ikke udvikle sig korrekt14,15,16. Selvom nyreorganoider in vitro indeholder nogle EC’er, undlader disse at danne et ordentligt vaskulært netværk og mindskes over tid17. Det er derfor ikke overraskende, at organoiderne forbliver umodne. Transplantation hos mus inducerer vaskularisering og modning af nyreorganoiderne 18,19,20,21. Desværre er dette en arbejdskrævende proces, der er uegnet til analyse af et stort antal organoider.
Kyllingembryoner er blevet brugt til at studere vaskularisering og udvikling i over et århundrede22. De er let tilgængelige, kræver lav vedligeholdelse, mangler et fuldt funktionelt immunsystem og kan udvikle sig normalt efter åbning af æggeskallen23,24,25,26. Transplantationen af organoider på deres chorioallantoic membran (CAM) har vist sig at føre til vaskularisering27. Imidlertid er varigheden af transplantationen på CAM såvel som niveauet for modning af transplantatet begrænset af CAM-dannelse, hvilket tager indtil embryonal dag 7 at fuldføre. Derfor blev der for nylig udviklet en metode til effektivt at vaskularisere og modne nyreorganoider gennem intracelomisk transplantation i kyllingembryoner28. Det celomiske hulrum af kyllingembryoner har siden 1930’erne været kendt for at være et gunstigt miljø for differentiering af embryonale væv29,30. Det kan tilgås tidligt i embryonal udvikling og giver mulighed for relativt ubegrænset udvidelse af transplantatet i alle retninger.
Dette papir skitserer en protokol til transplantation af hiPSC-afledte nyreorganoider i det celomiske hulrum i dag 4 kyllingembryoner. Denne metode inducerer vaskularisering og forbedret modning af organoiderne inden for 8 dage. Injektion af fluorescerende mærket linse culinaris agglutinin (LCA) inden ofring af embryonerne muliggør visualisering af perfunderede blodkar i organoiderne gennem konfokal mikroskopi.
Protocol
Representative Results
Discussion
I dette manuskript demonstreres en protokol for intracelomisk transplantation af hiPSC-afledte nyreorganoider i kyllingembryoner. Ved transplantation vaskulariseres organoider af perfunderede blodkar, der består af en kombination af humane organoidafledte og kyllingafledte EC’er. Disse spredes gennem organoiden og invaderer de glomerulære strukturer, hvilket muliggør interaktion mellem EC’erne og podocytterne. Det blev tidligere vist, at dette fører til forbedret modning af de organoide glomerulære og rørformede st…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker George Galaris (LUMC, Leiden, Holland) for hans hjælp med injektion af kyllingeembryoner. Vi anerkender støtten fra Saskia van der Wal-Maas (Institut for Anatomi & Embryologi, LUMC, Leiden, Holland), Conny van Munsteren (Institut for Anatomi & Embryologi, LUMC, Leiden, Holland), Manon Zuurmond (LUMC, Leiden, Holland) og Annemarie de Graaf (LUMC, Leiden, Holland). M. Koning støttes af »Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC«. Dette arbejde blev delvist støttet af Leiden University Fund “Prof. Jaap de Graeff-Lingling Wiyadhanrma Fund” GWF2019-02. Dette arbejde støttes af partnerne i Regenerative Medicine Crossing Borders (RegMedXB) og Health Holland, Top Sector Life Sciences & Health. C.W. van den Berg og T.J. Rabelink er støttet af The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Novo Nordisk Foundation Center for Stem Cell Cell Medicine er støttet af Novo Nordisk Fondens bevillinger (NNF21CC0073729).
Materials
| 0.2 µm filter: Whatman Puradisc 30 syringe filter 0.2 µm | Whatman | 10462200 | |
| 35 mm glass bottom dishes | MatTek Corporation | P35G-1.5-14-C | |
| Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma-Aldrich | A5177-5EA | Contains silicone tubes, mouth piece and connector |
| Confocal microscope: Leica White Light Laser Confocal Microscope | Leica | TCS SP8 | |
| Dissecting forceps, simple type. Titanium, curved, with fine sharp tips | Hammacher Karl | HAMMHTC091-10 | |
| Dissecting forceps, simple type. Titanium, straight, with fine sharp tips | Hammacher Karl | HAMMHTC090-11 | |
| Dissecting microscope | Wild Heerbrugg | 355110 | |
| Dissecting scissors, curved, OP-special, extra sharp/sharp | Hammacher Karl | HAMMHSB391-10 | |
| Donkey serum | Sigma-Aldrich | D9663 | |
| Donkey-α-mouse Alexa Fluor 488 | ThermoFisher Scientific | A-212-02 | dilution 1:500 |
| Donkey-α-sheep Alexa Fluor 647 | ThermoFisher Scientific | A-21448 | dilution 1:500 |
| Double edged stainless steel razor blades | Electron Microsopy Sciences | 72000 | |
| DPBS, calcium, magnesium (DPBS-/-) | ThermoFisher Scientific | 14040133 | |
| DPBS, no calcium, no magnesium (DPBS+/+) | ThermoFisher Scientific | 14190094 | |
| Egg cartons or custom made egg holders | NA | NA | |
| Fertilized white leghorn eggs (Gallus Gallus Domesticus) | Drost Loosdrecht B.V. | NA | |
| Incubator | Elbanton BV | ET-3 combi | |
| Lotus Tetragonolobus lectin (LTL) Biotinylated | Vector Laboratories | B-1325 | dilution 1:300 |
| Micro scissors, straight, sharp/sharp, cutting length 10 mm | Hammacher Karl | HAMMHSB500-09 | |
| Microcapillaries: Thin wall glass capillaries 1.5 mm, filament | World Precision Instruments | TW150F-3 | |
| Micropipette puller | Sutter Instrument Company | Model P-97 | We use the following settings: Heat 533, Pull 60, Velocity 150, Time 200 |
| Microscalpel holder: Castroviejo blade and pins holder, 12 cm, round handle, conical 10 mm jaws. | Euronexia | L-120 | |
| Mounting medium: Prolong Gold Antifade Mountant | ThermoFisher Scientific | P36930 | |
| Olivecrona dura dissector 18 cm | Reda | 41146-18 | |
| Parafilm | Heathrow Scientific | HS234526B | |
| Penicillin-streptomycin 5,000 U/mL | ThermoFisher Scientific | 15070063 | |
| Perforated spoon | Euronexia | S-20-P | |
| Petri dish 60 x 15 mm | CELLSTAR | 628160 | |
| Plastic transfer pipettes | ThermoFisher Scientific | PP89SB | |
| Purified mouse anti-human CD31 antibody | BD Biosciences | 555444 | dilution 1:100 |
| Rhodamine labeled Lens Culinaris Agglutinin (LCA) | Vector Laboratories | RL-1042 | This product has recently been discontinued. Vectorlabs does still produce Dylight 649 labeled LCA (DL-1048-1) and fluorescein labeled LCA (FL-1041-5) |
| Sheep anti-human NPHS1 antibody | R&D systems | AF4269 | dilution 1:100 |
| Sterile hypodermic needles, 19 G | BD microlance | 301500 | |
| Streptavidin Alexa Fluor 405 | ThermoFisher Scientific | S32351 | dilution 1:200 |
| Syringe 5 mL | BD Emerald | 307731 | |
| Transparent tape | Tesa | 4124 | Available at most hardware stores |
| Triton X | Sigma-Aldrich | T9284 | |
| Tungsten wire, 0.25 mm dia | ThermoFisher Scientific | 010404.H2 |
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