Generation af induceret pluripotente stamceller fra Muskelsvindfonden Patienter: Effektiv Integration-fri Omprogrammering af urin afledte celler
Summary
This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.
Abstract
Dystrofiske kardiomyopati er en dårligt forstået konsekvens af muskelsvind. Generering inducerede pluripotente stamceller (iPSCs) fra patienter med muskelsvind er en uvurderlig cellulær kilde til in vitro sygdom modelsystemer og kan bruges til narkotika screening studier. Patient-afledte urin celler er blevet anvendt i en vellykket omprogrammering i inducerede pluripotente stamceller med henblik på at modellere dystrofiske kardiomyopati 1. Varetagelse sikkerhedsproblemer integrere vektorsystemer præsenterer vi en protokol ved hjælp af en ikke-integrerende Sendaivirusvektor for transduktion af Yamanaka faktorer urin celler opsamlet fra patienter med muskelsvind. Denne protokol genererer fuldt omprogrammeres kloner inden for 2-3 uger. De pluripotente celler er vektor-fri ved passage-13. Disse dystrofiske iPSCs kan differentieres i cardiomyocytter og anvendes enten til at studere sygdom mekanismer eller til lægemiddelscreening.
Introduction
Kardiomyopati er den næsthyppigste dødsårsag hos patienter med Duchenne og Becker muskeldystrofi (MD). Selvom mutationer i X-bundet dystrophingen forekomme i 1: 3.500 mandlige fødsler, er meget lidt kendt om de molekylære og cellulære begivenheder, der fører til progressiv hjertemusklen skader. Menneskelige induceret pluripotente stamceller fra Muskelsvindfonden patienter har vist sig som et nyt redskab til at studere de underliggende sygdomsmekanismer og bruge til narkotika screening 1,2.
Den forventede ubehag hudbiopsier eller blodprøver kan afholde unge patienter og / eller deres værger til at give samtykke til undersøgelsen deltagelse. Urinprøver er en non-invasiv kilde af somatiske celler, der er amendable til omprogrammering metoder. Vi har for nylig vist, at urin-celler opsamlet fra muskelsvind patienter kan dyrkes og effektivt omprogrammeres i iPSCs hjælp retroviral transduktion med Yamanaka faktorer (Oct3 / 4, Sox2, Klf4, og c-Myc; OSKM) 1. Ulempen ved retroviral genlevering er den tilfældige integration af omprogrammering gener i værtskromosomerne. For at overvinde denne begrænsning, har vi brugt ikke-integrerende Sendaivirus for urin celle omprogrammering.
Denne protokol detaljer Sendaivirusset omprogrammering af isolerede urin celler fra Muskelsvindfonden patienter, som derefter kan differentieres i cardiomyocytter eller andre celletyper til yderligere undersøgelse. Denne protokol kan også tilpasses til andre patientpopulationer specifikke sygdomme.
Protocol
Representative Results
Discussion
Modeling kardiovaskulære sygdomme ved hjælp iPSCs bliver en fælles tilgang til at forstå den genetiske bidrag 4-6. Nogle uventede vanskeligheder ved at få celleprøver fra patienter, især små børn, kan undgås ved at tilbyde mulighed for en ikke-invasiv metode såsom en urinopsamling. I denne unge patientpopulation, er det ofte vanskeligt at indsamle en mængde urin tilstrækkelig til at give nok urin celler til omprogrammering. Coaching de unge patienter til at drikke 30 min forud for urinopsamling f…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Materials
| List of Materials | |||
| Name of Reagent/Material/Equipment | Company | Catalog Number | Comments |
| 4 Oz. Specimen Cup with Lid | STL Medical Supply | M9AMSAS340 | For Urine Sample Collection |
| 15 mL BD-Falcon Tubes | Fisher Scientific | 352097 | |
| 50 mL BD-Falcon Tubes | Fisher Scientific | 352098 | |
| Round Glass Coverslips | Fisher Scientific | 12-545-81 | |
| CytoTune-iPS Sendai Reprogramming Kit | Life Technologies | A1378-001 | |
| PBS, pH 7.4 | Life Technologies | 10010-023 | |
| Pen-Strep w/o Glutamine | Life Technologies | 15140-122 | Warm in 37 °C water bath before use |
| RPMI Medium 1640 | Life Technologies | 11875-093 | Warm in 37 °C water bath before use |
| B-27 Supplement w/Insulin (50x) | Life Technologies | 17504-044 | Warm in 37 °C water bath before use |
| B-27 Supplement w/o Insulin (50x) | Life Technologies | 0050129SA | Warm in 37 °C water bath before use |
| DMEM/F12 (1:1) | Life Technologies | 11330-032 | Warm in 37 °C water bath before use |
| Versene, 1:5000 | Life Technologies | 15040066 | Warm in 37 °C water bath before use |
| bFGF (10 µg) | Life Technologies | 13256-029 | Warm in 37 °C water bath before use |
| Cell Counter Cartridges | Life Technologies | C10228 | |
| Knockout Serum (500 mL Bottle) | Life Technologies | 10828-028 | Warm in 37 °C water bath before use |
| Recovery Cell Culture Freezing Medium | Life Technologies | 12648-010 | |
| Keratinocyte-SFM (1X), Liquid | Life Technologies | 17005-042 | Warm in 37 °C water bath before use |
| DMEM, High Glucose, Glutamax | Life Technologies | 10566-016 | Warm in 37 °C water bath before use |
| Ham's F-12 Nutrient Mix | Life Technologies | 11765-054 | Warm in 37 °C water bath before use |
| EGF Recombinant Human Protein, Liquid Form | Life Technologies | PHG0311L | Warm in 37 °C water bath before use |
| Insulin, Human Recombinant, Zinc Soln | Life Technologies | 12585-014 | Warm in 37 °C water bath before use |
| TeSR-E8 | Stem Cell Technologies | 5940 | Warm in 37 °C water bath before use |
| ROCK Inhibitor (Y-27632) | Selleck | S1049 | Warm in 37 °C water bath before use |
| Matrigel | BD Biosciences | 354277 | hESC Qualified Matrix, LVED Free |
| RNeasy Mini Kit | Qiagen | 74104 | |
| iScript cDNA Synthesis Kit | Bio-Rad | 170-8890 | |
| DreamTaq Green PCR Master Mix (2X) | Thermo-Scientific | K1081 | |
| FBS-Qualified | Sigma Aldrich | F6178 | Warm in 37 °C water bath before use |
| ADENINE BIOREAGENT | Sigma Aldrich | A2786-5G | Warm in 37 °C water bath before use |
| CHOLERA TOXIN FROM VIBRIO CHOLERAE | Sigma Aldrich | C8052-.5MG | Warm in 37 °C water bath before use |
| HYDROCORTISONE | Sigma Aldrich | H0888-1G | Warm in 37 °C water bath before use |
| HOLO-TRANSFERRIN FROM HUMAN | Sigma Aldrich | T0665-50MG | Warm in 37 °C water bath before use |
| 3,3',5-TRIIODO-L-THYRONINE SODIUM SALT | Sigma Aldrich | T6397-100MG | Warm in 37 °C water bath before use |
| DAPI | Santa Cruz Biotechnology | SC-3598 | |
| Fluoromount Aquous mounting medium | Sigma Aldrich | F4680 | |
| 16% Paraformaldehyde | Alfa-Aesar | 43368 | |
| List of Antibodies | |||
| Name of Reagent/Material/Equipment | Company | Catalog Number | Comments |
| Mouse anti CD44 – labelled with FITC | BD Biosciences | 560977 | |
| Mouse anti CD146 – labelled with PE | BD Biosciences | 561013 | |
| Mouse anti CD73 – labelled with PE | BD Biosciences | 561014 | |
| Mouse anti-α-smooth muscle actin | Sigma Aldrich | A2547 | |
| Mouse anti-Vimentin | Abcam | ab8978-100 | |
| Mouse Anti – TRA-1-81 | Life Technologies | 411100 | |
| Rabbit anti Oct 3/4 | Santa Cruz Biotechnology | SC-9081 | |
| Mouse Anti Dystrophin | Leica Biosystems | NCL-DYSB | |
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