Cinetica Misurazione e Tempo reale visualizzazione della riprogrammazione somatica
Summary
Il protocollo presentato in questo studio descrive metodi per il monitoraggio in tempo reale di progressione riprogrammazione tramite la misurazione cinetica dei marcatori di cellule staminali pluripotenti positivi e negativi usando analisi citofluorimetrica. Il protocollo include anche la valutazione di imaging basata su morfologia, e pennarello o espressione giornalista durante la generazione IPSC.
Abstract
Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources.
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.
Introduction
Le cellule staminali pluripotenti indotte paziente-derivato (iPSCs) sono strumenti promettenti per la terapia cellulare e lo screening di stupefacenti. Essi forniscono una fonte autologa di cellule per la terapia. Inoltre, essi comprendono una vasta gamma di background genetico, che permette una dettagliata analisi in vitro di malattie genetiche di là di quanto attuali linee di cellule staminali embrionali (ESC) consentirebbe. I recenti progressi hanno portato allo sviluppo di diversi metodi per generare iPSCs, compresi riprogrammazione con virus Sendai, plasmidi episomale o mRNA 1,2. In particolare, diversi metodi di riprogrammazione sono associati a livelli di efficienza e sicurezza variabile, ed è probabile che differiscono in altri modi che influenzano la loro adeguatezza per varie applicazioni. Con la disponibilità di una varietà di tecnologie di riprogrammazione, è diventato importante sviluppare metodi per valutare il processo di riprogrammazione. La maggior parte dei metodi esistenti si basano su l'ispezione qualitativa della morfologia o macchiecon coloranti e gli anticorpi specifici di cellule staminali. Un metodo recentemente sviluppato si avvale di giornalisti fluorescenza lentivirali che sono sensibili a specifici miRNA-PSC o mRNA specifico per cellulari differenziate 3. Tali metodi di monitoraggio facilitano la selezione e l'ottimizzazione delle tecniche di riprogrammazione per situazioni diverse. Ad esempio, CDy1 è stato utilizzato come una sonda fluorescente per i primi iPSCs al fine di schermo per modulatori di riprogrammazione 4. La capacità di osservare e confrontare diversi esperimenti di riprogrammazione è anche fondamentale per ottenere una migliore comprensione del processo stesso. Ad esempio, è ormai noto che alcuni tipi di cellule somatiche sono più facili da riprogrammare di altri 5, e che le cellule passare attraverso stati intermedi durante riprogrammazione 6-8. Purtroppo, i meccanismi alla base del processo di riprogrammazione non sono ancora del tutto chiare e, di conseguenza, le differenze esatte tra i metodi di riprogrammazione rimangono anche essere defined. Così, i metodi per il monitoraggio, la valutazione e confronto eventi riprogrammazione continuano ad essere critica per il campo delle cellule staminali.
I metodi descritti in questo protocollo consentono il controllo e la valutazione del processo di riprogrammazione e illustrano come queste tecniche possono essere utilizzati per confrontare diverse serie di reagenti di riprogrammazione. Il primo approccio prevede la citometria a flusso analizza utilizzando combinazioni di anticorpi contro cellule staminali pluripotenti positiva e negativa (PSC) marcatori. L'immagine in tempo reale secondo coppie di avvicinamento e la misurazione di confluenza totale (la superficie cento coperta dalle cellule) e confluenza dei segnali marcatori (la superficie percentuale coperta dai segnali fluorescenti).
Protocol
Representative Results
Discussion
This study provides strategies for monitoring and tracking of the reprogramming process using flow cytometry and real-time imaging-based analysis. The critical steps in the protocol are initiating reprogramming, measuring reprogramming progression based on marker expression and real-time monitoring of reprogramming. Any reprogramming method of choice can be used but here we focus on Sendai based reprogramming of human fibroblasts. The advantage of this method is the ease of use and consistent high efficiency of reprogram…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Gli autori ringraziano Chad MacArthur per le discussioni utili.
Materials
| DMEM, high glucose, GlutaMAXSupplement, pyruvate | Thermo Fisher Scientific | 10569-010 | |
| Fetal Bovine Serum, embryonic stem cell-qualified, US origin | Thermo Fisher Scientific | 16141-061 | |
| MEM Non-Essential Amino Acids Solution (100X) | Thermo Fisher Scientific | 11140-050 | |
| Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific | 25300-054 | |
| Mouse (ICR) Inactivated Embryonic Fibroblasts | Thermo Fisher Scientific | A24903 | |
| Attachment Factor Protein (1X) | Thermo Fisher Scientific | S-006-100 | |
| DMEM/F-12, GlutaMAX supplement | Thermo Fisher Scientific | 10565-018 | |
| KnockOut Serum Replacement | Thermo Fisher Scientific | 10828010 | |
| 2-Mercaptoethanol (55 mM) | Thermo Fisher Scientific | 21985-023 | |
| Collagenase, Type IV, powder | Thermo Fisher Scientific | 17104-019 | |
| TrypLE Select Enzyme (1X), no phenol red | Thermo Fisher Scientific | 12563-011 | |
| DPBS, no calcium, no magnesium | Thermo Fisher Scientific | 14190-144 | |
| Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix | Thermo Fisher Scientific | A1413302 | |
| Essential 8 Medium | Thermo Fisher Scientific | A1517001 | |
| FGF-Basic (AA 1-155) Recombinant Human Protein | Thermo Fisher Scientific | PHG0264 | |
| UltraPure 0.5M EDTA, pH 8.0 | Thermo Fisher Scientific | 15575-020 | |
| Bovine Albumin Fraction V (7.5% solution) | Thermo Fisher Scientific | 15260-037 | |
| HEPES (1 M) | Thermo Fisher Scientific | 15630-080 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140-122 | |
| InSolution Y-27632 | EMD Millipore | 688001 | |
| CytoTune-iPS Sendai Reprogramming Kit | Thermo Fisher Scientific | A1378001 | |
| CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo Fisher Scientific | A16517 | |
| Countess II Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | |
| Countess Cell Counting Chamber Slides | Thermo Fisher Scientific | C10228 | |
| BJ ATCC Human Foreskin Fibroblasts, Neonatal | ATCC | CRL-2522 | |
| DF1 Adult Human Dermal Fibroblast | Thermo Fisher Scientific | N/A | |
| BG01V/hOG Cells Variant hESC hOct4-GFP Reporter Cells | Thermo Fisher Scientific | R7799-105 | |
| IncuCyte ZOOM | Essen BioScience | ||
| SSEA-4 Antibody, Alexa Fluor 647 conjugate (MC813-70) | Thermo Fisher Scientific | SSEA421 | |
| SSEA-4 Antibody, Alexa Fluor 488 conjugate (eBioMC-813-70 (MC-813-70)) | Thermo Fisher Scientific | A14810 | |
| SSEA-4 Antibody (MC813-70) | Thermo Fisher Scientific | 41-4000 | |
| TRA-1-60 Antibody (cl.A) | Thermo Fisher Scientific | 41-1000 | |
| CD44 Rat Anti-Human/Mouse mAb (clone IM7), PE-Cy5 conjugate | Thermo Fisher Scientific | A27094 | |
| CD44 Alexa Fluor 488 Conjugate Kit for Live Cell Imaging | Thermo Fisher Scientific | A25528 | |
| CD44 Rat Anti-Human/Mouse mAb (Clone IM7) | Thermo Fisher Scientific | RM-5700 (no longer available) | |
| Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Thermo Fisher Scientific | A-11029 | |
| Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate | Thermo Fisher Scientific | A-11007 | |
| Alkaline Phosphatase Live Stain | Thermo Fisher Scientific | A14353 | |
| TRA-1-60 Alexa Fluor 488 Conjugate Kit for Live Cell Imaging | Thermo Fisher Scientific | A25618 | |
| CD24 Mouse Anti-Human mAb (clone SN3), FITC conjugate | Thermo Fisher Scientific | MHCD2401 | |
| beta-2 Microglobulin Antibody, FITC conjugate (B2M-01) | Thermo Fisher Scientific | A15737 | |
| EpCAM / CD326 Antibody, FITC conjugate (VU-1D9) | Thermo Fisher Scientific | A15755 | |
| CD73 / NT5E Antibody (7G2) | Thermo Fisher Scientific | 41-0200 | |
| VECTOR Red Alkaline Phosphatase (AP) Substrate Kit | Vector Laboratories | SK-5100 | |
| Zeiss Axio Observer.Z1 microscope | Carl Zeiss | 491912-0003-000 | |
| FlowJo Data Analysis Software | FLOJO, LLC | N/A | |
| Attune Accoustic Focusing Cytometer, Blue/Red Laser | Thermo Fisher Scientific | Use Attune NXT | |
| S3e Cell Sorter (488/561 nm) | BIO-RAD | 1451006 | |
| Falcon 12 x 75 mm Tube with Cell Strainer Cap | Corning | 352235 | |
| Falcon 15 mL, high-clarity, dome-seal screw cap | Corning | 352097 | |
| Falcon T-75 Flask | Corning | 353136 | |
| Falcon T-175 Flask | Corning | 353112 | |
| Falcon 6-well dish | Corning | 353046 | |
| HERAEUS HERACELL CO2 ROLLING INCUBATOR | Thermo Fisher Scientific | 51013669 | |
| Nonstick, RNase-free Microfuge Tubes, 1.5 mL | AM12450 | ||
| HulaMixer Sample Mixer | 15920D |
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