Kinetic Medição e visualização em tempo real de Reprogramação Somatic
Summary
O protocolo apresentado no presente estudo descreve métodos para a monitorização em tempo real da progressão reprogramação através da medição da cinética de marcadores de células estaminais pluripotentes positivos e negativos utilizando análise de citometria de fluxo. O protocolo também inclui a avaliação baseada em imagens da morfologia, e marcador ou expressão repórter durante a geração da 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
As células-tronco pluripotentes induzidas derivadas de doentes (iPSCs) são ferramentas promissoras para a terapia celular e triagem de drogas. Eles fornecem uma fonte de células autólogas para terapia. Além disso, elas abrangem um amplo conjunto de origens genéticas, permitindo uma análise detalhada in vitro de doenças genéticas além do que as linhas atuais células-tronco embrionárias (ESC) permitiria. Os avanços recentes levou ao desenvolvimento de vários métodos para gerar iPSCs, incluindo reprogramação com o vírus Sendai, plasmídeos epissomais ou ARNm 1,2. Notavelmente, os diferentes métodos de reprogramação são associados com diferentes níveis de eficiência e segurança, e é provável que diferem de outras formas que influenciam a sua adequação para várias aplicações. Com a disponibilidade de uma variedade de tecnologias de reprogramação, tornou-se importante o desenvolvimento de métodos para avaliar o processo de reprogramação. A maioria dos métodos existentes contam com a inspeção qualitativa da morfologia ou coloraçãocom corantes e anticorpos específicos de células-tronco. Um método recentemente desenvolvido faz uso de repórteres de fluorescência de lentivírus que são sensíveis à miARNs específicos do CPS ou de mRNAs específicos de células diferenciadas 3. Tais métodos de monitorização facilitar a selecção e optimização de técnicas de reprogramação para diferentes situações. Por exemplo, CDy1 foi usado como uma sonda fluorescente para os primeiros iPSCs, a fim de pesquisar moduladores de reprogramação 4. A capacidade de observar e comparar diferentes experiências de reprogramação também é crítico para a obtenção de uma melhor compreensão do próprio processo. Por exemplo, sabe-se agora que alguns tipos de células somáticas são mais fáceis de reprogramar 5 do que outros, e que as células passam por estados intermédios durante a reprogramação 6-8. Infelizmente, os mecanismos subjacentes ao processo de reprogramação ainda não estão completamente esclarecidos e, consequentemente, as diferenças exatas entre métodos de reprogramação também continuam a ser defined. Assim, métodos de monitorização, avaliação e comparação de eventos de reprogramação continuar a ser crítico para o campo das células estaminais.
Os métodos descritos no presente protocolo de permitir a monitorização e a avaliação do processo de reprogramação e ilustram como estas técnicas podem ser usadas para comparar diferentes conjuntos de reagentes de reprogramação. A primeira abordagem envolve a citometria de fluxo analisa utilizando combinações de anticorpos contra células-tronco pluripotentes positivo e negativo (PSC) marcadores. A segunda abordagem pares de imagens em tempo real e a medição da confluência total (área da superfície coberta por cento por as células) e confluência de sinais de marcador (por cento da área de superfície coberta pelos sinais fluorescentes).
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
Os autores agradecem Chad MacArthur para discussões úteis.
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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