체세포 재 프로그래밍의 운동 측정 및 실시간 시각화
Summary
본 연구에 제시된 프로토콜은 유세포 분석을 이용하여 양 및 음의 다 능성 줄기 세포 마커의 운동 측정을 통해 프로그래밍 진행의 실시간 모니터링 방법을 설명한다. 이 프로토콜은 또한 IPSC 생성시 형태, 마커 또는 기자의 표현의 영상 기반의 평가를 포함한다.
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
환자 유래 유도 만능 줄기 세포 (iPSCs)는 세포 치료 및 약물 검사를위한 유망한 도구입니다. 그들은 치료를위한 세포의자가 소스를 제공합니다. 또한, 이들은 현재 배아 줄기 세포 (ESC) 라인이 허용하는 것 이상의 유전 질환의 상세한 시험 관내 분석을 가능 유전 배경의 매우 광범위한 세트를 포함한다. 최근의 진보는 센다이 바이러스, 플라스미드 또는 에피 솜의 mRNA 1,2- 함께 프로그래밍 포함 iPSCs를 생성하는 여러 가지 방법의 개발을 이끌어왔다. 특히, 다양한 프로그래밍 방법은 효율성 및 안전성의 레벨 변화와 연관된 다양한 애플리케이션에 자신의 적합성에 영향을 미치는 다른 방법과 다를 가능성이있다. 프로그래밍 기술의 다양한 가용성, 상기 프로그래밍 프로세스를 평가하기위한 방법을 개발하는 것이 중요하게되었다. 대부분의 기존 방법은 형태 나 염색의 정성 검사에 의존줄기 세포 특이 염료 및 항체. 하나는 최근에 개발 된 방법은 PSC 별의 miRNAs 또는 분화 된 세포 고유의 mRNA 3에 민감한 렌티 바이러스 형광 기자를 사용합니다. 이러한 모니터링 방법은 선택과 다른 상황에 대한 재 프로그래밍 기술의 최적화를 용이하게한다. 예를 들어, 재 프로그래밍 CDy1 조절제 4 스크리닝하기 위해 초기 iPSCs하는 형광 프로브로서 사용되었다. 관찰하고 다른 프로그래밍 실험을 비교하는 기능은 프로세스 자체의 이해를 얻는데 중요하다. 예를 들어, 이제 일부 체세포 유형이 다른 5보다 쉽게 재 프로그래밍 것으로 알려져 있으며, 셀 6-8 재 프로그래밍 동안에 중간 상태를 끝까지. 불행하게도, 재 프로그래밍 절차의 기초가되는 메카니즘은 아직 완전히 이해되지 않으며, 따라서, 리 프로그래밍 방법의 정확한 차이 교리되도록 유지efined. 따라서, 모니터링 방법, 평가 및 재 프로그래밍 이벤트를 비교는 줄기 세포 분야에 대한 중요 할 것을 계속한다.
이 프로토콜에서 설명하는 방법은 모니터링을 허용하고, 프로그래밍 프로세스를 평가하고 이들 기술은 프로그래밍 시약의 상이한 세트를 비교하는 방법을 도시한다. 첫 번째 방법은 유세포 포함 양 및 음의 다 능성 줄기 세포 (PSC) 마커에 대한 항체의 조합을 사용하여 분석한다. 두 번째 방법 커플 실시간 영상 및 총 합류 (셀 적용 비율 표면적)와 마커 신호 (형광 신호에 의해 덮여 %의 표면적)의 합류 측정.
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
저자는 도움이 토론 차드 맥아더 감사합니다.
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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