体細胞の再プログラミングの反応速度測定とリアルタイムの可視化
Summary
この研究で提示されたプロトコルは、フローサイトメトリー分析を使用して、正および負の多能性幹細胞マーカーの動的測定によって再プログラミングの進行をリアルタイムでモニタリングするための方法を記載しています。プロトコルは、IPSCの生成時形態、およびマーカーまたはレポーター発現のイメージングに基づく評価を含みます。
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
患者由来の人工多能性幹細胞（iPS細胞）を細胞療法及び薬物スクリーニングのための有望なツールです。彼らは、治療のための細胞の自家源を提供します。さらに、それらは、胚性幹細胞（ESC）の行ができるようになり、現在のものを超えて遺伝病の詳細なインビトロ分析を可能にする、遺伝的背景の非常に広いセットを包含する。最近の進歩は、センダイウイルス、エピソームプラスミドまたはmRNAを1,2と再プログラミングを含む、iPS細胞を生成するためのいくつかの方法の開発につながっています。特に、別の再プログラミング方法は、効率性と安全性のレベルを変化させることに関連しており、様々な用途のためのそれらの適切性に影響を与える他の方法で異なる可能性が高いされています。再プログラミング技術の種々の利用可能性によれば、再プログラミングプロセスを評価するための方法を開発することが重要になってきています。ほとんどの既存の方法は、形態または染色の定性的な検査に依存します幹細胞特異的色素と抗体を用いました。一つの最近開発された方法は、PSC-特異的miRNAまたは分化細胞特異的なmRNA 3に敏感であるレンチウイルスの蛍光レポーターを利用します。このような監視方法は、さまざまな状況のための技術を再プログラミングの選択および最適化を容易にします。例えば、CDy1は再プログラミング変調器4をスクリーニングするために、初期のiPS細胞用の蛍光プローブとして使用されています。異なる再プログラミング実験を観察し、比較する能力はまた、プロセス自体のより良い理解を得るために重要です。例えば、現在、いくつかの体細胞タイプは他の5よりも再プログラムが容易であることが知られており、細胞を6-8再プログラミングの間の中間状態を通過すること。残念ながら、再プログラミングプロセスの基礎となるメカニズムはまだ完全には理解されておらず、その結果、再プログラミングの方法との間の正確な違いはまた、dをされずに残っていますefined。このように、監視のための方法、評価、および再プログラミングイベントを比較するには、幹細胞分野のために重要であり続けます。
このプロトコールに記載された方法は、監視を可能にし、再プログラミングプロセスを評価し、これらの技術は、再プログラミング試薬の異なるセットを比較するために使用することができる方法を示します。第1のアプローチは、フローサイトメトリーを含む正および負の多能性幹細胞（PSC）のマーカーに対する抗体の組合せを使用して分析します。第二のアプローチカップルリアルタイムイメージング及び合計コンフルエンス（細胞によって覆われたパーセント面積）を測定し、マーカ信号の合流（蛍光シグナルによって覆わパーセント面積）。
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
著者らは、有用な議論のためにチャドマッカーサーに感謝します。
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 |
Referenzen
- Yamanaka, S. Induced pluripotent stem cells: past, present, and future. Cell Stem Cell. 10, 678-684 (2012).
- Robinton, D. A., Daley, G. Q. The promise of induced pluripotent stem cells in research and therapy. Nature. 481, 295-305 (2012).
- Kamata, M., Liang, M., Liu, S., Nagaoka, Y., Chen, I. S. Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. PLoS One. 5, e11834 (2010).
- Vendrell, M., Zhai, D., Er, J. C., Chang, Y. T. Combinatorial strategies in fluorescent probe development. Chem Rev. 112, 4391-4420 (2012).
- Aasen, T., et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol. 26, 1276-1284 (2008).
- Quintanilla, R. H., Asprer, J. S., Vaz, C., Tanavde, V., Lakshmipathy, U. CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLoS One. 9, e85419 (2014).
- Papp, B., Plath, K. Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape. Cell Res. 21, 486-501 (2011).
- Nefzger, C. M., Alaei, S., Knaupp, A. S., Holmes, M. L., Polo, J. M. Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model. J Vis Exp. , e51728 (2014).
- Xu, C., et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells. 22, 972-980 (2004).
- International Stem Cell Initiative. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol. 25, 803-816 (2007).
- Singh, U., et al. Novel live alkaline phosphatase substrate for identification of pluripotent stem cells. Stem Cell Rev. 8, 1021-1029 (2012).
- Naujok, O., Lenzen, S. A critical re-evaluation of CD24-positivity of human embryonic stem cells differentiated into pancreatic progenitors. Stem Cell Rev. 8, 779-791 (2012).
- Ramirez, J. M., et al. Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal. Stem Cells. 29, 1469-1474 (2011).
- Huang, H. P., et al. Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming. J Biol Chem. 286, 33520-33532 (2011).
- Kolle, G., et al. Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling. Stem Cells. 27, 2446-2456 (2009).
- Thyagarajan, B., et al. Creation of engineered human embryonic stem cell lines using phiC31 integrase. Stem Cells. 26, 119-126 (2008).
