作者感谢乍得麦克阿瑟有益的讨论。

<ol>
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S., Vaz, C., Tanavde, V., Lakshmipathy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+is+a+negative+cell+surface+marker+for+pluripotent+stem+cell+identification+during+human+fibroblast+reprogramming.">CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming.</a> PLoS One. 9, e85419 (2014).</li><li>Papp, B., Plath, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+to+pluripotency:+stepwise+resetting+of+the+epigenetic+landscape.">Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape.</a> Cell Res. 21, 486-501 (2011).</li><li>Nefzger, C. M., Alaei, S., Knaupp, A. S., Holmes, M. L., Polo, J. 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所有作者[地区总部，JSTA，KS，和UL]是的Thermo Fisher Scientific，这产生了一些在这篇文章中所使用的试剂和仪器的员工。

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 reprogramming. 
To assess reprogramming progression, cells are harvested at discrete stages of reprogramming and subjected to an end-point flow cytometry analysis. Double staining of reprogramming cultures with the negative PSC marker CD44 and the positive PSC marker SSEA4 allows the assessment of reprogramming progression6. More specifically, the distribution of cells expressing either, neither or both markers at discrete stages of reprogramming enables the quantitative comparison of reprogramming kinetics under different conditions. This approach can demonstrate the differences in the kinetics and efficiency between different reprogramming methods and the media and matrices used during the reprogramming processes. The results obtained using different starting somatic cell samples confirms the importance of these factors in reprogramming efficiency and progression. They also demonstrate how this approach can be used to optimize reprogramming methods. The feasibility of monitoring reprogramming through flow cytometry using other combinations of pluripotent and non-pluripotent markers such as EPCAM, CD24, CD73, and B2M can be used in conjunction with the fore mentioned method14. Interestingly, these marker combinations show the formation of multiple cell populations through the course of reprogramming. Further studies will be required to fully characterize those populations and to determine their relevance in the reprogramming process. Regardless, the applicability of the flow cytometry approach with this diverse set of markers indicates that it can be applied more broadly, making use of different markers-of-interest and uncovering many other reprogramming intermediates. The limitation with this approach is that cells will need to be harvested for end point analysis. This limitation can be overcome with the use of live monitoring methods.
Monitoring of cells undergoing reprogramming in real time is done using the Live Cell Imaging system. Confluence, as measured under phase contrast, increased rapidly as colonies began to form. As such, measuring total confluence under phase contrast allows us to track the progression of reprogramming easily in real time, but because this measurement encompasses unreprogrammed and partially reprogrammed cells that are negative for AP, SSEA4 and TRA-1-60, this approach is only semi-quantitative. Linking the real-time phase contrast images to end-point positive marker staining with either dyes such as Live Alkaline Phosphatase or pluripotent markers such as SSEA4 or TRA-1-60 provides better insight into the kinetics of the reprogramming process11. However, this study shows that a more accurate and quantitative imaging-based method for tracking reprogramming would involve measuring fluorescence signal confluence while using an OCT4 reporter line16. Each of the methods presented here have their own advantages and disadvantages that make them appropriate for different situations. The flow cytometry approach is laborious and requires a lot of cells, but it provides the most accurate quantitative data. Measuring fluorescence confluence while using an OCT4 reporter line is easier and still quantitative, but it is not always feasible to obtain and use OCT4 reporter-containing parental cells. Finally, measuring total confluence under phase contrast is easy and applicable to any parental cell, but this approach is only semi-quantitative. Ultimately, the choice of method should depend on the available resources and the requirements of the actual experiments involved. 
The methods outlined here provide a platform for comparing new reprogramming technologies, optimizing current work flows and extending the use of reprogramming to novel primary cell types.

源自患者的诱导多能干细胞（iPS细胞）是用于细胞治疗和药物筛选有希望的工具。它们提供了细胞治疗自体来源。此外，它们包括非常广泛的一套遗传背景，使得遗传疾病超出当前的胚胎干细胞（ESC）的线路将允许进行详细的体外分析。最近的进展已经导致几种方法的发展，用于产生iPSCs的，包括与仙台病毒，附加型质粒或mRNA的1,2-重新编程。值得注意的是，不同的重编程方法具有不同的效率和安全性的水平相关，并有可能在影响其是否适宜用于各种用途的其他方法不同。随着各种重编程技术的可用性，它已开发用于评估重编程过程的方法变得很重要。大多数现有的方法依赖于形态和染色质检查用干细胞特异性的染料和抗体。一个最近开发的方法使用的慢病毒荧光记者，是对特定的PSC-miRNA的或分化的细胞特异性的mRNA 3敏感。这种监测方法便于选择和重新编程的技术对不同的情况下的优化。例如，CDy1已经用作早期的iPSC的荧光探针以筛选重新编程调制器4。观察和比较不同的重编程实验的能力也更好地了解这个过程本身的关键。例如，现在已知的是一些体细胞类型更容易比其它5重新编程，并且使细胞重新编程6-8中经过中间状态。不幸的是，重编程过程所依据的机制仍然不完全了解，因此，重编程方法之间的确切的差别也仍然为defined。因此，对于监测方法，评估和比较重编程事件仍然是干细胞领域的关键。 在这个协议中所描述的方法允许监控和重编程过程的评估，并说明这些技术如何可以用来比较不同组的重编程试剂。第一方法涉及流式细胞仪使用针对阳性和阴性的多能干细胞（PSC）标记的抗体的组合进行分析。第二种方式耦合实时成像和总合流（被细胞覆盖的百分比表面积）和标记信号（由荧光信号所覆盖的百分比表面积）的合流的测量。

<table border="0" cellpadding="0" cellspacing="0" width="795">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">DMEM, high glucose, GlutaMAXSupplement, pyruvate</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">10569-010</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Fetal Bovine Serum, embryonic stem cell-qualified, US origin</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">16141-061</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Trypsin-EDTA (0.05%), phenol red&#160;</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Mouse (ICR) Inactivated Embryonic Fibroblasts</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A24903</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Attachment Factor Protein (1X)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">S-006-100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">DMEM/F-12, GlutaMAX supplement</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">10565-018</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">KnockOut Serum Replacement</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:147px;">10828010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">2-Mercaptoethanol (55 mM)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Collagenase, Type IV, powder</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">TrypLE Select Enzyme (1X), no phenol red&#160;</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">12563-011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">DPBS, no calcium, no magnesium&#160;</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:297px;">Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A1413302</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Essential 8 Medium</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A1517001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">FGF-Basic (AA 1-155) Recombinant Human Protein</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">PHG0264</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Bovine Albumin Fraction V (7.5% solution)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">15260-037</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">HEPES (1 M)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">InSolution Y-27632</td>
			<td style="width:263px;">EMD Millipore</td>
			<td align="right" style="width:147px;">688001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A1378001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CytoTune-iPS 2.0 Sendai Reprogramming Kit</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A16517</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Countess II Automated Cell Counter</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Countess Cell Counting Chamber Slides</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">C10228</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">BJ ATCC Human Foreskin Fibroblasts, Neonatal</td>
			<td style="width:263px;">ATCC</td>
			<td style="width:147px;">CRL-2522</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">DF1 Adult Human Dermal Fibroblast</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">N/A</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">BG01V/hOG Cells Variant hESC hOct4-GFP Reporter Cells</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">R7799-105</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">IncuCyte ZOOM</td>
			<td style="width:263px;">Essen BioScience</td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">SSEA-4 Antibody, Alexa Fluor 647 conjugate (MC813-70)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">SSEA421</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:297px;">SSEA-4 Antibody, Alexa Fluor 488 conjugate (eBioMC-813-70 (MC-813-70))</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A14810</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">SSEA-4 Antibody (MC813-70)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">41-4000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">TRA-1-60 Antibody (cl.A)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">&#160;41-1000</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CD44 Rat Anti-Human/Mouse mAb (clone IM7), PE-Cy5 conjugate</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A27094</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CD44 Alexa Fluor 488 Conjugate Kit for Live Cell Imaging</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A25528</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CD44 Rat Anti-Human/Mouse mAb (Clone IM7)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">RM-5700 (no longer available)</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">&#160;A-11029</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">&#160;A-11007</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Alkaline Phosphatase Live Stain</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A14353</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">TRA-1-60 Alexa Fluor 488 Conjugate Kit for Live Cell Imaging</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A25618</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">CD24 Mouse Anti-Human mAb (clone SN3), FITC conjugate</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">MHCD2401</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">beta-2 Microglobulin Antibody, FITC conjugate (B2M-01)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A15737</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">EpCAM / CD326 Antibody, FITC conjugate (VU-1D9)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">A15755</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">CD73 / NT5E Antibody (7G2)</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">41-0200</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">VECTOR Red Alkaline Phosphatase (AP) Substrate Kit</td>
			<td style="width:263px;">Vector Laboratories</td>
			<td style="width:147px;">SK-5100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Zeiss Axio Observer.Z1 microscope&#160;</td>
			<td style="width:263px;">Carl Zeiss</td>
			<td style="width:147px;">491912-0003-000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">FlowJo Data Analysis Software</td>
			<td style="width:263px;">FLOJO, LLC</td>
			<td style="width:147px;">N/A</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Attune Accoustic Focusing Cytometer, Blue/Red Laser</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">Use Attune NXT&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">S3e\ Cell Sorter (488/561 nm)</td>
			<td style="width:263px;">BIO-RAD</td>
			<td align="right" style="width:147px;">1451006</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Falcon 12 x 75 mm Tube with Cell Strainer Cap</td>
			<td style="width:263px;">Corning</td>
			<td align="right" style="width:147px;">352235</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Falcon 15 mL, high-clarity, dome-seal screw cap</td>
			<td style="width:263px;">Corning</td>
			<td align="right" style="width:147px;">352097</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Falcon T-75 Flask</td>
			<td style="width:263px;">Corning</td>
			<td align="right" style="width:147px;">353136</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Falcon T-175 Flask</td>
			<td style="width:263px;">Corning</td>
			<td align="right" style="width:147px;">353112</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">Falcon 6-well dish</td>
			<td style="width:263px;">Corning</td>
			<td align="right" style="width:147px;">353046</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">HERAEUS HERACELL CO2 ROLLING INCUBATOR</td>
			<td style="width:263px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:147px;">51013669</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:297px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:263px;"></td>
			<td style="width:147px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">HulaMixer Sample Mixer</td>
			<td style="width:263px;"></td>
			<td style="width:147px;">15920D</td>
			<td></td>
		</tr>
	</tbody>
</table>

kinetic measurement and real time visualization of somatic reprogramming

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.

监测重新编程动力学采用流式细胞仪 CD44是一种纤维原细胞标记，而SSEA4是PSC标记6,10。从该表达模式预期，流式细胞仪BJ成纤维细胞显示了一个SSEA4 - CD44 +群体便于与未染色的样品组合象限门的创作。在DF1成纤维细胞的仙台病毒的重新编程，CD44逐渐丧失，而SSEA4缓慢表示。在与仙台病毒转导后第3天，有SSEA4的人口众多- CD44 +细胞和SSEA4 + CD44 +细胞中的一小人口变成7天（ 图1）更明显。状态-在13日，双阳性人群对SSEA4 + CD44过渡。 17日，行政长官有很大比例在文化LLS已经SSEA4 + CD44 - 。这个时间过程表明，与CD44和SSEA4流式细胞仪可用于监测重编程的进展和速率。 定量比较，证实了重新编程BJ成纤维细胞重编程的仙台病毒产生SSEA4 + CD44 -以不同速度比BJ成纤维细胞（ 图2A）的重编程细胞。该数据表明，PSC状SSEA4 + CD44的百分比的早期比较-细胞跟踪重新编程的进展。有趣的是，在DF1成纤维细胞重编程的第8天，分选和单独培养是在上调SSEA4过程细胞群和下调CD44生成AP +集落11（ 图2B）。与此相反，原始SSEA4 - CD44 +群体产生点UCH在重编程的第21天较少数量的AP +集落的，这是最后的集落分析的典型的一天。这表明，有可能量化和为了比较过渡人口预测连SSEA4 + CD44之前在重编程动力学差异-形成人口。这里要注意的重要一点是，重新编程是可变进程取决于几个因素，如起始体细胞群体，转导效率，介质等 ，但上述的表面标记的一般模式和进展是不同的重编程的实验和之间的一致性开始成纤维细胞。重编程动力学的分析也可以用不同的标记物进行，如新鉴定的阴性的PSC标记物CD73和B2M 6，以及已建立的PSC标记物CD24 12,13和EPCAM 14,15。以CD44，CD73和B2M类似在参数表示ental BJ成纤维细胞，但重编程的过程中下调，成为完全重新编程的iPSCs（ 图3A）检测不到。流式细胞仪使用CD44和CD73或CD44和B2M时间过程的流程表明CD44和CD73以类似的速度下调，而B2M比CD44（ 图3B）下调更快。在这两种情况下，重编程的速率可以通过测量双负人口的积累被观察到。相对于这些负PSC标记物，CD24和EPCAM是在成纤维细胞不存在，并且在iPSCs的（ 图3A）表示。在流式细胞仪时程的流程，CD24 - CD44 +和EPCAM - CD44 +成纤维细胞最终形成双阴性群后来过渡到CD24 + CD44 -和EPCAM + CD44 - PSC样细胞，分别为（ 图3B）。因此，重编程过程中，既有积极的PSC标记CD44被下调后表示。用SSEA4和CD44，形成与不同细胞群的积累所观察相似指示重新编程的进展。 跟踪重新编程使用实时成像分析 补播节目的殖民地逐渐形成（ 图4A），它对应于下相衬（ 图4B）的汇合对数增加后重新编程的文化实时成像。下相衬合流从而提供了用于定量地监测重编程其他指标，但它同时包括，是阳性的PSC标记和unreprogrammed或部分重编程的细胞（ 图4A，上面板）的持续增殖菌落。量化已染色多潜能标志物的区TRA-1-60，SSEA4及AP 6,10在第21天表示的iPSC只占小于总汇合（ 图4B）的一半。为了更严格测量重编程的进展，有可能使用报道线重新编程。这里，从该BG01V / HOG ESC线，其已被设计与OCT4-GFP报告16产生的二次成纤维细胞。 GFP被表达为细胞重新编程并作为菌落形成（ 图5A）。下相衬和绿色通道测量合流表明GFP的合流比总合流增加更慢，并且在第19天的GFP合流是小于总汇合（ 图5B）的一半，使人联想TRA-1-60的， SSEA4及AP染色在第21天。 <img alt="图1" src="/files/ftp_upload/54190/54190fig1.jpg" /> 图1. 墨nitoring重编程的使用流式细胞仪的研究进展。流式细胞仪散点图对DF1成纤维细胞与病毒仙台下无饲养条件下重新编程。收获细胞，并从第3天（D3）重编程过程的不同时间间隔19天（D19）与SSEA4和CD44抗体染色。在9日的文化（D9）起进行细胞在第7天（D7）进行了重新接种后进行分析。散点图显示8000汗衫。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54190/54190fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54190/54190fig2.jpg" /> 图 2. 在无饲养条件评估重编程动力学和效率 （A）图表显示SSEA4 + CD44的百分比变化-转导DF1 FIB后重新编程的细胞roblasts和BJ成纤维细胞的仙台病毒。细胞通过流式细胞术在第3天至19转导后进行分析，在9日培养起经历在节7（B）的伪彩色补种流式细胞地块为导仙台病毒并用SSEA4和CD44抗体染色DF1细胞。 SSEA4 - CD44 +细胞（黑圈）和SSEA4 +细胞（红圈）在8日进行了梳理，并允许染色碱性磷酸酶（红色）前生长，直到19日<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54190/54190fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54190/54190fig3.jpg" /> 图3. 无饲养文化的重新编程观测动力学使用不同正负PSC标记。（AD）染色直播的使用抗体为阳性PSC标记物CD24日间18 DF1衍生的重编程的文化和EPCAM和负PSC标记物CD44，CD73，和B2M。顶端板合并的绿色和红色荧光通道，而底板还包括相位对比图像。比例尺条为200μm的（E）流式细胞仪点阵图为收获并使用从9日同一标记染色以17（D9至D17）DF1衍生重新编程培养转导后。在分析之前，在培养7天再接种了点图显示8000汗衫。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54190/54190fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54190/54190fig4.jpg" /> 图4. 跟踪重编程动力学测量总合流。（A）实时IBJ的maging成纤维细胞无饲养条件下，用仙台病毒重新编程。同样的菌落相衬图像在7日被带到19，然后在21日与CD44和SSEA4，TRA-1-60或AP额外的染色。总合流（相差），为重编程的比例尺对应于400微米。（B）的定量从在第21天在第7天再接种到日21.总计合流进行过程中相比SSEA4，TRA-1-60和AP信号汇合（绿色通道）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54190/54190fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54190/54190fig5.jpg" /> 图5. 测量OCT4-GFP汇流评估重编程动力学。（A）和重新编程BG01V /猪胚胎干细胞成纤维细胞的实时成像仙台无饲养条件下的病毒。相衬，绿色荧光并且合并在第7天生成19.比例尺对应于400μm的相同菌落图像。（B）的总合流（相差）和OCT4-GFP融合（绿色通道）的定量为重编程的进展从7日重新播种到21日<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54190/54190fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Kinetic Measurement and Real Time Visualization of Somatic Reprogramming at JoVE.com

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

在本研究中提出的协议描述用于通过使用流式细胞仪分析阳性和阴性的多能干细胞标志物的动力学测定的重编程级数的实时监测的方法。该协议还包括的iPSC产生过程中的形态和标志物或报告表达的基于成像的评估。

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

kinetic-measurement-real-time-visualization-somatic

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.6K 次观看.  Thermo Fisher Scientific. 在本研究中提出的协议描述用于通过使用流式细胞仪分析阳性和阴性的多能干细胞标志物的动力学测定的重编程级数的实时监测的方法。该协议还包括的iPSC产生过程中的形态和标志物或报告表达的基于成像的评估。 

视频: Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

血管血管和血管生成的动力学分析体外

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...