Name: kinetic measurement and real time visualization of somatic reprogramming
Uploaded: 2016-07-30T13:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Kinetic Measurement and Real Time Visualization of Somatic Reprogramming at JoVE.com

作者感谢乍得麦克阿瑟有益的讨论。

<ol>
	<li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell Stem Cell. 10, 678-684 (2012).</li><li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481, 295-305 (2012).</li><li>Kamata, M., Liang, M., Liu, S., Nagaoka, Y., Chen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+monitoring+of+hiPSC+generation+and+differentiation+using+differential+expression+of+endogenous+microRNAs.">Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs.</a> PLoS One. 5, e11834 (2010).</li><li>Vendrell, M., Zhai, D., Er, J. C., Chang, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+strategies+in+fluorescent+probe+development.">Combinatorial strategies in fluorescent probe development.</a> Chem Rev. 112, 4391-4420 (2012).</li><li>Aasen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Quintanilla, R. H., Asprer, J. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+marker+mediated+purification+of+iPS+cell+intermediates+from+a+reprogrammable+mouse+model.">Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model.</a> J Vis Exp. , e51728 (2014).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+fibroblast-like+cells+derived+from+human+embryonic+stem+cells+support+undifferentiated+cell+growth.">Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth.</a> Stem Cells. 22, 972-980 (2004).</li><li>International Stem Cell Initiative. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nat Biotechnol. 25, 803-816 (2007).</li><li>Singh, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+live+alkaline+phosphatase+substrate+for+identification+of+pluripotent+stem+cells.">Novel live alkaline phosphatase substrate for identification of pluripotent stem cells.</a> Stem Cell Rev. 8, 1021-1029 (2012).</li><li>Naujok, O., Lenzen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+re-evaluation+of+CD24-positivity+of+human+embryonic+stem+cells+differentiated+into+pancreatic+progenitors.">A critical re-evaluation of CD24-positivity of human embryonic stem cells differentiated into pancreatic progenitors.</a> Stem Cell Rev. 8, 779-791 (2012).</li><li>Ramirez, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+report:+benchmarking+human+pluripotent+stem+cell+markers+during+differentiation+into+the+three+germ+layers+unveils+a+striking+heterogeneity:+all+markers+are+not+equal.">Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal.</a> Stem Cells. 29, 1469-1474 (2011).</li><li>Huang, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule+(EpCAM)+complex+proteins+promote+transcription+factor-mediated+pluripotency+reprogramming.">Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.</a> J Biol Chem. 286, 33520-33532 (2011).</li><li>Kolle, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+embryonic+stem+cell+surface+markers+by+combined+membrane-polysome+translation+state+array+analysis+and+immunotranscriptional+profiling.">Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling.</a> Stem Cells. 27, 2446-2456 (2009).</li><li>Thyagarajan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+engineered+human+embryonic+stem+cell+lines+using+phiC31+integrase.">Creation of engineered human embryonic stem cell lines using phiC31 integrase.</a> Stem Cells. 26, 119-126 (2008).</li></ol>

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 reprogram...

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

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

Universidad San Sebastian

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

Multifunctional Setup for Studying Human Motor Control Using Transcranial Magnetic Stimulation, Electromyography, Motion Capture, and Virtual Reality

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular function include transcranial magnetic stimulation, electromyography, and three-dimensional motion capture. The advent of readily available and cost-effective virtual reality solutions has expanded the capabilities of researchers in recreating &#8220;real-world&#8221; environments and movements in a laboratory setting. Naturalistic movement analysis will not only garner a greater understanding of motor control in healthy individuals, but also permit the design of experiments and rehabilitation strategies that target specific motor impairments (e.g. stroke). The combined use of these tools will lead to increasingly deeper understanding of neural mechanisms of motor control. A key requirement when combining these data acquisition systems is fine temporal correspondence between the various data streams. This protocol describes a multifunctional system&#8217;s overall connectivity, intersystem signaling, and the temporal synchronization of recorded data. Synchronization of the component systems is primarily accomplished through the use of a customizable circuit, readily made with off the shelf components and minimal electronics assembly skills.

Transcranial magnetic stimulation, electromyography, and 3D motion capture are commonly used non-invasive techniques for investigating neuromuscular function in humans. In this paper, we describe a protocol that synchronously samples data generated by all three of these tools along with the unique addition of virtual reality stimulus presentation and feedback.

多功能设置研究人类电机控制用经颅磁刺激，肌电图，运动捕捉和虚拟现实

The study of neuromuscular control of movement in humans is accomplished with numerous technologies. Non-invasive methods for investigating neuromuscular ...

科研

行为学

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

发育生物学

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

医学

Real-Time Proxy-Control of Re-Parameterized Peripheral Signals using a Close-Loop Interface

The fields that develop methods for sensory substitution and sensory augmentation have aimed to control external goals using signals from the central nervous systems (CNS). Less frequent however, are protocols that update external signals self-generated by interactive bodies in motion. There is a paucity of methods that combine the body-heart-brain biorhythms of one moving agent to steer those of another moving agent during dyadic exchange. Part of the challenge to accomplish such a feat has been the complexity of the setup using multimodal bio-signals with different physical units, disparate time scales and variable sampling frequencies.
In recent years, the advent of wearable bio-sensors that can non-invasively harness multiple signals in tandem, has opened the possibility to re-parameterize and update the peripheral signals of interacting dyads, in addition to improving brain- and/or body-machine interfaces. Here we present a co-adaptive interface that updates efferent somatic-motor output (including kinematics and heart rate) using biosensors; parameterizes the stochastic bio-signals, sonifies this output, and feeds it back in re-parameterized form as visuo/audio-kinesthetic reafferent input. We illustrate the methods using two types of interactions, one involving two humans and another involving a human and its avatar interacting in near real time. We discuss the new methods in the context of possible new ways to measure the influences of external input on internal somatic-sensory-motor control.

We present protocols and methods of analyses to build co-adaptive interfaces that stream, parameterize, analyze, and modify human body and heart signals in close-loop. This setup interfaces signals derived from the peripheral and central nervous systems of the person with external sensory inputs to help track biophysical change.

使用闭环接口实时代理控制重新参数化的外围信号

The fields that develop methods for sensory substitution and sensory augmentation have aimed to control external goals using signals from the central ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

神经科学

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

生物工程

Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). However, establishing the neural correlates associated with MT therapy have been challenging given that it is difficult to administer the therapy effectively within a magnetic resonance imaging (MRI) scanner environment. To characterize the functional organization of cortical regions associated with this rehabilitative strategy, we have developed a combined behavioral and functional neuroimaging protocol that can be applied in participants with a leg amputation. This novel approach allows participants to undergo MT within the MRI scanner environment by viewing real-time video images captured by a camera. The images are viewed by the participant through a system of mirrors and a monitor that the participant views while lying on the scanner bed. In this manner, functional changes in cortical areas of interest (e.g., sensorimotor cortex) can be characterized in response to the direct application of MT.

We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

MRI 实时视频显示在与镜治疗的恐惧症肢体疼痛相关的神经相关性特征中的应用

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

体细胞重编程的动力学测量和实时可视化

摘要

探索更多视频

此视频中的章节

多功能设置研究人类电机控制用经颅磁刺激，肌电图，运动捕捉和虚拟现实

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

目标移植的肌肉祖细胞的分子成像

使用闭环接口实时代理控制重新参数化的外围信号

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Assessing Cardiac Reprogramming using High Content Imaging Analysis

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

评估人体骨骼肌微组织骨骼肌健康的功能指标

MRI 实时视频显示在与镜治疗的恐惧症肢体疼痛相关的神经相关性特征中的应用

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

体细胞重编程的动力学测量和实时可视化

摘要

探索更多视频

此视频中的章节

多功能设置研究人类电机控制用经颅磁刺激，肌电图，运动捕捉和虚拟现实

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

目标移植的肌肉祖细胞的分子成像

使用闭环接口实时代理控制重新参数化的外围信号

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Assessing Cardiac Reprogramming using High Content Imaging Analysis

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

评估人体骨骼肌微组织骨骼肌健康的功能指标

MRI 实时视频显示在与镜治疗的恐惧症肢体疼痛相关的神经相关性特征中的应用

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性