The authors thank Chad MacArthur for helpful discussions.

<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>

All of the authors [RHQ, JSTA, KS, and UL] are employees of Thermo Fisher Scientific, which produces some of the reagents and instruments used in this article.

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...

Patient-derived induced pluripotent stem cells (iPSCs) are promising tools for cell therapy and drug screening. They provide an autologous source of cells for therapy. In addition, they encompass a very broad set of genetic backgrounds, enabling a detailed in vitro analysis of genetic diseases beyond what current embryonic stem cell (ESC) lines would allow. Recent advances have led to the development of several methods for generating iPSCs, including reprogramming with Sendai virus, episomal plasmids or mRNAs 1,2. Notably, different reprogramming methods are associated with varying levels of efficiency and safety, and are likely to differ in other ways that influence their appropriateness for various applications. With the availability of a variety of reprogramming technologies, it has become important to develop methods for assessing the reprogramming process. Most existing methods rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. One recently developed method makes use of lentiviral fluorescence reporters that are sensitive to PSC-specific miRNAs or differentiated cell-specific mRNAs 3. Such monitoring methods facilitate the selection and optimization of reprogramming techniques for different situations. For example, CDy1 has been used as a fluorescent probe for early iPSCs in order to screen for reprogramming modulators 4. The ability to observe and compare different reprogramming experiments is also critical for gaining a better understanding of the process itself. For instance, it is now known that some somatic cell types are easier to reprogram than others 5, and that cells go through intermediate states during reprogramming 6-8. Unfortunately, the mechanisms underlying the reprogramming process are still not completely understood and consequently, the exact differences between reprogramming methods also remain to be defined. Thus, methods for monitoring, assessing, and comparing reprogramming events continue to be critical for the stem cell field.
The methods described in this protocol permit the monitoring and assessing of the reprogramming process and illustrate how these techniques can be used to compare different sets of reprogramming reagents. The first approach involves flow cytometry analyses using combinations of antibodies against positive and negative pluripotent stem cell (PSC) markers. The second approach couples real-time imaging and the measurement of total confluence (the percent surface area covered by the cells) and confluence of marker signals (the percent surface area covered by the fluorescent signals).

<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.

Monitoring Reprogramming Kinetics Using Flow Cytometry
CD44 is a fibroblast marker while SSEA4 is a PSC marker 6,10. As expected from this expression pattern, flow cytometry of BJ fibroblasts shows an SSEA4- CD44+ population that facilitates the creation of quadrant gates in combination with the unstained sample. During reprogramming of DF1 fibroblasts with the Sendai viruses, CD44 is gr...

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

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

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

Research

JoVE Journal

Developmental Biology

6.6K Views.  Thermo Fisher Scientific. The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Video: 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.

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

Behavior

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.

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

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.

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

Medicine

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.

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.

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

Neuroscience

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.

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.

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

Bioengineering

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.

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.

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