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

The authors thank Chad MacArthur for helpful discussions.

1.Solution and Medium Preparation
<ol>
	<li>Basement Membrane Matrix (Purified from Engelbreth-Holm-Swarm Tumor)
	<ol>
		<li>Slowly thaw the basement membrane matrix (5 ml) on ice at 4 &#176;C overnight.</li>
		<li>Dilute the stock solution 1:1 with 5 ml of ice cold, sterile DMEM/F-12 medium in a sterile, pre-chilled 15 ml conical tube. Dispense aliquots into pre-chilled, 1.5 ml sterile micro centrifuge tubes and immediately store at -20 &#176;C.</li>
		<li>Prior to usage, thaw the frozen 1:1 basement ...

<ol>
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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 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.

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 ...

The overall goal of these procedures is to visualize and track the progression of somatic cell reprogramming into induced pluripotent stem cells or iPSC. This method can be used to answer key questions in the iPSC field, such as how reprogramming kinetics and reprogramming efficiencies are altered by the somatic tissues used, the reprogramming technologies applied, and the matrix and media combinations used with it. The main advantage of this method is that it utilizes cell surface markers to look at the transitions of somatic cells into iPSCs such that you can use this technology to be able to monitor in real time the reprogramming events.On day seven post transduction, wash the fibroblasts with two milliliters of DPBS per well and add 0.5 milliliters of 37 degree celsius 05%EDTA to each well for a three to five minute incubation at 37 degrees celsius. When the cells have rounded, stop the reaction with two milliliters of 37 degree celsius fibroblast medium per well and gently pipette the medium across the bottom of each well to dislodge the cells. Transfer the fibroblasts from each well into individual 15 milliliter conical tubes and collect them by centrifugation.Resuspend the pellets in two milliliters of fresh 37 degree celsius fibroblast medium and count the cells from each culture condition. Next, seed one times 10 to the fifth cells into pre-prepared feeder-independent conditioned wells in a six-well tissue culture plate and incubate the transduced cells in the cell culture incubator overnight. The next day, replace the medium in each well with the appropriate medium for the cell application.Then place the plate in the imager and set the software to capture continuous whole-well phase contrast and fluorescent images for each well every six to eight hours for the next 14 days. At the end of the reprogramming experiment, wash the wells one time with two milliliters of 37 degree celsius DMEM/F12 medium. Discard the wash and label the cells with one milliliter per well of the appropriate primary antibodies in fresh DMEM/F12 medium for 45 minutes at 37 degree celsius in the incubator.At the end of the incubation, wash the cells two times with 37 degree celsius DMEM/F12 medium. Then discard the wash and label the cells with one milliliter of the appropriate secondary antibodies in 37 degree celsius DMEM/F12 medium for 30 minutes at 37 degree celsius. At the end of the incubation, wash the cells two more times with DMEM/F12 medium.Then replace the wash in each well with two milliliters of fresh 37 degree celsius DMEM/F12 medium. Place the dish into the imager and open the imaging software. To set up scans on the imaging system software, first select the position of the tray to be used and select the type of dish to be scanned.Next, select Whole Well Imaging, the pattern for scanning the desired wells, and the appropriate fluorescent channels for the real-time identification of the surface markers of interest. Then label the experiment to be scanned and set the time points and intervals for the samples to be scanned each day. To analyze a set of scanned experiments, select the experiment of interest and select Launch New Analysis Job.Then select the appropriate processing definition for the type of analysis according to the channels used for the imaging, for example, PSC Phase Colony Count. Name the analysis job. And select the time range for the analysis and the wells to be analyzed.Then launch the analysis job. To measure the desired reprogramming time points from individual wells, wash the well of interest one time with two milliliters of DPBS. Then replace the wash with one milliliter of pre-warmed 37 degree celsius 05%trypsin-EDTA solution.After five minutes of 37 degree celsius, flush the trypsin against the bottom of the well to dissociate the cells into a single-cell suspension and transfer the cells into a 15 milliliter conical tube. Using three milliliters of DPBS, wash any of the remaining cells from the well and pull them in the 15 milliliter tube. Add an additional 10 milliliters of DPBS to the cells and spin them down by centrifugation.Resuspend the pellet in one milliliter of DMEM/F12 medium for counting. Then dilute the cell suspension to a one times tenth of the six cells per milliliter concentration and aliquot one milliliter of cells per tube for the appropriate number of samples. Next, label the cells with the primary antibodies of interest in one milliliter of DMEM/F12 medium for 30 to 45 minutes at room temperature with mixing.At the end of the incubation, wash the cells two times in 37 degree celsius DMEM/F12 medium. Resuspend the pellets in one milliliter of DPBS and transfer the samples into individual FACS tubes. Acquire the flow cytometry profiles of the reprogrammed plates.Then transfer the FCS files to a computer and import the files into the appropriate analysis software. Open the first control file in a forward by side scatter plot and set the y-axis to a logarithmic scale. Then create a polygonal gate around the live population.Next, create a side scatter area by scatter height plot with the live cells and set a rectangular gate to exclude the doublets. Then use these gates to analyze the experimental sample files under the appropriate fluorescent channels to identify the populations of interest in their respective quadrants. Reprogramming BJ fibroblasts with Sendai reprogramming viruses generates SSEA4+CD44-cells at a different rate than reprogramming DF1 fibroblasts, demonstrating that an early comparison of the percentage of pluripotent stem cell-like SSEA4+CD44-cells can be used to track the reprogramming progression.In a flow cytometry time course, CD24-CD44+and EpCAM negative CD44+fibroblasts form double negative populations that later transition into CD24+CD44-and EpCAM positive CD44-pluripotent stem cell-like cells respectively, further outlining the reprogramming progression. Real-time imaging of the reprogramming cultures after reseeding demonstrates a gradual colony formation corresponding to a logarithmic increase in their confluence and demonstrating the final pluripotent positive and fibroblast negative marker expression at the completion of the reprogramming. Further, using a reporter gene allows the confluence to be measured under phase contrast with a more gradual increase of the GFP confluence observed compared to the total confluence.Indeed, in this representative experiment, the GFP confluence at day 19 was less than half of the total confluence corresponding to the complete reprogramming. Once mastered, this technique could be used to elucidate information about reprogramming events and track the kinetics of human fibroblast reprogramming. This technique could also be used to look at different starting somatic cell tissues, the reprogramming technologies used, and the medium matrix combinations to elucidate reprogramming events.After watching this video, you should have a good understanding on how to utilize real-time monitoring and cell surface marker expression, to be able to track the reprogramming of human fibroblasts into iPSCs.

kinetic-measurement-real-time-visualization-somatic

Research

JoVE Journal

Developmental Biology

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.

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.

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.

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.

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).

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

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.

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.

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.

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

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...