Name: generation of ventricular-like hipsc-derived cardiomyocytes and high-quality cell preparations for calcium handling characterization
Uploaded: 2020-01-17T12:00:00
Description: Watch this Scientific Journal Video about Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization at JoVE.com

This research was supported by AHA Scientist Development Grant 17SDG33700093 (F.S.); Mount Sinai KL2 Scholars Award for Clinical and Translational Research Career Development KL2TR001435 (F.S.); NIH R00 HL116645 and AHA 18TPA34170460 (C.K.).

The experiments using adult rat cardiomyocytes in this study were conducted with approved Institutional Animal Care and Use Committee (IACUC) protocols of Icahn School of Medicine at Mount Sinai. The adult rat cardiomyocytes were isolated from Sprague Dawley rat hearts by the Langendorff-based method as previously described16.
1. Preparation of Media
<ol>
	<li>Prepare hiPSC media.
	<ol>
		<li>Equilibrate the supplement and the basal medium to room temperature (RT). En...

<ol>
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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=Cardiomyocytes+derived+from+pluripotent+stem+cells+recapitulate+electrophysiological+characteristics+of+an+overlap+syndrome+of+cardiac+sodium+channel+disease.">Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease.</a> Circulation. 125 (25), 3079-3091 (2012).</li><li>Fatima, A., 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=In+vitro+modeling+of+ryanodine+receptor+2+dysfunction+using+human+induced+pluripotent+stem+cells.">In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.</a> Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 28 (4), 579-592 (2011).</li><li>Novak, A., 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=Cardiomyocytes+generated+from+CPVTD307H+patients+are+arrhythmogenic+in+response+to+beta-adrenergic+stimulation.">Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to beta-adrenergic stimulation.</a> Journal of Cellular and Molecular Medicine. 16 (3), 468-482 (2012).</li><li>Lan, F., 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=Abnormal+calcium+handling+properties+underlie+familial+hypertrophic+cardiomyopathy+pathology+in+patient-specific+induced+pluripotent+stem+cells.">Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient-specific induced pluripotent stem cells.</a> Cell Stem Cell. 12 (1), 101-113 (2013).</li><li>Sun, N., 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=Patient-specific+induced+pluripotent+stem+cells+as+a+model+for+familial+dilated+cardiomyopathy.">Patient-specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy.</a> Science Translational Medicine. 4 (130), (2012).</li><li>Stillitano, F., 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=Modeling+susceptibility+to+drug-induced+long+QT+with+a+panel+of+subject-specific+induced+pluripotent+stem+cells.">Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells.</a> eLife. 6, (2017).</li><li>Matsa, E., Burridge, P. 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A., 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=Measuring+Cardiomyocyte+Contractility+and+Calcium+Handling+In+Vitro.">Measuring Cardiomyocyte Contractility and Calcium Handling In Vitro.</a> Methods in Molecular Biology. 1816, 93-104 (2018).</li><li>Kim, 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=Non-cardiomyocytes+influence+the+electrophysiological+maturation+of+human+embryonic+stem+cell-derived+cardiomyocytes+during+differentiation.">Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.</a> Stem Cells and Development. 19 (6), 783-795 (2010).</li><li>Lian, X., 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=Directed+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/beta-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions.</a> Nature Protocols. 8 (1), 162-175 (2013).</li><li>Patterson, A. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+fetal+heart+development.">Hypoxia and fetal heart development.</a> Current Molecular Medicine. 10 (7), 653-666 (2010).</li><li>Correia, 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=Combining+hypoxia+and+bioreactor+hydrodynamics+boosts+induced+pluripotent+stem+cell+differentiation+towards+cardiomyocytes.">Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.</a> Stem Cell Reviews. 10 (6), 786-801 (2014).</li><li>Tohyama, S., 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=Glutamine+Oxidation+Is+Indispensable+for+Survival+of+Human+Pluripotent+Stem+Cells.">Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells.</a> Cell Metabolism. 23 (4), 663-674 (2016).</li><li>Tohyama, S., 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=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li><li>Tu, C., Chao, B. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+Improving+the+Maturity+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes.">Strategies for Improving the Maturity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.</a> Circulation Research. 123 (5), 512-514 (2018).</li><li>Lundy, S. D., Zhu, W. Z., Regnier, M., Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+maturation+of+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">Structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells.</a> Stem Cells and Development. 22 (14), 1991-2002 (2013).</li><li>Hwang, H. 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Critical steps for using human iPSC-CMs as experimental models are: 1) generating high-quality cardiomyocytes (CMs) that can ensure the consistent performance and reproducible results; 2) allowing the cells to mature in culture for at least 90 days to adequately assess their phenotype; 3) performing electrophysiological studies, e.g. calcium (Ca2+) transient measurements, to provide a physiologically relevant functional characterization of human iPSC-CMs. We developed a monolayer-based differentiation method t...

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an attractive human-based platform to model a large variety of cardiac diseases in vitro1,2,3,4,5,6,7,8. Moreover, iPSC-CMs can be used for the prediction of patient responses to novel or existing drugs, to screen hit compounds, and develop new personalized drugs9,10. However, despite significant progress, several limitations and challenges need to be considered when using iPSC-CMs11. Consequently, methods to improve cardiac differentiation protocols, to enhance iPSC-CMs efficiency and maturation, and to generate specific cardiomyocyte subtypes (ventricular, atrial, and nodal) have been intensely studied and already led to numerous culture strategies to overcome these hurdles12,13,14,15.
Notwithstanding the robustness of these protocols, a major concern for the use of iPSC-CMs is the reproducibility of long and complex procedures to obtain high-quality cardiomyocytes that can ensure the same performance and reproducible results. Reproducibility is critical not only when comparing cell lines with different genetic backgrounds, but also when repeating cellular and molecular comparisons of the same cell line. Cell variability, such as well-to-well differences in iPSCs density, may affect cardiac differentiation, generating a low yield and poor-quality cardiomyocytes. These cells could still be used to perform experiments that do not require a pure population of CMs (e.g., when performing Ca2+ transient measurements). Indeed, when performing electrophysiological analysis, the non-CMs will not beat, neither spontaneously nor under electrical stimulation, so it will be easy to exclude them from the analysis. However, because of the poor quality, iPSC-CMs can show altered electrophysiological characteristics (e.g., irregular Ca2+ transient, low Ca2+ amplitude) which are not due to their genetic makeup. Therefore, especially when using iPSC-CMs to model cardiac disease, it is important not to confuse results from a poor-quality CM with the disease phenotype. Careful screening and exclusion processes are required prior to proceeding to electrophysiological studies.
This method includes optimized protocols to generate high-purity and high-quality cardiomyocytes and to assess their function by performing Ca2+ transient measurements using a calcium and contractility acquisition and analysis system. This technique is a simple, yet powerful, way to distinguish between high efficiency and low efficiency iPSC-CM preparations and provide a more physiologically relevant characterization of human iPSC-CMs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-Actin, &#945;-Smooth Muscle antibody, Mouse monoclonal</td>
			<td>Sigma Aldrich</td>
			<td>A5228</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti mouse</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti rabbit</td>
			<td>Invitrogen</td>
			<td>A21428</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27(-) insulin Supplement</td>
			<td>Gibco</td>
			<td>A18956-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI nuclear stain</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1X) + L- Glutamine + 15mM Hepes</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Ended Cell lifter, Flat blade and J-Hook</td>
			<td>Celltreat</td>
			<td>229306</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Multiwell Tissue Culture Plate, 6 well</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidic inline heater</td>
			<td>Live Cell Instrument</td>
			<td>IL-H-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2, AM</td>
			<td>Invitrogen</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC-qualified matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Matrigel Matrix</td>
		</tr>
		<tr>
			<td>hPSC media</td>
			<td>Gibco</td>
			<td>A33493-01</td>
			<td>StemFlex Basal Medium</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma Aldrich</td>
			<td>I0161</td>
			<td></td>
		</tr>
		<tr>
			<td>Live cell imaging chamber</td>
			<td>Live Cell Instrument</td>
			<td>EC-B25</td>
			<td></td>
		</tr>
		<tr>
			<td>MLC-2A, Monoclonal Mouse Antibody</td>
			<td>Synaptic Systems</td>
			<td>311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Myocyte calcium and contractility system</td>
			<td>Ionoptix</td>
			<td>ISW-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Light Chain 2 Antibody, Rabbit Polyclonal (MLC2V)</td>
			<td>Proteintech</td>
			<td>10906-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid Flow Sterile Disposable Filter units with PES Membrane</td>
			<td>ThermoFisher</td>
			<td>124-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Calcium and Magensium</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Glass Cover Slips</td>
			<td>Lab Scientific</td>
			<td>7807</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (-) D-glucose (1X)</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-lactate</td>
			<td>Sigma Aldrich</td>
			<td>L7022</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex Supplement</td>
			<td>Gibco</td>
			<td>A33492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazovivin</td>
			<td>Tocris</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution</td>
			<td>Boston Bioproducts</td>
			<td>BSS-355w</td>
			<td>Adjust pH at 7.2. Add 1.2mM Calcium Chloride</td>
		</tr>
	</tbody>
</table>

generation of ventricular-like hipsc-derived cardiomyocytes and high-quality cell preparations for calcium handling characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

The protocol described in Figure 1A generated highly pure cardiomyocytes that acquire a ventricular/adult-like phenotype with time in culture. As assessed by immunofluorescence staining for the atrial and ventricular myosin regulatory light chain 2 isoforms (MLC2A and MLC2V, respectively), the majority of the cells generated by this protocol were MLC2A-positive at day 30 after induction of cardiac differentiation, while MLC2V was expressed in much lower amounts at the same t...

Watch this Scientific Journal Video about Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization at JoVE.com

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Reproducibility is major concern when inducing stem cell-derived cardiomyocytes. Using these methods, high purity and high quality cardiomyocytes can be generated from iPS for their use in functional studies. The main advantage of this technique is that is a simple, reliable, cost-effective, and extremely powerful method for obtaining purified, high quality preparations of iPSC-derived cardiomyocytes.For cell passaging, rinse the cells with one milliliter of PBS without calcium and magnesium for seven to 10 minutes before replacing the PBS with one milliliter of passaging medium. Using a cell lifter, gently scrape the cells from the bottom of the wells, and use sterile two milliliter glass pipette to mechanically dissociate the detached cells. When the cells have lifted into small clusters as visible under the microscope add passaging medium to split the cells at a one to six ratio.Next, aspirate the human embryonic stem cell qualified matrix from the six well plate. Then add one milliliter of passaging medium and one milliliter of dissociated cells to each well, and let the cells grow until they reach 70 to 80%confluency before starting a cardiac differentiation. At least 30 minutes before dissociating the iPSC-CMs, wipe individual glass cover slips with 70%ethanol, and place the cover slips into individual wells of a sterile six well plate.When the cover slips have dried, add a 250 to 300 microliters of human embryonic stem cell qualified matrix solution directly onto the center of each cover slip, and place the six well plate in the tissue culture hood. To dissociate the metabolically selected iPSC-CM add one milliliter of sterile 0.25%trypsin with EDTA to each well for a five minute incubation at 35 degrees celsius, and use a 1000 microliter pipette to mechanically dissociate the cells until a single cell suspension has been achieved. Pull the cells in sterile 15 milliliter conical tube, and wash each well with two milliliters of RPMI 20 medium per well.Then add the washes to the tube and pellet the cells by centrifugation. Resuspend the cells in a sufficient volume of medium to achieve a three times 10 to the fifth cells per 250 microliters of cells concentration. Use a 1000 milliliter pipette tip to dissociate the cells until the solution appears to be homogenous.Then aspirate 250 microliters of cells into a 1000 microliter pipette tip, and slowly dispense the entire volume of solution onto one human embryonic stem cell qualified matrix coded cover slip. When all of the cells have been seeded, carefully place the plate into the cell culture incubator overnight. Gently adding two milliliters of D7 medium to each well the next morning.When the selected iPSC-CMs reach at least three months old treat the cells with two microliters of Fura-2 AM for 10 minutes at 37 degrees Celsius, and power on the calcium analysis system. Initiate the ArcLamp and connect the tubes from the pump to the appropriate inlet and outlets and the electronic wire from the stimulator to the chamber. Place the chamber onto the system.Fill the ProFusion tube that runs through the fluidic inlined heater with 37 degrees Celsius warmed Tyrode's solution, and adjust the camera and framing aperture dimensions to minimize the background area. Fasten the glass cover slip seeded with iPSC-CMs into the chamber, and gently add 500 microliters of Tyrode's solution directly on top of the cover slip. Begin profusing the chamber with Tyrode's solution at a 1.5 milliliter per minute rate, and use an electric stimulator to pace the iPC-CMs with 1 Hertz field stimulation at 10 volts every four milliseconds for three to five minutes.When all of the dye has been washed out of the chamber adjust the viewing window to the upper left region of the cover slip, and begin the recording. After collecting a consistent stream of five to 10 peaks click pause to temporarily stop the recording and move the microscope stage to an adjacent area for the opposite end of the cover slip before resuming the recording. After scanning the calcium transience across the entire cover slip in the same manner over a period of 10 minutes analyze the data with an appropriate florescence trace analysis software according to the manufacturer's instructions.This protocol generates highly pure cardiomyocytes that acquire a ventricular, adult-like phenotype in culture over time. As assessed by immuno fluorescent staining for the atrial and ventricular MLC2 isoforms, the majority of cells generated by this protocol are atrial MLC2 isoform positive at day 30 after the induction of cardiac differentiation. While ventricular MLC2 isoforms are expressed in much numbers at the same time point.As the time in culture increases, a complete switch to the MLC2 isoforms is observed. These data were also confirmed by flow cytometric quantification of the percentage of atrial and ventricular MLC2 marker expressing cells. The calcium amplitude is significantly increased in the iPSC-CMs at day 90, similar to that observed for adult rat CMs.It's important to note that contaminating non-cardiac cells may influence the functional maturation of the human iPSC-CMs during differentiation. Further, when record calcium transients in iPSC-CMs it's necessary to allow the cells to stabilize at 37 degrees Celsius under constant stimulation for at least 200 seconds, and to restrict the recordings to a specific time window to ensure reproducible results. Remember to confirm that the iPSCs exhibit a homogeneous morphology and to allow the cells to grow to 70 to 80%confluency before initiating a cardiac differentiation.Pure quality in immature iPS-derived cardiomyocytes might show altered functional characteristics, which do not reflect the real phenotype, but could be misinterpreted as the diseased phenotype. Therefore, obtaining pure and high quality cardiomyocytes is important to provide consistent and reproducible results.

generation-ventricular-like-hipsc-derived-cardiomyocytes-high-quality

Research

JoVE Journal

Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.