Name: assessing stem cell dna integrity for cardiac cell therapy
Uploaded: 2019-01-25T14:00:00
Description: Watch this Scientific Journal Video about Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy at JoVE.com

This work was supported in part by National Heart, Lung, and Blood Institute Grants RO1-HL-99507, HL-114120, HL-131017, HL138023, and UO1-HL-134764 (to J. Zhang) and by American Heart Association Scientific Development Grant 17SDG33670677 (to R. Kannappan).

1. Comet Assay
<ol>
	<li>Reagent preparation
	<ol>
		<li>For low-melting-point agarose, place 500 mg of low-melting agarose in 100 mL of DNA-/RNA-free water. Heat the bottle in a microwave oven until the agarose dissolves. Place the bottle in a 37 &#176;C water bath until needed. 
		NOTE: For further reading, see Azqueta et al., who studied the effects of agarose concentration on the DNA-unwinding time and several electrophoresis factors23.</li>
		<li>Dilut...

<ol>
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R., 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=Intracoronary+cardiosphere-derived+cells+for+heart+regeneration+after+myocardial+infarction+(CADUCEUS):+a+prospective,+randomised+phase+1+trial.">Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial.</a> The Lancet. 379 (9819), 895-904 (2012).</li><li>Tomita, Y., 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=Cardiac+neural+crest+cells+contribute+to+the+dormant+multipotent+stem+cell+in+the+mammalian+heart.">Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.</a> Journal of Cell Biology. 170 (7), 1135-1146 (2005).</li><li>Oyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+side+population+cells+have+a+potential+to+migrate+and+differentiate+into+cardiomyocytes+in+vitro+and+in+vivo.">Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo.</a> Journal of Cell Biology. 176 (3), 329-341 (2007).</li><li>Fischer, K. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochemical and Biophysical Research Communications. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. 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A., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transactivation+domain+of+p53+regulates+DNA+repair+and+integrity+in+human+iPS+cells.">Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.</a> American Journal of Physiology-Heart and Circulatory Physiology. , (2018).</li><li>Al-Baker, E. A., Oshin, M., Hutchison, C. J., Kill, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+UV-induced+damage+and+repair+in+young+and+senescent+human+dermal+fibroblasts+using+the+comet+assay.">Analysis of UV-induced damage and repair in young and senescent human dermal fibroblasts using the comet assay.</a> Mechanisms of Ageing and Development. 126 (6-7), 664-672 (2005).</li><li>Azqueta, A., Gutzkow, K. B., Brunborg, G., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+more+reliable+comet+assay:+optimising+agarose+concentration,+unwinding+time+and+electrophoresis+conditions.">Towards a more reliable comet assay: optimising agarose concentration, unwinding time and electrophoresis conditions.</a> Mutation Research. 724 (1-2), 41-45 (2011).</li><li>Carpenter, A. E., 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=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biology. 7 (10), R100 (2006).</li><li>Paradis, A. N., Gay, M. 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DNA integrity portrays cell integrity. Cells with damaged DNA are frequently in stress and eventually lose their integrity. The integrity of stem and stem-cell-derived cells that are being propagated for the purpose of transplantation is principal for the cells to perform their desired function. Transplanting cells with damaged DNA would result in a poor engraftment rate and performance of the cell8,20. Therefore, examining the DNA integrity prior to cell transpl...

Experimental and clinical evidence demonstrates that cell transplantation can moderately improve the left ventricle contractile performance of failing hearts1,2,3,4,5,6,7,8,9. Recent advances have opened further appealing opportunities for cardiovascular regeneration; these include the forced expression of reprogramming factors in somatic cells to induce pluripotency and differentiate these induced pluripotent stem cells (iPSC) into different cardiac lineages, importantly cardiomyocyte (CM)10,11,12,13. The hereditary material in every cell, including the artificially generated iPSCs and iPSC-derived CM (iPS-CM), is DNA. The genetic instructions stored in DNA dictates the growth, development, and function of cells, tissues, organs, and organisms. DNA is not inert; cell metabolites, such as reactive oxygen, carbonyl, and nitrogen species, and alkylating agents can cause DNA damage in vitro and in vivo14,15,16,17. Importantly, DNA damage occurs intuitively in every cell, with a significant frequency. If these damages are not corrected, it will lead to DNA mutation, cellular senescence, the loss of DNA and cell integrity, and possibly, diseases, including life-threatening cancers. Therefore, retaining DNA integrity is essential to any cell, especially iPSCs, that has enormous potential in the clinic.
For assessing the quantity and integrity of isolated genomic DNA, expensive equipment is available on the market. However, there are no simple and cost-effective methods to assess the DNA integrity in cells without isolating the cells. Moreover, user-induced DNA degradation during DNA isolation is one of the major drawbacks in using these methods. The single-cell gel electrophoresis (known as comet assay)18,19 and &#947;H2A.X immunolabeling8 techniques are fundamental approaches in research labs for assessing DNA damage. These two methods do not require expensive equipment or isolated genomic DNA to analyze DNA integrity8,20,21. Since, these techniques have been performed with whole cells; user-induced DNA/RNA/protein degradation during the sample preparation will not affect these protocols. Here, to assess the DNA damage and DNA damage response in stem and stem-cell-derived cells, we provide step-by-step protocols to perform both the comet assay and &#947;H2A.X immunolabeling. Moreover, combining these two approaches, we propose a naive assessment that can be used to evaluate the cell&#39;s suitability for transplantation.
The comet assay, or single-cell gel electrophoresis, measures the DNA breaks in cells. Cells embedded in low-melting agarose are lysed to form nucleoids containing supercoiled DNA. Upon electrophoresis, small pieces of fragmented DNA and broken DNA strands migrate through the agarose pores, whereas the intact DNA, due to their enormous size and their conjugation with the matrix protein, will have a restricted migration. The pattern of stained DNA under a fluorescence microscope mimics a comet. The comet head contains intact DNA and the tail is composed of fragments and broken DNA strands. The fraction of DNA damage can be measured by the fluorescence intensity of the damaged DNA (comet tail) relative to the intact DNA (comet head) intensity. The parameter tail moment can be calculated as shown in Figure 1.
DNA damage induces the phosphorylation of histone H2A.X (&#947;H2A.X) at Ser139 by ATM, ATR, and DNA-PK kinases. The phosphorylation and recruitment of H2A.X at DNA strand breaks is called DNA damage response (DDR) and happens rapidly after DNA is damaged. Following this process, checkpoint-mediated cell cycle arrest and DNA repair processes are initiated. After the successful completion of DNA repair, &#947;H2A.X is dephosphorylated and inactivated by phosphatases. Prolonged and multiple DNA strand breaks lead to the accumulation of &#947;H2A.X foci in DNA. This indicates the cell&#39;s inability to repair the DNA damage and the loss of DNA integrity. These &#947;H2A.X foci in DNA can be identified by and the number of DDR foci can be counted using the protocol in section 2.

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Corning</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Millicell</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stemcell Technologies</td>
			<td>7920</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure 
			Donkey Anti-Rabbit IgG</td>
			<td>Jackson Immuno 
			Research Laboratories</td>
			<td>711-545-152 
			(RRID: AB_2313584)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td>Jackson Immuno 
			Research Laboratories</td>
			<td>711-165-152 
			(RRID:AB_2307443)</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco B-27 Supplement, Serum Free,</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel growth factor reduced</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>PhalloidinA488</td>
			<td>Life Technology</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Histone H2A.X (Ser139) Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2577 (RRID: AB_2118010)</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I nucleic acid gel stain</td>
			<td>Sigma-Aldrich</td>
			<td>S9430</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade</td>
		</tr>
	</tbody>
</table>

assessing stem cell dna integrity for cardiac cell therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Human induced pluripotent stem cells were cultured, and the DNA damage and the tail moment, which were used as a measure of DNA integrity, were analyzed by comet assay. iPS cells were embedded in low-melting-point agarose and placed on a glass slide. The cells were, then, treated with lysis buffer, followed by an alkaline solution, to obtain supercoiled DNA. Nucleoids were electrophoresed and comets were visualized by DNA dye (Figure 1A-D). T...

Watch this Scientific Journal Video about Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy at JoVE.com

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

For neonatal diseases such as cardiomyopathy, regenerative therapy using induced pluripotent stem cell-derived cells has massive potential. However, in vitro accelerated cell growth leads to DNA damage by accumulated metabolites such as reactive oxygen species. Transplanting these DNA-damaged cells would result in poor engraftment and regeneration of the organ.Before transplantation, the quality of the cells should be assessed. Here we provide two step-by-step protocols to assess the DNA damage in stem cells. Demonstrating the procedures will be Jessica Miller, a researcher, Nikhil Mardhekar, a researcher, and Vasanthi Rajasekaran, a lab technician.To begin, place 500 milligrams of low-melting agarose in 100 milliliters of DNA-RNA free water. Heat the bottle in a microwave oven until the agarose dissolves. Then, place the bottle in a 37-degree-Celsius water bath until needed.To prepare pre-coated comet slides, place a few drops of 0.5%agarose on a glass slide. Immediately spread the agarose to form a thin layer of agarose coating using a coverslip. Working under low-light conditions to avoid ultraviolet light-induced cell damage, mix 10 microliters of cell suspension and 90 microliters of agarose solution in a tube.Place the tube in a 37-degree-Celsius water bath to prevent the agarose from solidifying. Mix the solution without introducing air bubbles, and pipette 70 microliters per spot onto the pre-coated comet slide. Using a pipette tip, spread the cell agarose mixture to form a thin layer.Place the slide at four degrees Celsius for 15 minutes. In a container, completely submerge the slide in cold lysis buffer. Place the container in the dark at four degrees Celsius for one hour.Replace the lysis buffer with cold alkaline solution. Make sure the slides are completely submerged. After leaving the container in the dark at four degrees Celsius for another 30 minutes, place the slide in a horizontal electrophoresis tank.Fill the tank with cold alkaline electrophoresis buffer until the slides are completely submerged. Apply voltage at one volt per centimeter for 15 to 30 minutes. In a container, completely submerge the slide in cold deionized water.After two minutes, remove the deionized water and add fresh deionized water, and then repeat this step a second time. Next, replace the deionized water with cold 70%ethanol. After five minutes, gently remove the slide without tilting it and let it air dry.Once dried, add 100 microliters of diluted DNA dye to each spot, and wait 15 minutes at room temperature. Using a FITC filter in an epifluorescence microscope, take images of 50 to 100 comets in total per sample. Culture iPS-derived cardiomyocytes as described in the text protocol.To see the iPS-derived cardiomyocytes in chamber slides, first discard the culture medium from the wells, and wash the cells three times with PBS. Add one milliliter of 0.25%Trypsin per well, and incubate at 37 degrees Celsius for five minutes. Check the plate under a microscope for cell detachment.Then add one milliliter of CMM to stop the Trypsin. Place the cell suspension in a 15-milliliter tube and centrifuge for five minutes at four degrees Celsius and 200 times g. Discard the medium, add one milliliter of CMM, and resuspend the cells.Count the cells and adjust the cell counts to obtain two million cells in 1.6 milliliters of CMM. Now seed 200 microliters of cell suspension per well of the eight-well chamber slide. Tap the slide gently to spread the cells around the well.Incubate the cells at 37 degrees Celsius. For the iPS-derived cardiomyocyte doxorubicin treatment, discard the culture medium from the iPS-derived cardiomyocyte wells, and wash the cells with PBS. Treat the cells with one-micromolar doxorubicin for four hours at 37 degrees Celsius.Aspirate the medium, and wash the cells with PBS. To perform immunolabeling, wash the cells three times with PBS and add 200 microliters of freshly-prepared 4%paraformaldehyde to each well. Fix the cells for 20 minutes in the dark at room temperature.After washing the cells with PBS, add 200 microliters of blocking buffer per well, and block for 30 minutes in a humidified chamber at room temperature. Remove the blocking buffer and add 200 microliters of primary antibody solution. After incubating for 45 minutes in a dark humidified chamber at room temperature, wash the wells three times with PBS.Remove the PBS and add 200 microliters of secondary antibody mix. Incubate for 45 minutes in a dark humidified chamber at room temperature. Then wash the wells three times with PBS.Wash with deionized water before mounting the slides with antifade. Place a cover glass, and store the slides in the dark at four degrees Celsius. Using a confocal microscope, take three to five images of random fields per sample and proceed to image analysis.To count the DNA damage response foci, download and install CellProfiler. Select file, import, pipeline from the file, and choose the pipeline file named puncti gamma-H2AX. Drag and drop the folder containing the acquired images of gamma-H2AX foci to the file list window in the input modules images section for analysis.Then follow the instructions as shown in the text protocol. To set the parameters for the images, drag the desired image for analysis onto the file list. Under input modules, select images.Also in input modules, select metadata, and then select names and types. For groups, select no. Working in analysis modules, select color to gray.Then select smooth, select identify primary objects, and again select smooth. Next select enhance or suppress features, and then select identify primary objects. Still working in analysis modules, select relate objects, then classify objects, and finally select export to spreadsheet.Click analyze images at the bottom left panel to perform image analysis. At the successful completion of the analysis, several images are generated. Note the CSV file that is generated and saved in the appropriate location on the computer.This file contains the quantitative data for further analysis. In the spreadsheet called my experiment image, column B will have the number of nuclei that have up to five puncti, and column D will have the number of nuclei that have more than five puncti. Add columns B and D to get the total number of nuclei.Repeat these steps for all images, changing the my experiment filename before every analysis. Representative micrographs of comets from doxo-treated and non-treated iPS cells are shown here. A basal amount of DNA damage was found in iPS cells, expressed as a fraction of DNA damage and tail moment.However, the doxo treatment increased the DNA damage in iPS cells as expected, indicating that the comet assay can be used to assess DNA integrity in pluripotent stem cells. Representative micrographs of gamma-H2AX immunolabeling are shown here, at lower magnification and at higher magnification. In the control iPS-derived cardiomyocytes, more than 90%of the cells had less than five DDR foci per nuclei.And a total of less than 10%of the cells had more than six DDR foci per nuclei. For iPS-derived cardiomyocytes cultured for six months, less than 90%of the cells had up to five DDR foci per nuclei, and a total of more than 13%of the cells had more than six DDR foci per nuclei. Whereas in the doxo-treated iPS-derived cardiomyocytes, less than 80%of the cells had up to five DDR foci per nuclei.And a total of about 24%of the cells had more than six DDR foci per nuclei. This data indicates that prolonged iPS-derived cardiomyocyte cell culture and doxo treatment induced significant DNA damage, making them unsuitable for cell transplantation. It is very important to avoid variables while mixing the cell suspension, as this might affect the electrophoresis.Following this procedure, you can perform the comet assays and the immunolabeling procedures with cells of various types. These techniques will allow for cells of various kinds to be assessed for DNA damage before being used in cell transplantation studies.

Research

JoVE Journal

Genetics

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...