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 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.
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 γH2A.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 γH2A.X immunolabeling. Moreover, combining these two approaches, we propose a naive assessment that can be used to evaluate the cell'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 (γH2A.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, γH2A.X is dephosphorylated and inactivated by phosphatases. Prolonged and multiple DNA strand breaks lead to the accumulation of γH2A.X foci in DNA. This indicates the cell's inability to repair the DNA damage and the loss of DNA integrity. These γH2A.X foci in DNA can be identified by and the number of DDR foci can be counted using the protocol in section 2.
1. Comet Assay
2. DNA Damage Response
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). The comets were, then, analyzed with ImageJ, using the comet assay plugin (Figure 2A-C).
Human iPS cells were treated with Doxo to induce DNA damage and to be used as a positive control. Representative micrographs of comets from Doxo-treated and nontreated iPS cells are shown in Figure 3A. 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 (Figure 3B,C). This shows that the comet assay can be used to assess DNA integrity not only in somatic cells18,19 but also in pluripotent stem cells21.
Freshly differentiated iPS cardiomyocytes (iPS-CMs), iPS-CMs cultured for 6 months (prolonged culture [PC]), and iPS-CMs treated with Doxo were subjected to γH2A.X immunolabeling. Representative micrographs of γH2A.X immunolabelling are shown in Figure 4A (lower-magnification) and Figure 4B (higher-magnification). The number of γH2A.X foci (DDR foci) (punctae) in each nucleus are quantified, using CellProfiler with custom pipeline modules (Figure 5A-L). The percentage of cells that are positive for γH2A.X are classified into nuclei with zero to five punctae and nuclei with more than 10 punctae. In the control iPS-CM, 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 (Figure 4C, control [Ctrl]). iPS-CMs cultured for 6 months had less than 90% cells with less than five DDR foci per nuclei, and a total of more than 13% of the cells had more than six DDR foci per nuclei (Figure 4C, PC), whereas the Doxo-treated iPS-CM showed less than 80% of the cells with less than five DDR foci per nuclei, and a total of about 24% of the cells had more than six DDR foci per nuclei (Figure 4C, Doxo). This data clearly shows that prolonged cell culture and Doxo treatment induce significant DNA damage in iPS-CMs and are not suitable for cell transplantation.
Figure 1: Schematic of the comet assay. (A) Mix the cell suspension with low-melting-point agarose and (B) place it on a glass slide. (C) Treat it with cell lysis buffer, followed by an alkaline solution, to get nucleoids containing supercoiled DNA. (D) Electrophorese and stain the DNA using SYBR green DNA dye. (E) Schematic of intact (left) and damaged DNA (right, comet shape). (F) Formula to calculate a fraction of the DNA damage and tail moment. Please click here to view a larger version of this figure.
Figure 2: DNA damage and tail moment quantification by ImageJ. (A–C) Screenshots of ImageJ, with the comet analysis plugin showing a selection of the comet head and tail. Please click here to view a larger version of this figure.
Figure 3: Doxorubicin induces DNA damage in human iPS cells. (A) DNA damage in doxorubicin-treated (Doxo) and nontreated (control) iPS cells, analyzed using the comet assay. (B) Fraction of DNA damage (n = 3) and (C) tail moment (n = 63 comets) quantified using the comet assay. Treatment with doxorubicin, a DNA-intercalating agent, significantly increased the fraction of DNA damage and tail moment in iPS cells. Data are means ± SEM. *P < 0.05, Student's unpaired t-test. Please click here to view a larger version of this figure.
Figure 4: DNA damage response in iPS-derived cardiomyocytes. (A) DNA damage response marker γH2A.X identified by immunofluorescence in doxorubicin-treated (Doxo) and nontreated (control) iPS-cardiomyocytes as well as prolonged culture iPS-cardiomyocytes (PC). (B) Higher magnification of γH2A.X (green punctae) labeling at the sites of DNA damage in iPS-CMs. (C) Quantification of DDR foci, analyzed using CellProfiler with custom pipeline modules. Data are means ± SD; n = 3 to 4. *P < 0.05, Student's unpaired t-test. Please click here to view a larger version of this figure.
Figure 5: Automated DNA damage response analysis by CellProfiler. (A–L) Screenshots of CellProfiler with specified settings for importing the images, and the automated identification and quantification of γH2A.X punctae in iPS-CMs. Please click here to view a larger version of this figure.
Figure 6: Automated DNA damage response analysis by CellProfiler. (A–F) Data images generated by CellProfiler at the completion of the image analysis. Please click here to view a larger version of this figure.
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 transplantation is a necessary quality control protocol. Here we describe two cost-effective approaches, namely the comet assay and γH2A.X immunofluorescence labeling, to assess the DNA damage and quality of stem and stem-cell-derived cells.
A major cause of cellular aging is thought to be the accumulation of DNA damage in cells. Using the comet assay, Al-Baker et al. analyzed the DNA damage in young and senescent human dermal fibroblasts22. Previously, we have shown that transplanting DNA damage-free cardiac progenitor cells isolated from a p53 transgenic mouse increased the rate of engraftment in the host organ8. Recently, we have shown the role of the p53 transactivation domain in DNA damage repair mechanisms in human iPS cells21. In both studies, we have employed both the comet assay and γH2A.X immunofluorescence labeling methodologies to assess the DNA damage in stem cells. Both these methods are cost-effective and can be performed with basic lab equipment. An annoying critical problem that we often experienced is agarose solidifying in the pipette and tubes. We overcome this issue by prewarming all the tubes and tips prior to pipetting agarose. While the comet assay takes up to 2 days to complete, the optimized γH2A.X immunofluorescence labeling procedure can be completed in under 4 h.
Human iPS cell cultures with more than 10% DNA damage, measured by the comet assay, did not efficiently differentiate into cardiomyocytes (data not shown) in vitro. The comet assay is sensitive and dynamic in assessing DNA damage in stem cells. However, DNA integrity assessments in certain cells, such as iPS-derived cardiomyocytes, are cumbersome by comet assay due to the nature of the cells. The cardiomyocytes are enriched with troponin, sarcomeric proteins, and the secreted extracellular matrix proteins. Denaturing these complex structures to make supercoiled DNA and its reproducibility are questionable. Most importantly, 25% to 40% of human cardiomyocytes are binucleated25, and these percentages vary in iPS-derived cardiomyocytes. Since the cell wall is disrupted in the comet assay, the precise assessment of the percentage of DNA-damaged cells is impossible. Therefore, in the case of iPS-CMs, the γH2A.X immunofluorescence labeling procedure to assess DNA damage is a simplistic alternative for the comet assay.
Based on these results and those from previous publications by our group8,20,21, we suggest that, if there is more than 10% DNA damage, assessed by the comet assay, or less than 90% of the cells have zero to five DDR foci, then the culture should be disqualified for cell transplantation.
The authors have nothing to disclose.
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).
0.25% Trypsin | Corning | 25200056 | |
8-well chamber slide | Millicell | PEZGS0816 | |
Accutase | Stemcell Technologies | 7920 | Cell detachment solution |
Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG |
Jackson Immuno Research Laboratories |
711-545-152 (RRID: AB_2313584) |
|
Bovine Serum Albumin | Sigma-Aldrich | A7906 | |
Cy3 AffiniPure Donkey Anti-Rabbit IgG | Jackson Immuno Research Laboratories |
711-165-152 (RRID:AB_2307443) |
|
DAPI | Sigma-Aldrich | D9564 | |
Gibco B-27 Supplement, Serum Free, | Gibco | 17504-044 | |
Matrigel growth factor reduced | Corning | 354230 | |
mTeSR1 | Stemcell Technologies | 85850 | |
PhalloidinA488 | Life Technology | A12379 | |
Phospho-Histone H2A.X (Ser139) Antibody | Cell Signaling Technology | 2577 (RRID: AB_2118010) | |
RPMI | Gibco | 11875-093 | |
SYBR Green I nucleic acid gel stain | Sigma-Aldrich | S9430 | |
Triton X-100 | Fisher scientific | BP151-100 | |
UltraPure Low melting Point Agarose | Invitrogen | 16520050 | |
Vectashield | Vector Laboratories | H-1000 | Antifade |