Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.
By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.
A histologic hallmark of heart failure is the loss of cardiomyocytes (CMs), either following injury, senescence, or apoptosis1. Replenishing lost or dysfunctional myocardium through tissue regeneration represents a potential therapeutic strategy for curing patients with heart failure. Over the past several decades, seminal advances in developmental and regenerative biology have unearthed a limited ability for the mammalian heart to replenish lost CMs2,3,4,5. This exciting work has raised the possibility that innate growth mechanisms can be deployed for regeneration. Because innate regenerative responses are functionally absent in the adult mammalian heart, methods to improve the robustness of endogenous repair are needed for therapeutic heart regeneration to be realized.
The mechanisms for innate heart regeneration appear to be conserved across species. Following injury, pre-existing CMs proliferate to generate new CMs in zebrafish6,7, newts8,9, mice4,10, rats11, and pigs12,13. Accordingly, many groups are seeking to identify mitogens capable of promoting cardiomyogenesis. However, such work is challenging. Not only is the task of getting adult mammalian CMs to proliferate daunting but being able to identify rare proliferative events is difficult1,14. The challenge of identifying rare cycling CMs is compounded by the tendency of adult mammalian CMs to preferentially undergo endomitosis. For example, after injury to the mouse heart, almost 25% of CMs in the border zone re-enter the cell cycle, but only 3.2% of CMs divide4. Because most cycling CMs duplicate their genome but fail to undergo cytokinesis, simply assaying for an increase in the numbers of cycling CMs is ambiguous to bona fide cardiomyogenesis. Thus, assays for nucleoside incorporation by CMs or for the presence of proliferative markers on CMs may not entirely indicate regeneration. As more candidate factors for heart regeneration emerge, assays to better identify CM hyperplasia are needed.
Clonal analysis by lineage tracing is a valuable approach to assay for cardiomyogenesis because it allows for the direct visualization of cells and their progeny. Traditional approaches for clonal analysis involve rare labeling of single cells with a reporter gene. However, single-color lineage tracing of rare cells may be of limited value for infrequent events such as CM proliferation because the chances of labeling a proliferating CM are low15. Alternatively, multicolor lineage tracing can increase the sensitivity for clonal analysis16. Briefly, individual cells are genetically labeled with one of several fluorescent proteins at random, such that proliferating cells will generate homogeneously colored clusters of cells that can be resolved from neighboring fluorescent cells. This method has been used to trace growth across a variety of organs, and has been more recently applied to studies of mammalian heart regeneration17,18. While multicolor lineage tracing can detect clonal expansion of CMs in embryonic and neonatal stages, innate regenerative responses are not easily detected in the adult mouse heart after cardiac injury17,19. One approach to enhance the sensitivity of multicolor lineage tracing would be to increase the level of labeling and increase the probability for visualizing rare events. However, wider labeling comes at the cost of not being able to distinguish similarly labeled cells as rising from a common ancestor versus cells that were independently labeled with the same fluorophore. Presented here is a protocol that uses nearest-neighbor modeling to identify clonally related CMs in the neonatal mouse heart. This method is unbiased, quantitative, and works over a range of labeling conditions.
All procedures for handling mice, performing survival surgeries, and for harvesting hearts require approval by a local institutional animal use committee.
1. Mice for clonal analysis of CMs
2. Cryoinjury and labeling of cardiomyocytes
3. Harvesting of hearts and processing for histological analysis
4. Imaging
NOTE: The steps below apply to the use of a commercial microscope (see Table of Materials) that has an upright widefield fluorescence system with filter cubes equipped to discriminate mCerulean, EGFP, mOrange, and mCherry fluorophores, and software associated with this set up (see Table of Materials). Additional steps will vary depending on the exact imaging system that is used.
5. Analysis of images to identify labeled CMs
6. Statistical analysis
NOTE: Cells carrying the same fluorophore are a mix of clonally derived cells (kin) and unrelated cells that underwent a random recombination event to express the same fluorophore (non-kin). Based on prior data17, kin cells are assumed to have a closer physical proximity than non-kin cells expressing the same fluorophore. Thus, kin and non-kin cells can be differentiated based on a distance threshold. However, in order to determine the threshold, the nearest neighbor distributions for kin and non-kin cells need to be deconvolved. Fortunately, nearest neighbor distributions for non-kin cells can be estimated by evaluating the nearest neighbor values from each cell to the closest cell carrying a different fluorophore. Here, methodology is presented for statistically determining a threshold distance to define clonality and for assigning a probability of kinship between cells.
Following the protocol for neonatal cryoinjury should yield P21 hearts with and without injury. Cryoinjured hearts have a well-circumscribed injury while the surface of sham hearts is smooth and homogeneous. In cryoinjured hearts, the area of injury should be consistent from heart to heart. After microscopy, images similar to Figure 1 should be obtained. Note that the image resolution allows for identification of individual CMs and imaging conditions allow for each fluorophore to be resolved. Additionally, note that the injuries are not transmural as regenerative responses can be diminished with larger injuries23,31. After image segmentation, files with CM coordinates were obtained for each fluorophore for each section (Figure 2C). For the analyses presented, a total of 81 sections were used (46 from cryoinjured hearts and 35 from sham hearts). The sections for each heart varied from 9 to 17 based on heart size, and were distributed at least 100 μm apart, covering the entire heart. Vigilant attention to organization and naming of files cannot be overemphasized. Image segmentation provides the raw data needed for calculation of nearest neighbor distances and the modeling described in Step 6.
The histograms in Figure 3A and Figure 3C show nearest neighbor distances for within-channel and between-channel pairs of CMs. While the histograms appear grossly similar, they differ at lower values, suggesting the presence of clustered kin cells in the within-channel distribution. The overall similarity of Figure 3A and Figure 3C can be explained by the rather small number of kin cells relative to non-kin cells, which are found in both within-channel and between-channel distributions. The log-normal distribution can be used to model within-channel and between-channel distributions (Figure 3B,D). To determine a threshold value for distinguishing kin and non-kin cells, the Bayesian model described in 6.4-6.7 was applied with 16 parallel chains to a representative heart during physiologic growth, yielding a kin threshold of 29.67 μm (Figure 5). This number is obtained by identifying the nearest neighbor distance at which the likelihood of a pair of cells being kin drops below 0.5. Importantly, this is the distance threshold for which adjacent cells carrying the same fluorophore have a greater probability of being clonally related than having arisen from independent recombination events in separate, un-related cells. Thus, it is possible to find kin pairs at distances greater than the threshold and non-kin pairs at distances less than the threshold. The distance estimates obtained from the model are listed in Supplementary Table 1. Representative results for nearest neighbor distance estimation are shown in Figure 4A,B,D,F. Step 6.6 provides a list of diagnostics to estimate the correctness of the model. The effective sample size and the Gelman-Rubin Convergence Diagnostic are listed in Supplementary Table 1 whereas the QQ plots are shown in Figure 4C,F. The autocorrelation plot obtained from Step 6.6 is shown in Supplementary Figure 1.
Using the presented methodology, results comparable to prior clonal analyses of CM expansion during physiologic growth and regeneration are obtained. For example prior work has determined that clonally related CMs are smaller in size compared to non-clonal CMs32. Indeed, after applying the presented approach to 81 sections, clonally related CMs were found to be smaller than non-clonally related CMs (165.20 ± 13.79 μm2 vs 252.37 ± 20.51 μm2, p = 0.007, Welch t-test, Figure 6A). Moreover, the size of kin cells across experimental conditions can be assessed. Interestingly, kin cells had a non-significant trend towards being larger in injured hearts compared to sham hearts (189.29 ± 16.07 μm2 vs 141.12 ± 10.57 μm2, p = 0.076, Welch t-test, Figure 6B), perhaps relating to some element of concomitant hypertrophy of CMs following injury. Prior work has also shown that CM proliferation is not enriched, and may even decrease in the area of injury, following cryoinjury to the neonatal mouse heart22,23,31. Accordingly, there was no significant enrichment in the percentage of CMs that are clonally related between injured and sham hearts when looking at entire sections (8.57 ± 2.69 % in cryoinjured hearts vs 9.45 ± 1.23 % in sham hearts, p=0.788, Welch t-test, Figure 6C).
An important aspect of this protocol is the reproducibility of results under different labeling conditions that arise stochastically. In our experiments, we observed a large degree of variation in labeling across sham and cryoinjured hearts. However, as shown in Figure 6D, the threshold distance is consistent (Line of fit: y = -0.0011x + 38.30289, standard error of fit: 5.082, r2=0.1911 – excluding the outlier) despite some hearts having more than a thousand cells labeled and other hearts being very sparsely labeled. The low r2 value indicates that there is a weak correlation between the number of labeled cells and the threshold distance, and thus points to the strength of the model. Additionally, the percentage of labeled CMs that are clonally related is also consistent across hearts regardless of labeling efficiency (Line of fit: y = -0.00068x + 12.58379, standard error of fit: 1.538). Together, these data demonstrate the robustness of this protocol across experimental conditions.
Figure 1: Multicolor labeling for clonal expansion. Tilescan image of cardiac sections from Myh6- MerCreMer; R26R-Rainbow mice after (A) sham injury with widespread labeling or (B) cryoinjury with limited labeling. Hearts carried fluorescent labels for mCerulean (blue), mCherry (red), and mOrange (green) CM, and EGFP (gray). The asterisk in panel B corresponds to the area of injury. Dashed boxes correspond to magnified insets. Arrows correspond to CMs that are clonally related based on their common fluorophore and close proximity. Such relationships are harder to assign by inspection alone for the more diffusely labeled heart in panel A. Scale bar is 500 mm. Please click here to view a larger version of this figure.
Figure 2: Segmentation of CMs for clonal analysis. (A) Segmentation of mCherry CMs using the trace tool. This tracing technique captures original shape and location allowing for the possibility to use the segmented data for further analysis on cell shape and size. Scale bar is 100 μm. (B) ROI manager window that records each segmented cell. (C) Results window that shows the saved measurements for each segmented cell including XY coordinates and the area. Please click here to view a larger version of this figure.
Figure 3: Distributions of nearest neighbor distances. (A) Density histogram of the distances between a cell and its nearest neighbor from another channel. (B) Natural logarithm of the nearest neighbor between-channel distances plotted in (A). (C) Density histogram of the distances between a cell and its nearest neighbor from the same channel. (D) Natural logarithm of the nearest neighbor within-channel distances plotted in (C). Please click here to view a larger version of this figure.
Figure 4: Modeled distribution of distances. (A) Density histogram of nearest neighbor kin distances. (B) Modeled distribution for nearest neighbor within-channel distances. (C) Q-Q plot showing goodness of fit of modeled within-channel distribution to actual data. (D) Density histogram of nearest neighbor non-kin distances. (E) Modeled distribution for nearest neighbor between-channel distances. (F) Q-Q plot showing goodness of fit of modeled between-channel distribution to actual data. Please click here to view a larger version of this figure.
Figure 5: Estimated probability of kin pairs as a function of distance. The estimated probability of obtaining a kin pair as a function of nearest neighbor distance for within-channel pairs. The distance at which the probability drops below 0.5 (dashed line at 29.67 mm for this heart) is considered as the threshold to determine kin pairs. By allowing two components of the normal mixture model to have different variances, the likelihood of being a kin pair can artifactually appear to increase at higher nearest-neighbor distances (> 100 mm). Such artifact is a well-known feature of quadratic discriminant analysis models, and the user is encouraged to focus on the descending limb of the curve to determine a threshold for defining kin pairs. Please click here to view a larger version of this figure.
Figure 6: Properties of kin and non-kin CMs. (A) Bar plot comparing mean cell area of kin cells (n = 1227 cells) with the entirety of the cells (n = 19733 cells). Error bars indicate SEM. (B) Normalized CM area for kin cells in injured (n = 579 cells) and sham hearts (n = 648 cells). Error bars indicate SEM. (C) Mean fraction of kin cells for injured (n = 3 mice) and sham hearts (n = 3 mice). (D) Kin threshold for different hearts (n = 6, 3 with injury, 3 with sham injury, 81 sections total) as a function of the number of cells imaged. The regression line is drawn excluding the outlier (marked by a plus). ** indicates p < 0.01 and NS indicates a non-significant difference. Please click here to view a larger version of this figure.
Supplementary Figure 1: Autocorrelation plot for the model parameters. Autocorrelation plots for the means and standard deviations of the distributions and the likelihood parameter obtained from MCMC sampling. The exponential decay in the autocorrelation points to a good sampling and trustworthy results. Please click here to download this figure.
Supplementary Table 1: Estimates of parameters from Bayesian Modeling using JAGS. The subscripts 1 and 2 indicate non-kin and kin respectively. Alpha, sigma, and beta stand for the mean, the standard deviation, and the kinship odds parameter respectively. Columns of note are the mean value, Rhat – which shows the Gelman Rubin Convergence Diagnostic, and Neff – which shows the effective sample size. Please click here to download this table.
Multicolor lineage tracing is a powerful approach to identify patterns of organ growth with single cell resolution. However, a major limitation to multicolor lineage tracing is the need for sparse labeling of cells, which can reduce the sensitivity for identifying rare events. For organs like the heart with notoriously low levels of parenchymal cell turnover, this can lead to underestimates of growth responses. Presented here is a step-by-step protocol for performing clonal analysis of CM expansion during growth and regeneration in the neonatal mouse. Most importantly, an analytical framework is provided for identifying clones by modeling clonal probability with nearest neighbor distributions. This approach assumes that proliferating cells are in close proximity to each other and may not be applicable to systems where extensive migration of kin cells is expected.
This approach offers several advantages for clonal analyses. In comparison to previous multicolor lineage tracing studies17, this protocol expands the sample size by imaging and segmenting entire heart sections, rather than analyzing only a subset of images per section. This method for segmentation also allows for ascertainment of physical features of CMs, including cell size and shape, providing a potential means to identify characteristics that can differentiate proliferating and non-proliferating cells. Finally, and most significantly, rather than assuming that clusters of cells carrying the same fluorophore are clonally related, the presented analysis provides an unbiased estimate of clonal probability that accounts for the density of cellular labeling.
Critical steps in the protocol include optimization of labeling efficiency, imaging, and segmentation. While this methodology works across a range of labeling conditions, extreme labeling conditions are likely to yield indeterminate results. Labeling that is too dense can limit the ability to deconvolve nearest neighbor distributions for kin and non-kin cells. By contrast, labeling that is too rare can limit the sensitivity for detecting rare events. Users are recommended to optimize labeling by testing a range of tamoxifen doses and ultimately selecting a dose that results in the largest amount of labeling while being able to deconvolve the kin and non-kin distributions. Sectioning and imaging conditions are also key to a successful experiment. High quality sections that enable a survey of anatomically distinct sets of cells are needed to avoid over-sampling of the same cells. Imaging conditions that result in a high signal-to-noise ratio for labeled cells is critical as systematic under-representation of fluorophores can bias estimates of non-kin nearest neighbor distances. Particular attention to the mCerulean fluorophore is needed as this fluorophore is especially sensitive to photobleaching and loss of signal. Finally, careful segmentation is needed because this method relies on accurate assessments of intercellular distances. When multiple users are performing segmentation, users should be issued images for segmentation that are randomized across treatment groups in order to minimize user bias.
Several aspects of this approach have the potential to be modified to address limitations. For example, after tissue harvesting and processing, the major bottleneck is image segmentation. With advances in machine learning and computer vision, automated methods for image segmentation are increasingly accurate and can increase the throughput of image segmentation. Because a file of cellular coordinates is all that is needed for this approach, the code provided is compatible with object identification using automated methods. Similarly, while this method is currently configured for 2-dimensional datasets, 3-dimensional datasets are likely to be more widely utilized with improvements in tissue clearing methodology and light sheet microscopy. This approach can easily accommodate such datasets by simply adjusting the code to compute 3-dimensional distance measures. Overall, future iterations of this approach are expected to be able to accommodate large datasets that encompass whole organs.
In summary, this protocol generates a pipeline for imaging and analyzing multicolor data that is scalable to large numbers of images. Prior work using multicolor lineage tracing for detecting cardiomyogenesis has relied on small scale assessment of samples for clonal analysis. The presented methodology allows for rapid and rigorous assessments of regenerative therapeutics using large data sets.
The authors have nothing to disclose.
This work was funded by an R03 HL144812 (RK), a Duke University Strong Start Physician Scientist Award (RK), a Mandel Foundation Seed Grant (RK), and a T32 HL007101 Training grant (DCC). We would additionally like to acknowledge Evelyn McCullough for assistance with mouse husbandry and Dr. Douglas Marchuk and Matthew Detter for helpful comments and discussion. Finally, we would like to thank Purushothama Rao Tata for kindly providing R26R-Rainbow mice.
#1.5 glass coverslip | FisherScientific | 12-544E | |
6-O prolene | Ethicon | 8706H | |
Anti-fade mounting medium | FisherScientific | 00-4958-02 | |
CO2 inhalational chamber | |||
Cold pack | |||
Cryomolds | VWR | 15160-215 | |
Cryoprobe | World Precision Instruments | 501313 | |
Filter cubes | |||
Gt(ROSA)26Sortm1(CAG-EGFP,-mCerulean,-mOrange,-mCherry)Ilw mice | |||
ImageJ software | https://imagej.net | ||
KCl 1M | FisherScientific | LC187951 | |
Leica CM3050 cryostat | |||
Liquid Nitrogen | |||
Microscissors, 6mm | World Precision Instruments | 14003 | |
Myh6-CreERT2 mice | The Jackson Laboratory | 005657 | |
Needler holder | World Precision Instruments | 14109 | |
Paraformaldehyde 4% | FisherScientific | AC416785000 | |
Phosphate buffered saline | |||
Python | https://www.python.org/ | ||
R | https://cran.r-project.org/ | ||
Rotating Shaker | |||
Stereoscope | |||
Sucrose 30% (wt/vol) | FisherScientific | BP220 | |
Surgical dissecting scissors | World Precision Instruments | 14393 | |
Syringe for tamoxifen | VWR | BD328438 | |
Tamoxifen, 20 μg | Sigma | T5648 | |
Tissue Freezing Media | VWR | 15148-031 | |
White Frosted/Plus slides | Globe Scientific | 1358W | |
Zeiss Axio Imager M1 upright widefield fluorescence system | |||
Zen 2.5 Blue software |
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