Fig 1.
EdU pulse chase experiment for quantification of cell proliferation.
Mice were injected intra-peritonealy with EdU at 0, 1, and 16 hours equivalent to a "pulse-chase-pulse" experiment. Thirty minutes after the last pulse, the mice were sacrificed, lymphoid organs removed, and cell suspensions were analysed by single-cell multicolour flow cytometry. The bi-dimensional dot plot reveals the intensity of EdU label (log scale) and DNA content (linear scale arbitrary unit) in lymphocytes. Manual gating allows identification of unlabelled cells in either G0/G1 (G) or G2/M (M) that have not transited through S phase during pulse, and EdU labelled cells (G'+S'+M') that have transited through S phase.
Fig 2.
Thymocyte proliferation in mice according to cell differentiation stage, age and genetic origin.
Cell proliferation was assessed in C57BL/6 and FVB mice of 2 or 18 months as in Fig 1. (A) Multi-colour flow cytometry and hierarchical gating in thymocytes showing EdU vs DNA content dot plot in 2-month-old mice. (B) Percentage of EdU+ labelled cells according to differentiation stages through time: from the most immature CD3-CD4-CD8- triple negative cells (TN), CD3loCD4+CD8+ double positive cells (DP CD3lo), CD3hiCD4+CD8+ double positive cells (DP CD3hi) to single positive mature CD4+CD3+ or CD8+CD3+stage. Each curve represents the %EdU uptake for one of the 4 mice per group.
Fig 3.
State transition model of cell cycle and EdU labeling.
According to the FACS dot plot, the evolution of cells through the cell cycle and EdU labelling can be represented by a transition diagram. Unlabelled cells can transit successively in the three phases of the cell cycle: G0/G1 (G), S (S), and G2/M (M). Terms in aX correspond to rates of entry into the next phase of cells in X phase (X being either G, S, or M). The exit of cells, either due to death, differentiation, or migration is represented by terms in dX. During the pulse-phase the cells in S phase incorporate EdU (red arrow) and enter into S’ with rate β and continue the cycle to M’ and G’. During the chase phase, in the absence of EdU, the labelled cells lose labelling upon several cell divisions (blue arrow) (de-labelling) with rate α. Unlabelled cells can enter into S phase but remain unlabelled.
Table 1.
Cell populations described in the model (Fig 3) and initial values used in the fit according to our hypotheses.
Table 2.
Model parameters and values used for the fit in our experimental conditions.
Table 3.
Biological and experimental hypotheses, allowing for constraint of parameter values.
Fig 4.
Proliferation rates and cell cycle phase durations in thymus and spleen.
Proliferation rates (%/day), G0/G1 and G2/M phase durations (in days) in B6 and FVB mice (n = 4 per group) according to age (2 and 18 months) in whole thymus (left) and whole spleen (right). Values are indicated in the box plot, the median is the 50th percentile, the lower and upper limits of the box are the 25th and 75th percentiles and the whiskers indicate 1.5x(interquartile range). *: p<0.05. **: p<0.01. (See Materials and Methods section for statistical tests used). Statistics are for proliferation rates only. Mean values and SD are given in S1 Table.
Fig 5.
Validation of the mathematical model and parameter values with pulse/chase experimental data from [15].
Young B6 mice received a single BrdU pulse, and within the BrdU-labelled cells the percentages of cells in GO/G1, S or G2/M is recorded over a nineteen hour chase period. The points and dashed lines represent Baron experimental data. Simulation of the same pulse chase conditions using our mathematical model, with the mean parameter values (S1 Table) obtained from our 2-month-old B6 mice (n = 4). The points and continuous lines represent the results of our simulation.
Fig 6.
Signatures of lymphocyte proliferation according to strains and ages.
The upper panel shows the correlation between the percentages of EdU+ cells observed 16 h after the pulse/chase/pulse experiment in thymus of B6 and FVB mice (80 values) and according to the age of mice (2 months: blue circle, 18 months: red circles). Hierarchical unsupervised clustering showing the clusters of mice according to the rates of proliferation. Values and statistics are given in S1, S2, S3 and S4 Tables. Proliferation rates/day are significantly different (p<0.05) comparing: In thymus: B6 2M vs B6 18M: 2M>18M in DP, CD8; FVB 2M vs FVB 18M: 2M>18M in DN and CD4; B6 2M vs FVB 2M:B6>FVB in DP CD3+, CD8; B6 18M vs FVB 18M:B6>FVB in CD8. In spleen: B6 2M vs B6 18M: 2M>18M in CD4 CD44hi; FVB 2M vs FVB 18M: 2M>18M in CD4 CD44hi;B6 2M vs FVB 2M:B6>FVB in CD4, CD4 Foxp3+; B6 18M vs FVB 18M:B6>FVB in CD4 Foxp3+, CD8 CD44hi
Fig 7.
Quantification of dividing cells during thymocyte differentiation.
For each stage of thymocyte differentiation, the number of cells and the number of EdU+ cells (in millions) is quantified by FACS after pulse/chase. The correlation between these numbers is shown according to strains and ages of mice (upper panel). Box plots represent the quartiles and the lines represent the evolution of the median values through stage of differentiation thus across time, in four mice per group (lower panel). DN CD3- cells are in yellow, DP cells are in red and decomposed into DPearly (CD4hiCD8hi) and DP late (CD4medCD8med). Then DP cells differentiate either into CD4+CD3+ (blue) or into CD8+CD3+(green) mature T cells.
Fig 8.
Quantification of cell proliferation and death in thymus.
For each stage of thymocyte differentiation, the total number of live cells (green), of accumulated EdU+ (red) cells, and of dead cells (blue) after the pulse/chase/pulse are shown (upper panel). The percentage of EdU+ cells (red) and dead cells (blue) among each differentiation stage identified is given (lower panel). Quantification is done by FACS and given as the median (line) of 4 mice per group (points). In DN2 and DN3 cells in some B6 mice death is so rare that quantification is below 10 cells/thymus and sometimes up to 0 and cannot be represented on the log scale. The ratio of EdU+/dead cells gives a performance of cell expansion.