Table 1. Cell Samples in ML Experiments. Mouse ML2 (26) Age: 10w ML4 (25) Age: 13w ML7 (67) Age: 5.5w Cell Type Satellite cell (10) Kidney SC (8) B-cell (8) Satellite cell (12) Mesenchymal SC (5) Hematopoietic SC (2) B-cell (5) NK-cell (1) Satellite cell (57) B-cell (10) Source EDL (2): Left (2 fibers) SOL (2): Left (1, 1 fiber) Right (1, 1 fiber) MAS (6): Left (4 fibers) Kidney Spleen GAS (8): Left (2 fibers) EDL (2): Right (1 fiber) MAS (2): Left (1 fiber) Right Tibia (3) Right Femur (2) Left Femur/Tibia Spleen Spleen GAS (46): Left (23, 4 fibers) Right (23, 5 fibers) EDL (11): Left (3, 2 fibers) Right (8, 3 fibers) Spleen Expansion Culture Culture WGA (G-Plex) Culture Culture Culture WGA (G-Plex) WGA (G-Plex) Culture WGA (G-Phi) % Mut. 9.8% 13.4% 18.2% 13.3% 8.5% 9.5% 16.3% 11.1% 12.9% 13.6% Cell Lineage Trees in Mice % Amp. 85.4% 38.7% 24.6% 69.7% 72.9% 92.8% 24.6% 13.4% 64.0% 69.3% Number of cells in each experiment from each cell type and organ source are indicated in brackets. Only cells which participated in lineage analysis are shown. % Amp. = percent of alleles which were successfully amplified on average from each cell type (based on manual analysis of signals); % Mut. = Percent of mutated alleles with respect to putative zygote (out of amplified alleles). w = weeks; SC = Stem Cell; EDL = Extensor Digitorum Longus muscle; SOL = Soleus muscle; GAS = Gastrocnemius muscle; MAS = Masseter muscle; WGA = Whole Genome Amplification; G-Plex = GenomePlex; G-Phi = GenomiPhi. doi:10.1371/journal.pone.0001939.t001 respectively) hence we preferred not to analyze them. For each muscle we created a lineage matrix (according to our two measures) and a physical distance matrix (according to the physical distance defined above), and checked whether they are significantly correlated (see Methods). We found that for both muscles there is a significant anti-correlation between the lineage measures and physical proximity (Figure 4G; p = 0.0018 and p = 0.0002 for Gastrocnemius left and right, respectively, accord- ing to the MRCA depth measure, and p = 0.0063 and p = 0.0259 according to the percent of common mutations measure). This anti-correlation demonstrates that satellite cells which are physically close to each other tend to share a longer common developmental path, and suggests a relatively coherent growth of satellite cells in myofibers within a muscle. Evaluation of DNA Amplification Fidelity The methods we employed for amplifying DNA from single cells, whether by ex vivo culturing or WGA, are not expected to introduce any substantial bias in our analyses, for the following reasons. When single cells are cultured ex vivo, mutations may occur during the clonal expansion, yet the identifier of a cell clone is with high probability identical to the identifier of the clone’s founder cell [1]. WGA may introduce artifact mutations [17], which cannot be distinguished from mutations that occurred in the mouse. To evaluate the level of WGA artifact mutations we created small colonies, each founded by a single RMA cell [18], and analyzed the cell identifier for each progeny cell (using the same set of MS loci). Progeny cells are assumed to have identical genomes, in the case of which mutations are attributed to WGA artifacts. The two WGA methods we used show 0.9%–1.2% mutations (see Tables S4). Since our MS are highly unstable, mutations may have occurred in culture, hence the actual percent of WGA artifact mutations is expected to be lower. In any case, this figure is significantly lower than the percentage of mutations in the analyzed cells (see Table 1), therefore the majority of mutations occurred in vivo. Lack of Mutations in ML7 Mesenchymal Stem Cells In contrast to most cells which showed a high percent of mutations (12.5% on average), 18 out of 20 mesenchymal stem cells (MSCs) in ML7 do not exhibit any mutations over the entire set of analyzed MS (although amplification levels of all cell types amplified in culture were normally high; two MSCs show a few mutations, which may be an artifact due to a slightly different protocol used in their culture, see Materials and Methods). We are currently investigating whether this result is reproducible, and whether it is related to the immortal strand hypothesis [19]. Discussion We reconstructed three lineage trees of cells of multiple types obtained from three adult mice based on the analysis of spontaneous somatic mutations accumulated in microsatellites. We analyzed correlations between lineage and cell type, and between lineage and the hierarchy of the anatomy of skeletal muscle. Such correlations are closely related to three basic questions in development: First, are lineage and biological function correlated, i.e. do cells sharing a long common developmental path tend to be of the same type and perform the same biological function? We found that for the cell types we analyzed there is no significant correlation between lineage and cell type. Specifically we showed that none of these cell types are a single exclusive clone. PLoS ONE | www.plosone.org 5 April 2008 | Volume 3 | Issue 4 | e1939