metastases and primary tumors, and the differential response of tumor subpopulation to cancer therapy. In stem cell research, depth analysis at successive timepoints can reveal the number of stem cells maintaining a niche, and their dynamics in physiological and pathological states. In developmental biology research, lineage analysis can reveal the developmental origin of various tissues. Here we performed a few such analyses, but we believe this is only the tip of the iceberg, and further analysis may yield deeper insights. For example, by applying tools and concepts used in the analysis of human populations to cell populations, it may be possible to derive quantitative estimates regarding the cell dynamics and the number of stem cells which maintain a tissue. Perhaps most importantly, the fact that this methodology is non- invasive theoretically allows analyzing humans, without being confined to analysis of model organisms. Materials and Methods Experiment mice Mlh1+/2 mice were obtained from Michael Liskay (described in [33]) and were maintained at our institute under C57Bl/6 and 129SvEv (kindly provided by Ari Elson, The Weizmann Institute of Science) backgrounds. Mlh1+/2C57Bl/6 and Mlh1+/2 129SvEv were mated to yield Mlh12/2 progeny of dual background, which were used for experiments. ML2, ML4 and ML7 were 10, 13 and 5.5 weeks old respectively when sacrificed, and were genotyped as Mlh12/2. All animal husbandry and euthanasia procedures were performed in accordance with the Institutional Animal Care and Use Committee at the Weizmann Institute of Science. Isolation and culture of satellite cells Satellite cells were isolated and cultured as described in [34]. Briefly, EDL, soleus, gastrocnemius and masseter muscles were digested in 0.2% (w/v) collagenase type I (Sigma-Aldrich) at 37uC. Collagenase was reconstituted in Dulbecco’s Modified Essential Medium (DMEM; high glucose, with L-glutamine, 110 mg/l sodium pyruvate, and pyridoxine hydrochloride; supplemented with 50 U/ml penicillin and 50 mg/ml streptomycin; GIBCO Invitrogen). Following digestion, the muscles were triturated with a wide-bore pipette to release single myofibers. Each single myofiber was transferred to a separate 60 ml dish and then to a tube containing 1ml DMEM. Single myofibers were triturated using a 20G needle mounted onto a 1 ml syringe, to disengage satellite cells. The resulting fiber suspension (in 1 ml DMEM) was then dispensed to 12 Matrigel pre-coated wells within a 24-well plate. Clones were observed every other day. Isolation and culture of mesenchymal stem cells (MSCs) MSCs were isolated and cultured as described in [35,36]. Briefly, femurs were cleaned off the soft tissue and epiphysis to allow bone marrow cells (BMC) collection. The BMC were flushed out with DMEM using syringe with 21G needle. To get Colony forming unit-fibroblast (CFU-F) single cell BMC suspensions were diluted to a concentration of 2.56106 cells/ml in DMEM supplemented with 10% FCS and plated in 24-wells plates. MSC clones from ML7 samples 104 and 105 were created slightly differently: due to uncertainty of a single cell origin of these clones, a second subcloning step was performed, and cells were allowed to proliferate for additional 4–5 days. Isolation and culture of kidney stem cells Kidney stem cells were isolated and cultured as described in [37]. Cell Lineage Trees in Mice Isolation and culture of hematopoietic stem cells Hematopoietic stem cells (SCA-1+ Lin2) were obtained by crushing tibiae and femurs and extracting cells from the bone marrow (BM). The BM cells suspension were passed through a nylon mesh twice to remove connective tissue and clumps of cells. Subsequently, the cells (206106) were washed and resuspended at 5ml PBS. BM cell suspensions were maintained on ice throughout the purification procedures. Antibodies used for immunomagnetic positive selection were SCA-1 conjugated to microbeads (SCA-1 multisort kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The following lineage marker antibodies conjugated to microbeads were used: CD45R/B220 for the B lineage, CD4/L3T4 and CD8a/Ly-2 for T cell lineage, and CD11b/Mac-1 for myelo- monocytic cells. SCA-1+ cells were positively selected using the MACSH magnetic bead system (Miltenyi). The microbeads were removed from the magnetic field, thereby allowing the SCA-1+ cells to be collected into the multisort release reagent (Miltenyi). Cytofluorimetric sorting of the isolated SCA-1+ cells was carried out by double immunofluorescent staining, using the following directly labeled antibodies (obtained from Pharmingen, San Diego, CA): PE-SCA-1/Ly-6A/E (clone E13-161.7), R-APC- CD45R/B220 (clone RA3-6B2), R-APC-CD4/L3T4 (clone GK 1.5), R-APC-CD8a/Ly-2 (clone 53-6.7) and R-APC-CD11b (clone M1/70). Stained cells were resuspended at 16106 cells / ml in buffer column at 4uC (on ice). Cells were then sorted for positive SCA-1-PE cells and lineage negative cells-APC (SCA-1+ Lin2 cells) into a sterile glass tube containing 500 ml/ml FCS sterile using ARIA sorter (BD Biosciences, Mountain View, CA). Immediately after purification, 103 cells per ml SCA-1+Lin2 cells were suspended in 10 ml of culture medium. Cells were counted and diluted to reach an average of 1 cell/600 ml culture medium and replated into 96-well culture plates (200 ml/well) to establish single cell clones. Single cell clones were cultured for 7 days in 96- wells plate (Nunc, Roskilde, Denmark) in culture medium made with Iscoves Modified Dulbeccos Medium (IMDM) containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ ml streptomycin. Isolation of single cells from a suspension Single cells were isolated by suspending a bulk of cells in PBS until a dilution of about 1 cell/0.5 ml was obtained. Then, 0.5 ml drops of this dilution were placed at the center of multiple wells of a 96-well (flat bottom) plate. Microscopic observation was used to identify wells with exactly one cell. Isolation of B-Cells, NK-Cells Spleens of experiment mice were dissected and crushed over a 1 mm mesh (A.D. Sinun, Israel) into a Petri dish obtaining a cell suspension. The cell suspension was transferred into a 50 ml tube, centrifuged (7 min, 1200 rpm) in a 5702 Eppendorf centrifuge. Cells were resuspended in 1 ml PBS and counted. Isolation of specifically desired cells was performed by magnetic sorting using the Miltenyi Biotec magnetic microbeads, MACS columns and MACS separator, according to the manufacturers instructions. CD45R (B220) microbeads were used for B cells, and CD49b (DX5) microbeads were used for NK cells. Single B-cells and NK- cells were obtained as described above. Isolation of oocytes Ovaries were removed and placed in Leibovitz’s L-15 tissue culture medium (Gibco), supplemented with 5% fetal bovine serum (Biolab, Jerusalem, Israel), penicillin (100 IU/ml) and streptomycin (100 mg/ml, Gibco). When isolated from the ovarian PLoS ONE | www.plosone.org 9 April 2008 | Volume 3 | Issue 4 | e1939