(B,E) Fluorescent images of 14-day-old clones labeled with anti-Myo-D (green) and nuclei visualized with DAPI (C,F; blue). The myogenic clone (A–C) is composed of spindle-like cells and myotubes, all nuclei are labeled with MyoD. The non-myogenic clone (D–F) is composed of fibroblast-like cells and none of the nuclei is labeled with MyoD. PCR reactions were performed using cDNA prepared MA-M and MA-NM clones (G–I), the latter clone contained cells with an adipogenic phenotype. MyoD and myogenin are skeletal muscle specific transcription factors and PPARγ is a key regulator of adipogenesis. mRNA of MyoD and myogenin were expressed only in MA-M clone and PPARγ was expressed only in the non-myogenic clone.

Cells from the left Gast muscle are depicted in green and from the and right Gast in red; cells from the right Masseter are depicted in purple. MSCs are depicted in brown. Myofiber-associated cells were significantly clustered according to the muscle they were extracted from with the p-values denoted in the figure.

These hypothetical scenarios are composed of the following types of cells: Myofiber-associated cells extracted from a specific muscle termed here as Muscle 1 (full blue circles represent myogenic progenitors, full light blue circles represent non-myogenic progenitors); myofiber-associated cells extracted from a different muscle, termed here Muscle 2 (full red circles represent myogenic and full pink circles represent non-myogenic progenitors); and MSCs (represented by full brown circles). In scenario (A) are depicted three independent stem cell lineages: MA-M cells, MA-NM stem cells, and MSCs; In scenario (B) MA-NM progenitors and MSCs belong to the same cell population, which has a developmental path distinct from MA-M cells [27], [28]; In scenario (C) satellite cells retain mesenchymal plasticity and may stochastically become myogenic or non-myogenic [20]. The last two scenarios postulate the existence of muscle-bound stem cells of a lineage distinct from MSCs that give rise to both myogenic and non-myogenic stem cells; In scenario (D) muscle-bound stem cells first differentiate into myogenic and non-myogenic stem cells, both of which migrate independently to the individual muscles and in scenario (E) muscle-bound stem cell first migrate to individual muscles and then differentiate into myogenic and non-myogenic stem cells within each muscle. Our work attempts to resolve which of these five scenarios hold.

(A) Lineage tree of 116 myofiber-associated cells (68 myogenic in blue, and 48 non-myogenic in light blue), and 29 MSC (brown) from a 330 day old mouse. Each terminal node (•) represents the ancestor of a single sampled clone. The vertical axis represents cell depth. Blue and brown lines indicate significant clustering of cells. The clustering of myofiber-associated cells is significantly different than that of MSCs (p<1e-26); (B) Boxplot of the depth of myofiber-associated and MCS cells extracted from a 330 days old mouse. The box represents the spread of the middle 50% data regarding the depth of all tested clones and the red lines represent the median value of depth. Whiskers at the ends of vertical lines indicate the minimum and maximum depth values. The range in the 25th to 75th percentiles of all data was 11.8–14.6 for MSCs, 19.9–26.2 and 23.7–29.7 for myofiber-associated, non-myogenic and myogenic cells, respectively.

The following 7 sets of cells were significantly clustered: MSC (brown), myogenic clones from the Gast left (dark green), non-myogenic clones from Gast left (bright green), myogenic clones from the right Gast (red), non-myogenic clones from the right Gast (pink), myogenic clones from the right Masseter (dark purple) and non-myogenic clones from the right Masseter (bright purple). Purple red and green boxes mark the different clustering by muscles (as in Figure 4).