Figure 1. Developmental origins of different connective tissues that have been reported to contain “MSCs”. Of note, bone originates from three different embryonic specifications (noted by a white star). Skin, muscle, tendons and ligaments, and bone arise from different specifications of the sclerotome. Bone marrow adipose tissue originates from paraxial mesoderm, lateral plate mesoderm and neural crest. While bone marrow adipose tissue arises from mesoderm and neural crest, other forms of fat originate from all three germ layers. “MSCs” are not a lineage. MSC: mesenchymal stem/stromal cell. Adapted from Bianco and Robey.2

Figure 2. Gold standard assays by which to assess differentiation capacity. Many of the currently used “standard” assays of differentiation are prone to artifact or misinterpretation. However, there are assays that can faithfully report differentiation capacity: 1) the in vitro cartilage pellet assay, whereby one can see chondrocytes lying in lacunae surrounded by extracellular matrix that stains purple with toluidine blue, 2) the in vivo transplantation assay whereby donor cells are able to make bone matrix, osteocytes, osteoblasts, and in some cases, support haematopoiesis and formation of marrow adipocytes (the latter two properties are not shared by all forms of skeletal stem cells), and 3) the in vitro myogenic assay, whereby myotubes are formed in the absence of exogenous myoblasts (which will spontaneously fuse with any fibroblastic population). Adapted in part from Sacchetti et al.30 BM: bone marrow; DAPI: diamidino-2-phenylindole; H&E: hematoxylin and eosin; hp: haematopoiesis; MSC: mesenchymal stem/stromal cell; MU: muscle; MyHC: myosin heavy chain.

Figure 3. Differences in cell morphology (A) and colony size and habit (B) of freshly isolated bone marrow stromal cell suspensions plated at clonal density. When individual colonies with different cell shapes and colony habits are expanded ex vivo and transplanted in vivo, neither parameter correlated with the formation of a bone/marrow organ (a measure of multipotency). Adapted from Satomura et al.43

Figure 4. Clonal analysis - an essential step in the determination of stem cell potency. (A) When colonies are allowed to grow beyond the 10–14 days usually used for colony forming efficiency, the individual colonies begin to spontaneously differentiate. The vast majority of the colonies are alkaline phosphatase positive (right panel), indicative of osteogenic and pre-adipogenic cells. Approximately 50% of the colonies were Alizarin Red positive (centre panel) and approximately 10% stained with oil red O (right panel, unpublished data). (B) When individual colonies were isolated, expanded ex vivo, and transplanted subcutaneously into immunocompromised mice with an hydroxyapatite/tricalcium phosphate scaffold, ~10% of the single colony-derived strains made a complete bone/marrow organ (multipotent), whereas ~50% formed only bone (unipotent), and the remainder formed only fibrous tissue. Adapted from Sworder et al.13 (C) In studies where colonies were first incubated with adipogenic medium, colonies that accumulated fat identifiable by inverted light microscopy were marked with a blue circle. When the medium was changed to an osteogenic medium, a number of the adipogenic colonies also became alizarin red positive, indicating that the original CFU-F was able to give rise to adipogenic cells, and then osteogenic cells; an indication of “flexibility” (unpublished data). CFU-F: colony forming units-fibroblast.

Figure 5. Changes in alkaline phosphatase based on position on the cell cycle. It has long been recognized that BMSC/SSC colonies are heterogeneous with respect to alkaline phosphatase activity (left panel, slide given to PGR by Alexander Friedenstein). Later, it was determined that when cultures of osteogenic cells are synchronized using an amphidicolin protocol, cells in S phase have high levels of activity. During G2 + M phase, alkaline phosphatase is cleaved from the cell surface and released into the medium. Cell surface activity is restored during the following G1 and S phases. Adapted in part from Fedarko et al.53 BMSC: bone marrow stromal cells; SSC: skeletal stem cell.