Tri-lineage differentiation of passage 2 (A) & passage 5 (B) Unselected and Stro-4 enriched ovine BMSCs and quantification of chondrogenic (Alcian Blue) and osteogenic (Alizarin Red) staining in passage 2 (C & D) and passage 5 (E & F) Unselected (Uns) and Stro-4 populations. An enhanced osteogenic response is seen in the Stro-4 population over unselected, Alizarin red stain (A). Chondrogenic response is comparable between Stro-4 and unselected cell groups with inherent chondrogenic activity noted in the Stro-4 population under basal conditions, Alcian blue stain (A). Adipogenic response in both groups was present but unconvincing, assessed by Oil Red O staining (A). Passage 5 cells response to osteogenic media was present in both cell groups although diminished compared to passage 2, Alizarin red (B). The chondrogenic response in unselected cells had diminished at passage 5 but was conserved in the stro-4 enriched population, Alcian blue (B). Adipogenic differentiation is absent in both populations. N = 4, both groups for each condition. Scale bar = 200 μm P2 Stro-4 enriched oBMSCs showed increased alcian blue staining intensity compared to Unselected oBMSCs Stro-4 enriched cells (C). Both P2 populations demonstrated a positive phenotypic change under osteogenic conditions but did not show a significant difference between unselected and enriched populations (D). In contrast to chondrogenic differentiation, no innate osteogenic response was noted under basal culture conditions (d). Under both basal and chondrogenic conditions, P5 Stro-4 enriched oBMSCs responded positively showing significant increases in staining intensity compared to Unselected cells (E,F). The response pattern between groups appears similar to that seen at passage 2 (E,F). Cellular response to osteogenic media diminished in late passage (F). Although an osteogenic response was present there was no significant difference in osteogenic response between conditioned and basal media (F). N = 4, error bar = mean ± SD. . *p < 0.05, **p < 0.005, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)