embryonic antigen-4 a marker for and in cultured and

One important challenge for regenerative medicine is to produce a clinically relevant number of cells with consistent tissue-forming potential. Isolation and expansion of cells from skeletal tissues results in a heterogeneous population of cells with variable regenerative potential. More consistent tissue formation could be achieved by identification and selection of potent progenitors based on cell surface molecules. In this study, we assessed the expression of stage-specific embryonic antigen-4 (SSEA-4), a classic marker of undifferentiated stem cells, and other surface markers in human articular chondrocytes (hAC), osteoblasts (hOB), and bone marrow-derived mesenchymal stromal cells (bmMSCs) and characterized their differentiation potential. Further, we sorted SSEA-4 expressing hACs and followed their potential to proliferate and to form cartilage in vitro . Cells isolated from cartilage and bone exhibited remarkably heterogeneous SSEA-4 expression profiles in expansion cultures. SSEA-4 expression levels increased up to ~5 population doublings but decreased following further expansion and differentiation cultures; levels were not related to the proliferation state of the cells. Although SSEA-4-sorted chondrocytes showed slightly better chondrogenic potential than their SSEA-4-negative counterparts, differences were insufficient to establish a link between SSEA-4 expression and chondrogenic potential. SSEA-4 levels in bmMSCs also did not correlate to the cells’ chondrogenic and osteogenic potential in vitro. SSEA-4 is clearly expressed by subpopulations of proliferating somatic cells with a MSC-like phenotype. However, the predictive value of SSEA-4 as a specific marker of superior differentiation capacity in progenitor cell populations from adult human tissue and even its usefulness as a stem cell marker appears questionable. H4C4, DSHB), CD90 (fluorescein isothiocyanate (FITC)-conjugated, 1:20, F15-42-1, Millipore), CD105 (1:40, P4A4, DSHB), SSEA-1 (1:40, MC-480, DSHB), SSEA-3 (1:40, MC-631, DSHB), SSEA-4 (1:80, MC-813-70, DSHB) or IgG isotype control (1:50, Invitrogen). Cells were washed twice with cold PBS and samples with unconjugated primary antibodies were incubated for 45 min in 3% BSA/PBS containing either FITC-labeled goat anti-mouse IgG antibody (1:80, CALTAG, Burlingame, CA) or DyLight™649-conjugated goat anti-rat IgG antibody (1:40, Jackson ImmunoResearch, West Grove, PA, USA). Finally, samples were washed with cold PBS and resuspended in 3% BSA/PBS. phenotype. Presentation of this surface antigen is remarkably heterogeneous and appears to be of transient nature during the cultivation period. Although SSEA-4 positive chondrocytes show slightly better chondrogenic potential compared to chondrocytes that do not present this surface molecule, the differences in the differentiation capacity of the two groups are not pronounced enough to link SSEA-4 expression to chondrogenic progenitors with superior differentiation potency. In addition, SSEA-4 levels in bmMSCs seem to be unrelated to the cells’ chondrogenic and osteogenic potential in vitro . These data suggest that SSEA-4 is not a reliable marker of multipotency in bmMSCs and is not specific to dedifferentiated mature cell populations with