Figure 7. Identification of fibroblast contamination and senescent cells.

Examples are shown of how to recognize cultures enriched in fibroblasts (left panels) and senescent cells (right panels) by immunostaining with cell type–specific markers (FN, green; S100B, red) and enzymatic SA-β-Gal staining (blue precipitate inside the hSCs), respectively. The cultures were grown in LP medium in the absence (control, A–B) or presence of 250 μM CPT-cAMP (Protocol 3C, C–D) for seven days before fixation and immunostaining (Protocol 1C). Even though hSCs differentiated effectively as denoted by S100B staining, the fibroblasts proliferated extensively (C–D). In E–H, hSCs (passage-5) were plated in HP medium, fixed, and stained with SA-β-Gal after three days (Protocol 3D). Observe the differential behavior of the cells in areas of low (E–F) and high (G–H) density. Senescent hSCs are migratory and have the capacity to form aggregates that are prone to detachment. Recommendations: (1) purify the cells from A–D; (2) use or cryopreserve the cells from E–H but do not continue the culturing, as nearly all hSCs are senescent.