Reversion of SDF-1–enhanced EPC differentiation by mitogenic stimulation of c-kit⁺ cells. (A) Culturing c-kit⁺ cells in serum-free medium in the presence of IL-3/IL-6, FL, and SCF dramatically enhanced the number of EPCs either in the presence or absence of SDF-1. *P < .05; n = 3. (B) These cells, however, failed to differentiate both in the presence or absence of SDF-1 when cultured onto FN substrate. *P < .05; n = 3. Error bars indicate standard errors. C indicates untreated cells or cells treated with cytokine cocktail without SDF-1. (C) To assess whether c-kit⁺ cells cultured in the presence of cytokines express stem cell markers CXCR4 and integrin receptors, we performed a FACS analysis for c-kit, Sca-1, CXCR4, and α₄ and α₅ integrins. Results are given as average of 3 independent experiments ± SE. (D) RNAse protection assay showing expression of CXCR4 mRNA in freshly isolated c-kit⁺ cells and its up-regulation following culture in mitogenic condition for 7 days. exp indicates expression.