Isolation, characterization, and differentiation of c-kit⁺ cells in culture. (A) FACS analysis showing the purity of c-kit⁺ cells obtained by MACS sorting and relative CXCR4 and CD34 expression in these cells. (B) Percentage of c-kit^–, CXCR4^–, and CD34-expressing cells in MACS-sorted cells (n = 3). tot indicates total. (C) Quantification of differentiated EPC number (Ac-LDL-DiI⁺ cells) on the indicated substrates in the presence or absence of SDF-1. *P < .05; n = 5. (D) In contrast to SDF-1, VEGF does not act as a differentiation factor for c-kit⁺ cells onto Coll I. *P < .05; n = 3. C indicates control-untreated cells. (E) Phase contrast image of clusters of c-kit⁺–derived cells after 3 days of culture. (F) Double staining of Hoechst 33258 and Ac-LDL-DiI⁺ in differentiated EPCs after 7 days in culture. (G-H) Immunohistochemistry showing VWF (G) and KDR (H) expression in clusters of differentiated EPCs after SDF-1 stimulation in culture for 7 days.