Recently,gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting(MACS) showed remarkable periodontal regenerative potential in vivo.As a second-stage investigation,the present study’s aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive(MACS~+) and STRO-1-negative(MACS~-) cell populations from the human free gingival margin.Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies.Subsequently,the MACS~+ and MACS~- cell fractions were characterized by flow cytometry for expression of CD14,CD34,CD45,CD73,CD90,CD105,CD146/MUC18 and STRO-1.Colony-forming unit(CFU) and multilineage differentiation potential were assayed for both cell fractions.Mineralisation marker expression was examined using real-time polymerase chain reaction(PCR).MACS~+ and MACS- cell fractions showed plastic adherence.MACS~+ cells,in contrast to MACS- cells,showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs(P<0.01).More than 95%of MACS~+ cells expressed CD105,CD90 and CD73;lacked the haematopoietic markers CD45,CD34 and CD14,and expressed STRO-1 and CD146/MUC18.MACS- cells showed a different surface marker expression profile,with almost no expression of CD14 or STRO-1,and more than 95%of these cells expressed CD73,CD90 and CD146/MUC18,as well as the haematopoietic markers CD34 and CD45 and CD105.MACS~+ cells could be differentiated along osteoblastic,adipocytic and chondroblastic lineages.In contrast,MACS- cells demonstrated slight osteogenic potential.Unstimulated MACS~+ cells showed significantly higher expression of collagen I(P<0.05) and collagen III(P<0.01),whereas MACS~- cells demonstrated higher expression of osteonectin(P<0.05;MannWhitney).The present study is the first to compare gingival MACS~+ and MACS- cell populations demonstrating that MACS~+ cells,in contrast to MACS- cells,harbour stem/progenitor cell characteristics.This study also validates the effectiveness of the STRO-l/MACS~+technique for the isolation of gingival stem/progenitor cells.Human free gingival margin-derived STRO-1/MACS~+ cells are a unique renewable source of multipotent stem/progenitor cells. Recently, gingival margin-derived stem / progenitor cells isolated via STRO-1 / magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study was aim was to perform in vitro characterization and comparison of the stem / progenitor cell characteristics of sorted STRO-1-positive (MACS ~ +) and STRO-1-negative (MACS ~ -) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies using the MACS ~ + and MACS ~ - fractional fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146 / MUC18 and STRO -1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR) .MACS ~ + and MACS- cell fractions showed plastic adherence. ~ + cells, in contrast to MACS- cells, showed all of the predefined mesenchymal stem / progenitor cell characteristics and a significantly higher number of CFUs (P <0.01) .More than 95% of MACS ~ + cells expressed CD105, CD90 and CD73 ; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146 / MUC18. MACS-cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed as CD73, CD90 and CD146 / MUC18, as well as the haematopoietic markers CD34 and CD45 and CD 105. MACS ~ + cells could be differentiated along osteoblastic, adipocytic and chondroblastic lines. In contrast, MACS-cells did slight osteogenic potential. The present study was the first to compare (P <0.05) and collagen III (P <0.01), while MACS ~ - cells demonstrated higher expression of osteonectin (P <0.05; Mann Whitney) gingival MACS ~ + and MACS-cell populations demonstrating that MACS ~ + cells, in contrast to MACS- cells, harbor stem / progenitor cell characteristics. this study also validates the effectiveness of the STRO-1 / MACS ~ + technique for the isolation of gingival stem / progenitor cells. Human free gingival margin-derived STRO-1 / MACS ~ + cells are a unique renewable source of multipotent stem / progenitor cells.