ABSTRACT: 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; Mann-Whitney). 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-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.
