Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS- cell populations

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.