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

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.

Retinol/inflammation affect stemness and differentiation potential of gingival stem/progenitor cells via Wnt/β‐catenin

“…27 Stem/progenitor cells migration could be stimu- reparative/regenerative behavior. 15,16 In the present investigation, G-MSCs, similar to earlier in vitro studies, 20,30 showed all predefined stem/progenitor cells characteristics, with CFUs, the distinctive expression/absence of predefined surface markers and a multilineage differentiation aptitude. 31 The G-MSCs were challenged by a proinflammatory cytokines "cocktail" (IL-1β, TNFα, and IFNγ), 15 via retinol 22 or their combination, and their pluripotency, proliferation, and differentiation capacity evaluated.…”

“…On day 12, cells were fixed with ice‐cold 100% methanol, stained with 0.1% crystal violet and CFUs evaluated. Second passage G‐MSCs were characterized for CD14, CD34, CD45, CD73, CD90, and CD105 expression, employing FACSCalibur E6370 and FACSComp 5.1.1 software (Becton Dickinson, Franklin Lakes, NJ) …”

Stem cell‐like populations and immunoregulatory molecules in periodontal granulation tissue

“…The GT biopsies and the disease‐free connective tissue specimens contained MSC‐like cells that expressed CD90, CD73, CD105, CD106, CD146 (MSC markers), as well as SSEA‐4 (embryonic marker). Cells at passage 2 expressed low levels of STRO‐1 (<2%‐3%), albeit also found by other studies, and of CD105 (49.2%‐61.3%) . The tissues demonstrated a high inter‐subject variability regarding the expression of these markers possibly reflecting heterogeneity of the cell population and respective variability in “stemness” properties .…”

Mesenchymal stem cells and biologic factors leading to bone formation

“…When first described, human GMSC were found to have high proliferative rates, a stable morphology and retained stable karyotype and telomerase activity over sustained passage in culture (Tomar et al., ). These cells have been repeatedly demonstrated to have osteogenic potential in vitro (El‐Sayed et al., ; Fournier et al., , Mitrano et al., ; Tomar, et al., ). While early in vivo studies failed to demonstrate osteogenic capacity of GMSC when implanted subcutaneously (Zhang et al., ), subsequent studies have shown that these cells can promote bone formation in vivo when implanted into periodontal defects and calvarial critical size defects (Wang et al., ; Yu et al., ).…”