ABCG2 is a selectable marker for enhanced multilineage differentiation potential in periodontal ligament stem cells

Abstract

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth.

FIG. 1.

Periodontal ligament stem cell (PDLSC) isolates express the ATP-binding cassette subfamily G member 2 (ABCG2) transporter at different levels. (A) Expression was detected with antibody labeling: dotted lines mark isotype controls and continuous lines the 5D3-antibody-labeled cell populations. (B) Detection of the side-population (SP) with DyeCycle Violet (DCV) extrusion method: rectangular gate marks the DCV excluding SP cells inhibitable with Ko143 or verapamil. (C) The ABCG2 mRNA expression determined by RT-qPCR.

FIG. 2.

Alizarin red S staining of differentiated PDL cells induced for 14 days in osteogenic medium (A), and the colorimetric quantitation of Alizarin red S stain (B). The calcium accumulation of the differentiated cells shows positive correlation with ABCG2 expression (C).

FIG. 3.

The trilineage differentiation capacity of sorted PDLSC samples. The ABCG2 expression of sorted PDLSCs measured by flow cytometer: dotted lines mark isotype controls and continuous lines the 5D3-antibody-labeled cell populations (A). The demonstration of osteogenic differentiation potential of sorted PDLSCs with Alizarin red S staining after 14 days of induction into osteogenic lineage (B), and the quantitation of the stain (C). Elevation of alkaline phosphatase mRNA expression of cell populations in the early stage of bone differentiation (D). The glycosaminoglycan pattern of PDLSCs differentiated into chondrogenic direction (E), and SOX9 expression of the sorted cells (F). Oil red O staining of cells induced for 14 days into adipogenic lineage (G), the quantitation of the stain (H), and the changes of peroxisome-proliferator-activated receptor gamma (PPARγ) mRNA expression after 7 days in adipogenic medium (I). Student's t-test was performed between ABCG2^high versus ABCG2^low and ABCG2^high versus parental cell populations; P values of <0.05 were considered significant (*).

FIG. 4.

Bone differentiation marker expression of sorted PDLSCs in the early stage of osteogenic induction—presented on a representative sample. The mRNA level of osteocalcin (A), osterix (B), and the cementogenesis marker cementum protein 1 (C). Student's t-test was performed between ABCG2^high versus ABCG2^low and ABCG2^high versus parental cell populations; P values of <0.05 were considered significant (*).