Dedifferentiated Fat Cells Isolated From the Human Buccal Fat Pad

Objectives: We previously reported that dedifferentiated fat (DFAT) cells isolated from subcutaneous adipose tissue in F344 rats are a potential cell source for periodontal tissue regeneration (2014 AADR). The purpose of this study is to clarify whether human buccal fat pad could be an alternative cell source for DFAT cell preparation.
Methods: Approximately 10 g of human buccal fat pad was harvested from 10 patients, minced into small pieces, and then dissociated into individual cells with collagenase solution. DFAT cells were separated from mature adipocytes using a ceiling culture technique based on buoyancy. Isolated DFAT cells were characterized at passage 3: i.e., cell surface marker expression, specific gene expression, colony-forming unit fibroblast (CFU-F), proliferation activity, and osteogenic and adipogenic differentiation potential.
Results: Mature adipocytes floated up and adhered to the ceiling of culture flask. The adhered cells divided asymmetrically and generated DFAT cells. Those DFAT cells began to proliferate extensively after inversion of the flasks. In CFU-F assay, colony formation (about 60/well) was observed in DFAT cells culture 10 days after plating. Flow cytometric analysis showed a high level of expression of MSC markers such as CD13, CD73, CD90, and CD105. mRNA expression of c-Myc, Klf-4, Oct3/4, Runx2, Pparγ2, and Sox9 was demonstrated in DFAT cells by RT-PCR. DFAT cells under osteogenic induction exhibited calcium deposition positive in alizarin red S staining. Also shown was intracellular accumulation of oil red O-positive lipid droplets in DFAT cells after adipogenic induction.
Conclusions: DFAT cells prepared from human buccal fat pad possessed cellular characteristics and differentiation potentials equivalent to those of DFAT cells from ordinary subcutaneous fat pad. These results indicate that buccal fat pad could be an another potential cell source for DFAT cell preparation.