Cells that can be harvested from subcutaneous tissues are possible candidates for use in regenerative medicine because of the safety of harvesting and the abundance of donor tissues. Two different cell types can be harvested from subcutaneous adipose tissue following collagenase treatment and fractionation by centrifugation. The first population, found in the dense fraction, contains adipose-derived stem cells (ASCs). The other population is termed “ceiling culture-derived preadipocytes” (ccdPAs), which are cultured from the low density fraction by ceiling culture methods first reported by Sugihara et al..
Both ASCs and ccdPAs have potentials for multi-lineage differentiation. Recently, it was reported that human ceiling culture-derived cells have a higher osteogenic potential than ASCs. However, the underlying physiologic basis that determines the differences between ASCs and ccdPAs is unknown.
Epigenetics play a key role in cell differentiation. DNA methylation of CpG sites is one such type of epigenetic modification. CpG methylation in the promoter has a strong effect on silencing gene transcription. Histone modification is another type of epigenetic control. Trimethylation of histone H3 at lysine 4 (H3K4me3) within the promoter correlates with activation of gene expressions. However, little is known about the relationship between cellular phenotypes and epigenetic modifications in ASCs and ccdPAs.
Purpose of this study was to clarify the osteogenic potential and underlying epigenetic status of ASCs and ccdPAs.
Materials and Methods
ASCs and ccdPAs were primarily cultured from abdominal subcutaneous fat tissues of metabolically healthy four lean females. After seven weeks of culture, cellular responses to osteogenic differentiation media were examined. To evaluate the osteogenic potentials of undifferentiated ASCs and ccdPAs, two types of epigenetic assessments were performed using next generation sequencing (NGS). One was DNA methylation assays with the 450K BeadChip and the other utilized chromatin immunoprecipitation assays (ChiP-Seq) for H3K4me3.
Even after 7 weeks of culture, ccdPAs showed higher osteogeic differentiation than ASCs, with higher RUNX2 expression, BGLAP (osteocalcin) secretion, and alkaline phosphatase stain (ALP) stain. Focusing on the promoters of two osteogenic master regulator genes (RUNX2 P1 and RUNX2 P2), we found that CpG methylation was higher in ASCs than in ccdPAs. In RUNX2 P2, H3K4me3 levels were higher in ccdPAs than in ASCs. Those results indicated that ccdPAs were more likely to transcribe their RUNX2 genes than were ASCs. Other osteogenic related genes (SP7, ATF4 and BGLAP) also showed differences in CpG methylation or H3K4me3 that were consistent with cellular functional differences.
Our analyses coupled with NGS showed that ASCs and ccdPAs differed in DNA methylation and H3K4me3 levels in osteogenesis-related genes. The underlying epigenetic differences between ASCs and ccdPAs were consistent with the cellular functional differences. Both ASCs and ccdPAs are important cells in regenerative medicine. Plastic surgeons using cells harvested from subcutaneous fat tissue must characterize ASCs and ccdPAs at a fundamental level for their reliable application in regenerative medicine. Our results enhance our understanding of these cell populations and will facilitate further application of ASCs and ccdPAs in regenerative medicine.