Sex as a biological variable in human dental pulp stem cells: An exploratory epigenomic and transcriptomic comparison

To determine whether baseline epigenomic and transcriptional profiles of human dental pulp stem cells (DPSCs) differ by sex, and to assess sex dependence of key markers related to growth and stemness.

Primary DPSCs from nine young adult donors were isolated and cultured under standard growth conditions. Genome-wide DNA methylation was screened by Illumina Infinium Methylation EPIC v2.0. Bulk RNA sequencing was performed with poly A selected libraries on an Illumina NovaSeq X Plus sequencer. Additionally, a targeted RT-qPCR array covering canonical proliferation, stemness (multi- and pluri-potency, neural crest markers), MSC identity, and WNT and NOTCH signaling markers was tested.

Global differences between male- and female-derived samples were predominantly confined to the sex chromosomes. DNA methylation profiling revealed sex-dependent patterns, with mild autosomal hypermethylation in males and marked hypermethylation of the X chromosome in females. This pattern was accompanied by female-specific expression of XIST, supporting X-chromosome inactivation as a major contributor to the observed sex-chromosome signal. Autosomal methylation differences were generally modest (|Δβ| < 0.2). At the transcriptomic level, only a small fraction of genes (52 of 17,204) was differentially expressed, with most mapping to the sex chromosomes. Consistently, the RT-qPCR array indicated minimal differences between male and female DPSCs, with no consistent sex dependence across targets except for CCNE1.

Under basal culture conditions, DNA methylation and transcriptional profiles in DPSCs are largely similar between sexes. The limited differential signal is associated predominantly with sex chromosomes, while autosomal effects are few and modest. These findings provide an initial molecular baseline and motivate larger and context specific studies.