Obesity is a significant public health concern, frequently linked to cardiovascular disease. Visceral fat accumulation, in particular, predicts negative metabolic outcomes and cardiovascular burden. White adipose tissue (WAT) expands through adipocyte hypertrophy (increase in size) or hyperplasia (new adipocyte recruitment and differentiation from adipose progenitor cells (APCs)). Visceral adipose tissue (VAT) expansion is influenced by intrinsic and environmental factors, with evidence suggesting functionally distinct APC subsets within VAT in humans and mice. Prior research has highlighted visceral APC heterogeneity and their *in vitro* adipogenic potential. Single-cell and single-nucleus transcriptomic data have advanced understanding of VAT cellular composition, but the identification and functional relevance of APC markers require further exploration. This study utilizes single-cell RNA sequencing (scRNA-seq) and publicly available single nuclei RNA sequencing (snRNA-seq) data to identify novel APC markers and adipogenic modulators in human and mouse VAT, also investigating the influence of sex and obesity on visceral APC adipogenic potential in mice.

Existing research indicates a heterogeneous population of APCs within VAT, exhibiting varying adipogenic potential *in vitro*. Studies using single-cell and single-nucleus transcriptomics have provided insights into the cellular composition of VAT, identifying major cell types such as fibro-adipogenic progenitors (FAPs), immune cells, mesothelial cells, and endothelial cells. However, a comprehensive understanding of the functional roles of these cell populations and the identification of specific markers for APCs remain incomplete. Previous work has explored the use of markers like CD34 to distinguish adipogenic and non-adipogenic APCs, but more refined markers are needed for precise APC isolation and characterization. The impact of sex and obesity on the cellular composition and adipogenic capacity of VAT also requires further investigation.

The study employed scRNA-seq on omental fat samples from lean and obese human subjects (Table 1) and scRNA-seq on perigonadal white adipose tissue (eWAT) from male and female C57BL/6J mice fed either a normal chow diet (NCD) or high-fat diet (HFD) for 8 weeks. Publicly available snRNA-seq data of human omental adipose tissue from lean and obese subjects was also analyzed. Rigorous quality filtering removed low-quality cells and those with high mitochondrial DNA content. Data integration combined human and mouse data independent of sex or diet, identifying four major clusters (FAPs, immune cells, mesothelial cells, endothelial cells) based on differential gene expression. Further analyses explored sex differences by re-clustering cell types by sex, independent of obesity. Differential gene expression and pathway analyses were performed between male and female samples. PDGFRA+ FAPs were re-clustered to identify transcriptionally distinct subpopulations. The adipogenic potential of murine visceral APCs was investigated *in vitro* by isolating APCs via FACS and differentiating them in the presence of differentiation media (DM). Lipid accumulation and expression of adipogenic genes (Pparg, Fabp4) were assessed. The study also examined BMPER expression in human and murine VAT APCs and adipocytes, utilizing scRNA-seq and snRNA-seq data. The role of BMPER in adipogenesis was investigated using siRNA knockdown in 3T3-L1 preadipocytes and by generating Bmperfl/fl mice using CRISPR/Cas9 technology. APCs from these mice were infected with adenoviruses expressing either GFP (control) or Cre-GFP (to knock out Bmper) before differentiation, assessing lipid accumulation and adipogenic gene expression. Statistical analysis employed unpaired two-tailed t-tests, two-way ANOVAs, and Tukey's post hoc tests as appropriate.

The study revealed sex-specific and diet-specific effects on visceral APC composition and adipogenic potential. In humans, no significant differences in cell proportions were observed between lean and obese groups, possibly due to small sample size; however, analysis of the Rosen group's snRNA-seq data showed obesity significantly reduced the proportion of adipose progenitors and increased macrophages. Sex differences in VAT cellularity were more pronounced in mice than in humans. Human males had more FAPs and endothelial cells, while females had more immune cells. In mice, females had more FAPs and fewer immune cells. Transcriptional differences between sexes were more prominent in mice. Male mouse FAPs showed a pro-adipogenic/pro-lipogenic profile compared to females. Re-clustering of PDGFRA+ cells identified seven FAP clusters common between humans and mice, representing uncommitted, committed APCs, and fibro-progenitors. Obesity increased committed progenitors and fibro-progenitors in human omental fat, while in mice, HFD increased uncommitted APCs. *In vitro* studies showed that male APCs from NCD-fed mice were highly adipogenic, while female APCs were refractory to adipogenesis. Obesity decreased adipogenic potential in male APCs. BMPER was identified as a conserved marker for APCs and adipocytes in human and mouse VAT, highly enriched in lineage-negative stromal vascular cells. BMPER expression peaked at day four post-differentiation in 3T3-L1 preadipocytes. Knockdown of BMPER in 3T3-L1 cells and knockout in mouse APCs significantly reduced adipogenesis.

The findings demonstrate sex- and diet-specific differences in VAT cellular composition and adipogenic capacity, more pronounced in mice. Male mice exhibited a pro-adipogenic/pro-inflammatory transcriptional profile compared to females. The identification of BMPER as a positive regulator of adipogenesis provides a novel insight into the mechanisms controlling adipogenesis. BMPER's role in early differentiation stages suggests it may influence clonal expansion and migration. Given BMPER's modulation of BMP signaling and BMP4's known role in adipogenesis, BMPER likely modulates BMP signaling in APCs to facilitate adipogenic commitment. The decrease in PPARγ and Fabp4 expression in Bmper-deleted cells suggests BMPER acts upstream of these adipogenic regulators, potentially by modulating BMP concentration. Further research is needed to fully elucidate the mechanisms by which BMPER regulates adipogenesis.

This study provides a comprehensive cellular atlas of human and mouse VAT, revealing sex- and diet-dependent variations in cellular composition and adipogenic potential. The identification of BMPER as a novel marker for APCs and a positive regulator of adipogenesis represents a significant advancement in our understanding of adipogenesis. Future research should focus on elucidating the precise mechanisms by which BMPER regulates adipogenesis and exploring its potential as a therapeutic target for obesity and related metabolic disorders.

The study's limitations include the relatively small number of human subjects, potentially limiting the statistical power of some analyses and the generalizability of findings to the broader population. The use of menopausal women in the human cohort might also have influenced the results. Differences in fat depot location between humans and mice (omental vs. gonadal) and potential influences of sex hormones should also be considered. The study primarily focused on VAT; future research should investigate other adipose tissue depots.