A single-cell-based identification and characterisation of Aregs, an inhibitory subpopulation of adipose stem and precursor cells

Adipose tissue is an essential element in energy conservation mechanisms. Its unique plasticity is driven by the ability of adipocytes to accumulate and liberate lipids as a function of the energetic status of the organism. Given the recent rise in the global incidence of obesity, there is great interest in understanding the mechanisms behind disturbed energy balance. However, the heterogeneity of adipose tissue and of the somatic stem cells giving rise to mature adipocytes, makes it extremely challenging to characterise the cellular and molecular identity of fat depots. Single-cell RNA sequencing has recently enabled ground-breaking insights into the composition of complex cell populations. Our single cell-based dissection of adipogenic precursors revealed the existence of distinct cell sub-populations within the murine subcutaneous fat depot. We demonstrated that one of these populations, characterised by a high expression of F3 gene (encoding CD142), showed a completely non-adipogenic phenotype. Moreover, it revealed to be regulatory towards other adipose stem and precursor cells by supressing their ability to form adipocytes in a paracrine manner. These adipogenic regulatory cells, which we termed Aregs, proved to maintain their inhibitory properties in vivo and were shown phenotypically conserved in humans. We next established the robustness of Aregs as a novel cell sub-type in the context of various isolation strategies, the strength of the adipogenic cue and sex-based differences. Interestingly, we observed that Aregs isolated from the bourgeoning subcutaneous depots of new-born mice had high adipogenic propensity, suggesting that the appearance of classical phenotypical and functional properties of Aregs is development-dependent. In the light of the considerable implications of Aregs in adipose tissue composition and plasticity and, subsequently metabolic health, it is critical to understand the mechanism of their inhibitory nature. Integration of transcriptomic and proteomic datasets allowed us to identify a comprehensive set of highly specific candidates, which we validated in the context of Aregs’ identity and function. Our findings revealed a few potentially involved molecular actors. These include secreted factors CD142, GDF10 and MGP, which proved to be directly capable of inhibiting adipogenesis of Areg-depleted adipose stem and precursor cells, and transcription factors PKNOX2 and MEOX2, whose inactivation in Aregs compromised their ability to inhibit adipogenesis of co-cultured differentiating pre-adipocytes. Collectively, these molecules point out to a potential functional relationship of Aregs with vasculature as well as evoke a plausible association of Aregs’ phenotype to visceral-like non-adipogenic properties. We are now integrating these findings and completing them by investigating the transcriptional regulation and signalling pathways underlying the elusive mechanism of Aregs’ activity.