Adipocyte development and differentiation play an important role in the origin of obesity and many related diseases such as type 2 diabetes and cancer. Fat tissue has a remarkable capacity for growth. It can expand in two ways: by increasing the size of the individual fat cells (adipocytes) and by expanding the pool of fat cells from progenitor fat stem cells through adipogenesis.
The origin of the body's fat cells has been poorly understood and research has been complicated by the fact that fat tissue is heterogenic, unstructured and contains many cell types other than adipocytes. In addition, adipocytes at different locations around the body derive from different precursor cells.
Historically, classical approaches such as FACS analysis, which separate cells based on specific cell-surface markers and employing Cre recombinase models, were utilized to study adipose tissues. These approaches, however, allowed only for isolation of pools containing mixtures of adipocyte stem and progenitor cells (ASPCs).
To identify molecular profiles of stems cells within the pools, Schwalie et al., used the resolving power of single-cell transcriptomics.
This study was performed on a pre-selected subpopulation of Lin-(CD31-CD45-TER119-) CD29+CD34+SCA1+ ASPCs cells from the subcutaneous stromal vascular fraction of mouse fat tissue.
208 high-quality purified cells were isolated, which were subjected to the single-cell RNA sequencing to determine which genes were expressed in each cell.
Using bioinformatics analysis, cells were clustered according to their gene-expression profiles. The authors identified three different subpopulations within ASPC pool (designated P1, P2 and P3), characterized by the expression of hundreds of genes.
Virtually all cells from P1 pool scored high on the stem cells scale whilst only the P2 fraction showed enrichment in genes for known adipogenesis-related functionality, including the adipogenic master regulator Pperg and Fabp4.
Interestingly, P3 pool, which made up only about 10% of the total isolated cell population, showed a distinct molecular profile from the other two pools.
Surface proteins markers for the individual populations were identified, Cd55 and Il13ra1 for P1, Aoc3 (VAP1) and Adam12 for P2, and F3 (CD142) and Abcg1 for P3.
To assign adipogenic functionality to the three major subpopulations, new cells were isolated by FACS and validated by qPCR for their specific markers. Cell populations were induced to differentiate in vitro with a white fat differentiation cocktail and lipid accumulation was quantified.
The P2 population had no significant difference in lipid droplet accumulation to ASPCs. The P1 population had increased accumulation, suggesting more differentiated cells, confirmed by a corresponding increase in adipogenic markers.
P3 cells had a lower level of fat droplet accumulation that corresponded with decreased adipocyte marker expression suggesting that they did not form mature adipocytes when induced to differentiate in a cell-culture dish. Most importantly, removing P3 cells from the ASPC pool improved the ability of the other cells in the dish to differentiate into adipocytes, suggesting that these cells inhibit adipogenesis.
Further analysis of this novel low-abundance group, which was named “Adepogenesis-regulatory cells” (Aregs) using transcriptomic profiling demonstrated low expression of adipogenesis-associated genes such as Pparg, Fabp4, Lpl and Fabp12. Genes associated with blood vessels and especially hedgehog signaling were expressed at high levels.
Transwell experiments confirmed that the inhibitory effects of Aregs on adipogenesis were mediated by a paracrine mechanism and knockout of candidate genes by RNAi implicated Rtp3 and, to a lesser extent, Spink2 and Vit in this signaling process.
Matrigel-containing cells implanted into mouse abdomen demonstrated that SVF and isolated ASPCs lacking Aregs resulted in more adipocytes compared total SVF and ASPCs. The authors also observed that obese ob/ob mice have significantly more Aregs than lean mice suggesting a role for Areg in adipogenesis in vivo.
Aregs staining by IHC demonstrated a perivascular localization linking them spatially to blood vessels, consistent with the gene expression data.
The authors repeated a number of these experiments using human cells and demonstrated that Aregs exist in human fat and a paracrine signal was also detected implying that fat-development mechanisms are conserved between mice and humans.
In summary a single-cell transcriptomics-based approach using a mouse model revealed three different adipocyte progenitor cell populations. The largest one showed enrichment of adipogenic stem cell makers such as CD34 and high adipogenic differentiation capacity in vitro, while the smallest CD142+ subpopulation (Aregs) suppressed adipocyte formation in vitro and in vivo.
The use of state-of-the-art technology for single-cell gene expression profiling has proven to be a powerful strategy for identifying new cell populations with distinctive genetic profiles and their role in modulating plasticity of fat tissue during its formation and maintenance. This research sheds a new light into origins of obesity and obesity-related conditions.