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 Aregsâ 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 Aregsâ 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 Aregsâ activity.

The role and transcriptional regulation of FAM46A in adipocyte differentiation

Ann-Kristin Hov

Obesity is currently a leading global health concern. Treatments include undergoing a weight-loss regimen. However, successful weight-loss is variable, where the heritability of obesity is considered part of the cause. This patient variability to weight-loss was addressed by Carayol et al. (2017), where protein plasma level changes was associated to genetic variability and changes in BMI under a low-calorie diet intervention. A highly significant association region was found to regulate downstream gene FAM46A; coding for a nucleotidyl-transferase protein. Loss of function mutations in FAM46A have previously been discovered in multiple human diseases, such as osteogenesis imperfecta. However, there is a lack of understanding of FAM46Aâs function, especially in the context of adipose tissue and metabolism. This thesis aimed to follow up on the Carayol et al. study by investigating the transcriptional regulation of FAM46A and how the associated region is influencing this regulation, as well as how FAM46A is involved in adipose tissue function. When mapping the physical DNA interaction network around the associated region using chromosome conformation capture (3C) in human subcutaneous adipocytes (SGBS cells), no interactions between the FAM46A promoter and selected loci of the associated region were found. Interestingly, significant interaction peaks downstream of the promoter overlapped with regions of open chromatin and were enriched for transcription factor (TF) motifs that are involved in stem cell differentiation and TGFÎ² signalling. SMAD4, part of the TGFÎ² signalling pathway, binds to this region, indicating a potential regulator of FAM46A. To address FAM46Aâs potential role in adipocyte function, FAM46A expression was disrupted in vitro in differentiating adipocytes. Reduction of FAM46A expression in SGBS cells resulted in decreased expression of PPARG and CEBPA, the master regulators of adipogenesis, complemented by a reduction in lipid accumulation. Follow up studies in murine mesenchymal stem cells where Fam46a was over-expressed were controversial as this led to a decrease in adipogenic markers (Adipoq and PPARðŸ). Overall, disruption of FAM46A expression led to opposing changes in adipogenic potential, suggesting the requirement for a more stable cell system to study FAM46A in vitro. To further characterise the role of Fam46a, metabolic phenotyping of a Fam46a knockout (KO) mouse model under hyper-caloric stress was performed in collaboration with the IMG in Prague. This model revealed a significantly smaller mouse, and increased plasma alkaline phosphatase, as previously observed. When fed a high-fat diet, KO mice had reduced overall fat mass, improved glucose clearance and reduced adipocyte size in both subcutaneous and visceral white adipose tissue (subWAT/visWAT) depots. Transcriptomic analysis of these two depots revealed an up-regulation of genes expressed in ribosome related pathways in the subWAT, whereas the visWAT showed the up-regulation of fatty acid metabolism pathways, suggesting that the KO adipose tissue is more metabolically active. Other down-regulated pathways involved stem cell differentiation and BMP/TGFÎ²-signalling. In conclusion, this study found the possible regulation of the FAM46A promoter by TFs involved in adipogenesis regulation, such as SMAD proteins, as well as strengthening its possible functional involvement in TGFÎ² signalling by enrichment of this pathway in KO studies.

EPFL2021

Deciphering Metabolic Regulation of Hematopoietic Stem Cell Fate

Mukul Girotra

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. This unique characteristic makes them an ideal candidate for cell-based therapies to treat various hematological malignancies. Their extensive use in the clinic is often hampered due to insufficient number of cells obtained from donors. Countless attempts to expand HSCs in vitro have failed, primarily due to our inability to recapitulate key features of the native bone marrow microenvironment, termed niche, in a dish. The absence of important niche signals in vitro results in rapid proliferation of HSCs with a concomitant loss of their long-term multi lineage blood reconstitution potential. The niche in the bone marrow involves a highly complex network of physical and biochemical signals that, in concert with cell-intrinsic mechanisms, is believed to control HSC fate choices. Moreover, the hypoxic conditions in the niche presents an extreme metabolic environment, imposing HSCs to attain a distinct metabolic identity as compared to their differentiated progeny. However, despite decades of research it is currently very poorly understood how HSCs take the decision to either undergo self-renewal or differentiation. Insights into the mechanisms regulating HSC fate choices are key to design better strategies for HSC maintenance and expansion in vitro for use in clinical transplantations. The overall goal of this thesis is to employ innovative experimental tools to explore the role of metabolism in regulating HSC fate choices. In the first part of this thesis a versatile cell-tracking assay was developed to follow HSC divisions in vitro. A combination of cell tracking and immunostaining was used to systematically map phenotypic changes in HSCs up to four divisions, under defined culture conditions imposing specific fates. We found that the proportion of cells maintaining an HSC phenotype decreased with increasing number of cell divisions, supporting the notion that faster cycling results in HSC exhaustion. In the second part of this thesis, we for the first time identify a link between mitochondrial metabolism and HSC fate decision. Using flow cytometry and long-term blood reconstitution assays low mitochondrial activity was established as a reliable marker of functional HSCs, independent of their cell cycle state. Consequently, we could use this marker to reliable identify self-renewing HSCs from heterogeneous in vitro cultures. Strikingly, we found that HSC fate could be altered by artificially modulating their mitochondrial activity in vitro. These results suggest that mitochondrial activity is a determinant of HSC fate. The last part of this thesis describes an experimental paradigm to analyze in vivo niche-instructed fate choices in paired HSC daughter cells. Live single cell imaging revealed a significant increase in asynchronous divisions in niche activated HSCs compared to control cells, suggesting a possible involvement of niche-instructed asymmetric cell division program. Indeed, a significantly higher level of asymmetric gene expression was found in paired daughter cells arising from niche-instructed HSCs. This analysis led to the identification of 12 asymmetrically expressed genes, among them were key enzymes belonging to glycolytic and mitochondrial TCA cycle metabolic pathways. Altogether, this thesis successfully employed unique experimental strategies to provide an intriguing link between metabolism and HSC fate choices.

EPFL2015

Gene regulatory dynamics underlying brown fat cell differentiation

Rachana Narendra Pradhan

Adipose tissue is increasingly recognized as a dynamic, endocrine organ responsible for regulating metabolic homeostasis via the collective action of its two principal subtypes: white adipose tissue (WAT) and brown adipose tissue (BAT). Recently, the discovery of metabolically active brown fat reserves and brown in white fat (also known as "brite" or "beige" fat) in adult humans has prompted fundamental research to understand the development and function of brown adipocytes. The functional property of brown adipocytes is executed by the mitochondrial protein, Un- coupling Protein 1 (UCP1). UCP1 is responsible for uncoupling oxidative phosphorylation from ATP production thereby dissipating heat. The gene expression of Ucp1 is orchestrated by a multitude of transcriptional regulators and co-regulators that are distinct from those governing white adipocyte differentiation and function. However, in contrast to its counter- part, the white adipocyte, the gene regulatory mechanisms and transcriptional drivers of the brown adipocyte differentiation program remain elusive. Thus the focus of this thesis is to elucidate the gene regulatory network underlying brown fat cell differentiation and to decou- ple differentiation from the thermogenesis program using an integrative genomics approach. The main contributions of this work are as follows (1) construction of a comprehensive map integrating signaling, transcriptional and metabolic modules necessary for the activation of a brown adipocyte, (2) systematic, comprehensive analysis of transcriptional and chromatin state dynamics during brown fat cell differentiation leading to the identification of novel transcriptional regulators, (3) genomic characterization of the TF BCL6 during brown fat cell differentiation and (4) phenotypic characterization of a novel conditional BCL6 knockout mouse model.