Reconstructing human Brown Fat developmental trajectory in vitro

Brown adipocytes represent a specialized type of mammalian adipocytes able to uncouple nutrients catabolism and ATP generation to dissipate energy as heat. They play an important role in mammals, allowing non-shivering thermogenesis to regulate body temperature in response to cold exposure. In humans, the brown fat tissue is dispersed in small depots found throughout the neck and trunk region. Increasing brown fat activity either with drug treatment or with cell therapy approaches are considered as potential approaches for the treatment of metabolic syndrome and obesity. The recent development of in vitro differentiation strategies relying on human pluripotent stem cells (hPSCs) offers in theory the possibility to produce unlimited amounts of BAT. A strategy efficiently applied to several tissues is to recapitulate step by step the development of the tissue of interest by exposing hPSCs to the signaling cues used during normal embryonic development. However, this strategy has proven difficult to implement for brown fat as the development of this tissue is poorly characterized. Here, we first used single cell RNA sequencing to characterize the development of interscapular brown fat in mouse. Our analysis identified a previously unrecognized population of brown adipocytes precursors characterized by expression of the transcription factor GATA6. We showed that this precursor population can be efficiently generated from hPSCs by modulating the signaling pathways identified our transcriptomic analysis in paraxial mesoderm precursors differentiated in vitro. These precursors can in turn be efficiently converted into functional brown adipocytes which can respond to adrenergic stimuli by increasing their metabolism resulting in heat production. Wildtype pluripotent stem cell line was differentiated into brown adipocytes as described in Rao et. al. iPSCs, terminally differentiated iPSC-derived brown adipocytes and human fetal brown adipose tissues were analyzed to investigate the characterstics of developing brown adipocytes in vitro and in vivo. To evaluate the differentation protocol performance we compare our terminally differentiated iPSC-derived brown adipocytes with the ones produced by Carobbio et al. (10.1016/j.stemcr.2021.01.013, GSE168387) and Zhang et al (10.1016/j.stem.2020.07.013, GSE131169). Moreover, to demostrate the specificity of the differentiation, we compared these samples to iPSCs differentiated to a late myogenic stage previously generated by us as reported in Al Tanoury et al. (https://doi.org/10.1073/pnas.2022960118, GSE164874).