A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells.

Beige/brite/brown-like adipocytes (BAs), dispersed in white adipose tissue, represent promising cell targets to counteract obesity and associated diseases. However, there are major limitations for a BA-based treatment of obesity, among which the main ones are the rareness of BAs in adult humans and the lack of a relevant cell culture condition for modeling the development of BAs. We describe in this chapter the capacity of human induced pluripotent stem cells-derived BA progenitors (hiPSC-BAPs) to self-organize in spheroids and a method for their differentiation at a high efficiency in hiPSC-derived 3D adipospheres containing UCP1-expressing cells. Enrichment of adipospheres with human dermal microvascular endothelial cells (HDMECs) allows to better mimic native adipose tissue. To observe the accumulation of lipid droplets, organization of the extracellular matrix and expression of adipogenic markers on the surface of hiPSC-adipospheres, we detail how to combine Oil Red O staining with immunostaining both imaged by fluorescence microscopy. Furthermore, to have a global view of pre-vascularized network formed by HDMECs inside of hiPSC-adipospheres, we describe a method which consists of the whole adipospheres fixation, multicolor immunostaining, clearing, and imaging.