Université Côte d'Azur, INSERM, CNRS, iBV, Nice, France.
Methods Mol Biol. 2022;2454:495-507. doi: 10.1007/7651_2021_360.
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.
米色/亮色/棕色样脂肪细胞（BAs）分散在白色脂肪组织中，是对抗肥胖及其相关疾病的有前途的细胞靶点。然而，基于 BA 的肥胖治疗存在重大局限性，其中主要的局限性是成人中 BA 的稀有性以及缺乏相关的细胞培养条件来模拟 BA 的发育。在本章中，我们描述了人诱导多能干细胞衍生的 BA 祖细胞（hiPSC-BAP）在球体中自我组织的能力，以及一种在含有表达 UCP1 的细胞的 hiPSC 衍生的 3D 脂肪球中高效分化它们的方法。富含人类真皮微血管内皮细胞（HDMEC）的脂肪球可更好地模拟天然脂肪组织。为了观察 hiPSC 脂肪球表面的脂质滴积累、细胞外基质的组织和表面脂肪生成标志物的表达，我们详细介绍了如何结合油红 O 染色和免疫荧光染色进行观察。此外，为了全面观察 HDMEC 在内的 hiPSC 脂肪球中形成的预血管化网络，我们描述了一种方法，包括整个脂肪球固定、多色免疫染色、透明化和成像。
