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

Area of Science:

• Cell Biology
• Stem Cell Research
• Metabolic Diseases

Background:

• Beige/brite/brown-like adipocytes (BAs) are key targets for combating obesity.
• Limited BAs in adults and lack of suitable culture models hinder BA-based therapies.
• Human induced pluripotent stem cells (hiPSCs) offer a potential source for BA progenitors.

Purpose of the Study:

• To describe the self-organization of hiPSC-derived BA progenitors (hiPSC-BAPs) into spheroids.
• To present a method for efficient differentiation into hiPSC-derived 3D adipospheres with UCP1 expression.
• To detail techniques for characterizing these adipospheres, mimicking native adipose tissue.

Main Methods:

• hiPSC-BAPs self-organization into spheroids.
• High-efficiency differentiation into 3D adipospheres containing UCP1-expressing cells.
• Enrichment with human dermal microvascular endothelial cells (HDMECs) for tissue mimicry.
• Oil Red O and immunostaining for lipid droplets and adipogenic markers.
• Multicolor immunostaining, clearing, and imaging for vascular network visualization.

Main Results:

• hiPSC-BAPs successfully self-organized into spheroids.
• Efficient differentiation into 3D adipospheres expressing UCP1 was achieved.
• Adipospheres enriched with HDMECs better mimicked native adipose tissue.
• Combined staining and microscopy visualized lipid accumulation, ECM, and adipogenic markers.
• Advanced imaging techniques revealed pre-vascularized networks within adipospheres.

Conclusions:

• hiPSC-derived 3D adipospheres provide a robust model for studying beige adipocyte development.
• This model system facilitates the investigation of obesity and related metabolic diseases.
• The described methods enable detailed characterization and visualization of engineered adipose tissue.