Polarized Distribution of Lipid Droplets with Long Acyl Chains and Unsaturation are Hallmarks of Human Intestinal Enteroid Differentiation

Cell polarization and differentiation require increased energy mobilization and cell membrane synthesis, whereby mitochondria and lipid droplets (LDs) play key roles. However, how these metabolic organelles organize at the subcellular level to efficiently meet energy demands in human intestinal organoids is unclear. To address this, we introduce coherent anti-Stokes Raman scattering (CARS) microscopy multiplexed with confocal fluorescence microscopy to spatially map LDs and mitochondria throughout cell differentiation in human intestinal enteroids. The results show an overall decrease in LDs over time, though less pronounced for cells positive for proliferation or stemness markers. The LD depletion was observed in the apical region, resulting in a polarized distribution to the basal side. A similar mitochondrial polarization pattern was also observed in differentiated enteroids. Spectral CARS further shows that LDs postdifferentiation contain lipids with signatures of longer acyl chains and a higher degree of unsaturation. These observations demonstrate that polarized metabolic and lipid supply infrastructures are formed to support intestinal cell differentiation in organoid cultures.