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Abstract: SA-PO348

Benchmarking Mitochondrial Organization and Abundance in Organoids Against Fetal and Newborn Kidney Tissue

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine


  • Khan, Mohsina Anjum, Rogosin Institute, New York, New York, United States
  • Oxburgh, Leif, Rogosin Institute, New York, New York, United States

Proximal tubule (PT) epithelia are some of the most energy demanding cells in the body, and rely on mitochondria for a large variety of ATP-dependent functions. Fidelity of stem cell-derived kidney tissue depends on achieving similar mitochondrial function. Our previous investigations revealed a paucity of mitochondria in PT epithelia of human stem cell-derived organoids. In this study we benchmarked synthetic PT epithelia against natural PT epithelia, and used a screening approach to boost mitochondrial mass in organoid epithelia.


Mitochondria were localized by staining with anti-TOMM20, LTL and anti-HNF4A were used for PT. TMRM was used to identify energetically active mitochondria in kidney tissue.


Human fetal kidney (HFK) and newborn mouse kidneys were stained for mitochondria and PT. Newly formed PT stain weakly for HNF4A and LTL have characteristic apically arranged mitochondria. The mitochondria translocate basolaterally as these cells differentiate and mitochondrial mass increases several-fold, suggesting an abrupt activation of biogenesis. Live tissue staining of newborn mouse kidney with TMRM reveals undetectable mitochondrial polarization in apical mitochondria of nascent epithelial cells, while cells with basolateral mitochondria show strong mitochondrial polarization. Presumptive PT epithelia in stem cell-derived organoids have almost exclusively apically located mitochondria, suggesting that these cells may be functionally immature despite HNF4A and LTL staining. Single cell data from HFK and organoids were mined for mitochondrial biogenesis, mitophagy and mitocytosis pathways. Compounds selected based on this analysis were tested for their capacity to modify mitochondrial mass and function in a flow cytometry-based screening assay using organoids with fluorescent reporters for mitochondria and for PT. As expected, modifiers of the biogenesis regulator AMPK modulate mitochondrial mass, but we also found unanticipated effects of modulating LRRK2, which determines mitochondrial positioning within the cell.


Subcellular localization of mitochondria serves as a hallmark of epithelial cell functional maturity in human stem cell derived kidney organoids, and modulating mitochondrial mass is an important step in developing high-fidelity human kidney organoids.