Abstract: FR-OR095
Organelles in Organoids: Live Imaging of Human Mini-Kidneys Using Targeted Tracers Reveals Dynamic Intracellular Responses to Kidney Injury
Session Information
- Stem Cells to Organoids: How We Get There
November 03, 2017 | Location: Room 385, Morial Convention Center
Abstract Time: 04:30 PM - 04:42 PM
Category: Developmental Biology and Inherited Kidney Diseases
- 402 Stem Cells
Authors
- Churchill, Angela J, University of Washington, Seattle, Washington, United States
- Himmelfarb, Jonathan, Kidney Research Institute, Seattle, Washington, United States
- Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
Background
Cellular subcompartments, known as organelles, play critical roles in cell biology, but little is known about the dynamics of organelles in complex tissues and organs. Mini-kidney organoids derived from human pluripotent stem cells (hPSCs) are complex structures in vitro that resemble nephrons and can be genetically modified to study regeneration and disease. We used human mini-kidneys to model organelle dynamics in complex tissues and acute kidney injury.
Methods
CRISPR-modified hPSC lines encoding fluorescently-tagged microtubules, nuclear envelopes, mitochondria, or desmosomes were differentiated into kidney organoids. Whole organoids were imaged every 5 minutes with a spinning disk confocal microscope over the course of 16 hours, and organelle dynamics were quantified. Organoids were physically disrupted or chemically treated to simulate kidney injury. Organelle distributions in tubule and podocyte populations were compared to fixed organoids and kidney tissue in vivo by fluorescence and electron microscopy.
Results
Live movies of human kidney organoids captured tubule movements, cell rearrangements, and mitotic events at high temporal and spatial resolution. Organelle distribution in diverse nephron segments closely resembled kidney tissue in vivo. Organelles exhibited a range of behaviors, from highly dynamic microtubules and nuclear envelopes, to relatively static desmosomes forming regularly-spaced foci. Quantitative time-lapse analysis revealed extended cell division duration and fragmented mitochondrial morphology under acute kidney injury conditions.
Conclusion
Live imaging of fluorescently-labeled kidney organoids reveals that organelle distributions and dynamics depend on nephron segment identity and environmental cues, approximating tissues in vivo. Furthermore, kidney injury induces changes in cell cycle length and mitochondrial structure, recapitulating and revealing aspects of the tubular injury/repair process. The study of organelles in organoids provides a new framework for elucidating the intracellular mechanisms underlying kidney injury, disease, and regeneration.
Funding
- NIDDK Support – Northwest Kidney Centers (unrestricted gift)