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Abstract: TH-PO636

Fluorescent Reporters in Kidney Organoids Generated with Cheap Mini-Bioreactors for High-Throughput Modeling of Kidney Fibrosis

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Sun, Zhao, Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
  • Uchimura, Kohei, Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
  • Wu, Haojia, Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
  • Donnelly, Erinn L., Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
  • Humphreys, Benjamin D., Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
Background

Kidney organoids from human pluripotent stem cells (hPSCs) provide a unique opportunity to study human kidney development and kidney diseases. Current protocols are low throughput, generate immature and off-target cell types, and are costly. We tested the ability of spinning bioreactors to overcome these limitations.

Methods

We 3D-printed spinning mini-bioreactors that fit onto 12-well plates, as described (Qian et.al. Cell, 2016). We adapted the Takasato organoid differentiation protocol to the spinning mini-bioreactors by initially differentiating hPSC in a mono-layer for 7 days, followed by low-attachment plate culturing to generate spheroids for another 5 days, and then transferring the spheroids to a mini-bioreactor for maturation for 7- 42 days. We optimized the throughput and characterized resulting kidney organoids by qPCR, immunofluorescence and single cell RNA-seq (scRNA-seq). Finally, we generated and validated three novel fluorescent reporter hPSC lines relevant to the study of fibrosis in kidney organoids (Fibronectin1-GFP, Col1a1-GFP and Gli1-turboGFP).

Results

One 12-well plate could generate 200-600 kidney organoids, representing a more than 100 – fold increase compared to the original Takasato protocol. scRNA-seq analysis revealed that when compared to the standard organoid protocol using a transwell plate, the kidney organoids generated with the mini-bioreactor exhibit more mature nephron structures (including ureteric bud), and more closely resemble the adult human kidney by Pearson correlation. We validated that Fibronectin1-GFP, CollA1-GFP and Gli1-turboGFP all express in the correct stromal cell type in mini-bioreactor-derived organoids. Furthermore, kidney organoids generated by the mini-bioreactor method mount a fibrotic response when treated with 20 µM cisplatin for 2 days by using RPMI medium for organoid differentiation.

Conclusion

We used a 3D-printed spinning mini-bioreactor to increase kidney organoid throughput by at least 100-fold. The protocol is cheaper and results in better differentiation of nephron structures when compared to the standard protocol using a transwell plate. We have generated and validated novel fluorescent reporter hPSC lines relevant to studying fibrosis. This work will accelerate adoption of kidney organoid protocols for the research community.

Funding

  • NIDDK Support