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Abstract: TH-PO630

Brain Derived Neurotrophic Factor (BDNF) Inhibition Dramatically Reduces Off-Target Cells in Kidney Organoid

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

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

Authors

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


Kidney organoids differentiated from pluripotent stem cells hold great promise for disease modeling and ultimately as a source of replacement tissue, however we have shown that all protocols also generate substantial off-target cell types, primarily neurons. To eliminate off-target cells without altering kidney cell maturation, we performed single cell RNA-seq and analyzed ligand - receptor pairs expressed exclusively within the neuronal lineages. We identified 19 receptors with 24 cognate ligands, and determined that the neuron survival factor BDNF and its receptor NTRK2 was expressed exclusively in the neuronal lineage. We tested whethyer inhibition of BDNF-NTRK2 signaling during organoid differentiation would reduce neuronal populations without altering kidney differentiation.

Methods


A range of doses of the NTRK2 inhibitor K252a was screened as well as different time schedules during organoid differentiation from human induced pluripotent stem cells. We assessed marker expression by qPCR and immunofluorescence, as well as scRNA-seq on control and K252a-treated organoids. We sequenced 3,314 cells to a final read depth of 50K mapped reads/cell with 3,056 transcripts and 1,524 unique genes detected per cell.

Results

Out of 16 different dosing and timing protocols tested, we found that 250 nM, of K252a added at day 12 did not alter nephron formation according to marker expression by qPCR and immunofluorescence. By contrast, neuronal markers were reduced by almost ten-fold. scRNA-Seq of control vs. K252a-treated organoids showed that all major kidney cell types remained present with K252a treatment, but the neuronal lineage was reduced by over 90%, from 29.8% of total organoid cells before treatment to 2.1% after treatment.

Conclusion

We employed a rational approach based on organoid scRNA-seq to identify BDNF signaling as a potential neuronal survival pathway contributing to the growth of off-target cells in during kidney organoid differentiation. Inhibition of this pathway reduced off-target cells by more than an order of magnitude without affecting kidney differentiation, illustrating the power of scRNA-seq to improve organoid differentiation protocols.

Funding

  • NIDDK Support