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Abstract: PO1679

A Highly Efficient and Reproducible Differentiation Protocol for Induced Pluripotent Stem Cell-Derived Podocytes

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Kyrychenko, Sergii, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Gallagher, Justin Anthony, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Norman, Timothy A., Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Fanelli, Alyssa, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Pan, Xiang, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Fast, Eva, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Gallagher, Rachel, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Gustafson, Thomas A., Goldfinch Bio Inc, Cambridge, Massachusetts, United States
  • Lalioti, Maria, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
Background

Podocyte processes intertwine to create a slit diaphragm, which, when compromised leads to filtration dysfunction, proteinuria, and eventually to renal failure. It is critical to be able to study the disease and test therapeutic interventions in patient-derived cells, and assess genetic and environmental aspects. Current protocols for differentiation of iPSCs into podocytes (iPodos) suffer from a lack of podocyte maturity or low reproducibility. Our goal was to test and optimize multiple protocols to establish a more translatable, physiologically relevant, and reproducible method.

Methods

We compared two distinct published protocols [Ciampi 2016; Musah 2018]. Additional conditions were tested, including varying the extracellular matrices, media, and length of differentiation. Podocyte signature was evaluated by IF, flow cytometry, and Nanostring analysis. For models of injury, we utilized protamine sulfate (PS) or puromycin aminonucleoside (PAN) treatment. A mouse podocyte cell line was used as a control.

Results

Both protocols generated iPodos with similar efficiency, as measured by synaptopodin, nephrin and podocin staining. iPodos generated from protocol-1 could be maintained in culture up to 14 days but remained relatively immature, based on the expression of collagen α1α2α1(IV) and lack of α3α4α5(IV). Response to PS and PAN treatment was variable compared to mouse podocytes. Altering the matrix from collagen to laminin did not improve reproducibility. iPodos from protocol-2 developed more filopodia and complex cell-cell junctions and appeared more homogeneous, with extended survival up to 4 weeks post-differentiation. PS treatment induced a significant and reproducible dose- and time-dependent decrease in synaptopodin expression, and a more robust accumulation of phalloidin aggregation. Both effects were effectively prevented by cyclosporin A, a calcineurin inhibitor, in a similar manner as in mouse podocytes. iPodos from protocol-2 also showed a more consistent dose-dependent response to PAN injury.

Conclusion

We achieved a more robust and translatable iPodos platform utilizing human iPSCs. Patient-derived iPodos will be an invaluable tool to enable a precision medicine-based approach to validate new therapeutic approaches for podocytopathy-driven kidney disease.

Funding

  • Commercial Support