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Abstract: PO1262

TSC2 Loss-of-Heterozygosity Mutations Drive Cystogenesis in Kidney Organoids Generated from Tuberous Sclerosis Complex Patient-Derived Induced Pluripotent Stem Cells

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Author

  • Lemos, Dario R., Brigham and Women's Hospital, Boston, Massachusetts, United States
Background

Kidney cysts are the second most common renal manifestation of TSC, accounting for 50% of kidney lesions. The genetic mechanisms driving TSC-associated cystic kidney disease remain poorly understood.

Methods

We induced nephric differentiation of a series of TSC patient-derived iPSC lines that included a line carrying a heterozygous microdeletion in the TSC2 locus (TSC2+/-), and a TALEN-engineered isogenic cell line carrying microdeletions in both TSC2 alleles (TSC2−/−).

Results

Analysis of Day-21 two-dimentsional cell cultures showed that nephrons derived from TSC2+/- and TSC2+/+ hiPSCs presented normal morphology, and were sequentially segmented into distal tubules expressing cadherin 1 (CDH1), proximal tubules containing brush borders labeled by lotus tetranoglobus lectin (LTL) and glomeruli containing podocytes expressing podocalyxin 1 (PODXL). By Day-21 of differentiation TSC2-/- hiPSC-derived kidney tissues showed cavitated structures resembling cysts. Analysis of nephron segments showed positive signal for both CDH1 and LTL, indicating the cyst lining could comprise distal tubule and/or proximal tubule cells. The observed frequency of cyst formation was ~4.7 cysts/well for TSC2-/- cultures, compared with 0 and ~0.5 cysts/well for TSC2+/+ and TSC2+/+ cultures, respectively. Of note, 2-D cyst-like structures developed in a spontaneous manner, that is without addition of cAMP modulators. Analysis of the three-dimensional anatomy of TSC2-/- organoids also showed that while individual tubule segments were involved in cyst lining, in certain cases a combination of both proximal and distal tubule cells were detected, suggesting improper segmentation mechanisms in the absence of TSC2. While single-cell lining layers were associated with well-defined tubule segments, mixed-cell lining regions were multi-layered, resembling the columnar epithelium observed in the renal cysts of TSC patients. The distribution of LTL staining, in proximal tubules of TSC2-/- organoids indicated loss of cell polarity in contrast to the polarized cells observed in TSC2+/- tubules. Cyst formation in TSC2-/- organoids did not involve loss of polycystin 1 (PKD-1) expression.

Conclusion

Our kidney organoid models provide evidence supporting LOH events resulting in complete TSC2 inactivation in nephron tubule cells as a mechanism driving kidney cyst formation in TSC.

Funding

  • NIDDK Support