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Abstract: FR-PO298

A scRNAseq Approach to Define Early Events in Cyst Formation and Stepwise Transformation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1101 Genetic Diseases of the Kidneys: Cystic


  • Clerici, Sara, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Spies, Daniel, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy

We have previously generated and described a mouse model carrying kidney-specific inactivation of the Tsc1 gene using a Cadherin16-Cre line (KspCre). The peculiarity of the Tsc1f/f;KspCre model is that it develops a slowly progressive cystic kidney disease model, triggered by mTORC1-dependent downregulation of polycystin-1 (PC-1), which progressively transform into cystadenomas and carcinomas (Pema et al, Nat Comms, 2016; Drusian et al, Cell Reports, 2018). As they transform, the renal lesions lose the expression of tubule-specific markers.


In order to track all KO cells within the kidney, we generated mT/mG;Tsc1f/f;KspCre mice. Indeed, only KspCre expressing cells would excise mTomato (mT) in favor of mGFP (mG), while the surrounding tissue ubiquitously expresses mTomato. Mutants were characterized through histological and immunofluorescent analyses. Single cell suspension from control and mutant kidney was obtained through mechanical and enzymatic dissociation with Miltenyi kits and validated by flow cytometry confirming viability and aggregates requirements for single-cell sequencing.


Histological characterization of mT/mG;Tsc1f/f;KspCre confirmed the progression from cysts (at P20) to cystadenomas and carcinomas (at P50 and P80) with the previously reported frequency. Furthermore, careful characterization of the mT/mG;Tsc1f/f;KspCre revealed that cystic structure, papillae, cystadenomas and carcinomas displayed mG-positivity in all lesions of all stages of the phenotype. A dissociation protocol was set up obtaining 75% viability in both control and mutant kidneys. Flow cytometry confirmed the 25-30% mG+ cells expected in controls, which expanded to 65% in mutant kidneys, in line with the robust proliferation observed in these mutants. scRNAseq analysis of control mT/mG and mT/mG;Tsc1f/f;KspCre kidneys is being set-up at different time points (P10, precystic; P20 cystic; P50 and P80 transformed).


We generated a mouse model allowing to separate and study Tsc1 mutant cells and the surrounding healthy tissue in a mouse model of progressive cysts and renal cell carcinoma (RCC) development. Single-cell sequencing analysis will help defining early versus late events in cystogenesis and in transformation towards RCC.


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