FC042: Generation of Patient-Specific Hipsc-Podocytes to Study Phenotype-Specific Alterations and Test Therapeutic Targets Ex Vivo
Authors: Rose Victoria , Sopel Nina , Ohs Alexandra , Warnecke Christina , Schiffer Mario , Müller-Deile Janina 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Genetic focal segmental glomerulosclerosis (FSGS) is related to irreversible damage of the filtration barrier and podocyte effacement. More than 40 genes with mutations, involved in slit diaphragm formation, actin cytoskeleton synthesis, podocyte adhesion or metabolic pathways, are known to induce FSGS. Up to 17% of all cases of autosomal-dominant FSGS are associated with mutations in the inverted formin 2 (INF2). To study the patient-specific alterations of the podocyte phenotype and to investigate potential treatment effects on INF2 mutant cells ex vivo we generated human induced pluripotent human stem cell (hiPSC)-derived podocytes from a patient holding an INF2 mutation and a healthy control. Wild-type and INF2 mutant hiPSCs-podocytes were compared regarding morphology, podocyte-specific marker expression and actin cytoskeleton and treated with different immunosuppressive drugs and actin modulating agents to analyse cell response.

METHOD
To generate hiPSCs-podocytes, fibroblasts were outgrown from a skin punch biopsy, reprogrammed into hiPSCs and subsequently stepwise differentiated into podocytes. The generated hiPSCs-podocytes were characterized by bulk sequencing, qPCR, western blot analysis and immunofluorescence staining regarding morphology (e.g. cell size and filopodia length), podocyte-specific markers and actin cytoskeleton development. Moreover, wild-type and INF2 mutant cells were treated with different stimuli [e.g. Solu-Decortin H (SDH), tacrolimus, Bis-T and ROCK inhibitor Y-27 632] and compared regarding their response. Especially filopodia length and cytoskeletal alterations were observed by quantification of total fluorescence of F-actin and cortactin and classified regarding actin filament thickness (type-A: thick bundles, type-B: thin filaments no bundles and type-C: disrupted cytoskeleton without filaments).

RESULTS
Marker genes regarding pluripotency and podocytes were compared at different timepoints during differentiation of hiPSCs into podocytes and compared to human immortalized podocytes. For example, synaptopodin gene expression increased during differentiation and was highest in terminally differentiated hiPSC-podocytes. Phenotypical characterization showed a distinct network of filopodia in wild-type hiPSCs-derived podocytes. Differentiated cells from healthy controls were compared to patient-specific podocytes holding an INF2 mutation, generated with the same protocol. Patient-specific cells showed no distinct net of foot processes and a fragmented actin cytoskeleton. Untreated wild-type cells mostly represented classification type-A actin whereas untreated INF2 mutated cells mostly showed type-B and -C actin filaments. First experiments indicated that treatment of INF2 mutated cells with Y-27 632 lead to an even more impaired phenotype whereas stimulation with SDH improved cytoskeleton rearrangements with up to 20% of type-A actin. The small acting modulating agent Bis-T did not alter the actin fibers in INF2 mutated hiPSC-podocytes but increased total fluorescence of cortactin. Tacrolimus had no effect.

CONCLUSION
Podocytes could be generated from hiPSCs reprogrammed from a patient's skin punch biopsy and enabled to study phenotype alterations of patient-specific podocytes holding an INF2 mutation ex vivo with the potential to test medications and find new potential therapeutic targets in the future.