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Abstract: SA-OR40

Glomerular 3D Co-Culture to Study Podocyte Disease Ex Vivo in a Personalized Manner

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Rose, Victoria, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Sopel, Nina, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Ohs, Alexandra, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Warnecke, Christina, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Schiffer, Mario, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Müller-Deile, Janina, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
Background

As a cause for glomerular disease, podocyte damage and related changes in the glomerular filtration barrier are typical findings. However, podocyte cell culture is challenging due to the inability of terminally end differentiated primary podocytes to proliferate and due to altered morphology and expression of cell-specific markers in immortalized podocytes. Besides, classical mono-cultures limit paracrine cell-cell-contact and communication by 2-dimensionality. To investigate cell-cell-interaction and to improve cell culture conditions we want to generate a 3D co-culture model of glomerular cells. Moreover, we want to study podocyte disease by personalizing the 3D co-culture model using patient-derived podocytes.

Methods

In order to generate 3D glomerular spheroids, immortalized differentiated podocytes, glomerular endothelial cells and mesangial cells were co-cultured in a hanging media droplet or via agarose microwells. Time laps experiments displayed spheroid formation and spheroids were characterized regarding extracellular matrix proteins and cell-specific marker expression by qPCR, histological sections, immunostainings and electron microscopy. Patient-specific podocytes and podocytes from healthy controls were generated from skin fibroblasts via reprogramming into human induced pluripotent stem cells (hiPSCs) and subsequent differentiation into hiPSC-podocytes.

Results

Spheroid formation could be imaged in time laps experiments and sectioning showed encapsulation of the spheroid. SEM could display the ultrastructure and produced extracellular matrix and collagen IV and laminin were quantified by immunostaining and qPCR. In the 3D model podocytes formed protrusions, shown by TEM, that were not seen in 2D. After reprogramming of fibroblasts, generated hiPSCs showed an increased expression of common pluripotency markers. Subsequently differentiated cells were positive for podocyte specific markers and developed distinct primary and secondary foot processes.

Conclusion

3D co-culture is a model enabling paracrine cell-cell-contact and communication and providing better physiological conditions. Patient-specific hiPSC derived podocytes have the potential to personalize the 3D co-culture.