Abstract
Background and Aims
The smallest filtration unit of the kidney, the glomerulus, is composed of capillaries formed by glomerular endothelial cells (GEC), glomerular basement membrane and podocytes. Furthermore, glomerular mesangial cells (GMC) between the capillary loops give structural support. It is known that loss of podocyte foot processes is leading to a dysfunctional glomerular filtration barrier and is seen in glomerular diseases like focal segmental glomerulosclerosis and other podocytopathies.
Indeed, it is hard to investigate podocytes in culture because podocytes are terminally end-differentiated cells that do not proliferate, lack foot processes and cell type-specific markers. Although conditionally immortalized human podocytes regained the capacity of proliferation, marker expression and behaviour differ between cell lines. The overarching aims of this study are to generate a 3D glomerular co-culture model that better reflects the in vivo phenotype of glomerular cell types. We want to investigate cell-cell contact, interaction and communication and extracellular matrix production in 3D glomerular co-cultures. Furthermore, patient-derived hiPSC-podocytes will be used in the glomerular co-cultures to investigate podocyte disease in a personalized manner and to identify potential therapeutic targets.
Method
The hanging droplet method was used to produce 3D glomerular spheroids. Therefore, human differentiated immortalized podocytes, human GECs and human GMCs were inserted in a medium-droplet hanging from the lid of a petri dish and harvested at different time points. Fluorescent cell lines of the different glomerular cell types were tracked in a time-lapse experiment to study if cell attachment and spheroid formation undergoes a specific order and structure. Glomerular spheroids were further characterized regarding the expression of podocyte-specific markers and extracellular matrix synthesis by immunohistochemistry, electron microscopy and qPCR and were compared to human cells isolated from glomeruli. Furthermore, scRNA-sequencing analysis was performed in 2D mono-cultures of human GECs, GMCs and immortalized podocytes and on 3D co-cultures to see if this change in culture conditions leads to transcriptomic alterations. For the generation of patient-derived podocytes, skin fibroblast of patients with podocyte mutations (INF2 mutation and WT1 mutation) and from healthy controls were reprogramed in iPSCs and differentiated into podocytes that keep the patient’s mutation.
Results
First time-lapse experiments of glomerular co-cultures showed that human podocytes and human glomerular endothelial cells attach to each other (Fig. 1a) and histological sections revealed that the glomerular spheroids are encapsulated by a monolayer of cells (Fig. 1b). SEM allowed ultrastructural characterization of the 3D spheroid-like structures (Fig. 1c). TEM revealed cell protrusions of podocytes that were not seen in monocultures (Fig. 1d, e). We could also demonstrate production of extracellular matrix by the cells (Fig. 1f). Immunohistochemistry and qPCR showed expression of collagen-IV and laminin. During the reprogramming of patient-derived fibroblasts, size of the generated hiPSC decreased and the nuclei to cell body ratio increased. HiPSCs formed colonies with distinct boarders and the proliferation rate increased. Furthermore, generated hiPSC showed similar gene expression of pluripotency markers compared to a commercial hiPSC control cell line and podocytes derived from these hiPSC expressed synaptopodin (Fig. 2).
Conclusion
We generated a 3D co-culture model that better represents the complexity of the glomerulus ex vivo. It is indicated that this model provides better physiological conditions. By an insertion of patient-specific hiPSC-derived podocytes in the 3D co-culture we will investigate glomerular diseases in a personalized manner in the future.
Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis