Abstract
Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation.
Highlights
In vivo formation of foot processes in podocytes is characterized by highly arborized processes from the cell body and the interdigitation of neighboring processes at high confluence [1,2]
There have been no reports on the successful induction of process formation in high-confluence in vitro cultures of podocytes induced from pluripotent stem cells [4e6]
This study provides the new insight that the combination of serum depletion, the scaffold of L521, and the inhibiting Rho kinase cascade promotes process formation in conditionally immortalized podocytes
Summary
In vivo formation of foot processes in podocytes is characterized by highly arborized processes from the cell body and the interdigitation of neighboring processes at high confluence [1,2]. The reproduction of podocyte foot process formation in vitro would be significant for the development of a powerful evaluation system for drug discovery research. It would allow the evaluation of the drug cytotoxicity of candidate compounds in podocytes by phase contrast observation. There have been no reports on the successful induction of process formation in high-confluence in vitro cultures of podocytes induced from pluripotent stem cells [4e6]. In 2018, Yaoita et al reported a novel culture method to reproduce the interdigitating process formation of podocytes, at 100% confluence, whereas until process induction methods were only successful under sparse cell density conditions [7e10]. The culture method developed by Yaoita et al has high novelty, the applicable cell source is limited to rat primary podocytes; the method could not be applied to conditionally immortalized podocytes [9]
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