Abstract
The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.
Highlights
The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis
Several groups have reported the generation of three-dimensional self-organizing kidney organoids structures from iPSC19–23. induced pluripotent stem cells (iPSC)-derived renal organoids contain nephron-like structures, including glomerular structures, proximal tubular cells, loop of Henle cells, distal tubular cells and in some protocols collecting duct structures, in a highly structured and organized way
Megalin is a large endocytic receptor that is highly expressed in the renal proximal tubule and together with cubilin it is responsible for the reabsorption of filtered proteins and several other ligands from the glomerular filtrate[33]
Summary
The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). IPSC-derived renal organoids contain nephron-like structures, including glomerular structures, proximal tubular cells, loop of Henle cells, distal tubular cells and in some protocols collecting duct structures, in a highly structured and organized way. We developed a rapid and simple protocol to drive iPSC into proximal tubular like cells (PTL) within 14 days using temporal addition of a combination of small molecules and growth factors. Some functional assays were used for characterization including albumin uptake, ABCB1 mediated extrusion and hormonal sensitivity
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