Abstract 2051 A CTNS Mutation Alters the differentiation of Human-induced Pluripotent Stem Cells (hiPSCs) into Renal Proximal Tubules

Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). It is not understood how a defect in the lysosomal cystine transporter ultimately results in malfunctioning of the renal proximal tubule. Here we examine the hypothesis that the malfunctioning of the RPT in cystinosis arises from defective differentiation. In order to examine this hypothesis, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts derived from an individual with infantile cystinosis, as well as a normal individual, as described by Okita, K. et al. (Nat. Method (2011) 8, 409). Fluorescence microscopy was employed to examine a) the expression of pluripotent markers in the hiPSCs, b) the embryoid bodies, derived from the hiPSCs as described by Lin, Y. & Chen, G. (2014, doi/10.3824/stembook.1.98.1), as well as c) the organoids which formed from the hiPSCs following a protocol which induces the differentiation of the hiPSCs into RPTs, as described by Lam, A. et al. (JASN (2014) 25,1211). Our results indicate that both the cystinotic and normal hiPSCs are pluripotent, as indicated by the expression of pluripotent markers, including SSEA4, Oct4, SOX2 and TRA-1-60. Pluripotency was also indicated by the ability of normal and cystinotic hiPSCs to form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to the differentiation regime of Lam et al. that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). However, the formation of tubules with lumens was impaired in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an alteration in differentiation and/or other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death. This work received support from The Cystinosis Research Foundation and WNYSTEM.