SAT-115 Generation and characterisation of induced pluripotent stem cells derived from patients with Fabry disease

Abstract

Fabry disease (FD) is an X-linked inherited disorder resulting in accumulation of globotriaosylceramide (Gb3) that ultimately progresses to end-stage renal disease. Although the genetic basis of FD is well established, the pathophysiological mechanisms by which Gb3 storage leads to the FD phenotype remains unclear. It is well defined that the Gb3 accumulation in FD causes podocyte damage leading to fibrosis. To date, modelling of renal diseases and drug screening has utilised immortalised kidney cell lines which retain only limited characteristics of their somatic cell counterparts. Alternatively, primary podocytes are difficult to establish and maintain in culture. We therefore generated induced pluripotent stem cells (iPSC) by reprogramming human somatic cells from FD patients, with varying known FD mutations, to generate iPSCs followed by directed differentiation to podocytes that can be maintained long-term. Skin fibroblasts obtained from punch biopsies of patients with mapped FD mutations (n=3) compared to non-FD control patients (n=3) were obtained under informed consent. A NM-RNA reprogramming protocol that delivers transcription factors OCT3/4, TRA-1-60 and SSEA4 in lipofectamines through RNA transfection, was used to generate iPSCs. Pluripotency was confirmed by alkaline phosphatase staining and immunofluorescence staining performed using Oct4 and CSTEM 27, 28, 31. FD and control iPSCs were differentiated to podocytes following the addition of retinoic acid, bone morphogenic protein 7 and activin A. Day 10 iPSC-podocytes were immunostained for the expression of podocyte-specific markers – podocin and synaptopodin. Scanning electron microscopy (SEM) was performed on FD patient-derived iPSCs and control lines to observe podocyte morphological following differentiation and quantification of Gb3 performed using mass spectrometry (MS). Skin biopsy-derived dermal fibroblasts from FD and non-FD control patients were grown as monolayers and reprogrammed into iPSCs over 14 days, with pluripotency evident by alkaline phosphatase staining with normal karyotype confirmed. Colonies showed positive expression of pluripotency markers - Oct4 and CSTEM 27, 28 and 31. Directed differentiation of iPSCs resulted in the generation of podocytes over a period of 10 days that had an acquired typical podocyte morphology, consisting of either a single or multi-nucleated cell bodies and prominent foot processes. Day 10 iPSC-podocytes showed positive expression of podocyte-specific markers - podocin and synaptopodin. SEM imaging of differentiated iPSC-podocytes revealed primary and secondary foot processes, characteristic of kidney podocytes with no major morphological differences apparent between FD and control lines. Gb3 quantification using MS showed a varied increase in FD iPSC podocytes reflective of varied mutations associated with a range of pathologies and severity of disease, compared to non-FD cells. This study has generated iPSC from patients with FD that were differentiated to podocytes confirmed by morphological characteristics and podocyte marker localization. Understanding how mutations cause disease in FD using iPSC-derived kidney cells is a fundamental step for future studies to correct the genetic defects in these cells and as a valuable tool for use in disease modeling and toxicology screening.