Generation of patient-specific human induced pluripotent stem cells to analyze mutation- and cell-specific pathomechanisms in Fabry disease

Abstract

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (GLA), leading to intracellular globotriaosylceramide (Gb3) accumulation. Induced human pluripotent stem cells (iPSCs) provide a powerful tool to analyze cellular pathomechanisms caused by a rare disease. Here we demonstrate the successful non-invasive generation of iPSCs from urinary cells of four male FD patients with individual GLA mutations. Urinary cells from 4 male patients with the GLA mutations p.S126G, p.N215S, p.L294S, and IVS2+1C>T were isolated and cultivated for pluripotency induction. iPSCs were induced by a transient triple transfection with pCXLE-hUL, pCXLE-hSK, and pCXLE-hOCT3/4-shp53 (all Addgene). Q-PCR demonstrated a stable OCT3/4, NANOG, and SOX2 expression in all IPSCs over time (> passage 25), which was confirmed by flow cytometric analysis and fluorescence microscopy. Induction of embryoid bodies confirmed pluripotency demonstrating increasing marker expression for the three germ layers of mesoderm (FOXF1, HAND1), endoderm (FOXA2, HNF4a), and ectoderm (PAX6), while marker expression for self-renewal (SOX2, NANOG, OCT4) decreased in parallel. Neuronal induction of iPSCs to generate neural progenitor cells resulted in an increased mRNA expression of NCAM and SOX1, as well as a high protein expression for NCAM and nestin. Mesodermal induction of early mesoderm resulted in an increased brachyury (T) and NCAM expression. FD-specific characterization of IPSCs demonstrated different endogenous GLA activities and amenability to migalastat, depending on the existing genotype. Here we demonstrate the successful and non-invasive generation of patient- and mutation-specific iPSCs from urine of male Fabry patients. This approach can now be used to analyze the cellular pathology of FD and response to present and future therapeutically options in different primary cells of interest in a GLA mutation-dependent manner.