Induced pluripotent stem cells derived from a patient with familial idiopathic basal ganglia calcification (IBGC) caused by a mutation in SLC20A2 gene

Idiopathic basal ganglia calci ﬁ cation (IBGC), also known as Fahr disease or primary familial brain calci ﬁ cations (PFBC), is a rare neurodegenerative disorder characterized by calcium deposits in basal ganglia and other brain regions, causing neuropsychiatric and motor symptoms. We established human induced pluripotent stem cells (iPSCs) from an IBGC patient. The established IBGC-iPSCs carried SLC20A2 c.1848G N A mutation (p.W616* of translated protein PiT2), and also showed typical iPSC morphology, pluripotency markers, normal karyotype, andtheabilityof in vitro differentiationintothree-germlayers.TheiPSClinewillbeusefulforfurther elucidating the pathomechanism and/or drug development for IBGC. © 2017 The Authors. Published by Elsevier B.V. This is an


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IBGC is a rare disease, characterized by massive calcification around brain vessels and various neurological symptoms, the mechanism of which is unknown.IBGC-iPSC will provide endothelial cells or neural cells and help the elucidation of the pathomechanism in the neurovascular system, one of key targets to treat neurological disorders.

Resource details
Idiopathic basal ganglia calcification (IBGC) is a rare genetic condition characterized by symmetric calcification in the basal ganglia and other brain regions.The clinical symptoms of IBGC include neuropsychiatric symptoms, dementia, psychosis, seizures, or chronic headache, and normal serum levels of calcium, phosphate, alkaline phosphatase and parathyroid hormone.Typical age at clinical onset is between 20 and 50 years, and most individuals are asymptomatic (Nicolas et al., 2015).The neuropathological hallmark of IBGC is calcification of peri-and intra-vascular space of capillaries in the basal ganglia, dentate nuclei of the cerebellum and white matter.IBGC is frequently inherited in an autosomal dominant manner.Approximately half of the cases of familial IBGC associate with mutations in SLC20A2, which encodes the type III Na + -dependent inorganic phosphate transporter PiT2 (Yamada et al., 2014).The mutations are predicted to result in a loss of function of inorganic phosphate transport.SLC20A2 knockout mice show cerebrovascular calcification.We generated induced pluripotent stem cells (iPSCs) from an IBGC patient with SLC20A2 mutation (c.1848GNA, p.W616* of translated protein PiT2) Table 1.Established IBGC-iPSCs showed positive staining of pluripotency markers (Fig. 1A).Three germ layer differentiation capacity was showed in the in vitro differentiation followed by immunohistochemistry staining of the mesodermal marker (alpha smooth muscle actin: αSMA), the endodermal marker (SOX17), and the ectodermal marker (βIII-tubulin) (Fig. 1B), Table 2. Established IBGC-iPSCs also maintained a normal karyotype after reprogramming process (Fig. 1C), and retained SLC20A2 mutation (c.1848GNA, p.W616* of translated protein PiT2) (Fig. 1D.By flow cytometry, 66.1% of iPSCs were positive for SSEA-4 (Fig. 1E).

Ethics statements
Generation and use of human iPSCs was approved by the Ethics Committee of the Department of Medicine and Graduate School of Medicine, Kyoto University and Gifu University, and all methods were performed in accordance with the approved guidelines.Formal informed consent was obtained from the patient.

Genotyping by sequencing analysis
Genomic DNA was extracted from PBMCs and iPSCs with Purelink Genomic DNA Kits (Invitrogen, Thermo Fisher Scientific, Waltham, MA).The SLC20A2 coding region was amplified by using KOD-plus-Neo (Toyobo, Osaka, Japan) and Veriti Thermal Cycle (Thermo Fisher Scientific), followed by direct sanger sequence (3700 Genetic Analyzer; formerly Applied Biosystems, Thermo Fisher Scientific).

Karyotype analysis
Karyotyping was performed by LSI Medience Corporation (Tokyo, Japan).

Table 1
Characterization and validation.