Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks

Orphanet Journal of Rare Diseases

Moritz J Frech¹  Arndt Rolfs¹  Christine Klein²  Philip Seibler²  Rayk Hübner¹  Michaela Trilck¹ 

[1] Albrecht-Kossel-Institute for Neuroregeneration (AKos), University of Rostock, Gehlsheimer Strasse 20, D-18147 Rostock, Germany;Institute of Neurogenetics, University of Lübeck, Maria-Goeppert-Strasse 1, 23562 Lübeck, Germany

关键词: Neural differentiation;    Neural progenitor cells;    Lysosomal storage disorder;    Induced pluripotent stem cells;    Niemann-Pick Type C1;   

Others  :  863539
DOI  :  10.1186/1750-1172-8-144

 received in 2013-07-03, accepted in 2013-09-15,  发布年份 2013

【 摘 要 】

Background

Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired, resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However, the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore, a human model using patient-specific induced pluripotent stem cells provides a promising approach.

Methods

We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation.

Results

The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81 and SSEA4. Using the embryoid body assay, the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice, demonstrating their pluripotency. In addition, neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels, spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural progenitor cells, but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay, demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient.

Conclusions

We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts, providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease.

【 授权许可】

   
2013 Trilck et al.; licensee BioMed Central Ltd.

【 图 表 】

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