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Open Access Research

A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient’s skin

Natascha Bergamin1, Andrea Dardis2, Antonio Beltrami1, Daniela Cesselli1, Silvia Rigo1, Stefania Zampieri2, Rossana Domenis1, Bruno Bembi2 and Carlo Alberto Beltrami1*

  • * Corresponding author: Carlo Alberto Beltrami beltrami@uniud.it

  • † Equal contributors

Author Affiliations

1 Department of Medical and Biological Sciences (DSMB), University of Udine, P.le Kolbe 3, 33100, Udine, Italy

2 Regional Coordinator Centre for Rare Diseases, University Hospital “Santa Maria della Misericordia”, Udine, Italy

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Orphanet Journal of Rare Diseases 2013, 8:34  doi:10.1186/1750-1172-8-34

Published: 21 February 2013

Abstract

Background

Niemann Pick C (NPC) disease is a neurovisceral lysosomal storage disorder due to mutations in NPC1 or NPC2 genes, characterized by the accumulation of endocytosed unesterified cholesterol, gangliosides and other lipids within the lysosomes/late endosomes. Even if the neurodegeneration is the main feature of the disease, the analysis of the molecular pathways linking the lipid accumulation and cellular damage in the brain has been challenging due to the limited availability of human neuronal models.

Objective

The aim of this study was to develop a human neuronal model of NPC disease by inducing neuronal differentiation of multipotent adult stem cells (MASC) isolated from NPC patients.

Methods

Stem cells were isolated from 3 NPC patients and 3 controls both from skin biopsies and previously established skin fibroblast cultures. Cells were induced to differentiate along a neuronal fate adapting methods previously described by Beltrami et al, 2007. The surface immunophenotype of stem cells was analyzed by FACS. Stem cell and neuronal markers expression were evaluated by immunofluorescence. Intracellular accumulation of cholesterol and gangliosides were assessed by filipin staining and immunofluorescence, respectively. A morphometric analysis was performed using a Neurite outgrowth image program.

Results

After 3 passages in selective medium, MASC isolated either from skin biopsies or previously established skin fibroblast cultures displayed an antigenic pattern characteristic of mesenchymal stem cells and expressed the stem cell markers Oct-4, Nanog, Sox-2 and nestin. A massive lysosomal accumulation of cholesterol was observed only in cells isolated from NPC patients. After the induction of neural differentiation, remarkable morphologic changes were observed and cells became positive to markers of the neuronal lineage NeuN and MAP2. Differentiated cells from NPC patients displayed characteristic features of NPC disease, they showed intracellular accumulation of unesterified cholesterol and GM2 ganglioside and presented morphological differences with respect to cells derived from healthy donors.

In conclusion, we generated a human neuronal model of NPC disease through the induction of differentiation of stem cells obtained from patient’s easily accessible sources. The strategy described here may be applied to easily generate human neuronal models of other neurodegenerative diseases.

Keywords:
Niemann Pick C; Human neuronal model; Adult stem cells; Neuronal differentiation