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

Abstract

BackgroundNiemann 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.ObjectiveThe 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.MethodsStem 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.ResultsAfter 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.

Complex lipid trafficking in Niemann‐Pick disease type C

“…Considering differences in levels of cholesterol storage and sphingolipid metabolism between systemic cells and neurons, data obtained on NPC1 mutant CHO cells, mutant human skin fibroblasts or normal systemic cells treated with U18666A (which has side effects) may not reflect what happens in the brain. Patient-specific iPS cells or other neuro-like cells derived from skin (Bergamin et al 2013) should offer new possibilities. Similarly, the most widely used natural Balb/c murine model Npc1 NIH is a null mutant, and corresponds to the most severe early infantile neurological onset form of the disease.…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…An advantage of these nonmammalian models is that genetic manipulation is relatively straightforward. Recently, studies have been initiated in human stem cells isolated from individual NPC patients, particularly stem cells that were differentiated into neuron-like cells ( 43,(83)(84)(85). It is likely that these cells will be useful for evaluation of drug candidates for NPC disease.…”

Role of LIMP-2 in the intracellular trafficking of β-glucosidase in different human cellular models

“…However, the study of the molecular bases of neurodegeneration in humans is challenging because of the lack of suitable cellular models. We have recently described a method of generating a human neuronal model of Niemann Pick C disease through the induction of differentiation of hSKIN‐MASCs (23). hSKIN‐MASCs exhibit a mesenchymal stem cell immunophenotype, express pluripotent state‐specific transcription factors, and are clonogenic and multipotent.…”

“…First‐strand cDNA synthesis and quantitative RT‐PCR of choline acetyltransferase (CHAT), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and dopamine active transporter (DAT) were performed with a LightCycler 480 Real‐Time PCR System and LightCycler 480 SYBR Green I Master Mix (Roche) (23). GAPDH was used as the internal control for normalization.…”