title: Niemann-Pick type C1 patient-specific induced pluripotent stem cells display disease specific hallmarks.
abstract: 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.
authors.affiliation: Albrecht-Kossel-Institute for Neuroregeneration (AKos), University of Rostock, Gehlsheimer Strasse 20, D-18147 Rostock, Germany. moritz.frech@med.uni-rostock.de.
authors.forename: Michaela
authors.initials: M
authors.lastname: Trilck
authors.forename: Rayk
authors.initials: R
authors.lastname: Hübner
authors.forename: Philip
authors.initials: P
authors.lastname: Seibler
authors.forename: Christine
authors.initials: C
authors.lastname: Klein
authors.forename: Arndt
authors.initials: A
authors.lastname: Rolfs
authors.forename: Moritz J
authors.initials: MJ
authors.lastname: Frech
mesh_terms: D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D052556:Niemann-Pick Disease, Type C / Q000378:metabolism* / Q000473:pathology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D020598:Proto-Oncogene Proteins c-myb; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors
references.citation: Hepatology. 2010 May;51(5):1810-9
references.pmid: 20432258
references.citation: Biochim Biophys Acta. 2009 Jul;1791(7):659-70
references.pmid: 19416638
references.citation: Cell Stem Cell. 2010 Oct 8;7(4):521-31
references.pmid: 20887957
references.citation: Biochim Biophys Acta. 2004 Oct 11;1685(1-3):48-62
references.pmid: 15465426
references.citation: J Neurosci. 2011 Apr 20;31(16):5970-6
references.pmid: 21508222
references.citation: J Biol Chem. 2000 Aug 11;275(32):24367-74
references.pmid: 10821832
references.citation: Science. 1997 Jul 11;277(5323):232-5
references.pmid: 9211850
references.citation: Stem Cells. 2006 Feb;24(2):292-8
references.pmid: 16099992
references.citation: Hum Mol Genet. 2011 Sep 15;20(18):3653-66
references.pmid: 21685203
references.citation: Nat Biotechnol. 2008 Jul;26(7):795-7
references.pmid: 18568017
references.citation: Development. 2005 Nov;132(22):5115-24
references.pmid: 16221727
references.citation: Cell. 2007 Nov 30;131(5):861-72
references.pmid: 18035408
references.citation: Hum Mol Genet. 2012 Jun 15;21(12):2651-62
references.pmid: 22437840
references.citation: Cell. 2008 Sep 5;134(5):877-86
references.pmid: 18691744
references.citation: Cell Prolif. 2012 Apr;45(2):91-100
references.pmid: 22324506
references.citation: Biochim Biophys Acta. 1980 Sep 8;619(3):669-79
references.pmid: 6257302
references.citation: Cell Stem Cell. 2007 Sep 13;1(3):253-8
references.pmid: 18371359
references.citation: Stem Cells. 2009 Apr;27(4):806-11
references.pmid: 19350680
references.citation: J Cell Mol Med. 2009 Sep;13(9B):3786-96
references.pmid: 18774957
references.citation: J Neurophysiol. 2001 Oct;86(4):1632-43
references.pmid: 11600626
references.citation: J Cell Biol. 1989 May;108(5):1625-36
references.pmid: 2715172
references.citation: Nature. 2008 Jan 10;451(7175):141-6
references.pmid: 18157115
references.citation: Biochem Biophys Res Commun. 1999 Aug 2;261(2):493-8
references.pmid: 10425213
references.citation: Orphanet J Rare Dis. 2010 Jun 03;5:16
references.pmid: 20525256
references.citation: Neurobiol Dis. 2011 Jan;41(1):209-18
references.pmid: 20883783
references.citation: Cell. 2009 Jun 26;137(7):1213-24
references.pmid: 19563754
references.citation: Biochem Biophys Res Commun. 2009 Sep 25;387(3):482-8
references.pmid: 19615974
references.citation: JIMD Rep. 2012;4:29-37
references.pmid: 23430894
references.citation: Blood. 2009 Dec 24;114(27):5473-80
references.pmid: 19797525
references.citation: Circ Res. 2009 Feb 27;104(4):e30-41
references.pmid: 19213953
references.citation: Cell Res. 2009 Apr;19(4):429-38
references.pmid: 19255591
references.citation: Cell Stem Cell. 2013 May 2;12(5):573-86
references.pmid: 23642366
references.citation: PLoS Curr. 2010 Oct 28;2:RRN1193
references.pmid: 21037797
references.citation: Am J Hum Genet. 2001 Jun;68(6):1361-72
references.pmid: 11349231
references.citation: J Clin Invest. 2010 Sep;120(9):3127-36
references.pmid: 20739751
references.citation: Pediatr Res. 2008 Nov;64(5):544-9
references.pmid: 18614965
references.citation: J Physiol. 2007 Mar 1;579(Pt 2):413-29
references.pmid: 17170046
references.citation: J Neurosci. 1997 Aug 15;17(16):6075-85
references.pmid: 9236219
references.citation: Stem Cells Int. 2012;2012:738910
references.pmid: 22550517
references.citation: Cell Stem Cell. 2013 May 2;12(5):497-9
references.pmid: 23642355
references.citation: J Biol Chem. 1988 Mar 5;263(7):3411-7
references.pmid: 3277970
references.citation: PLoS One. 2011 Feb 10;6(2):e17084
references.pmid: 21347327
references.citation: Orphanet J Rare Dis. 2013 Feb 21;8:34
references.pmid: 23433359
references.citation: J Lipid Res. 2011 Apr;52(4):813-25
references.pmid: 21245028
references.citation: Mol Genet Metab. 2009 Sep-Oct;98(1-2):152-65
references.pmid: 19647672
references.citation: Science. 1997 Jul 11;277(5323):228-31
references.pmid: 9211849
references.citation: Genes Dev. 2008 Jan 15;22(2):152-65
references.pmid: 18198334
references.citation: Mol Ther. 2004 Jul;10(1):27-36
references.pmid: 15233939