title: In vitro modelling of familial amyloidotic polyneuropathy allows quantitative detection of transthyretin amyloid fibril-like structures in hepatic derivatives of patient-specific induced pluripotent stem cells.
abstract: The transthyretin protein is thermodynamically destabilised by mutations in the transthyretin gene, promoting the formation of amyloid fibrils in various tissues. Consequently, impaired autonomic organ function is observed in patients suffering from transthyretin-related familial amyloidotic polyneuropathy (FAP). The influence of individual genetic backgrounds on fibril formation as a potential cause of genotype-phenotype variations needs to be investigated in order to ensure efficient patient-specific therapies. We reprogrammed FAP patient fibroblasts to induced pluripotent stem (iPS) cells and differentiated these cells into transthyretin-expressing hepatocyte-like cells (HLCs). HLCs differentiated from FAP iPS cells and healthy control iPS cells secreted the transthyretin protein in similar concentrations. Mass spectrometry revealed the presence of mutant transthyretin protein in FAP HLC supernatants. In comparison to healthy control iPS cells, we demonstrated the formation of transthyretin amyloid fibril-like structures in FAP HLC supernatants using the amyloid-specific dyes Congo red and thioflavin T. These dyes were also applicable for the quantitative determination of in vitro formed transthyretin fibril-like structures. Moreover, we confirmed the inhibition of fibril formation by the TTR kinetic stabiliser diclofenac. Thioflavin T fluorescence intensity measurements even allowed the quantification of amyloid fibril-like structures in 96-well plate formats as a prerequisite for patient-specific drug screening approaches.
authors.forename: Jeannine
authors.initials: J
authors.lastname: Hoepfner
authors.forename: Mandy
authors.initials: M
authors.lastname: Kleinsorge
authors.forename: Oliver
authors.initials: O
authors.lastname: Papp
authors.forename: Susanne
authors.initials: S
authors.lastname: Alfken
authors.forename: Robin
authors.initials: R
authors.lastname: Heiringhoff
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
authors.forename: Vanessa
authors.initials: V
authors.lastname: Sauer
authors.forename: Andree
authors.initials: A
authors.lastname: Zibert
authors.forename: Gudrun
authors.initials: G
authors.lastname: Göhring
authors.forename: Hartmut
authors.initials: H
authors.lastname: Schmidt
authors.forename: Malte
authors.initials: M
authors.lastname: Sgodda
authors.forename: Tobias
authors.identifier: 0000-0002-1382-9577
authors.initials: T
authors.lastname: Cantz
mesh_terms: D000368:Aged; D000682:Amyloid / Q000737:chemistry*; D028227:Amyloid Neuropathies, Familial / Q000378:metabolism / Q000473:pathology*; D001483:Base Sequence; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D007700:Kinetics; D008099:Liver / Q000166:cytology*; D008297:Male; D008875:Middle Aged; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
chemical_list: D000682:Amyloid; D011228:Prealbumin
keywords: disease modelling; familial amyloidotic polyneuropathy; hepatic differentiation; induced pluripotent stem cells; transthyretin amyloid fibrils