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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.

Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).

In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.