Here we describe the generation of ATTR patient specific iPS

Here, we describe the generation of ATTR patient-specific iPSCs and the directed differentiation of these pkc inhibitor into hepatic, neuronal, and cardiac lineages thereby modeling the three major tissue types involved in this disease (Figure 1). Our data are consistent with the clinical trial findings that known small molecule TTR stabilizers can inhibit TTR fibril formation in vivo, validating patient-specific iPSCs as a platform for testing therapeutics.
Results
Discussion Human iPSC-based disease modeling provides an opportunity to recapitulate a disease phenotype in the context of a patient’s exact genetic background. In the case of ATTR, patients with the same genetic mutations have been shown to exhibit variable patterns of disease progression (Planté-Bordeneuve and Said, 2011), highlighting the involvement of genetic modifiers of disease. ATTR is an unusual hereditary disease that manifests clinically in adulthood with no reproductive pressure against transmission of the disease. Until recently, diagnostics were not available, and carriers could not be identified before clinical disease onset, which typically occurs in the fourth decade or later. In patients affected with the TTRL55P mutation, the age of disease onset is earlier and the clinical outcome more severe (Jacobson et al., 1992), characteristics that reflect greater instability of the mutant TTR tetramer (Lashuel et al., 1998, 1999). Our work documents the successful modeling of ATTR in vitro with the use of iPSC technology, demonstrating that it is possible to model a long-term, complex, multisystem disease in a relatively short space of time, using lineage-specified cells derived from patient-specific stem cells. iPSCs have been utilized to model genetic diseases in a single lineage in which the variant protein functions, for example, Fanconi anemia (Raya et al., 2009), alpha 1 antitrypsin deficiency (Rashid et al., 2010), and amyotrophic lateral sclerosis (Dimos et al., 2008). Our work demonstrates that iPSC technology can be used to model a multisystem disease in which the effects of variant protein produced by one organ manifests disease in other target tissues. The mechanism by which variant TTR adversely affects cells is still being elucidated. It is postulated that abundant extracellular amyloid fibril deposits seen in patient biopsies have a disruptive effect on tissue architecture and organ function, but recent evidence suggests that TTR monomers and/or oligomeric prefibrillar protein structures are toxic to cells (Reixach et al., 2004; Sousa et al., 2001a, 2002). We have recapitulated a part of this process in a patient-specific fashion in vitro. In our iPSC-based disease model, the lack of visible protofilament/aggregate material in ATTRL55P hs cultures suggests that the negative effect of ATTRL55P hs is likely to be due to monomer or oligomer-induced cytotoxicity, reported by others to be a mechanism of cellular damage (Reixach et al., 2004; Sousa et al., 2002). Although extended exposure to ATTRL55P hs increased apoptosis in both neuronal and cardiac cells, we were interested in examining some of the molecular changes in the exposed target-lineage cells. Based on the previously discussed microarray data obtained through the comparison of hepatic cells derived from normal or ATTRL55P-specific iPSCs (Figure S2), we focused on expression of hsp70 family genes that are involved in protein folding and upregulated in response to cellular stressors. These genes have been studied in a range of protein-misfolding disorders, including Alzheimer’s disease (Hoshino et al., 2011). Intriguingly, an ATTR mouse model with impaired heat shock response exhibits a comparatively accelerated amyloid deposition pattern, implicating the importance of these genes for the regulation of the process of ATTR amyloidogenesis (Santos et al., 2010). We also examined the expression of markers previously reported to be associated with ATTR in other model systems and patient tissues, such as M-CSF, p21, and antioxidant enzyme HO1 (Sousa et al., 2001a, 2001b, 2005), and the receptor for advanced glycation end products (RAGE), a marker that has been postulated as a mechanism through which variant TTR targets certain tissues for amyloid deposition and cytotoxic effects (Sousa et al., 2001b). Through the examination of these markers in the exposed target-lineage cells, we were able to determine a pattern of gene expression upregulation in ATTRL55P hs-exposed cells that was accordant with previous observations. This iPSC-based, in vitro system may therefore prove to be a valuable model system in which to further dissect the molecular mechanisms through which aberrant TTR affects ATTR disease target cells and tissues. Moreover, it is interesting that in our system, iPSC-derived ATTRL55P hepatic cells exhibit gene expression differences compared to control cells because the livers of patients with ATTR are thought to be comparatively normal despite being the site of aberrant TTR production. Recent findings from a murine SSA model also suggest that the liver has a role to play in the degree of amyloidogenesis in target tissues, through a novel long-range chaperoning effect (Buxbaum et al., 2012). The iPSC-based system may prove to be an ideal platform in which to further investigate the role of the liver in ATTR amyloidogenesis.