Induced pluripotent stem cell modeling of multisystemic, hereditary transthyretin amyloidosis

Abstract

Familial transthyretin amyloidosis (ATTR) is an autosomal-dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe the directed differentiation of ATTR patient-specific iPSCs into hepatocytes that produce mutant TTR, and the cardiomyocytes and neurons normally targeted in the disease. We demonstrate that iPSC-derived neuronal and cardiac cells display oxidative stress and an increased level of cell death when exposed to mutant TTR produced by the patient-matched iPSC-derived hepatocytes, recapitulating essential aspects of the disease in vitro. Furthermore, small molecule stabilizers of TTR show efficacy in this model, validating this iPSC-based, patient-specific in vitro system as a platform for testing therapeutic strategies.

Figure 1

Schematic Summary of the In Vitro iPSC Approach to Study ATTR The cytokines that were used in each differentiation protocol are indicated. FAP, familial amyloid polyneuropathy; FAC, familial amyloid cardiomyopathy.

Figure 2

Generation and Characterization of ATTR^L55P iPSC-Hepatic Lineage Cells (A) Schematic diagram details the stepwise differentiation protocol used to obtain hepatic-lineage cells from iPSCs. (B) Cells at day 5 of differentiation express the definitive endoderm cell surface markers CXCR4 and cKit, as determined by FACS analysis. (C) Hepatic markers AFP, ALB, APOA1, and TTR are upregulated in hepatic-lineage cells. These markers are not expressed in iPSCs or (precursor cell type) definitive endoderm cells. All values are normalized to GAPDH. Error bars represent the SD (n = 3 up to six independent biological samples). (D) Hepatic-lineage cells derived from ATTR^L55P iPSCs morphologically resemble hepatocytes and are capable of glycogen storage as demonstrated by PAS staining. (E) ATTR^L55P iPSC hepatic-lineage cells produce and secrete TTR protein as demonstrated by immunoblot analysis with media supernatants. Recombinant human TTR (25 ng) and serum-free media alone serve as positive and negative controls, respectively. The lower band, seen in the ATTR^L55P hs and media-only lanes, is a result of the nonspecific cross-reactivity of the TTR antibody to a product in the media formulation. (F) Mass spectrometry analysis of control and ATTR^L55P hs for TTR species is shown. hs generated from ATTR^L55P hepatic-lineage cells contained both WT (13,759 kDa) and L55P (13,742 kDa) forms of TTR protein (labeled black and red arrows, respectively), whereas only the former was detectable in control samples.

Figure 3

Generation of Beating Cardiomyocytes from ATTR^L55P iPSCs (A) Day 14 contracting EBs are presented. (B) Quantitative PCR analysis of ATTR^L55P iPSC and ATTR^L55P day 14 cEB cultures is shown. The pluripotency marker Oct4 is downregulated, and cardiac markers (Nkx2.5, Tbx5, Islet1, cardiac actin, and the end-stage marker cardiac troponin) are upregulated in the latter sample type. All values are normalized to GAPDH. Independent sample sizes are iPSC n = 2 and cEB n = 3. Error bars, SD. (C) Intracellular FACS was used to quantitate the proportion of cells that express the cardiac marker sarcomeric anti-actinin (blue peak) in day 13 cEB cultures. The isotype control peak is shown in red.

Figure 4

Derivation of Neuronal-Lineage Cells from ATTR^L55P iPSCs that Are Capable of TTR Internalization (A) Phase-contrast image shows neuronal cells derived from ATTR^L55P iPSCs. (B) Immunofluorescence staining presents ATTR^L55P iPSC-derived neuronal cells for the neuronal marker TUJ1 (red). Nuclei were stained with DAPI (blue). (C) Immunofluorescence staining shows ATTR^L55P iPSC-derived neuronal cells for the MAP2 (red). Nuclei were stained with DAPI (blue). (D) Quantitative PCR analysis of derived neuronal-lineage cells compared to undifferentiated iPSCs and human brain cDNA is shown. Neuronal cells exhibit upregulated levels of TUJ1 and motor neuron homeobox transcription factor HB9. All independent biological samples (n = 3) were normalized to GAPDH. Error bars, SD. (E) ATTR^L55P iPSC neuronal-lineage cells are capable of TTR (both WT and L55P) internalization as demonstrated by exposure to AF488-labeled human recombinant proteins. Foci of labeled proteins (green fluorescence) are visible in the cells. Nuclei were stained with DAPI (blue).

Figure 5

iPSC-Based Modeling of ATTR In Vitro; ATTR^L55P hs Deleteriously Affects iPSC-Derived Target Cells (A) Mature ATTR^L55P iPSC neuronal-lineage cells were exposed to hs from either control iPSCs or ATTR^L55P iPSC hepatic-lineage cells. No discernable differences in cell morphology and no visible protein aggregation were seen after 10 days. Images, 20× magnification. (B) Cardiomyocytes derived from ATTR^L55P iPSCs were exposed to hs for 6 days. There were no visible differences in the cultures containing control hs and ATTR^L55P hs (20× magnification). (C and D) Gene expression analysis of day 12-dosed ATTR^L55P neuronal cells (C) and day 10-dosed ATTR^L55P cardiac cells (D) for a panel of markers associated with stress response and protein folding. Error bars, SD. Asterisks (^∗, ^∗∗) denote t test significance at the 5% and 1% level, respectively. Sample size is n = 2 from independent experiments. ATTR^L55P samples were normalized to controls for each experiment. (E) ATTR neuronal cells cultured in media containing hs for 19 days were analyzed for the uptake of PI and Hoechst 33342. Cultures dosed with ATTR^L55P hs contained fewer live cells (forward versus side scatter gate; Hoechst-positive gate, annotated) compared to control hs cultures. (F) ATTR^L55P cardiac cells dosed for 10 days with media containing ATTR^L55P hs exhibited a decrease in cell viability compared to control cultures.

Figure 6

The Deleterious Effects of ATTR^L55P hs Can Be Abrogated by Small Molecule TTR-Stabilizing Agents (A) ATTR^L55P iPSC-derived neuronal-lineage cells were exposed to control and ATTR^L55P hs in the presence or absence of diflunisal (20 μM) and flufenamic acid (10 μM). Cells were harvested and examined for their H-PI positivity by flow cytometry. Increased apoptosis and cell death seen in ATTR^L55P hs cultures were rescued in cultures also containing the small molecule compounds. (B) Neuronal live-cell proportions in cultures dosed with ATTR^L55P hs for 12 days, ± compounds, compared to control hs-dosed cells. Error bars, SD. Asterisks (^∗, ^∗∗) denote t test significance at the 5% and 1% level, respectively. p Values are denoted in the graph. Sample sizes (from independent experiments) are ATTR^L55P n = 4, ATTR^L55P + Diflu n = 3, and ATTR^L55P + Fluf n = 3. n.s., not significant. (C) ATTR^L55P iPSC-derived cardiac cells were dosed for 6 days and analyzed for Hoechst-PI positivity in the same manner. (D) Cardiac live-cell proportions in cultures dosed with ATTR^L55P hs, ± compounds, compared to control hs-dosed cells. Error bars, SD. Asterisks (^∗, ^∗∗) denote t test significance at the 5% and 1% level, respectively. p Values are denoted in the graph. Sample sizes (from independent experiments) are ATTR^L55P n = 3, ATTR^L55P + Diflu n = 3, and ATTR^L55P + Fluf n = 3.