Human iPSC-based modeling of late-onset disease via progerin-induced aging

Abstract

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

Figure 1. Old Donor Fibroblasts Lose Age-Associated Markers Following Reprogramming to the Pluripotent State

(A) Immunocytochemistry for markers identifying the nuclear lamina (Lamin A/C), a lamina-associated protein (LAP2α) and peripheral heterochromatin (H3K9me3, HP1γ) in young fibroblasts (11-year-old donor) compared to old fibroblasts (82-year-old donor). Percentages indicate the proportion of cells with folded and/or blebbed nuclear morphologies. (B) Quantification of the markers depicted in (A) demonstrates the ability of the selected age-associated markers to stratify young versus old donor fibroblasts and the similarity of old donor fibroblasts to HGPS patient fibroblasts. The data are plotted as frequency distributions of relative fluorescence intensity for 100 cells from single fibroblast lines that were passage-matched. a.u., arbitrary units. (C) Similar to HGPS patient fibroblasts, old donor fibroblasts have higher levels of DNA damage (measured by γH2AX immunocytochemistry) and higher levels of mitochondrial reactive oxygen species (ROS; measured by flow cytometry using the superoxide indicator MitoSOX) than young donor fibroblasts. n= 3 independent experiments. (D) Immunocytochemistry for age-associated markers in passage 10 (P10) iPSCs derived from the young and old donor fibroblasts. (E) Quantification of staining in (D) indicates loss of age-associated markers during reprogramming of old donor-derived iPSCs (similar to HGPS-derived iPSCs). n= 300 cells each (100 cells from 3 independent iPSC clones). (F) DNA damage and mitochondrial superoxide levels are reset upon reprogramming. n= 3 independent clones. n.s. not significant, *p<0.05, **p<0.01, ****p<0.0001 according to Kolmogorov-Smirnov tests (B and E) or Student’s t tests (C and F). Bar graphs represent mean ± SEM. Scale bars: 50 μm (A), 100 μm (C). See also Figure S1 and S7 & Tables S1-S2 and S4-S5.

Figure 2. iPSC-Fibroblasts from Old Donors Do Not Regain Age-Associated Markers

(A) Immunocytochemistry for age-associated markers. Percentages indicate the proportion of cells with folded and/or blebbed nuclear morphologies. (B) Quantification of the markers shown in (A) indicates the high degree of overlap between iPSC-derived fibroblasts (iPSC-fibroblasts) from young and old donors compared to HGPS iPSC-fibroblasts, which reestablish an age-like phenotype. (C) Analysis of DNA damage (left) and mitochondrial superoxide (right) further show that iPSC-fibroblasts from old donors have been reset to a “young”-like state. n.s. not significant, **p<0.01, ****p<0.0001 according to Kolmogorov-Smirnov tests (B) or Student’s t tests (C). n= 3 differentiations of independent iPSC clones performed at different times. Bar graphs represent mean ± SEM. Scale bar: 50 μm. See also Figure S2 and S7 & Tables S2 and S4.

Figure 3. Progerin Overexpression Induces Age-Associated Changes in iPSC-Fibroblasts Regardless of Donor Age

(A) Modified-RNA was transfected into iPSC-fibroblasts on three consecutive days prior to analysis on day 4. (B) Overexpression of progerin (GFP-progerin) in iPSC-fibroblasts causes changes in nuclear morphology (as seen by GFP), expression of the lamina-associated protein (LAP2α), levels of DNA damage (γH2AX), and chromatin organization (H3K9me3; HP1γ), which were not observed with overexpression of a nuclear-localized GFP control (nuclear-GFP). Percentages indicate the proportion of cells with folded and/or blebbed nuclear morphologies. (C) Quantification of data shown in (B). Frequency distribution plots represent the fluorescence intensity of 100 cells from 3 independent RNA transfections of iPSC-fibroblasts derived from independent iPSC clones. (D) Flow cytometry analysis of the mitochondrial superoxide indicator MitoSOX suggests a dramatic increase in mitochondrial dysfunction with progerin overexpression. n= 3 independent RNA transfections of iPSC-fibroblasts derived from independent iPSC clones. *p<0.05, **p<0.01, ****p<0.0001 according to Kolmogorov-Smirnov tests (LAP2α, H3K9me3, HP1γ) or Student’s t-tests (γH2AX, MitoSOX). Bar graphs represent mean ± SEM. Scale bar: 25 μm. See also Figure S2 and S7 & Tables S2 and S4-S5.

Figure 4. Progerin Overexpression Induces a Subset of the Fibroblast Age-Associated Signature in iPSC-mDA Neurons derived from both Young and Old Donors

(A) Modified-RNA was transfected into iPSC-derived mDA neurons (iPSC-mDA neurons) on five consecutive days prior to analysis on day 6. (B) Western blot analysis of transgene expression. A GFP band at 100 kDA denotes the GFP-progerin fusion protein while a GFP band at 27 kDA represents the nuclear-GFP transgene. All lamin A isoforms including the transgene were recognized by a single antibody. Note that progerin overexpression levels exceed endogenous lamin A levels (arrows). iPSC-mDA neurons do not appear to express detectable levels of progerin protein endogenously. n, nuclear-GFP; p, GFP-progerin. (C) Progerin overexpression enhances nuclear folding and blebbing (as seen by lamin B2, pink) and increases DNA damage accumulation (γH2AX) in both young and old donor-derived iPSC-mDA neurons. Percentages indicate the proportion of cells with enhanced nuclear folding and/or blebbing or the proportion of cells with >3 enlarged γH2AX foci. (D) Flow cytometry analysis of mitochondrial superoxide levels (MitoSOX) demonstrates increased mitochondrial dysfunction with progerin overexpression. n= 3 independent RNA transfections of iPSC-mDA neurons derived from independent iPSC clones (E) Quantification of immunocytochemistry for LAP2α, H3K9me3 and HP1γ shows no difference between iPSC-mDA neurons transfected with GFP-progerin or nuclear-GFP, unlike the phenotype observed in iPSC-fibroblasts (see Figure 3). Fluorescence intensities were normalized to the intensities observed in nuclear-GFP-treated cells. *p<0.05 according to Student’s t tests (D). Bar graphs represent mean ± SEM. Scale bars: 10 μm (C, bottom), 25 μm (C, top). See also Figure S3 and S7 & Tables S2 and S4-S5.

Figure 5. Progerin Overexpression Elicits Features Consistent with Neuronal Aging in iPSC-mDA Neurons

(A) Immunocytochemistry for the pan-neuronal marker TUJ1 shows a loss of the established neuronal network in day 70 iPSC-mDA neurons overexpressing progerin but not iPSC-mDA neurons overexpressing nuclear-GFP. (B) MAP2 immunocytochemistry reveals reduced intact dendrite lengths following overexpression of progerin in most but not all (inset) iPSC-mDA neurons derived from both young and old donors. Frequency distributions display total dendrite length measurements from 3 independent RNA transfections (50 cells each, non-apoptotic nuclei only). (C) Principal component analysis of RNA-seq gene expression data further corroborates the reprogramming-induced reset of age that results in the high similarity of iPSC-mDA neurons from both young and old donors. Progerin overexpression induces similar changes in mDA neurons independent of donor age. (D) The top 20 upregulated (left) and downregulated (right) genes in progerin-treated compared to control nuclear-GFP-treated young donor (green) and old donor (blue) iPSC-mDA neurons. Genes are ranked according to iPSC-mDA neurons derived from the old donor. Red denotes uncharacterized genes and orange denotes non-coding RNAs. Dotted line indicates the threshold for significance. (E) Pie charts representing the proportion of the significantly differentially expressed transcripts that are coding, non-coding, or uncharacterized. ****p<0.0001 according to Kolmogorov-Smirnov tests. Scale bars: 200 μm (A), 50 μm (B). See also Figure S4 and S7 & Tables S2-S5.

Figure 6. Progerin Overexpression Reveals Disease-Specific Phenotypes In Vitro in iPSC-Based Models of Genetic PD

(A and B) Quantification of NURR1+ cells (A) and western blot analysis of TH protein levels (B) do not reveal significant differences with transfection of GFP-progerin modified-RNA. n, nuclear-GFP; p, GFP-progerin. Numbers below the western blot indicate the ratio of GFP-progerin:nuclear-GFP expression of TH normalized to GAPDH. (C) Analysis of GFP+ cells undergoing cell death following RNA transfection as identified by condensed nuclear morphologies. Images display a representative example of cleaved caspase-3 immunocytochemistry in cells treated with progerin. (D) Immunocytochemistry for the dendrite marker MAP2. (E) Quantification of total dendrite lengths per GFP+ neuron shows accelerated dendrite shortening in PD mutant iPSC-mDA neurons compared to apparently healthy controls (C1-4) in response to progerin overexpression. (F and G) Western blot analysis of AKT pathway signaling (F) demonstrates genotype-specific responses to progerin overexpression. For quantification phospho-specific bands (G) were normalized to total protein before calculating the ratio of the levels expressed following progerin versus nuclear-GFP treatment. Dotted line indicates an equal amount of phospho protein in both treatment conditions. Quantification represents 3 independent cell isolates for each genotype. *p<0.05, ** p<0.01, *** p<0.001 according to one-way ANOVA with Dunnett’s tests (n= 3 independent differentiations and modified-RNA transfections in all cases). Bar graphs represent mean ± SEM. n, nuclear-GFP; p, GFP-progerin; C1-4, lines derived from apparently healthy donors; (R), iPSC derived using retroviral factors; (S), iPSC derived using Sendai viral factors. Scale bars: 25 μm. See also Figure S5 and S7 & Table S2 and Table S4-S5.

Figure 7. Long-Term Progerin Overexpression In Vivo Reveals a Severe Degenerative Phenotype in PD Mutant Cells

(A) Schematic illustration of the transplantation studies into 6-OHDA lesioned Parkinsonian mice. (B) Rotational behavior analysis of lesioned mice transplanted with control or PD mutant iPSC-mDA neurons expressing hSyn::nuclear-GFP or hSyn::GFP-progerin. Mice were lesioned and tested for amphetamine-induced rotation behavior twice prior to grafting. Dotted line indicates threshold for successful lesioning. Pink symbols identify successfully lesioned animals that did not show recovery. n= 3-5 animals per treatment group. (C) Assessment at 3 months post transplant revealed a dramatic loss of TH+ mDA neurons in PD mutants overexpressing progerin. (D) Quantification of the percentage of GFP+ cells that are TH+. Data are presented as mean ± SEM. n= 3 mice per condition. (E-G) Ultrastructural analysis 6 months after transplantation revealed accumulation of neuromelanin with lipofuscin deposits (E, yellow arrowheads) in grafts with progerin overexpression. Strikingly, the PINK1 mutant graft with progerin displayed enlarged mitochondria (F, compare representative mitochondria indicated by orange arrows in +nuclear-GFP and +GFP-progerin groups) while the Parkin mutant graft with progerin had large multilamellar bodies (G, pink arrows). These phenotypes were not observed in any other treatment groups. Asterisks in (E) and (G) indicate a fibrillar body. *p<0.05, ** p<0.01 according to Student’s t-tests. Bar graph represents mean ± SEM. Scale bars: 200 μm (C), 500 nm (E-G). See also Figure S6-S7 & Tables S2 and S4-S5.