Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

Figure 1. Generation and characterization of HGPS-iPSCs

a, Immunofluorescence analysis performed on HGPS (left) and BJ (right) fibroblasts at p17 with the indicated antibodies. b, Immunofluorescence analysis of the indicated pluripotent markers in HGPS-iPSCs (left) and BJ-iPSCs (right). Nuclei were visualized with Hoechst (blue). Scale bar, 20 μm.

Figure 2. HGPS-associated nuclear defects are reset in HGPS-iPSCs

a, RT-PCR analysis of Progerin, Lamin A and Lamin B1 in the specific cell lines (n=3). b, Immunoblotting analysis of the indicated proteins. Emerin used as loading control. Asterisk denotes progerin (Δ50 lamin A). Arrowheads denote lamin A (upper) and lamin C (lower). c, Immunofluorescence analysis performed on BJ-iPSCs and HGPS-iPSCs for detection of the indicated proteins. Scale bar, 10 μm. d, Hierarchical clustering of genome-wide DNA methylation profiles.

Figure 3. SMCs expressing progerin show nuclear defects and accelerated senescence

a, Immunostaining of calponin and lamin A in iPSC-derived SMCs (p5). Arrowheads denote dysmorphic nuclei. Scale bar, 20μm. b, Percentage of calponin-positive cells demonstrating dysmorphic nuclei, (n=3, p<0.001). c, Immunostaining of H3K9Me3 and calponin in iPSC-derived SMCs (p5). Nuclei were visualized with Hoechst (blue). Arrowheads denote decreased nuclear H3K9Me3 (percentage in corner). Scale bar, 20 μm. d–e, Senescence-associated (SA)-β-Gal staining of iPSC-derived SMCs, p<0.05. f, Southern blot analysis of SMCs showing telomere length (left). Quantified average of telomere length (right, n=2). g, Percentage of Ki67-positive cells in iPSC-derived SMCs (calponin-positive, p3), **p<0.01. h–i, Cell proliferation analysis of iPSC-derived SMCs (n=3), *p<0.05 (h) or primary vascular SMCs (overexpressing GFP or GFP-progerin, n=3), **p<0.01 (i). Typical GFP-progerin-positive nucleus showing abnormal morphology (upper). j, Immunoblotting of the indicated proteins in shRNA-modified HGPS-iPSCs after 21 days of EB-mediated differentiation. Asterisks denote progerin (Δ50 laminA). k, Cell proliferation analysis of the SMCs derived from shRNA-modified HGPS-iPSCs (p2, n=3), **p<0.01.

Figure 4. Decreased expression of DNAPK holoenzyme correlates with premature cell aging

a, Extracts from BJ fibroblasts expressing GFP, GFP-Lamin A, or GFP-Progerin, were immunoprecipitated with a GFP antibody and examined by immunoblotting analysis. b, DNAPKcs staining in the indicated cell lines. c–d, Immunoblot analysis of DNAPKcs expression. e–f, Immunostaining of the indicated proteins in iPSCs-derived SMCs (e) or primary vascular SMCs overexpressing progerin (f). Arrowheads denote decreased DNAPKcs or Ku80. Nuclei were visualized with Hoechst (blue). Scale bar, 20μm.