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

Journal name:
Nature
Volume:
472,
Pages:
221–225
Date published:
DOI:
doi:10.1038/nature09879
Received
Accepted
Published online

Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease1, 2, 3, 4, 5, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs)6, 7, 8. 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 heterochromatin9, 10, 11, 12. 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 ageing6, 12, 13, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

At a glance

Figures

  1. Generation of iPSCs from HGPS fibroblasts.
    Figure 1: Generation of iPSCs from HGPS fibroblasts.

    a, Immunofluorescence analysis performed on HGPS (left) and BJ (right) fibroblasts at passage 17 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 stain (blue). Scale bar, 20μm.

  2. HGPS-associated nuclear defects are reset in HGPS-iPSCs.
    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). BJ- and HGPS-fib, BJ and HGPS fibroblasts. b, Immunoblotting analysis of the indicated proteins. Emerin was used as loading control. Asterisk denotes progerin (Δ50 lamin A). Arrowheads denote lamin A (top) and lamin C (bottom). c, Immunofluorescence analysis performed on BJ-iPSCs and HGPS-iPSCs for detection of the indicated proteins. Scale bar, 10μm. d, Hierarchical clustering of genome-wide DNA methylation profiles.

  3. SMCs expressing progerin show nuclear defects and accelerated senescence.
    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 showing dysmorphic nuclei, (n = 3, P<0.001). p2, p3 and p5, passage 2, 3 and 5, respectively. c, Immunostaining of H3K9me3 and calponin in iPSC-derived SMCs (p5). Nuclei were visualized with Hoechst stain (blue). Arrowheads denote decreased nuclear H3K9me3 (percentage in corner). Scale bar, 20μm. 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 (inset). j, Immunoblotting of the indicated proteins in shRNA-modified HGPS-iPSCs after 21days of EB-mediated differentiation. Asterisks denote progerin (Δ50 lamin A). k, Cell proliferation analysis of the SMCs derived from shRNA-modified HGPS-iPSCs (p2, n = 3), **P<0.01.

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

    a, Extracts from BJ fibroblasts expressing GFP, GFP–lamin A, or GFP–progerin, were immunoprecipitated (IP) with a GFP antibody and examined by immunoblotting analysis (IB). 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 stain (blue). Scale bar, 20μm.

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Gene Expression Omnibus

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Author information

Affiliations

  1. Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA

    • Guang-Hui Liu,
    • Basam Z. Barkho,
    • Sergio Ruiz,
    • Jing Qu,
    • Sheng-Lian Yang,
    • Athanasia D. Panopoulos,
    • Keiichiro Suzuki,
    • Leo Kurian,
    • Christopher Walsh,
    • Ignacio Sancho-Martinez &
    • Juan Carlos Izpisua Belmonte
  2. Department of Bioengineering, University of California at San Diego, La Jolla, California 92093, USA

    • Dinh Diep,
    • Ho Lim Fung &
    • Kun Zhang
  3. Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA

    • James Thompson &
    • John Yates III
  4. Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain

    • Stephanie Boue &
    • Juan Carlos Izpisua Belmonte

Contributions

G.-H.L. and J.C.I.B. conceived the experiments; G.-H.L., B.Z.B., S.R., D.D., J.Q., S.-L.Y., A.D.P., K.S., L.K., C.W., J.T. and H.L.F. performed the experiments and analysed the data; S.B., I.S.-M., K.Z., J.Y. and J.C.I.B. analysed the data; G.-H.L., S.R., B.Z.B., A.D.P., K.Z. and J.C.I.B. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Microarray data have been deposited in NCBI-GEO under the accession number GSE24487.

Author details

Supplementary information

PDF files

  1. Supplementary Figures (11.4M)

    This file contains Supplementary Figures 1-18 with legends.

Word documents

  1. Supplementary Table 1 (30K)

    This table contains the DNA methylation parameters.

  2. Supplementary Table 4 (58K)

    This table contains the peptides identified by MudPIT for the indicated candidate progerin-associated partners.

  3. Supplementary Table 5 (101K)

    This table contains the primers used in this study.

Excel files

  1. Supplementary Table 2 (356K)

    This table contains the genes within 10kb of a differentially methylated region (DMR) found between BJ and HGPS fibroblasts.

  2. Supplementary Table 3 (77K)

    This table contains the genes within 10kb of a differentially methylated region (DMR) found between BJ-iPSCs and HGPS-iPSCs.

Movies

  1. Supplementary Movie 1 (6.3M)

    In this movie we see the contracting area derived from BJ-iPSC colony following directed differentiation into cardiac tissue. Movies were taken at day 12 of embryoid body development.

  2. Supplementary Movie 2 (9.7M)

    In this movie we see HGPS-iPSCs develop into contractile cardiac tissue with pacemaker activity in vitro. Movies were taken at day 12 of embryoid body development.

Comments

  1. Report this comment #21030

    Bobby Baum said:

    AICAR has been shown to induce 2 (Klf4 & Myc) of the 4 genes that produce iPSCs (BMC Pharmacology 9:2 – available at www.biomedcentral.com/1471-2210/9/2). It would be interesting to see if AICAR has any effect on progerin production. Most likely it wouldn't, but it's worth a try.
    Bobby Baum

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