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Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome

Abstract

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems1,2. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

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Figure 1: Gene expression profile in LEOPARD syndrome iPSCs is similar to human ES cells.
Figure 2: LEOPARD syndrome iPSCs differentiate in vitro and in vivo into all three germ layers.
Figure 3: Cardiomyocytes derived from LEOPARD syndrome iPSCs show hypertrophic features.
Figure 4: Phosphoproteomic and MAPK activation analyses.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in NCBI-GEO under the accession number GSE20473.

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Acknowledgements

We thank T. James, X. Niu and D. York for their technical support and laboratory management, and B. MacArthur for support in microarray analysis. We also would like to thank K. Moore and her laboratory, and S. Mulero-Navarro from B.D.G.’s laboratory for their help, and V. Fuster and A. Bernad for their support. This research was funded by grants from the National Institutes of Health (NIH) to I.R.L (5R01GM078465), the Empire State Stem Cell Fund through New York State Department of Health (NYSTEM) C024410 to I.R.L. and C.S., C024176 (HESC-SRF) to I.R.L. and S.L.D., C024407 to B.D.G., American College of Cardiology/Pfizer Research Fellowship to E.D.A., and ERA-Net for research programmes on rare diseases 2009 to M.T. X.C.-V. is a recipient of a Postdoctoral Fellowship from the Ministerio de Ciencia e Innovacion/Instituto de Salud Carlos III, D.-F.L. is a New York Stem Cell Foundation Stanley and Fiona Druckenmiller Fellow and S.P. is a recipient of a Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant (T32).

Author information

Authors and Affiliations

Authors

Contributions

X.C.-V. (iPSC establishment, project planning, experimental work and preparation of manuscript); A.S., S.L.D., Y.-S.A., L.Y., A.D.K., E.D.A., D.-F.L., A.W., B.C., J.S. and S.P. (experimental work); R.R. and Y.G. (microarray analysis); N.C. and L.J.E. (karyotype analysis); K.D.L. and M.T. (obtaining of fibroblast samples from patients); C.S. (project planning, experimental work); B.D.G. and I.R.L. (project planning, preparation of manuscript).

Corresponding authors

Correspondence to Xonia Carvajal-Vergara or Ihor R. Lemischka.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12 with legends and Supplementary Table 1. (PDF 2117 kb)

Supplementary Movie 1

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line HES2. (MOV 5661 kb)

Supplementary Movie 2

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L1-iPS6. (MOV 5718 kb)

Supplementary Movie 3

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L1-iPS13. (MOV 2601 kb)

Supplementary Movie 4

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2-iPS6. (MOV 5762 kb)

Supplementary Movie 5

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2-iPS10. (MOV 6553 kb)

Supplementary Movie 6

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2iPS16. (MOV 15213 kb)

Supplementary Movie 7

This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line S3-iPS4. (MOV 6356 kb)

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Carvajal-Vergara, X., Sevilla, A., D’Souza, S. et al. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465, 808–812 (2010). https://doi.org/10.1038/nature09005

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