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Generation of induced pluripotent stem cell lines from 3 distinct laminopathies bearing heterogeneous mutations in lamin A/C.

Ho JC, Zhou T, Lai WH, Huang Y, Chan YC, Li X, Wong NL, Li Y, Au KW, Guo D, Xu J, Siu CW, Pei D, Tse HF, Esteban MA - Aging (Albany NY) (2011)

Bottom Line: More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them.These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts.Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, China.

ABSTRACT
The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.

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Nuclear abnormalities are restored in iPSC-derived fibroblasts containing mutations in lamin A/C(A) Immunofluorescence photographs show expression of fibronectin and vimentin, and distribution of actin in stress fibers, in iPSC-derived fibroblasts from the 3 diseases. Abnormalities of the nuclear membrane are shown on the right panels. Scale bars indicate 10 μm (lamin A/C) and 100 μm (rest). (B) Western blot for lamin A/C in iPSC clones from the 3 diseases and the derived secondary fibroblasts. (C) Quantification of cells displaying abnormal nuclear membrane by immunofluorescence microscopy. The mean of 3 independent experiments +/− SD is shown. (D) Electron microscopy photographs show abnormalities of the nuclear membrane in more detail for all types of secondary fibroblasts. Arrows point to more obvious defects. Scale bars indicate 1 μm.
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Figure 4: Nuclear abnormalities are restored in iPSC-derived fibroblasts containing mutations in lamin A/C(A) Immunofluorescence photographs show expression of fibronectin and vimentin, and distribution of actin in stress fibers, in iPSC-derived fibroblasts from the 3 diseases. Abnormalities of the nuclear membrane are shown on the right panels. Scale bars indicate 10 μm (lamin A/C) and 100 μm (rest). (B) Western blot for lamin A/C in iPSC clones from the 3 diseases and the derived secondary fibroblasts. (C) Quantification of cells displaying abnormal nuclear membrane by immunofluorescence microscopy. The mean of 3 independent experiments +/− SD is shown. (D) Electron microscopy photographs show abnormalities of the nuclear membrane in more detail for all types of secondary fibroblasts. Arrows point to more obvious defects. Scale bars indicate 1 μm.

Mentions: To evaluate the potential of our iPSC cell lines for understanding the complexity of the 3 laminopathies, we differentiated them into secondary fibroblasts. The acquisition of mesenchymal-like characteristics in the iPSC-derived fibroblasts was validated by immunofluorescence microscopy for actin stress fibers, the intermediate filament vimentin and the extracellular matrix protein fibronectin. All secondary fibroblasts, including those from a non-affected iPSC clone, displayed the expected staining pattern (Figure 4A). Western blotting verified the up-regulation of lamin A/C compared to iPSCs (Figure 4B), and the acquisition of nuclear blebbing was demonstrated by immunofluorescence (Figure 4A and C) and electron microscopy (Figure 4D). Notably, the latter technique reproduced the more subtle differences in pore size and membrane thickening observed with the primary cells. Then we analyzed whether these changes were associated with cell senescence (Figure 5A-C) and slow proliferation (Figure 5D-E), as observed with the respective primary fibroblasts. Both parameters were significantly increased in secondary fibroblasts from the 3 diseases, though the tendency to display a more marked phenotype in HGPS was less obvious than in primary cells. This is probably related to the earlier onset of disease in HGPS patients and the moment in which the primary cells were collected. Likewise, we exposed primary and secondary fibroblasts to electrical stimulation because this physical stress has been shown to increase nuclear blebbing and apoptosis in vascular smooth muscle cells derived from HGPS iPSCs [19]. Primary and secondary fibroblasts displayed higher basal apoptosis compared to controls and there was synergistic increase upon electrical stimulation without significant bias among the 3 laminophaties (Figure 5F-G). Other stresses like hypoxia or gamma irradiation showed no noticeable difference between normal and diseased secondary fibroblasts (data not shown). In addition, we did not detect differential apoptosis after electrical stimulation between normal and affected iPSCs (data not shown), in agreement with their low levels of lamin A/C. Hence, reprogramming of somatic cells with mutations in lamin A/C reverses their abnormal phenotype thanks to reduction in protein expression, but differentiation back into the original donor cell type restores the alteration.


Generation of induced pluripotent stem cell lines from 3 distinct laminopathies bearing heterogeneous mutations in lamin A/C.

Ho JC, Zhou T, Lai WH, Huang Y, Chan YC, Li X, Wong NL, Li Y, Au KW, Guo D, Xu J, Siu CW, Pei D, Tse HF, Esteban MA - Aging (Albany NY) (2011)

Nuclear abnormalities are restored in iPSC-derived fibroblasts containing mutations in lamin A/C(A) Immunofluorescence photographs show expression of fibronectin and vimentin, and distribution of actin in stress fibers, in iPSC-derived fibroblasts from the 3 diseases. Abnormalities of the nuclear membrane are shown on the right panels. Scale bars indicate 10 μm (lamin A/C) and 100 μm (rest). (B) Western blot for lamin A/C in iPSC clones from the 3 diseases and the derived secondary fibroblasts. (C) Quantification of cells displaying abnormal nuclear membrane by immunofluorescence microscopy. The mean of 3 independent experiments +/− SD is shown. (D) Electron microscopy photographs show abnormalities of the nuclear membrane in more detail for all types of secondary fibroblasts. Arrows point to more obvious defects. Scale bars indicate 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117453&req=5

Figure 4: Nuclear abnormalities are restored in iPSC-derived fibroblasts containing mutations in lamin A/C(A) Immunofluorescence photographs show expression of fibronectin and vimentin, and distribution of actin in stress fibers, in iPSC-derived fibroblasts from the 3 diseases. Abnormalities of the nuclear membrane are shown on the right panels. Scale bars indicate 10 μm (lamin A/C) and 100 μm (rest). (B) Western blot for lamin A/C in iPSC clones from the 3 diseases and the derived secondary fibroblasts. (C) Quantification of cells displaying abnormal nuclear membrane by immunofluorescence microscopy. The mean of 3 independent experiments +/− SD is shown. (D) Electron microscopy photographs show abnormalities of the nuclear membrane in more detail for all types of secondary fibroblasts. Arrows point to more obvious defects. Scale bars indicate 1 μm.
Mentions: To evaluate the potential of our iPSC cell lines for understanding the complexity of the 3 laminopathies, we differentiated them into secondary fibroblasts. The acquisition of mesenchymal-like characteristics in the iPSC-derived fibroblasts was validated by immunofluorescence microscopy for actin stress fibers, the intermediate filament vimentin and the extracellular matrix protein fibronectin. All secondary fibroblasts, including those from a non-affected iPSC clone, displayed the expected staining pattern (Figure 4A). Western blotting verified the up-regulation of lamin A/C compared to iPSCs (Figure 4B), and the acquisition of nuclear blebbing was demonstrated by immunofluorescence (Figure 4A and C) and electron microscopy (Figure 4D). Notably, the latter technique reproduced the more subtle differences in pore size and membrane thickening observed with the primary cells. Then we analyzed whether these changes were associated with cell senescence (Figure 5A-C) and slow proliferation (Figure 5D-E), as observed with the respective primary fibroblasts. Both parameters were significantly increased in secondary fibroblasts from the 3 diseases, though the tendency to display a more marked phenotype in HGPS was less obvious than in primary cells. This is probably related to the earlier onset of disease in HGPS patients and the moment in which the primary cells were collected. Likewise, we exposed primary and secondary fibroblasts to electrical stimulation because this physical stress has been shown to increase nuclear blebbing and apoptosis in vascular smooth muscle cells derived from HGPS iPSCs [19]. Primary and secondary fibroblasts displayed higher basal apoptosis compared to controls and there was synergistic increase upon electrical stimulation without significant bias among the 3 laminophaties (Figure 5F-G). Other stresses like hypoxia or gamma irradiation showed no noticeable difference between normal and diseased secondary fibroblasts (data not shown). In addition, we did not detect differential apoptosis after electrical stimulation between normal and affected iPSCs (data not shown), in agreement with their low levels of lamin A/C. Hence, reprogramming of somatic cells with mutations in lamin A/C reverses their abnormal phenotype thanks to reduction in protein expression, but differentiation back into the original donor cell type restores the alteration.

Bottom Line: More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them.These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts.Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, China.

ABSTRACT
The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.

Show MeSH
Related in: MedlinePlus