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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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Increased senescence and susceptibility to apoptosis in iPSC-derived fibroblasts containing lamin A/C mutations(A) Phase contrast photographs after β-galactosidase staining of primary and secondary fibroblasts from the 3 diseases. Samples were measured in triplicate. Scale bars indicate 100 μm. (B-C) Quantification of β-galactosidase positive cells. Secondary fibroblasts from H1 ESCs were included (also in E). The mean of 3 independent experiments +/− SD is shown (also in F-G). * indicates p value <0.05. (D-E) Proliferation of the indicated cell types measured at different time points; a representative experiment is shown. (F) Electrical stimulation (E-Stim) significantly elevated the apoptotic rate in primary fibroblasts of the 3 patients compared with control fibroblasts as assessed by Annexin V apoptotic assay. (G) A similar trend of induced apoptosis was observed after electrical stimulation of the secondary fibroblasts from the 3 diseases.
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Figure 5: Increased senescence and susceptibility to apoptosis in iPSC-derived fibroblasts containing lamin A/C mutations(A) Phase contrast photographs after β-galactosidase staining of primary and secondary fibroblasts from the 3 diseases. Samples were measured in triplicate. Scale bars indicate 100 μm. (B-C) Quantification of β-galactosidase positive cells. Secondary fibroblasts from H1 ESCs were included (also in E). The mean of 3 independent experiments +/− SD is shown (also in F-G). * indicates p value <0.05. (D-E) Proliferation of the indicated cell types measured at different time points; a representative experiment is shown. (F) Electrical stimulation (E-Stim) significantly elevated the apoptotic rate in primary fibroblasts of the 3 patients compared with control fibroblasts as assessed by Annexin V apoptotic assay. (G) A similar trend of induced apoptosis was observed after electrical stimulation of the secondary fibroblasts from the 3 diseases.

Mentions: We characterized selected iPSC colonies for all 3 diseases by standard procedures [26]. They stained positively for markers such as alkaline phosphatase (AP), SSEA-4, TRA-1-60, TRA-1-81, and the transcription factor Nanog (Figure 2A). Moreover, they displayed normal nuclear morphology as assessed by immunofluorescence for the nuclear membrane marker LAP2 (Lamina-Associated Polypeptide 2) (Figure 2A). This correlated with high expression of ESC-transcription factors (endogenous Oct4 and Sox2, plus Nanog) and hTERT (human telomerase reverse transcriptase) comparable to human ESCs and iPSCs produced from fibroblasts of a normal individual (Figure 2B). However, lamin A/C mRNA levels were much lower in all pluripotent cell lines than in control fibroblasts (Figure 2C). The latter may explain why DCM and aWS fibroblasts, and with lower efficiency also HGPS fibroblasts, could be reprogrammed in spite of their slow proliferation and increased senescence relative to the donor cells (see below Figure 5). Indeed, ESCs are known to have low expression of lamin A/C, which increases sharply upon differentiation [27]. Besides, others [19] and we (data not shown) have observed reduction of lamin A/C mRNA early after transduction with the exogenous factors, likely allowing the recovery of normal nuclear morphology after the initial phase of reprogramming. The diseased iPSCs also displayed normal karyotype (Figure 3A), had low levels of CpG methylation in the proximal Oct4 promoter (Figure 3B), and produced derivatives of the 3 germ layers when growth as teratomas in immunocompromised mice (Figure 3C). Therefore, we have successfully produced pluripotent cell lines by reprogramming of somatic cells from 3 independent diseases associated with mutations in lamin A/C mutations and they seem undistinguishable from normal ESCs/iPSCs.


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)

Increased senescence and susceptibility to apoptosis in iPSC-derived fibroblasts containing lamin A/C mutations(A) Phase contrast photographs after β-galactosidase staining of primary and secondary fibroblasts from the 3 diseases. Samples were measured in triplicate. Scale bars indicate 100 μm. (B-C) Quantification of β-galactosidase positive cells. Secondary fibroblasts from H1 ESCs were included (also in E). The mean of 3 independent experiments +/− SD is shown (also in F-G). * indicates p value <0.05. (D-E) Proliferation of the indicated cell types measured at different time points; a representative experiment is shown. (F) Electrical stimulation (E-Stim) significantly elevated the apoptotic rate in primary fibroblasts of the 3 patients compared with control fibroblasts as assessed by Annexin V apoptotic assay. (G) A similar trend of induced apoptosis was observed after electrical stimulation of the secondary fibroblasts from the 3 diseases.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Increased senescence and susceptibility to apoptosis in iPSC-derived fibroblasts containing lamin A/C mutations(A) Phase contrast photographs after β-galactosidase staining of primary and secondary fibroblasts from the 3 diseases. Samples were measured in triplicate. Scale bars indicate 100 μm. (B-C) Quantification of β-galactosidase positive cells. Secondary fibroblasts from H1 ESCs were included (also in E). The mean of 3 independent experiments +/− SD is shown (also in F-G). * indicates p value <0.05. (D-E) Proliferation of the indicated cell types measured at different time points; a representative experiment is shown. (F) Electrical stimulation (E-Stim) significantly elevated the apoptotic rate in primary fibroblasts of the 3 patients compared with control fibroblasts as assessed by Annexin V apoptotic assay. (G) A similar trend of induced apoptosis was observed after electrical stimulation of the secondary fibroblasts from the 3 diseases.
Mentions: We characterized selected iPSC colonies for all 3 diseases by standard procedures [26]. They stained positively for markers such as alkaline phosphatase (AP), SSEA-4, TRA-1-60, TRA-1-81, and the transcription factor Nanog (Figure 2A). Moreover, they displayed normal nuclear morphology as assessed by immunofluorescence for the nuclear membrane marker LAP2 (Lamina-Associated Polypeptide 2) (Figure 2A). This correlated with high expression of ESC-transcription factors (endogenous Oct4 and Sox2, plus Nanog) and hTERT (human telomerase reverse transcriptase) comparable to human ESCs and iPSCs produced from fibroblasts of a normal individual (Figure 2B). However, lamin A/C mRNA levels were much lower in all pluripotent cell lines than in control fibroblasts (Figure 2C). The latter may explain why DCM and aWS fibroblasts, and with lower efficiency also HGPS fibroblasts, could be reprogrammed in spite of their slow proliferation and increased senescence relative to the donor cells (see below Figure 5). Indeed, ESCs are known to have low expression of lamin A/C, which increases sharply upon differentiation [27]. Besides, others [19] and we (data not shown) have observed reduction of lamin A/C mRNA early after transduction with the exogenous factors, likely allowing the recovery of normal nuclear morphology after the initial phase of reprogramming. The diseased iPSCs also displayed normal karyotype (Figure 3A), had low levels of CpG methylation in the proximal Oct4 promoter (Figure 3B), and produced derivatives of the 3 germ layers when growth as teratomas in immunocompromised mice (Figure 3C). Therefore, we have successfully produced pluripotent cell lines by reprogramming of somatic cells from 3 independent diseases associated with mutations in lamin A/C mutations and they seem undistinguishable from normal ESCs/iPSCs.

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