Generation of human iPS cell line ihFib3.2 from dermal fibroblasts

Mesquita, Fernanda; Kasai-Brunswick, Taís Hanae; Gubert, Fernanda; Borgonovo, Tamara; Silva-dos-Santos, Danubia; Araújo, Dayanada Silva de; Carvalho, Antônio Carlos Campos de; Carvalho, Adriana Bastos

doi:10.1016/j.scr.2015.09.001

Cited by 8 publications

(4 citation statements)

References 3 publications

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“…For spontaneous differentiation into the three embryonic germ layers, EBs were cultured in suspension for 7 days in basal culture medium. Then, EBs were transferred back to adherent plates and cultured for another 7 days for immunofluorescence 40 .…”

Section: Methodsmentioning

confidence: 99%

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Mesquita

Arantes

Kasai-Brunswick

et al. 2019

Sci Rep

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Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.

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Section: Methodsmentioning

confidence: 99%

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Mesquita

Arantes

Kasai-Brunswick

et al. 2019

Sci Rep

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“…Cell viability was determined by staining with Trypan blue and counting cells in a hemocytometer. 29 The cardiomyocytes generated in this study were obtained by differentiating the hiPSCs using the STEMdiff™ Cardiomyocyte Differentiation Kit (StemCell Technologies Inc., Vancouver, Canada) (ESI Fig. 1b †).…”

Section: Human Ipsc Expansion and Cardiac Differentiationmentioning

confidence: 99%

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes

Mesquita¹,

Morrissey²,

Lee³

et al. 2021

Biomater. Sci.

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“…Induced pluripotent stem cells derived cardiomyocytes were generated as summarized in Figure 1A . Two iPSC lineages (we used four independent clones for each cell line investigated) were used in this work, one derived from a healthy donor previously described by our group ( Mesquita et al, 2015 ), used as control, and the other, obtained from The Progeria Research Foundation/Cell and Tissue Bank, derived from a patient with HPGS. The experimental protocol was approved by the research ethics committee (IRB) of the National Institute of Cardiology under number 24138414.1.0000.5272.…”

Section: Methodsmentioning

confidence: 99%

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient

et al. 2022

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Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

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Generation of human iPS cell line ihFib3.2 from dermal fibroblasts

Abstract: The human ihFib3.2 iPS cell line was generated from dermal fibroblasts obtained from a healthy donor. Lentiviral particles were produced with the polycistronic hSTEMCCA vector with Oct4, Sox2, cMyc and Klf4 as reprogramming factors.

Cited by 8 publications

References 3 publications

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient

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