RESUMO
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.
Assuntos
Canal de Potássio ERG1/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Síndrome do QT Longo/genética , Mutação/genética , Miócitos Cardíacos/fisiologia , Transporte Proteico/genética , Potenciais de Ação/genética , Adolescente , Adulto , Membrana Celular/genética , Feminino , Edição de Genes/métodos , Humanos , Leucócitos Mononucleares/fisiologia , Masculino , Fenótipo , Adulto JovemRESUMO
Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Esclerose Lateral Amiotrófica/metabolismo , Brasil , Linhagem Celular , Humanos , Cariótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Induced pluripotent stem cell (iPSC) lines were generated from erythroblasts of two patients with amyotrophic lateral sclerosis (ALS) and two healthy individuals. One familial and one sporadic ALS patients were used, both with genetic alterations in VAPB gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the iPSC cell lines. The four iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.