Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes

Nijak, Aleksandra; Simons, Eline; Vandendriessche, Bert; Sande, Dieter Van de; Fransén, Erik; Sieliwonczyk, Ewa; Gucht, Ilse Van; Craenenbroeck, Emeline Van; Saenen, Johan; Heidbüchel, Hein; Ponsaerts, Peter; Labro, Alain J.; Snyders, Dirk J.; Vos, Winnok H. De; Schepers, Dorien; Alaerts, Maaike; Loeys, Bart

doi:10.1242/bio.059016

Cited by 4 publications

(2 citation statements)

References 55 publications

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“…Reprogramming to iPSCs was performed with the CytoTune™-iPS 2.0 Sendai Reprogramming Kit and Essential 8 Flex media (Life Technologies), according to the manufacturers’ protocol. Immunocytochemistry staining of the iPSCs showed the presence of pluripotency markers Nanog, Oct4, Tra1-60 and Tra1-81 and RT-PCR proven absence of Sendai virus vectors [ 18 ]. Trilineage differentiation potential of the iPSCs was confirmed by embryoid body formation followed by qPCR showing expression of endoderm, mesoderm and ectoderm markers.…”

Section: Methodsmentioning

confidence: 99%

“…Differentiation to iPSC-CMs was performed according to a protocol adapted from Burridge et al [ 18 , 19 ]. Briefly, mesodermal differentiation was induced by CHIR99021 (6 μM, Axon Medchem) in cardiomyocyte medium consisting of RPMI1640 (Life Technologies) media supplemented with 2% B27 supplement without insulin (Life Technologies) on day 0 for 48 h. On day 2, cardiomyocyte medium supplemented with Wnt-C59 (2 μM, SelleckChem) was added to the cells for 48 h. Next, cells were maintained in cardiomyocyte medium, with addition of T3 starting from day 8.…”

Section: Methodsmentioning

confidence: 99%

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Clinical and functional characterisation of a recurrent KCNQ1 variant in the Belgian population

Sieliwonczyk

Alaerts

Simons

et al. 2023

Orphanet J Rare Dis

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Background The c.1124_1127delTTCA p.(Ile375Argfs*43) pathogenic variant is the most frequently identified molecular defect in the KCNQ1 gene in the cardiogenetics clinic of the Antwerp University Hospital. This variant was observed in nine families presenting with either Jervell-Lange-Nielsen syndrome or long QT syndrome (LQTS). Here, we report on the molecular, clinical and functional characterization of the KCNQ1 c.1124_1127delTTCA variant. Results Forty-one heterozygous variant harboring individuals demonstrated a predominantly mild clinical and electrophysiological phenotype, compared to individuals harboring other KCNQ1 pathogenic variants (5% symptomatic before 40 years of age, compared to 24% and 29% in p.(Tyr111Cys) and p.(Ala341Val) variant carriers, respectively, 33% with QTc ≤ 440 ms compared to 10% in p.(Tyr111Cys) and p.(Ala341Val) variant carriers). The LQTS phenotype was most comparable to that observed for the Swedish p.(Arg518*) founder mutation (7% symptomatic at any age, compared to 17% in p.(Arg518*) variant carriers, 33% with QTc ≤ 440 ms compared to 16% in p.(Arg518*) variant carriers). Surprisingly, short tandem repeat analysis did not reveal a common haplotype for all families. One KCNQ1 c.1124_1127delTTCA harboring patient was diagnosed with Brugada syndrome (BrS). The hypothesis of a LQTS/BrS overlap syndrome was supported by electrophysiological evidence for both loss-of-function and gain-of-function (acceleration of channel kinetics) in a heterologous expression system. However, BrS phenotypes were not identified in other affected individuals and allelic KCNQ1 expression testing in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) showed nonsense mediated decay of the c.1124_1127delTTCA allele. Conclusions The c.1124_1127delTTCA frameshift variant shows a high prevalence in our region, despite not being confirmed as a founder mutation. This variant leads to a mild LQTS phenotype in the heterozygous state. Despite initial evidence for a gain-of-function effect based on in vitro electrophysiological assessment in CHO cells and expression of the KCNQ1 c.1124_1127delTTCA allele in patient blood cells, additional testing in iPSC-CMs showed lack of expression of the mutant allele. This suggests haploinsufficiency as the pathogenic mechanism. Nonetheless, as inter-individual differences in allele expression in (iPSC-) cardiomyocytes have not been assessed, a modifying effect on the BrS phenotype through potassium current modulation cannot be excluded.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Clinical and functional characterisation of a recurrent KCNQ1 variant in the Belgian population

Sieliwonczyk

Alaerts

Simons

et al. 2023

Orphanet J Rare Dis

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A generated induced pluripotent stem cell (iPSC) line (CMGANTi005-A) of a Marfan syndrome patient with an FBN1 c.7754T > C (p.Ile2585Thr) variation

et al. 2023

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In situ monolayer patch clamp of acutely stimulated human iPSC-derived cardiomyocytes promotes consistent electrophysiological responses to SK channel inhibition

Butler,

Ascione,

Marrion

et al. 2024

Sci Rep

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Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent an in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit electrophysiological heterogeneity which hinders their utility in the study of certain cardiac currents. In the healthy adult heart, the current mediated by small conductance, calcium-activated potassium (SK) channels (ISK) is atrial-selective. Functional expression of ISK within atrial-like iPSC-CMs has not been explored thoroughly. The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of ISK. iPSCs were differentiated using retinoic acid (RA) to produce iPSC-CMs which exhibited an atrial-like phenotype (RA-iPSC-CMs). Only 18% of isolated RA-iPSC-CMs responded to SK channel inhibition by UCL1684 and isolated iPSC-CMs exhibited substantial cell-to-cell electrophysiological heterogeneity. This variability was significantly reduced by patch clamp of RA-iPSC-CMs in situ as a monolayer (iPSC-ML). A novel method of electrical stimulation was developed to facilitate recording from iPSC-MLs via In situ Monolayer Patch clamp of Acutely Stimulated iPSC-CMs (IMPASC). Using IMPASC, > 95% of iPSC-MLs could be paced at a 1 Hz. In contrast to isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to UCL1684, with APD50 being prolonged by 16.0 ± 2.0 ms (p < 0.0001; n = 12). These data demonstrate that in conjunction with IMPASC, RA-iPSC-MLs represent an improved model for the study of ISK. IMPASC may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs and in pharmacological studies.

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Morpho-functional comparison of differentiation protocols to create iPSC-derived cardiomyocytes

Cited by 4 publications

References 55 publications

Clinical and functional characterisation of a recurrent KCNQ1 variant in the Belgian population

Clinical and functional characterisation of a recurrent KCNQ1 variant in the Belgian population

A generated induced pluripotent stem cell (iPSC) line (CMGANTi005-A) of a Marfan syndrome patient with an FBN1 c.7754T > C (p.Ile2585Thr) variation

In situ monolayer patch clamp of acutely stimulated human iPSC-derived cardiomyocytes promotes consistent electrophysiological responses to SK channel inhibition

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