Time-regulated transcripts with the potential to modulate human pluripotent stem cell-derived cardiomyocyte differentiation

Abstract

Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising disease model, even though hiPSC-CMs cultured for extended periods display an undifferentiated transcriptional landscape. MiRNA–target gene interactions contribute to fine-tuning the genetic program governing cardiac maturation and may uncover critical pathways to be targeted. Methods We analyzed a hiPSC-CM public dataset to identify time-regulated miRNA–target gene interactions based on three logical steps of filtering. We validated this process in silico using 14 human and mouse public datasets, and further confirmed the findings by sampling seven time points over a 30-day protocol with a hiPSC-CM clone developed in our laboratory. We then added miRNA mimics from the top eight miRNAs candidates in three cell clones in two different moments of cardiac specification and maturation to assess their impact on differentiation characteristics including proliferation, sarcomere structure, contractility, and calcium handling. Results We uncovered 324 interactions among 29 differentially expressed genes and 51 miRNAs from 20,543 transcripts through 120 days of hiPSC-CM differentiation and selected 16 genes and 25 miRNAs based on the inverse pattern of expression (Pearson R-values < − 0.5) and consistency in different datasets. We validated 16 inverse interactions among eight genes and 12 miRNAs (Person R-values < − 0.5) during hiPSC-CMs differentiation and used miRNAs mimics to verify proliferation, structural and functional features related to maturation. We also demonstrated that miR-124 affects Ca2+ handling altering features associated with hiPSC-CMs maturation. Conclusion We uncovered time-regulated transcripts influencing pathways affecting cardiac differentiation/maturation axis and showed that the top-scoring miRNAs indeed affect primarily structural features highlighting their role in the hiPSC-CM maturation.

YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFβ uptake in human pluripotent stem cell-derived cardiomyocytes

“…Employing Traction Force Microscopy (TFM), we measured contractile force generation of single patterned hiPSC-CMs on physiological stiffness (10 kPa) polyacrylamide hydrogels (Supplement 2A) 57,58 . Isolated D28 hiPSC-CMs were cultured on hydrogels for 2 days prior to traction force measurements (performed on D30, standard maturity labeled by enhanced expression of MYH7 and TNNI3 and depletion of proliferative mark EdU 59 ). The TFM platform enabled the assessment of traction forces for 53-112 cardiomyocytes from three differentiation batches per cell line (four mutant and four respective control lines) 57 .…”

Engineering the maturation of stem cell-derived cardiomyocytes

“…After overexpression of let-7 family members in SC-CMs, cell size, sarcomere length, force of contraction and respiratory capacity were improved significantly ( Kuppusamy et al, 2015 ). Recently, a bioinformatic analysis of the SC-CM dataset uncovered the positive effect of miR-124 on SC-CM maturation ( Muñoz et al, 2022 ).…”

Extended voltage imaging in cardiomyocytes with a triplet state quencher-stabilized silicon rhodamine

“…1 Voltage sensitive indicators offer a complementary method for studying electrophysiology and have led to progress in whole-heart optical mapping and understanding of cardiac arrhythmias. 3,4 More recently, fluorescent indicators have been used in the characterization of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), [5][6][7][8] as well as in screening assays for arrhythmias and drug toxicity, including screening for COVID-therapeutics. [9][10][11][12] Fluorescence imaging of cardiac electrophysiology dates to 1976 with the study of frog hearts with merocyanine 540.…”