EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival.

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1-4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.