Significance Stem-cell microenvironment has been identified as an important modulator of plasticity, self-renewal, and differentiation. This work details the development of a hydrogel system tailored to promote human pluripotent stem cell (HPSC) self-renewal with a simple chemical microenvironmental switch to direct differentiation. Furthermore, the timing of switching post hydrogel fabrication can promote specific lineage differentiation as in vivo. This system highlights the role of microenvironment on fate choices of pluripotent cells and demonstrates that it may be tailored to control differentiation in vitro. Importantly, this approach may improve the generation of fully differentiated tissues, as demonstrated for cardiogenic differentiation. Our combination of hydrogels allows dense tissue structures to be produced from HPSCs by using a single-step process inaccessible to any current methodology.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

The use of materials to impose tissue-like architecture at cell resolution will be important if engineered functional replacements for damaged cardiovascular, pulmonary, renal or digestive tissues are to be authentically engineered. Here, we demonstrate a coordinated system for the fabrication and subsequent culture of tubular tissues composed of multiple layers, cell-types and materials with physiological dimensions and defined architectures at cell resolution. We developed an automated tube fabricator that rolls 2D-matrices into 3D-tubular constructs directly from cells, hydrogels and scaffold biomaterials. Coordinated use of surface modification strategies allows 2D cell sheets and cell/biomaterial composites (i.e. hydrogels or electrospun scaffolds) to be fabricated which may be transferred into a perfusion bioreactor in a rapid and standardized procedure. To exemplify our strategy we fabricated structures resembling human mammary artery and gut; these can be imaged in situ and real-time electrical resistance measurements performed of the vessel walls, allowing non-invasive assessment of viability and functionality. Our system allows patterning at cellular resolution with variable tissue thickness, length, luminal diameter, and constituent biomaterial. This inherent flexibility will allow the recapitulation of the complex hierarchical biological architectures and generate functionality found natively in vivo.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

Long QT syndrome (LQTS) is characterized by a prolonged QT-interval on electrocardiogram and by increased risk of sudden death. One of the most common and potentially life-threatening electrolyte disturbances is hypokalemia, characterized by low concentrations of K+. Using a multielectrode array platform and current clamp technique, we investigated the effect of low extracellular K+ concentration ([K+]Ex) on the electrophysiological properties of hiPSC-derived cardiomyocytes (CMs) generated from a healthy control subject (WT) and from two symptomatic patients with type 1 of LQTS carrying G589D (LQT1A) or IVS7-2A>G mutation (LQT1B) in KCNQ1. The baseline prolongations of field potential durations (FPDs) and action potential durations (APDs) were longer in LQT1-CMs than in WT-CMs. Exposure to low [K+]Ex prolonged FPDs and APDs in a concentration-dependent fashion. LQT1-CMs were found to be more sensitive to low [K+]Ex compared to WT-CMs. At baseline, LQT1A-CMs had more prolonged APDs than LQT1B-CMs, but low [K+]Ex caused more pronounced APD prolongation in LQT1B-CMs. Early afterdepolarizations in the action potentials were observed in a subset of LQT1A-CMs with further prolonged baseline APDs and triangular phase 2 profiles. This work demonstrates that the hiPSC-derived CMs are sensitive to low [K+]Ex and provide a platform to study acquired LQTS.

Microelectrode arrays (MEAs) are widely used to assess the electrophysiology of human pluripotent stem cell-derived cardiomyocytes (hPS-CMs). Traditionally, MEAs have been used to record data at the cell population level, but it would be beneficial to be able to analyze also at the single-cell level using MEAs. To realize this, we present a special MEA platform for recording field potential from single beating cardiomyocytes. The size and location of transparent indium tin oxide (ITO) electrodes have been optimized to make noninvasive studies of the electrophysiological activity of cardiomyocytes at the single-cell level possible and also to enable simultaneous video imaging through transparent electrodes and thus image-based analysis of the mechanical beating behavior of the same cardiomyocytes. Because of these characteristics, this novel platform provides a powerful tool for assessing the functionality of cardiomyocytes in basic cardiac research, disease modeling, as well as drug development and toxicology.

We study complex scaling properties of RR and QT intervals of electrocardiograms (ECGs) with their equivalences at the cellular level, that is, inter-beat intervals (IBI) and field potential durations (FPD) of spontaneously beating human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) aggregates. Our detrended fluctuation analysis and Poincaré plots reveal remarkable similarities between the ECG and hiPSC-CM data. In particular, no statistically significant difference was found in the short- and long-term scaling exponents α 1 and α 2 of RR and QT intervals and their cellular equivalences. Previously unknown scaling properties of FPDs of hiPSC-CM aggregates reveal that the increasing scaling exponent of QT intervals as a function of the time scale, is an intrinsic feature at the cellular level.

Exosomes are small extracellular nanovesicles that are released by cells, and their potential has been explored for use in cosmetics, skin care, tissue regeneration, and dermatological diseases. The therapeutic value of exosomes lies in their ability to modulate the microenvironment of cells, regulate gene expression, and induce cell differentiation, which can have a positive impact on skin health. In terms of cosmetics, exosomes have been used to reduce wrinkles, improve skin texture and hydration, and enhance skin elasticity, as well as to reduce inflammation and damage caused by UV radiation. Furthermore, exosomes have been used to promote tissue regeneration in skin wounds and to treat dermatological diseases such as systemic lupus erythematosus, psoriasis, atopic dermatitis, systemic sclerosis, pigment regulation, vitiligo, and hair growth. In this review, the therapeutic value of exosomes in the field of cosmetics, skin care, tissue regeneration, and dermatological diseases, has been elaborated. The existing literature demonstrated that with further research, exosomes may become a viable therapeutic option for many skin conditions.