Background Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics. Methods We directly compared the p.β-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines. Results Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.β-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs. Conclusions Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a ‘one-size fits all’ treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy.

Stem cell therapy has emerged as a promising alternative for replacing lost cells involved in neurodegenerative diseases. High efficiency of differentiation and full cell viability are actual challenges to achieve the translation of cell therapies to the clinic. To address this, the construction of aqueous two-phase systems in three-dimensional (ATPS-3D) cultures has been proposed. This technique involves the combination of two polymers in which cells are confined in dextran droplets immersed over a substrate located in a poly(ethylene glycol) phase. The controlled placement of cells in a defined pattern promotes intercellular communication. This review aims to provide insight into the techniques used to enhance neural differentiation and current challenges to achieve the implementation of cell therapies. Cell density, colony size, interconnectivity and an appropriate substrate to modulate paracrine signaling are factors that determine neural differentiation efficiency during the construction of ATPS-3D cultures. Hence, this contact-free technique enables the design of neural niches to recapitulate in vivo environments more accurately.

Stem cells are characterized by their self‐renewal and differentiation potential, emerging as a promising strategy for developing cell therapies to treat degenerative diseases. Stem cell culture under in vitro conditions involves the addition of growth factors to the media to stimulate their proliferation. However, most of these growth factors are of animal origin, hindering the implementation of the cultured cells into the clinic. Therefore, the replacement of animal‐derived growth factors for plant‐derived compounds has been proposed to accomplish the guidelines of good manufacturing practices. Plants are rich sources of bioactive compounds implicated in mainly anti‐inflammatory, anti‐oxidative and immunomodulatory mechanisms. Thus, for many years they have been used to prevent and treat many human diseases. Regarding stem cell culture, plant‐derived compounds act as regulators of signaling pathways involved in proliferation; therefore, their use as supplements in culture media represents a lower cost alternative with greater reproducibility to potentialize cell proliferation under in vitro conditions. Hence, this review aims to discuss plant‐derivative effects on the proliferation of stem cells with special interest in three main mechanisms of actions: (i) growth factors and their proliferation signaling effect, (ii) mitogens and their cell‐cycle regulation effect, and (iii) survival factors and their anti‐apoptotic effect. © 2021 Society of Chemical Industry (SCI).

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BACKGROUND: Cell-based high-throughput platforms using aqueous two-phase systems (ATPS) have led to the construction of 3D cultures using ATPS in which stem cells are confined into polymer-polymer droplets, giving rise to viable spheroids for their further proliferation and differentiation. This work aims to characterize polymer-polymer ATPS droplet generation based on diameter size, morphology and uniformity, using basic laboratory equipment.RESULTS: Dextran (DEX), including DEX 500 000 and 70 000 g mol −1 , as well as UCON were used as the droplet phase (0.5, 1, 2 ∼L). Polyethylene glycol (PEG), including PEG 8 000, 10 000 and 35 000 g mol −1 , as well as DEX 75 000 g mol −1 and Ficoll were used as the bulk phase (50, 100 ∼L). Different ATPS compositions (molecular weights and concentrations) with phosphate-buffered saline as ATPS solvent and construction strategies (droplet added, immersed or covered) were evaluated in 96-well plates. ATPS droplets were generated in PEG-DEX ATPS. Diameter size and morphology were susceptible to ATPS compositions, construction strategies and DEX droplet phase volumes with no significant difference in PEG bulk phase volumes, whereas higher uniformity of ATPS droplets was correlated to lower PEG bulk volumes. Therefore, 1 ∼L DEX droplets immersed at the bottom of a well filled with 50 ∼L PEG resulted in uniform ATPS droplets with a mid-range diameter of 1.35 ± 0.23 mm. CONCLUSIONS: According to the results, this work demonstrates simplified and practical alternatives for the generation of reproducible PEG-DEX ATPS droplets that could be adapted for the encapsulation of different cell types for further 3D culture applications.