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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. © 2020 Society of Chemical Industry (SCI)

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., Graphical abstract Unlabelled Image, Highlights • Isogenic models of hiPSC-CMs bearing sarcomeric mutations address HCM complexity. • Calcium reporter lines and optogenetics enabled refined phenotypic analyses of HCM. • Different mutations showed phenotypic and mechanistic similarities and differences. • Arrhythmias in both models were rescued by combining ranolazine and dantrolene. • Variable phenotypes support mechanism-driven development of effective therapeutics.