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Marfan syndrome is an autosomal dominant genetic disorder resulting from pathogenic variants in FBN1 gene. FBN1 encodes for fibrillin-1, an important extracellular matrix protein. Impaired fibrillin-1 affects multiple organ systems, including the cardiovascular system. We generated an iPSC line carrying a heterozygous variant c.7754 T > C (p.Ile2585Thr, missense) in FBN1 from a patient with Marfan syndrome. Also, an isogenic control is generated, where the pathogenic variant is repaired using CRISPR-Cas9. This isogenic pair provides a valuable resource for in vitro disease modelling.

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS.

Three-dimensional (3D) cell culturing has several advantages over 2D cultures. 3D cell cultures more accurately mimic the in vivo environment, which is vital to obtain reliable results in disease modelling and toxicity testing. With the introduction of the Yamanaka factors, reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) became available. This iPSC technology provides a scalable source of differentiated cells. iPSCs can be programmed to differentiate into any cell type of the body, including cardiomyocytes. These heart-specific muscle cells, can then serve as a model for therapeutic drug screening or assay development. Current methods to achieve multicellular spheroids by 3D cell cultures, such as hanging drop and spinner flasks are expensive, time-consuming and require specialized materials and training. Hydrophobic powders can be used to create a micro environment for cell cultures, which are termed liquid marbles (LM). In this procedure we describe the first use of the LM technology for 3D culturing in vitro derived human cardiomyocytes which results in the formation of cardiospheres within 24h. The cardiospheres could be used for several in depth and high-throughput analyses., Graphical abstract Image, graphical abstract

Nanoparticle-mediated photoporation is used to temporarily permeabilize cell membranes for intracellular delivery of macromolecules, but cell exposure to nanoparticles might cause cellular damage and hamper application of the technique to therapeutic cell engineering. Here the authors show that, under photothermal heating, nanofibre-embedded iron oxide nanoparticles can be used to deliver effector macromolecules to different types of cells, in a contactless manner, with no cellular toxicity or diminished therapeutic potency. Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR-Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.