Improving cardiac differentiation of human pluripotent stem cells by targeting ferroptosis.

Generation of cardiomyocytes from human pluripotent stem cells (hPSCs) is of high interest for disease modelling and regenerative medicine. hPSCs can provide an unlimited source of patient-specific cardiomyocytes that are otherwise difficult to obtain from individuals. Moreover, the low proliferation rate of adult cardiomyocytes and low viability ex vivo limits the quantity of study material. Most protocols for the differentiation of cardiomyocytes from hPSCs are based on the temporal modulation of the Wnt pathway. However, during the initial stage of GSK-3 inhibition, a substantial number of cells are lost due to detachment. In this study, we aimed to increase the efficiency of generating cardiomyocytes from hPSCs. We identified cell death as a detrimental factor during this initial stage of in vitro cardiomyocyte differentiation. Through pharmacological targeting of different types of cell death, we discovered that ferroptosis was the main cell death type during the first 48 h of the in vitro differentiation procedure. Inhibiting ferroptosis using ferrostatin-1 during cardiomyocyte differentiation resulted in increased robustness and cell yield.

Three-dimensional co-culturing of stem cell-derived cardiomyocytes and cardiac fibroblasts reveals a role for both cell types in Marfan-related cardiomyopathy.

Pathogenic variants in the FBN1 gene, which encodes the extracellular matrix protein fibrillin-1, cause Marfan syndrome (MFS), which affects multiple organ systems, including the cardiovascular system. Myocardial dysfunction has been observed in a subset of patients with MFS and in several MFS mouse models. However, there is limited understanding of the intrinsic consequences of FBN1 variants on cardiomyocytes (CMs). To elucidate the CM-specific contribution in Marfan's cardiomyopathy, cardiosphere cultures of CMs and cardiac fibroblasts (CFs) are used. CMs and CFs were derived by human induced pluripotent stem cell (iPSC) differentiation from MFS iPSCs with a pathogenic variant in FBN1 (c.3725G>A; p.Cys1242Tyr) and the corresponding CRISPR-corrected iPSC line (Cor). Cardiospheres containing MFS CMs show decreased FBN1, COL1A2 and GJA1 expression. MFS CMs cultured in cardiospheres have fewer binucleated CMs in comparison with Cor CMs. 13% of MFS CMs in cardiospheres are binucleated and 15% and 16% in cardiospheres that contain co-cultures with respectively MFS CFs and Cor CFs, compared to Cor CMs, that revealed up to 23% binucleation when co-cultured with CFs. The sarcomere length of CMs, as a marker of development, is significantly increased in MFS CMs interacting with Cor CF or MFS CF, as compared to monocultured MFS CMs. Nuclear blebbing was significantly more frequent in MFS CFs, which correlated with increased stiffness of the nuclear area compared to Cor CFs. Our cardiosphere model for Marfan-related cardiomyopathy identified a contribution of CFs in Marfan-related cardiomyopathy and suggests that abnormal early development of CMs may play a role in the disease mechanism.

Photoporation-mediated spatial intracellular delivery of stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are promising candidates for disease modeling and therapeutic purposes, however, non-viral intracellular delivery in these cells remains challenging. Gold nanoparticle (AuNP)-sensitized photoporation creates transient pores in the cell membrane by vapor nanobubble formation, allowing diffusion of extracellular biomolecules. This non-viral technique was employed to test and optimize its distinct physical mode of action in iPSC-CMs. Photoporation optimization was aimed at achieving high delivery rates while minimizing cell death. Various AuNP concentrations, in conjunction with different laser fluences, were explored to facilitate the intracellular delivery of 10 kDa and 150 kDa FITC-labelled dextran as model macromolecules. Cardiomyocyte viability was assessed using the CellTiter-Glo® viability assay, while the delivery efficiency was quantified through flow cytometry. On 30 day-old cardiomyocytes, AuNP photoporation was able to yield ∼60 % delivery efficiency while maintaining a high cell viability (∼70 %). Overall, higher AuNP concentrations resulted in greater delivery efficiencies, albeit at the expense of lower cell viability. Finally, photoporation was capable of patterning a geometric shape, demonstrating its exceptional selective resolution in delivering molecules to spatially restricted regions of the cell culture. In conclusion, AuNP-photoporation exhibits considerable potential as an effective and gentle non-viral method for intracellular delivery in iPSC-CMs.•AuNP-photoporation is a non-viral intracellular delivery method suitable for iPSC-CMs with high efficiency and cell viability•This method is capable of spatially resolved intracellular delivery with excellent resolution.

Generation of human induced pluripotent stem cell line UGENTi001-A from a patient with Marfan syndrome carrying a heterozygous c.7754 T > C variant in FBN1 and the isogenic control UGENT001-A-1 using CRISPR/Cas9 editing.

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.

Photothermal nanofibres enable safe engineering of therapeutic cells.

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.

Use of Transparent Liquid Marble: Microbioreactor to Culture Cardiospheres.

Cells have a remarkable ability to self-organize and rearrange in functional organoids, this process was greatly boosted by the recent advances in 3D culture technologies and materials. Presently, this approach can be applied to model human organ development and function "in a dish" and can be used to predict drug response in a patient specific fashion.Here we describe a protocol that allows for the derivation of functional cardiac mini organoids consisting of cocultured cardiomyocytes and cardiac fibroblast. Cells are suspended in a drop of medium and encapsulated with hydrophobic fumed silica powder nanoparticles. These nanoparticles are treated with hydrophobic chemicals, hexamethyldisilazane (nHMDS), and result in the formation of microbioreactors. These microenvironments are defined as "liquid marbles," stimulating cell coalescence and 3D aggregation. Then nHMDS shell ensures optimal gas exchange between the interior liquid and the surrounding environment. This microbioreactor makes working in smaller volumes possible and is therefore amenable for higher throughput applications. Moreover, the properties of liquid marble microbioreactors makes it an excellent culture technique for cocultures. Here we demonstrate how cocultures of cardiac fibroblast and cardiomyocytes in a cardiosphere can be a valuable tool to model cardiac diseases in vitro and to assess cell interactions to decipher disease mechanisms.

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome.

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.

Liquid marble technology to create cost-effective 3D cardiospheres as a platform for in vitro drug testing and disease modelling.

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.