Enhancing the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes with an n-Type Organic Semiconductor Coating.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a promising cell source for cardiac regenerative medicine and in vitro modeling. However, hPSC-CMs exhibit immature structural and functional properties compared with adult cardiomyocytes. Various electrical, mechanical, and biochemical cues have been applied to enhance hPSC-CM maturation but with limited success. In this work, we investigated the potential application of the semiconducting polymer poly{[N,N'-bis(2-octyldodecyl)-naphthalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]-alt-5,5'-(2,2'-bithiophene)} (P(NDI2OD-T2)) as a light-sensitive material to stimulate hPSC-CMs optically. Our results indicated that P(NDI2OD-T2)-mediated photostimulation caused cell damage at irradiances applied long-term above 36 µW/mm2 and did not regulate cardiac monolayer beating (after maturation) at higher intensities applied in a transient fashion. However, we discovered that the cells grown on P(NDI2OD-T2)-coated substrates showed significantly enhanced expression of cardiomyocyte maturation markers in the absence of a light exposure stimulus. A combination of techniques, such as atomic force microscopy, scanning electron microscopy, and quartz crystal microbalance with dissipation monitoring, which we applied to investigate the interface of the cell with the n-type coating, revealed that P(NDI2OD-T2) impacted the nanostructure, adsorption, and viscoelasticity of the Matrigel coating used as a cell adhesion promoter matrix. This modified cellular microenvironment promoted the expression of cardiomyocyte maturation markers related to contraction, calcium handling, metabolism, and conduction. Overall, our findings demonstrate that conjugated polymers such as P(NDI2OD-T2) can be used as passive coatings to direct stem cell fate through interfacial engineering of cell growth substrates.

Protocol to measure calcium spikes in cardiomyocytes obtained from human pluripotent stem cells using a ready-to-use media.

The derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) is a powerful tool to investigate early cardiogenesis and model diseases in vitro. Here, we present an optimized protocol to obtain contracting hPSCs-derived cardiomyocytes using a ready-to-use kit. We describe steps for hPSC culture and differentiation to cardiomyocytes including the identification of key parameters such as starting cell confluency and temperature. We then detail immunofluorescence, flow cytometry, and the quantification of cardiomyocytes' calcium spikes using live imaging. For complete details on the use and execution of this protocol, please refer to Astro et al.1.

Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism.

The histone demethylase KDM1A is a multi-faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A-/- hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a-/- hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells.

Heart in a Dish: From Traditional 2D Differentiation Protocols to Cardiac Organoids.

Human pluripotent stem cells (hPSCs) constitute a valuable model to study the complexity of early human cardiac development and investigate the molecular mechanisms involved in heart diseases. The differentiation of hPSCs into cardiac lineages in vitro can be achieved by traditional two-dimensional (2D) monolayer approaches or by adopting innovative three-dimensional (3D) cardiac organoid protocols. Human cardiac organoids (hCOs) are complex multicellular aggregates that faithfully recapitulate the cardiac tissue's transcriptional, functional, and morphological features. In recent years, significant advances in the field have dramatically improved the robustness and efficiency of hCOs derivation and have promoted the application of hCOs for drug screening and heart disease modeling. This review surveys the current differentiation protocols, focusing on the most advanced 3D methods for deriving hCOs from hPSCs. Furthermore, we describe the potential applications of hCOs in the pharmaceutical and tissue bioengineering fields, including their usage to investigate the consequences of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV2) infection in the heart.

Delivery of Endothelial Cell-Laden Microgel Elicits Angiogenesis in Self-Assembling Ultrashort Peptide Hydrogels In Vitro.

Blood vessel generation is an essential process for tissue formation, regeneration, and repair. Notwithstanding, vascularized tissue fabrication in vitro remains a challenge, as current fabrication techniques and biomaterials lack translational potential in medicine. Naturally derived biomaterials harbor the risk of immunogenicity and pathogen transmission, while synthetic materials need functionalization or blending to improve their biocompatibility. In addition, the traditional top-down fabrication techniques do not recreate the native tissue microarchitecture. Self-assembling ultrashort peptides (SUPs) are promising chemically synthesized natural materials that self-assemble into three-dimensional nanofibrous hydrogels resembling the extracellular matrix (ECM). Here, we use a modular tissue-engineering approach, embedding SUP microgels loaded with human umbilical vein endothelial cells (HUVECs) into a 3D SUP hydrogel containing human dermal fibroblast neonatal (HDFn) cells to trigger angiogenesis. The SUPs IVFK and IVZK were used to fabricate microgels that gel in a water-in-oil emulsion using a microfluidic droplet generator chip. The fabricated SUP microgels are round structures that are 300-350 µm diameter in size and have ECM-like topography. In addition, they are stable enough to keep their original size and shape under cell culture conditions and long-term storage. When the SUP microgels were used as microcarriers for growing HUVECs and HDFn cells on the microgel surface, cell attachment, stretching, and proliferation could be demonstrated. Finally, we performed an angiogenesis assay in both SUP hydrogels using all SUP combinations between micro- and bulky hydrogels. Endothelial cells were able to migrate from the microgel to the surrounding area, showing angiogenesis features such as sprouting, branching, coalescence, and lumen formation. Although both SUP hydrogels support vascular network formation, IVFK outperformed IVZK in terms of vessel network extension and branching. Overall, these results demonstrated that cell-laden SUP microgels have great potential to be used as a microcarrier cell delivery system, encouraging us to study the angiogenesis process and to develop vascularized tissue-engineering therapies.

Optimization of a 3D bioprinting process using ultrashort peptide bioinks.

The field of three-dimensional (3D) bioprinting is rapidly emerging as an additive manufacturing method for tissue and organ fabrication. The demand for tissues and organ transplants is ever increasing, although donors are not as readily available. Consequently, tissue engineering is gaining much attention to alleviate this problem. The process of achieving well-structured 3D bioprinted constructs using hydrogel bioinks depends on symmetrical precision, regulated flow rates, and viability of cells. Even with the mentioned parameters optimized, the printed structures need additional refining by removing excessive liquids, as peptide hydrogel bioprints encapsulate water. However, it is challenging to eliminate the confined fluids without compromising the printing process. In this paper, we introduced a vacuum system to our 3D bioprinting robotic arm and thus optimized the printing quality for complex and refined 3D scaffolds. Moreover, the proposed vacuum system supports printing with cells. Our results show improved printing resolution which facilitates the printing of higher and more stable structures.

Novel antimalarial chloroquine- and primaquine-quinoxaline 1,4-di-N-oxide hybrids: Design, synthesis, Plasmodium life cycle stage profile, and preliminary toxicity studies.

Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di-N-oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii, Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di-N-oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei, respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di-N-oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di-N-oxide hybrids as new potential dual-acting antimalarials for further investigation.

New hydrazine and hydrazide quinoxaline 1,4-di-N-oxide derivatives: In silico ADMET, antiplasmodial and antileishmanial activity.

We report the design (in silico ADMET criteria), synthesis, cytotoxicity studies (HepG-2 cells), and biological evaluation of 15 hydrazine/hydrazide quinoxaline 1,4-di-N-oxide derivatives against the 3D7 chloroquine sensitive strain and FCR-3 multidrug resistant strain of Plasmodium falciparum and Leishmania infantum (axenic amastigotes). Fourteen of derivatives are novel quinoxaline 1,4-di-N-oxide derivatives. Compounds 18 (3D7 IC50=1.40µM, FCR-3 IC50=2.56µM) and 19 (3D7 IC50=0.24µM, FCR-3 IC50=2.8µM) were identified as the most active against P. falciparum, and they were the least cytotoxic (CC50-values>241µM) and most selective (SI>86). None of the compounds tested against L. infantum were considered to be active. Additionally, the functional role of the hydrazine and hydrazide structures were studied in the quinoxaline 1,4-di-N-oxide system.