Stefania Rapino

Human pluripotent stem cells (hPSCs), derived from embryos or reprogrammed from somatic cells, represent a key resource for preclinical research and personalized medicine due to their capacity for indefinite self-renewal and differentiation into all three germ layer derivatives. However, incomplete maturation of hPSC-derived cells limits their translational potential across multiple fields, including cardiovascular medicine. In particular, cardiomyocytes differentiated from hPSCs (hPSC-CMs), although valuable models to recapitulate physio-pathological traits of heart development and disease, retain fetal-like properties that constrain their utility in late-onset disease modelling, cell therapy and drug testing. While several strategies have been proposed to enhance maturation, all present some technical or biological limitations. Recently, optically active semiconductors, both organic and inorganic, have emerged as gene-free tools to modulate cellular function with high spatial and temporal precision. Here, we investigate the effects of long-term photostimulation using the far-red absorbing conjugated polymer (PCPDTBT) on hPSC-CMs. We show that 6-hour pulsed photostimulation significantly enhances maturation across transcriptional, functional, structural, and metabolic levels without inducing phototoxicity. Overall, our findings establish a simple and effective approach to improve hPSC-CM maturation, thereby expanding their applicability in disease modelling and therapeutic development.

Two-dimensional cell culture may be insufficient when it comes to understanding human disease. The redox behavior of complex, three-dimensional tissue is critical to understanding disease genesis and propagation. Unfortunately, few measurement tools are available for such three-dimensional models to yield quantitative insight into how reactive oxygen species (ROS) form over time. Here, we demonstrate an imaging platform for the real-time visualization of H₂O₂ formation for mammalian spheroids made of noncancerous human embryonic kidney cells (HEK-293) and metastatic breast cancer cells (MCF-7 and MDA-MB-231). We take advantage of the luminol and H₂O₂ electrochemiluminescence reaction on a transparent tin-doped indium oxide electrode. The luminescence of this reaction as a function of [H₂O₂] is linear (R ² = 0.98) with a dynamic range between 0.5 μM to 0.1 mM, and limit of detection of 2.26 ± 0.58 μM. Our method allows for the observation of ROS activity in growing spheroids days in advance of current techniques without the need to sacrifice the sample postanalysis. Finally, we use our procedure to demonstrate how key ROS pathways in cancerous spheroids can be up-regulated and downregulated through the addition of common metabolic drugs, rotenone and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Our results suggest that the Warburg Effect can be studied for single mammalian cancerous spheroids, and the use of metabolic drugs allows one to implicate specific metabolic pathways in ROS formation. We expect this diagnostic tool to have wide applications in understanding the real-time propagation of human disease in a system more closely related to human tissue.

Melanoma is a heterogeneous tumor composed of many interacting cellular populations and highly plastic melanoma cells that pass through distinct cell states to adapt to the surrounding microenvironment. Slow cycling is a transient state that defines a minor population of cells with cancer-initiating features. These cells are enriched upon drug therapy and can trigger cancer relapse and metastasis dissemination when they acquire proliferative potential. This population is still not entirely characterized. Here we provide evidence of the existence of a slow cycling melanoma population isolated in vivo from melanoma PDXs using the H2B-GFP system. These cells display a highly invasive phenotype and are able to dynamically respond to cancer microenvironmental stimuli. Single cell transcriptomic analysis unveils a significant transcriptional heterogeneity of GFP-retaining slow cycling cells, defining a quiescent subpopulation of cells. These cells show a different phenotype in primary tumors and matched metastases, suggesting that tumor niche pressure drives a transcriptional reprogramming of quiescent cells during melanoma progression.

The design of soft and nanometer-scale photoelectrodes able to stimulate and promote the intracellular concentration of reactive oxygen species (ROS) is searched for redox medicine applications. In this work, we show semiconducting polymer porous thin films with an enhanced photoelectrochemical generation of ROS in human umbilical vein endothelial cells (HUVECs). To achieve that aim, we synthesized graft copolymers, made of poly(3-hexylthiophene) (P3HT) and degradable poly(lactic acid) (PLA) segments, P3HT-g-PLA. In a second step, the hydrolysis of sacrificial PLA leads to nanometer-scale porous P3HT thin films. The pore sizes in the nm regime (220-1200 nm) were controlled by the copolymer composition and the structural arrangement of the copolymers during the film formation, as determined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The porous P3HT thin films showed enhanced photofaradaic behavior, generating a higher concentration of ROS in comparison to non-porous P3HT films, as determined by scanning electrochemical microscopy (SECM) measurements. The exogenous ROS production was able to modulate the intracellular ROS concentration in HUVECs at non-toxic levels, thus affecting the physiological functions of cells. Results presented in this work provide an important step forward in the development of new tools for precise, on-demand, and non-invasive modulation of intracellular ROS species and may be potentially extended to many other physiological or pathological cell models.

AlgMa bioink obtainedviaa green synthetic route and a dual crosslinking process is characterized by rheology to study the effect of crosslinking on cell viability and to fabricate bioprinted tissue models with spatially controlled stiffness.

Current cancer research is limited by the availability of reliable in vivo and in vitro models that are able to reproduce the fundamental hallmarks of cancer. Animal experimentation is of paramount importance in the progress of research, but it is becoming more evident that it has several limitations due to the numerous differences between animal tissues and real, in vivo human tissues. 3D bioprinting techniques have become an attractive tool for many basic and applied research fields. Concerning cancer, this technology has enabled the development of three-dimensional in vitro tumor models that recreate the characteristics of real tissues and look extremely promising for studying cancer cell biology. As 3D bioprinting is a relatively recently developed technique, there is still a lack of characterization of the chemical cellular microenvironment of 3D bioprinted constructs. In this work, we fabricated a cervical tumor model obtained by 3D bioprinting of HeLa cells in an alginate-based matrix. Characterization of the spheroid population obtained as a function of culturing time was performed by phase-contrast and confocal fluorescence microscopies. Scanning electrochemical microscopy and platinum nanoelectrodes were employed to characterize oxygen concentrations—a fundamental characteristic of the cellular microenvironment—with a high spatial resolution within the 3D bioprinted cervical tumor model; we also demonstrated that the diffusion of a molecular model of drugs in the 3D bioprinted construct, in which the spheroids were embedded, could be measured quantitatively over time using scanning electrochemical microscopy.

The ability to exploit energy autonomously is one of the hallmarks of life. Mastering such processes in artificial nanosystems can open technological opportunities. In the last decades, light‐ and chemically driven autonomous systems have been developed in relation to conformational motion and self‐assembly, mostly in relation to molecular motors. In contrast, despite electrical energy being an attractive energy source to power nanosystems, its autonomous harnessing has received little attention. Herein we consider an operation mode that allows the autonomous exploitation of electrical energy by a self‐assembling system. Threading and dethreading motions of a pseudorotaxane take place autonomously in solution, powered by the current flowing between the electrodes of a scanning electrochemical microscope. The underlying autonomous energy ratchet mechanism drives the self‐assembly steps away from equilibrium with a higher energy efficiency compared to other autonomous systems. The strategy is general and might be extended to other redox‐driven systems.