Exposure of the embryo to ionizing radiation (IR) is detrimental as it can cause genotoxic stress leading to immediate and latent consequences such as functional defects, malformations, or cancer. Human embryonic stem (hES) cells can mimic the preimplantation embryo and help to assess the biological effects of IR during early development. In this study, we describe the alterations H9 hES cells exhibit after X-ray irradiation in respect to cell cycle progression, apoptosis, genomic stability, stem cell signaling, and their capacity to differentiate into definitive endoderm. Early postirradiation, hES cells responded with an arrest in G2/M phase, elevated apoptosis, and increased chromosomal aberrations. Significant downregulation of stem cell signaling markers of the TGF beta-, Wnt-, and Hedgehog pathways was observed. Most prominent were alterations in the expression of activin receptors. However, hES cells responded differently depending on the culture conditions chosen for maintenance. Enzymatically passaged cells were less sensitive to IR than mechanically passaged ones showing fewer apoptotic cells and fewer changes in the stem cell signaling 24 h after irradiation, but displayed higher levels of chromosomal aberrations. Even though many of the observed changes were transient, surviving hES cells, which were differentiated 4 days postirradiation, showed a lower efficiency to form definitive endoderm than their mock-irradiated counterparts. This was demonstrated by lower expression levels of SOX17 and microRNA miR-375. In conclusion, hES cells are a suitable tool for the IR risk assessment during early human development. However, careful choice of the culture methods and a vigorous monitoring of the stem cell quality are mandatory for the use of these cells. Exposure to IR influences the stem cell properties of hES cells even when immediate radiation effects are overcome. This warrants consideration in the risk assessment of radiation effects during the earliest stages of human development.

Cognitive dysfunction induced by ionizing radiation remains a major concern in radiation therapy as well as in space mission projects. Both fields require sophisticated approaches to improve protection of the brain and its neuronal circuits. Radiation therapy related research focusses on advanced techniques imposing maximal effect on the tumor while minimizing toxicity to the surrounding tissue. Research for example has led to the revival of spatially fractionated radiation therapy (SFRT) and the advent of FLASH radiotherapy. To investigate the influence of the space radiation environment on brain cells, low dose, high LET radiation in addition to simulated microgravity have to be studied. Both research areas, however, call for cutting-edge cellular systems that faithfully resemble the architecture of the human brain, its development and its regeneration to understand the mechanisms of radiation-induced neurotoxicity and their prevention. In this review, we discuss the proposed mechanisms of neurotoxicity such as the loss of complexity within the neuronal networks, vascular changes, or neuroinflammation. We compare the current in vivo and in vitro studies of neurotoxicity including animal models, animal and human neural stem cells, and neurosphere models. Particularly, we will address the new and promising technique of generating human brain organoids and their potential use in radiation biology.

The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1–2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture.

Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid^®s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account.

Human embryonic stem cells (hESCs) differentiated into cardiomyocytes (CM) often develop into complex 3D structures that are composed of various cardiac cell types. Conventional methods to study the electrophysiology of cardiac cells are patch clamp and microelectrode array (MEAs) analyses. However, these methods are not suitable to investigate the contractile features of 3D cardiac clusters that detach from the surface of the culture dishes during differentiation. To overcome this problem, we developed a video-based motion detection software relying on the optical flow by Farnebäck that we call cBRA (cardiac beat rate analyzer). The beating characteristics of the differentiated cardiac clusters were calculated based on the local displacement between two subsequent images. Two differentiation protocols, which profoundly differ in the morphology of cardiac clusters generated and in the expression of cardiac markers, were used and the resulting CM were characterized. Despite these differences, beat rates and beating variabilities could be reliably determined using cBRA. Likewise, stimulation of β-adrenoreceptors by isoproterenol could easily be identified in the hESC-derived CM. Since even subtle changes in the beating features are detectable, this method is suitable for high throughput cardiotoxicity screenings.