Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported “2× genome” and the “1.2× genome” infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.

In Vitro Methods for Measuring the Permeability of Cell Monolayers

Cell monolayers, including endothelial and epithelial cells, play crucial roles in regulating the transport of biomolecules to underlying tissues and structures via intercellular junctions. Moreover, the monolayers form a semipermeable barrier across which leukocyte transmigration is tightly regulated. The inflammatory cytokines can disrupt the epithelial and endothelial permeability, thus the reduced barrier integrity is a hallmark of epithelial and endothelial dysfunction related with numerous pathological conditions, including cancer-related inflammation. Therefore, the assessment of barrier function is critical in in vitro models of barrier-forming tissues. This review summarizes the commercially available in vitro systems used to measure the permeability of cellular monolayers. The presented techniques are separated in two large groups: macromolecular tracer flux assays, and electrical impedance measurement-based permeability assays. The presented techniques are briefly described and compared.

Assessment of Collagen-Based Nanostructured Biomimetic Systems with a Co-Culture of Human Bone-Derived Cells

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and other factors, is considered a potential alternative to conventional treatments. Innovative scaffolds need to be tested in in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs), the two main players of bone remodeling, is required to mimic their crosstalk and molecular cooperation. To this aim, two composite materials were developed, based on type I collagen, and containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. The developed nanostructured systems underwent genipin chemical crosslinking and were then tested with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors, for 2–3 weeks. The favorable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of cells, encouraging a further investigation of the developed bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

Animal Models in Human Adenovirus Research

Human adenovirus (HAdV) infections cause a wide variety of clinical symptoms, ranging from mild upper respiratory tract disease to lethal outcomes, particularly in immunocompromised individuals. To date, neither widely available vaccines nor approved antiadenoviral compounds are available to efficiently deal with HAdV infections. Thus, there is a need to thoroughly understand HAdV-induced disease, and for the development and preclinical evaluation of HAdV therapeutics and/or vaccines, and consequently for suitable standardizable in vitro systems and animal models. Current animal models to study HAdV pathogenesis, persistence, and tumorigenesis include rodents such as Syrian hamsters, mice, and cotton rats, as well as rabbits. In addition, a few recent studies on other species, such as pigs and tree shrews, reported promising data. These models mimic (aspects of) HAdV-induced pathological changes in humans and, although they are relevant, an ideal HAdV animal model has yet to be developed. This review summarizes the available animal models of HAdV infection with comprehensive descriptions of virus-induced pathogenesis in different animal species. We also elaborate on rodent HAdV animal models and how they contributed to insights into adenovirus-induced cell transformation and cancer.

Insights into in vivo follicle formation: a review of in vitro systems

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.

Development of a self-limiting model of methotrexate-induced mucositis reinforces butyrate as a potential therapy

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005–50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

Review: Challenges of In Vitro CAF Modelling in Liver Cancers

Primary and secondary liver cancer are the third cause of death in the world, and as the incidence is increasing, liver cancer represents a global health burden. Current treatment strategies are insufficient to permanently cure patients from this devastating disease, and therefore other approaches are under investigation. The importance of cancer-associated fibroblasts (CAFs) in the tumour microenvironment is evident, and many pre-clinical studies have shown increased tumour aggressiveness in the presence of CAFs. However, it remains unclear how hepatic stellate cells are triggered by the tumour to become CAFs and how the recently described CAF subtypes originate and orchestrate pro-tumoural effects. Specialized in vitro systems will be needed to address these questions. In this review, we present the currently used in vitro models to study CAFs in primary and secondary liver cancer and highlight the trend from using oversimplified 2D culture systems to more complex 3D models. Relatively few studies report on the impact of cancer (sub)types on CAFs and the tumour microenvironment, and most studies investigated the impact of secreted factors due to the nature of the models.