Herpes simplex virus infection induces necroptosis of neurons and astrocytes in human fetal organotypic brain slice cultures.

BACKGROUND: Herpes simplex virus (HSV) encephalitis (HSE) is a serious and potentially life-threatening disease, affecting both adults and newborns. Progress in understanding the virus and host factors involved in neonatal HSE has been hampered by the limitations of current brain models that do not fully recapitulate the tissue structure and cell composition of the developing human brain in health and disease. Here, we developed a human fetal organotypic brain slice culture (hfOBSC) model and determined its value in mimicking the HSE neuropathology in vitro. METHODS: Cell viability and tissues integrity were determined by lactate dehydrogenase release in supernatant and immunohistological (IHC) analyses. Brain slices were infected with green fluorescent protein (GFP-) expressing HSV-1 and HSV-2. Virus replication and spread were determined by confocal microscopy, PCR and virus culture. Expression of pro-inflammatory cytokines and chemokines were detected by PCR. Cell tropism and HSV-induced neuropathology were determined by IHC analysis. Finally, the in situ data of HSV-infected hfOBSC were compared to the neuropathology detected in human HSE brain sections. RESULTS: Slicing and serum-free culture conditions were optimized to maintain the viability and tissue architecture of ex vivo human fetal brain slices for at least 14 days at 37 °C in a CO2 incubator. The hfOBSC supported productive HSV-1 and HSV-2 infection, involving predominantly infection of neurons and astrocytes, leading to expression of pro-inflammatory cytokines and chemokines. Both viruses induced programmed cell death-especially necroptosis-in infected brain slices at later time points after infection. The virus spread, cell tropism and role of programmed cell death in HSV-induced cell death resembled the neuropathology of HSE. CONCLUSIONS: We developed a novel human brain culture model in which the viability of the major brain-resident cells-including neurons, microglia, astrocytes and oligodendrocytes-and the tissue architecture is maintained for at least 2 weeks in vitro under serum-free culture conditions. The close resemblance of cell tropism, spread and neurovirulence of HSV-1 and HSV-2 in the hfOBSC model with the neuropathological features of human HSE cases underscores its potential to detail the pathophysiology of other neurotropic viruses and as preclinical model to test novel therapeutic interventions.

Axons of cortical basket cells originating from dendrites develop higher local complexity than axons emerging from basket cell somata.

Neuronal differentiation is regulated by neuronal activity. Here, we analyzed dendritic and axonal growth of Basket cells (BCs) and non-Basket cells (non-BCs) using sparse transfection of channelrhodopsin-YFP and repetitive optogenetic stimulation in slice cultures of rat visual cortex. Neocortical interneurons often display axon-carrying dendrites (AcDs). We found that the AcDs of BCs and non-BCs were, on average, the most complex dendrites. Further, the AcD configuration had an influence on BC axonal development. Axons originating from an AcD formed denser arborizations with more terminal endings within the dendritic field of the parent cell. Intriguingly, this occurred already in unstimulated BCs, and complexity was not increased further by optogenetic stimulation. However, optogenetic stimulation exerted a growth-promoting effect on axons emerging from BC somata. The axons of non-BCs neither responded to the AcD configuration nor to the optogenetic stimulation. The results suggest that the formation of locally dense BC plexuses is regulated by spontaneous activity. Moreover, in the AcD configuration, the AcD and the axon it carries mutually support each other's growth.

Non-Polio Enterovirus C Replicate in Both Airway and Intestine Organotypic Cultures.

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99.

ST2-Conditioned Medium Fosters Dorsal Horn Cell Excitability and Synaptic Transmission in Cultured Mouse Spinal Cord.

Conditioned medium obtained from bone marrow-derived stem cells has been proposed as a novel cell-free therapy in spinal cord injury and neuropathic pain, yet the direct effect on spinal neuron function has never been investigated. Here, we adopted spinal cord organotypic cultures (SCOCs) as an experimental model to probe the effect of ST2 murine mesenchymal stem cells-conditioned medium (ST2-CM) on dorsal horn (DH) neuron functional properties. Three days of SCOC exposure to ST2-CM increased neuronal activity measured by Fos expression, as well as spontaneous or induced firing. We showed that the increase in neuronal excitability was associated with changes in both intrinsic membrane properties and an enhanced excitatory drive. The increased excitability at the single-cell level was substantiated at the network level by detecting synchronous bursts of calcium waves across DH neurons. Altogether, SCOCs represent a viable tool to probe mesenchymal cells' effect on intact neuronal networks. Our findings indicate that ST2-CM enhances neuronal activity and synaptic wiring in the spinal dorsal horn. Our data also support the trophic role of mesenchymal cells CM in maintaining network activity in spinal circuits.

In Vitro Culture and Histological Evaluation of 3D Organotypic Cultures.

Organotypic cultures allow cells to grow in a system which mimics in vivo tissue organization. Here we describe a method for establishing 3D organotypic cultures (using intestine as an example system), followed by methods for demonstrating cell morphology and tissue architecture using histological techniques and molecular expression analysis using immunohistochemistry, though the system is also amenable to molecular expression analysis, such as by PCR, RNA sequencing, or FISH.

LPS-stimulated microglial cells promote ganglion cell death in organotypic cultures of quail embryo retina.

During development microglia colonize the central nervous system (CNS) and play an important role in programmed cell death, not only because of their ability to remove dead cells by phagocytosis, but also because they can promote the death of neuronal and glial cells. To study this process, we used as experimental systems the developing in situ quail embryo retina and organotypic cultures of quail embryo retina explants (QEREs). In both systems, immature microglia show an upregulation of certain inflammatory markers, e.g., inducible NO synthase (iNOS), and nitric oxide (NO) under basal conditions, which can be further enhanced with LPS-treatment. Hence, we investigated in the present study the role of microglia in promoting ganglion cell death during retinal development in QEREs. Results showed that LPS-stimulation of microglia in QEREs increases (i) the percentage of retinal cells with externalized phosphatidylserine, (ii) the frequency of phagocytic contacts between microglial and caspase-3-positive ganglion cells, (iii) cell death in the ganglion cell layer, and (iv) microglial production of reactive oxygen/nitrogen species, such as NO. Furthermore, iNOS inhibition by L-NMMA decreases cell death of ganglion cells and increases the number of ganglion cells in LPS-treated QEREs. These data demonstrate that LPS-stimulated microglia induce ganglion cell death in cultured QEREs by a NO-dependent mechanism. The fact that phagocytic contacts between microglial and caspase-3-positive ganglion cells increase suggests that this cell death might be mediated by microglial engulfment, although a phagocytosis-independent mechanism cannot be excluded.

A Microfluidic Device for Long-Term Maintenance of Organotypic Liver Cultures.

Liver cultures may be used for disease modeling, testing therapies and predicting drug-induced injury. The complexity of the liver cultures has evolved from hepatocyte monocultures to co-cultures with non-parenchymal cells and finally to precision-cut liver slices. The latter culture format retains liver's native biomolecular and cellular complexity and therefore holds considerable promise for in vitro testing. However, liver slices remain functional for ~72 h in vitro and display limited utility for some disease modeling and therapy testing applications that require longer culture times. This paper describes a microfluidic device for longer-term maintenance of functional organotypic liver cultures. Our microfluidic culture system was designed to enable direct injection of liver tissue into a culture chamber through a valve-enabled side port. Liver tissue was embedded in collagen and remained functional for up to 31 days, highlighted by continued production of albumin and urea. These organotypic cultures also expressed several enzymes involved in xenobiotic metabolism. Conversely, matched liver tissue embedded in collagen in a 96-well plate lost its phenotype and function within 3-5 days. The microfluidic organotypic liver cultures described here represent a significant advance in liver cultivation and may be used for future modeling of liver diseases or for individualized liver-directed therapies.

The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The ΔBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

Patient-Derived In Vitro Models of Ovarian Cancer: Powerful Tools to Explore the Biology of the Disease and Develop Personalized Treatments.

Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy worldwide due to a late diagnosis caused by the lack of specific symptoms and rapid dissemination into the peritoneal cavity. The standard of care for OC treatment is surgical cytoreduction followed by platinum-based chemotherapy. While a response to this frontline treatment is common, most patients undergo relapse within 2 years and frequently develop a chemoresistant disease that has become unresponsive to standard treatments. Moreover, also due to the lack of actionable mutations, very few alternative therapeutic strategies have been designed as yet for the treatment of recurrent OC. This dismal clinical perspective raises the need for pre-clinical models that faithfully recapitulate the original disease and therefore offer suitable tools to design novel therapeutic approaches. In this regard, patient-derived models are endowed with high translational relevance, as they can better capture specific aspects of OC such as (i) the high inter- and intra-tumor heterogeneity, (ii) the role of cancer stem cells (a small subset of tumor cells endowed with tumor-initiating ability, which can sustain tumor spreading, recurrence and chemoresistance), and (iii) the involvement of the tumor microenvironment, which interacts with tumor cells and modulates their behavior. This review describes the different in vitro patient-derived models that have been developed in recent years in the field of OC research, focusing on their ability to recapitulate specific features of this disease. We also discuss the possibilities of leveraging such models as personalized platforms to design new therapeutic approaches and guide clinical decisions.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Xeno-free self-assembling peptide scaffolds for building 3D organotypic skin cultures.

Organotypic skin cultures represent in vitro models of skin which can be used for disease modeling, tissue engineering, and screening applications. Non-human collagen is currently the gold standard material used for the construction of the supporting matrix, however, its clinical applications are limited due to its xenogeneic origin. We have developed a novel peptide hydrogel-based skin construct that shows a pluristratified epidermis, basement membrane, and dermal compartment after 3 weeks of in vitro culture. Peptide-based constructs were compared to collagen-based constructs and stratification marker expression was histologically higher in peptide constructs than in collagen constructs. Transepithelial electrical resistance also showed mature barrier function in peptide constructs. This study presents a novel application of the self-assembling peptide hydrogel in a defined xeno-free in vitro system.

Hamster organotypic kidney culture model of early-stage SARS-CoV-2 infection highlights a two-step renal susceptibility.

Kidney pathology is frequently reported in patients hospitalized with COVID-19, the pandemic disease caused by the Severe acute respiratory coronavirus 2 (SARS-CoV-2). However, due to a lack of suitable study models, the events occurring in the kidney during the earliest stages of infection remain unknown. We have developed hamster organotypic kidney cultures (OKCs) to study the early stages of direct renal infection. OKCs maintained key renal structures in their native three-dimensional arrangement. SARS-CoV-2 productively replicated in hamster OKCs, initially targeting endothelial cells and later disseminating into proximal tubules. We observed a delayed interferon response, markers of necroptosis and pyroptosis, and an early repression of pro-inflammatory cytokines transcription followed by a strong later upregulation. While it remains an open question whether an active replication of SARS-CoV-2 takes place in the kidneys of COVID-19 patients with AKI, our model provides new insights into the kinetics of SARS-CoV-2 kidney infection and can serve as a powerful tool for studying kidney infection by other pathogens and testing the renal toxicity of drugs.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

Cancer Associated Fibroblast (CAF) Regulation of PDAC Parenchymal (CPC) and CSC Phenotypes Is Modulated by ECM Composition.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancers, having one of the lowest five-year survival rates. One of its hallmarks is a dense desmoplastic stroma consisting in the abnormal accumulation of extracellular matrix (ECM) components, especially Collagen I. This highly fibrotic stroma embeds the bulk cancer (parenchymal) cells (CPCs), cancer stem cells (CSCs) and the main producers of the stromal reaction, the Cancer Associated Fibroblasts (CAFs). Little is known about the role of the acellular ECM in the interplay of the CAFs with the different tumor cell types in determining their phenotypic plasticity and eventual cell fate. METHODS: Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals by incubating PDAC CPCs and CSCs grown on ECM mimicking early (low collagen I levels) and late (high collagen I levels) stage PDAC stroma with conditioned medium from primary cultured CAFs derived from patients with PDAC in a previously described three-dimensional (3D) organotypic model of PDAC. RESULTS: We found that CAFs (1) reduced CPC growth while favoring CSC growth independently of the ECM; (2) increased the invasive capacity of only CPCs on the ECM mimicking the early tumor; and (3) favored vasculogenic mimicry (VM) especially of the CSCs on the ECM mimicking an early tumor. CONCLUSIONS: We conclude that the CAFs and acellular stromal components interact to modulate the tumor behaviors of the PDAC CPC and CSC cell types and drive metastatic progression by stimulating the phenotypic characteristics of each tumor cell type that contribute to metastasis.

Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters.

BACKGROUND: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. METHODS: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. RESULTS: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. CONCLUSIONS: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Microglia in a Dish-Which Techniques Are on the Menu for Functional Studies?

Microglia build the first line of defense in the central nervous system (CNS) and play central roles during development and homeostasis. Indeed, they serve a plethora of diverse functions in the CNS of which many are not yet fully described and more are still to be discovered. Research of the last decades unraveled an implication of microglia in nearly every neurodegenerative and neuroinflammatory disease, making it even more challenging to elucidate molecular mechanisms behind microglial functions and to modulate aberrant microglial behavior. To understand microglial functions and the underlying signaling machinery, many attempts were made to employ functional in vitro studies of microglia. However, the range of available cell culture models is wide and they come with different advantages and disadvantages for functional assays. Here we aim to provide a condensed summary of common microglia in vitro systems and discuss their potentials and shortcomings for functional studies in vitro.

Looking at Interleukin-22 from a New Dermatological Perspective: From Epidermal Homeostasis to Its Role in Chronic Skin Diseases.

Twenty years after the cloning, characterization, and identification of interleukin (IL)-22 in 2000, the precise biological role of this cytokine in healthy and unhealthy skin is not completely known. The aim of this review is to provide an overview on the recent knowledge available in literature about the origin, sources, targets, molecular mechanism of action, and clinical issues regarding IL-22. Last but not least, recent experimental evidence obtained in a 3D model of organotypic culture of normal human skin highlights its homeostatic role and will be discussed in detail, as personal observations. As most of the data concerning IL-22 immunomodulating activity are obtained from mouse models, this work offers a new perspective on its clinical role. The hypothesis herein advanced is that IL-22 profoundly affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.

Donor-to-donor variability of a human three-dimensional bronchial epithelial model: A case study of cigarette smoke exposure.

Three-dimensional (3D) cultured primary cells are used to predict the toxicity of substances towards humans because these 3D cultures closely mimic the physiological architecture of tissues. Nonetheless, it is important to consider primary-cell-specific variability for endpoint selection and appropriate evaluation of toxicity because donor-dependent characteristics may be retained even in in vitro cell cultures. In this report, 3D differentiated bronchial epithelial cells from three donors were used to investigate donor-to-donor variability, with an aqueous extract of cigarette smoke (CS) used as the test substance. Ciliary function, cytokine secretion, and histopathology, which are affected by CS, were examined, and transcriptomic analysis was also performed. The results revealed that interleukin-8 secretion and oxidative stress-related gene expression were consistently altered for all donors; however, their amplitudes varied. Moreover, one of the donors showed unique responses to CS, suggesting that this donor was an outlier. This donor showed intrinsic differences in histology, cytokine secretion, and gene expression profile. Such donors may help evaluate potential toxicological concerns and aid our understanding of disease pathogenesis. Conversely, these donors may confound toxicological assessment and endpoint selection. Fit-for-purpose handling of inter-donor variability is warranted.

Technologies to Assess Drug Response and Heterogeneity in Patient-Derived Cancer Organoids.

Patient-derived cancer organoids (PDCOs) are organotypic 3D cultures grown from patient tumor samples. PDCOs provide an exciting opportunity to study drug response and heterogeneity within and between patients. This research can guide new drug development and inform clinical treatment planning. We review technologies to assess PDCO drug response and heterogeneity, discuss best practices for clinically relevant drug screens, and assert the importance of quantifying single-cell and organoid heterogeneity to characterize response. Autofluorescence imaging of PDCO growth and metabolic activity is highlighted as a compelling method to monitor single-cell and single-organoid response robustly and reproducibly. We also speculate on the future of PDCOs in clinical practice and drug discovery.Future development will require standardization of assessment methods for both morphology and function in PDCOs, increased throughput for new drug development, prospective validation with patient outcomes, and robust classification algorithms.