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Lower back pain continues to be a global epidemic, limiting quality of life and ability to work, due in large part to symptomatic disc degeneration. Development of more effective and less invasive biological strategies are needed to treat disc degeneration. In vitro models such as macro- or micro-bioreactors or mechanically active organ-chips hold great promise in reducing the need for animal studies that may have limited clinical translatability, due to harsher and more complex mechanical loading environments in human discs than in most animal models. This review highlights the complex loading conditions of the disc in situ, evaluates state-of-the-art designs for applying such complex loads across multiple length scales, from macro-bioreactors that load whole discs to organ-chips that aim to replicate cellular or engineered tissue loading. Emphasis was placed on the rapidly evolving more customizable organ-chips, given their greater potential for studying the progression and treatment of symptomatic disc degeneration. Lastly, this review identifies new trends and challenges for using organ-chips to assess therapeutic strategies.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

There is substantial evidence that both N-methyl-D-aspartate receptor (NMDAR) hypofunction and dysfunction of GABAergic neurotransmission contribute to schizophrenia, though the relationship between these pathophysiological processes remains largely unknown. Although models using cell-type-specific genetic deletion of NMDARs have been informative, they display overly pronounced phenotypes extending beyond those of schizophrenia. Here, we used the serine racemase knockout (SRKO) mice, a model of reduced NMDAR activity rather than complete receptor elimination, to examine the link between NMDAR hypofunction and decreased GABAergic inhibition. The SRKO mice, in which there is a >90% reduction in the NMDAR coagonist d-serine, exhibit many of the neurochemical and behavioral abnormalities observed in schizophrenia. We found a significant reduction in inhibitory synapses onto CA1 pyramidal neurons in the SRKO mice. This reduction increases the excitation/inhibition balance resulting in enhanced synaptically driven neuronal excitability without changes in intrinsic excitability. Consistently, significant reductions in inhibitory synapse density in CA1 were observed by immunohistochemistry. We further show, using a single-neuron genetic deletion approach, that the loss of GABAergic synapses onto pyramidal neurons observed in the SRKO mice is driven in a cell-autonomous manner following the deletion of SR in individual CA1 pyramidal cells. These results support a model whereby NMDAR hypofunction in pyramidal cells disrupts GABAergic synapses leading to disrupted feedback inhibition and impaired neuronal synchrony.NEW & NOTEWORTHY Recently, disruption of excitation/inhibition (E/I) balance has become an area of considerable interest for psychiatric research. Here, we report a reduction in inhibition in the serine racemase knockout mouse model of schizophrenia that increases E/I balance and enhances synaptically driven neuronal excitability. This reduced inhibition was driven cell-autonomously in pyramidal cells lacking serine racemase, suggesting a novel mechanism for how chronic NMDA receptor hypofunction can disrupt information processing in schizophrenia.

Rapid death of infected cells is an important antiviral strategy. However, fast decisions that are based on limited evidence can be erroneous and cause unnecessary cell death and subsequent tissue damage. How cells optimize their death decision making strategy to maximize both speed and accuracy is unclear. Here, we show that exposure to TNF, which is secreted by macrophages during viral infection, causes cells to change their decision strategy from "slow and accurate" to "fast and error-prone". Mathematical modeling combined with experiments in cell culture and whole organ culture show that the regulation of the cell death decision strategy is critical to prevent HSV-1 spread. These findings demonstrate that immune regulation of cellular cognitive processes dynamically changes a tissues' tolerance for self-damage, which is required to protect against viral spread.

With 1.5-2.0 million new cases annually worldwide, corneal injury represents a common cause of vision loss, often from irreversible scarring due to surface corneal defects. In this study, we assessed the use of hepatocyte growth factor (HGF) loaded into an in situ photopolymerizable transparent gelatin-based hydrogel for the management of corneal defects. In vitro release kinetics showed that, in regard to the total amount of HGF released over a month, 55 ± 11% was released during the first 24 h, followed by a slow release profile for up to one month. The effect of HGF was assessed using an ex vivo model of pig corneal defect. After three days of organ culture, epithelial defects were found to be completely healed for 89% of the corneas treated with HGF, compared to only 11% of the corneas that had fully re-epithelialized when treated with the hydrogel without HGF. The thickness of the epithelial layer was found to be significantly higher for the HGF-treated group compared to the group treated with hydrogel without HGF (p = 0.0012). Finally, histological and immunostaining assessments demonstrated a better stratification and adhesion of the epithelial layer in the presence of HGF. These results suggest that the HGF-loaded hydrogel system represents a promising solution for the treatment of persistent corneal defects at risk of scarring.

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death in the US. CRC frequently metastasizes to the liver and these patients have a particularly poor prognosis. The infiltration of immune cells into CRC tumors and liver metastases accurately predicts disease progression and patient survival. Despite the evident influence of immune cells in the CRC tumor microenvironment (TME), efforts to identify immunotherapies for CRC patients have been limited. Here, we argue that preclinical model systems that recapitulate key features of the tumor microenvironment-including tumor, stromal, and immune cells; the extracellular matrix; and the vasculature-are crucial for studies of immunity in the CRC TME and the utility of immunotherapies for CRC patients. We briefly review the discoveries, advantages, and disadvantages of current in vitro and in vivo model systems, including 2D cell culture models, 3D culture systems, murine models, and organ-on-a-chip technologies.

Angiogenesis is a complex process that is required for development and tissue regeneration and it may be affected by many pathological conditions. Chemicals and drugs can impact formation and maintenance of the vascular networks; these effects may be both desirable (e.g., anti-cancer drugs) or unwanted (e.g., side effects of drugs). A number of in vivo and in vitro models exist for studies of angiogenesis and endothelial cell function, including organ-on-a-chip microphysiological systems. An arrayed organ-on-a-chip platform on a 96-well plate footprint that incorporates perfused microvessels, with and without tumors, was recently developed and it was shown that survival of the surrounding tissue was dependent on delivery of nutrients through the vessels. Here we describe a technology transfer of this complex microphysiological model between laboratories and demonstrate that reproducibility and robustness of these tissue chip-enabled experiments depend primarily on the source of the endothelial cells. The model was highly reproducible between laboratories and was used to demonstrate the advantages of the perfusable vascular networks for drug safety evaluation. As a proof-of-concept, we tested Fluorouracil (1-1,000 μM), Vincristine (1-1,000 nM), and Sorafenib (0.1-100 μM), in the perfusable and non-perfusable micro-organs, and in a colon cancer-containing micro-tumor model. Tissue chip experiments were compared to the traditional monolayer cultures of endothelial or tumor cells. These studies showed that human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs. The data from the 3D models confirmed advantages of the physiological environment as compared to 2D cell cultures. We demonstrated how these models can be translated into practice by verifying that the endothelial cell source and passage are critical elements for establishing a perfusable model.

ObjectiveCancer patient-derived organoids (PDOs) grow as three dimensional (3D) structures in the presence of extracellular matrix and have been found to represent the original tumor's genetic complexity. In addition, PDOs can be grown and subjected to drug sensitivity testing in a shorter time course and with lesser expense than patient-derived xenograft models. Many patients with recurrent ovarian cancer develop malignant effusions that become refractory to chemotherapy. Since these same patients often present for palliative aspiration of ascites or pleural effusions, there is a potential opportunity to obtain tumor specimens in the form of multicellular spheroids (MCS) present in malignant effusion fluids. Our objective was to develop a short duration culture of MCS from ovarian cancer malignant effusions in conditions selected to support organoid growth and use them as a platform for empirical drug sensitivity testing.MethodsIn this study, malignant effusion specimens were collected from patients with high-grade serous ovarian carcinoma (HGSOC). MCS were recovered and subjected to culture conditions designed to support organoid growth. In a subset of specimens, RNA-sequencing was performed at two time points during the short-term culture to determine changes in transcriptome in response to culture conditions. Organoid induction was also characterized in these specimens using Ki67 staining and histologic analysis. Drug sensitivity testing was performed on all specimens.ResultsOur model describes organoids formed within days of primary culture, which can recapitulate the histological features of malignant ascites fluid and can be expanded for at least 6 days. RNA-seq analysis of four patient specimens showed that within 6 days of culture, there was significant up-regulation of genes related to cellular proliferation, epithelial-mesenchymal transition, and KRAS signaling pathways. Drug sensitivity testing identified several agents with therapeutic potential.ConclusionsShort duration organoid culture of MCS from HGSOC malignant effusions can be used as a platform for empiric drug sensitivity testing. These ex vivo models may be helpful in screening new or existing therapeutic agents prior to individualized treatment options.

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole-cell electrophysiological recordings with laser-scanning photostimulation to test whether peripheral nerve injury in the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, yet there are no established treatments for preventing maternal-fetal transmission. During first trimester, HCMV replicates in basal decidua that functions as a reservoir for virus and source of transmission to the attached placenta and fetal hemiallograft but also contains immune cells, including natural killer cells, macrophages, and T cell subsets, that respond to pathogens, protecting the placenta and fetus. However, the specific cellular and cytokine responses to infection are unknown, nor are the immune correlates of protection that guide development of therapeutic strategies. Here we survey immune cell phenotypes in intact explants of basal decidua infected with a clinical pathogenic HCMV strain ex vivo and identify specific changes occurring in response to infection in the tissue environment. Using 4-color immunofluorescence microscopy, we found that at 3 days postinfection, virus replicates in decidual stromal cells and epithelial cells of endometrial glands. Infected cells and effector memory CD8+ T cells (TEM) in contact with them make IFN-γ. CD8+ TEM cells produce granulysin and cluster at sites of infection in decidua and the epithelium of endometrial glands. Quantification indicated expansion of two immune cell subtypes-CD8+ TEM cells and, to a lesser extent, iNKT cells. Approximately 20% of immune cells were found in pairs in both control and infected decidua, suggesting frequent cross-talk in the microenvironment of decidua. Our findings indicate a complex immune microenvironment in basal decidua and suggest CD8+ TEM cells play a role in early responses to decidual infection in seropositive women.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/β-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Neonatal hypoxia-ischemia (HI) in the preterm human results in damage to subcortical developing white matter and cognitive impairments. Subplate neurons (SPNs) are among the first-born cortical neurons and are necessary for normal cerebral development. While moderate or severe HI at P1 in rats leads to SPN loss, it is unclear if HI, esp. forms not associated with overt cell loss lead to altered SPN circuits. Thus, we used two HI models with different severities in P1 rats. Cauterization of the common carotid artery (CCA) causes a largely transient and thus milder ischemia (HI-Caut) while CCA ligation causes more severe ischemia (HI-Lig). While HI-Lig caused subplate damage, HI-Caut did not cause overt histological damage on the light microscopic level. We used laser-scanning photostimulation (LSPS) in acute thalamocortical slices of auditory cortex during P5-10 to study the functional connectivity of SPNs. Both HI categories resulted in hyperconnectivity of excitatory and inhibitory circuits to SPNs. Thus, alterations on the circuit level are present in the absence of cell loss. Our results show that SPN circuits are uniquely susceptible to HI. Given the key developmental role of SPNs, our results suggest that altered SPN circuits might underlie the abnormal development of cortical function after HI.

SEC14 and Spectrin domain-1 (Sestd1) is a synapse protein that exhibits a striking shift from the presynaptic to postsynaptic space as neurons mature postnatally in the mouse hippocampus. Hippocampal pyramidal neurons from mice with global genetic deletion of Sestd1 have reduced dendrite arbors, spines, and excitatory synapses. Electrophysiologically this correlates with cell-autonomous reductions in both AMPA- and NMDA-excitatory postsynaptic currents in individual hippocampal neurons from which Sestd1 has been deleted in vivo. These neurodevelopmental and functional deficits are associated with increased activation of the Rho family GTPases Rac1 and RhoA. Co-immunoprecipitation and mass spectrometry reveal that the Breakpoint Cluster Region protein, a Rho GTPase activating protein (GAP), forms complexes with Sestd1 in brain tissue. This complements earlier findings that Sestd1 can also partner with other Rho family GAPs and guanine nucleotide exchange factors. Our findings demonstrate that Sestd1 is a developmentally dynamic synaptic regulator of Rho GTPases that contributes to dendrite and excitatory synapse formation within differentiating pyramidal neurons of the forebrain.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT.

Hair cells of the auditory and vestibular systems are capable of detecting sounds that induce sub-nanometer vibrations of the hair bundle, below the stochastic noise levels of the surrounding fluid. Furthermore, the auditory system exhibits a highly rapid response time, in the sub-millisecond regime. We propose that chaotic dynamics enhance the sensitivity and temporal resolution of the hair bundle response, and we provide experimental and theoretical evidence for this effect. We use the Kolmogorov entropy to measure the degree of chaos in the system and the transfer entropy to quantify the amount of stimulus information captured by the detector. By varying the viscosity and ionic composition of the surrounding fluid, we are able to experimentally modulate the degree of chaos observed in the hair bundle dynamics in vitro. We consistently find that the hair bundle is most sensitive to a stimulus of small amplitude when it is poised in the weakly chaotic regime. Further, we show that the response time to a force step decreases with increasing levels of chaos. These results agree well with our numerical simulations of a chaotic Hopf oscillator and suggest that chaos may be responsible for the high sensitivity and rapid temporal response of hair cells.

BackgroundHomeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.MethodsDifferential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures.ResultsWe identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures.ConclusionsTcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments.

Neuromodulators are important regulators of synaptic transmission throughout the brain. At the presynaptic terminal, neuromodulation of calcium channels (CaVs) can affect transmission not only by changing neurotransmitter release probability, but also by shaping short-term plasticity (STP). Indeed, changes in STP are often considered a requirement for defining a presynaptic site of action. Nevertheless, some synapses exhibit non-canonical forms of neuromodulation, where release probability is altered without a corresponding change in STP. Here, we identify biophysical mechanisms whereby both canonical and non-canonical presynaptic neuromodulation can occur at the same synapse. At a subset of glutamatergic terminals in prefrontal cortex, GABAB and D1/D5 dopamine receptors suppress release probability with and without canonical increases in short-term facilitation by modulating different aspects of presynaptic CaV function. These findings establish a framework whereby signaling from multiple neuromodulators can converge on presynaptic CaVs to differentially tune release dynamics at the same synapse.

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-γ-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-γ expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-γ production, but also the protective function of iNKT cells in arthritis.

Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) around MNs in a VGLUT1-dependent manner. Loss of astrocyte-encoded Kir4.1 selectively altered FαMN size and function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation might uncouple symptoms of muscle weakness from MN cell death in diseases like ALS.

The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.

Variants of TREM2 are associated with Alzheimer's disease (AD). To study whether increasing TREM2 gene dosage could modify the disease pathogenesis, we developed BAC transgenic mice expressing human TREM2 (BAC-TREM2) in microglia. We found that elevated TREM2 expression reduced amyloid burden in the 5xFAD mouse model. Transcriptomic profiling demonstrated that increasing TREM2 levels conferred a rescuing effect, which includes dampening the expression of multiple disease-associated microglial genes and augmenting downregulated neuronal genes. Interestingly, 5xFAD/BAC-TREM2 mice showed further upregulation of several reactive microglial genes linked to phagocytosis and negative regulation of immune cell activation. Moreover, these mice showed enhanced process ramification and phagocytic marker expression in plaque-associated microglia and reduced neuritic dystrophy. Finally, elevated TREM2 gene dosage led to improved memory performance in AD models. In summary, our study shows that a genomic transgene-driven increase in TREM2 expression reprograms microglia responsivity and ameliorates neuropathological and behavioral deficits in AD mouse models.

The biology of aging is challenging to study, particularly in humans. As a result, model organisms are used to approximate the physiological context of aging in humans. However, the best model organisms remain expensive and time-consuming to use. More importantly, they may not reflect directly on the process of aging in people. Human cell culture provides an alternative, but many functional signs of aging occur at the level of tissues rather than cells and are therefore not readily apparent in traditional cell culture models. Organoids have the potential to effectively balance between the strengths and weaknesses of traditional models of aging. They have sufficient complexity to capture relevant signs of aging at the molecular, cellular, and tissue levels, while presenting an experimentally tractable alternative to animal studies. Organoid systems have been developed to model many human tissues and diseases. Here we provide a perspective on the potential for organoids to serve as models for aging and describe how current organoid techniques could be applied to aging research.

PurposeNitrogen mustard (NM), which simulates the effects of sulfur mustard (SM), is a potent vesicant known to cause irreversible corneal damage. This study investigates the mechanisms by which NM induces corneal damage by examining the impact of NM exposure on the morphology and lipidome of the cornea.MethodsIntact ex vivo rabbit eyes were placed in serum-free DMEM organ culture. NM (0, 1, 2.5, 5 or 10 mg/ml) was applied to the central cornea for 5, 10 or 15 min using a 5 mm filter disk and subsequently rinsed with DMEM. Corneas were then cultured for 3, 24, or 48 h before being fixed for morphological analysis or for 24 h before being snap frozen for lipidomic analysis.ResultsNo morphological changes were detected 3 h after NM exposure. Twenty-four h after exposure, 1 mg/ml NM caused erosion of the corneal epithelium, but no damage to the underlying stroma. Damage caused by 2.5 mg/ml NM extended almost two thirds through the corneal stroma, while 5 mg/ml completely penetrated the corneal stroma. An altered lipid profile occurred 24 h after corneas were exposed to NM. Specific sphingomyelins, ceramides, and diacylglycerols were increased up to 9-, 60- and 10-fold, respectively.ConclusionsNM induces concentration- and exposure time-dependent damage to the cornea that increases in severity over time. Alterations in the sphingomyelin-ceramide pathway may contribute to the damaging effects of NM exposure.

The emergence of congenital Zika virus (ZIKV) disease, with its devastating effects on the fetus, has prompted development of vaccines and examination of how ZIKV breaches the maternal-fetal barrier. Infection of placental and decidual tissue explants has demonstrated cell types at the uterine-placental interface susceptible to infection and suggests routes for transmission across the placenta and amniochorionic membrane. ZIKV replicates in proliferating Hofbauer cells within chorionic villi in placentas from severe congenital infection. Explants of anchoring villi recapitulate placental architecture and early-stage development and suggest infected Hofbauer cells disseminate virus to fetal blood vessels. ZIKV infection of explants represents a surrogate human model for evaluating protection against transmission by antibodies in vaccine recipients and passive immune formulations and novel therapeutics.

Selective modifications in the expression or function of dendritic ion channels regulate the propagation of synaptic inputs and determine the intrinsic excitability of a neuron. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open upon membrane hyperpolarization and conduct a depolarizing inward current ( I h ). HCN channels are enriched in the dendrites of hippocampal pyramidal neurons where they regulate the integration of synaptic inputs. Synaptic plasticity can bidirectionally modify dendritic HCN channels in excitatory neurons depending on the strength of synaptic potentiation. In inhibitory neurons, however, the dendritic expression and modulation of HCN channels are largely unknown. In this study, we systematically compared the modulation of I h by synaptic potentiation in hippocampal CA1 pyramidal neurons and stratum radiatum (sRad) interneurons in mouse organotypic cultures. I h properties were similar in inhibitory and excitatory neurons and contributed to resting membrane potential and action potential firing. We found that in sRad interneurons, HCN channels were downregulated after synaptic plasticity, irrespective of the strength of synaptic potentiation. This suggests differential regulation of I h in excitatory and inhibitory neurons, possibly signifying their distinct role in network activity., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Herstel and Wierenga.)

Background: Inhibitors of epidermal growth factor receptor (EGFRi) or mitogen-activated kinase (MEKi) induce a folliculitis in 75-90% of patients, the pathobiology of which remains insufficiently understood., Objectives: To characterize changes in the skin immune status and global transcriptional profile of patients treated with EGFRi; to investigate whether EGFRi affects the hair follicle's (HF) immune privilege (IP); and to identify early proinflammatory signals induced by EGFRi/MEKi in human scalp HFs ex vivo., Methods: Scalp biopsies were taken from patients exhibiting folliculitis treated long term with EGFRi ('chronic EGFRi' group, n = 9) vs. healthy scalp skin (n = 9) and patients prior to commencing EGFRi treatment and after 2 weeks of EGFRi therapy ('acute EGFRi' group, n = 5). Healthy organ-cultured scalp HFs were exposed to an EGFRi (erlotinib, n = 5) or a MEKi (cobimetinib, n = 5). Samples were assessed by quantitative immunohistomorphometry, RNA sequencing (RNAseq) and in situ hybridization., Results: The 'chronic EGFRi' group showed CD8+ T-cell infiltration of the bulge alongside a partial collapse of the HF's IP, evidenced by upregulated major histocompatibility complex (MHC) class I, β2-microglobulin (B2 M) and MHC class II, and decreased transforming growth factor-β1 protein expression. Healthy HFs treated with EGFRi/MEKi ex vivo also showed partial HF IP collapse and increased transcription of human leucocyte antigen (HLA)-A, HLA-DR and B2 M transcripts. RNAseq analysis showed increased transcription of chemokines (CXCL1, CXCL13, CCL18, CCL3, CCL7) and interleukin (IL)-26 in biopsies from the 'chronic EGFRi' cohort, as well as increased IL-33 and decreased IL-37 expression in HF biopsies from the 'acute EGFRi' group and in organ-cultured HFs., Conclusions: The data show that EGFRi/MEKi compromise the physiological IP of human scalp HFs and suggest that future clinical management of EGFRi/MEKi-induced folliculitis requires HF IP protection and inhibition of IL-33., Competing Interests: Conflicts of interest D.R. is part funded by Roche/Genentech. R.P. is president of a contract research organization that also engages in analyses of drug skin toxicity (www.monasteriumlab.com), and chief executive officer of a company that develops new actives for skin and hair diseases (www.cutaneon.com), but declares no relevant conflicts of interest related to this study., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Synaptic plasticity is an essential process encoding fine-tuned brain functions, but models to study this process in adult human systems are lacking., Objective: We aim to test whether ex vivo organotypic culture of post-mortem adult brain explants (OPABs) retain synaptic plasticity., Methods: OPABs were seeded on 3D microelectrode arrays to measure local field potential (LFP). Paired stimulation of distant electrodes was performed over three days to investigate our capacity to modulate specific neuronal connections., Results: Long-term potentiation (LTP) or depression (LTD) did not occur within a single day. In contrast, after two and three days of training, OPABs showed a significant modulation of the paired electrodes' response compared to the non-paired electrodes from the same array. This response was alleviated upon treatment with dopamine., Conclusion: Our work highlights that adult human brain explants retain synaptic plasticity, offering novel approaches to neural circuitry in animal-free models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Alzheimer's disease (AD) features progressive neurodegeneration and microglial activation that results in dementia and cognitive decline. The release of soluble amyloid (Aβ) oligomers into the extracellular space is an early feature of AD pathology. This can promote excitotoxicity and microglial activation. Microglia can adopt several activation states with various functional outcomes. Protective microglial activation states have been identified in response to Aβ plaque pathology in vivo. However, the role of microglia and immune mediators in neurotoxicity induced by soluble Aβ oligomers is unclear. Further, there remains a need to identify druggable molecular targets that promote protective microglial states to slow or prevent the progression of AD., Methods: Hippocampal entorhinal brain slice culture (HEBSC) was employed to study mechanisms of Aβ 1-42 oligomer-induced neurotoxicity as well as the role of microglia. The roles of glutamate hyperexcitation and immune signaling in Aβ-induced neurotoxicity were assessed using MK801 and neutralizing antibodies to the TNF-related apoptosis-inducing ligand (TRAIL) respectively. Microglial activation state was manipulated using Gi-hM4di designer receptor exclusively activated by designer drugs (DREADDs), microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX3397, and microglial repopulation (PLX3397 withdrawal). Proteomic changes were assessed by LC-MS/MS in microglia isolated from control, repopulated, or Aβ-treated HEBSCs., Results: Neurotoxicity induced by soluble Aβ 1-42 oligomers involves glutamatergic hyperexcitation caused by the proinflammatory mediator and death receptor ligand TRAIL. Microglia were found to have the ability to both promote and restrain Aβ-induced toxicity. Induction of microglial Gi-signaling with hM4di to prevent pro-inflammatory activation blunted Aβ neurotoxicity, while microglial depletion with CSF1R antagonism worsened neurotoxicity caused by Aβ as well as TRAIL. HEBSCs with repopulated microglia, however, showed a near complete resistance to Aβ-induced neurotoxicity. Comparison of microglial proteomes revealed that repopulated microglia have a baseline anti-inflammatory and trophic phenotype with a predicted pathway activation that is nearly opposite that of Aβ-exposed microglia. mTORC2 and IRF7 were identified as potential targets for intervention., Conclusion: Microglia are key mediators of both protection and neurodegeneration in response to Aβ. Polarizing microglia toward a protective state could be used as a preventative strategy against Aβ-induced neurotoxicity., (© 2024. The Author(s).)

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the broad application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Using a holistic approach, we studied organotypic whole hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a 5-wk period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed multielectrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by 2 wk in culture, followed by the transition of spontaneous activity toward more complex patterns including high-frequency oscillations. However, culturing weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders. NEW & NOTEWORTHY The development of the mouse neocortex in vitro mimics the in vivo development. Mouse brain cultures can serve as a model system for cortical development for the first 2 wk in vitro and as a model system for the adult cortex from 2 to 4 wk in vitro. Mouse organotypic brain slice cultures develop high-frequency network oscillations at γ frequency after 2 wk in vitro. Mouse brain cultures exhibit increased heterogeneity and variability after 4 wk in culture.

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 μm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Endothelial cell density (ECD) changes long after penetrating keratoplasty (PKP) of organ-cultured corneas have been little studied. We aim to calculate the point when ECD decline stabilises following PKP with organ culture stored corneas. Methods: This is an observational study of first-ever PKPs and first-ever re-grafts, performed over 17 years under a single surgeon. ECDs were acquired at 3 and 6 months, 1 year post-graft and annually thereafter by specular microscopy. Time-dependent ECD data was fitted to a log-biexponential model. Results: We studied 465 first-ever grafts and 128 re-grafts. Mean recipient age was 59 years (range 0–96 years; SD 22). Median follow-up was 5.7 (range 0.2–17.1) years. Probability of ED at 5 years in first grafts and re-grafts was 4.4% (2.6–7.1%) and 14.8% (8.3–23.2%). In first grafts, ECD loss reached 0.6% per annum at 7.9 (6.2–9.6) years post-operatively. The half-lives of ECD loss during the immediate post-operative period for first grafts, re-grafts, dystrophies, ectasias, and previous ocular surgery are 20.1 (14.9–30.9), 12.8 (6.9–79.4), 19.5 (13.1–37.7), 26.2 (16.2–68), and 11.6 (6.7–41.3) months, respectively. The half-life during this rapid phase of ECD loss has an inverse correlation with graft survival at 10 years (r = − 0.89, p = 0.02). Conclusions: Rate of endothelial decompensation is higher in first grafts than re-grafts. ECD decline stabilises 7.9 years post-operatively in first grafts but then becomes lower than the physiological loss expected. Further work is needed to verify whether organ-cultured grafts reach physiological levels of ECD loss faster than hypothermically stored grafts. [ABSTRACT FROM AUTHOR]

Background and Objectives: Cardiovascular disease is one of the leading causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources of cell-based therapies in heart regeneration. There are several approaches to differentiate MSCs into cardiac-like cells, such as genetic modification. In addition, using of 3D culture, such as hydrogels, increases the efficiency of differentiation. Methods: In the present study, lentiviruses containing microRNA 1 (miR- 1) and myocardium (Myocd) were co-transducted to mouse adipose-derived MSCs. Three days after, transduced MSCs were transferred to a hydrogel containing chitosan and collagen. After 21 days, the differentiation of encapsulated cells was evaluated. In this regard, the expression of cardiac markers such as NK2 homeobox 5 (Nkx2-5), GATA binding protein 4 (Gata4) and troponin T type 2 (Tnnt2) at the level of gene and protein were investigated. Results: The results of real-time quantitative polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by transfer to composite hydrogel increased the expression of cardiac markers. Conclusion: The use of 3D culture such as chitosan/collagen hydrogel improves the differentiation of MSCs and subsequently obtains more mature cells for use in cell-based regenerative medicine

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.

Dopaminergic modulation of prefrontal cortex (PFC) is thought to play key roles in many cognitive functions and to be disrupted in pathological conditions, such as schizophrenia. We have previously described a phenomenon whereby dopamine D2 receptor (D2R) activation elicits afterdepolarizations (ADPs) in subcortically projecting (SC) pyramidal neurons within L5 of the PFC. These D2R-induced ADPs only occur following synaptic input, which activates NMDARs, even when the delay between the synaptic input and ADPs is relatively long (e.g., several hundred milliseconds). Here, we use a combination of electrophysiological, optogenetic, pharmacological, transgenic, and chemogenetic approaches to elucidate cellular mechanisms underlying this phenomenon in male and female mice. We find that knocking out D2Rs eliminates the ADP in a cell-autonomous fashion, confirming that this ADP depends on D2Rs. Hyperpolarizing current injection, but not AMPA receptor blockade, prevents synaptic stimulation from facilitating D2R-induced ADPs, suggesting that this phenomenon depends on the recruitment of voltage-dependent currents (e.g., NMDAR-mediated Ca2+ influx) by synaptic input. Finally, the D2R-induced ADP is blocked by inhibitors of cAMP/PKA signaling, insensitive to pertussis toxin or β-arrestin knock-out, and mimicked by Gs-DREADD stimulation, suggesting that D2R activation elicits the ADP by stimulating cAMP/PKA signaling. These results show that this unusual physiological phenomenon, in which D2Rs enhance cellular excitability in a manner that depends on synaptic input, is mediated at the cellular level through the recruitment of signaling pathways associated with Gs, rather than the Gi/o-associated mechanisms that have classically been ascribed to D2Rs.SIGNIFICANCE STATEMENT Dopamine D2 receptors (D2Rs) in the prefrontal cortex (PFC) are thought to play important roles in behaviors, including working memory and cognitive flexibility. Variation in D2Rs has also been implicated in schizophrenia, Tourette syndrome, and bipolar disorder. Recently, we described a new mechanism through which D2R activation can enhance the excitability of pyramidal neurons in the PFC. Here, we explore the underlying cellular mechanisms. Surprisingly, although D2Rs are classically assumed to signal through Gi/o-coupled G-proteins and/or scaffolding proteins, such as β-arrestin, we find that the effects of D2Rs on prefrontal pyramidal neurons are actually mediated by pathways associated with Gs-mediated signaling. Furthermore, we show how, via this D2R-dependent phenomenon, synaptic input can enhance the excitability of prefrontal neurons over timescales on the order of seconds. These results elucidate cellular mechanisms underlying a novel signaling pathway downstream of D2Rs that may contribute to prefrontal function under normal and pathological conditions.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between β-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates β-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and β-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.

ObjectivePatient-derived organoids (PDOs), used in multiple tumor types, have allowed evaluation of tumor characteristics from individual patients. This study aimed to assess the feasibility of applying PDO in vitro culture for endocrine-based and drug sensitivity testing in endometrial cancer.MethodsEndometrial cancer cells were enzymatically dissociated from tumors retrieved from fresh hysterectomy specimens and cultured within basement membrane extract in serum-free medium. An organoid growth assay was developed to assess the inhibitory effects of a variety of drugs including endocrine treatments. Organoid cultures were also prepared for histological and immunohistochemical comparison to the tumors of origin.ResultsFifteen endometrial cancer specimens were successfully cultured as PDOs. Small spherical structures formed within 24 hours, and many continued to grow to larger, denser organoids, providing the basis for an organoid growth assay. The STAT3 transcription factor inhibitor, BBI608 (Napabucasin), strongly inhibited growth in almost all PDO cultures, suggesting that stemness programing is involved in organoid formation and/or growth. Inhibition by different growth factor receptor tyrosine kinase inhibitors was observed in several PDO specimens. Four cultures were inhibited by fulvestrant, implying the importance of estrogen-receptor signaling in some PDO cultures. Organoids closely resembled their tumors of origin in both histomorphology and immunohistochemical expression.ConclusionsThe use of endometrial cancer PDO cultures for development of drug sensitivity testing for individual patient tumors is feasible. The potential value of the PDO model for clinical decision making will require clinical trial evaluation.

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall. The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics. In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

The bacterial pathogen Listeria monocytogenes causes foodborne systemic disease in pregnant women, which can lead to preterm labor, stillbirth, or severe neonatal disease. Colonization of the maternal decidua appears to be an initial step in the maternal component of the disease as well as bacterial transmission to the placenta and fetus. Host-pathogen interactions in the decidua during this early stage of infection remain poorly understood. Here, we assessed the dynamics of L. monocytogenes infection in primary human decidual organ cultures and in the murine decidua in vivo A high inoculum was necessary to infect both human and mouse deciduas, and the data support the existence of a barrier to initial colonization of the murine decidua. If successful, however, colonization in both species was followed by significant bacterial expansion associated with an inability of the decidua to mount appropriate innate cellular immune responses. The innate immune deficits included the failure of bacterial foci to attract macrophages and NK cells, cell types known to be important for early defenses against L. monocytogenes in the spleen, as well as a decrease in the tissue density of inflammatory Ly6Chi monocytes in vivo These results suggest that the infectivity of the decidua is not the result of an enhanced recruitment of L. monocytogenes to the gestational uterus but rather is due to compromised local innate cellular immune responses.

PurposePhotoreceptor degeneration in the retina is a major cause of blindness in humans. Elucidating mechanisms of degenerative and neuroprotective pathways in photoreceptors should afford identification and development of therapeutic strategies.MethodsWe used mouse genetic models and improved methods for retinal explant cultures. Retinas were enucleated from Mef2d+/+ and Mef2d-/- mice, stained for MEF2 proteins and outer nuclear layer thickness, and assayed for apoptotic cells. Chromatin immunoprecipitation (ChIP) assays revealed MEF2 binding, and RT-qPCR showed levels of transcription factors. We used AAV2 and electroporation to express genes in retinal explants and electroretinograms to assess photoreceptor functionality.ResultsWe identify a prosurvival MEF2D-PGC1α pathway that plays a neuroprotective role in photoreceptors. We demonstrate that Mef2d-/- mouse retinas manifest decreased expression of PGC1α and increased photoreceptor cell loss, resulting in the absence of light responses. Molecular repletion of PGC1α protects Mef2d-/- photoreceptors and preserves light responsivity.ConclusionsThese results suggest that the MEF2-PGC1α cascade may represent a new therapeutic target for drugs designed to protect photoreceptors from developmental- and age-dependent loss.

Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.

Juvenile hormone (JH) controls many biological activities in insects, including development, metamorphosis, and reproduction. In the Aedes aegypti mosquito, a vector of dengue, yellow fever, chikungunya, and zika viruses, the metabolic tissue (the fat body, which is an analogue of the vertebrate liver) produces yolk proteins for developing oocytes. JH is important for the fat body to acquire competence for yolk protein production. However, the molecular mechanisms of how JH promotes mosquito reproduction are not completely understood. In this study we show that stimulation of the JH receptor methoprene-tolerant (Met) activates expression of genes encoding the regulator of ribosome synthesis 1 (RRS1) and six ribosomal proteins (two ribosomal large subunit proteins, two ribosomal small subunit proteins, and two mitochondrial ribosomal proteins). Moreover, RNAi-mediated depletion of RRS1 decreased biosynthesis of the ribosomal protein L32 (RpL32). Depletion of Met, RRS1, or RpL32 led to retardation of ovarian growth and reduced mosquito fecundity, which may at least in part have resulted from decreased vitellogenin protein production in the fat body. In summary, our results indicate that JH is critical for inducing the expression of ribosomal protein genes and demonstrate that RRS1 mediates the JH signal to enhance both ribosomal biogenesis and vitellogenesis.

The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.

BackgroundLiver organoids show potential for development as a tissue replacement therapy for patients with end-stage liver disease, but efficient methods for introducing organoids into host livers have not been established. In this study, we aimed to develop a surgical technique to implant hepatic organoids into the liver and assess their engraftment.MethodsDonor hepatocytes were isolated from ROSA26 C57BL/6 mice, so that engrafted cells, when implanted into wild-type mice, could be easily identified by X-gal staining. Hepatic organoids were generated by three-dimensional culture in rotating wall vessel bioreactors. We qualitatively and quantitatively compared organoid engraftment to that of single-cell hepatocyte transplants. In addition, we determined the effect of adding stellate cells to hepatocytes to form co-aggregated organoids and the effect of partial hepatectomy of the host liver on organoid engraftment.ResultsDirect orthotopic implantation of hepatic organoids within a hepatotomy site resulted in local engraftment of exogenous hepatocytes with limited durability. Hepatocyte-stellate cell organoids produced more extracellular matrix but did not significantly improve engraftment compared with hepatocyte-alone organoids. Partial hepatectomy of the host liver led to significantly decreased engraftment of organoids. Survival of organoids was limited by the presence of apoptotic hepatocytes within organoids as early as 1 h after implantation. Organoids eventually became necrotic and elicited a chronic inflammatory giant cell reaction similar to a foreign body response.ConclusionsWith additional organoid and host factor optimization, direct orthotopic implantation of hepatic organoids may be an approach to introduce large numbers of exogenous hepatocytes into recipient livers.