To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

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.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7 days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5 Hz, 1 Hz, and 2 Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24 h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7 d. Pacing rates above 1 Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7 d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1 d and 7 d, while Ca2+ transient duration was prolonged after 7 d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7 d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.

Reproductive processes are dynamic and involve extensive morphological remodeling and cell-cell interactions. Live imaging of organs enhances our understanding of how biological processes occur in real time. Slice culture is a type of organ culture where thick slices are collected from an organ and cultured for several days. Slice culture is a useful and easy-to-implement technique for live imaging of reproductive events at cellular resolution. Here we describe a pipeline of live imaging on slice culture to visualize the process of urethra closure in mouse embryonic penis as a proof of principle. In combination with genetic reporter mice, nuclear stains, and exposure experiments, we demonstrate the feasibility of slice culture on a reproductive organ. We also provide a step-by-step protocol and troubleshooting guide to facilitate the adoption of slice culture with live imaging in other reproductive organs. Lastly, we discuss potential utilities and experiments that could be implemented with slice culture in reproductive sciences.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

BACKGROUND: StrataGraft® (allogeneic cultured keratinocytes and dermal fibroblasts in murine collagen-dsat) is an FDA-approved viable bioengineered allogeneic cellularized construct for adult patients with deep partial-thickness burns requiring surgery. We characterized the structural and functional properties of StrataGraft to improve product understanding by evaluating extracellular matrix (ECM) molecule distribution and secreted protein factor expression in vitro.
METHODS: ECM protein expression was determined using indirect immunofluorescence on construct cross sections using commercial antibodies against collagen III, IV, VI, laminin-332, and decorin. Human collagen I expression was verified by enzyme-linked immunosorbent assay (ELISA) for collagen I C-terminal propeptide. Soluble protein factor secretion was quantified by multiplex biomarker assays and singleplex ELISA in conditioned media from meshed constructs.
RESULTS: StrataGraft cellular components produced collagen I, collagen III, collagen VI, and decorin in patterns indicating an organized ECM. Distributions of collagen IV and laminin-332 indicated formation of basement membranes and dermal-epidermal junctions. Soluble protein factors were observed in the pg/cm2/h range from 1 h to the experiment end at 168 h.
CONCLUSIONS: The organization of the ECM proteins was like human skin and the viable cellular components provided sustained secretion of soluble wound healing factors, making StrataGraft an attractive option for treating severe burns.

We are witnessing the revival of the CAM model, which has already used been in the past by several researchers studying angiogenesis and anti-cancer drugs and now offers a refined model to fill, in the translational meaning, the gap between in vitro and in vivo studies. It can be used for a wide range of purposes, from testing cytotoxicity, pharmacokinetics, tumorigenesis, and invasion to the action mechanisms of molecules and validation of new materials from tissue engineering research. The CAM model is easy to use, with a fast outcome, and makes experimental research more sustainable since it allows us to replace, reduce, and refine pre-clinical experimentation (\"3Rs\" rules). This review aims to highlight some unique potential that the CAM-assay presents; in particular, the authors intend to use the CAM model in the future to verify, in a microenvironment comparable to in vivo conditions, albeit simplified, the angiogenic ability of functionalized 3D constructs to be used in regenerative medicine strategies in the recovery of skeletal injuries of critical size (CSD) that do not repair spontaneously. For this purpose, organotypic cultures will be planned on several CAMs set up in temporal sequences, and a sort of organ model for assessing CSD will be utilized in the CAM bioreactor rather than in vivo.

Podoplanin (PDPN) is widely used as a marker of lymphatic endothelial cells. PDPN is also involved in tumor progression, and upregulated PDPN expression is often found in various cancers. In this study, we first immunohistochemically examined PDPN expression in 87 cases of Bowen disease. Positive expression was detected in 64.4% of Bowen disease specimens, and the positive cells were exclusively located in the basal layer and corresponded to palisaded basal cells (PBCs). PBCs have been considered to be residual normal keratinocytes so far, but PDPN expression in cancers is generally associated with poor clinical outcomes. We also examined PDPN expression in 27 cases of Bowen carcinoma. Diffuse and strong PDPN expression was detected in 22.2% of Bowen carcinoma specimens, and another 22.2% showed PDPN expression at the leading edges of tumor nests. These results prompted us to determine whether PDPN-positive cells are more tumorigenic than PDPN-negative cells. We cultured Bowen disease cells using a three-dimensional (3D) cell culture system and examined PDPN expression. In the cultured Bowen disease tissue, PDPN expression was again detected in the basal layer. Then, we isolated 1.2 × 105 PDPN-positive and -negative cells from the 3D organotypic culture of Bowen disease by fluorescence-activated cell sorting analysis and compared their tumorigenicity using 3D culture. The PDPN-positive tumor cells were able to regenerate Bowen disease tissue, but the PDPN-negative tumor cells were not. In addition, the regenerated Bowen disease tissue derived from the PDPN-positive cells exhibited PDPN expression in its basal layer, as the parental Bowen disease tissue did. These results indicate that PDPN-positive cells include tumor cells with cancer stem cell properties. Although the precise mechanism through which PDPN expression is involved in the pathogenesis of Bowen disease needs to be determined, PDPN may be a novel druggable target for Bowen disease.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.