Co-culture Studies Research Articles

Astrocyte TrkB.T1 deficiency disrupts glutamatergic synaptogenesis and astrocyte-synapse interactions.

Perisynaptic astrocyte processes (PAPs) contact pre- and post-synaptic elements to provide structural and functional support to synapses. Accumulating research demonstrates that the cradling of synapses by PAPs is critical for synapse formation, stabilization, and plasticity. The specific signaling pathways that govern these astrocyte-synapse interactions, however, remain to be elucidated. Herein, we demonstrate a role for the astrocyte TrkB.T1 receptor, a truncated isoform of the canonical receptor for brain derived neurotrophic factor (BDNF), in modulating astrocyte-synapse interactions and excitatory synapse development. Neuron-astrocyte co-culture studies revealed that loss of astrocyte TrkB.T1 disrupts the formation of PAPs. To elucidate the role of TrkB.T1 in synapse development, we conditionally deleted TrkB.T1 in astrocytes in mice. Synaptosome preparations were employed to probe for TrkB.T1 localization at the PAP, and confocal three-dimensional microscopy revealed a significant reduction in synapse density and astrocyte-synapse interactions across development in the absence of astrocytic TrkB.T1. These findings suggest that BDNF/TrkB.T1 signaling in astrocytes is critical for normal excitatory synapse formation in the cortex and that astrocyte TrkB.T1 serves a requisite role in astrocyte synapse interactions. Overall, this work provides new insights into the molecular mechanisms of astrocyte-mediated synaptogenesis and may have implications for understanding neurodevelopmental disorders and developing potential therapeutic targets.

Zinc and gallium doped borate bioactive glasses influence in-vitro angiogenesis: New evidence in cell co-culture studies

Borate bioactive glasses doped with 1 mol% ZnO or Ga2O3 were produced by melt-quenching. The cytocompatibility of fibroblast and endothelial cells in response to glass extracts was assessed. The angiogenic potential was examined through fibroblast-derived vascular endothelial growth factor secretion and tubular formation analysis in an indirect co-culture of fibroblast and endothelial cells. The results confirm that Zn and Ga ion-releasing bioactive glasses are attractive biomaterials with angiogenic properties.

Neuroprotective Role of AQP4 Knockdown in Astrocytes After Oxygen-Glucose Deprivation.

Aquaporin-4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post-stroke neuroinflammation is not fully understood. Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post-stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post-MCAO. Additionally, primary astrocytes were isolated for quantitative real-time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co-culture studies. MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co-culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD-treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(-) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post-OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post-OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN-020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. Our findings underscore AQP4's pivotal role in modulating post-stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia-induced neuronal damage.

Evaluating the Role of Nrf-2/HO-1 Pathway in Glioblastoma Treatment Efficacy: A Co-Culture Study

Objective: Glioblastoma (GB) is a highly lethal form of brain tumor. Although standard therapy appears to be effective, the survival time is quite short, and the recurrence rate is high. Bortezomib (BTZ), is a proteasome inhibitor, used in GB therapies and resulted in serious off-target effects. Carfilzomib (CFZ), is an alternative for BTZ, has known with nonserious off-target effects. This study aimed to examine the potential off-target effects caused by BTZ and CFZ in terms of the therapy related activation of antioxidant mechanisms regarding to Nuclear factor (erythroid-derived 2)-like 2 (Nrf-2)/Heme Oxygenase-1 (HO-1)-dependent response. Methods: GB cells were co-cultured with heathy astrocyte (HA) cells to mimic the tumor microenvironment in some extent. Cell viability was determined following ionizing radiation (IR), temozolomide (TMZ), BTZ and CFZ alone and in combination. Nrf-2 and HO-1 protein expressions were analyzed by western blotting assay. Results: Co-culture results showed that the GB cells in the BTZ-treated groups expressed higher levels of Nrf-2 and HO-1 than in the CFZtreated groups. In the HAs, the group treated with CFZ showed higher Nrf-2 expression than the group treated with BTZ alone, while the same groups in combination with TMZ&IR showed exactly opposite results. HO-1 expression was also not seen in any of the HA groups. Conclusion: The significant increase in Nrf-2 levels in the CFZ-treated group in the HAs could also be interpreted as CFZ promoting the defence of healthy cells against therapy-induced stress conditions. Although further studies are needed, these preliminary results show that the evaluation of CFZ as a second-line therapy could be a milestone for the treatment of GB.

Enhancement of Triple-Negative Breast Cancer-Specific Induction of Cell Death by Silver Nanoparticles by Combined Treatment with Proteotoxic Stress Response Inhibitors.

Metal nanoparticles have been tested for therapeutic and imaging applications in pre-clinical models of cancer, but fears of toxicity have limited their translation. An emerging concept in nanomedicine is to exploit the inherent drug-like properties of unmodified nanomaterials for cancer therapy. To be useful clinically, there must be a window between the toxicity of the nanomaterial to cancer and toxicity to normal cells. This necessitates identification of specific vulnerabilities in cancers that can be targeted using nanomaterials without inducing off-target toxicity. Previous studies point to proteotoxic stress as a driver of silver nanoparticle (AgNPs) toxicity. Two key cell stress responses involved in mitigating proteotoxicity are the heat shock response (HSR) and the integrated stress response (ISR). Here, we examine the role that these stress responses play in AgNP-induced cytotoxicity in triple-negative breast cancer (TNBC) and immortalized mammary epithelial cells. Furthermore, we investigate HSR and ISR inhibitors as potential drug partners to increase the anti-cancer efficacy of AgNPs without increasing off-target toxicity. We showed that AgNPs did not strongly induce the HSR at a transcriptional level, but instead decreased expression of heat shock proteins (HSPs) at the protein level, possibly due to degradation in AgNP-treated TNBC cells. We further showed that the HSR inhibitor, KRIBB11, synergized with AgNPs in TNBC cells, but also increased off-target toxicity in immortalized mammary epithelial cells. In contrast, we found that salubrinal, a drug that can sustain pro-death ISR signaling, enhanced AgNP-induced cell death in TNBC cells without increasing toxicity in immortalized mammary epithelial cells. Subsequent co-culture studies demonstrated that AgNPs in combination with salubrinal selectively eliminated TNBCs without affecting immortalized mammary epithelial cells grown in the same well. Our findings provide additional support for proteotoxic stress as a mechanism by which AgNPs selectively kill TNBCs and will help guide future efforts to identify drug partners that would be beneficial for use with AgNPs for cancer therapy.

The Role of Breastmilk in Macrophage-Tumour Cell Interactions in Postpartum Breast Cancer

Background: Lactation is associated with long-term reduced risk of breast cancer. However, there is a transient increased risk of breast cancer in the 5 to 10 years postpartum and this is associated with a high incidence of metastasis and mortality. Breastmilk is a physiological fluid secreted by the mammary glands intimately connected with breast cells and the microenvironment that may affect postpartum breast cancer development and progression. This study aims to investigate the effect of breastmilk on interactions between breast cancer cells and macrophages in vitro. Methods: Human breastmilk from healthy donors (n = 7) was pooled and incubated with breast cancer (MCF-7 and MDA-MB-231) and macrophage (RAW264.7) cell lines to assess cell proliferation, viability, migration, and expression of key genes associated with epithelial-mesenchymal transition (EMT) and macrophage phenotype. Indirect co-culture studies assessed the effect of breastmilk on interactions between breast cancer cells and macrophages. Results: Breastmilk increased the proliferation and viability of breast cancer cells, reduced EMT markers, and reduced cell migration in MDA-MB-231 cells. Breastmilk decreased mRNA expression of interleukin 1B (IL1B) and interleukin 10 (IL10) in macrophages. Reduced EMT marker expression was observed in breast cancer cells co-cultured with macrophages pre-treated with breastmilk. Macrophages co-cultured with breast cancer cells pre-treated with breastmilk exhibited increased expression of a pro-inflammatory cytokine tumor necrosis factor A (TNFA) and pro-inflammatory nitric oxide synthase 2 (NOS2), and reduced expression of cytokines IL10 and transforming growth factor B1 (TGFB1) which are associated with the alternatively-activated macrophage phenotype. Conclusions: Breastmilk has the potential to promote breast cancer proliferation, however, it can also reduce breast cancer progression through inhibition of breast cancer cell migration and regulation of macrophage polarisation. These findings suggest that breastmilk has potential to shape the tumour microenvironment in postpartum breast cancer.

Neutrophil membrane-coated multifunctional biomimetic nanoparticles for spinal cord injuries

Spinal cord injury (SCI) is one of the most complex diseases. After SCI, severe secondary injuries can cause intense inflammatory storms and oxidative stress responses, leading to extensive neuronal apoptosis. Effective regulation of inflammation and oxidative stress after SCI remains an unresolved challenge. In this study, resveratrol-loaded nanoparticles coated with neutrophil membranes (NMR) were prepared using the emulsion-solvent evaporation method and membrane encapsulation technology. Multifunctional biomimetic nanoparticles retain neutrophil membrane-related receptors and possess a strong adsorption capacity for inflammatory factors. As a drug carrier, NMR can sustainably release resveratrol for >72 h. Moreover, co-culture studies in vitro show that the NMR help regulate macrophage polarization to relieve inflammatory response, reduce intracellular reactive oxygen species by approximately 50%, and improve mitochondrial membrane potential to alleviate oxidative stress. After injecting NMR into the injury site, it reduces early apoptosis, inhibit scar formation, and promote neural network recovery to improve motor function. This study demonstrates the anti-inflammatory, antioxidant, and neuroprotective effects of NMR, thus providing a novel therapeutic strategy for SCI.

ST2 + T-Regulatory Cells in Renal Inflammation and Fibrosis after Ischemic Kidney Injury.

Inflammation is a major cause of kidney injury. The Interleukin (IL)-1 family cytokine IL-33 is released from damaged cells and modulates the immune response through its receptor ST2 expressed on many cell types, including regulatory T-cells (Tregs). While a proinflammatory role of IL-33 has been proposed, exogenous IL-33 expanded Tregs and suppressed renal inflammation. However, the contribution of endogenous IL-33/ST2 for the role of Tregs in the resolution of kidney injury has not been investigated. We used murine renal ischemia-reperfusion injury and kidney organoids to delineate the role of the ST2 and amphiregulin (AREG) specifically in Treg cells using targeted deletion. Bulk and single-cell RNA sequencing was performed on flow-sorted Tregs from spleen and CD4 T-cells from post-ischemic kidneys respectively. The protective role of ST2-sufficient Tregs was analyzed using a novel co-culture system of syngeneic kidney organoids and Treg cells under hypoxic conditions. Bulk RNA-sequencing of splenic and single-cell-RNA-sequencing of kidney T-cells showed that ST2+ Tregs are enriched for genes related to Treg proliferation and function. Genes for reparative factors such as AREG were also enriched in ST2+ Tregs. Treg-specific deletion of ST2 or AREG exacerbated kidney injury and fibrosis in the unilateral ischemia reperfusion injury model. In co-culture studies, WT but not ST2-deficient Tregs preserved the hypoxia-induced loss of kidney-organoid viability, which was restored by AREG supplementation. Our study identified the role of the IL-33/ST2 pathway in Tregs for resolution of kidney injury. The transcriptome of ST2+ Tregs was enriched for reparative factors including AREG. Lack of ST2 or AREG in Tregs worsened kidney injury. Tregs protected kidney organoids from hypoxia in ST2 and AREG-dependent manner. Thus Treg-based approaches could be of benefit for resolution of renal injury.

Suppression of inner blood-retinal barrier breakdown and pathogenic Müller glia activation in ischemia retinopathy by myeloid cell depletion

Ischemic retinopathies including diabetic retinopathy are major causes of vision loss. Inner blood-retinal barrier (BRB) breakdown with retinal vascular hyperpermeability results in macular edema. Although dysfunction of the neurovascular unit including neurons, glia, and vascular cells is now understood to underlie this process, there is a need for fuller elucidation of the underlying events in BRB dysfunction in ischemic disease, including a systematic analysis of myeloid cells and exploration of cellular cross-talk. We used an approach for microglia depletion with the CSF-1R inhibitor PLX5622 (PLX) in the retinal ischemia-reperfusion (IR) model. Under non-IR conditions, PLX treatment successfully depleted microglia in the retina. PLX suppressed the microglial activation response following IR as well as infiltration of monocyte-derived macrophages. This occurred in association with reduction of retinal expression of chemokines including CCL2 and the inflammatory adhesion molecule ICAM-1. In addition, there was a marked suppression of retinal neuroinflammation with reduction in expression of IL-1b, IL-6, Ptgs2, TNF-a, and Angpt2, a protein that regulates BRB permeability. PLX treatment significantly suppressed inner BRB breakdown following IR, without an appreciable effect on neuronal dysfunction. A translatomic analysis of Müller glial-specific gene expression in vivo using the Ribotag approach demonstrated a strong suppression of Müller cell expression of multiple pro-inflammatory genes following PLX treatment. Co-culture studies of Müller cells and microglia demonstrated that activated microglia directly upregulates Müller cell-expression of these inflammatory genes, indicating Müller cells as a downstream effector of myeloid cells in retinal IR. Co-culture studies of these two cell types with endothelial cells demonstrated the ability of both activated microglia and Müller cells to compromise EC barrier function. Interestingly, quiescent Müller cells enhanced EC barrier function in this co-culture system. Together this demonstrates a pivotal role for myeloid cells in inner BRB breakdown in the setting of ischemia-associated disease and indicates that myeloid cells play a major role in iBRB dysregulation, through direct and indirect effects, while Müller glia participate in amplifying the neuroinflammatory effect of myeloid cells.

Advancing Bioactive Material for Mandibular Bone Regeneration: Transformation of Fibrous Mat into 3D Matrix Cotton for Enhanced Shape Retention and Rapid Hemostasis.

Transformation of a fibrous mat into a three-dimensional (3D) scaffold opens up abundant innovative prospects in biomedical research, particularly for studying both soft as well as hard tissues. Electrospun nanofibers, which mimic the extracellular matrix have attracted significant attention in various studies. This research focuses on rapidly converting a fibrous mat made of polycaprolactone (PCL)/pluronic F-127 (PF-127) with different percentages of monetite calcium phosphate (MCP) into desirable 3D matrix cotton using a unique gas foaming technology. These matrix cottons possess biomimetic properties and have oriented porous structures. Using this innovative technique, various shapes of 3D matrix cotton, such as squares, hollow tubes, and other customizable forms, were successfully produced. Importantly, these 3D matrix cottons showed a consistent distribution of monetite particles with total porosity ranging from 90% to 98%. The structure of the 3D matrix cotton, its water/blood absorption capacity, the potential for causing non-hemolysis, and rapid hemostatic properties were thoroughly investigated. Additionally, periodontal cells were cultured on the 3D matrix cotton to assess their viability and morphology, revealing promising results. Furthermore, a coculture study involving NIH-3T3 and MG-63 cells on the 3D matrix cotton showed spheroidal formation within 24 h. Notably, in vitro assessments indicated that the matrix cotton containing 15% monetite (PCL-MMC15%) exhibited superior absorbent capabilities, excellent cell viability, and rapid hemostatic characteristics. Subsequently, the effectiveness of PCL-MMC15% in promoting mandibular bone regeneration was evaluated through an in vivo study on rabbits using a mandibular injury model. The results demonstrated that PCL-MMC15% facilitated the resolution of defects in the mandibular region by initiating new bone formation. Therefore, the presented 3D matrix cotton (PCL-MMC15%) shows significant promise for applications in both mandibular bone regeneration and hemostasis.

The Influence of Sulfation Degree of Glycosaminoglycan-Functionalized 3D Collagen I Networks on Cytokine Profiles of In Vitro Macrophage-Fibroblast Cocultures.

Cell-cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell-cell communication of the fibroblast-macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

Exopolysaccharides from Limosilactobacillus reuteri: their influence on in vitro activation of porcine monocyte-derived dendritic cells - brief report

The aim of this study was to evaluate the immunomodulatory potential of two α-D-glucans from Limosilactobacillus reuteri L26 Biocenol™ (EPS-L26) and L. reuteri DSM17938 (EPS-DSM17938), with respect to their influence on in vitro activation of porcine dendritic cells (DCs). We used immature DCs differentiated from porcine blood monocytes under in vitro conditions. Based on the surface expression of MHC II and costimulatory CD80/86 molecules, we showed that both used EPSs favour the maturation of monocyte-derived DCs (MoDCs) similarly to the commonly used stimulant tumour necrosis factor α (TNF-α). In contrast to TNF-α stimulation, MoDCs treated with both used EPSs significantly up-regulated the mRNA levels not only for interleukin (IL)-10 (P < 0.0001 for EPS-DSM17938; P = 0.0037 for EPS-L26), but also for IL-12 (P = 0.0176 for EPS-DSM17938; P = 0.0019 for EPS-L26). These cytokines are known to regulate T-cell kinetics and play a key role in maintaining immune homeostasis. Interestingly, only relatively linear α-D-glucan (EPS-DSM17938) significantly increased gene expression of the major pro-inflammatory cytokine IL-1β (P = 0.0011) and the “SOS” cytokine IL-6 (P = 0.0127). However, it is important to highlight the need for further studies aimed at cytokine kinetics in DCs, as well as a co-culture study with allogenic T-lymphocytes.

Gentiopicroside improves NASH and liver fibrosis by suppressing TLR4 and NLRP3 signaling pathways

BackgroundNon-alcoholic steatohepatitis (NASH) and liver fibrosis are progressive conditions associated with non-alcoholic fatty liver disease (NAFLD), characterized by hepatocyte pyroptosis and hepatic stellate cell (HSC) activation. Gentiopicroside (GPS) has emerged as a potential treatment for NASH, yet its underlying mechanism remains unclear. AimTo confirm that GPS can improve NASH and liver fibrosis by blocking the NLRP3 signaling pathway Study designInitially, different animal models were used to study the effects and mechanisms of GPS on NASH and fibrosis. Subsequent in vitro experiments utilized co-cultures and other techniques to delve deeper into its mechanism, followed by validation of the findings in mouse liver tissues. MethodsC57BL/6 mice were fed high-fat, high-cholesterol (HFHC), or methionine-choline-deficient (MCD) diets to induce NASH and fibrosis. RAW264.7 cells and born marrow bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP to induce inflammation, then co-cultured with primary hepatocytes and HSCs, treated with GPS, and its efficacy and mechanism were analyzed. ResultsIn vivo, GPS alleviated NASH and liver fibrosis by inhibiting the NLRP3 pathway. In vitro, GPS attenuated inflammation induced by BMDMs by inhibiting TLR4 and NLRP3 signaling pathways, and Co-culture studies suggested that GPS reduced hepatocyte pyroptosis and HSC activation, which was also confirmed in liver tissues ConclusionGPS improves NASH and liver fibrosis by inhibiting the TLR4 and NLRP3 signaling pathways. The specific mechanism may be related to the suppression of macrophage-mediated inflammatory responses, thereby reducing hepatocyte pyroptosis and HSC activation.

Enhancing the treatment of Staphylococcus aureus infections: A nanosystem with including dual antimicrobial peptide

The unique properties of nanoparticles (NPs) and their modification to selectively target infected tissues or specific pathogens could be an effective approach to revolutionize the treatment of infectious diseases. The objective of this study was to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with an antimicrobial peptide (HF-18) and decorated with an antimicrobial peptide (P6.2) for selective targeting against Staphylococcus aureus (S. aureus). Applying the Box-Behnken experimental design, HF-18 loaded PLGA nanoparticles were produced and optimized to have minimum particle size and polydispersity index (PDI) value by selecting primary water phase volume, organic phase volume and secondary water phase volume as independent parameters. Secondary water phase volume had the most significant effect on particle size and PDI. OPT-NP was more effective than HF-18 against most bacterial strains. The non-targeted (OPT-NP) and targeted (P6.2-OPT-NP) systems were shown to be biocompatible at levels that exhibited antibacterial activity. The P6.2-OPT-NP was predominantly taken up by S. aureus, while Escherichia coli (E. coli) used as a negative control showed minimal uptake. Furthermore, co-culture studies indicated that P6.2-OPT-NP was drastically uptake by S. aureus, while no internalized nanoparticles were visualized in fibroblast cells indicating active targeting of bacterial cells by P6.2-OPT-NP. Thus, these findings demonstrate the development of a formulation that selectively targets S. aureus, presenting promising prospects for future therapeutic applications.

Administration of Human-Derived Mesenchymal Stem Cells Activates Locally Stimulated Endogenous Neural Progenitors and Reduces Neurological Dysfunction in Mice after Ischemic Stroke.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Recent insights from human induced pluripotent stem cell models into the role of microglia in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, primarily leading to the degeneration of motor neurons. The traditional focus on motor neuron-centric mechanisms has recently shifted towards understanding the contribution of non-neuronal cells, such as microglia, in ALS pathophysiology. Advances in induced pluripotent stem cell (iPSC) technology have enabled the generation of iPSC-derived microglia monocultures and co-cultures to investigate their role in ALS pathogenesis. Here, we briefly review the insights gained from these studies into the role of microglia in ALS. While iPSC-derived microglia monocultures have revealed intrinsic cellular dysfunction due to ALS-associated mutations, microglia-motor neuron co-culture studies have demonstrated neurotoxic effects of mutant microglia on motor neurons. Based on these findings, we briefly discuss currently unresolved questions and how they could be addressed in future studies. iPSC models hold promise for uncovering disease-relevant pathways in ALS and identifying potential therapeutic targets.

Abstract PO5-25-02: Microenvironment based co-culture dependence of breast cancer and immune cell interactions on functional outcomes

Abstract The tumour microenvironment (TME) includes physical forces from interstitial fluid flow and cell-cell interactions that modulate cancer development and progression through activation of multiple oncogenic pathways leading to tumor growth and metastatic spreading. We previously reported on the role of physical forces on epithelial to mesenchymal transition (EMT) and S100 gene family expression and cell to cell adhesion properties with implications to normal breast development and cancer. Signals from the tumor exit the local tumor environment through interstitial fluid transport and cell migration where they can be found in the blood stream. We developed a whole blood RNA gene expression biomarker panel coupled with proprietary software capable of identifying the presence of an active breast cancer signature at early stages of disease with clinical study results previously reported. Here we report on results from transcriptomic and function studies with cell interaction models. Cancer cells can influence immune cells through direct contact or via secreted molecules causing the immune cells to undergo functional changes. To investigate mechanisms involved in the induction of these alterations in a model system, we performed co-culture studies with human monocytic cells and breast cancer cells. Interaction of human monocytic cells with the breast cancer cells caused significant changes in both behavior and transcriptomes. Moreover, using RNAseq and pathway analysis we demonstrated that the changes induced through the exposure to the more aggressive triple negative (TNBC) phenotype were distinct from the alterations triggered upon contact with an ER+ breast cancer cell line. We also show that cancer-immune crosstalk triggers EMT as well as activation of multiple oncogenic pathways associated with cell migration and invasion, cell chemotaxis and cell-to-cell interactions. As the interplay between cancer and immune cells plays an important role in cancer progression, these findings provide important insight into key mechanisms controlling these outcomes. Citation Format: Karen Norek, Jacob Kennard, Kenneth Fuh, Robert Shepherd, Kristina Rinker, Olesya Kharenko. Microenvironment based co-culture dependence of breast cancer and immune cell interactions on functional outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-02.

Cell Instructive Behavior of Composite Scaffolds in a Co-Culture of Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.

Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

The artificial sweetener neotame negatively regulates the intestinal epithelium directly through T1R3-signaling and indirectly through pathogenic changes to model gut bacteria.

Recent studies have indicated considerable health risks associated with the consumption of artificial sweeteners. Neotame is a relatively new sweetener in the global market however there is still limited data on the impact of neotame on the intestinal epithelium or the commensal microbiota. In the present study, we use a model of the intestinal epithelium (Caco-2) and microbiota (Escherichia coli and Enterococcus faecalis) to investigate how physiologically-relevant exposure of neotame impacts intestinal epithelial cell function, gut bacterial metabolism and pathogenicity, and gut epithelium-microbiota interactions. Our findings show that neotame causes intestinal epithelial cell apoptosis and death with siRNA knockdown of T1R3 expression significantly attenuating the neotame-induced loss to cell viability. Similarly, neotame exposure results in barrier disruption with enhanced monolayer leak and reduced claudin-3 cell surface expression through a T1R3-dependent pathway. Using the gut bacteria models, E. coli and E. faecalis, neotame significantly increased biofilm formation and metabolites of E. coli, but not E. faecalis, reduced Caco-2 cell viability. In co-culture studies, neotame exposure increased adhesion capacity of E. coli and E. faecalis onto Caco-2 cells and invasion capacity of E. coli. Neotame-induced biofilm formation, E.coli-specific Caco-2 cell death, adhesion and invasion was identified to be meditated through a taste-dependent pathway. Our study identifies novel pathogenic effects of neotame on the intestinal epithelium or bacteria alone, and in co-cultures to mimic the gut microbiome. These findings demonstrate the need to better understand food additives common in the global market and the molecular mechanisms underlying potential negative health impacts.