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Endocytosis of nerve growth factor by 'differentiated' PC12 cells studied by quantitative ultrastructural autoradiography.
The endocytosis of [125I]nerve growth factor (NGF) by rat pheochromocytoma cells (PC12 line), previously exposed to the growth factor ('differentiated' or 'primed' cells), was studied by ultrastructural quantitative autoradiography. Cells previously grown in the presence of NGF were incubated at 37 degrees C with [125I]NGF for periods of up to 24 h. Under these culture conditions, PC12 cells have a rich network of neurites. At the commencement of the experiment, after incubation of cells with [125I]NGF for 1 min at room temperature, the plasma membranes of perikarya and processes showed similar levels of labeling by [125I]NGF of 0.186 +/- 0.03 grains/micron and 0.152 +/- 0.013 grains/micron respectively. The density of grains per micron of plasma membrane of perikarya reached a plateau between 15 min to 2 h of incubation of cells at 37 degrees C with [125I]NGF (0.58 +/- 0.15 grains/micron and 0.65 +/- 0.18 grains/micron, respectively). The endocytosis of [125I]NGF in perikarya of cells incubated for 6 h at 37 degrees C was studied by the 'mask' analysis method of Salpeter et al.22. At this time, the greatest amount of endocytosis was observed, corresponding to 28.4% of total grain counts. The following optimized computed source densities, or relative specific activities +/- standard errors of measurement (S.E.M.), were obtained: plasma membrane, 16.52 +/- 0.86; multivesicular bodies, 9.58 +/- 2.84; endosomes, 5.00 +/- 0.97; smooth vesicles and tubules, 1.66 +/- 0.38; lysosomes, 1.13 +/- 0.20; mitochondria, 0.46 +/- 0.10; nuclear membranes or envelopes, 0.32 +/- 0.14; nuclei, 0.06 +/- 0.01; the Golgi apparatus, 0.08 +/- 0.06; and other cytoplasmic elements 0.07 +/- 0.03. Our findings indicate that smooth vesicles and tubules, endosomes, multivesicular bodies and lysosomes are part of the pathway(s) of endocytosis of NGF, while all other cytoplasmic and nuclear elements, including the nuclear membrane, are not. The heavy plasma membrane labeling of NGF and the relatively low degree of its endocytosis are consistent with the hypothesis that the NGF action is mediated through plasma membrane activated second messenger(s).
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Potassium channel dysfunction in human neuronal models of Angelman syndrome.
Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.
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Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy.
Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients' primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.
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Oncoprotein-specific molecular interaction maps (SigMaps) for cancer network analyses.
Tumor-specific elucidation of physical and functional oncoprotein interactions could improve tumorigenic mechanism characterization and therapeutic response prediction. Current interaction models and pathways, however, lack context specificity and are not oncoprotein specific. We introduce SigMaps as context-specific networks, comprising modulators, effectors and cognate binding-partners of a specific oncoprotein. SigMaps are reconstructed de novo by integrating diverse evidence sources-including protein structure, gene expression and mutational profiles-via the OncoSig machine learning framework. We first generated a KRAS-specific SigMap for lung adenocarcinoma, which recapitulated published KRAS biology, identified novel synthetic lethal proteins that were experimentally validated in three-dimensional spheroid models and established uncharacterized crosstalk with RAB/RHO. To show that OncoSig is generalizable, we first inferred SigMaps for the ten most mutated human oncoproteins and then for the full repertoire of 715 proteins in the COSMIC Cancer Gene Census. Taken together, these SigMaps show that the cell's regulatory and signaling architecture is highly tissue specific.
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A gut-on-a-chip incorporating human faecal samples and peristalsis predicts responses to immune checkpoint inhibitors for melanoma.
Patient responses to immune checkpoint inhibitors can be influenced by the gastrointestinal microbiome. Mouse models can be used to study microbiome-host crosstalk, yet their utility is constrained by substantial anatomical, functional, immunological and microbial differences between mice and humans. Here we show that a gut-on-a-chip system mimicking the architecture and functionality of the human intestine by including faecal microbiome and peristaltic-like movements recapitulates microbiome-host interactions and predicts responses to immune checkpoint inhibitors in patients with melanoma. The system is composed of a vascular channel seeded with human microvascular endothelial cells and an intestinal channel with intestinal organoids derived from human induced pluripotent stem cells, with the two channels separated by a collagen matrix. By incorporating faecal samples from patients with melanoma into the intestinal channel and by performing multiomic analyses, we uncovered epithelium-specific biomarkers and microbial factors that correlate with clinical outcomes in patients with melanoma and that the microbiome of non-responders has a reduced ability to buffer cellular stress and self-renew. The gut-on-a-chip model may help identify prognostic biomarkers and therapeutic targets.
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BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.
Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.
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Microfluidic engineering of polymer-based drug delivery systems: From particle synthesis to organ-on-a-chip validation and application.
The convergence of microfluidic engineering and organ-on-a-chip (OoC) technology is redefining the development and preclinical validation of polymer-based drug delivery systems and biomedical preparations. This review presents a bidirectional framework: first, how microfluidics enables precision synthesis of polymer particles with controlled size, morphology and multifunctionality; second, how OoC platforms serve as physiologically relevant testbeds to evaluate these particles under dynamic, human-mimetic conditions. We examine droplet microfluidics principles for generating monodisperse polymer particles (spheres, Janus, core-shell, porous architectures) from natural and synthetic polymers. These particles are explored as functional additives within OoC systems, as embedded sensors for real-time oxygen/pH monitoring, as controlled-release depots for localized growth factor delivery and as building blocks (microscaffolds, bioinks, spheroid templates) for constructing three-dimensional tissue microenvironments. Conversely, we critically analyze how OoC platforms, including vascularized tumor models, liver-kidney multi-organ chips and blood-brain barrier systems, enable more physiologically relevant assessment of micro- and nanoparticle transport, extravasation, toxicity, immunogenicity and metabolism under fluidic shear and multi-cellular complexity, which are capabilities inaccessible to static cultures. Current challenges (scalability, GMP compliance, standardization, regulatory context-of-use) and emerging opportunities (smart responsive particles, personalized screening using patient-derived organoids, AI-driven automation and closed-loop optimization) are critically discussed. This review demonstrates that microfluidic particle engineering and OoC technology together form an iterative framework for advancing next-generation biomaterials and supporting their preclinical-to-clinical development pathway.
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IL-6 and IL-8, secreted by myofibroblasts in the tumor microenvironment, activate HES1 to expand the cancer stem cell population in early colorectal tumor.
Interaction between a tumor and its microenvironment is important for tumor initiation and progression. Cancer stem cells (CSCs) within the tumor interact with a microenvironmental niche that controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts into the microenvironment of early colorectal cancer tumors and its molecular mechanism. By messenger RNA microarray analysis, expression of HES1, a Notch signaling target, significantly increased in Caco-2 cells cocultured with 18Co cells (pericryptal myofibroblasts), compared to its expression in Caco-2 cells cultured alone. Caco-2 cells cultured in 18Co-conditioned media (CM) showed a significant increase in CD133+CD44+ cells and HES1 expression compared to that in Caco-2 cells cultured in regular media. Significant amounts of interleukin-6 (IL-6) and IL-8 were detected in 18Co-CM compared to levels in regular media. The 18Co-CM-induced increase in CD133+CD44+ cells was attenuated by IL-6- and IL-8-neutralizing antibodies. Furthermore, these neutralizing antibodies and inhibitors of STAT3 and gamma-secretase reduced the expression of HES1 induced in Caco-2 cells cultured in 18Co-CM. Immunohistochemical analysis of human tissues revealed that IL-6, IL-8, and HES1 expression increased from normal to adenoma, and from adenoma to cancer tissues. In addition, IL-6 and HES1 expression was positively correlated in early colorectal cancer tissues. In conclusion, the increase of CSCs by myofibroblasts could be mediated by IL-6/IL-8-induced HES1 activation in the tumor microenvironment. Based on these data, the IL-6/IL-8-mediated Notch/HES1 and STAT3 pathway, through which CSCs interact with their microenvironment, might be a potential target for the prevention and treatment of colorectal tumors.
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Patient-derived three-dimensional cortical neurospheres to model Parkinson's disease.
There are currently no preventive or disease-modifying therapies for Parkinson's Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.
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ETV2 Mediated Differentiation of Human Pluripotent Stem Cells Results in Functional Endothelial Cells for Engineering Advanced Vascularized Microphysiological Models.
Patient-specific microphysiological models have become a valuable tool for broad applications, revolutionizing biomedical research. However, limitations persist, with functional vasculature being a significant challenge. With the discovery of ETV2's determinant role in specifying EC lineages during differentiation, researchers have adopted techniques involving ETV2 overexpression to produce h-iECs more efficiently and consistently. Here, we generated multiple h-iPSC lines with inducible ETV2 expression, and subsequently differentiated them into h-iECs, which were validated functionally and by key endothelial markers and RNA-seq analysis. These cells are capable of reproducibly self-organizing into stable microvascular networks (MVNs) in a microfluidic chip, forming lumenized and functional vessels that mimic the in vivo capillary bed in both morphology and function-a result not achieved using h-iECs differentiated with previous two-step methods using the same h-iPSC lines. Complex microphysiological models featuring perfusable vasculature were also successfully developed using ETV2-activated h-iECs, demonstrated with vascularized tumor and blood-brain barrier (BBB) models. Additionally, by pooling genetically engineered h-iPSCs with inducible ETV2, we employed an orthogonally induced differentiation approach to enhance vascularization of an organoid model. Our methodology opens avenues in precision medicine, leading to personalized microphysiological models with perfusable vasculature for various applications.