establishing-three-dimensional-coculture-module-epithelial-cells

Establishing a Three-dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

Ajou University

The p90 ribosomal S6 kinase family (RSK1-4) of Ser/Thr kinases is a downstream component of the Ras-MAPK cascade responsible for regulating various cellular processes. Here, we examined the potential involvement of RSKs in regulating mitosis by transfecting HeLa cells with siRNAs targeting RSK1 and -2, which are the major isoforms. Depletion of RSK1 but not RSK2 triggered a significant accumulation of binucleated cells compared to control cells (0.5% vs. 10.5%, respectively); this was rescued by expression of exogenous RSK1 but not a kinase-defective mutant. Monitoring of cell division by time-lapse imaging revealed that the observed binucleation mainly stemmed from a failure to form and ingress the cleavage furrow during early cytokinesis. Immunocytochemical analysis of RhoA and anillin, the two principal regulators of cleavage furrow formation and ingression, showed that these proteins were abnormally localized during anaphase in RSK1-depleted cells. Furthermore, RSK1-depleted cells seemed to have impairments in midzone microtubule formation, as suggested by morphological changes and lengthening of the midzone (15.2 ± 1.7 μm vs. 17.4 ± 1.7 μm in control cells). We also observed shortening of the pole-to-polar-cortex distance in RSK1-depleted cells (4.30 ± 1.37 μm vs. 2.80 ± 0.84 μm in control cells) and scanty distribution of microtubules at the periphery of the equatorial region during anaphase, suggesting an aberrant distribution of astral microtubules. Taken together, these results suggest that RSK1 is specifically required for cleavage furrow formation and ingression during cytokinesis. This may occur via the involvement of RSK1 in proper midzone and astral microtubule structure formation during anaphase, which is essential for the correct localization of anillin and RhoA.

p90 ribosomal S6 kinase 1 (RSK1) isoenzyme specifically regulates cytokinesis progression.

Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKα(Thr172) signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKα(Thr172) signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα (Thr172) signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase.

CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells.

Tetraploidy, a potential precursor of cancer-associated aneuploidy, is produced either by cell fusion or failure of cytokinesis. In this study, low p53-expressing HeLa cells were used to address the fate of cancer cells after fusion. We found that massive cell death or growth arrest occurred a few days after fusion. Interestingly, cells with larger nuclei preferentially died after fusion, suggesting that a larger deviation of DNA content is a strong inducer of apoptosis. Notably, a fraction of cells escaped cell death. Also, the stability of survivin increased, and its localization changed preferentially to the cytosol in fused cells. Knockdown of survivin decreased the survival of fused cells, more than observed in unfused cells, showing increased dependency of fused cells on survivin. Collectively, after cancer cell fusion, some fused cells avoid the apoptotic crisis partly owing to survivin, and continue to proliferate, a process that contributes to human cancer progression. [BMB Reports 2017; 50(7): 361-366].

Survivin protects fused cancer cells from cell death.

In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform.  and phagocytes were able to adhere to the nanofibers and phagocytes engulfed  in the PCL-NM. The migration of phagocytes to  was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. -infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.

Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane.

Lipid droplets (LDs) are lipid-abundant organelles found in most cell lines and primarily consist of neutral lipids. They serve as a repository of various lipids and are associated with many cellular metabolic processes, including energy storage, membrane synthesis, and protein homeostasis. LDs are prominent in a variety of diseases related to lipid regulation, including obesity, fatty liver disease, diabetes, and atherosclerosis. To monitor LD dynamics in live samples, we developed a highly selective two-photon fluorescent tracker for LDs (). It exhibited outstanding sensitivity with a remarkable two-photon-action cross section (Φδ > 600 GM), photostability, and low cytotoxicity. In human hepatocytes and  mouse liver tissue imaging,  showed very bright fluorescence with high LD selectivity and minimized background signal to evaluate the stages of nonalcoholic fatty liver disease. Interestingly, we demonstrated that the liver sinusoid morphology became narrower with increasing LD size and visualized the dynamics including fusion of the LDs . Moreover, real-time and dual-color TPM imaging with  and a two-photon lysosome tracker could be a useful predictive screening tool in the drug development process to monitor impending drug-induced liver injury inducing drug candidates.

Highly Sensitive Two-Photon Lipid Droplet Tracker for  Screening of Drug Induced Liver Injury.

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.

In Vivo Two-Photon Imaging Analysis of Dynamic Degradation of Hepatic Lipid Droplets in MS-275-Treated Mouse Liver.

The plasma membrane, which is a phosphoglyceride bilayer at the outer edge of the cell, plays diverse and important roles in biological systems. Visualization of the plasma membrane in live samples is important for various applications in biological functions. We developed an amphiphilic two-photon (TP) fluorescent probe () to selectively monitor the plasma membrane in live samples. This probe exhibited red emission (620-700 nm), large TP absorption cross sections (δ > 790 GM), and high selectivity to the plasma membrane. In cultured cells and  hepatic tissue imaging,  showed bright TP-excited fluorescence (TPEF) and remarkable selectivity for the plasma membrane. Furthermore, simultaneous  imaging with  and a TP lipid droplet probe could serve as an efficient tool to monitor morphological and physiological changes in the plasma membrane and lipid droplets.

 Simultaneous Imaging of Plasma Membrane and Lipid Droplets in Hepatic Steatosis using Red-Emissive Two-Photon Probes.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. -GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with  and liver capsule macrophages labeled with GFP in vivo. , a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP liver capsule macrophages (LCMs) in vivo in the liver capsule of -GFP mice. In the NAFLD mouse model,  LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images.  will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

An ideal extracellular matrix (ECM) replacement scaffold in a three-dimensional cell (3D) culture should induce in vivo-like interactions between the ECM and cultured cells. Highly hydrophilic polyvinyl alcohol (PVA) nanofibers disintegrate upon contact with water, resulting in the loss of their fibrous morphology in cell cultures. This can be resolved by using chemical crosslinkers and post-crosslinking. A crosslinked, water-stable, porous, and optically transparent PVA nanofibrous membrane (NM) supports the 3D growth of various cell types. The binding of cells attached to the porous PVA NM is low, resulting in the aggregation of cultured cells in prolonged cultures. PVA NMs containing integrin-binding peptides of fibronectin and laminin were produced to retain the blended peptides as cell-binding substrates. These peptide-blended PVA NMs promote peptide-specific cell adherence and growth. Various cells, including epithelial cells, cultured on these PVA NMs form layers instead of cell aggregates and spheroids, and their growth patterns are similar to those of the cells cultured on an ECM-coated PVA NM. The peptide-retained PVA NMs are non-stimulatory to dendritic cells cultured on the membranes. These peptide-retaining PVA NMs can be used as an ECM replacement matrix by providing in vivo-like interactions between the matrix and cultured cells.

Synthetic Extracellular Matrix of Polyvinyl Alcohol Nanofibers for Three-Dimensional Cell Culture.

The natural polyphenolic compound known as Rosmarinic acid (RosA) can be found in various plants. Although its potential health benefits have been extensively studied, its effect on osteoarthritis (OA) progression and cartilage regeneration function still needs to be fully elucidated in OA animal models. This study elucidated the effect of RosA on OA progression and cartilage regeneration.

In-Jeong Lee

3D Immune System Imaging Core Center

Ajou University

Establishing a Three-dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

In-Jeong Lee

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}3D Immune System Imaging Core Center

Ajou University

Establishing a Three-dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

3D Immune System Imaging Core Center