Cyclic uniaxial mechanical stretching of cells using a LEGO® parts-based mechanical stretcher system.

Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO® parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.

The polycomb protein Ezh2 regulates differentiation and plasticity of CD4(+) T helper type 1 and type 2 cells.

After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.

Tau modulates Schwann cell proliferation, migration and differentiation following peripheral nerve injury.

Tau protein (encoded by the gene microtubule-associated protein tau, Mapt) is essential for the assembly and stability of microtubule and the functional maintenance of the nervous system. Tau is highly abundant in neurons and is detectable in astrocytes and oligodendrocytes. However, whether tau is present in Schwann cells, the unique glial cells in the peripheral nervous system, is unclear. Here, we investigated the presence of tau and its coding mRNA, Mapt, in cultured Schwann cells and find that tau is present in these cells. Gene silencing of Mapt promoted Schwann cell proliferation and inhibited Schwann cell migration and differentiation. In vivo application of Mapt siRNA suppressed the migration of Schwann cells after sciatic nerve injury. Consistent with this, Mapt-knockout mice showed elevated proliferation and reduced migration of Schwann cells. Rats injected with Mapt siRNA and Mapt-knockout mice also exhibited impaired myelin and lipid debris clearance. The expression and distribution of the cytoskeleton proteins α-tubulin and F-actin were also disrupted in these animals. These findings demonstrate the existence and biological effects of tau in Schwann cells, and expand our understanding of the function of tau in the nervous system.

Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence†.

In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A two-dimensional attachment mouse secondary follicle culture system was used to assess the oocyte's capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral (PA) and early antral (EA) follicles was performed. Established endpoints were the oocyte's potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from PA and EA cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections with no significant differences in meiotic and developmental competence compared with those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as nonhuman chorionic gonadotropin triggered in vitro maturation.

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1.

Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.

Demonstration of the first-pass metabolism in the skin of the hair dye, 4-amino-2-hydroxytoluene, using the Chip2 skin-liver microphysiological model.

We used TissUse's HUMIMIC Chip2 microfluidic model, incorporating reconstructed skin models and liver spheroids, to investigate the impact of consumer-relevant application scenarios on the metabolic fate of the hair dye, 4-amino-2-hydroxytoluene (AHT). After a single topical or systemic application of AHT to Chip2 models, medium was analysed for parent and metabolites over 5 days. The metabolic profile of a high dose (resulting in a circuit concentration of 100 µM based on 100% bioavailability) of AHT was the same after systemic and topical application to 96-well EpiDerm™ models. Additional experiments indicated that metabolic capacity of EpiDerm™ models were saturated at this dose. At 2.5 µM, concentrations of AHT and several of its metabolites differed between application routes. Topical application resulted in a higher Cmax and a 327% higher area under the curve (AUC) of N-acetyl-AHT, indicating a first-pass effect in the EpiDerm™ models. In accordance with in vivo observations, there was a concomitant decrease in the Cmax and AUC of AHT-O-sulphate after topical, compared with systemic application. A similar alteration in metabolite ratios was observed using a 24-well full-thickness skin model, EpiDermFT™, indicating that a first-pass effect was also possible to detect in a more complex model. In addition, washing the EpiDermFT™ after 30 min, thus reflecting consumer use, decreased the systemic exposure to AHT and its metabolites. In conclusion, the skin-liver Chip2 model can be used to (a) recapitulate the first-pass effect of the skin and alterations in the metabolite profile of AHT observed in vivo and (b) provide consumer-relevant data regarding leave-on/rinse-off products.

The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression.

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38 alpha is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38 alpha activation is linked to cell type-specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38 elicit inflammatory responses, yet p38 alpha signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38 alpha limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38 alpha in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal-7 microRNA.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell-derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)-/- mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2-/- mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2-/- mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Type I interferon inhibits interleukin-1 production and inflammasome activation.

Type I interferon (IFN) is a common therapy for autoimmune and inflammatory disorders, yet the mechanisms of action are largely unknown. Here we showed that type I IFN inhibited interleukin-1 (IL-1) production through two distinct mechanisms. Type I IFN signaling, via the STAT1 transcription factor, repressed the activity of the NLRP1 and NLRP3 inflammasomes, thereby suppressing caspase-1-dependent IL-1ß maturation. In addition, type I IFN induced IL-10 in a STAT1-dependent manner; autocrine IL-10 then signaled via STAT3 to reduce the abundance of pro-IL-1α and pro-IL-1ß. In vivo, poly(I:C)-induced type I IFN diminished IL-1ß production in response to alum and Candida albicans, thus increasing susceptibility to this fungal pathogen. Importantly, monocytes from multiple sclerosis patients undergoing IFN-ß treatment produced substantially less IL-1ß than monocytes derived from healthy donors. Our findings may thus explain the effectiveness of type I IFN in the treatment of inflammatory diseases but also the observed "weakening" of the immune system after viral infection.

Conversion of human urine-derived cells into neuron-like cells by small molecules.

Neural cell transplantation is an effective way for treatment of neurological diseases. However, the absence of transplantable human neurons remains a barrier for clinical therapies. Human urine-derived cells, namely renal cells and urine stem cells, have become a good source of cells for reprogramming or trans-differentiation research. Here, we show that human urine-derived cells can be partially converted into neuron-like cells by applying a cocktail of small molecules. Gene expression analysis has shown that these induced cells expressed some neuron-specific genes, and a proportion of the cells are GABAergic neurons. Moreover, whole-cell patch clamping recording has shown that some induced cells have neuron-specific voltage gated Na+ and K+ currents but have failed to generate Ca2+ currents and action potentials. Taken together, these results suggest that induced neuronal cells from human urine-derived cells may be useful for neurological disease modelling, drug screening and cell therapies.

Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells.

Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.

Isolation and culture of vascular smooth muscle cells from rat placenta.

We developed a new separation method for isolating placental vascular smooth muscle cells (PVSMCs) from a rat in this study. Our method used the magnetic force between a magnet and ferrous ferric oxide (Fe3 O 4 ) to make the separation and extraction processes easier and more efficient. From the first to sixth generation, the cells isolated using this protocol were identified as smooth muscle cells (SMCs) by their immunoreactivity to the SMC markers and by the "hill and valley" morphology. PVSMCs were exposed to angiotensin II (1 µmol/L) and resulted in sharply increased intracellular Ca 2+ concentration. Furthermore, activation of protein kinase C (PKC) increased concomitantly with a decrease in calponin expression. These results indicate that the isolated cells had biological activity. Our method of isolating PVSMCs from rat leads to isolation of cultured cells with activity and high purity. The approach will be useful in research studies on placental vascular diseases.

Addressing the impact of different fetal bovine serum percentages on mesenchymal stem cells biological performance.

Human mesenchymal stem cells (MSCs) are presently on the top of hierarchy in the field of stem cell therapy, due to their miraculous therapeutic abilities in diminishing the symptoms of many chronic diseases and initiating regeneration and repair for various damaged tissues and organs. The foremost initial step to reach high success rate in any MSCs based study is the optimization of culture growth media by establishing a suitable fetal bovine serum (FBS) percentage that suits the purpose of MSCs based experiment. Choosing the suitable FBS percentage is a controversial issue and merely depends on the researchers experience and suggested recommendations by the suppliers. Despite the huge improvements in overall MSCs investigating approaches, there are no definite protocols that set up a range of FBS percentages that can be followed. Toward achieving this objective, we evaluate in the present report the effect of using various FBS percentages (5-20%) added to DMEM low glucose media, on the biological behaviour of MSCs. Growing MSCs in high FBS percentages containing culture media (15% and 20% FBS) increase the proliferation and expansion rate of MSCs, although it decreases the immunosuppressive properties. On the other hand, adding low FBS percentage (7% FBS) to MSCs culture media enhanced the immunosuppression characteristics of MSCs, even though the proliferation rate was moderately reduced. 7% FBS is the cut off percentage that can be used without negatively altering major MSCs biological properties in which using 5% FBS will cause a tremendous decrease in the proliferation capacity and immunosuppressive properties. This report may assist other researchers in choosing appropriate FBS percentage when preparing MSCs culture media that serve the purpose of their MSCs based studies.

Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors.

Currently, there is no widely accepted technique to efficiently and reproducibly grow stem and progenitor cells in vitro. Stem cells require contact with extracellular matrices as well as signals from growth factors to proliferate and to retain their stemness. We have shown a novel tissue culture platform (StemTrix cultureware) that transforms standard tissue culture plasticware into a multi-functional chitosan-based scaffold that supports the expansion of neural stem cells. The StemTrix scaffold is comprised of chitosan with immobilized heparin which in turn tethers heparin-binding growth factors. The scaffold is also coated with an adhesive ECM protein. Here we demonstrate that fibronectin or the RGD peptide contained in fibronectin are equally effective in promoting the adhesion, viability and growth of rat and human neural stem cells. When FGF-2 and heparin-binding EGF are tethered to the StemTrix cultureware neural stem cells grow ∼3 times faster and remain in a more primitive state as determined by both Western Blot and gene expression analyses. Another important feature of this new culture platform is that the NSCs remain in a primitive and proliferative state for 4days without refreshing the culture medium or providing new growth factors, which represents a 20-fold extension of FGF-2's biological activity vs when it is freely soluble in the medium. To test the utility of this scaffold for propagating other types of stem cells and progenitors we tethered platelet-derived growth factor (PDGF) and FGF-2 alone and in combination to the scaffold and tested the efficacy of this platform to maintain primary oligodendrocyte progenitors or the CG-4 cell line in a primitive state. Oligodendrocyte progenitors plated onto this multifunctional film proliferated for at least 3days without providing soluble growth factors while inhibiting the expression of the differentiation marker myelin-basic protein. Oligodendrocyte progenitors proliferated 3 times more rapidly than cells maintained on fibronectin-coated culture substrates in culture medium supplemented with soluble FGF-2 and PDGF. Finally, we show that StemTrix cultureware can be produced using clinical grade components, providing users with a fully defined platform suitable for clinical use that maintains stem cells or progenitors in a more uniform and primitive state.

The insulin receptor is expressed and functional in cultured blood-brain barrier endothelial cells but does not mediate insulin entry from blood to brain.

Insulin and its receptor are known to be present and functional in the brain. Insulin cerebrospinal fluid concentrations have been shown to correlate with plasma levels of insulin in a nonlinear fashion, indicative of a saturable transport pathway from the blood to the brain interstitial fluid. The aim of the present study was to investigate whether insulin was transported across brain endothelial cells in vitro via an insulin receptor-dependent pathway. The study showed that the insulin receptor was expressed at both the mRNA and protein levels in bovine brain endothelial cells. Luminally applied radiolabeled insulin showed insulin receptor-mediated binding to the endothelial cells. This caused a dose-dependent increase in Akt-phosphorylation, which was inhibited by coapplication of an insulin receptor inhibitor, s961, demonstrating activation of insulin receptor signaling pathways. Transport of insulin across the blood-brain barrier in vitro was low and comparable to that of a similarly sized paracellular marker. Furthermore, insulin transport was not inhibited by coapplication of an excess of unlabeled insulin or an insulin receptor inhibitor. The insulin transport and uptake studies were repeated in mouse brain endothelial cells demonstrating similar results. Although it cannot be ruled out that culture-induced changes in the cell model could have impaired a potential insulin transport mechanism, these in vitro data indicate that peripheral insulin must reach the brain parenchyma through alternative pathways rather than crossing the blood-brain barrier via receptor mediated transcytosis.

Sleep-disordered breathing, circulating exosomes, and insulin sensitivity in adipocytes.

BACKGROUND: Sleep-disordered-breathing (SDB), which is characterized by chronic intermittent hypoxia (IH) and sleep fragmentation (SF), is a prevalent condition that promotes metabolic dysfunction, particularly among patients suffering from obstructive hypoventilation syndrome (OHS). Exosomes are generated ubiquitously, are readily present in the circulation, and their cargo may exert substantial functional cellular alterations in both physiological and pathological conditions. However, the effects of plasma exosomes on adipocyte metabolism in patients with OHS or in mice subjected to IH or SF mimicking SDB are unclear. METHODS: Exosomes from fasting morning plasma samples from obese adults with polysomnographically-confirmed OSA before and after 3 months of adherent CPAP therapy were assayed. In addition, C57BL/6 mice were randomly assigned to (1) sleep control (SC), (2) sleep fragmentation (SF), and (3) intermittent hypoxia (HI) for 6 weeks, and plasma exosomes were isolated. Equivalent exosome amounts were added to differentiated adipocytes in culture, after which insulin sensitivity was assessed using 0 nM and 5 nM insulin-induced pAKT/AKT expression changes by western blotting. RESULTS: When plasma exosomes were co-cultured and internalized by human naive adipocytes, significant reductions emerged in Akt phosphorylation responses to insulin when compared to exosomes obtained after 24 months of adherent CPAP treatment (n = 24; p < 0.001), while no such changes occur in untreated patients (n = 8). In addition, OHS exosomes induced significant increases in adipocyte lipolysis that were attenuated after CPAP, but did not alter pre-adipocyte differentiation. Similarly, exosomes from SF- and IH-exposed mice induced attenuated p-AKT/total AKT responses to exogenous insulin and increased glycerol content in naive murine adipocytes, without altering pre-adipocyte differentiation. CONCLUSIONS: Using in vitro adipocyte-based functional reporter assays, alterations in plasma exosomal cargo occur in SDB, and appear to contribute to adipocyte metabolic dysfunction. Further exploration of exosomal miRNA signatures in either human subjects or animal models and their putative organ and cell targets appears warranted.

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis.

Hepatocyte apoptosis in nonalcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, which permanently damage the liver. Understanding the regulation of hepatocyte apoptosis is therefore important to identify therapeutic targets that may prevent the progression of NASH to fibrosis. Recently, increasing evidence has shown that long noncoding (lnc) RNAs are involved in various biological processes and that their dysregulation underlies a number of complex human diseases. By performing gene expression profiling of 4,383 lncRNAs in 82 liver samples from individuals with NASH (n = 48), simple steatosis but no NASH (n = 11), and healthy controls (n = 23), we discovered a liver-specific lncRNA (RP11-484N16.1) on chromosome 18 that showed significantly elevated expression in the liver tissue of NASH patients. This lncRNA, which we named lnc18q22.2 based on its chromosomal location, correlated with NASH grade (r = 0.51, P = 8.11 × 10-7 ), lobular inflammation (r = 0.49, P = 2.35 × 10-6 ), and nonalcoholic fatty liver disease activity score (r = 0.48, P = 4.69 × 10-6 ). The association of lnc18q22.2 to liver steatosis and steatohepatitis was replicated in 44 independent liver biopsies (r = 0.47, P = 0.0013). We provided a genetic structure of lnc18q22.2 showing an extended exon 2 in liver. Knockdown of lnc18q22.2 in four different hepatocyte cell lines resulted in severe phenotypes ranging from reduced cell growth to lethality. This observation was consistent with pathway analyses of genes coexpressed with lnc18q22.2 in human liver or affected by lnc18q22.2 knockdown. CONCLUSION: We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808).

Influence of Bovine Serum Lipids and Fetal Bovine Serum on the Expression of Cell Surface Markers in Cultured Bovine Preadipocytes.

To establish the influence of fetal bovine serum (FBS) and bovine serum lipids (BSL) on cell differentiation marker expression, bovine adipose-derived stem cells from subcutaneous tissue were incubated for 14 days in 4 types of differentiation media containing 10% FBS and 10 µL/mL BSL (TRT-1), no FBS and 10 µL/mL of BSL (TRT-2), 10% FBS and no BSL (TRT-3), or no supplements (TRT-4). Cells were subjected to Nile red staining, immunocytochemistry (CD73, CD90, CD105, DLK1, FabP4), and quantitative real-time PCR (CD73, CD90, CD105, FabP4). The number of cells presenting FabP4 and the percentage of mature adipocytes with large lipid droplets were increased in TRT-2, accompanied by a robust increase in FabP4 mRNA abundance and a decrease in DLK1-positive cells. In preadipocytes, CD73 was present around the nucleus and translocated towards cell membranes during differentiation. Although the percentage of CD73-positive cells was not different among treatments, its mRNA abundance, immunocytochemical staining intensity, and translocation towards cell membranes were decreased when the medium contained no FBS (TRT-2 and TRT-4). All cells showed a diffuse distribution of CD90 and CD105 and remained positive for these markers irrespective of the treatment. However, the CD90 and CD105 mRNA abundance was decreased in TRT-2 and TRT-4; i.e., in media containing no FBS. The presence of FBS increased the absolute number of cell nuclei as assessed by DAPI fluorescence. Our results suggest that bovine subcutaneous preadipocytes display typical stem cell markers. The differentiation into mature adipocytes is promoted by BSL, whereas FBS endorses cell proliferation.