Establishment and characterization of immortalized sweat gland myoepithelial cells.

Sweat glands play an important role in thermoregulation via sweating, and protect human vitals. The reduction in sweating may increase the incidence of hyperthermia. Myoepithelial cells in sweat glands exhibit stemness characteristics and play a major role in sweat gland homeostasis and sweating processes. Previously, we successfully passaged primary myoepithelial cells in spheroid culture systems; however, they could not be maintained for long under in vitro conditions. No myoepithelial cell line has been established to date. In this study, we transduced two immortalizing genes into primary myoepithelial cells and developed a myoepithelial cell line. When compared with primary sweat gland cells, the immortalized myoepithelial cells (designated "iEM") continued to form spheroids after the 4th passage and expressed α-smooth muscle actin and other proteins that characterize myoepithelial cells. Furthermore, treatment with small compounds targeting the Wnt signaling pathways induced differentiation of iEM cells into luminal cells. Thus, we successfully developed an immortalized myoepithelial cell line having differentiation potential. As animal models are not useful for studying human sweat glands, our cell line will be helpful for studying the mechanisms underlying the pathophysiology of sweating disorders.

Production of Murine Macrophages from Hoxb8-Immortalized Myeloblasts: Utility and Use in the Context of Salmonella Infection.

Salmonella enterica is a Gram-negative intracellular pathogen that causes a range of life-threatening diseases in humans and animals worldwide. In a systemic infection, the ability of Salmonella to survive/replicate in macrophages, particularly in the liver and spleen, is crucial for virulence. Transformed macrophage cell lines and primary macrophages prepared from mouse bone marrow are commonly used models for the study of Salmonella infection. However, these models raise technical or ethical issues that highlight the need for alternative methods. This chapter describes a technique for immortalizing early hematopoietic progenitor cells derived from wild-type or transgenic mice and using them to produce macrophages. It validates, through a specific example, the interest of this cellular approach for the study of Salmonella infection.

Abrogation of IFN-γ Signaling May not Worsen Sensitivity to PD-1/PD-L1 Blockade.

Programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockade is a promising therapy for various cancer types, but most patients are still resistant. Therefore, a larger number of predictive biomarkers is necessary. In this study, we assessed whether a loss-of-function mutation of the interferon (IFN)-γ receptor 1 (IFNGR1) in tumor cells can interfere with anti-PD-L1 therapy. For this purpose, we used the mouse oncogenic TC-1 cell line expressing PD-L1 and major histocompatibility complex class I (MHC-I) molecules and its TC-1/A9 clone with reversibly downregulated PD-L1 and MHC-I expression. Using the CRISPR/Cas9 system, we generated cells with deactivated IFNGR1 (TC-1/dIfngr1 and TC-1/A9/dIfngr1). In tumors, IFNGR1 deactivation did not lead to PD-L1 or MHC-I reduction on tumor cells. From potential inducers, mainly IFN-α and IFN-ß enhanced PD-L1 and MHC-I expression on TC-1/dIfngr1 and TC-1/A9/dIfngr1 cells in vitro. Neutralization of the IFN-α/IFN-ß receptor confirmed the effect of these cytokines in vivo. Combined immunotherapy with PD-L1 blockade and DNA vaccination showed that IFNGR1 deactivation did not reduce tumor sensitivity to anti-PD-L1. Thus, the impairment of IFN-γ signaling may not be sufficient for PD-L1 and MHC-I reduction on tumor cells and resistance to PD-L1 blockade, and thus should not be used as a single predictive marker for anti-PD-1/PD-L1 cancer therapy.

Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.

PURPOSE: To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line. METHODS: iHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs. RESULTS: The iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days. CONCLUSION: The iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.

Lifetime extension of humpback whale skin fibroblasts and their response to lipopolysaccharide (LPS) and a mixture of polychlorinated biphenyls (Aroclor).

Marine mammals, such as whales, have a high proportion of body fat and so are susceptible to the accumulation, and associated detrimental health effects, of lipophilic environmental contaminants. Recently, we developed a wild-type cell line from humpback whale fibroblasts (HuWa). Extensive molecular assessments with mammalian wild-type cells are typically constrained by a finite life span, with cells eventually becoming senescent. Thus, the present work explored the possibility of preventing senescence in the HuWa cell line by transfection with plasmids encoding the simian virus large T antigen (SV40T) or telomerase reverse transcriptase (TERT). No stable expression was achieved upon SV40 transfection. Transfection with TERT, on the other hand, activated the expression of telomerase in HuWa cells. At the time of manuscript preparation, the transfected HuWa cells (HuWaTERT) have been stable for at least 59 passages post-transfection. HuWaTERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWaTERT cells to an immune stimulant (lipopolysaccharide (LPS)) and an immunotoxicant (Aroclor1254) was assessed by measurement of intracellular levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1ß and tumour necrosis factor (TNF)-α. HuWaTERT cells constitutively express IL-6, IL-1ß and TNFα. Exposure to neither LPS nor Aroclor1254 had an effect on the levels of these cytokines. Overall, this work supports the diverse applicability of HuWa cell lines in that they display reliable long-term preservation, susceptibility to exogenous gene transfer and enable the study of humpback whale-specific cellular response mechanisms.

Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source.

Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.

Fibroblast activation protein alpha is expressed by transformed and stromal cells and is associated with mesenchymal features in glioblastoma.

Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP+ host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

Inhibiting cortical protein kinase A in spinal cord injured rats enhances efficacy of rehabilitative training.

Elevated levels of the second messenger molecule cyclic adenosine monophosphate (cAMP) are often associated with neuron sprouting and neurite extension (i.e., neuroplasticity). Phosphokinase A (PKA) is a prominent downstream target of cAMP that has been associated with neurite outgrowth. We hypothesized that rehabilitative motor training following spinal cord injuries promotes neuroplasticity via PKA activation. However, in two independent experiments, inhibition of cortical PKA using Rp-cAMPS throughout rehabilitative training robustly increased functional recovery and collateral sprouting of injured corticospinal tract axons, an indicator of neuroplasticity. Consistent with these in vivo findings, using cultured STHdh neurons, we found that Rp-cAMPS had no effect on the phosphorylation of CREB (cAMP response element-binding protein), a prominent downstream target of PKA, even with the concomitant application of the adenylate cyclase agonist forskolin to increase cAMP levels. Conversely, when cAMP levels were increased using the phosphodiesterase inhibitor IBMX, Rp-cAMPS potently inhibited CREB phosphorylation. Taken together, our results suggest that an alternate cAMP dependent pathway was involved in increasing CREB phosphorylation and neuroplasticity. This idea was supported by an in vitro neurite outgrowth assay, where inhibiting PKA did enhance neurite outgrowth. However, when PKA inhibition was combined with inhibition of EPAC2 (exchange protein directly activated by cAMP), another downstream target of cAMP in neurons, neurite outgrowth was significantly reduced. In conclusion, blocking PKA in cortical neurons of spinal cord injured rats increases neurite outgrowth of the lesioned corticospinal tract fibres and the efficacy of rehabilitative training, likely via EPAC.

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1.

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with ß-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line.

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

INTRODUCTION: Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. METHODS: Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor ß (TGFß). RESULTS: The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFß induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. CONCLUSION: We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis.

Oxidative stress is a cause of inflammation-related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H2O2) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H2O2-resistant cell line from an immortal cholangiocyte cell line (MMNK1) by chronic treatment with low-concentration H2O2 (25 µM). After 72 days of induction, H2O2-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H2O2-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 µM H2O2). These findings suggest that H2O2-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes.

Mechanisms of the induction of apoptosis mediated by radiation-induced cytokine release.

The aim of the present work was to investigate the mechanisms of radiation-induced bystander signalling leading to apoptosis in non-irradiated co-cultured cells. Cultured non-transformed cells were irradiated, and the effect on the apoptosis rate on co-cultured non-irradiated malignant cells was determined. For this, two different levels of the investigation are presented, i.e. release of signalling proteins and transcriptomic profiling of the irradiated and non-irradiated co-cultured cells. Concerning the signalling proteins, in this study, the attention was focussed on the release of the active and latent forms of the transforming growth factor-ß1 protein. Moreover, global gene expression profiles of non-transformed and transformed cells in untreated co-cultures were compared with those of 0.5-Gy-irradiated non-transformed cells co-cultured with the transformed cells. The results show an effect of radiation on the release of signalling proteins in the medium, although no significant differences in release rates were detectable when varying the doses in the range from 0.25 to 1 Gy. Moreover, gene expression results suggest an effect of radiation on both cell populations, pointing out specific signalling pathways that might be involved in the enhanced induction of apoptosis.

Formation of covalently closed circular DNA in Hep38.7-Tet cells, a tetracycline inducible hepatitis B virus expression cell line.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection.

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

Development of a conditionally immortalized human pancreatic ß cell line.

Diabetic patients exhibit a reduction in ß cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ß cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ß cell line (EndoC-ßH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-ßH1 cells display many functional properties of adult ß cells, including expression of ß cell markers and insulin secretion following glucose stimulation; however, unlike primary ß cells, EndoC-ßH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ß cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-ßH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ß cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-ßH2 cells are highly representative of human ß cells and should be a valuable tool for further analysis of human ß cells.

Ligand-dependent stabilization of androgen receptor in a novel mouse ST38c Sertoli cell line.

Mature Sertoli cells (SC) are critical mediators of androgen regulation of spermatogenesis, via the androgen receptor (AR) signaling. Available immortalized SC lines loose AR expression or androgen responsiveness, hampering the study of endogenous AR regulation in SC. We have established and characterized a novel clonal mouse immortalized SC line, ST38c. These cells express some SC specific genes (sox9, wt1, tjp1, clu, abp, inhbb), but not fshr, yet more importantly, maintain substantial expression of endogenous AR as determined by PCR, immunocytochemistry, testosterone binding assays and Western blots. Microarrays allowed identification of some (146) but not all (rhox5, spinlw1), androgen-dependent, SC expressed target genes. Quantitative Real-Time PCR validated regulation of five up-regulated and two down-regulated genes. We show that AR undergoes androgen-dependent transcriptional activation as well as agonist-dependent posttranslational stabilization in ST38c cells. This cell line constitutes a useful experimental tool for future investigations on the molecular and cellular mechanisms of androgen receptor signaling in SC function.

Eternity and functionality - rational access to physiologically relevant cell lines.

In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.

Establishment and characterization of immortalized human amniotic epithelial cells.

Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine.

TLR4 activation under lipotoxic conditions leads to synergistic macrophage cell death through a TRIF-dependent pathway.

Macrophage dysfunction in obesity and diabetes may predispose to the development of diabetic complications, such as infection and impaired healing after tissue damage. Saturated fatty acids, such as palmitate, are present at elevated concentrations in the plasma of patients with metabolic disease and may contribute to the pathogenesis of diabetes and its sequelae. To examine the effect of lipid excess on macrophage inflammatory function, we determined the influence of palmitate on LPS-mediated responses in peritoneal macrophages. Palmitate and LPS led to a profound synergistic cell death response in both primary and RAW 264.7 macrophages. The cell death had features of apoptosis and necrosis and was not dependent on endoplasmic reticulum stress, ceramide generation, or reactive oxygen species production. Instead, we uncovered a macrophage death pathway that required TLR4 signaling via TRIF but was independent of NF-κB, MAPKs, and IRF3. A significant decrease in macrophage lysosomal content was observed early in the death pathway, with evidence of lysosomal membrane damage occurring later in the death response. Overexpression of the transcription factor TFEB, which induces a lysosomal biogenic program, rescued the lysosomal phenotype and improved viability in palmitate- and LPS-treated cells. Our findings provide new evidence for cross-talk between lipid metabolism and the innate immune response that converges on the lysosome.