Cells In Explant Cultures Research Articles

Isolation, identification and osteogenic capability analysis of mesenchymal stem cells derived from different layers of human maxillary sinus membrane.

To discover the populations of mesenchymal stem cells (MSCs) derived from different layers of human maxillary sinus membrane (hMSM) and evaluate their osteogenic capability. hMSM was isolated into a monolayer using the combined method of physical separation and enzymatic digestion. The localization of MSCs in hMSM was performed by immunohistological staining and other techniques. Lamina propria layer-derived MSCs (LMSCs) and periosteum layer-derived MSCs (PMSCs) from hMSM were expanded using the explant cell culture method and identified by multilineage differentiation assays, colony formation assay, flow cytometry and so on. The biological characteristics of LMSCs and PMSCs were compared using RNA sequencing, reverse transcription and quantitative polymerase chain reaction, immunofluorescence staining, transwell assay, western blotting and so forth. LMSCs and PMSCs from hMSMs were both CD73-, CD90- and CD105-positive, and CD34-, CD45- and HLA-DR-negative. LMSCs and PMSCs were identified as CD171+/CD90+ and CD171-/CD90+, respectively. LMSCs displayed stronger proliferation capability than PMSCs, and PMSCs presented stronger osteogenic differentiation capability than LMSCs. Moreover, PMSCs could recruit and promote osteogenic differentiation of LMSCs. This study identified and isolated two different types of MSCs from hMSMs. Both MSCs served as good potential candidates for bone regeneration.

CREB activation drives acinar to ductal reprogramming and promote pancreatic cancer progression in animal models of alcoholic chronic pancreatitis.

In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic Kras G12D/+ (Kras*) in promoting pancreatic cancer progression with ACP remains unexplored. Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion (Creb fl/fl ) in Ptf1a CreERTM/+ ;LSL-Kras G12D+/-(KC) genetic mouse models (KCC-/-). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1a CreERTM+/-, KC and KCC -/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.

Türk Civanperçemi (Achillea gypsicola Hub. Mor.) Hücre Süspansiyon Kültürlerinde Fenilpropanoid Metabolitlerin Işık Aracılığıyla Biosentezi

Turkish yarrow (Achillea gypsicola Hub. Mor.) contains medicinally valuable bioactive metabolites. Light plays a key role in many physiological events in plants such as the accumulation of phenylpropanoid metabolites, particularly photosynthesis. The aim of this study was to determine the effects of light on the biosynthesis of phenylpropanoid metabolites and biomass growth in Turkish yarrow cell suspension cultures. In vitro plantlets obtained from seeds were used as explant sources and cell cultures of A. gypsicola were established using stem segment-derived callus tissues. Then, 8-day-old cell cultures kept in a B5 medium supplemented with 0.5 mg/L BA (benzylaminopurine)+0.5 mg/L NAA (naphthalene acetic acid) were incubated in light and dark environments. Cells were harvested five times (1st, 2nd, 3rd, 6th, and 12th day) in total. The amount of camphor was determined by gas chromatography-mass spectrometry (GC–MS). Other parameters were calculated using the spectrophotometric method. The total phenolic amount increased depending on the sampling time in dark and light conditions. Phenolic accumulation was increased by 29.25% compared to the initial culture. Also, light application increased the amount of camphor 1.12 times, the amount of flavanol 4.9 times, and the amount of flavonol 0.5 times. These results clearly demonstrate the effect of light on cell growth and phenylpropanoid metabolite accumulation in A. gypsicola cell cultures.

Does the ovarian surface epithelium differentiate into primordial follicle and primary follicle precursor structures?

Amaç: Ovaryum yüzey epiteli hücrelerinin farklılaşma kapasitelerini hem hücre kültürü şartlarında hem de ovaryum doku kesitlerinde araştırmaktır.
 Gereç ve Yöntem: İki tane puberte öncesi dönemdeki (4 haftalık) dişi sıçanların ovaryumları küçük parçalara ayrılarak eksplant hücre kültürü oluşturuldu. Miks hücre kültüründe overyan stromal hücrelerle birlikte çoğalan ovaryum yüzey epiteli izole edilerek çoğaltıldı. Bununla birlikte ovaryum dokusunun histolojik kesitlerinde ovaryum yüzey epiteli incelenerek mikroskop altında görüntüleri alındı.
 Bulgular: Ovaryum yüzey epitelinin morfolojik görünümünün parke taşı (cobblestone) şeklinde olduğu görüldü. Faz kontrast mikroskobisi altında yapılan sayımda kültür kaplarında sırasıyla 2x106 ve 3x106 hücrenin ürediği izlendi. Petri kaplarının bazı alanlarında primordial folikül benzeri yapıların oluştuğu görüldü. Histolojik kesitlerde ise bazal membranın üzerinde primordial ve primer folikül öncüsü yapıların olduğu gözlemlendi.
 Sonuç: Hem hücre kültürlerinde, hem de histolojik kesitlerde oosit belirteçlerini (Gdf-9, C-Mos, Zpc, Stella) ve germ hücre belirteçlerini (Dazl,Vasa,Blimp1,Fragilis) göstermek bu hücrelerin farklılaşma kapasitelerini izlememiz açısından bizlere değerli bilgiler verebilir.

Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma.

Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC. The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic-activated cell sorting. Colony-forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD-L 1). Freshly isolated cells containing tumor cells and cancer-associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6cm petri dish in 5-7days. Anti-epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM- cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM- population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD-L 1 was identified in most subpopulation of CD44+ cells at low passage numbers. Efficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.

Hypoxic stress enhances extension and branching of dorsal root ganglion neuronal outgrowth.

It has been shown that painful intervertebral discs (IVDs) were associated with a deeper innervation. However, the effect of the disc's degenerative microenvironment on neuronal outgrowth remains largely unknown. The focus of this study was to determine the influence of hypoxia on dorsal root ganglion (DRG) neurite outgrowth. Toward this aim, the DRG‐derived cell line ND7/23 was either directly subjected to 2% or 20% oxygen conditions or exposed to conditioned medium (CM) collected from IVDs cultured under 2% or 20% oxygen. Viability and outgrowth analysis were performed following 3 days of exposure. Results obtained with the cell line were further validated on cultures of rabbit spinal DRG explants and dissociated DRG neurons. Results showed that hypoxia significantly increased neurite outgrowth length in ND7/23 cells, which was also validated in DRG explant and primary cell culture, although hypoxia conditioned IVD did not significantly increase ND7/23 neurite outgrowth. While hypoxia dramatically decreased the outgrowth frequency in explant cultures, it significantly increased collateral sprouting of dissociated neurons. Importantly, the hypoxia‐induced decrease of outgrowth frequency at the explant level was not due to inhibition of outgrowth branching but rather to neuronal necrosis. In summary, hypoxia in DRG promoted neurite sprouting, while neuronal necrosis may reduce the density of neuronal outgrowth at the tissue level. These findings may help to explain the deeper neo‐innervation found in the painful disc tissue.HighlightsHypoxia promoted elongation and branching of neurite outgrowth at single cell level, but reduced outgrowth density at tissue level, possibly due to hypoxia‐induced neuronal necrosis; these findings may help to explain the deeper neo‐innervation found in clinically painful tissues.

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

Ex vivo culture of oral keratinocytes using direct explant cell culture technique.

Background:Culture of cells and tissues is a standard research method practiced in many laboratories. In most of the cases, these cultures are being used as substrates for cell products or as investigative tools for delving the mechanism of gene expression, cell proliferation and transformation. Primary monolayer cell culture has been beneficial to study the general biology of both oral and skin keratinocytes. There are two different techniques of primary cell cultures followed, which include direct explant and enzymatic techniques.Aims:The aim of the study was to optimize the culture of keratinocytes obtained from patients with normal oral mucosa by direct explant technique.Materials and Methods:Keratinocytes were isolated from 15 patients and were cultured in vitro and observed under an inverted microscope. The cultured cells were characterized by immunocytochemistry method using pan-cytokeratin.Results:The total success rate of primary culture of the oral epithelial cells by direct explant technique was 88.6%. No contamination of microorganisms in the primary cell cultures was obtained.Conclusion:Within the limited numbers of samples used in the current pilot study, we conclude that the direct explant technique appears to be a simple and successful technique for the isolation of oral mucosal keratinocytes if we follow the appropriate protocol.

Helicase-Driven Activation of NFκB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment.

Presence of cytotoxic CD8+ T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:C12U) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to ex vivo-treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB- and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity.Significance: This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. Cancer Res; 78(15); 4292-302. ©2018 AACR.

No effect of thymosin beta-4 on the expression of the transcription factor Islet-1 in the adult murine heart.

The transcription factor Islet‐1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet‐1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta‐4 (TB4), a 43‐aminoacid peptide, was associated with increased Islet‐1 immunostaining, suggesting the induction of an Islet‐1‐positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1‐nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet‐1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet‐1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1‐nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet‐1 expression in adult mouse ventricle.

Comparison of culture media indicates a role for autologous serum in enhancing phenotypic preservation of rabbit limbal stem cells in explant culture.

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Reduction of c-kit positive cardiac stem cells in patients with atrial fibrillation

BackgroundWe aimed to determine expression patterns of cardiac stem cells in the left atrium (LA) tissue from patients with atrial fibrillation. MethodsLA appendages were obtained during open-heart surgery and processed for explant cell culture and tissue analysis (n=319). The total number of grown cells and c-kit positive cells were analyzed by flow cytometry after 4 weeks of culture. The remaining tissue was used for Masson's trichrome staining to determine the area of the fibrosis. ResultsThe diameter of the LA, as measured by echocardiography, was significantly larger in the AF group than in the sinus rhythm group. Reverse transcription polymerase chain reaction analysis revealed higher expression of collagen in the AF group and an increase in the expression of basic fibrosis growth factor and transforming growth factor-2 and -3. Masson's trichrome staining showed progression of fibrosis in the AF tissue. In addition, the expression of apoptosis-related genes were significantly higher in AF group. There was no difference in the expression of connexin-40 between groups, while the expression of connexin-43 was decreased and that of connexin-45 was increased in the AF group. The total numbers of grown cells as well as c-kit positive cells after 4 weeks of cardiac tissue culture were significantly lower in the AF group. ConclusionProgression of remodeling in LA tissue was observed in AF patients. The number of c-kit positive cells cultured from LA appendages was reduced in AF patients, suggesting impairments in self-renewal.

Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle.

Damage-activated stem/progenitor cells play important roles in regenerating lost cells and in tissue repair. Previous studies reported that the mouse utricle has limited hair cell regeneration ability after hair cell ablation. However, the potential progenitor cell population regenerating new hair cells remains undiscovered. In this study, we first found that Lgr5, a Wnt target gene that is not usually expressed in the neonatal mouse utricle, can be activated by 24 h neomycin treatment in a sub-population of supporting cells in the striolar region of the neonatal mouse utricle. Lineage tracing demonstrated that these Lgr5-positive supporting cells could regenerate new hair cells in explant culture. We isolated the damage-activated Lgr5-positive cells with flow cytometry and found that these Lgr5-positive supporting cells could regenerate hair cells in vitro, and self-renew to form spheres, which maintained the capacity to differentiate into hair cells over seven generations of passages. Our results suggest that damage-activated Lgr5-positive supporting cells act as hair cell progenitors in the neonatal mouse utricle, which may help to uncover a potential route to regenerate hair cell in mammals.

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Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.

Impairment of endothelial-mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos.

Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events.

Rapamycin improves peripheral nerve myelination while it fails to benefit neuromuscular performance in neuropathic mice

Charcot–-Marie–Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy characterized by progressive demyelination and distal muscle weakness. Abnormal expression of peripheral myelin protein 22 (PMP22) has been linked to CMT1A and is modeled by Trembler J (TrJ) mice, which carry the same leucine to proline substitution in PMP22 as affected pedigrees. Pharmacologic modulation of autophagy by rapamycin in neuron-Schwann cell explant cultures from neuropathic mice reduced PMP22 aggregate formation and improved myelination. Here we asked whether rapamycin administration by food supplementation, or intraperitoneal injection, could alleviate the neuropathic phenotype of affected mice and improve neuromuscular performance. Cohorts of male and female wild type (Wt) and TrJ mice were assigned to placebo or rapamycin treatment starting at 2 or 4months of age and tested monthly on the rotarod. While neither long-term feeding (8 or 10months) on rapamycin-enriched diet, or short-term injection (2months) of rapamycin improved locomotor performance of the neuropathic mice, both regimen benefited peripheral nerve myelination. Together, these results indicate that while treatment with rapamycin benefits the myelination capacity of neuropathic Schwann cells, this intervention does not improve neuromuscular function. The observed outcome might be the result of the differential response of nerve and skeletal muscle tissue to rapamycin.

Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

Tyrosine phosphatase inhibitor triggers rodlet cell discharge in sunfish scale epidermis cultures

AbstractEpidermal rodlet cells were evaluated after treatment with the tyrosine phosphatase inhibitor pervanadate. Treatment of sunfish explant cell cultures with the inhibitor triggered a contraction of the rodlet cells and expulsion of cell contents. Time‐lapse video differential interference contrast (DIC) microscopy was used to evaluate rodlet cell contraction and rodlet discharge. Three general steps in pervanadate triggered discharge were identified. First the rodlet cell undergoes a constriction of the midsection. Constriction is followed by a rapid forward movement of rodlets and sacs to the apical end of cell, culminating in discharge of rodlets and other cellular contents, including the nucleus. A ring‐shaped structure around the apical pore was identified with DIC microscopy. Fluorescent‐labeled phalloidin and antibodies to alpha‐actinin and phosphotyrosine strongly stained the apical ring. A diffuse granular staining for both antibodies was also observed throughout the fibrous capsule. The results suggest that tyrosine kinases play a role in rodlet cell contraction. Alpha‐actinin is a known substrate for tyrosine kinases and is a potential target for triggering rodlet cell contraction and rodlet ejection. Modification of alpha‐actinin tyrosines could also be a mechanism for regulating the structural integrity of the fibrous capsule.

Activation of Smad‐mediated TGF‐β signaling triggers epithelial–mesenchymal transitions in murine cloned corneal progenitor cells

Epithelial-mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum-free medium. Single cell-derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E-cadherin and β-catenin from intercellular junctions, coupled with nuclear translocation of β-catenin and LEF-1. Large-colony-forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α-SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α-SMA was promoted by high-density cultures and their conditioned media, which contained cell density-dependent levels of TGF-β1, TGF-β2, GM-CSF, and IL-1α. Exogenous TGF-β1 induced α-SMA positive cells in a low-density culture, while TGF-β1 neutralizing antibody partially inhibited α-SMA expression in a high-density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad-mediated TGF-β-signaling.

The endogenous retroviral envelope protein syncytin-1 inhibits LPS/PHA-stimulated cytokine responses in human blood and is sorted into placental exosomes

ObjectivesTo examine whether syncytin-1 has immune regulatory functions and is carried by human placental exosomes. Further, to examine whether corticotropin-releasing hormone (CRH) can induce the production of syncytin-1. Study designHuman placental exosomes were isolated from placental explant, primary trophoblast and BeWo cell cultures. The presence of exosomes was confirmed by transmission electron microscopy and western blotting. Exosomal protein was probed with 3 separate antibodies targeting syncytin-1.Syncytin-1 immunosuppression was tested, using either a syncytin-1 recombinant ectodomain protein or a synthetic peptide with the human syncytin-1 immunosuppressive domain sequence, in an in vitro human blood culture system immune challenged with LPS or PHA. The inhibition of cytokine production by syncytin-1 was determined by ELISA of TNF-α, IFN-γ and CXCL10.BeWo cells were stimulated with CRH or vehicle for 24 h. mRNA and Protein was extracted from the cells for real-time PCR and western blotting analysis while exosomes were extracted from conditioned media for analysis by western blotting. ResultsProtein expression of syncytin-1 was detected in exosomes isolated from placental explants, primary trophoblast and BeWo cell cultures. Syncytin-1 recombinant ectodomain was also shown to inhibit the production of the Th1 cytokines TNF-α and IFN-γ as well as the chemokine, CXCL10 in human blood cells. Finally, this study showed that syncytin-1 can be stimulated by CRH. ConclusionsThe presence of syncytin-1 in placental exosomes provides a mechanism for syncytin-1 to reach and interact with target cells of the maternal immune system and represents a novel mechanism of endogenous retroviral mediated immunosuppression that may be relevant for maternal immune tolerance.