Co-culture Medium Research Articles

Single Chain Cationic Polymer Dot as a Fluorescent Probe for Cell Imaging and Selective Determination of Hepatocellular Carcinoma Cells.

This letter describes formation of single chain cationic polymer dots (Pdots) made of poly[1,4-dimethyl-1-(3-((2,4,5-trimethylthiophen-3-yl)oxy)propyl)piperazin-1-ium bromide] conjugated polyelectrolyte (CPE). The single chain Pdot formation relies on a simple process which is a rapid nanophase separation between CPE solution of ethylene glycol and water. Pdots show narrow monodisperse size distribution with a 3.6 nm in diameter exhibiting high brightness and excellent colloidal and optical stability. It has been demonstrated that photoluminescent Pdots provide selective nuclear translocation to hepatocellular carcinoma cells as compared to healthy liver cells. The Pdot labeling effectively discriminates cancer cells in the coculture media. Pdots hold great promise as a luminescent probe to diagnose cancer cells in histology and may guide surgeons during operations to precisely separate out cancerous tissue due to augmented fluorescence brightness.

Microvesicles Derived from TGF-β1 Stimulated Hepatic Stellate Cells Aggravate Hepatocellular Injury.

Hepatic stellate cells (HSCs) are liver-specific cells playing critical roles in liver physiological and pathophysiological processes. Transforming growth factor-β1 (TGF-β1) is an inflammatory cytokine secreted by both hepatocytes and HSCs. We have previously shown that microvesicles (MVs) derived from quiescent HSCs protect hepatocyte functions. In this study, we investigated the effects of MVs released from TGF-β1-stimulated HSCs (HSC-MVs) on xenobiotic-injured hepatocytes. Two hepatocyte cell lines (BRL-3A and HL-7702) were treated with N-acetyl-p-aminophenol or H2O2 to build the injury models. Different concentrations of HSC-MVs were used to coculture with injured hepatocytes. MTT, Hochest33258 staining, and flow cytometry were used to determine their effects on the viability and apoptosis of hepatocytes. Liver injury indicators, alanine aminotransferase (ALT) and aspartate amino transferase (AST), were assessed by enzyme-linked immune sorbent assay kits. The phosphoinositide 3-kinase (PI3K) activator (740Y-P) and extracelluar signal regulated kinase (Erk)1/2 activator (platelet-derived growth factor-BB) were used for pathway analysis. The expression levels of p-PI3K/PI3K, p-Akt/Akt, and activated caspase-3 were measured by western blot. Results showed that (i) HSC-MVs dose dependently impaired the viability of hepatocytes in both injury models, (ii) moreover, HSC-MVs dose dependently increased the apoptosis in those cell models, (iii) HSC-MVs also elevated the levels of ALT and AST in the coculture media, and (iv) these effects were accompanied by a decrease in p-PI3K/PI3K and p-Akt/Akt, which could be partially abolished by 740Y-P. Meanwhile, the proapoptotic effect of HSC-MVs was associated with p-Erk1/2/Erk1/2 downregulation and activated caspase-3 upregulation, and could be inhibited by Erk1/2 activation. Our findings demonstrate that HSC-MVs are involved in inflammatory hepatocytes injury probably through the PI3K/Akt, Erk1/2, and caspase-3 pathways.

Near-infrared fluorescent probe for fast track of cyclooxygenase-2 in Golgi apparatus in cancer cells

Cyclooxygenase-2 (COX-2) has been used as an excellent traceable biomarker, and exists maximally in Golgi apparatus (Cancer cells). Celecoxib (CCB) is a selective inhibitor for COX-2, and has been used as one of non-steroidal anti-inflammatory drug. Herein we report the conjugation of nile blue (NB) with CCB via a six-carbon linkage to form a fluorescence probe NB-C6-CCB for the detection of COX-2. NB-C6-CCB displays strong fluorescence with the emission peak centered at near-infrared wavelength (700 nm) in tumor cells or tumor tissues with high expression of COX-2. Importantly, NB-C6-CCB can discriminate cancer cells (MCF-7) fluorescence intensity from normal ones (COS-7) in the co-culture medium under confocal microscope. Subcellular localization of the NB-C6-CCB preferentially points to the Golgi apparatus and increases the fluorescent intensity. The competitive analysis (with CCB) and Native-PAGE analysis confirmed that NB-C6-CCB shows selective binding affinity towards COX-2 enzyme. Competitive analysis with CCB (flow cytometry assay) revealed the fluorescence intensity fluctuation due to pretreatment of CCB with different concentrations, indicating that the NB-C6-CCB is a precise or sensitive probe for the COX-2. Tumor tissue (depth: 500 µm), organs and mice imaging tests show excellent near-infrared visualization, specific localization and identification of tumors.

Crosstalk between tumor cells and lymphocytes modulates heparanase expression

BackgroundHeparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes.MethodsLymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR.ResultsThe medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer.ConclusionsThe results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes.

Distinct Roles of Direct Transduction Versus Exposure to the Tumor Secretome on Murine Endothelial Cells After Melanoma Gene Therapy with Interferon-β and p19Arf.

Tumor vasculature plays a central role in tumor progression, making it an attractive therapeutic target. In this study, we explore the antiangiogenic potential of our melanoma gene therapy approach combining interferon β (IFNβ) and p19Arf gene transfer. Since these proteins are modulators of tumor vasculature, we explore the impact of IFNβ and p19Arf gene transfer on murine endothelial cells (tEnd). Adenovirus-mediated gene transfer of p19Arf to tEnd cells inhibited proliferation, tube formation, migration, and led to increased expression of genes related to the p53 cell death pathway, yet IFNβ gene transfer had no significant impact on tEnd viability. Alternatively, tEnd cells were exposed to the factors generated by transduced B16 (mouse melanoma) cells using either coculture or conditioned medium. In either case, transduction of B16 cells with the IFNβ vector, whether alone or in combination with p19Arf, resulted in endothelial cell death. Strikingly, treatment of tEnd cells with recombinant IFNβ did not induce death, demonstrating that additional factors produced by B16 cells contributed to the demise of tEnd cells. In this work, we have shown that our melanoma gene therapy strategy produces desirable negative effects on endothelial cells, possibly correlating with antiangiogenic activity.

Macrophage-derived MMP-8 determines smooth muscle cell differentiation from adventitia stem/progenitor cells and promotes neointima hyperplasia.

Emerging evidence has suggested that adventitia stem/progenitor cells (AdSPCs) migrate into the intima of arteries in response to injury, where they differentiate towards smooth muscle cells (SMCs) and participate in neointimal hyperplasia. We have previously identified matrix metalloproteinase-8 (MMP8) as a key player in atherogenesis. In this study, we aimed to investigate the functional roles of macrophage-derived MMP8 in AdSPC differentiation and injury-induced arterial remodelling. We first observed an important role for MMP8 in SMC differentiation from embryonic stem cells, but this effect was not seen in AdSPCs. Instead, through macrophages/AdSPCs co-culture and macrophage conditional culture medium studies, we have demonstrated that the MMP8 protein secreted from macrophages promotes SMC differentiation from AdSPCs. Mechanistically, we showed that macrophage-derived MMP8 promotes SMC differentiation from AdSPCs through modulating transforming growth factor-β activity and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10)/Notch1 signalling. We further demonstrated that the binding site for CBF1, Suppressor of Hairless, and Lag-1 (CSL) within SMC gene promoters is responsible for Notch1 mediated SMC differentiation. Finally, we demonstrated that macrophage-derived MMP8 increased injury-induced neointimal SMC hyperplasia by activating ADAM10/Notch1 signalling. We have identified macrophage-derived MMP8 as a regulator in SMC differentiation from AdSPCs and neointimal SMC hyperplasia in response to injury. Our data provide new insights into the roles of MMP8 in AdSPC differentiation and the pathogenesis of neointima formation in the context of angiographic restenosis, and therefore may aid in the development of novel therapeutic agents for the prevention of this disease.

The role of the vasculature niche on insulin-producing cells generated by transdifferentiation of adult human liver cells

BackgroundInsulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells.MethodsIPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the “building blocks” of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture.ResultsCo-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINβα, which play a key role in pancreatic differentiation.ConclusionsVasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.

Pancreatic Stellate Cells Serve as a Brake Mechanism on Pancreatic Acinar Cell Calcium Signaling Modulated by Methionine Sulfoxide Reductase Expression.

Although methionine sulfoxide reductase (Msr) is known to modulate the activity of multiple functional proteins, the roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT-PCR, Western blot and immunocytochemistry. Msr over-expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co-cultured with pancreatic acinar cells AR4-2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura-2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over-expressions of MsrA, B1 or B2 were found to enhance ATP-stimulated calcium increase but decreased reactive oxygen species generation and lipopolysaccharide-elicited IL-1 production. Pancreatic stellate cell-co-culture with AR4-2J blunted cholecystokinin- and acetylcholine-stimulated calcium increases in AR4-2J, depending on acinar/stellate cell ratio, this inhibition was reversed by MsrA, B1 over-expression in stellate cells or by Met supplementation in the co-culture medium. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression.

Pancreatic stellate cells activated by mutant KRAS-mediated PAI-1 upregulation foster pancreatic cancer progression via IL-8.

Background: The dense fibrotic stroma enveloping pancreatic tumors is a major cause of drug resistance. Pancreatic stellate cells (PSCs) in the stroma can be activated to induce intra-tumor fibrosis and worsen patient survival; however, the molecular basics for the regulation of PSC activation remains unclear.Methods: The in vitro coculture system was used to study cancer cell-PSC interactions. Atomic force microscopy was used to measure the stiffness of tumor tissues and coculture gels. Cytokine arrays, qPCR, and Western blotting were performed to identify the potential factors involved in PSC activation and to elucidate underlying pathways.Results: PSC activation characterized by α-SMA expression was associated with increased pancreatic tumor stiffness and poor prognosis. Coculture with cancer cells induced PSC activation, which increased organotypic coculture gel stiffness and cancer cell invasion. Cancer cells-derived PAI-1 identified from coculture medium could activate PSCs, consistent with pancreatic cancer tissue microarray analysis showing a strong positive correlation between PAI-1 and α-SMA expression. Suppression by knocking down PAI-1 in cancer cells demonstrated the requirement of PAI-1 for coculture-induced PSC activation and gel stiffness. PAI-1 could be upregulated by KRAS in pancreatic cancer cells through ERK. In PSCs, inhibition of LRP-1, ERK, and c-JUN neutralized the effect of PAI-1, suggesting the contribution of LRP-1/ERK/c-JUN signaling. Furthermore, activated PSCs might exacerbate malignant behavior of cancer cells via IL-8 because suppression of IL-8 signaling reduced pancreatic tumor growth and fibrosis in vivo.Conclusions: KRAS-mutant pancreatic cancer cells can activate PSCs through PAI-1/LRP-1 signaling to promote fibrosis and cancer progression.

Selective deletion of hepatocyte platelet-derived growth factor receptor \u03b1 and development of liver fibrosis in mice

BackgroundPlatelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs.MethodsHepatocytes were isolated from normal and thioacetamide (TAA)-induced cirrhotic livers for assessment of PDGFRα expression. Conditional knock-out (KO) C57BL/6 mice, in which PDGFRα was selectively deleted in hepatocytes, were generated. Liver fibrosis was induced by injecting TAA for 8 weeks. Hep3B cells were transfected with a small interfering RNA (siRNA) (PDGFRα or control) and co-cultured with LX2 cells.ResultsPDGFRα expression was increased in hepatocytes from fibrotic livers compared to normal livers. Conditional PDGFRα KO mice had attenuated TAA-induced liver fibrosis with decreased HSC activation and proliferation. Immunoblot analyses revealed decreased expression of phospho-p44/42 MAPK in TAA-treated KO mice; these mice also showed almost complete suppression of the upregulation of mouse double minute 2. Although KO mice exhibited increased expression of transforming growth factor (TGF)-β and Smad2/3, this was compensated for by increased expression of inhibitory Smad7. LX2 cells co-cultured with PDGFRα siRNA-infected Hep3B cells showed decreased PDGFRα, α smooth muscle actin, collagen α1(I), TGFβ, and Smad2/3 expression. LX2/PDGFRα-deleted hepatocyte co-culture medium showed decreased PDGF-BB and PDGF-CC levels.ConclusionsDeletion of PDGFRα in hepatocytes attenuated the upregulation of PDGFRα in HSCs after TAA treatment, resulting in decreased liver fibrosis and HSC activation. This suggests that in the event of chronic liver injury, PDGFRα in hepatocytes plays an important role in liver fibrosis by affecting PDGFRα expression in HSCs.

High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Cichorium intybus L. via removing macronutrients

Transformed hairy roots technology provides new opportunities for mass production of pharmaceutical metabolites. Hairy roots culture has been considered as an alternative method for producing medicinal biomolecules in plants. Chicory (Cichorium intybus L.) is a medicinal herb from Asteraceae family. It contains many important metabolites including chicoric acid, inulin, scoline, coumarin, and flavonoids. In this study, for the first time, a reliable gene transfer system via modifying co-cultured medium using different strains of Agrobacterium rhizogenes and explants were established for C. intybus. Different co-culture media were used for this purpose, including MS basal medium and MS medium without either of KNO3, NH4NO3, KH2PO4, CaCl2, and MgSO4. The results showed increases in hairy roots induction percentage, biomass production, and phenolic content of the hairy roots. Maximum hairy root induction percentage obtained by using A4 strain when a KNO3-free MS medium was used. High frequency of hairy root induction percentage with ATCC11325 and ATCC15834 strains was achieved when MS medium had KH2PO4 removed. In all of the experiments, very low hairy root induction was observed when NH4NO3 was removed from co-culture MS medium. Molecular confirmation of transgenic hairy roots was done with PCR using gene-specific primers for rolB gene. Total phenols, flavonoids, anthocyanins and chicoric acid contents of hairy root clones and the control (untransformed) roots were also determined. The results indicated significant increases in total metabolites content of hairy root clones induced by A4, ATCC11325 and ATCC15834 strains with some macronatrient–free co-culture MS media and also directly-regenerated shoots from hairy roots were spontaneous without using any plant growth regulators.

Effects of VEGFR1+ hematopoietic progenitor cells on pre-metastatic niche formation and in vivo metastasis of breast cancer cells

The pre-metastatic niche has been shown to play a critical role in tumor metastasis, and its formation is closely related to the tumor microenvironment. However, the underlying molecular mechanisms remain unclear. In the present study, we successfully established a mouse model of lung metastasis using luciferase-expressing MDA-MB-435s cells. In this model, recruitment of vascular endothelial growth factor receptor-1 (VEGFR1)+CD133+ hematopoietic progenitor cells (HPCs) was gradually increased in lung but gradually decreased after the formation of tumor colonies in lung. We also established a highly metastatic MDA-MB-435s (MDA-MB-435s-HM) cell line from the mouse model. Changes in protein profiles in different culture conditions were investigated by protein microarray analysis. The levels of CXC chemokine ligand 16, interleukin (IL)-2Rα, IL-2Rγ, matrix metalloproteinase (MMP)-1, MMP-9, platelet-derived growth factor receptor (PDGFR)-α, stromal cell-derived factor (SDF)-1α, transforming growth factor (TGF)-β, platelet endothelial cell adhesion molecule (PECAM)-1 and vascular endothelial (VE)-cadherin were significantly greater (> fivefold) in the culture medium from MDA-MB-435s-HM cells than in that from MDA-MB-435s cells. Moreover, the levels of MMP-9, PDGFR-α, and PECAM-1 were significantly greater in the co-culture medium of MDA-MB-435s-HM cells and CD133+ HPCs than in that from MDA-MB-435s-HM cells. Differentially expressed proteins were validated by enzyme-linked immunosorbent assay, and expression of their transcripts was confirmed by quantitative real-time polymerase chain reaction. Moreover, inhibition of MMP-9, PDGFR-α, and PECAM-1 by their specific inhibitors or antibodies significantly decreased cell migration, delayed lung metastasis, and decreased recruitment of VEGFR1+CD133+ HPCs into lung. Intra-hepatic growth of HPCs enhanced the invasive growth of MDA-MB-435s-HM cells in the liver. Our data indicate that VEGFR1+CD133+ HPCs contribute to lung metastasis.

Neuroprotection by Human Dental Pulp Mesenchymal Stem Cells: From Billions to Nano.

Mesenchymal Stem Cell (MSC) therapy in recent years has gained significant attention. Though the functional outcomes following MSC therapy for neurodegenerative diseases are convincing, various mechanisms for the functional recovery are being debated. Nevertheless, recent studies convincingly demonstrated that recovery following MSC therapy could be reiterated with MSC secretome per se thereby shifting the dogma from cell therapy to cell "based" therapy. In addition to various functional proteins, stem cell secretome also includes extracellular membrane vesicles like exosomes. Exosomes which are of "Nano" size have attracted significant interest as they can pass through the bloodbrain barrier far easily than macro size cells or growth factors. Exosomes act as a cargo between cells to bring about significant alterations in target cells. As the importance of exosomes is getting unveil, it is imperial to carry out a comprehensive study to evaluate the neuroprotective potential of exosomes as compared to conventional co-culture or total condition medium treatments. Thus, the present study is designed to compare the neuroprotective potential of MSC derived exosomes with MSC-condition medium or neuron-MSC-co-culture system against kainic acid induced excitotoxicity in in vitro condition. The study also aims at comparing the neuroprotective efficacy of exosomes/condition medium/co-culture of two MSC viz., neural crest derived human Dental Pulp Stem Cells (hDPSC) and human Bone-Marrow Mesenchymal Stem Cells (hBM-MSC) to identify the appropriate MSC source for treating neurodegenerative diseases. Our results demonstrated that neuroprotective efficacy of MSC-exosomes is as efficient as MSC-condition medium or neuron-MSC co-culture system and treating degenerating hippocampal neurons with all three MSC based approaches could up-regulate host's endogenous growth factor expressions and prevent apoptosis by activating cell survival PI3K-B-cell lymphoma-2 (Bcl-2) pathway. Thus, the current study highlights the possibilities of treating neurodegenerative diseases with "Nano" size exosomes as opposed to transplanting billions of stem cells which inherit several disadvantages.

Supplementation of 17β-estradiol and progesterone in the co-culture medium of bovine oviductal epithelial cells and ovine spermatozoa reduces the sperm kinematics and capacitation

This study investigated the effect that bovine oviductal epithelial cell (BOEC) and ovine spermatozoa co-culture exposed to different hormonal environments had on ram sperm function over the course of a 24-h incubation period. Ram cooled-stored spermatozoa were selected by swim-up and then co-cultured separately for 24 h at 38.5 °C under 5% CO2 with either: (1) Fert-TALP medium (positive control [POSControl]), (2) Fert-TALP medium supplemented with 17β-estradiol (E2) and progesterone (P4) at concentrations similar to follicular phase (Follicular NEGControl), (3) Fert-TALP medium supplemented with E2 and P4 concentrations similar to luteal phase (Luteal NEGControl), (4) BOEC cultured in the same medium as that of the Follicular NEGControl group (Follicular BOEC group), or (5) BOEC cultured in the same medium as that of the Luteal NEGControl group (Luteal BOEC group). The sperm kinematics, capacitation status, and plasma membrane (PM) integrity were evaluated at different intervals. Sperm PM integrity was not affected (P ˃ 0.05) by BOEC co-culture, regardless of the phase of the estrous cycle. After 4 h of incubation, the Luteal BOEC group presented lower (P < 0.05) progressive motility and total motility than the Luteal NEGControl group while the Follicular BOEC group showed lower (P < 0.05) velocimetric parameters and progressive motility than the Follicular NEGControl group. Throughout the incubation period, both BOEC co-culture groups showed a decrease (P < 0.05) in their capacitation rate in comparison to the POSControl group. Conversely, the Luteal BOEC group presented a higher (P < 0.05) non-capacitated rate than both the POSControl and Luteal NEGControl groups. In conclusion, BOEC co-culture with ovine spermatozoa at either the follicular or luteal phase decreases sperm kinematics and delays sperm capacitation.

Development of an organotypic stem cell model for the study of human embryonic palatal fusion.

Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1,000 births worldwide. The secondary palate is formed by paired palatal shelves, consisting of a mesenchymal core with an outer layer of epithelial cells that grow toward each other, attach, and fuse. One of the mechanisms that can cause CP is failure of fusion, that is, failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme confluence. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical-derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEKs). Organoids were incubated in co-culture medium with or without hEGF and assessed for cell proliferation and time to fusion. Osteogenic differentiation in hMSC spheroids was highest by Day 13. hEGF delayed fusion of organoids after 12 and 18 hr of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone. Our results show that this model of human palatal fusion appropriately mimics the morphology of the developing human palate and responds to hEGF as expected.

Differentiation of naive T cells into Th2 cells induced by nuocyte cells in mice with allergic rhinitis

Objective:The aim of this study is to examine whether nuocytes induced differentiation of primary T cells into Th2 cells in AR mice in vitro. Method:A murine model of AR on the background of BALB/c was established using ovalbumin, and nuocytes were sorted and purified from the mouse nasal associated lymphoid tissue (NALT) and cultured in vitro. Then, we assessed the expression of IL-4 in these cells. Mouse peripheral blood mononuclear cells were isolated and Th2 cells and T cells were isolated for in vitro culture, and the percentage of T cells in Th2 cells was detected. Then, NALT-derived nuocyte cells cultured in vitro were added to the above-mentioned mouse T cell culture medium for co-culture. Result:Numbers of sneezing, nasal rubbing and eosinophils in nasal lavage fluid were all enhanced in AR mice compared to normal ones. We used flow cytometry analysis to identify nuocytes from mice NALT as CD3CD4CD8CD19CD11bCD11cFcεR1 (lineage)-ICOS+, and also found that the cells expressed IL-4, and its protein and mRNA were all increased in AR mice versus normal mice. After nuocytes were co-cultured with T-cell cultures, we determined the percentage of Th2 cells in total T cells, and found that the percentage was increased significantly. Conclusion:nuocyte cells may induce the differentiation of primary T cells into Th2 cells in AR mice through IL-4 pathway.

Fusion of Dendritic Cells and Cancer-Associated Fibroblasts for Activation of Anti-Tumor Cytotoxic T Lymphocytes

Here we explored the fusion of dendritic cells (DCs), potent antigen-presenting cells that initiate primary immune responses, with cancer-associated fibroblasts (CAFs), which are a stromal component needed for tumor progression, with the aim of stimulating T cells to inhibit tumor growth. Dendritic cells from the bone marrow of BALB/c mice were co-cultured with CAFs from H22 mouse hepatoma cells. CAFs were found to express fibroblast activation protein and α-smooth muscle actin by flow cytometry, Western blotting and immunofluorescence. Polyethylene glycol was added to the co-culture medium to encourage fusion, and the ability of the resulting fusion cells to produce TNF-α, IL-1β, IL-6, and IL-12p70 was confirmed using ELISA. These fusion cells efficiently stimulated T lymphocytes in vitro, causing them to generate IFN-α and IFN-γ. T cells activated by DC/CAF fusion cells led to strong CTL response against CAFs in vitro. The activated T cells also inhibited growth of H22 xenografts in vivo. These results indicate that DC/CAF fusion cells show potential for stimulating T cells as a novel anti-tumor vaccine.

Enhancement of docosahexaenoic acid (DHA) and beta-carotene production in Schizochytrium sp. using symbiotic relationship with Rhodotorula glutinis

The aim of the study is to enhance the docosahexaenoic acid (DHA) and beta-carotene production yields in Schizochytrium sp. by forming a symbiotic relationship with yeast cell Rhodotorula glutinis. Schizochytrium sp. is one of the most studied microalgae for DHA production. On the other hand, Rhodotorula glutinis is well known as carotenoid-producing yeast. Here, mixed culture of Schizochytrium sp. and Rhodotorula glutinis was used, for the first time, to enhance the yields for DHA and beta-carotene. The cells were recovered, biomass, lipid content, DHA and beta-carotene yields were analyzed. GC-FID and HPLC were used for lipid content analysis and beta-carotene yield, respectively. Cell dry weight was increased ∼2.6 fold while DHA and β-carotene yields were increased ∼1.18 and ∼1.76 fold respectively in co-culture medium compared to Schizochytrium-monoculture. The results highlight the possibility of using heterotroph microalgae and yeast cells in co-culture environment to enhance DHA yield along with β-carotene production.

Activated gastric cancer-associated fibroblasts contribute to the malignant phenotype and 5-FU resistance via paracrine action in gastric cancer

BackgroundCancer-associated fibroblasts (CAFs) play important roles in tumor progression. However, the behaviors of activated CAFs in gastric cancer remain to be determined. The aim of the present study was to investigate the correlations between activated gastric CAFs and the prognosis of patients with gastric cancer, and to determine the effects of activated CAFs on the malignant phenotype and 5-fluorouracil resistance in this cancer.MethodsNinety-five patients with primary gastric cancer were enrolled in this study. Activation states of gastric CAFs were evaluated by immunohistochemistry. A modified method for the primary culture of gastric CAFs was employed. Types of CAFs and activation states were identified by immunocytochemical and immunofluorescent staining. Cell co-culture and gastric CAF conditioned medium transfer models were established to investigate the paracrine effects of activated CAFs on the migration and invasion of gastric cell lines. The half maximal inhibitory concentration of 5-fluorouracil and levels of cell apoptosis were examined using cell viability assay and flow cytometry, respectively. Protein expression levels of associated molecules were measured by Western blotting.ResultsKaplan–Meier survival curves showed that activated gastric CAFs identified via fibroblast activation protein were significantly related to poorer cumulative survival in gastric cancer patients. Five strains of CAFs were successfully cultured via the modified culture method, and three gastric CAFs strains were identified as activated gastric CAFs. The migration and invasion abilities of gastric cells were significantly enhanced in both the co-culture group and the conditioned medium group. The half maximal inhibitory concentration for 5-fluorouracil in BGC-823 cells was elevated after treatment with conditioned medium, and early apoptosis was inhibited. Additionally, an obvious elevation of epithelial–mesenchymal transition level was observed in the conditioned medium group.ConclusionsActivated gastric CAFs correlate with a poor prognosis of cancer patients and may contribute to the malignant phenotype and the development of resistance to 5-fluorouracil via paracrine action in gastric cancer. Gastric CAFs with a specific activation state might be used as a tumor biomarker within the microenvironment for prognosis and as a new therapeutic target for chemoresistant gastric cancer.

Bone marrow derived mesenchymal stem cells ameliorate inflammatory response in an in vitro model of familial hemophagocytic lymphohistiocytosis 2

BackgroundFamilial hemophagocytic lymphohistiocytosis 2 (FHL2) is the most common familial type of hemophagocytic lymphohistiocytosis with immune dysregulation. FHL2 patients have mutations in the perforin gene which cause overactivation and proliferation of cytotoxic T lymphocytes and natural killer cells. Perforin is the key component of the cytolytic granule response function of cytotoxic T lymphocytes and natural killer cells. Perforin dysfunction causes a cytotoxic immune deficiency with a clinical outcome of uncontrolled and continuous immune stimulation response. This excessive stimulation leads to continuous systemic inflammation and, ultimately, multiorgan failure. Radical therapy is hematopoietic stem cell transplantation which is limited by the availability of a donor. Exacerbations of inflammatory attacks require a palliative immunosuppressive regimen. There is a need for an alternative or adjuvant therapy to maintain these patients when immunosuppression is ineffective or a donor is not available. Beneficial actions of mesenchymal stem cells (MSCs) have been shown in autoimmune diseases in clinical trials and are attributed to their immune-modulatory properties. This study aimed to assess the immune-modulatory effect of MSCs in an in-vitro model of FHL2.MethodsWe generated a targeted mutation in the perforin gene of NK92 cells to create an in-vitro FLH2 model using Crispr/Cas technology. A coculture setup was employed to assess the immunomodulatory efficacy of MSCs.ResultsEngineered NK92 clones did not show PRF1 mRNA expression and failed to secrete perforin upon phorbol myristate acetate–ionomycin stimulation, providing evidence for a valid FHL2 model. Coculture media of the engineered cells were investigated for the abundance of several cytokines. Coculture with MSCs revealed a reduction in major proinflammatory cytokines and an induction in anti-inflammatory and immunomodulatory cytokines compared to the parental NK92 cells.ConclusionsThis study shows the ameliorating effect of MSCs as an adjuvant immune modulator toward the therapy of FHL2 patients. MSCs are supportive therapy candidates for FHL2 patients under circumstances where prolonged immunosuppression is required to gain time before allogeneic hematopoietic stem cell transplantation.