Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures

Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells （BMSCs） and endothelial cells （ECs） contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs （HUVECs）. The expression levels of CD31, von Willebrand factor, osteonectin （ON） and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs.

New Antibacterial Dihydropyrones Induced by Co-Culture of Penicillium crustosum PRB-2 and Penicillium citrinum HDN11-186

Two new dihydropyrones,rhytismatones C(1)and D(2),and a known compound,penicillenol A1(3),were isolated from the co-culture broth of the deep-sea-derived fungus Penicillium crustosum PRB-2 and Suaeda salsa-derived endophytic fungus Peni-cillium citrinum HDN11-186.Their structures were elucidated through comprehensive analysis of nuclear magnetic resonance(NMR)spectra and mass spectra.The absolute configurations of new compounds were determined by calculating the electronic circular di-chroism(ECD)spectrum.UPLC-MS data showed that compounds 1–3 could only be detected in the media of co-culture,suggesting new biosynthetic pathways were activated in the co-cultured fungi.Compound 1 showed obvious antibacterial activities against Pro-teus sp.MMBC-1002 and Bacillus subtilis MMBC-1004 with minimum inhibitory concentration(MIC)both at 25μmolL^(-1).

Brain-derived neurotrophic factor protects neurons from GdCl_3-induced impairment in neuron-astrocyte co-cultures

Gadolinium (Gd3+) complexes are important contrast agents in medical magnetic resonance imaging (MRI) and of great potential value in brain research. In order to better understand the mechanisms of the action of Gd3+ on neurons in the complex central nervous system (CNS), the neurotoxic actions of GdCl3 have been investigated in both neuron monoculture and astrocyte-neuron co-culture systems. Measurements of lactate dehydrogenase release showed that GdCl3 causes significant cell death of monocultured neurons as a result of reactive oxygen species (ROS) generation and down-regulation of brain-derived neurotrophic factor (BDNF). However, GdCl3 does not affect the viability and BDNF expression of astrocytes. Both co-culturing of neurons with astrocytes and addition of BDNF ameliorated GdCl3-induced neurotoxicity by decreasing ROS generation and facilitating recovery of BDNF levels. The results obtained suggest that astrocytes in the CNS may protect neurons from GdCl3-induced impairment through secreting BDNF and thus up-regulating BDNF expression and interfering with Gd3+-induced cell signaling in neurons. A possible molecular mechanism is suggested which should be helpful in understand- ing the neurotoxic actions of gadolinium probes .

“Co-culture Engineering”for Enhanced Phytoremediation of Metal Contaminated Soils

A co-culture of two plant materials, Astragalus sinicus L., a leguminous plant with concomitant nodules, and Elsholtzia splendens Naki-a Cu accumulator, along with treatments of a chelating agent (EDTA), root excretions (citric acid), and a control with E. splendens only were used to compare the mobility of heavy metals in chelating agents with a co-culture and to determine the potential for co-culture phytoremediation in heavy metal contaminated soils. The root uptake for Cu, Zn, and Pb in all treatments was significantly greater (P ＜ 0.05) than that of the control treatment. However with translocation in the shoots, only Cu, Zn, and Pb in plants grown with the EDTA treatment and Zn in plants cocropped with the A. sinicus treatment increased significantly (P ＜ 0.05). In addition, when a co-culture in soils with heavy and moderate contamination was compared, for roots in moderately contaminated soils only Zn concentration was significantly less (P ＜ 0.05) than that of heavily contaminated soils, however, Cu, Zn, and Pb concentrations of shoots were all significantly lower (P ＜ 0.05). Overall, this 'co-culture engineering' could be as effective as or even more effective than chelating agents, thereby preventing plant metal toxicity and metal leaching in soils as was usually observed in chelate-enhanced phytoremediation.

Rice-duck co-culture benefits grain 2-acetyl-1-pyrroline accumulation and quality and yield enhancement of fragrant rice

Rice-duck co-culture is an integrated farming technology that benefits rice production, grain quality, and ecological sustainability in paddy fields. However, little is known about the effects of rice-duck co-culture on enzyme activity involved in the biosynthesis of 2-acetyl-1-pyrroline (2-AP), the volatile that gives fragrant rice its' distinctive and sought-after aroma. The present study aimed to examine the influence of rice-duck co-culture on the photosynthesis, yield, grain quality, rice aroma, and the enzymes involved in 2-acetyl-1-pyrroline biosynthesis in the cultivar Meixiangzhan 2 during the early and late rice growing seasons of 2016 in Guangzhou, China. We compared the rice grown in paddy fields with and without ducks. We found that rice-duck co-culture not only improved the yield and quality of fragrant rice grain, but also promoted the precursors of 2-AP biosynthesis formation and 2-AP accumulation in the grain. Grain 2-AP content in rice-duck co-culture was noticeably increased with 9.60% and 20.81% in early and late seasons, respectively. Proline and pyrroline-5-carboxylic acid (P5C) (precursors of 2-AP biosynthesis) and the activity of enzymes such as proline dehydrogenase (ProDH), ornithine aminotransferase (OAT) and Δ1 pyrroline-5-carboxylic acid synthetase (P5CS) were all improved by 10.15%–12.99%, 32.91%–47.75%, 17.81%–26.71%, 6.25%–21.78%, and 10.58%–38.87% under rice-duck co-culture in both seasons, respectively. Overall, our results suggest that rice-duck co-culture is an environmentally-friendly and sustainable approach to improving rice aroma and grain quality of fragrant rice.

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

AIM： To investigate cytokine production and cell surface phenotypes of dendritic cells （DC） in the presence of epithelial cells stimulated by probiotics.METHODS： Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or in- verted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis ADO 11 （BL）, Bifidobacterium bilfidum BGN4 （BB）, Lactobacillus casei IBS041 （LC）, and Lactobacillus acidophilus AD031 （LA）, for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent as say and phenotypic analysis of DC was investigated by flow cytometry.RESULTS： BB and LC in singlecultured DC increased the expression of I-Ad, CD86 and CD40 （I-Ad, 18.51 vs 30.88, 46.11, CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P 〈 0.05）. All of the experimental probiot-ics increased the production of inflammatory cytokines, interleukin （IL）-6 and tumor necrosis factor （TNF）-α. However, in the normal coculture systems, LC and LA decreased the expression of I-A^α （39.46 vs 30.32, 33.26, P 〈 0.05）, and none of the experimental probiotics increased the levels of IL-6 or TNF-α. In the inverted coculture systems, LC decreased the expression of CD40 （1.36 vs -2.27, P 〈 0.05）, and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 （103.8 vs 166.0, P 〈 0.05） and LC and LA increased transforming growth factor-13 secretion （235.9 vs 618.9, 607.6, P 〈 0.05）.CONCLUSION： These results suggest that specific pro- biotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.

Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells

AIM: To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes. METHODS: Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC- assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone. RESULTS: The mRNA expression of α-fetoprotein, albumin (ALB), α-1-antitrypsin, and hepatocyte nuclear factor 4α was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storageand urea synthesis were also demonstrated. CONCLUSION: MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells

Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms.However,to date,no unified method has been described for establishing a blood-brain barrier model.Here,we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-μm pores,and conducted transepithelial electrical resistance measurements,leakage tests and assays for specific bloodbrain barrier enzymes.We show that the permeability of our model is as low as that of the bloodbrain barrier in vivo.Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

Effects of Rice-Fish Co-culture on Oxygen Consumption in Intensive Aquaculture Pond

Rice-fish co-culture has gained increasing attention to remediate the negative environmental impacts induced by intensive aquaculture. However, the effect of rice-fish co-culture on oxygen depletion has rarely been investigated. We constructed a rice-fish co-culture system in yellow catfish(Pelteobagrus fulvidraco) and freshwater shrimp(Macrobrachium nipponense) ponds using a new high-stalk rice variety, and conducted a field experiment to investigate the effect of rice-fish co-culture on water parameters and oxygen consumption. The results showed that rice-fish co-culture reduced the nutrients(total nitrogen, ammonia-N, total phosphorous and potassium) and the dissolved oxygen content in fish and shrimp ponds. However, they showed similar seasonal change of dissolved oxygen in the water of fish and shrimp ponds. Rice-fish co-culture reduced the total amount of oxygen consumption and optimized the oxygen consumption structure in pond. The respiration rates in water and sediment were significantly reduced by 66.1% and 31.7% in the catfish pond, and 64.4% and 38.7% in the shrimp pond, respectively, by additional rice cultivation. Rice-fish co-culture decreased the proportions of respiration in sediment and water, and increased the proportion of fish respiration. These results suggest that rice-fish co-culture is an efficient way to reduce hypoxia in intensive culture pond.

Impact of Rice-Catfish/Shrimp Co-culture on Nutrients Fluxes Across Sediment-Water Interface in Intensive Aquaculture Ponds

Exchange of nitrogen and phosphorus across sediment-water interface plays an important role in the management of nutrient recycling in the aquaculture pond. In this study, a plot experiment was conducted to study the effect of rice-catfish/shrimp co-culture on the micro-profile of oxygen (O2), pH and nutrient exchange across sediment-water interface in the intensive culture ponds. The results showed that rice-catfish co-culture increased the concentration and penetrating depth of O2, but decreased the pH value across the sediment-water interface, compared with catfish monoculture. Additional rice cultivation significantly reduced the flux rates of ammonium (NH4+) and nitrate (NO3-) across sediment-water interface in the catfish and shrimp ponds. The flux rates of NO2 - and soluble phosphorus (PO43-) showed no significant difference between rice-catfish/shrimp co-culture ponds and catfish/shrimp monoculture ponds. Rice only affected the dissolved inorganic nitrogen and phosphorus fractions in the sediment. The concentrations of NH4 + were significantly lower in the sediment of co-culture ponds than in the monoculture ponds. Additional rice cultivation also significantly reduced the content and percentage of dissolved inorganic phosphorus in the sediment of catfish ponds.

Advances in tumor-endothelial cells co-culture and interaction on microfluidics

The metastasis in which the cancer cells degrade the extracellular matrix （ECM） and invade to the sur- rounding and far tissues of the body is the leading cause of mortality in cancer patients, With a lot of advancement in the field, yet the biological cause of metastasis are poorly understood, The microfluidic system provides advanced technology to reconstruct a variety of in vivo-like environment for studying the interactions between tumor ceils （TCs） and endothelial ceils （ECs）. This review gives a brief account of both two-dimensional models and three-dimensional microfluidic systems for the analysis of TCs-ECs co- culture as well as their applications to anti-cancer drug screening, Furthermore, the advanced methods for analyzing cell-to-cell interactions at single-cell level were also discussed,

Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system

The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer’s disease, Parkinson’s disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astro-cytesin vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identiifed and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by lfuorescence spectrophotometry and lfuorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addi-tion, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neu-rons than that observed in astrocytes.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Neural differentiation of human placenta-derived mesenchymal stem cells following neural cell co-culture

We induced human placenta-derived mesenchymal stem cells （hPMSCs） to differentiate into neural cells by adding chemical reagents, despite the fact that toxic chemicals induce cell shrinkage or cytoskeletal formation, which does not represent a proper cell differentiation process. The present study established a co-culture system with hPMSCs and neural cells and analyzed the influence of neural cells on hPMSC differentiation in a co-culture system, hPMSCs were isolated and purified from human full-term placenta using collagenase digestion. Fetal neural cells were co-cultured with hPMSCs for 48 hours using the Transwell co-culture system, hPMSCs co-cultured with neural cells exhibited a slender morphology with a filament. After 96 hours, hPMSCs expressed neuron-specific enolase, which suggested that co-culture of hPMSCs and neural cells induced neural differentiation of hPMSCs.

Osteogenic Differentiation of Bone Mesenchymal Stem Cells Regulated by Osteoblasts under EMF Exposure in a Co-culture System

This study examined the osteogenic effect of electromagnetic fields （EMF） under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells （BMSCs） and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit （CCK-8）. For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase （ALP） staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure （2 h/day） could obviously promote prolifera- tion of BMSCs and osteoblasts, while long-term EMF （8 h/day） could promote osteogenic differen- tiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when ceils were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differ- entiation of BMSCs.

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

Science Letters:Cellular growth under hydrostatic pressure using bovine aortic EC-SMC co-cultured ePTFE vascular graft

High blood pressure (hypertension) is implicated in the development of atherosclerosis. Blood vessels are constantly subjected to stretch due to blood pressure and changes in stretch usually instigate adaptive vascular remodeling, including abnormal growth and proliferation of vascular smooth muscle cells (VSMCs) as well as extracellular matrix (ECM). In this experiment, we used bovine aortic endothelial cells and smooth muscle cells (EC-SMC) co-cultured ePTFE vascular grafts subjected to normal atmospheric pressure (as a control), and 100 mmHg hydrostatic pressure for 7 d. The increase of cell layer thickness was observed. When measured, the cell layer thickness increased by 116.2%. The increase of collagen (Type Ⅳ)synthesis was also observed in the immunohistochemistry assay. When stained with toluidine blue, the cells showed metachromatic phenomenon.

Osteoblast integration of dental implant materials after challenge by sub-gingival pathogens: a co-culture study in vitro

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium （Ti）, titanium-zirconium （TiZr） alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.