An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P < 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P > 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P < 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P < 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P < 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P < 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.     

Neuroprotection of aucubin in primary diabetic encephalopathy

Hippocampal neuronal apoptosis accompanied by impairment of cognitive function occurs in primary diabetic encephalopathy. In this study, we investigated the neuroprotective mechanism of the iridoid glycoside, aucubin, using rats (n=8). Diabetes mellitus was induced in the rats by intraperitoneal (i.p.) injection of streptozotocin (60 mg/kg body weight). After 65 d, half of the DM rats were administered aucubin (5 mg/kg; i.p.) for 15 d, yielding treatment DM A. A third group of rats received no strepto- zotocin or aucibin, and served as controls (CON). Encephalopathy was assessed using Y-maze be- havioral testing. Rats were euthanized on Day 87, and hippocampi were excised for visual (light and transmission electron microscopic) and immunochemical (Western blot; immunohistochemical) as- sessments of the CA1 subfield for apoptosis and expression of regulatory proteins Bcl-2 and Bax. Treatment responses to all the parameters examined (body weight, plasma glucose, Y-maze error rates, pyramidal cell ultrastructure, proportions of apoptotic cells, levels of expression of Bcl-2 and Bax, and survivability of neuronal cells) were identical: there were highly significant differences between DM and CON groups (P<0.001), but the effects were significantly moderated (P<0.01) in DM A compared with DM. These findings confirm the association of apoptosis with the encephalopathic effects of diabetes mellitus, and suggest a major role of the expression levels of Bcl-2 and Bax in the regulation of apop- totic cell death. All of the results suggest that aucubin could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.     

Effect of LDL concentration polarization on the uptake of LDL by human endothelial cells and smooth muscle cells co-cultured

To substantiate our hypothesis that concentration polarization of low-density Upoprotein （LDL） plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate （or perfusion pressure） on the luminal surface LDL concentration （cw） and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cyto-metry method. The results indicated that the uptake of fluorescent labeled LDL （DiI-LDL） by the co-cultured cells was positively correlated with Cw in a non-linear fashion. When cw was low, the uptake increased very sharply with increasing Cw. Then the increase became gradual and the uptake was seemingly leveled out when Cw reached beyond 160 μg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.     

Transplanted neuronal precursors migrate and differentiate in the devel-oping mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of prolife     

Proliferation and tenogenic differentiation of bone marrow mesenchymal stem cells in a porous collagen sponge scaffold

BACKGROUND Collagen is one of the most commonly used natural biomaterials for tendon tissue engineering.One of the possible practical ways to further enhance tendon repair is to combine a porous collagen sponge scaffold with a suitable growth factor or cytokine that has an inherent ability to promote the recruitment,proliferation,and tenogenic differentiation of cells.However,there is an incomplete understanding of which growth factors are sufficient and optimal for the tenogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in a collagen sponge-based 3D culture system.AIM To identify one or more ideal growth factors that benefit the proliferation and tenogenic differentiation of rat BMSCs in a porous collagen sponge scaffold.METHODS We constructed a 3D culture system based on a type I collagen sponge scaffold.The surface topography of the collagen sponge scaffold was observed by scanning electron microscopy.Primary BMSCs were isolated from Sprague-Dawley rats.Cell survival on the surfaces of the scaffolds with different growth factors was assessed by live/dead assay and CCK-8 assay.The mRNA and protein expression levels were confirmed by quantitative real-time polymerase chain reaction and Western blot,respectively.The deposited collagen was assessed by Sirius Red staining.RESULTS Transforming growth factorβ1(TGF-β1)showed great promise in the tenogenic differentiation of BMSCs compared to growth differentiation factor 7(GDF-7)and insulin-like growth factor 1(IGF-1)in both the 2D and 3D cultures,and the 3D culture enhanced the differentiation of BMSCs into tenocytes well beyond the level of induction in the 2D culture after TGF-β1 treatment.In the 2D culture,the proliferation of the BMSCs showed no significant changes compared to the control group after TGF-β1,IGF-1,or GDF-7 treatment.However,TGF-β1 and GDF-7 could increase the cell proliferation in the 3D culture.Strangely,we also found more dead cells in the BMSC-collagen sponge constructs that were treated with TGF-β1.Moreover,TGF-β1 promoted more collagen deposition in both the 2D and 3D cultures.CONCLUSION Collagen sponge-based 3D culture with TGF-β1 enhances the responsiveness of the proliferation and tenogenic differentiation of rat BMSCs.     

The characteristics of the spindle fiber attachments（SFAs） enriched fraction from mouse nuclei

The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunofluorescence,test of the activity of microtubule organizing center(MTOC),SDS-PAGE,and fluorescence in situ hybridization.Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity.The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascions of the primary constrictions of all chromosomes of ascites cells.The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD.Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.     

The presence of ANP in rat peritoneal mast cells

Atrial natriuretic peptide （ANP） is an important component of the natriuretic peptide system. A great role in many regulatory systems is played by mast cells. Meanwhile involvement of these cells in ANP activity is poorly studied. In this work, we have shown the presence of ANP in rat peritoneal mast cells. Pure fraction of mast cells was obtained by separation of rat peritoneal cells on a Percoll density gradient. By Westem blotting, two ANP-immunoreactive proteins of molecular masses of 2.5 kDa and 16.9 kDa were detected in lysates from these mast cells. Electron microscope immunogold labeling has revealed the presence of ANP-immunoreactive material in storage, secreting and released granules of mast cells. Our findings indicate the rat peritoneal mast cells to contain both ANP prohormone and ANP. These both peptides are located in mast cell secretory granules and released by mechanism of degranulation. It is discussed that many mast cell functions might be due to production of natriuretic peptides by these cells.     

Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood

AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product. METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential. RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34 + cells increased 43-fold. The granulocyte-macrophage colonyforming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells. CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.     

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.     

Characterization of a novel α4/4-conotoxin,Qcl.2, from vermivorous Conus quercinus

As part of continuing studies of the identification of gene organization and cloning of novel α-conotoxins, the first α4/4-conotoxin identified in a vermivorous Conus species, designated Qcl.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds （Ⅰ-Ⅲ, Ⅱ-Ⅳ connectivity） in a native globular configuration. The mature peptide of Qcl.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qcl.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qcl.2 was shown to selectively inhibit both rat neuronal α3β2 and α3β4 subtypes of nicotinic acetylcholine receptors with low potency. A block of -63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized α-conotoxin members, the unusual structural features in Qcl.2 that confer to its receptor recognition profile are addressed.     

Transplated neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone(SVZ),lining the lateral ventricle in forebrain,retains a population of neuronal precursors with the ability of proliferation in adult mammals.To test the potential of neuronal precursors in adult mice,we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages.The preliminary results indicated that the grafted cells were able to survive and migrate into multiople regions of the recipient brain,including SVZ, the third ventricle,thalamus,superior colliculus,inferior colliculus,cerebellum and olfactory bulb etc;and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain.Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker.Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation,which is likely to be induced by some environmental factors.     

Priming with magnesium-deficient media inhibits preadipocyte differentiation via potential upregulation of tumor necrosis factor-α

The effect of priming stromal-vascular cells in primary cultures with magnesium-deficient (MgD) media on preadipocyte differentiation was studied. Cultures were derived from dorsal subcutaneous fat tissue of young pigs and maintained 3 d in serum-free control or MgD Dulbecco’s modified Eagle’s medium, 3 d in 10% fetal bovine serum and dexamethasone, and 6 d in insulin. At d 12 of culture, immunocytochemical and glycerophosphate dehydrogenase assays indicated depressed adipocyte differentiation in the MgD groups. Cultures were enriched for preadipocytes up to 50% of total cells. On the third day of treatment with control and MgD medium, total cell lysates were isolated and 50 μg of them were run on two-dimensional gel electrophoresis. The separated proteins from both treatment groups showed similar patterns. However, spots of proteins with predicted molecular weight in the range of 25.8–37.4 kDa and pI of 5.39–5.85 were sixfold denser from the MgD 10 groups than from the controls. These proteins migrate similarly to tumor necrosis factor-α (TNF-α). The amount of TNF-α in cell lysates from the MgD group was about 2.35 times greater than controls determined by TNF-α-ELISA. It is likely that proteins upregulated by MgD medium are TNF-α isoforms.     

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells weresuccessfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.     

Microarray analysis of the developing rat mandible

To analyze the molecular events that occur in the developing mandible, we examined the expression of 8803 genes from samples taken at different time points during rat postnatal mandible development. Total RNA was extracted from the mandibles of 1-day-old, 1-week-old, and 2-week-old rats. Complementary RNA (cRNA) was synthesized from cDNA and biotinylated. Fragmented cRNA was hybridized to RGU34A GeneChip arrays. Among the 8803 genes tested, 4344 were detectable. We identified 148 genes with significantly increased expression, and 19 genes with significantly decreased expression. A comprehensive analysis appears to be an effective method of studying the complex process of development.     

Perfused transcapillary smooth muscle and endothelial cell co-culture—a novelin vitro model

Summary As mostin vitro endothelial cell (EC)-vascular smooth muscle cell (SMC) co-culture studies have been performed utilizing static culture conditions, none have successfully mimicked the physical environment of these cellsin vivo. EC covering the inner surface of blood vessels are continuously exposed to a hemodynamically imposed mechanical stress resulting from the flow of blood, while SMC are affected by pressure, a flow-related force acting perpendicular to the surface. We have developed a perfused transcapillary co-culture system that permits the chronic exposure of EC and SMC to physiological shear stresses and pressures. SMC and EC co-cultures were successfully established and maintained in long-term culture (7 wk) on an enclosed perfused bundle of semipermeable polypropylene capillaries. By altering flow rate and/or viscosity, shear stresses of 0.07–20 dyn/cm² can be readily achieved in this system. Electron microscopic analysis revealed that SMC formed multilayers around the outside of the capillaries, whereas EC, subjected to 3 dyn/cm² shear stress, formed an intact closely adherent monolayer lining the capillary lumen. EC and SMC exhibited characteristic ultrastructural and gross morphology. EC were separated from SMC by the capillary wall (pore size 0.5 μm, width 150 μM) and while no direct cell-cell contact was evident some cells were seen to migrate into the capillary wall. Both EC and SMC are exposed to the same culture medium, allowing the interaction of substances released in both directions. Yet separate populations of cells are maintained and can be individually harvested for further analysis. This co-culture system that mimics the architecture and physical environment of the vessel wall should have many potential applications in vascular biology.     

The characteristics of the spindle fiber attachments (SFAs) enriched fraction from mouse nuclei

The characteristics of the particulate mouse centromere enriched fraction from isolated nuclei obtained in our laboratory were investigated by indirect immunofiuorescence, test of the activity of microtubule organizing center(MTOC), SDS-PAGE, and fluorescence in situ hybridization. Most of the particles of the fraction are complexes of DNA and kinetochore proteins and show MTOC activity. The DNA isolated from the fraction can hybridize with DNA in the regions of the primary constrictions of all chromosomes of ascites cells. The kinetochore proteins isolated from the fraction are mainly those with molecular weight of 55 KD and 59 KD. Results suggested that the fraction obtained is a centromere enriched nuclear fraction as indicated in our previous report.     

Inhibition of phagocytosis reduced the classical activation of BV2 microglia induced by amyloidogenic fragments of beta-amyloid and prion proteins

The inflammatory responses in Alzheimer＇s disease and prion diseases are dominated by microglia activation. Three different phenotypes of microglial activation, namely classical activation, alternative activation, and acquired deactivation, have been described. In this study, we investigated the effect of amyloido- genic fragments of amyloid 13 and prion proteins （Aβ1_42 and PrP106-126） on various forms of microglial activation. We first examined the effect of Aβ1_42 and PrP106-126 stimulation on the mRNA expression levels of several markers of microglial activation, as well as the effect of cytochalasin D, a phagocytosis inhibitor, on microgllal activation in Aβ1_42- and PrP106-126- stimulated BV2 microglla. Results showed that Aβ1-42 and PrPlo6_126 induced the classical activation of BV2 microglia, decreased the expression level of alternative expression markers, and had no effect on the expression of acquired de- activation markers. Cytochalasin D treatment significantly reduced Aβ1_42- and PrP106-26-induced up-regulation of proinflammatory factors, but did not change the expression profile of the markers of alternative activation or acquired de- activation in BV2 cells which were exposed to Aβ1-42 and PrPlo6_126. Our results suggested that microglia interact with amyloidogenic peptides in the extraceilular milieu-stimulated microglial classical activation and reduce its alternative activa- tion, and that the uptake of amyloidogenic peptides from the extracellular milieu amplifies the classical microglial activation.     

Interactions of primary neuroepithelial progenitor and brain endothelial cells： distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

Neurovascular interactions are crucial for the normal development of the central nervous system. To study suchinteractions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelialcell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favormaintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differ-entiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellularinteractions needed for the proper development of the brain.