CUMS and dexamethasone induce depression-like phenotypes in mice by differentially altering gut microbiota and triggering macroglia activation.

BACKGROUND: Although the link between gut microbiota and depression has been suggested, changes of gut microbiota vary largely among individuals with depression. AIMS: Explore the heterogeneity of microbiota-gut-brain axis and new pathogenic characteristics in murine models of depression. METHODS: Adolescent female mice were randomly divided into control (CON) group (n=10), chronic unexpected mild stress (CUMS) group (n=15) and dexamethasone (DEX) group (n=15). Mice in the DEX group were gavaged twice a day with 0.2 mg/kg of DEX for 5 weeks, whereas CON mice were given the same amount of solvent. Mice in the CUMS group were exposed to stressors. After behavioural evaluations, all mice were sacrificed for harvesting tissues and blood samples. Enzyme-linked immunosorbent assay (ELISA) was conducted for measuring levels of corticosterone (CORT) and interleukin-1ß (IL-1ß) in sera, whereas levels of protein expression in colon and hippocampal tissues were examined by western blot. Faecal microbial communities were analysed by sequencing 16S rDNAs. RESULTS: Mice in CUMS and DEX groups exhibited severe depression-like behaviours. Compared with CON mice, CUMS-exposed mice showed a significant increase in both α and ß diversity. Prevotellaceae and Desulfovibrio were enriched, whereas Bacilli were decreased in the faeces of mice in the CUMS group. DEX-treated mice had a decrease in the abundance of Clostridium XVIII. Levels of occludin in colon tissue of DEX-treated mice were reduced. Relative to mice in the CON and CUMS groups, DEX-treated mice contained higher serum levels of CORT and IL-1ß. Compared with CON mice, mice in the DEX and CUMS groups had higher levels of IL-1ß in sera and lower levels of glial fibrillary acidic protein (GFAP), Nestin, Synapsin-1 and P2Y12 receptor in the hippocampus. CONCLUSIONS: Changes of gut microbiota diversity, intestinal integrity and neuroinflammation in the brain contribute to CUMS-induced depression, whereas pathobionts and excessive immunosuppression with damaged neuronal synapses is a basis of the DEX-induced depression.

Comparative study of three types of mesenchymal stem cell to differentiate into pancreatic ß-like cells in vitro.

Type 1 diabetes (TID) is a chronic metabolic disease where the body produces insufficient or no insulin. Stem cells with multi-directional differentiation potential are transplanted and differentiate into ß-like cells in vivo to replace pancreatic ß cells, which has become a novel treatment strategy. The aim of the present study was to investigate the ability of three types of adult mesenchymal stem cell (MSC) to differentiate into pancreatic ß-like cells in vitro in order to identify suitable sources for the treatment of diabetes. The three MSC types were menstrual blood-derived MSCs (MENSCs), umbilical cord-derived MSCs (UCMSCs) and dental pulp MSCs (DPSCs). The differentiation method used in the present study was divided into three steps and the MSCs were differentiated into pancreatic ß-like cells in vitro. Among these MSCs, MENSCs had a greater ability to differentiate into islet ß-like cells in vitro, while UCMSCs and DPSCs exhibited a similar differentiation potency, which was relatively lower compared with that of MENSCs. The present results indicated that MENSCs may be a suitable cell source for the curative treatment of TID.

Amyloid-ß1-42 dynamically regulates the migration of neural stem/progenitor cells via MAPK-ERK pathway.

Neural stem/progenitor cell (NSPC) based therapy represents an attractive treatment for Alzheimer's disease (AD), the most common neurodegenerative disorder with no effective treatment to date. This can be achieved by stimulating endogenous NSPCs and/or administrating exogenously produced NSPCs. Successful repair requires the migration of NSPCs to the loci where neuronal loss occurs, differentiation and integration into neural networks. However, the progressive loss of neurons in the brain of AD patients suggests that the repair by endogenous NSPCs in the setting of AD may be defective. The production and deposition of amyloid-ß1-42 (Aß1-42) peptides is thought to be a central event in the pathogenesis of AD. Here we report that Aß1-42 peptides inhibit the migration of in vitro cultured NSPCs by disturbing the ERK-MAPK signal pathway. We found that the migratory capacity of NSPCs was compromised upon treatment with oligomeric Aß1-42; the inhibitory effect occurred in a dose-dependent manner. Our previous studies have shown that Aß1-42 triggers the expression of GRK2 by unknown mechanism. Herein we found that the Aß1-42 evoked upregulation of GRK2 expression was attenuated upon treatment with the ERK inhibitor SCH772984 at 2.5 µM, but not with inhibitors for p38 or JNK. We detected a dose-dependent increase in levels of phosphorylated ERK1/2 after incubation of cells with oligomeric Aß1-42 peptides for 3 days. We observed that an increase in the phosphorylation of p38 and JNK coincided with reduced phosphorylation of ERK1/2 upon treatment with Aß1-42 for 6 and/or 9 days. We hypothesize that the divergence of the activation of the MAPK family of pathways may contribute to the inhibition of NSPCs migration after the long-term incubation with Aß1-42. Pretreatment with 1  µM MEK inhibitor U0126 reversed the effects of Aß1-42 on GRK2 expression of and NSPC migration. Together, our results suggest that Aß1-42 oligomers compromise the migratory capacity of NSPCs through the MEK-ERK pathway.

Improved osteogenic differentiation of human dental pulp stem cells in a layer-by-layer-modified gelatin scaffold.

Dental pulp stem cell is a new type of mesenchymal stem cell that has a potential for tissue regeneration. Gelatin sponges are often used for hemostasis in dental surgery. In this study, we aimed to evaluate the dental pulp stem cells' proliferation and osteogenic differentiation in different layer-by-layer-modified gelatin sponge scaffolds including the G, G + P (gelatin sponge+ poly-l-lysine modification), G + M (gelatin sponge + mineralization modification), and G + M + P (gelatin sponge + mineralization modification + poly-l-lysine modification) groups in vitro and assessed them in vivo. The results showed that dental pulp stem cells had a great potential for osteogenic differentiation. In vitro, the G + M + P group not only enhanced the adhesion and proliferation of dental pulp stem cells but also facilitated their osteogenic differentiation. However, alkaline phosphatase activity was prohibited after modification. In vivo, both dental pulp stem cells and cells from nude mice grew well on the scaffold, and G + M and G + M + P groups could promote the mineralization deposit formation and the expression of osteocalcin in osteogenic differentiation of dental pulp stem cells. In conclusion, the combination of dental pulp stem cells and G + M + P scaffold has a great potential for bone tissue engineering.

Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture.

Charge heterogeneity has been broadly studied as a critical quality attribute during monoclonal antibody (mAb) production that may subsequently affect product stability and biopotency. However, the charge variation distribution is poorly controlled, so methods of more effective control need to be explored. In this study, the combined effects of temperature shift (37-34, 37-32, or 37-30 °C) and hydrolysate addition (0.100 g/L) to culture feed on the charge heterogeneity of anti-IgE mAb were investigated. The results showed that the distribution of charge variation was significantly regulated by the combination of hydrolysate addition with a highly sub-physiological temperature (34 °C). In addition, under this condition, the main peak content significantly increased, and the acidic peak content significantly decreased. Furthermore, we explored Lys variant content, which is the major basic variant content, as well as its relationship with temperature shift and hydrolysate addition. Lys variant levels were positively related to the Lys and Arg concentrations in the medium and negatively related to carboxypeptidase B and carboxypeptidase H transcript levels. The combination of temperature shift and hydrolysate addition can thus effectively improve anti-IgE mAb charge heterogeneity and significantly increase main variant levels and decrease acidic variant levels.

Different fermentation processes produced variants of an anti-CD52 monoclonal antibody that have divergent in vitro and in vivo characteristics.

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.

Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. METHODS: Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. RESULTS: A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 106 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. DISCUSSION: In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 106 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

In vitro proliferation and osteogenic differentiation of human dental pulp stem cells in injectable thermo-sensitive chitosan/ß-glycerophosphate/hydroxyapatite hydrogel.

Injectable thermo-sensitive hydrogels have a potential application in bone tissue engineering for their sensitivities and minimal invasive properties. Human dental pulp stem cells have been considered a promising tool for tissue reconstruction. The objective of this study was to investigate the proliferation and osteogenic differentiation of dental pulp stem cells in injectable thermo-sensitive chitosan/ß-glycerophosphate/hydroxyapatite hydrogel in vitro. The chitosan /ß-glycerophosphate hydrogel and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel were prepared using the sol-gel method. The injectability of chitosan /ß-glycerophosphate hydrogel and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel was measured using a commercial disposable syringe. Scanning electron microscopy was used to observe the inner structure of hydrogels. Then dental pulp stem cells were seeded in chitosan /ß-glycerophosphate hydrogel and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel, respectively. The growth of dental pulp stem cells was periodically observed under an inverted microscope. The proliferation of dental pulp stem cells was detected by using an Alamar Blue kit, while cell apoptosis was determined by using a Live/Dead Viability/Cytotoxicity kit. The osteogenic differentiations of dental pulp stem cells in chitosan /ß-glycerophosphate hydrogel and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel were evaluated by alkaline phosphatase activity assay and mRNA expression of osteogenesis gene for 21 days in osteogenic medium. The results indicated that there was no significant difference between chitosan /ß-glycerophosphate hydrogel and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel in injectability. Cells within the chitosan/ß-glycerophosphate/hydroxyapatite hydrogel displayed a typical adherent cell morphology and rapid proliferation with high cellular viability after 14 days of culture. Dental pulp stem cells seeded in chitosan/ß-glycerophosphate/hydroxyapatite hydrogels had a higher alkaline phosphatase activity and better up-regulation of gene expression levels of Runx-2, Collagen I, alkaline phosphatase and osteocalcin than in chitosan /ß-glycerophosphate hydrogels after osteogenic differentiation. These results demonstrated that the chitosan/ß-glycerophosphate/hydroxyapatite hydrogel had excellent cellular compatibility and the superiority in promoting dental pulp stem cells osteogenic differentiation in vitro, showing that the combination of dental pulp stem cells and chitosan/ß-glycerophosphate/hydroxyapatite hydrogel has the potential to be used for bone tissue engineering.

Comparative Analysis of Human Mesenchymal Stem Cells from Umbilical Cord, Dental Pulp, and Menstrual Blood as Sources for Cell Therapy.

Although mesenchymal stem cells (MSCs) based therapy has been considered as a promising tool for tissue repair and regeneration, the optimal cell source remains unknown. Umbilical cord (UC), dental pulp (DP), and menstrual blood (MB) are easily accessible sources, which make them attractive candidates for MSCs. The goal of this study was to compare the biological characteristics, including morphology, proliferation, antiapoptosis, multilineage differentiation capacity, and immunophenotype of UC-, DP-, and MB-MSCs in order to provide a theoretical basis for clinical selection and application of these cells. As a result, all UC-, DP-, and MB-MSCs have self-renewal capacity and multipotentiality. However, the UC-MSCs seemed to have higher cell proliferation ability, while DP-MSCs may have significant advantages for osteogenic differentiation, lower cell apoptosis, and senescence. These differences may be associated with the different expression level of cytokines, including vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, and hepatocyte growth factor in each of the MSCs. Comprehensively, our results suggest DP-MSCs may be a desired source for clinical applications of cell therapy.

Salt ions and related parameters affect PEI-DNA particle size and transfection efficiency in Chinese hamster ovary cells.

Transfection efficiency is directly associated with the expression level and quantity of recombinant protein after the transient transfection of animal cells. The transfection process can be influenced by many still-unknown factors, so it is valuable to study the precise mechanism and explore these factors in gene delivery. Polyethylenimine (PEI) is considered to have high transfection efficiency and endosome-disrupting capacity. Here we aimed to investigate optimal conditions for transfection efficiency by setting different parameters, including salt ion concentration, DNA/PEI ratio, and incubation time. We examined the PEI-DNA particle size using a Malvern particle size analyzer and assessed the transfection efficiency using flow cytometry in Chinese hamster ovary-S cells. Salt ions, higher amounts of PEI tended to improve the aggregation of PEI-DNA particles and the particle size of PEI-DNA complexes and the transfection efficiency were increased. Besides, the particle size was also found to benefit from longer incubation time. However, the transfection efficiency increased to maximum of 68.92 % at an incubation time of 10 min, but decreased significantly thereafter to 23.71 %, when incubating for 120 min (P < 0.05). Besides, PEI-DNA complexes formed in salt-free condition were unstable. Our results suggest DNA and PEI incubated in 300 mM NaCl at a ratio of 1:4 for 10 min could achieve the optimal transfection efficiency. Our results might provide guidance for the optimization of transfection efficiency and the industrial production of recombinant proteins.

Study of differential effects of TGF-beta3/BMP2 on chondrogenesis in MSC cells by gene microarray data analysis.

In order to explore the differential effects of TGF-beta3 and BMP2 on chondrogenesis in mesenchymal stem cells (MSCs), the gene expression profiles of MSC treated with TGF-beta3 and BMP2 were subjected to systematic analysis on the gene and functional level. The gene expression profiles of MSCs (downloaded from Gene Expression Omnibus database) in the early and later stages, induced with TGF-beta2 and BMP2, were analyzed using packages within R software and the differentially expressed genes (DEGs) were screened. The DEGs both in the two experimental groups were subjected to Gene Ontology and pathway enrichment analysis. The protein-protein interaction (PPI) networks of the DEGs were constructed using cytoscape software. Among the DEGs, 1,194 genes were up-regulated and 580 genes were down-regulated. The up-regulated genes were mainly enriched in the TGF-beta and cell cycle signaling pathways and down-regulated genes were mainly enriched in the insulin-mediated signal pathway, metabolic pathway of fructose and mannose, and glycolysis/gluconeogenesis pathway. Based on the PPI network analysis, the genes of KIAA0101, NEDD4, and TINF2 were confirmed to be important on chondrogenesis. The analysis of DEGs both in TGF-beta3 and BMP2 treated MSCs indicates that the genes are mainly involved in the cell cycle and intracellular signaling pathways. Also the similar gene expression profile can be achieved by transcription factors or microRNAs (miR-199a-5p and miR-31-5p) based on our prediction, which can provide a new approach for the treatment of cartilage injury.

Multi-level factorial analysis of Ca2+/Pi supplementation as bio-instructive media for in vitro biomimetic engineering of three-dimensional osteogenic hybrids.

We report on the in vitro use of Ca(2+)/P(i) supplementation as a bio-instructive medium to drive human periosteum-derived cells (hPDCs) toward osteogenic differentiation on three-dimensional (3D) porous Ti6Al4V scaffolds. Through a multilevel factorial analysis, we have systematically investigated the biological effect and interactions of Ca(2+) or P(i) supplementation in three selected media preparations (i.e., basic growth medium, osteogenic medium [OM], and osteogenic medium without ß-glycerophosphate [OM(-)]) and have identified specific conditions which induce proliferation and significant osteogenic differentiation of two-dimensional (2D) hPDC cultures. These findings were translated from 2D to 3D cultures conditions to instruct hPDCs to populate porous Ti6Al4V scaffolds and to differentiate into the osteoblast lineage with collagenous matrix production and subsequent matrix mineralization on the 3D structures. These osteogenic hybrids may potentially serve as a clinically relevant customizable bone reparative unit, providing a biomimetic template to more effectively mediate in vivo bone regeneration.

Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures.

DNA measurement and RNA extraction are two frequently used methods for cell characterization. In the conventional protocols, they require similar, but separate samples and in most cases, different pretreatments. The few combined protocols that exist still include time-consuming steps. Hence, to establish an efficient combined RNA extraction and DNA measurement protocol for two-dimensional (2D) and three-dimensional (3D) cell cultures, a PicoGreen-based DNA measurement was integrated in an existing RNA extraction protocol. It was validated by analysis of the influence of different lysis buffers, RLT, RA1, or Trizol, used for RNA extraction on the measured DNA concentration. The DNA cell yield was evaluated both in cell suspensions (2D) and on 3D cell-seeded scaffolds. Results showed that the different RNA lysis buffers caused a concentration-dependent perturbation of the PicoGreen signal. The measured DNA concentrations in 2D and 3D using RLT and RA1 buffer were comparable, also to the positive control. We, therefore, concluded that RNA extraction protocols using RA1 or RLT buffer allow the integration of a DNA quantification step without the buffer influencing the results. Hence, the combined DNA measurement and RNA extraction offer an alternative for DNA measurement techniques that is time and sample saving, for both 2D cell cultures and specific 3D constructs.

Characterization and optimization of cell seeding in scaffolds by factorial design: quality by design approach for skeletal tissue engineering.

Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.

Enhancement of osteogenic gene expression for the differentiation of human periosteal derived cells.

The osteogenic differentiation of progenitor populations allows analysis of cell functionality as well as creating a platform for investigating stem cells for bone tissue engineering. Protocols used for osteogenic differentiation of progenitor cells are often identical to those detailed for bone marrow mesenchymal stem cells, however this may be flawed due to cell populations residing in different niches and being in distinct stages of differentiation. We herein describe the individual and combined effects of known osteo-inductive agents; dexamethasone (Dex), 1,25-dihydroxyvitamin D3 (VitD3), all trans-retinoic acid (atRA), cyclic AMP (cAMP) and bone morphogenic protein 2 (BMP2) in combination with fetal bovine serum (FBS) on osteogenesis of human periosteal derived cells (hPDCs). The addition of Dex&FBS was essential for the transition of hPDCs to an ALP positive cell population. Subsequently, atRA, Dex&FBS and BMP2 were required for the expression of transcription factors governing osteogenesis and hence differentiation towards a mature osteoblast. It is also hypothesized that Dex has no direct effect on the differentiation of hPDCs, instead its effect is to augment differentiation in combination with other factors. These data provide a comprehensive assessment of known osteogenic factors, in a novel multiplex system, to evaluate their effect on progenitor cell differentiation.

Controlled cell-seeding methodologies: a first step toward clinically relevant bone tissue engineering strategies.

The repair of large and complex bone defects could be helped by a cell-based bone tissue engineering strategy. A reliable and consistent cell-seeding methodology is a mandatory step in bringing bone tissue engineering into the clinic. However, optimization of the cell-seeding step is only relevant when it can be reliably evaluated. The cell seeding efficiency (CSE) plays a fundamental role herein. Results showed that cell lysis and the definition used to determine the CSE played a key role in quantifying the CSE. The definition of CSE should therefore be consistent and unambiguous. The study of the influence of five drop-seeding-related parameters within the studied test conditions showed that (i) the cell density and (ii) the seeding vessel did not significantly affect the CSE, whereas (iii) the volume of seeding medium-to-free scaffold volume ratio (MFR), (iv) the seeding time, and (v) the scaffold morphology did. Prolonging the incubation time increased the CSE up to a plateau value at 4 h. Increasing the MFR or permeability by changing the morphology of the scaffolds significantly reduced the CSE. These results confirm that cell seeding optimization is needed and that an evidence-based selection of the seeding conditions is favored.

Effects of sinusoidal magnetic field observed on cell proliferation, ion concentration, and osmolarity in two human cancer cell lines.

Low frequency magnetic fields have previously been shown to affect cell functions. In this article, the effects of 20 mT, 50 Hz sinusoidal magnetic field on cell proliferation, ion concentration, and osmolarity in two human cancer cell lines (HL-60 and SK-Hep-1) were investigated. Inhibition of cell growth was observed. On the other hand, the exposure also increased the Na+, K+ ion concentration and osmolarity in cell supernatant compared to the control group. To our knowledge, this is the first study on cancer cells where magnetic fields affect osmolarity in cell supernatant. In addition, a model of cells exposed to the oscillating magnetic field is described as well as the characteristics of ions in and out of cells. The experimental data appears to be consistent with the theoretical analysis. The results are also discussed in terms of the relationships among cell growth, ion concentration, and osmolarity. Magnetic field inhibitions of cell growth in vitro may relate to changes in cell ion concentration and osmolarity.