Introduction: Neurodegeneration resulting from pathogen invasion or tissue damage has been associated with activation of microglia, and exacerbated by the release of neurotoxic mediators such as pro-inflammatory cytokines, chemokines and reactive oxygen species. Activation of microglia stimulated by lipopolysaccharide is mediated in part by GSK-3 signaling molecule. Induced IL-10 expression via GSK-3 inhibition is noteworthy since IL-10 has been remarkably shown to suppress inflammation. Objectives: We aimed to inactivate microglia through inhibition of GSK-3 signaling and to determine its effects on the production of pro- and anti-inflammatory mediators. Methods: LPS-stimulated BV-2 cells were treated with a GSK-3 inhibitor (LiCl, NP12, SB216763 or CHIR99021). Inhibition of GSK-3 was determined by the phosphorylation status of GSK-3β. The effects of GSK-3 inhibition on microglial inflammatory response were investigated by examining various mediators and CD200R marker. Production of nitric oxide (NO), glutamate and pro- and anti-inflammatory cytokines were measured using flow cytometry, Griess assay, glutamate assay and Cytometric Bead Array (CBA) respectively. Results: GSK-3β signaling in LPS-stimulated microglia was blocked by GSK-3 inhibitor through increased phosphorylation at Serine 9 residue. GSK-3 inhibitors had also led to reducing in microglia activity via increased expression of CD200R. Inhibition of GSK-3 also diminished inflammatory mediators such as nitric oxide (NO), glutamate, pro-inflammatory cytokines (TNF-α and IL-6) and chemokine, MCP-1. Reduction of pro-inflammatory mediators by GSK-3 inhibitor was coincided with increased IL-10 production. Conclusions: Suppression of microglia-mediated inflammatory response was facilitated by GSK-3 inhibition with associated increased in IL-10 production.
Microglia

Introduction: The vast majority of in vitro research on microglia are based on cells isolated from neonatal animals (3-5 days of age). Studying microglia of adults has been limited by the lack of a suitable culture system that supports their growth. In this study, we describe a protocol for growing microglia of adults based on modifications of the technique for culturing microglia isolated from neonatal rats. Methods: Mixed glia isolated from adult rats (age range of 1 month to 3 years old) were seeded in culture flasks coated with poly-L-lysine. Cells were maintained in DMEM media supplemented with insulin-transferrin-selenium (ITS) and recombinant human macrophage colony-stimulating factor (M-CSF). Mild trypsinisation was carried out to isolate microglia from mixed glia culture. Results: Microglia cells of adult rats were successfully grown in vitro. For the expansion of adult microglia, it was observed that coating the cell culture flasks with poly-L-lysine was crucial to encourage cell adherence. The substitution of insulin in culture media with ITS was found to improve cell yield and reduced the number of days required for culture from 28 days to 14 days. Addition of M-CSF to cell culture medium, along with the improvisations described above provided the best adult microglia cell yield (2.91 ± 0.56 x 10⁶ cells) compared to the technique of replating cells (0.91 ± 0.65 x 10⁶ cells; p<0.05). Conclusion: Optimisation of primary cell culture technique by coating culture flasks with poly-L-lysine,supplementation of culture medium with ITS and M-CSF allowed microglia of adult rats to be successfully cultured in vitro

Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 µg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/ well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0µg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.
Nitric Oxide

Introduction:Mesenchymal stem cells (MSCs) hold a great therapeutic potential for regenerative medicine and tissue engineering due to inherent immunomodulatory and reparative properties. Hence, it necessitates a readily available supplyof MSCs to meet the clinical demands adequately. Although, a human placenta can produce MSCs, the in vitro culture-mediated cellular senescence often affect the quality of cell product. Thus, the current study has explored the feasibility of basic fibroblast growth factor (bFGF) to enhance the growth of placenta-derived MSCs (PLC-MSCs). Methods:The basic fibroblast growth factor (bFGF) was supplemented to optimise the growth of MSCs. The effects of bFGF on morphology, growth kinetics and cytokine secretion of PLC-MSCs were assessed. Results: The bFGF supplementation increased the proliferation of PLC-MSCs in a dose-dependent manner and 40 ng/ml showed a high trophism effect on PLC-MSC’s growth. In the presence of bFGF, PLC-MSCs acquired a small and well-defined morphology that reflect an active proliferative status. BFGF has induced PLC-MSCs to achieve a shorter doubling time (45 hrs) as compared to the non-supplemented PLC-MSCs culture (81 hrs). Furthermore, bFGF impelled PLC-MSCs into cell cycle machinery where a substantial fraction of cells was driven to S and G2/M phases. Amongst, 36 screened cytokines, bFGF had only altered the secretion of IL-8, IL-6, TNFR1, MMP3 and VEGF. Conclusion:The present study showed that bFGF supplementation promotes the growth of PLC-MSCs without significantly deviating from the standard criteria of MSCs. Thus, bFGF could be considered as a potential mitogen to facilitate the large-scale production of PLC-MSCs.
Mesenchymal Stromal Cells

Introduction: During the last three decades hematopoietic stem cell transplantation (HSCT) has become a well-established treatment for many hematologic malignancies. The most important limitation for HSC transplantation is the low number of hematopoietic stem cells (HSC) that can lead to delayed engraftment or graft failures. Numerous attempts have been made to improve in vitro HSC expansion via optimization of various methods such as isolation techniques, supplementing with growth factors, utilizing stromal cells as feeder layer and other culture conditions. Objective: This project is aimed to decipher the efficiency of an isolation technique and retrieval of culture expanded HSC from feeder layer using two different harvesting methods. Materials and Methods: Hematopoietic stem cells from human umbilical cord blood were isolated via MACS mediated CD34+ double sorting. Then, the cells were cultured onto MSC feeder layer for 3 and 5 days. Culture expanded cells were harvested using two different harvesting method namely cell aspiration and trypsinization methods. Hematopoietic stem cell expansion index were calculated based on harvesting methods for each time point. Results: The numbers of HSC isolated from human umbilical cord blood were 1.64 x 106 and 1.20 x106 cells at single and double sortings respectively. Although the number of sorted cells diminished at the second sorting yet the yield of CD34+ purity has increased from 43.73% at single sorting to 81.40% at double sorting. Employing the trypsinization method, the HSC harvested from feeder layer showed a significant increase in expansion index (EI) as compared to the cell aspiration harvesting method (p≤ 0.05). However, the purity of CD34+ HSC was found higher when the cells were harvested using aspiration method (82.43%) as compared to the trypsinization method (74.13%). Conclusion: A pure population of CD34+ HSC can be retrieved when the cells were double sorted using MACS and expanded in culture after being harvested using cell aspiration method.
Hematopoietic Stem Cells

Introduction: The potential immunoregulatory effects of tocotrienols, the less studied form of vitamin E, had not been determined for microglia until our last publication showcased primary evidence of palm tocotrienols limiting microglia activation, explicitly by inhibiting nitric oxide (NO) production. Here we further explored the nitrite scavenging activity of the two most potent NO-reducing tocotrienol isoforms - δ- tocotrienol and Tocomin50% (contains a spectrum of tocotrienols and α-tocopherol) based on their inhibitory effects on NO production and also their effects on CD40 (a microglial co-stimulator molecule) expression of BV2 microglia. Methods: BV2 cells were treated with two different doses of tocotrienols (δ-tocotrienol: 3.96 μg/mL and 19.80 μg/mL; Tocomin50%: 47.50 μg/mL and 237.50 μg/mL) followed by stimulation with 1 μg/mL of lipopolysaccharide (LPS). A chemical scavenging assay was conducted to study the nitrite scavenging activity of δ- tocotrienol. Together with Tocomin50%, we also determined their effects on CD40 expression of BV2 microglia via flow cytometry. Results: We demonstrate that the inhibitory effect of tocotrienols on NO production by microglia is not attributed to their nitrite scavenging activity. Additionally, tocotrienols also reduced the expression of the microglial co-stimulator molecule, CD40. Conclusions: Our data aids the further characterisation of the actions of tocotrienols on microglia, offering insight into the potential modulatory properties of palm tocotrienols on microglial inflammatory responses within the central nervous system (CNS).

Introduction: Group B Streptococcus (GBS), infection and recurrence in newborns and pregnant women can lead to chronic medical illness resulting in significant morbidity, and mortality. Pathogenesis of GBS may be due to reasons such as activation of the immune system, followed by the production of inflammatory markers and toxic components by immune cells including macrophages. Methods: The studies on invasive and colonizing GBS strains inoculated either with peripheral or brain macrophages, the expression of nitric oxide (NO), cell viability, and CD40 were also measured by Griess assay, methyl tetrazolium assay (MTT), and flow cytometry, respectively. Furthermore, the clinical manifestations of the selected patients were also assessed for this study. Results: Outcome of inflammatory markers studies, after GBS inoculation indicated that, invasive GBS strains induced higher inflammatory markers in comparison to colonizing GBS strains. Furthermore, patients’ clinical data showed that patients with invasive GBS infections had severe condition unlike among patients with colonizing GBS strains. The fatality rate in patients with invasive GBS strain were 30.8% while there was no death among carriers. Conclusion: This study, aimed to understand the immune response to GBS, and strengthen the knowledge on GBS pathogenesis. It was concluded that invasive GBS strains not only showed higher expression of inflammatory markers on immune cells but also had higher pathogenesis effect in comparison to colonizing GBS strains.
Streptococcus agalactiae ; Pregnancy

Microglia-induced neurotoxicity occurs when inflammation mediated by microglia causes loss of neuronal structures or functions in the central nervous system implicated in stroke, spinal cord injury, sepsis, neurodegenerative diseases and even psychiatric illnesses. Various co-culture in vitro microglia-induced neurotoxicity (MINT) models have been established to enable an in-depth study of this process and yet there is a dearth of information regarding usages, advantages and limitations of each of the components of this model. In this review, we examined 56 MINTs for the cells, stimuli, parameters, methods of neurotoxicity measurement and formats of co-culture used in their construction. We aim to provide foundational information, overall guideline and framework for the novice researcher to develop his/her own model and for the advancement of improved, novel and more representative MINT models.

Introduction: Monocytes are essential phagocytic cells of the innate immune system as they are required for the maintenance of tissue homeostasis. However, accumulation of monocytes is implicated in various chronic inflammatory diseases like coronary heart disease, atherosclerosis and in autoimmune disorders. Therefore, the number of monocytes must be carefully regulated to avoid monocyte induced inflammatory disorders. Mesenchymal stem cells (MSCs) have shown to be effective against various inflammatory diseases due to their immunosuppressive properties. The present study was designed to evaluate the less understood immunomodulatory effect of MSCs on monocyte proliferation and survival. Method: Primary monocytes were isolated from peripheral human blood using CD14+ monocyte isolation kit. The in house produced umbilical cord MSCs were co-cultured with monocytes at different ratio and time; assessed for the monocyte viability, proliferation and cell cycle. Results: Mesenchymal stem cells suppressed monocyte proliferation in a dose-dependent manner. The antiproliferative effect of MSCs was mediated by cell cycle arrest, whereby monocytes were arrested in the G0/G1 phase of the cell cycle by preventing them from progress into S and G2/M phases. Although cell cycle arrest could potentially lead to apoptosis; however, MSCs significantly enhanced the monocytes survival and inhibited apoptosis. Conclusion: Human MSCs inhibit the stimulated monocyte proliferation without inducing cellular apoptosis at in vitro. These results reveal that MSCs can be utilised to control monocytes’ quantity during an unwanted immune response to maintain homeostasis.

Introduction: Preclinical studies on mesenchymal stromal cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. In recent years, conflicting data have been reported regarding various aspects of their characteristics, development and differentiation potential, which may be due to arrange of factors. Among the factors worth investigating is the culture medium formulation. Methods: Here we have made a comparative characterization of mouse bone marrow mesenchymal stromal cells (mBM-MSC) that were cultured using two common supplements, MesenCult™ Stimulatory Supplement (MSS) and fetal bovine serum (FBS), under the same experimental conditions at different passages. Clonogenic potential, cumulative population doubling level (CPDL), population doubling time (PDT), immunophenotyping, differentiation, immunosuppression potentials and chromosome analysis of early and late passages mBM-MSC were assessed. Results: Our findings showed that the CPDL, immunophenotype and immunosuppression potential of mBM-MSC were similar. However, variations were seen in their clonogenicity, population doubling time and differentiation efficacy whereby all of these were enhanced in DMSS. These observations suggest that their genetic make-up may be affected by both supplements upon prolonged culture. Interestingly, this conjecture was supported when chromosomal analysis revealed genetic instability of mBM-MSCs cultured in both supplements. Conclusion: In conclusion, culture medium formulation was shown to cause variations and spontaneous transformation in mBM-MSCs raising concerns on the usage of late passages mBMMSCs in fundamental and preclinical downstream experiments.