Deciphering the anti-tumour activity of human mesenchymal stem cells on haematopoietic tumour cell lines

Cancer becomes a progressive disease where it necessitates the emergence of new treatment modalities such as immune and stem cell therapies. The recent development in mesenchymal stem cell research promises a new anti-cancer therapy via a possible mode of cell cycle arrest, apoptosis and immunosuppressive activity. The present study aims to investigate the inhibitory activity of UC-MSCs on tumour cells’ (HL-60, K562, Jurkat and BV173 cell lines) proliferation at in vitro by elucidating the signalling mechanisms and the relevant gene expression profiles. The effect of UC-MSCs on tumour cells’ activities namely proliferation, apoptosis, cell cycle arrest, gene expression, biological process and signalling pathways were further evaluated. In the presence of UC-MSCs, tumour cell proliferation was profoundly inhibited in a dose-dependent manner as measured by 3H-thymidine uptake and MTS Assay. The dramatic reduction of proliferation was not due to induction of apoptosis yet, a substantial number of tumour cells were arrested in G0/G1 and S phase of the cell cycle of tumour cells. Subsequent microarray analysis of whole genome using BV173 cell line showed that approximately 2000 and 1019 genes were up and down-regulated respectively in the presence of UC-MSCs. Ontology analysis revealed that the various biological processes such as cell adhesion, vasculature development, regulation of cell proliferation and regulation of cell migration were mainly affected by by dysregulated genes. In addition, the microarray analysis showed the most significant signalling pathways such as focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, MAPK signalling pathway, p53 signalling pathway and TGF-beta signalling pathway were dysregulated by UC-MSCs in BV173. The present study indicated that UC-MSCs inhibits tumour cells proliferation by arresting in a G0/G1 phase of cell cycle, and this effect is mediated by cell-to-cell contact. Further gene profiling and signalling pathways revealed that anti-proliferative activity of UC-MSCs on tumour cell proliferation is associated with genes which are involved in cell cycle (CDK6, CCNE2 and CDKN1A), cell adhesion (TGFβI, MYO7A and CD82), metastasis (CCL2, TFPI2 and CXCR3), angiogenesis (PDGF, CXCL1 and PTX3) and regulation of cell proliferation (HECW1, SCIMP and ANXA1). The generalized tumour cells inhibition by UC-MSCs could be potentially exploited to treat various tumours. However, this anti-proliferative activity needs to be tested in in-vivo model and at protein level for their better understandings and validation

Immunomodulatory potential of mesenchymal stem cells on microglia

It is becoming increasingly evident that inflammatory reactions of microglia contribute to the pathology of neurodegenerative diseases. Although the focus for rescuing neurones previously lied on minimising direct insult (including limiting aggregation of misfolded proteins and antagonising the effects of glutamate), therapeutic approaches now include moderating the ensuing inflammatory responses of microglia. Microglia responses in the central nervous system (CNS) are diverse and their involvement in both neuroprotection and neurotoxicity may seem paradoxical. Accordingly, management of neuroinflammation must include an understanding of conditions that trigger neurotoxic responses by microglia and deciphering strategies to maintain their neuroprotective phenotype. Mesenchymal stem cells (MSC) are stem cells with great capacity for immunomodulation on a wide range of immune cells. Evidence presented here highlights the potential of using MSC to modulate the inflammatory responses of microglia. The mechanisms underlying the ability of MSC to moderate microglia responses are also explained in this review. Although many aspects of this approach will require defined characterisation, MSC serve as a potential cell-based therapy that may slow or halt the progression of inflammatory CNS diseases

Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer

Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over other types of viruses due to their capacity to transduce non-dividing cells. An optimized generation of lentiviruses carrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) or CMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by co-transfecting lentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines. Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine (PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvested at days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFP harvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally, the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transient and stable GFP expression. It was found that the GFP expression declined overtime. However, the transduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentiviruses carrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells at different efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to the subsequent analysis of the effect of regulatory elements in future study

The crosstalk between mesenchymal stem cells and damaged cartilage in osteoarthritis

Human cartilage contains multipotent stem cells, namely mesenchymal stem cells (MSCs) which are progenitors of connective tissue that play homeostatic and reparative roles. Although the major constituent cells in the cartilage are chondrocytes, they possess a limited regenerative ability, and as a result, spontaneous cartilage repair by chondrocytes leads to the synthesis of fibrocartilage. Similarly, MSCs derived from articular cartilage of osteoarthritis patients have demonstrated inadequacy in cartilage repair. The role of MSCs in the pathophysiology of osteoarthritis (OA) is not entirely understood, whether the inflammatory milieu associated with OA joints affects the reparative properties of MSCs or the inherent defects of OA cartilage-derived MSCs impair the proper execution of the required immunosuppressive and reparative functions. Therefore, the current review explores the biological characteristics and features of MSCs derived from physiological state and OA condition with the aim of identifying how OA affects MSC functions as well as the role of MSCs in the pathophysiology of OA

Basic fibroblast growth factor enhances the expansion and secretory profile of human placenta-derived mesenchymal stem cells

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

Human mesenchymal stem cells protect neutrophil from serum deprived cell death.

We have previously shown that human MSC (mesenchymal stem cells) inhibit the proliferation of most of the immune cells. However, there are innate immune cells such as neutrophils and other PMN (polymorphonuclear) cells that do not require an extensive proliferation prior to their effector function. In this study, the effect of MSC on neutrophils in the presence of complete and serum-deprived culture media was investigated. In the presence of MSC, the viability of neutrophils increase as measured in 24 h of incubation at various supplementation of serum concentration. We have utilized Annexin V and PI (propidium iodide) staining to confirm whether the enhancement of neutrophil's viability is due to a reduction in PCD (programmed cell death). MSC significantly rescue neutrophils from apoptosis at 1, 5 and 10% of FBS (fetal bovine serum) supplementation. The fractions of viable and dead cells were increased and decreased respectively in the presence of MSC. Our results indicate MSC rescue neutrophils from nutrient- or serum-deprived cell death. However, whether this effect is exerted through a specific signalling pathway or confining neutrophils in resting state by MSC requires further investigation

Growth, photoluminescence, lifetime, and laser damage threshold studies of 1, 3, 5-triphenylbenzene (TPB) single crystal for scintillation application

The 1, 3, 5-triphenylbenzene (TPB) single crystal has been grown using slow cooling seed rotation technique. Optical transmittance of the grown crystal was obtained from UV-Visible analysis. The grown TPB crystal has good transmission in the entire visible region with a lower cutoff wavelength of 330 nm. The solubility of TPB material was determined using toluene as a solvent with different temperatures. The full width at half maximum is 18 arcsec, which indicates that the crystal is of good quality. The TPB crystal was excited (lambda (exc)) at 307 nm, and the corresponding emission (lambda (em)) has been observed at 352 nm. The laser-induced damage threshold (LDT) value of grown crystal is 1.25 GW/cm(2). Third-order nonlinear optical susceptibility chi (3) is determined using the Z-scan technique as 3.07422x10(-09) esu. The TPB crystal proves its suitability for scintillation applications and optoelectronic device fabrications

The bahasa Melayu version of cornell musculoskeletal discomfort questionnaire (CMDQ): reliability and validity study in Malaysia

BACKGROUND: The Cornell Musculoskeletal Discomfort Questionnaire (CMDQ) was developed to assess the level of musculoskeletal discomfort among office workers related to their ergonomic situation. OBJECTIVE: The primary objective of this initial study is to analyze the validity and dependability of the Malay translation of the Cornell Musculoskeletal Discomfort Questionnaire. METHODS: The questionnaire was self-administered two times, with an interval of two weeks in order to evaluate the accuracy of the original findings with a retest. The study involved 115 participants. RESULTS: The range of Cronbach Alpha coefficient showed a considerable consistency of the items for each sub-scale (Cronbach’s a > 0.95). The range of Kappa coefficients was between (ICC = 0.690–0.949, p < 0.001), (ICC = 0.801–0.979, p < 0.001) and (ICC = 0.778–0.944, p < 0.001) for frequency, severity and interference scales. CONCLUSIONS: This research, introduced the Malay-language version of the CMDQ (CMDQ-M) as the first formal validation of the CMDQ, and confirmed a high reliability and validity for the evaluation of musculoskeletal discomfort among the study population

Qualitative flow cytometric analysis of Malaysian myelodysplastic syndromes (MDS) patients.

Myelodysplastic syndromes (MDS) are a group of haematological malignancies categorized by ineffective hematopoiesis that result in dysplasia. Although morphological diagnosis is a traditional and standard technique that is used for the diagnosis of MDS, the heterogeneous blood and bone marrow characteristics of MDS patients can potentially obscure the right diagnosis. Thus, we have utilized flow cytometric immunophenotyping as a supportive mechanism to obtain a more accurate and faster method for detection of abnormal markers in MDS. Flow cytometry was used for analyzing bone marrow samples from newly diagnosed MDS patients to investigate the abnormal antigen expression patterns in granulocytic, monocytic, erythroid, lymphoid lineages and myeloid precursors. The results were compared with those obtained from cases that had Idiopathic Thrombocytopenic Purpura (ITP) as a control. The most common abnormality found in the granulocytic lineage was the decrease of CD10. Low expressions of CD13 were the most frequent abnormality in the monocytic lineage. The erythroid lineage was found to have low expression of CD235A+/CD71+, reduce of CD71 and decreased CD235a. In conclusion, this method is useful for confirming cases in which it is difficult to make a diagnosis by morphology

Establishing a culture system that supports in vitro expansion of adult 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 × 10 6 cells) compared to the technique of replating cells (0.91 ± 0.65 × 10 6 cells; p<O.O5). 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