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

Immunosuppresive activity of human umbilical cord and placenta derived mesenchymal stem cells on lymphocyte proliferation

Current research in mesenchymal stem cells (MSC) concur its potential to be used in therapies to treat various inflammatory diseases and degenerative disorders. In the present study, human umbilical cord (UC) and placenta (PLC) derived MSC were generated and their immunosuppressive activity was assessed using human adaptive and innate lymphocytes. CD3/CD28 micro-beads activated T cells, pokeweed stimulated B cells and NK-92MI cell lines were cultured in the presence or absence of UC-MS and PLC-MSC. The proliferation and cell cycle status of responder cells was measured by tritiated thymidine assay and flow cytometer analysis respectively. Both, UC-MSC and PLC-MSC significantly exerted a significant dose dependent inhibition on lymphocytes proliferation. Further cell cycle analysis showed that T cells were arrested at G0 phase and NK-92MI cells were halted at G1 by preventing them transit from G1→ S phase (p<0.05). Transwell assay revealed that the immunosuppressive activity of MSC was mediated by a direct cell-to-cell contact than soluble factors (p<0.05). Although both UC and PLC derived MSC exerted a profound anti-proliferative activity on lymphocytes yet UC-MSC express the higher magnitude of immunosuppression in all tested assays

Evaluation of metabolic and immunological changes in streptozotocin-nicotinamide induced diabetic rats

Type 2 diabetes is a chronic disease with growing public health concern globally. Finding remedies to assist this health issue requires recruiting appropriate animal model for experimental studies. This study was designated to evaluate metabolic and immunologic changes in streptozotocin-nicotinamide induced diabetic rats as a model of type 2 diabetes. Male rats were induced diabetes using nicotinamide (110 mg/kg) and streptozotocin (65 mg/kg). Following 42 days, biochemical and immunological tests showed that diabetic rats had higher levels of blood glucose, WBC, certain abnormalities in lipid profile and insufficient mitogenic responses of lymphocytes (p < 0.05). However, the status of the total antioxidant, inflammatory biomarkers and other parameters of full blood count (except HCT) were not significantly altered. Phenotyping assay indicated insignificant lymphocyte subtype imbalances excluding a significant rise in the level of CD4+CD25+ marker (p < 0.05). This model of diabetic animals may represent some but not all symptoms of human type 2 diabetes

Human mesenchymal stromal cells modulate T-cell immune response via transcriptomic regulation

Background aims: Mesenchymal stromal cells (MSCs) have been identified as pan-immunosuppressant in various in vitro and in vivo inflammatory models. Although the immunosuppressive activity of MSCs has been explored in various contexts, the precise molecular signaling pathways that govern inhibitory functions remain poorly elucidated. Methods: By using a microarray-based global gene expression profiling system, this study aimed to decipher the underlying molecular pathways that may mediate the immunosuppressive activity of umbilical cord–derived MSCs (UC-MSCs) on activated T cells. Results: In the presence of UC-MSCs, the proliferation of activated T cells was suppressed in a dose-depended manner by cell-to-cell contact mode via an active cell-cycle arrest at the G0/G1 phase of the cell cycle. The microarray analysis revealed that particularly, IFNG, CXCL9, IL2, IL2RA and CCND3 genes were down-regulated, whereas IL11, VSIG4, GFA1, TIMP3 and BBC3 genes were up-regulated by UC-MSCs. The dysregulated gene clusters associated with immune-response-related ontologies, namely, lymphocyte proliferation or activation, apoptosis and cell cycle, were further analyzed. Conclusions: Among the nine canonical pathways identified, three pathways (namely T-helper cell differentiation, cyclins and cell cycle regulation, and gap/tight junction signalling pathways) were highly enriched with these dysregulated genes. The pathways represent putative molecular pathways through which UC-MSCs elicit immunosuppressive activity toward activated T cells. This study provides a global snapshot of gene networks and pathways that contribute to the ability of UC-MSCs to suppress activated T cells

Metabolic and immunologic alterations of ginger rhizome among streptozotocin-nicotinamide induced diabetic rats

Introduction: This study was conducted to determine immunological and metabolic effects of different concentrations of ginger rhizome (Zingiber officinale Roscoe) in streptozotocin (STZ)-nicotinamide (NA) induced diabetic rats. Methods: Forty-eight fasted male Sprague-Dawley rats were induced diabetes using a single intraperitoneal injection of NA(110 mg/kg b.w.) and STZ (65 mg/kg b.w, 15 min after NA). Diabetic rats orally received either different concentrations (250, 500 and 750 mg/kg body weight) of ginger rhizome suspension or glibenclamide (10 mg/kg body weight) for 6 weeks. Two control diabetic and normal groups were gavaged with only distilled water as a vehicle. Results: The results indicated that the lower concentrations of ginger modulated body weight, fasting blood glucose, level of triglyceride and tumor necrosis factor-α (TNF-α) (p0.05). Conclusion: Ginger indicated better impact on metabolic and immunologic parameters in lower doses of supplementation compared with high doses of treatment

Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells

Overcoming the challenge of transduction of human T-cells with chimeric antigen receptor (CAR) specific for ERBB2 antigen

Breast cancer is one of the most common malignancies among woman. Decades of scientific study have linked the overexpression of ERBB2 antigen to aggressive tumors. To target aggressive breast cancer, chimeric antigen receptor (CAR) technology can be utilized. For this, human T-cells are transduced with a gene sequence encoding a CAR that is specific for tumor-associated antigens (TAAs). These genetically-engineered CAR transduced T-cells (CAR-T cells) are able to target the tumor antigen without the need for major histocompatibility complex (MHC) recognition, rendering it a potentially universal immunotherapeutic option. However, efficient transduction of therapeutic gene into human T-cells and further cell expansion are challenging. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The constructed CAR gene was inserted into a lentiviral plasmid containing the green fluorescent protein (GFP) tag and lentiviral particles were produced. These lentiviral particles were used to transduce activated T-cells by spinoculation. T-cells were activated using Dynabead-conjugated CD3/CD28 human T-cell activator and interleukin-2 (IL-2) before transduction. CD3+ T-cells were selected and GFP expression, which indicated transduction, was observed. Future studies will focus on in vitro and in vivo models to determine the efficiency of CAR-T cells in specifically targeting ERBB2-expressing cells

Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition

Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7–10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications