MSCs (mesenchymal stem cells) promise a great potential for regenerative medicine due to their unique properties of self-renewal, high plasticity, modulation of immune response and the flexibility for genetic modification. Therefore, the increasing demand for cellular therapy necessitates a larger-scale production of MSC; however, the technical and ethical issues had put a halt on it. To date, studies have shown that MSC could be derived from human UC (umbilical cord), which is once considered as clinical waste. We have compared the two conventional methods which are classic enzymatic digestion and explant method with our newly tailored enzymatic-mechanical disassociation method to generate UC-MSC. The generated UC-MSCs from the methods above were characterized based on their immunophenotyping, early embryonic transcription factors expression and mesodermal differentiation ability. Our results show that enzymatic-mechanical disassociation method increase the initial nucleated cell yield greatly (approximately 160-fold) and maximized the successful rate of UC-MSC generation. Enzymatic-mechanical disassociation-derived UC-MSC exhibited fibroblastic morphology and surface markers expression of CD105, CD73, CD29, CD90 and MHC class I. Furthermore, these cells constitutively express early embryonic transcription factors (Nanog, Oct-4, Sox-2 and Rex-1), as confirmed by RT-PCR, indicating their multipotency and high self-renewal capacity. They are also capable of differentiating into osteoblasts and adipocytes when given an appropriate induction. The present study demonstrates a new and efficient approach in generating MSC from UC, hence serving as ideal alternative source of mesenchymal stem cell for clinical and research use.
The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats. Rats (24-26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation. Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutrophils that could potentially harm multiple biological systems in aged individuals.
BackgroundWe report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma.MethodsBV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison.ResultsBV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 μg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (P < .05). BV2 microglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation.ConclusionsIn summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation.
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