Synthesis and Characterization of Giant Plasma Membrane Vesicles and its Effect on Human Mesenchymal Stem Cells

Abstract

Differentiation of mesenchymal stem cells to osteogenic lineage is a crucial step of in vivo bone remodelling and bone regeneration. In bone tissue engineering, numerous effort has been made for directed osteogenic differentiation of mesenchymal stem cells. A critical analysis of the literature revealed that in vivo, osteoblast itself induce the differentiation of mesenchymal stem cells towards osteogenic lineage. Keeping that perspective in mind here we hypothesized that giant plasma membrane vesicles (GPMV) derived from osteoblast can be as a ‘packaged cues’ for osteogenic differentiation of mesenchymal stem cells.

Here we have reported the synthesis of giant plasma membrane vesicles (GPMVs) from the osteoblast MG-63 using a combination of paraformaldehyde (PFA) / dithiotheritol (DTT). To analyse the effect of different cellular constituents on the physico-chemical properties of the GPMV, osteoblast cells were treated with an actin blocker (cytochalacin-D) and a cholesterol sequester (methyl-β-cyclo dextrin) prior to GPMV generation. Size, shape and structural complexicty of the GPMV was characterized by image based analysis (phase contrast microscopy, fluorescence microscopy and electron microscopy) and by flow cytometry. The lipid extracted from the GPMVs was analysed by biochemical assay and NMR spectroscopy. Further, we fused the GPMV with the hMSCs and the fusion efficiency was analysed by confocal microscopy and flow cytometry. More than 15% of Fusion efficiency was observed. Proliferation of hMSC after fusion was analysed by MTT assay, PI based live/dead assay and confocal microscopy. The osteogenic differentiation was investigated by checking the activity of alkaline phosphatase, extent of biomineralization and collagen synthesis. The RT-PCR based analysis of the expression of osteogenic markers revealed that hMSCs cultured in the presence of GPMVs shows increased expression of early and late osteogenic markers.