Dielectrophoretic discrimination of pluripotent myoblast with Raman spectroscopic analysis of the cell plasma membrane for application in Huntington's disease

Abstract

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre -clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. This thesis demonstrates that the biomarker -free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast pluripotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co- cultured with GFP- expressing fibroblasts of comparable size distributions (mean diameter -10 gm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.These high levels of discrimination raised more questions about the cell plasma membrane characteristics that may be responsible for the dielectrophoretic response. This prompted to extend the work to a specific neurodegenerative disease, Huntington's disease. Several studies have been revealing the association between plasma membrane dysregulation and Huntington's disease. In particular the feasibility to use peripheral fibroblasts cells from donors affected by the disease, as a forecasting model marker for Huntington. Although there are substantial evidences about the indirect effect of the disease on the plasma membrane, a non -invasive technique that can discriminate and characterise a cell sample is not available. Raman spectroscopy with associated statistical multivariate analysis was used to characterise sub -cellular differences in extracted plasma membranes from peripheral fibroblastic cells in order to elucidate the differences between cells affect and non - affected by the disease. The results clearly showed that indeed the plasma membrane carries differences that can be attributed to the presence of the disease making the plasma membrane an amenable and novel biomarker for Huntington's diseas