Date of Award

12-2023

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Bournias-Vardiabasis, Nicole

Abstract

Human mesenchymal stem cells (hMSCs) have gained popularity in clinical trials due to their multipotent differentiation characteristics, ability to secrete bioactive molecules, migrate into diseased or damaged tissues, and their immunosuppressive properties. HMSC cultures are heterogeneous, containing stem cells, partially differentiated progenitor cells, and fully differentiated cells. One of the major challenges with hMSCs therapeutic potential is the inability to select specific cell subpopulations due to an insufficient number of biomarkers. Often the biomarkers used, like those for fluorescence-activated cell sorting, are not sufficient to define hMSCs because they overlap with other cell types. Consequently, there is a need to develop alternative biomarkers and sorting technology to reduce hMSCs heterogeneity through cell identification and selection. Recently, electrophysiological properties such as membrane capacitance and cytoplasm conductivity have emerged as biomarkers to identify subsets of stem cells using dielectrophoresis (DEP), a label-free cell analysis technique. DEP uses electric fields to align ions around the surface of cells and induces cell movement. One key feature of the cell surface is the glycocalyx, a biointerface composed of glycolipids and glycoproteins surrounding the membrane of cells. This work focuses on modifying the cell-specific glycosylation patterns that make up the glycocalyx using treatments of either Kifunensine or N-Acetylglucosamine to further develop membrane capacitance and cytoplasm conductivity as contenders for label-free biomarkers. Measurements collected by modifications made to the glycocalyx using DEP to investigate the contributions of the glycocalyx to membrane capacitance and cytoplasm conductivity. Adipose tissue, (AT) was assessed throughout this experiment to determine if the changes undergone by treatment and differentiation can be accessed. Transient slope, a new parameter from DEP measurements, is evaluated as a third potential label-free biomarker. Modified and unmodified hMSCs will be differentiated into adipocytes and osteocytes, and RT-qPCR analysis of differentiation-related genes was performed to determine if the glycocalyx impacts cell fate. The results demonstrate that DEP can be used as a good engineering tool that provides label-free biomarkers, membrane capacitance, cytoplasm conductivity, and transient slope, as identifiers of hMSCs subpopulations.

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