THE EFFECT OF MICROFIBER MORPHOLOHY AND CELL DENSITY ON THE LOCALIZATION OF YAP PROTEIN IN MESENCHYMAL STEM CELLS

Open Access
Author:
Steltz, Austin E
Area of Honors:
Biomedical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Justin Lee Brown, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Yong Wang, Faculty Reader
Keywords:
  • mesenchymal stem cell
  • msc
  • YAP
  • Yes Associated Protein
  • Mechanotransduction
  • Transduce
  • biology
  • biomedical engineering
  • electrospinning
  • fibers
  • flat
Abstract:
Mesenchymal stem cells (MSCs), a non-hematopoietic adult derived stem cell found in the bone marrow of mammals, are capable of differentiating into many cell types of the mesenchyme, including osteoblasts, chondrocytes, adipocytes, and myoblasts. The ability to easily harvest and grow these stem cells has caused MSCs to become a large focus of research in regenerative medicine. Past studies have shown that combinations of chemical and mechanical stimulus, including specific growth factors and extracellular fiber morphology, can be used to influence the differentiation of MSCs into specific cell types. However, the underlying signaling pathways controlling how MSCs respond to different extracellular signals are not as well understood. Recently, Yes-associated protein (YAP), a well-known regulator and coactivator in the Hippo Signaling Pathway, has been shown to function as a nuclear transducer of mechanical signals. YAP is now known to transduce many mechanical signals, including ECM rigidity, cell density, and tensional forces, into cell responses. In this study, the effect of microfiber morphology and cell confluence on the localization and concentration of YAP protein in mouse mesenchymal stem cells (mMSCs) was investigated. mMSCs were grown on both flat PMMA surfaces and PHEMA surfaces coated in PMMA microfibers at different cell densities. The cells were allowed to incubate for 24 hours, then were examined using immunofluorescence and DIC microscopy, as well as Western Blot to quantify YAP expression and localization. In a follow up experiment, the effect of different fiber diameters as well as cell confluency on YAP expression and localization was examined. In addition to immunofluorescence and DIC microscopy, samples were separated into cytoplasmic and nuclear components. Western blotting was then used to quantify changes in nuclear and cytoplasmic YAP expression as a function of fiber diameter. Results suggested that cell-cell contact and cell microenvironment have an effect on mMSC YAP localization. Cell microenvironment was also shown to affect the overall YAP concentration in mMSCs.