Mc Neill, Eoin (2019-12). Extracellular Matrix Secreted by Osteogenically Enhanced Mesenchymal Stem Cells Recapitulates the Osteogenic Microenvironment. Doctoral Dissertation. Thesis uri icon

abstract

  • Extracellular matrix (ECM) is a primary component of the osteogenic microenvironment, providing support for attachment, proliferation, and differentiation to facilitate bone growth and maintenance. Previously we have shown that ECM secreted by osteogenically enhanced mesenchymal stem cells (OEhMSCs) resembles an anabolic bone microenvironment and promotes bone healing by OEhMSCs in vivo. OEhMSCs are usually obtained from bone marrow, a finite source, and are subject to reduced efficacy with extensive expansion in culture. This limits their widespread clinical use. This issue can be overcome by using induced pluripotent stem cells (iPSCs) as a source of MSCs (ihMSCs). The ihMSCs are highly osteogenic and can be osteogenically enhanced (OEihMSCs) by upregulating canonical Wnt signaling. OEihMSCs secrete a dense ECM rich in collagens VI and XII. When implanted into a murine calvarial defect this ECM promotes significant levels of bone healing, with 4-6 times more bone generated than bone morphogenetic protein 2 (BMP-2). Furthermore, unlike ECM from OEhMSCs, OEihMSC-derived ECM possesses intrinsic osteogenic activity, not requiring co-administration with exogenous osteoprogenitors to promote bone healing. Malignant bone disease (MBD) is characterized by the formation of osteolytic lesions, which promote tumor relapse and resistance to chemotherapy. Standard tissue culture techniques to study MBD do not accurately represent the bone microenvironment and the complex bone-tumor interactions therein. These interactions can be studied more accurately by co-culturing osteoprogenitors with osteosarcoma (OS) cells on OEhMSC-derived ECM-coated microcarriers in a 3D culture system that better recapitulates the topological and biochemical characteristics of bone tissue. This platform captured biochemical markers indicative of osteoinhibition via Dkk-1 secretion from the OS cells and unlike monolayer culture, migration of osteoprogenitors and OS cells was readily visualized. On OEhMSC-derived ECM, OS cells proliferated rapidly, displacing the osteoprogenitors, ultimately leading to their death, providing an experimentally accessible system to simultaneously examine how the osteogenic microenvironment can drive OS cell migration, engraftment, proliferation, bone destruction and ultimately disease progression. Recapitulation of the osteogenic microenvironment is appealing both as a therapeutic to promote bone healing, and as a substrate to study interactions within bone. This study presents a novel osteoinductive scaffold with profound ability to promote bone healing, that is obtained from a theoretically infinite pluripotent cell source. Furthermore, a system involving co-culture of osteoprogenitors and OS cells on ECM-coated microcarriers in a 3D culture system has the capacity to provide new and quantifiable insights in the multiple pathological mechanisms of MBD, that are not readily measurable with current techniques.

publication date

  • December 2019