hESC- and iPSC-Derived Fibroblasts for Skin Engineering and Repair.
Abstract: The unlimited biological potential of pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as seen by their ability to differentiate into any cell type in the human body, makes them promising candidates for regenerative medicine applications. Novel differentiation approaches are continuously being developed to generate therapeutic... read moreally-relevant cell types from hESCs and iPSCs. However, before these pluripotent stem cell-derivatives can be implemented in regenerative medicine, thorough testing of their phenotypic stability and functional abilities are required. I hypothesized that pluripotent stem cells could be directed to differentiate to mesenchymal lineage fates, and that these cells would recapitulate the essential functional features of dermal fibroblasts within three-dimensional (3D) in vitro tissue models. Generation of mesenchymal cells from hESCs was first explored using two distinct differentiation protocols. The first protocol was based on spontaneous differentiation of hESCs followed by selective isolation of CD73-positive mesenchymal cells. The second protocol was based on a directed differentiation approach by culturing hESCs under defined substrate and media condition and exposure to bone morphogenic protein 4 (BMP4). I further studied the therapeutic potential of these cells using a spectrum of phenotypic assays and 3D tissue models to assess their contribution to wound re-epithelialization, production and assembly of extracellular matrix, and regulation of angiogenesis. While both differentiation approaches generated mesenchymal cells with similar morphology, surface marker, and gene expression profiles, there were marked differences in functional properties. Cells that were generated using the directed differentiation approach demonstrated functionality of dermal fibroblasts as seen by their capacity to support development of stratified squamous epithelia and re-epithelialization of wounds generated in bioengineered 3D skin tissues that was mediated by production of soluble growth factors. In addition, hESC- and iPSC-derived fibroblasts generated using the directed differentiation approach produced and assembled thick extracellular matrices reminiscent of skin stromal tissue, induced vascular sprouting, and promoted the formation and stabilization of vascular networks in vitro in 3D models of dermal regeneration and angiogenesis. Furthermore, the administration of hESC-derived fibroblasts into severe mouse hindlimb ischemia model, prevented autoamputation of ischemic limbs, attenuated tissue necrosis, and improved blood perfusion, thus demonstrating the repair-competent phenotype of these cells in vivo. These findings highlight the robust therapeutic potential of hESC- and iPSC-derived fibroblasts, and demonstrate our ability to characterize the functional properties of these cells following differentiation in unique 3D assays in vitro and in vivo.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Jonathan Garlick.
Committee: James Schwob, David Kaplan, John Castellot, and David Mooney.
Keywords: Developmental biology, Cellular biology, and Molecular biology.read less