Innovative approaches to regenerative medicine: Tissue repair and generation of stratified squamous epithelia.

摘要

Cell-based therapeutics generated from somatic or pluripotent stem cell sources have the potential to revolutionize the treatment of debilitating diseases and medical conditions previously believed to be untreatable. This dissertation describes a multifaceted investigation of regenerative medicine approaches ranging from basic to translational research in the field of cutaneous wound repair employing both somatic and pluripotent stem cell sources.;Allogeneic/autologous chimeric bioengineered skin substitutes offer an innovative approach for the treatment of severe cutaneous trauma. Mixed populations of green fluorescent protein-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric cultures. These proof-of-principle studies demonstrated that organotypic co-culture of NIKS and primary keratinocytes generated a fully-stratified epidermal component of composite skin substitute tissue, providing wound coverage and restoring cutaneous barrier function while delivering "autologous" keratinocytes to the wound site.;Initial studies examining the therapeutic potential of pluripotent stem cells for skin regeneration and repair highlighted the technical challenges associated with cultivating pluripotent cell types. A continuous, uniform, adventitious agent-free feeder layer cell source (3T3CG) was evaluated for the ability to support human embryonic stem cell serial cultivation. 3T3 CG feeder layers sustained long-term serial cultivation of karyotypically normal, undifferentiated human embryonic stem cells that retained the ability to differentiate into ectodermal, mesodermal, and endodermal cell types. Use of this cell source would facilitate basic stem cell research and support early-stage development of stem cell-derived therapeutics.;Although several studies describe the generation of human embryonic stem cell-derived keratinocyte-like cells, no laboratory has reported the successful generation of three-dimensional tissue structure from human embryonic stem-derived cell sources. This dissertation describes the morphogenesis of a stratified squamous epithelium from human embryonic stem cell-derived keratinocytes. This tissue exhibited keratinocyte terminal differentiation up to the final formation of a definitive cornified layer with epidermal-specific gene and protein expression patterns, although a functional epidermal permeability barrier was not established and tissue did not stably engraft in an in vivo model of skin transplantation. These findings represent a significant advance in the generation of human tissues and organs from human embryonic stem cell-derived progenitor cells.