Abstract
For patients suffering from extensive burns or chronic wounds, allogeneic cell-based therapies provide a means to restore viability and function to severely damaged cutaneous tissues. Pluripotent stem cells have been proposed as an allogeneic source for regenerative medicine applications, such as those required for burn and wound management. Ultimately the clinical utility of pluripotent stem cells relies on the ability to direct differentiation into the desired cell lineage, facilitate appropriate tissue formation, and confirm tissue-specific biological activity. We examined the capacity of human embryonic stem cell-derived keratinocytes (hES-DK) to undergo morphogenesis and form biologically active interfollicular epidermis using methods routinely employed to engineer skin substitutes for clinical applications. Throughout directed differentiation, the orderly sequence of epidermal gene expression mimicked the progression of fetal skin development. When introduced into three-dimensional organotypic culture, hES-DK cells formed a pluristratified tissue with architecture similar to that of the interfollicular epidermis. In hES-DK tissue the expression and localization of cell-cell adhesion proteins, markers of both early- and late-stage keratinocyte terminal differentiation, and host defense peptides were comparable to the patterns observed in keratinized stratified squamous epithelia generated from epidermal keratinocytes. Despite similar tissue morphology, functional analysis revealed that hES-DK tissues did not display robust cutaneous barrier function. However, hES-DK tissues were shown to possess antimicrobial activity, which represents the first demonstration of biological activity in a keratinized stratified squamous epithelium generated from a pluripotent stem cell-derived source. The successful generation of biologically active hES-derived stratified squamous epithelia represents a significant advance in the development of a hES-derived bioengineered human organ for clinical use.
Highlights
The utility of pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, for regenerative medicine applications relies on the cells’ ability to differentiate into clinically relevant cell populations and form constructs that recapitulate the structure and function of the intended organ system
MRNA expression of the intermediate filament nestin remained unchanged in retinoic acid (RA)-treated cells, a reduction was observed for the neural ectoderm differentiation marker Sox 1. mRNA expression of both ΔNp63, a transcription factor expressed in the proliferative compartment of epithelial tissues, and keratin 18 (K18), an intermediate filament primarily associated with simple epithelia [23], increased with RA treatment
Keratinocyte type 1 transglutaminase (Tgase1) was properly localized to the cell membrane of the spinous and granular layers (Figures 3Q and 3R). These results demonstrated that key structural proteins of stratified squamous epithelia, and proteins associated with both earlyand late-stage keratinocyte terminal differentiation, were appropriately expressed and localized in human embryonic stem cell-derived keratinocytes (hES-DK) tissues relative to stratified squamous epithelia generated from epidermal keratinocytes
Summary
The utility of pluripotent stem cells, such as human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells, for regenerative medicine applications relies on the cells’ ability to differentiate into clinically relevant cell populations and form constructs that recapitulate the structure and function of the intended organ system. Stem cell therapy in burn and wound management requires a consistent source of keratinocytes with the capacity to fully restore functional epidermis. The resulting heterogeneous populations displayed sporadic differentiation into a keratinocyte lineage as evidenced by expression of markers such as keratin 14 (K14) or yielded keratinocyte-like cells with limited expansion potential. Serial cultivation yielded a relatively pure population of keratinocyte-like cells which expressed several proteins associated with early-stage keratinocyte terminal differentiation in monolayer culture. In 2011, a differentiation protocol employing RA and BMP4 was reported to facilitate the generation of K14-expressing keratinocytes from hiPS cells [7]. Differentiation methods using RA and Activin [8], as well as ascorbic acid and BMP4 [9], have been shown to generate K14+ keratinocytes from hES cells provided the specified cultivation steps were adhered to
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
