Abstract
IntroductionPluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation.MethodsIn this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs.ResultsWhile both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF.ConclusionsOur findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation.
Highlights
Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies
We have demonstrated that human embryonic stem (hES) cells give rise to fibroblast-like cells [18]; we have not determined if hES-derived cells can manifest the functional properties of dermal fibroblasts that can support the organization and development of three dimensional (3D) skinlike tissues known as human skin equivalents (HSEs) through epithelial-mesenchymal cross-talk
To ensure the fibroblast-like phenotype of EDK cells, several cell lines were derived from hES-cells using the same differentiation conditions and were screened for the secretion of paracrine factors known to be secreted at high levels in dermal fibroblasts keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF); data not shown) [5,6]
Summary
Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies Such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation. Despite the critical impact of this reciprocal cross-talk between stromal fibroblasts and epithelial cells on tissue homeostasis, little is known about the identity and maturational development of the precursor cells that give rise to these fibroblasts. Homoeostasis, and repair of many tissues depends on interactions between epithelial cells and their adjacent stromal fibroblasts [3,4,5,6], the functional analysis of hES-derived fibroblasts could best be accomplished in such engineered HSEs that demonstrate many features of their in vivo counterparts
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