The overall objective of this project was to develop novel acellular matrices derived from differentiating embryonic stem cells (ESCs) undergoing morphogenesis. The central hypothesis was that embryonic matrices contain complex mixtures of extracellular factors that, when isolated, retain bioactivity and enhance wound healing in an adult environment.
To further elucidate the expression profile of endogenous ECM synthesized by differentiating ESCs, we analyzed the temporal gene profile of matrix molecules and growth factors expressed in ESCs and EBs using Extracellular Matrix (84 adhesion molecules, basement membrane constituents, structural components, and proteases) and Growth Factor PCR arrays. Interestingly, by the end of the differentiation time course examined, almost no molecules decreased significantly compared to earlier time points, all together suggesting that an onset of specification events later in EB differentiation is coincident with a surge of matrix synthesis. Importantly, gene ontology analysis of ECM and growth factor expression values highlighted that matrix synthesis alone is a reliable indicator of differentiation events occurring within the EBs, further emphasizing the underlying role of ECM in ESC differentiation.
While gene array studies provided global insight into ESC matrix synthesis, additional in-depth investigation of versican, which appeared in network analyses, was performed to more specifically examine its different isoforms. Of the four splice variants, gene expression of V0 and V1 dominated over the course of differentiation and increased significantly with time. Interestingly, the glycosaminoglycan hyaluronan (HA) was found to co-localize with versican, and the HA/versican spatial localization patterns in EBs strengthened the notion that ECM molecules play a role in cell differentiation events. Importantly, the cell morphology in areas rich in HA and versican appeared to be distinctly different from those excluded from the region, and when examined more closely, those cells were found to be N-cadherin-positive, indicative of mesenchymal cells. Cells excluded from the versican-rich region stained positive for the epithelial marker E-cadherin. These results suggested that HA and versican are associated with an epithelial-mesenchymal transition within the EBs, which more broadly implies that the temporal expression of ECM molecules is linked to cell differentiation events.
In order to isolate the complex matrices synthesized by differentiating ESCs, a variety of reagents were tested to assess their efficacy in decellularizing EBs. Based on established protocols developed for decellularizataion of other tissue types, Triton X-100, peracetic acid (PAA), and sodium dodecyl sulfate (SDS) were tested alone and in combination with DNase. A combination of Triton X-100 and DNase significantly reduced cell viability and DNA content while maintaining a greater protein:mass ratio than either PAA or SDS treatments. More in-depth studies indicated that longer Triton durations significantly reduced protein content, and lower DNase concentrations were less efficient at DNA removal. In addition, greater solvent volumes were effective for decellularizing a range of EBs, up to ∼6000 EBs.
The matrix resulting from the decellularization of EBs was then examined in an in vivo dermal wound healing model. Acellular EBs were applied to wounds either alone or using fibrin as a delivery vehicle and were compared to wounds left untreated or treated with fibrin alone. Matrix-treated wounds (delivered with fibrin) exhibited significantly greater percent wound closure during the 2-week post-wounding examination period than untreated wounds. In addition, compared to both fibrin-treated and untreated groups, wounds treated with matrix alone had significantly greater blood vessels per area that extravasated towards the epithelial layer. Similar observations in other studies have been linked to improved healing, which, along with the wound closure data, demonstrates that matrix derived from differentiating ESCs significantly improve dermal wound healing compared to untreated wounds. These promising results suggest that acellular EB-derived matrices can be a clinically relevant therapy for a variety of wound healing applications. (Abstract shortened by UMI.)