Wharton's jelly mesenchymal stem cells (WJMSCs) are being increasingly recognized for their ectodermal differentiation potential. Previously, we demonstrated that when WJMSC were seeded onto an acellular matrix material derived from Wharton's jelly and cultured in osteogenic induction media, generated CK19 positive cells and hair-like structures indicative of ectodermal differentiation of WJMSCs. In this manuscript, we examine the underlying mechanism behind this observation using a variety of microscopy and molecular biology techniques such as western blotting and qPCR. We demonstrate that these hair-like structures are associated with live cells that are positive for epithelial and mesenchymal markers such as cytokeratin-19 and alpha-smooth muscle actin, respectively. We also show that up regulation of beta-catenin and noggin, along with the expression of TGF-beta and SMAD and inhibition of BMP4 could be the mechanism behind this ectodermal differentiation and hair-like structure formation. (C) 2016 Elsevier Inc. All rights reserved.