**4. ECad-Fc as a cell-recognizable biomaterial**

As a biomaterial, ECad-Fc was first applied as plate-coating materials for hepatocyte differ‐ entiation experiments [50]. It was observed that differentiated hepatocytes can efficiently adhere with the cell culture plate coated with ECad-Fc. The adhered cells demonstrated comparable molecular characteristics e.g., low DNA synthesizing activity and maintenance of tryptophan oxygenase (TO) expression like those of spheroid-form hepatocytes. As well, the hepatocyte cultured on ECad-Fc-coated plate supported the differentiation of hepatocytes in culture. These results suggested important roles of ECad-Fc matrix for the maintenance of differentiating hepatocytes. This was the first report of ECad-mediated matrix dependability, as a biomaterial, for any cell type in regenerative medicine. After a while, Nagaoka *et al*. published the landmark report regarding the application of ECad-Fc cell-cooking plate (since target cell can be obtained on such type of biomaterial-coated plate without additional cell purification method therefore named so) as a defined matrix for successful maintenance of murine stem cells without any feeder layer in 2006 [55]. This report signified the alluring potential of ECad-Fc as a biomaterial for practical application in stem cell technology and regenerative medicine.

Xenogeneic-agent free stem cell culture method is extremely critical if the objective of the relevant protocol is to apply the relevant products in regenerative medicine. Since MEF secrets many unidentified molecules, which are potential xenogeneic elements for human subject therefore feeder-cell-based early methodologies are not considerable for applying in regener‐ ative medicine. Matrigel is also produced from mouse carcinoma tissue and ill-defined therefore causing serious known and unknown hazards of xenogeneic contamination in experimentations. An immunogenic sialic acid (NeuGc) has been identified in a co-culture experiment for human ESCs applying MEF and animal derivatives as serum replacement [24,56]. This is specifically worrying as such kind of non-human sialic acid can initiate immunogenic processes in human triggering complete graft rejection and consequential complexities. Non-human animal-derived products also can be a possible cause for myco‐ plasma contamination, which can directly infect the cells in culture and either damage them totally or can change their properties, and thereby directly or indirectly initiate complicacies for regenerative medicine protocols. Human feeder-cells and serum have been recommended for culturing human ESCs to evade xenogeneic compound in experimental system for regenerative medicine. However, this is associated with a high risk of microbial contamination, for example retroviral components, and hence are not as suitable for *in vivo* application. Therefore it is a prime importance to establish completely defined human stem cell culture system for safe application of relevant products in regenerative medicine.
