**5. ECad-Fc is a unique defined matrix for ESC and MSC**

to address this issue; after a decade, it has been proven to be a suitable material for stem cell

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

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.

technology and regenerative medicine.

regenerative medicine.

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

142 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

The study of Nagaoka *et al.* [55] revealed that murine ESCs can maintain their pluripotency on ECad-Fc-coated surface for extended culture periods (Fig. 2). Cells cultured on such type of substratum were later successfully used to generate germline-competent chimeric mouse [57]. Consistent with the findings, a separate study using mouse mesenchymal cell lines STO and NIH3T3 stably expressed with ECad as feeder-cell showed higher level of stem cell marker expression with standard colony-forming phenotype compare to the cells cultured on normal MEF-feeder-cell layer [58]. A number of feeder-free culture methods for ESCs have been reported where ESCs grow with their standard tightly-bound colony phenotype [4,11,13,22,24,56,59]. This type of tight colony formation generates heterogeneous cell popu‐ lation within a colony, which potentially affects homogenous accessibility of cytokines to these cells as well as creates heterogeneous niches. As a result stem cells in a colony differentiate heterogeneously and produce various kinds of cells as contamination with the desired type of cells, a major drawback that regenerative medicine has to overcome. In this respect, ECad-Fc matrix drives murine stem cells out of the colony to form a normal monolayer of cells, where stem cell resides as single cell condition [55]. This is a ground breaking technology that provides an exciting solution for overcoming the inherent colony forming phenotype-linked cellular heterogeneity. Biochemical analyses revealed that these cells bear all the signatures of pluripotent stem cells, and can form all three germ layers in a teratoma forming assay, and as mentioned earlier can generate germline-competent chimeric mouse. Additionally, they require lower amounts of LIF for maintenance of pluripotency, reducing costs related to ESCs culture. The monolayer-type single cell ESCs was also associated with higher proliferation ability and greater transfection efficiency compared to the colony-forming cells cultured on other substratum. Such improved proliferation ability could be extremely helpful for quick amplification of iPSCs on ECad-Fc substratum, which could mean shorter waiting periods for patients to receive cell therapy. The higher transfection efficiency of stem cells on ECad-Fc cooking plate could be exploited for targeted delivery of desired extracellular cargo for example, transgene products or drug molecules, into these cells for better outcomes.

This type of cooking-plate technology, where ECad-Fc provides basal support to the cells, and other immobilized factors for example, LIF-Fc [57] which satisfy specific needs, can be very advantageous for (1) ensuring undifferentiated state of stem cell in culture, (2) cost reduction associated with cytokines, and (3) hassle-free working condition without the necessity of regular media change, which is a standard time-consuming practice for stem cell culture.

The single-cell phenotype seen for ESCs was also observed for other stem cells for example, mouse embryonal carcinoma cells F9 and P19 but not for differentiated cells for example, NMuMG mouse mammary gland cells, MDCK kidney epithelial cells and isolated mouse primary hepatocytes [60]. This result indicated that ECad-Fc-mediated cellular migratory behaviors are most likely specific for embryonic stem cells. Reportable that ECad-facilitated cell-cell adhesion is often rearranged during initial stages of embryogenesis to control cell migration, cell sorting, and tissue function, which is suggesting a close cooperativity of stem cell maintenance, proliferation, and differentiation with ECad [39,48,49,61,62]. However, there

ECad-Fc cooking plate with a completely defined media named mTeSR1 (Stemcell Technolo‐ gies), and that made the culture method completely defined and xenogeneic-agent free, which is a significant achievement in regenerative medicine. The stem cells cultured on ECad-Fc cooking-plate were practically identical to those cultured on Matrigel-coated plate including cell morphology, proliferation rate, preservation of undifferentiated phenotype, and ability of differentiation into multiple cell types in embryoid bodies as well as in teratoma assay [56]. Interestingly, contrasting with the single-cell phenotype for mouse ESCs, human ESCs produced normal colony forming phenotype on ECad-Fc cooking-plate. The mechanism underlying the difference for this observation was not completely understood though.

Cadherin-Fc Chimeric Protein-Based Biomaterials: Advancing Stem Cell Technology and...

http://dx.doi.org/10.5772/58287

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Human and mouse ESCs have been shown to demonstrate significant disparities in expression of cell surface markers, transcription factors, cytokines, and proteins in them. The difference was evidently recognized by the fact that mouse ESC can be maintained in undifferentiated state with the addition of LIF devoid of feeder-cell but human ESC cannot [14]. It has been shown that the inhibition of Rho-ROCK signaling pathway generates cell scattering in human ESCs suggesting direct connection between cell scattering and signaling pathways [63]. While both mouse and human ESCs express ECad, however, it appears there are diverse additional factors involved to define ECad-mediated activities in these cells and additional investigations are required to reveal the complete molecular circuitry associated to this phenomenon.

MSC is a type of ASCs, and can be collected from donor by satisfying approved ethical issues. These cells have been considered as potential starting materials for regenerative medicine and tissue engineering. They must be expanded *in vitro* before dispensing for specific applications to accomplish anticipated therapeutic effects. MSCs also need xenogeneic agent-free culture method for maintaining their differentiation potency over the culture period. ECad-Fc cooking-plate technology was effectively applied for this reason as well [43]. The cultured MSCs on human ECad-Fc (hECad-Fc) matrix exhibited superior attachment on culture plate compare with standard tissue culture plate and gelatin-coated plate. The MSCs cultured on hECad-Fc showed comparable level of CD 105 and significantly greater level of β-catenin and ECad expression. It has been reported that β-catenin enhances the activity of Oct-4, which is one of the principal Yamanaka factors that plays critical function during the regulation of selfrenewal of ESC [45,64], on conjecture it can be suggested that MSCs maintained on ECad-FC cooking-plate might preserve superior stem-ness compare to the MSCs maintained on tissue culture-treated plate and gelatin-coated plate, and therefore possess greater applicability for

**6. ECad-Fc in directed differentiation and** *in-situ* **cell sorting of stem cell**

Targeted differentiation of stem cells and enrichment of desired cell for example, hepatocytes, from the pool of differentiated cells are very important steps towards use of the cells for regenerative medicine. Functionally matured hepatocytes derived from stem cells can be a potential remedy for various hepatic diseases. There have been several hepatic differentiation protocols reported from ESCs using orthodox techniques including embryonic body (EB)

regenerative medicine.

**Figure 2.** ECad-Fc is a defined matrix for culturing monolayer of iPS cells. Mouse EB3 cells were successfully cultured on ECad-Fc-coated surface that showed monolayer phenotype (C and D) compare with compact colony phenotype (A and B) for general protocol, which was significantly advantageous for faster growth (E), and higher transfection efi‐ ciency (F).

is no such suitable system to explore the necessary signaling pathways to address these questions. Nevertheless, since ESC does not form colony on ECad-Fc cell-cooking plate therefore this can be a perfect tool for obtaining single cell model system of stem cells to investigate relevant signaling pathways necessary for stem cell maintenance, proliferation, and differentiation. Our recent study successfully exploited this single-cell phenotype for monitoring cell cycle properties of stem cells on cell-cooking plate (unpublished), indicating the importance of this system for cell biology experiments designed to reveal their individual characteristics. The findings could be invaluable for regulating stem cells for desired applica‐ tion in regenerative medicine.

Most of the stem cell innovations, comprising generation of ESCs and iPSCs, were primarily established in mouse model, and then applied in human models. Similarly, ECad-Fc cellcooking plate technology was first developed and established for murine stem cells [55,57]. Thereafter, ECad-Fc cooking-plate was successfully applied for human ESC culture following similar methodologies with additional consideration for mild enzymatic treatment during the cell dissociation and seeding steps [56]. A strong protease cocktail Accutase (Millipore) was used for murine ESC culture; however, Accutase treatment was found detrimental to human ESCs, which was recuperated by using enzyme-free proprietary preparation named, Cell Dissociation Buffer (Life Technologies). It is reportable that the human ESCs were cultured on ECad-Fc cooking plate with a completely defined media named mTeSR1 (Stemcell Technolo‐ gies), and that made the culture method completely defined and xenogeneic-agent free, which is a significant achievement in regenerative medicine. The stem cells cultured on ECad-Fc cooking-plate were practically identical to those cultured on Matrigel-coated plate including cell morphology, proliferation rate, preservation of undifferentiated phenotype, and ability of differentiation into multiple cell types in embryoid bodies as well as in teratoma assay [56]. Interestingly, contrasting with the single-cell phenotype for mouse ESCs, human ESCs produced normal colony forming phenotype on ECad-Fc cooking-plate. The mechanism underlying the difference for this observation was not completely understood though.

Human and mouse ESCs have been shown to demonstrate significant disparities in expression of cell surface markers, transcription factors, cytokines, and proteins in them. The difference was evidently recognized by the fact that mouse ESC can be maintained in undifferentiated state with the addition of LIF devoid of feeder-cell but human ESC cannot [14]. It has been shown that the inhibition of Rho-ROCK signaling pathway generates cell scattering in human ESCs suggesting direct connection between cell scattering and signaling pathways [63]. While both mouse and human ESCs express ECad, however, it appears there are diverse additional factors involved to define ECad-mediated activities in these cells and additional investigations are required to reveal the complete molecular circuitry associated to this phenomenon.

MSC is a type of ASCs, and can be collected from donor by satisfying approved ethical issues. These cells have been considered as potential starting materials for regenerative medicine and tissue engineering. They must be expanded *in vitro* before dispensing for specific applications to accomplish anticipated therapeutic effects. MSCs also need xenogeneic agent-free culture method for maintaining their differentiation potency over the culture period. ECad-Fc cooking-plate technology was effectively applied for this reason as well [43]. The cultured MSCs on human ECad-Fc (hECad-Fc) matrix exhibited superior attachment on culture plate compare with standard tissue culture plate and gelatin-coated plate. The MSCs cultured on hECad-Fc showed comparable level of CD 105 and significantly greater level of β-catenin and ECad expression. It has been reported that β-catenin enhances the activity of Oct-4, which is one of the principal Yamanaka factors that plays critical function during the regulation of selfrenewal of ESC [45,64], on conjecture it can be suggested that MSCs maintained on ECad-FC cooking-plate might preserve superior stem-ness compare to the MSCs maintained on tissue culture-treated plate and gelatin-coated plate, and therefore possess greater applicability for regenerative medicine.

is no such suitable system to explore the necessary signaling pathways to address these questions. Nevertheless, since ESC does not form colony on ECad-Fc cell-cooking plate therefore this can be a perfect tool for obtaining single cell model system of stem cells to investigate relevant signaling pathways necessary for stem cell maintenance, proliferation, and differentiation. Our recent study successfully exploited this single-cell phenotype for monitoring cell cycle properties of stem cells on cell-cooking plate (unpublished), indicating the importance of this system for cell biology experiments designed to reveal their individual characteristics. The findings could be invaluable for regulating stem cells for desired applica‐

**Figure 2.** ECad-Fc is a defined matrix for culturing monolayer of iPS cells. Mouse EB3 cells were successfully cultured on ECad-Fc-coated surface that showed monolayer phenotype (C and D) compare with compact colony phenotype (A and B) for general protocol, which was significantly advantageous for faster growth (E), and higher transfection efi‐

144 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

Most of the stem cell innovations, comprising generation of ESCs and iPSCs, were primarily established in mouse model, and then applied in human models. Similarly, ECad-Fc cellcooking plate technology was first developed and established for murine stem cells [55,57]. Thereafter, ECad-Fc cooking-plate was successfully applied for human ESC culture following similar methodologies with additional consideration for mild enzymatic treatment during the cell dissociation and seeding steps [56]. A strong protease cocktail Accutase (Millipore) was used for murine ESC culture; however, Accutase treatment was found detrimental to human ESCs, which was recuperated by using enzyme-free proprietary preparation named, Cell Dissociation Buffer (Life Technologies). It is reportable that the human ESCs were cultured on

tion in regenerative medicine.

ciency (F).
