**3. Cadherins in cell biology**

not biologically compatible at a desired level and may generate pathophysiological compli‐

Proteins are native elements of cells and natural ECM scaffolds [27] and therefore recombinant proteins could be one of the best candidates to design superior biomaterial for application in regenerative medicine and tissue engineering. Recent progress in biochemistry, molecular biology, bioinformatics, and engineering provides the prospect of expressing and purifying desired recombinant protein with high yield (g/L is achievable) in large scale [28], which can eventually be applied (directly or with modification) as novel, simplified, and bio-active macromolecules in regenerative medicine and tissue engineering [29,30,31]. Such proteins can be generated from a genetic template by natural cellular read-out process namely, DNA>RNA>protein that ensures excellent uniformity and reproducibility of the designed biomaterial depending on cellular conditions, where the production is executed. The native biological production process confirms high degree of reproducibility, which is not realistic by traditional chemosynthetic or mechanosynthetic processes. On the contrary, protein science has its own negative issues for example, highly efficient expression system for the desired protein, convenient purification of the target protein, proper folding of the purified protein, stability of the functional protein, mode of application of experimental protein etc. Chimeric protein technology has long been considered one of the potential methodologies to overcome many of these issues including higher productivity, better stability, and efficient purification of a target protein for bulk scale. Fc-chimeric protein is one such engineered protein that was introduced in 1989, and has been showing great promise for comparatively convenient production efficiency of chimeric protein with functional integrity and long-term stability, and therefore successful applcation in diverse fields of biomedical sciences [32,33,34]. An illustra‐

tion of Fc-chimeric protein is shown in Fig. 1 with ECad-Fc as a model.

**Figure 1.** Schematics of Fc-chimeric protein, and its molecular function. (A) Functional domain of target protein is fused as N-terminal with the Fc domain of IgG. ECad is shown here as an example. (B) Plasma-membrane localized ECad dimer can interact with apposing ECad dimer and form high affinity binding that makes cell-cell and cell-surface

cations in the long term in the body.

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

adhesion.

The cadherins is a large family of single transmembrane proteins with more than 100 members. Out of these we will be focusing on epithelial cadherin (ECad) and neural cadherin (NCad) in this report. They are the member of classical cadherin family, and both of them are glycosylated in their extracellular domain. They have the ability to function as adhesion molecules for the relevant protein-expressing cells. Generally cadherin forms homophilic dimer, and the dimeric forms of cadherins take part in Ca+2-dependent coupling from apposing cells that mediates cell-cell adhesion. These single transmembrane-domain plasma membrane-resident proteins are not only necessary for cell-cell adhesion but also involved in indispensible signaling cascades, which are critical for the development-to-homeostasis-to-demise of cells and organisms.

The extracellular N terminal region of ECad consists of 5 structural domains, which are the signature motifs for ECad and are responsible for the homophilic binding between two neighboring as well as apposing molecules, while the C-terminal intracellular region of ECad interacts with several intracellular proteins such as β-catenin/Armadillo and p-120 catenin [35,36,37]. The p-120 catenin is associated with the targeted transport and stabilization of the adhesion complexes on the plasma membrane. Beside, β-catenin interacts with α-catenin, which in turn initiates actin filament formation *via* interaction with formin at the adherens junction [38,39,40,41,42]. However, how cadherin-catenin complexes are connected with cytoskeletal components *e.g.*, actin is not clearly known.

ECad has been shown linked with many early-to-late developmental and differentiation processes *in vivo* and *in vitro* systems including ESCs, MSCs, iPSCs, and whole embryo [43,44,45,46,47]. ECad knock out mouse was reported embryonic lethal [48,49], which is a direct evidence of its critical importance in stem cell biology and regenerative medicine. Our lab first envisioned the application of ECad as a novel cell-recognizable biomaterial little over a decade ago while Nagaoka *et al.* endeavored to improve the differentiation and maturation efficiency of hepatocyte in an *in vitro* system [50]. The idea was conceived from the fact that Fc domain of IgG can bind directionally with an appropriate surface *via* hydrophobic interaction, and the fused protein stretches out directionally to offer interaction with a suitable partner [51]. At that period, several reports suggested that ECad is indispensable for tissue morphogenesis, and is also required for maintenance of matured tissues. Awata et al. showed that ECad-mediated cell-cell interaction is necessary for hepatocytes to maintain their differentiated phenotypes by forming 3D spheroid structure, or multi-layer cell aggregates [52]. Further it was reported that high cell density culture of fetal liver cells [53,54], which most likely is an ECad-dependent characteristics, enhanced hepatocyte maturation in culture. These findings suggested that cellcell interaction may directly influence hepatocyte maturation as well as maintenance of differentiated phenotypes. There was, however, no substantial information regarding the role of ECad in the relevant processes, and to reveal the answer it was essential to have a suitable tool or methodology that can expedite cell-cell interaction analysis in a controlled manner. ECad-Fc was designed and deployed as a novel biomaterial in the regenerative medicine field to address this issue; after a decade, it has been proven to be a suitable material for stem cell technology and regenerative medicine.

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

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

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

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

143

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
