**2. Rationale for using protein as biomaterials**

was successfully removed from the culture system by introducing MEF-conditioned media (MEF-CM) that has made the protocol more suitable for experimentation targeting regenera‐ tive medicine but not up to the desired standard due to the presence of xenogeneic agents in the system [11,12,13]. MEF-CM is enriched cell culture media with MEF-secreted molecules that functions as a depot for the necessary cytokines for the healthy maintenance of stem cells. However MEF-CM alone were not adequate to upkeep ESC and iPSC survival and growth thereby suggesting that MEF cells are not only providing necessary nutrients and cytokines, in addition they are also backing as physicochemical supports through the ECM to these cells. However, technically it remains elusive to point out the essential factors, required to maintain stem cell culture, present in the MEF-CM due to the inconsistency in expression and secretion of biological factors between experiments and batches. Moreover, it has been shown that not only proliferation of these cells but the secretion of necessary biomolecules and deposition of ECM components were also directly related to the gamma-irradiation [11,12,13]. Such factors directly influence properties of stem cells in culture, and instigate restriction for application of relevant protocols for regenerative medicine and tissue engineering. Therefore suitable cellrecognizable biomaterials are highly desired to overcome the dependency of cell-based basal

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

Matrigel was one of the first biomaterials that was effectively applied as plate-coating materials for *in vitro* culture of human ESC and iPSC with the aid of MEF-CM as culture medium [14,15]. This was a significant advancement in stem cell technology to make stem cells free from undesirable feeder-layer cells. Matrigel is a product from decellularization of Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, and a cocktail of laminin, collagen IV, entactin, heparin sulfate proteoglycans, and known and unknown growth factors with variable compositions [16,17,18,19]. It closely resembles the embryonic basement membrane in consistency and activity as well as providing a biologically functional complex [17,19]. However, Matrigel is not a defined material with high purity and incorporated with substantial lot to lot variation in constituents both in qualitative and quantitative measures. It has also been reported contaminated with Lactate Dehydrogenage Elevating Virus, and has raised additional concerns for safe application of this material in stem cell culture [20]. Such kinds of issues are strongly demanding a more defined culture condition under good manufacturing practice (GMP) for safe application of stem cell protocols or methodologies if the ultimate

objective is to employ stem cells in regenerative medicine or tissue engineering.

The individual components of Matrigel provide specific functional queues to ESCs and iPSCs. For example, ESC exhibits normal growth when cultured on laminin-coated plate, which was not observed on either fibronectin- or collagen IV-coated surface [21,22,23,24]. It was also reported that specific laminin isoforms have distinctive effects on stem cells; for instance, laminin-111, -332, -511 support adhesion and proliferation of stem cells but isoforms -211 and -411 of laminin do not [22]. The information suggested that designing a defined matrix for stem cell culture requires special biomaterials that can deliver concurrent supports for cell adhesion, proliferation and differentiation. In fact, effective stem cell culture condition with high pluripotency was occasionally achieved in spite of introducing several synthetic and semi‐

supports for stem cell culture.

An ideal chemically defined xenogeneic-agent free stem cell culture system might be consists of chemically known matrix for plate coating that would provide structural basal support to the stem cells and defined media that is supplemented with highly pure recombinant proteins as functional cytokines. The system should essentially be free from serum or feeder-cells or any other animal products. Even though it is very demanding however, designing and preparing a completely defined stem cell culture system is highly challenging. One worthwhile goal is to design a defined plate-coating material that can successfully replace Matrigel. Since stem cells are essentially dependent on cell-cell or cell-surface interaction for survival, which are mainly mediated by extracellular matrix protein (ECM), a cell-recognizable biomaterial should preferably mimic ECM protein(s).

Such kind of biomaterials can either be employed as a scaffolding molecule that may provide structural support of the growing cells, or as functional effector molecules that can target cellular signal recognition machineries like cell surface receptors or channels to trigger or maintain signaling cascades necessary for survival, proliferation, and differentiation of experimental cells [25]. To act as an artificial ECM the biomaterial under consideration should mimic the physicochemical and biological properties of native components of ECM to facilitate targeted functionalities of cell for example, adhesion, proliferation, differentiation, etc [26]. Similarly, the candidate effector molecules should have physicochemical signature of the comparable native molecules for recognition as functional substrate to endogenous receptors or channels of experimental cells. Synthetic biomaterials have limitations for providing perfect biochemical structural motif for effective recognition by the cellular recognition machineries to execute necessary cellular function, and therefore are generally not efficient enough for practical applications for *in vivo* condition. Moreover, many of these synthetic biomaterials are not biologically compatible at a desired level and may generate pathophysiological compli‐ cations in the long term in the body.

**3. Cadherins in cell biology**

organisms.

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

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

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

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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

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

cytoskeletal components *e.g.*, actin is not clearly known.

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 adhesion.
