**1. Introduction**

'Stem cell' – the term was first coined by Russian histologist Alexander Maksimov in 1908 to herald the existence of special cells those have capacity to generate blood cell. Stem cells are the core materials of regenerative medicine and tissue engineering. Although there are multiple types of stem cells available based on their origin and functionality; however, scientifically they can be classified into four well-defined classes– (1) embryonic stem cell (ESC), (2) adult stem cells (ASC) for example, muscle satellite cells are muscle-specific adult stem cell, (3) induced pluripotent stem cell (iPSC), and (4) pathological stem cells (PSC) for example, cancer stem cells (CSC) [1]. Out of these 4 types, ESC and ASCs are true physiological stem cells, iPSCs are engineered stem cells and PSCs are conditional stem cells. Among them, ESC and iPSC are being considered true pluripotent stem cells, which have the capacity for unlimited self-renewal and differentiation into all the specialized cell types of the body. Therefore these cells have been considered the most favorable cells for using in regenerative medicine and tissue engineering [2,3,4,5,6,7,8].

Stem cells need a special environment for their survival, maintenance and growth. During the early stage of establishing the culture methodologies for stem cells, it was realized that they need support from other cells for example, mouse embryonic fibroblast (MEF). Co-culture methodology with gamma-irradiated MEF cells used as feeder-cells and enriched culture media with fetal bovine serum (FBS) were successfully utilized for establishing *in vitro* stem cell culture [9,10]. However, using a second non-related cell type (although growth restricted) is not suitable for differentiation studies – particularly, for 3D cell culture. Later, the MEF layer

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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 supports for stem cell culture.

synthetic biomaterials alone or as a blend as cell-culture substrate, and therefore, designing

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

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

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Pioneering work from our laboratory introduced Fc-chimeric protein in stem cell technology approximately a decade ago, and over the years we and others have established multiple Fcchimeric proteins as significantly favorable cell-recognizable biomaterials in stem cell tech‐ nology. These works with varieties of Fc-chimeric proteins spanning from ECM component protein [for example, E-cadherin (ECad)] to cytokine [for example, hepatocyte growth factor (HGF)] have shown tremendous potential to overcome the major barriers in stem cell tech‐ nology, namely defined condition for stem cell culture, selective differentiation to the target lineages, convenient purification of the desired cells etc., for the application of stem cell technology targeting to regenerative medicine. In this article we will focus on ECad-Fc and NCad-Fc chimeric proteins as novel cell-recognizable biomaterials in stem cell technology

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

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

such a biomaterial remains a challenging but ultimately rewarding task.

towards application in regenerative medicine.

should preferably mimic ECM protein(s).

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

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‐ synthetic biomaterials alone or as a blend as cell-culture substrate, and therefore, designing such a biomaterial remains a challenging but ultimately rewarding task.

Pioneering work from our laboratory introduced Fc-chimeric protein in stem cell technology approximately a decade ago, and over the years we and others have established multiple Fcchimeric proteins as significantly favorable cell-recognizable biomaterials in stem cell tech‐ nology. These works with varieties of Fc-chimeric proteins spanning from ECM component protein [for example, E-cadherin (ECad)] to cytokine [for example, hepatocyte growth factor (HGF)] have shown tremendous potential to overcome the major barriers in stem cell tech‐ nology, namely defined condition for stem cell culture, selective differentiation to the target lineages, convenient purification of the desired cells etc., for the application of stem cell technology targeting to regenerative medicine. In this article we will focus on ECad-Fc and NCad-Fc chimeric proteins as novel cell-recognizable biomaterials in stem cell technology towards application in regenerative medicine.
