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
Matrigel was one of the first biomaterials that was effectively applied as plate-coating materials for
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 semisynthetic 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 Fc-chimeric proteins as significantly favorable cell-recognizable biomaterials in stem cell technology. 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 technology, 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.
2. Rationale for using protein as biomaterials
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
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 illustration of Fc-chimeric protein is shown in Fig. 1 with ECad-Fc as a model.
3. Cadherins in cell biology
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
ECad has been shown linked with many early-to-late developmental and differentiation processes
4. ECad-Fc as a cell-recognizable biomaterial
As a biomaterial, ECad-Fc was first applied as plate-coating materials for hepatocyte differentiation 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
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 regenerative 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 mycoplasma 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
5. ECad-Fc is a unique defined matrix for ESC and MSC
The study of Nagaoka
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 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 application 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 cell-cooking 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 Technologies), 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
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) formation, and clustered colony formation on gelatin- or feeder-cell-coated plates [52,54]. However, these protocols come with many drawbacks, for example, heterogeneous cell population, spontaneous differentiation, xenogeneic contamination, inefficient conversion to hepatocytes, requirement for enrichment of target cell population etc. Our group has effectively applied ECad-Fc as a cell-recognizable plate-coating materials that facilitated good quality mouse ESCs in culture with superior proliferative activities and single-cell phenotype. Similarly, the cell-recognition property of such Cadherin-Fc chimeric protein was exploited for the possibility of facilitated differentiation of ESCs to specific cells for example, hepatocytes and neural cells [29,30,50,65]. Remarkably, ECad-Fc substratum favored progressive differentiation of ESCs to cells with features of definitive endoderm, hepatic progenitor cells, and finally phenotypical as well as functional hepatocytes-like cells [30,50]. The ECad-Fc-coated substratum stimulated selective hepatocyte differentiation in association with ectopic hepatocyte-producing cocktail resulting around 55% hepatic endoderm cells devoid of neuroectoderm and mesoderm markers [30]. High level of (approximately 98%) ECad and developing-hepatocyte marker α-fetoprotein (FTP) were co-expressed in these cells. Since these differentiating hepatocytes express high level of ECad on the plasma membrane therefore ECad-Fc was employed for on-site one-step enrichment of
The enhanced differentiation and cell-recognizable properties were also observed with ECad-Fc and NCad-Fc-based mixed biomaterial cooking-plate for neural cells [65], and is discussed in detail under NCad-Fc section. Such kind of ECad-Fc and NCad-Fc hybrid cooking-plate can be applied for either generation of large number of homogeneous cell population, which can be applied for therapeutic evaluation, or for analyzing the signaling pathways related to nerve generation at a single cell level.
7. ECad-Fc is a superior matrix for iPSC
iPSCs are commonly derived from somatic cells by ectopic and forced expression of common transcription factors Oct4, Sox2, and Nanog along with protocol-dependent treatments with cocktails of some other transcription factors, and even miRNA or small molecules [10,66,67,68,69,70,71,72,73]. Despite the existence of many protocols for generating iPSCs, the required time and efficiency of iPSC generation is still not practical for application of the technology to a mass scale. As per recent published information, depending on protocol, it may take somewhere between 2~4 weeks to get a 1% conversion of cells to iPSCs. During the reprograming process, starting cells experience mesenchymal-to-epithelial transitions (METs) as a natural requirement [74]. This fact was further proved by the findings that MET happens during the initial stage of reprograming process [71,74,75]. Recent evidence further suggested significant functional roles of ECad and other cell adhesion molecules in METs.
ECad interacts with cytoskeletal components
The protocols for generating ESCs or iPSCs as well as differentiation to target cells from these cells require cell isolation step either by mechanical process or in combination with enzymatic treatment [79]. These types of methodologies require skilled labor, specialized instrumentation, additional time and cost, and distinct morphologic and phenotypic features. Several protocols have been described recently for enzyme-selective passage of specific cells; however, they are not globally applicable and very often appeared with unwanted cells. Enzymatic treatment also caused karyotypic anomalies compared with manual passaging [66,80]. FACS protocol has been applied for cell sorting based on surface marker recognition. However, relevant protocols need enzymatic treatment, application of foreign molecules, and mechanical processes involving severe stress on experimental cells [81,82], which are highly unfavorable for cells. ECad-Fc cooking-plate, advantageously, neither needs any kind of mechanical sorting nor any harsh chemical or enzymatic treatment. The experimental cells can selectively and strongly make homophilic binding with ECad-Fc matrix in a Ca+2-dependent manner subjected to the differential expression pattern of ECad in them during the transformation process. The cells with no or low level expression of ECad cannot and does not firmly bind with ECad-Fc substratum and can be washed off with suitable buffer thus offering a unique, robust, and stress-free cell enrichment system. Such a protocol ensures quicker, cheaper and convenient cell enrichment system for
8. NCad in cell biology
N-cadherin (NCad) or neural cadherin is also known as Cadherin-2, which is encoded in human by
During embryogenesis cells undergo an epithelial-mesenchymal transition (EMT) initiating upregulation of NCad and the downregulation of ECad in the mesoderm [88]. It has been suggested that NCad expression is essential for morphogenesis of the mesodermal germ layer during gastrulation [89]. NCad expression pattern has been found complementary to that of ECad in epidermal ectoderm [88,90,91]. NCad expression has been detected in mesoderm and notochord in the early phase of embryonic development, which is later also evident in neural tissue, lens placode [92], some epithelial tissues, myocardium of heart [93], epiblast of skeletal muscle [94], endothelial cells, osteoblasts, mesothelium, limb cartilage, and primordial germ cells [95,96].
NCad is found to be present in the early hematopoietic progenitor CD34+CD19+ cells, and it was proposed that NCad plays critical role for the hematopoietic cell differentiation as well as the early retention of this subpopulation in bone marrow [97]. During skeletal muscle formation mesodermal precursors exit from the cell cycle, and differentiate into myoblasts that terminally differentiates into multinucleate myofibers [98]. Cell cycle arrest and the expression of skeletal muscle–specific genes are the critical checkpoints for this developmental process [99]. All the epiblast cells undergoing skeletal myogenesis express the skeletal muscle-specific transcription factor MyoD, among them only the cells expressing NCad but not ECad can differentiate into skeletal muscle [94]. NCad function-perturbing antibodies showed that it plays a significant role in interaction between myoblasts in myotube formation and in myofibrillogenesis [100,101,102]. NCad is also found to be involved in myoblast migration in limb bud [103].
Cartilage is formed from the vertebrate embryonic limb by a highly synchronized and systematic event of cell commitment, condensation and chondrogenic differentiation of mesenchymal cells to chondrogens, and by the production of cartilaginous matrix. SOX9, an essential transcription factor for chondrocyte differentiation and cartilage formation, binds to the SOX9-binding motif in NCad promoter [104] that facilitates expressing of NCad gene products to play necessary roles in cellular condensation [105]. Prolonged expression of NCad due to the missexpression of wnt7a stabilizes NCad-mediated cell-cell adhesion resulting inhibition of chondrogenesis from mesenchymal chondrogenic culture [106]. The level of NCad mRNA was found increases during osteoblast differentiation and decreased during adipogenic differentiation thus suggesting their involvement in relevant differentiation processes [107]. NCad expression is increased in osteoblasts by BMP-2, FGF-2 and phorbol ester (e.g., PMA) in PKC-dependent manner, whereas factors like TNFα and IL-1 reduce the expression of NCad [108].
Migratory cell populations, also known as neural crest cells, are pluripotent cells those originate from dorsal part of neural tube and play important roles in embryonic development and pathophysiological conditions. These cells express NCad when they are associated with neural tube; however, NCad expression is down-regulated after EMT process and the relevant cells started to migrate over long distance, and finally transform into different types of tissues and cell populations, such as peripheral nervous system, cartilage, bone and melanocytes. Slug plays here important roles in down-regulating NCad that leads to a loss of cell-cell adhesion and allowing the cells to migrate. The dorso-ventral migratory cells re-express NCad during dorsal root and sympathetic ganglia developmental steps and promotes cell aggregation; thereafter, only dermal melanocytes express NCad [109,110]. This observation is suggesting critical involvement of NCad in the development of relevant tissues.
Several proteins can interact with NCad
EMT of squamous epithelial cells ectopically expressed specific amino acid sequences of EC4 of NCad induces motility. The cell motility behavior and adhesion is independent to each other, as antibody against the aforementioned relevant amino acid sequence of NCad inhibits cell motility but the cell-cell adhesion phenomena was uninterrupted [114]. The influence of NCad mediated cell migration is cell type specific, as it was found that NCad can inhibit LM8 mouse osteosarcoma cell migration but it did not have any significant effect on the movement of MDA-MB-435 cells [115]. The cytoplasmic domain of NCad form complexes with various types of molecules, such as p120, β-catenin, α-catenin and GAP-43, and regulate various cytoskeletal dynamics. All of these interactions are critically involved in tissue-to-animal development, morphogenesis and maturation, and is suggesting the possibility of exploiting this gene product for regenerative medicine.
9. NCad-Fc as biomaterial in regenerative medicine
NCad-Fc was introduced by Lambert et al. in 2000, and the study revealed that NCad-Fc not only induced the recruitment of NCad on the plasma membrane but also other components of the cadherin/catenin complex. This work for the first time demonstrated that NCad-Fc can mimic natural cell-cell contact formation and signal transduction [116]. Pioneering work from our lab has introduced NCad-Fc as cell-coating biomaterials for stem cell culture. NCad-Fc protein was collected from ‘pRC-NCFC’ plasmid, which was constructed by inserting the N terminal extracellular domain of mouse NCad into pRC/CMV (Invitrogen) plasmid [29]. The expression and purification methodologies of NCad-Fc are similar like ECad-Fc and have been described in details in relevant publications [50,55,117]. Over recent years our laboratory work revealed significant advantages of NCad-Fc in neural differentiation from stem cells. Early work was performed with mouse embryonic carcinoma cell P19 and neural stem cell MEB5 because of their easy management over the ESCs. It was observed that culturing these cell lines on NCad-Fc substratum can maintain the undifferentiated state and scattering morphology compare with other control substratum such as gelatin, fibronectin, laminin or poly-L- ornithine. P19 and MEB5 cells were differentiated effectively to neural lineage on this defined matrix in presence of retinoic acid supplemented with insulin-transferrin-selenium commercial preparation (ITS, Invitrogen). Interestingly, P19 cells showed higher level of
Later, the findings were extrapolated to MEF-dependent mouse embryonic stem cell ST1 and mouse iPSCs to evaluate whether the effect is restricted to specific pre-committed cell lines or it is globally applicable [65]. Since during EMT conversion ECad is downregulated and NCad is upregulated therefore a hybrid matrix of ECad-Fc and N-Cad-Fc was designed to exploit the stage-specific cadherin switching phenomenon. The concept was that, initially the ESCs and iPSCs would bind to ECad-Fc through cell-resident ECad, however, during and after neuroectoderm formation cadherin switching will cater for cellular NCad in place of ECad that would bind to NCad-Fc. The cadherin switching was experimentally confirmed in house during neural differentiation protocol (Fig. 3A), where Dkk-1, a Wnt signaling pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist were used for triggering neural differentiation. Specific markers for primitive ectoderm, primitive neural stem cells, neural stem and progenitor cells were checked. Along with, promisingly, the efficiency of neural progenitor differentiation from mouse ESCs on cadherin-Fc chimeric matrix was significantly higher compare to the cells cultured on other standard substratum as evaluated by the higher level of expression of neural progenitor marker Nestin gene products. Furthermore, the differentiated cells exhibited greater levels expression of βIII-tubulin (Tuj1) (Fig. 3B), microtubule associated protein 2 (MAP2), Pax6, and tyrosine hydroxylase but not GFAP, which is a marker of glial cell, signifying the presence of a lineage confined to neural cells.
Culturing of ESCs and iPSCs on ECad-Fc and NCad-Fc hybrid substratum not only developed scattered cell morphology as reported for ECad-Fc substratum but higher cell proliferation rate and enhanced differentiation efficiency were also noted. Along with these phenomena significant higher degree of homogeneity and enhanced differentiation efficiency were also observed, which is a remarkable advantage for harvesting target neuronal cells from
Some cells release 90 kDa fragment of soluble NCad (sNCad), and NCad-Fc was used to mimic sNCad response on neurite development [118]. Application of NCad-Fc by Doherty
Not only for neuronal population related regenerative medicine, NCad-Fc also showed potentials for application in other tissues as well, for example myogenesis related issues. Charrasse
10. Conclusion
To design an efficient biomaterial capable of maintaining and stewarding specific cell phenotypes critical for the development, homeostasis, differentiation, and regeneration of tissues, the material must have a high degree of selective recognition property to the desired cells. As well, such a biomaterial should be devoid of unexpected stimulation characteristics to the cells that can be hazardous to them or to the desired results of the protocols. Being the intrinsic component of cellular milieu, proteins are highly desirable molecules to be used in regenerative medicine and tissue engineering technology. Their 3D conformation made them perfectly fit in the cell-biology and ensuring that only specific function to the experimental cells has been achieved. The natural homeostasis properties of cells can adequately remove these proteins once they are used up without exerting any unnatural effect or stress to the cells. Expressing and purifying large protein with proper 3D conformation is extremely challenging therefore mimetic peptide technology has been becoming popular. These small peptide sequences represent small functional domain of the relevant proteins, albeit not with the native 3D structure of the parent protein molecule. While most cases they are being generated using artificial synthetic technology in test tubes, however, their purity, reproducibility and yield are major concerns for their confident application in stem cell technology. Additional limitations for mimetic peptides are (1) the restricted size of desired peptides, and (2) inability to provide native post-translational modifications, most of which are critical for proper bio-functionality of the relevant molecule. Therefore mimetic peptides cannot and do not behave identically as their natural parent protein. On the contrary, Fc-chimeric proteins can be generated with high degree of reproducibility with identical molecular properties using the natural cellular readout process from the DNA template. The additional stability of the target protein instigated by the presence of Fc domain is significantly advantageous for higher yield of the tailored chimeric protein. The intrinsic property of Fc domain to form homodimer is beneficial to keep the target chimeric protein in soluble form. On the other hand, the natural affinity of Fc domain to bind with Protein A or Protein G is a technical boon for convenient purification of the target protein without fusion of any secondary bait to the amino acid sequence, which often create complex situation for getting rid of them at the later stage of the processing to harvest only the desired designed protein. Directional binding of Fc domain with the polystyrene or hydrophobic surface and catering the functional protein outwards is also an intrinsic benefit for using this class of chimeric proteins for obtaining higher functional efficacy of the applied biomaterials. Since the specific homophilic interactions between cadherins mediate cell attachment therefore specific cadherin isoform-expressing cells can be purified by using the relevant cadherin-Fc biomaterial as surface-coating materials. For example, iPS cells express high level of ECad and neuronal cells express NCad therefore, by employing these matrices in different time points of differentiation protocol, the target cells can be purified
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