**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 effec‐ tively 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 differen‐ tiation 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 hepa‐ tocyte-producing cocktail resulting around 55% hepatic endoderm cells devoid of neuroecto‐ derm and mesoderm markers [30]. High level of (approximately 98%) ECad and developinghepatocyte 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 *de novo* hepatocyte-like cells. Practically, 92% albumin expressing cells were successfully harvested on ECad-Fc cookingplate without any harsh enzymatic treatment or mechanical cell sorting, which are usually detrimental for cells [30]. Therefore the technology can be successfully applied for quick and stress-free cell purification, which will be useful in regenerative medicine.

ECad interacts with cytoskeletal components *via* various intracellular molecules for example, α-catenin, β-catenin, and p-120 [38]. ECad-mediated signaling was found associated with cytoskeletal remodeling processes through Rho activation [41,63,76]. ECad has been estab‐ lished as an essential factor for maintaining typical colony-forming phenotype of ESCs and iPSCs. Recent studies, remarkably, revealed that forced expression of ECad can significantly enhance the effectiveness of relevant iPSCs-generation protocol [45]. A separate study revealed that ECad expression was enhanced upon treatment with small molecules resulting in enhanced efficiency for the relevant iPSC-generation protocol [77]. This enhanced productivity for iPSCs was successfully reproduced by the application of N terminal extracellular domains of ECad, which suggested that the phenomenon is mainly mediated by the extracellular functional domains of this protein [77]. Most importantly, ECad was sufficient to generate iPSCs with only three Yamanaka factors –KLF4, SOX2, and c-MYC from murine fibroblasts without OCT4 [45]. This study indicated that the spatial and mechanical input exerted by ECad has a critical role in driving cell fate. However, it is not clearly understood how ECad can compensate for OCT4. Since many studies showed that where it was possible to skip other factors of Yamanaka-cocktail for reprograming of somatic cells to iPSCs but OCT4 was hardly indispensable [10,70,78], further studies are warranted to determine the underlying mecha‐ nism. One potential explanation might be that ECad and KLF4 together initiated an early MET process of the experimental cells, and then SOX2 and KLF4 operated co-operatively to propel pluripotency genes to induce initiation of reprogramming [74]. The hypothesis is favored by the fact that cells those already express ECad, for example keratinocytes, can be reprogrammed more effectively and quicker because the MET process is not required [71]. Since the extracel‐ lular domain of ECad is adequate to produce ECad-mediated influences related to the reprogramming of somatic cells to iPSCs we have therefore assumed that ECad-Fc could significantly enhance the reprogramming efficiency. Our preliminary observation suggested that indeed co-transfection of ECad-Fc-expressing plasmid with Yamanaka factors enhanced reprograming efficiency of mouse fibroblast (unpublished). Enhanced reprogramming efficiency was further witnessed while the Yamanaka-cocktail-transfected starting cells were cultured on ECad-Fc-coated plate compare to gelatin-coated plate. However, further experi‐ ments are necessary for providing detail quantitative and qualitative information for these observations. Nonetheless this finding is highly promising regarding enhanced and efficient

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

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generation of iPSCs using a biomaterial as substratum.

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 instrumenta‐ tion, 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

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.
