**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 *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 generation of iPSCs using a biomaterial as substratum.

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

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

stress-free cell purification, which will be useful in regenerative medicine.

generation at a single cell level.

**7. ECad-Fc is a superior matrix for iPSC**

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

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.

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 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 *in vitro* culture without risk of additional contamination and cellular alteration, and therefore, is highly advantageous for application in regenerative medicine and tissue engineering to achieve desired therapeutic effect with minimal adverse consequences.

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

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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

Several proteins can interact with NCad *via* intracellular and extracellular domains and influence subsequent signaling pathways. The functions of NCad in controlling neurite outgrowth, synaptic plasticity and guidance in synapse formation have been proposed [111]. These functions may involve interaction with other membrane bound molecules, such as fibroblast growth factor receptor (FGFR), which was confirmed by blocking the FGFR by pharmacological inhibitor [112]. NCad directly interacts with FGFR *via* HAV epitope of FGFR with IDPVNGQ epitope of EC4 of NCad [112], and this interaction between NCad and FGFR can be of both ligand dependent and independent [113] suggesting wider cooperative

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,

critical involvement of NCad in the development of relevant tissues.

functional significance of this duo in relevant development and physiology.

expression of NCad [108].

## **8. NCad in cell biology**

N-cadherin (NCad) or neural cadherin is also known as Cadherin-2, which is encoded in human by *CADH2* gene [83,84]. Like ECad, it is also a cell-cell adhesion molecule composed of five extracellular cadherin domains, a transmembrane domain and a highly conserved cytoplasmic region. NCad can exist either as strand dimers or in an alternate monomeric form [85]. NCad typically forms homotypic homophilic interactions between two neighbouring cells for example, Sertoli cells and spermatides, and also heterotypic homophilic and heterophilic interactions, such as interaction between N- and R-cadherin in transfected L cells [86]; such interactions are Ca+2 dependent [87], and can be reversed by withdrawing Ca+2 from the system.

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 forma‐ tion 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].

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 *in vitro* culture without risk of additional contamination and cellular alteration, and therefore, is highly advantageous for application in regenerative medicine and tissue engineering to achieve desired therapeutic effect with minimal adverse consequences.

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

N-cadherin (NCad) or neural cadherin is also known as Cadherin-2, which is encoded in human by *CADH2* gene [83,84]. Like ECad, it is also a cell-cell adhesion molecule composed of five extracellular cadherin domains, a transmembrane domain and a highly conserved cytoplasmic region. NCad can exist either as strand dimers or in an alternate monomeric form [85]. NCad typically forms homotypic homophilic interactions between two neighbouring cells for example, Sertoli cells and spermatides, and also heterotypic homophilic and heterophilic interactions, such as interaction between N- and R-cadherin in transfected L cells [86]; such interactions are Ca+2 dependent [87], and can be reversed by withdrawing Ca+2 from the system.

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

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 forma‐ tion 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

**8. NCad in cell biology**

cells [95,96].

limb bud [103].

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 *via* intracellular and extracellular domains and influence subsequent signaling pathways. The functions of NCad in controlling neurite outgrowth, synaptic plasticity and guidance in synapse formation have been proposed [111]. These functions may involve interaction with other membrane bound molecules, such as fibroblast growth factor receptor (FGFR), which was confirmed by blocking the FGFR by pharmacological inhibitor [112]. NCad directly interacts with FGFR *via* HAV epitope of FGFR with IDPVNGQ epitope of EC4 of NCad [112], and this interaction between NCad and FGFR can be of both ligand dependent and independent [113] suggesting wider cooperative functional significance of this duo in relevant development and physiology.

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.

level of expression of neural progenitor marker Nestin gene products. Furthermore, the differentiated cells exhibited greater levels expression of βIII-tubulin (Tuj1) (Fig. 3B), micro‐ tubule associated protein 2 (MAP2), Pax6, and tyrosine hydroxylase but not GFAP, which is a

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**Figure 3.** NCad-Fc, and ECad-Fc promote directed differentiation of target lineage from iPSCs. (A) Western blot data revealed ECad to NCad switching occurs during neuronal differentiation. The expression level was normalized using house-keeping gene, β-actin. (B) βIII-tubulin expression was significantly higher on E/NCad-Fc matrix compared to gel‐

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 *in vitro*

atin.

marker of glial cell, signifying the presence of a lineage confined to neural cells.

#### **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-Lornithine. P19 and MEB5 cells were differentiated effectively to neural lineage on this defined matrix in presence of retinoic acid supplemented with insulin-transferrin-selenium commer‐ cial preparation (ITS, Invitrogen). Interestingly, P19 cells showed higher level of *Neurog1* expression on NCad-Fc-coated surface compare with gelatin-coated surface. Additionally, MEB5 differentiated on NCad-Fc matrix, compared to fibronectin-coated surface, showed complete neuronal differentiation phenomena and significantly higher expression levels of neural markers, such as *Neurog1* and *MAP2*. These results clearly suggested the superiority of NCad-Fc substratum over the other experimental substratum for neuronal differentiation process.

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), micro‐ tubule 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.

morphogenesis and maturation, and is suggesting the possibility of exploiting this gene

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-Lornithine. P19 and MEB5 cells were differentiated effectively to neural lineage on this defined matrix in presence of retinoic acid supplemented with insulin-transferrin-selenium commer‐ cial preparation (ITS, Invitrogen). Interestingly, P19 cells showed higher level of *Neurog1* expression on NCad-Fc-coated surface compare with gelatin-coated surface. Additionally, MEB5 differentiated on NCad-Fc matrix, compared to fibronectin-coated surface, showed complete neuronal differentiation phenomena and significantly higher expression levels of neural markers, such as *Neurog1* and *MAP2*. These results clearly suggested the superiority of NCad-Fc substratum over the other experimental substratum for neuronal differentiation

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

**9. NCad-Fc as biomaterial in regenerative medicine**

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

product for regenerative medicine.

process.

**Figure 3.** NCad-Fc, and ECad-Fc promote directed differentiation of target lineage from iPSCs. (A) Western blot data revealed ECad to NCad switching occurs during neuronal differentiation. The expression level was normalized using house-keeping gene, β-actin. (B) βIII-tubulin expression was significantly higher on E/NCad-Fc matrix compared to gel‐ atin.

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 *in vitro*

system that can later be applied for regenerative medicine protocol. Although EB-based protocols are being relatively well-practiced for neural differentiation, however, the inconsis‐ tency of the embryoid body (EB) size and shape, and the asynchronous distribution of growth factors throughout the EBs give rise to heterogeneous products. Besides, monitoring cell morphology during differentiation process for EB-based differentiation protocols is incon‐ venient. Culturing ESCs or iPSCs in scattered single cell condition, on the contrary, can effectively overcome these issues. Interestingly, this blend of Cadherin-Fc matrices maintained a complete homogeneous cell population for murine ESCs and iPSCs for several passages. Highly homogeneous population of primitive ectoderm and neural progenitor cells were routinely generated on such a hybrid-type cooking-plate [65]. Enriched population of neuro‐ ectoderm progenitor cells can be obtained within 4 to 6 days by using E/NCad-Fc based monolayer-forming ESCs and iPSCs culture protocol and standard neurogenic cocktail treatment, which is a great advantage for quick generation of the target cells for application in regenerative medicine.

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 regenera‐ tive 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 limita‐ tions 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 biofunctionality 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 *in situ* without the necessity of any harsh enzymatic or mechanical treatments. Some of these chimeric proteins are commercially available for application and some are in pipeline, which can be obtained from our laboratory under proper regulatory affairs. Collec‐ tively, Fc-chimeric protein-based biomaterials provide distinct advantages for overcoming many existing challenges in stem cell technology and significantly advancing the regenerative

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medicine and tissue engineering field towards practical application.

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 *et al.* with cerebral neurons showed that NCad-Fc initiated neurite outgrowth in a FGF receptor depend‐ ed manner [111,119] suggesting that NCad-Fc can be utilized for controlling FGF receptor signaling pathway to facilitate relevant neuronal development events. Using mouse E12.5 ventral spinal cord explants as a convenient model Marthiens *et al*. showed that the axons formed contacts along the axon-shaft by long filopodia-like processes on NCad-Fc matrix [120]. They further showed that growth cones preferentially interact with cad-11 or NCad-Fc when progressing on this substratum whereas it differs on laminin. This study proved direct involvement of cadherin-11 and NCad in peripheral nervous system establishment from embryonic tissues [120].

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 *et al*. used NCad-Fc to mimic NCad binding effect for myogenic differentiation [121]. They showed that NCad-Fc based NCad–dependent cell–cell adhesion triggers RhoA GTPase activity, which is essential for myogenic differentiation. Activity and expression of SRF, a transcription factor that binds to the promoter regions of muscle-specific genes [122,123] and controls the expression of MyoD, is controlled by RhoA. In turn, MyoD binds to the promoter region of skeletal muscle activating genes in mesenchymal cells and convert them to skeletal myoblasts [124,125,126]. These findings demonstrated that N-cadherin–dependent adhesion event that regulates the RhoA/SRF pathway to trigger myogenesis can be harnessed by NCad-Fc matrix and therefore such technology is holding great promises for using in relevant regenerative medicine protocols.

## **10. Conclusion**

To design an efficient biomaterial capable of maintaining and stewarding specific cell pheno‐ types 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 regenera‐ tive 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 limita‐ tions 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 biofunctionality 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 *in situ* without the necessity of any harsh enzymatic or mechanical treatments. Some of these chimeric proteins are commercially available for application and some are in pipeline, which can be obtained from our laboratory under proper regulatory affairs. Collec‐ tively, Fc-chimeric protein-based biomaterials provide distinct advantages for overcoming many existing challenges in stem cell technology and significantly advancing the regenerative medicine and tissue engineering field towards practical application.

system that can later be applied for regenerative medicine protocol. Although EB-based protocols are being relatively well-practiced for neural differentiation, however, the inconsis‐ tency of the embryoid body (EB) size and shape, and the asynchronous distribution of growth factors throughout the EBs give rise to heterogeneous products. Besides, monitoring cell morphology during differentiation process for EB-based differentiation protocols is incon‐ venient. Culturing ESCs or iPSCs in scattered single cell condition, on the contrary, can effectively overcome these issues. Interestingly, this blend of Cadherin-Fc matrices maintained a complete homogeneous cell population for murine ESCs and iPSCs for several passages. Highly homogeneous population of primitive ectoderm and neural progenitor cells were routinely generated on such a hybrid-type cooking-plate [65]. Enriched population of neuro‐ ectoderm progenitor cells can be obtained within 4 to 6 days by using E/NCad-Fc based monolayer-forming ESCs and iPSCs culture protocol and standard neurogenic cocktail treatment, which is a great advantage for quick generation of the target cells for application in

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

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 *et al.* with cerebral neurons showed that NCad-Fc initiated neurite outgrowth in a FGF receptor depend‐ ed manner [111,119] suggesting that NCad-Fc can be utilized for controlling FGF receptor signaling pathway to facilitate relevant neuronal development events. Using mouse E12.5 ventral spinal cord explants as a convenient model Marthiens *et al*. showed that the axons formed contacts along the axon-shaft by long filopodia-like processes on NCad-Fc matrix [120]. They further showed that growth cones preferentially interact with cad-11 or NCad-Fc when progressing on this substratum whereas it differs on laminin. This study proved direct involvement of cadherin-11 and NCad in peripheral nervous system establishment from

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 *et al*. used NCad-Fc to mimic NCad binding effect for myogenic differentiation [121]. They showed that NCad-Fc based NCad–dependent cell–cell adhesion triggers RhoA GTPase activity, which is essential for myogenic differentiation. Activity and expression of SRF, a transcription factor that binds to the promoter regions of muscle-specific genes [122,123] and controls the expression of MyoD, is controlled by RhoA. In turn, MyoD binds to the promoter region of skeletal muscle activating genes in mesenchymal cells and convert them to skeletal myoblasts [124,125,126]. These findings demonstrated that N-cadherin–dependent adhesion event that regulates the RhoA/SRF pathway to trigger myogenesis can be harnessed by NCad-Fc matrix and therefore such technology is holding great promises for using in relevant

To design an efficient biomaterial capable of maintaining and stewarding specific cell pheno‐ types critical for the development, homeostasis, differentiation, and regeneration of tissues,

regenerative medicine.

embryonic tissues [120].

regenerative medicine protocols.

**10. Conclusion**
