Cadherin-based biomaterials:Inducing stem cell fate towards tissue construction and therapeutics

Yn Zhng ,Zheng Qin ,Zhnyun Qu ,Min Ge ,Jun Yng,*

a The Key Laboratory of Bioactive Materials,Ministry of Education,College of Life Science,Nankai University,Tianjin,300071,China b State Key Laboratory of Medicinal Chemical Biology,Nankai University,Tianjin,300350,China

Keywords:Cadherin Mimic cell-cell interactions Biomaterials Stem cell Tissue construction

ABSTRACT The precise regulation of cell fate by biomaterials is one of the core focuses in tissue engineering and regenerative therapeutics.Cell fate is controlled by the niche signaling cues,including growth factors/morphogens,direct cell-cell contact,and extracellular matrix components.Here,we discuss how to bio-mimic cell-cell interaction by tailoring the interface of biomaterials to design effective scaffolds.Cadherins,as a key cell membrane protein family mediating the intercellular adherens junction,have attracted extensive attention in the functional modification of biomaterials.Therefore,cadherin-based biomaterials,their ability to modulate stem cell fate,and their applications in regenerative medicine are reviewed.Furthermore,we provide an outlook for exploiting the bio-mimic biomaterials in advanced cell therapeutics.

1.Introduction

Given the severe shortage of donor organs in solid organ transplant and engineered human tissues for solid organ transplantation and patient-focused drug development,respectively,the construction of threedimensional (3D) tissue and the regulation of stem cell fate based on biomaterial scaffolds have always been hotspots in the fields of regenerative medicine [1].Nowadays,highly mimicked tissue or organs can be engineered with the advances of stem cell research,intelligent biomaterials,and biofabrication strategies [2].Among them,biomaterials,as the template of the extracellular microenvironment delivering chemical,physical,and biological signals,are the core focus in simulating the stem cell niche and supporting the complicated processes of tissue development and repair [3].

There are three basic elements of the stem cell niche:growth factors/morphogens,direct cell-cell contact,and extracellular matrix(ECM) components (Fig.1) [4].Growth factors/morphogens,as important biochemical cues,are dynamically produced and coordinated in time and space during embryonic development and wound healing[5].Intercellular interactions coordinate cell recognition and classification,cellular signal transduction,and the generation of functional cell polarity,which are essential for the development of stem cells [6].ECM has been primarily understood as the adhesive substrate that anchors cells within their microenvironments [7].Multiple bio-artificial ECMs based on biomaterials are currently designed,synthesized and used in tissue engineering scaffolds,which have the advantages of bioactivity,biocompatibility,and similar mechanical properties as native tissues[8].Many growth factors/morphogens and ECM proteins are immobilized in the scaffolds,which is beneficial (i) to increase the local concentration by continuously releasing and hindering diffusion and(ii) to enhance cell adhesion [9].They can spatially integrate different types of signals in a specific microenvironment in vivo and have been considered as an important determinant of stem cell fate [10].However,according to the principle of bionics,the low cell density at the initial stage of establishing the complex of the cells-tissue engineering scaffolds would limit the biological effect mediated by cell-cell interactions,which is still an important challenge in the field of tissue engineering [11].

The extracellular microenvironment transforms from the initial state rich in cell-cell interactions to cell-ECM interactions in the process of tissue development in vivo (Fig.2A) [12].Ultrastructural studies of intercellular interactions have revealed that cells are directly connected by multiple junctional complexes,including tight junctions,adherens junctions,gap junctions,and desmosomes(Fig.2B),which is necessary for the division,self-renewal and differentiation [13].Among them,adherens junctions make key contributions to embryogenesis and tissue homeostasis [6].As a core protein of adherens junctions,the cadherin contributes to the dynamic regulation of adhesive contacts that are associated with diverse morphogenetic processes,such as epithelialmesenchymal transformation [14],the formation and maintenance of epithelial polarity [15],large-scale cell migration [16],and synaptic and vascular formation in mature tissues[17].In the past 10 years,the studies on cadherins,as a large family of intercellular adhesion molecules,have made great progress in tissue engineering.It is worth noting that new biomaterials based on cadherin have been developed to trigger cell-cell interactions when cells attach to the scaffolds,which stimulates the activation of adherens junctions to improve cell biological functions[18,19].In light of the increasing applications of cadherin in new areas of tissue engineering and the increasing knowledge on structural characteristics of cadherin,a more detailed review is now warranted.

Fig.1.Composition of the stem cell niche.

Fig.2.Role of cell-cell interactions during tissue development.A.Schematic representation of the evolution of the mechanical microenvironment during mesenchymal development [12].B.Classification and functions of cell-cell interactions.

In this review,we first briefly introduce the structure and role of cadherin,which are closely related to its application in tissue engineering.Next,we review the applications of different types of cadherin-based biomaterials in tissue engineering.Finally,we look at further developments in cell therapy.

2.Cadherins and tissue dynamics

There are more than 100 members with similar sequence and structure,mediating the homotypic cell patterning behavior in specific tissues [20].Cadherins are Ca2+dependent cell-cell adhesion molecules,whose extracellular domain consists of five extracellular cadherin(EC) repeats [21].According to the presence or absence of the HAV(His-Ala-Val)cell recognition sequence in the EC1 domain,the classical cadherins are classified into type I (such as E-and N-cadherins) and type II cadherins (such as cadherin-11 and cadherin-6B) [22].The ECs mediate homophilic interactions between cells expressing identical cadherins and selective heterophilic interactions between cells expressing different cadherins,which induce the cell segregation during tissue morphogenesis and drive cell aggregation [23].These processes not only map exclusively to protein-level differences in cadherin adhesion,but are highly related to the graded differences between the homophilic and heterophilic cadherin dimerization affinities [24].

The intracellular regions of cadherins interact with several intracellular proteins (α-catenin,β-catenin,and p-120 catenin) to form cadherin-catenin complexes [25].Recent research works revealed that the cytoplasmic regions of cadherins bind directly to β-and p120-catenins,and indirectly to the actin filaments(F-actin)through α-catenin[26].They mediate the mechanotransduction at cadherin adhesions,which enables cells to sense and respond to the physical changes in the extracellular microenvironment[27].The dynamic link of the cadherincatenin complex to F-actin contributes to the mechanical coupling that drives many morphogenetic events and tissue repair.Moreover,there is bidirectional regulation between cadherin and several growth factors,such as vascular endothelial growth factor (VEGF),fibroblast growth factor (FGF),and epidermal growth factor (EGF).The growth factors can influence cadherin endocytosis,while cadherin endocytosis can modulate growth factor signaling [28].The cadherins can activate multiple pathways,including the high-pressure-preferred orientation(Hippo),wingless/integrated (Wnt),receptor tyrosine kinase (RTK),Rho-family of small GTPases,mitogen-activated protein kinase(MAPK),nuclear factor-kappa-B (NF-κB),yes-associated protein 1 (YAP1),and Hedgehog pathways,which not only maintain tissue cohesion but also allow cell rearrangement during organ development (Fig.3) [29].Therefore,cadherin can not only mediate cell adhesion but also actively tune cell signaling in response to multiple extracellular cues.

Fig.3.Cadherin-catenin complexes and their role in the regulation of intracellular signal transduction [127].

During in vitro culture of stem cells,the proliferation,secretion,differentiation,and other cell behaviors are related to the types and expression levels of cadherins.For instance,E-cadherin is crucial for the pluripotency and generation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),and has been proved to replace OCT4 during somatic cell reprogramming [30].The expression level of N-cadherin can distinguish the reserved versus primed states of hematopoietic stem cells,mesenchymal stromal cells,etc.,and regulate the maintenance,proliferation,and differentiation of neural progenitor cells during development[31].Furthermore,T Passanha et al.reported that the proliferating mesenchymal stem cells (MSCs) in monolayer culture expressed cadherin-2 and switched to cadherin-11 over time,which was related to the differentiation tendency of adipogenic or osteogenic differentiation [32].Together,the quantity and affinity of cadherins could be used to control tissue segregation and cell assembly through the specification of the types and relative intensities of cell-cell adhesions [33].

3.Cadherin-based biomaterials for cell fate regulation

3.1.Mimicking cell-cell interactions at the cell-matrix interface

Cell-ECM adhesion has been reported to be the determinant of stem cell fate during the in vitro culture [34].Surface modification of the biomaterials by intercellular interaction molecules would make the control of cellular differentiation more applicable and cell-specific.Several biomaterials that introduce direct cell-cell contact onto the surface of matrices are listed in Table 1.In addition,another successful strategy was discovered to incorporate ordinarily lipid-bound molecules on the cell membrane to the surface of biomaterials,simulating the signals provided by adjacent cells [35].For instance,Wall et al.explored the immobilization and/or oligomerization of Shh molecules on biomaterials and found that they effectively stimulate angiogenesis[36].The ligands of the Notch pathway,one of the classic signaling pathways mediating cell-cell interactions,have also been used for the modification of biomaterials to culture neural stem cells and direct the differentiation of bone marrow-derived hematopoietic stem cells [37].

Akaike's group first envisioned the application of E-cadherin as a novel cell-recognizable biomaterial by fusing the extracellular domain of E-cadherin with the Fc domain of immunoglobulin G (IgG) for stem cell expansion and differentiation [49].The Fc domain of IgG can bind with an appropriate surface of biomaterials via hydrophobic interaction,and the fused cadherin stretches out directionally to offer interaction with a suitable partner,including cadherins and growth factor receptors on the membrane of directly contacted cells [50].Over the last decade,Akaike's group has constructed several Fc-chimeric proteinbased biomaterials and applied them for the surface modification of cell culture plates to(i)establish xenogeneic-agent free monolayer stem cell culture and their maintenance,(ii)enhance the directed differentiation of stem cells to specific lineages and the non-enzymatic on-site one-step purification of target cells [51].To date,a variety of cadherin-based biomaterials have been developed to achieve cell fate direction in vitro and in vivo (Table 2).Out of these,our efforts have mainly focused on the biomaterials mimicking the cell-cell interactions,and we have constructed a series of human cadherin-Fc fusion proteins (hE/N/VEcadherin-Fc) with high binding ability to hydrophobic surfaces for the culture and differentiation of stem cells.

Table 1 Several biomaterials mimicking cell-cell interactions.

Table 2 Different cadherin-based biomaterials for regulating cell fate.

3.2.E-cadherin-based biomaterials

Epithelial cadherin (E-cadherin) is considered as the prototype cadherin due to its early identification and exhaustive characterization.It functions in the cell rearrangements at both local and tissue levels during epithelial morphogenesis and in the maintenance of epithelial cell morphology during adulthood [82].The homophilic ligation of Ecadherin regulates cell growth and the density-dependent contact inhibition as well as the cellular response to epidermal growth factors independently of other cell interactions[83].E-cadherin expression can be considered as one of the performance indicators of feeder cells in the culture of ESCs[84].Nagaoka et al.attempted to modify the surface of culture plates with mouse E-cadherin-Fc,and found that this extended the period of maintenance of the pluripotency and the monolayer phenotype of murine ESCs without any feeder cells,which was significantly advantageous for faster growth and higher transfection effi-ciency[85].The human E-cadherin-Fc(hE-cadherin-Fc)constructed by our group was able to effectively self-assemble into a protein monolayer with nano-structures (5–7 nm in height on average) on surfaces ofpolystyrene (PS) plates,which significantly enhanced the adhesion,proliferation,and paracrine functions of human MSCs (Fig.4) [69].Meanwhile,the E-cadherin-Fc-modified plates were applied for the induction of directed differentiation of ESCs to epithelial tissue.The results showed that E-cadherin-Fc substratum favored the progressive differentiation of ESCs into cells with features of definitive endoderm,hepatic progenitor cells,and finally phenotypical as well as functional hepatocyte-like cells [78].

Recently,the incorporation of microparticles into multicellular aggregates has advanced the development of complex 3D tissue/disease models in tissue engineering and drug screening platforms [86].Both the traditional application of the microparticles,i.e.,drug delivery reservoirs and microcarriers in bioreactors,and this new strategy require biocompatible (and/or targeted) interaction of microparticles with the cell membrane.E-cadherin is the major cause of stem cell aggregation and is tightly linked to several major signaling networks that control cell apoptosis,self-renewal,differentiation,and gene expression in response to external stimuli [87].It can be speculated that surface modification with E-cadherin on the microparticles strengthens the contact and recognition of microparticles with target cells.

Kutsuzawa et al.embedded E-cadherin in carbonate-apatite DNA carriers,which improved their affinity interactions with the surface of ESCs and accelerated the transgene delivery significantly compared with a commercially available lipofection system [88].Moreover,our results indicated that the hE-cadherin-Fc-coated poly-(lactic-co-glycolic acid) (PLGA) microparticles (hE-cad-PLGAs) (i) were able to distribute more evenly within the aggregates and (ii) improved the proliferation and secretion of MSCs through upregulating the phosphorylation of EGF receptors and activating the Akt and ERK1/2 signaling pathways in the MSCs [60].The incorporation of hE-cad-PLGAs also induced the expression of a large number of genes,especially long non-coding RNAs and genes encoding transcription factors,mediating posttranscriptional regulation,modulating the organization of protein complexes,and regulating cell-cell signaling.Under the stimulation of hepatocyte directional differentiation medium,hE-cad-PLGAs in MSC aggregates significantly promoted endoderm differentiation,and strongly supported the hepatic differentiation in vitro and in vivo(Fig.5).Furthermore,the differentiated MSC/hE-cad-PLGA aggregates showed better hepatic-specific functions compared with MSC aggregates,including the expression of cell polarity as well as energy and drug metabolism markers [77].These findings suggest that the combination of cadherin-based biomaterials with other culture conditions may produce additional biological effects that possess greater applicability for tissue construction.

3.3.N-cadherin-based biomaterials

In the process of embryogenesis,N-cadherin mediates the interaction between cells through activating several pathways,like Rho GTPase and tyrosine kinase signaling,and plays a crucial role in mesenchymal chondrogenesis and neural crest development [89].Furthermore,N-cadherin participates in the lineage determination process during the in vitro differentiation of stem cells [90].As mentioned above,the type I classical cadherins contain an evolutionarily conserved HAV sequence.This sequence provides a homologous cell adhesion recognition site that mediates cell adhesion in N-cadherin,but is not involved in E-cadherin-mediated adhesion [91].Moreover,among these cadherin subtypes,only N-cadherin shows very little sequence divergence between species [92].These facts may increase the enthusiasm for the extraction and applications of N-cadherin sequence features in peptide/protein hybrid materials.

Fig.4.Surface modification of hE-cadherin-Fc for culturing MSCs [69].A.Schematic representation of surface modification with hE-cadherin-Fc for the culture of MSCs.B.Topography of hE-cadherin-Fc-modified PS surfaces and surface roughness investigated by atomic force microscopy.C.Western blot results of the expression levels of E-cadherin and α-/β-catenin/p120-catenin in MSCs cultured on PS and collagen-and hE-cadherin-Fc-modified surfaces for 4 and 48 h.D Western blot results revealing the activation of EGFR,Akt and ERK pathways in MSCs cultured on PS and collagen-and hE-cadherin-Fc-modified surfaces for 4 and 48 h.

Bian et al.polymerized HAV mimetic peptides into HA hydrogels,and found that this significantly enhanced the early differentiation of MSCs into cartilage and the production of the cartilage-specific matrix(Fig.6)[93].Thereafter,Kwon et al.optimized the timing and duration of HAV peptide presentation and regulated the MSC chondrogenesis and cartilage matrix production within hydrogels [94].Additionally,Cimenci et al.modified HAV peptide onto nanofibers,which promoted chondrogenic differentiation of MSCs in conjunction with chondrogenic factors as a synthetic ECM system [79].As the N-cadherin specificity and its crosstalk with fibroblast growth factor signaling are determined by amino acids Asp-Ile(DI),which flank the HAV motif[95],Lim et al.applied His-Ala-Val-Asp-Ile (HAVDI) in polyethylene glycol dimethacrylate hydrogels,and observed its concentration-dependent effects on mouse ESC behavior,including cell survival and neural differentiation[79].Therefore,although mimetic peptides provide convenient and diverse methods for the modification of biomaterials,it is still impossible to completely replace the fusion proteins constructed with the full-length sequences of the cadherin extracellular domain.

During the development of the nervous system,N-cadherin promotes cell adhesion in neural progenitor cells (NPCs) and neurons and regulates the balance between NPC self-renewal and differentiation,thereby controlling the size and structure of the nervous system [31].Lambert et al.produced a dimeric N-cadherin-Fc chimera to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction in 2000 [75].After that,Utton et al.found that the immobilized N-cadherin-Fc promoted neurite adhesion and outgrowth following affinity capture to a tissue culture substrate,whereas soluble N-cadherin-Fc could also stimulate neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein [96].Akaike's group constructed a mouse N-cadherin-Fc fusion protein and was the first to apply it for the culture and differentiation of ESCs and iPSC-derived NPCs[61].The results showed that N-cadherin-Fc immobilized on the culture plates promoted the differentiation of P19 and MEB5 cells into neural cells at a single cell level when induced with proper conditions(Fig.7)[61].Moreover,the combined application of N-cadherin-Fc and other adhesion factors,including E-cadherin [81] and L1 [97],also showed a significant synergistic effect on promoting neural differentiation,neurite extension and neuroprotection of ESCs and iPSCs.

In terms of MSCs,it was reported that N-cadherin adhesive interactions modulate the micro/nanotopography induced indirect mechanotransduction,and strongly affect the secretion profile as well as cellular fate commitment [98].Our data confirmed that there were significant differences in protein secretion of MSCs cultured on hE-and N-cadherin-Fc fusion proteins.Additionally,the differentiation effi-ciency of MSCs to chondrocytes on hN-cadherin-Fc was even stronger than that of the commonly used collagen matrix (data unpublished).Compared with ESCs and iPSCs,MSCs are much more easily accessible.We believe that hN-cadherin-Fc coupled with MSCs would provide an exciting solution to the problem of cartilage repair.

3.4.VE-cadherin-based biomaterials

Rapid and sufficient functional angiogenesis is one of the prerequisites for constructed tissues to survive and exert biological functions in vivo [99].The interaction between endothelial cells and the artificial matrix plays an important role in regulating the formation of new blood vessels [100].Vascular endothelial cadherin (VE-cadherin)is a strict endothelial-specific adhesion molecule located at the junction between endothelial cells,which is essential for maintaining and controlling endothelial cell contact [101].In addition,VE-cadherin is involved in the regulation of various cellular processes,such as cell proliferation and apoptosis [102] as well as mechanical signaling[103],and its crosstalk with the vascular endothelial growth factor receptor plays a vital role in vascular permeability and angiogenesis[104].Furthermore,the expression of VE-cadherin mediates the epigenetic regulation of endothelial gene expression extensively,and upregulates the genes with key roles in endothelial differentiation and stability [105].

Fig.5.Construction of MSC aggregates incorporating hE-cad-PLGAs inducing the hepatic differentiation of MSCs [60,77].A.Schematic representation of the formation of MSC/hE-cad-PLGA aggregates and the stepwise protocol of induced differentiation of aggregates to hepatocyte-like cells.B.Hematoxylin-eosin staining images of MSC,MSC/PLGA,and MSC/hE-cad-PLGA aggregates.C.CCK-8 assay of MSC,MSC/PLGA,and MSC/hE-cad-PLGA aggregates after being cultured for 1,3,and 5 days.D.Immunofluorescence staining of E-cadherin of MSC,MSC/PLGA,and MSC/hE-cad-PLGA groups after having differentiated for 21 days;E.Flow cytometry analysis of albumin expression of hepatocyte-like cells in MSC,MSC/PLGA,and MSC/hE-cad-PLGA groups after having differentiated for 21 days.F.Immunofluorescence staining of CYP3A4 of hepatocyte-like cells in MSC,MSC/PLGA,and MSC/hE-cad-PLGA groups after having differentiated for 21 days.

Our group biosynthesized and characterized an hVE-cadherin-Fc fusion protein and applied it as the cell culture matrix for the first time.The human umbilical vein endothelial cells(HUVECs)cultured on hVEcadherin-Fc showed a fibroblast morphology,and their adhesion,proliferation,and migration were also significantly improved,which was mediated by the activation of the VE-cadherin/catenin complex and the intracellular signaling pathway of focal adhesion kinase(FAK)/Akt/Bcl-2 (Fig.8).Moreover,the adhesion of human vascular smooth muscle cells on the hVE-cadherin-Fc surface was significantly limited compared with HUVECs,which should be beneficial to achieve selective cell adhesion and proliferation on the biomaterials during vascularization[57].Yang et al.designed a recombinant mussel foot protein (Mfp-5)fused with the VE-cadherin extracellular domain EC1-2,which increased the adhesion and endogenous VE-cadherin expression as well as the endothelial eNOS content in HUVECs,but there was no significant difference in the number of captured SMCs between VE-M and other substrates [106].

The differentiation and organization of endothelial cells is an early step in the formation of the extraembryonic and intraembryonic vasculature.In mouse embryos,VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5),whereas the expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells at E9.5 [107].VEcadherin is a coordinator of the key cellular signaling cascades controlling the behavior of endothelial cells,which runs through the entire process of tissue morphogenesis and regeneration [108].We have evaluated the effects of hVE-cadherin-Fc on regulating the differentiation of ESCs.The results showed that hVE-cadherin-Fc could activate the Stat 3 signaling pathway in ESCs,leading to the upregulation of the stemness-related markers SSEA-1 and alkaline phosphatase.Moreover,hVE-cadherin-Fc recovered the expression of Oct4,c-Myc,Nanog,Sox 2,Tbx3,and Klf4 in differentiated ES cells and enhanced the pluripotency of ES cells [109].

Fig.6.Application of an N-cadherin mimic peptide in the induction of cartilage differentiation of MSCs[128].A.Hydrogel design to harness cell-cell interactions by using an N-cadherin mimic peptide.B and C.GAG and total collagen content normalized by(B)sample wet weight(C)equilibrium modulus indicated that N-cadherin bio-mimetic peptides enhance neocartilage formation by MSCs in vitro.D.and E.Immunohistochemical staining results for type II collagen(COL2)of MSC-laden HA hydrogel constructs after 28 days of in vitro culture in differentiation media.＊P ＜0.05 vs.Scrambled or Control group (n=4).Scale bars=50 μm.

Fig.7.Application of N-cadherin-Fc in the induced neural differentiation of MSCs [61].A.The cloning of N-cadherin-Fc;B.The scheme of differentiation of MEB5 cells;C.The expression of Nestin (red) and βIII-tubulin (green) after the differentiation induction of MEB5 cells on different matrices was analyzed by immunostaining.Scale bar=50 μm;D.mRNA levels of specific neural markers(Nestin,Neurog1,Map 2,and Gfap),as was detected by RT-PCR,after the differentiation induction of MEB5 cells on fibronectin or 10 μg/ml N-cadherin-Fc-coated surfaces.

Fig.8.Application of VE-cadherin-Fc in the culture of HUVECs [57].A.hVE-cadherin-Fc activated intracellular signaling in HUVECs.B.Western blotting of total cellular Vwf protein in HUVECs after culturing on different matrices for 4,24,and 48 h.C.The release of NO from HUVECs cultured on different matrices for 4,24,or 48 h.D.VE-cadherin distributed in HUVECs on different matrices at 24 h,as analyzed by confocal laser scanning microscopy.Scale bars=25 μm.

In the fundamental processes of regulating cell behavior and driving organization with engineered biomaterials,the bionic signals from vascular endothelium seem to be indispensable [110].Morini et al.found that the expression and clustering of VE-cadherin at cell-cell contacts relieved the inhibitory effect of the Forkhead box protein O1(FoxO1)/β-catenin complex on the expression of the tight junction protein claudin-5,which inferred that VE-cadherin could cause the crosstalk between different cell-cell interactions [111].Based on our previous results,our group has attempted to introduce the endothelial bionic signal of hVE-cadherin-Fc into the formation of liver organoid.The optimized combination of multiple cadherins(hE-/VE-cadherin-Fc)and inducing factors significantly promoted the simultaneous directional differentiation of hMSCs into hepatocyte-,cholangiocyteand endothelium-like cells (data unpublished).We believe that these cadherin-based biomaterials hold great promise for use in regulating stem cell fate during tissue construction.

4.Cadherin-based biomaterials modulate cellular matrix mechanosensing

In addition to biochemical motifs or signals,the substrate stiffness of the ECM has long been known to drive stem cells to differentiate into the desired phenotype or to regulate somatic cell reprogramming[112].Zoldan et al.proposed that,the scaffolds with a stiffness greater than 6 MPa are conducive to maintain the undifferentiated state of hESCs,whereas the scaffolds with an elastic modulus of 1.5–6 MPa,0.1–1 MPa,and ＜0.1 MPa would lead the hESCs to differentiate into mesoderm-,endoderm-,and ectoderm-like cells,respectively [113].Cells rely on adhesion receptors,such as integrins and cadherins,to probe and make sense of the milieu of the mechanical microenvironment.Force is transmitted between integrin and cadherin through the actin network,thereby forming the mechanical structure of the cell,and the distinct site-specific tyrosine phosphorylation of vinculin distinguishes the mechanical transductions in cadherin-and integrin-related pathways[114].

Moreover,there is reciprocal regulation between the cadherin-and integrin-related pathways,and these crosstalk signals can also be modulated by the cadherin-based biomaterials.A mechanosensory complex comprising VE-cadherin,PECAM1,and VEGFR signals upstream of integrins has been proved to activate PI3K and Rac in response to shear flow,while the activation of the FAK-Rac signaling pathway at focal adhesions in response to substrate stiffness stimulates N-cadherin gene expression [114].In our previous study,the hVEcadherin-Fc coated on the polystyrene plate significantly triggered the phosphorylation of FAK (P-FAK) at Tyr397 and of Akt at Ser 473,and promoted the protein interactions between FAK and endogenous VEcadherin in HUVECs [57].Moreover,Mui et al.showed that mechanosensitive mediators of substrate stiffness sensing in focal adhesions(e.g.,focal adhesion kinase,FAK) could interact with and control the activation of Rac 1,altering cellular stiffening and controlling N-cadherin expression [115].

The activity of transcription factors (i.e.,YAPs),is correlated with the stability of the actin cytoskeleton and cell tension,and is the core molecule of the above mechanical transduction pathways (Fig.9A)[116,117].Therefore,our group has constructed a thiolated hyaluronic acid (PAMAM/HS-HA,P/H) hydrogel functionalized by hVE-cadherin-Fc that significantly elevated the paracrine and immunomodulation of hMSCs,which promoted the endothelial differentiation of hMSCs,host cell recruitment,and phenotype transformation of macrophages from M1 to M2,and synergistically led to enhanced vascularization.In this culture system,the hVE-cadherin-Fc-modified P/H hydrogel stimulated the FAK/KAT signaling pathways in MSCs and inhibited the amount of YAP entering the nuclei of MSCs (nuc/cyt=0.6 ± 0.1) compared with P/H (nuc/cyt=1.1 ± 0.1) and RGD (an integrin adhesion sequence)-P/H (nuc/cyt=1.0 ± 0.1) hydrogels (Fig.9B and C) [19].Cosgrove et al.designed a hyaluronic acid hydrogel system and showed that,across a physiological range of ECM stiffness,the N-Cadherin mimetic peptides altered the interpretation of ECM stiffness and subsequently changed cell proliferation and differentiation(Fig.9D and E)[12].Although the role of this mechanism in the complex and timeevolving tissue construction remains to be determined,these findings confirm the application potential of cadherin-based biomaterials to (i)modulate the cellular response to substrate stiffness and(ii)direct stem cell behavior.

5.Limitations and future perspectives

Of the stem cells mentioned above,MSCs have emerged as an extremely promising seed cell in tissue engineering and therapeutic agent in regenerative medicine because of their easy preparation,low immunogenicity and potent paracrine capability[118].Specifically,MSCs can secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues [119].This flexibility provides a particular advantage in many regenerative,anti-inflammatory and autoimmune applications,however,the exogenous factors involved in MSC isolation and ex vivo preparation may greatly impact the biological properties of MSCs and eventually on their therapeutic abilities [120].

Pre-conditioning of MSCs under artificial microenvironment biomimicking physiological conditions prior to transplantation has been shown to improve their therapeutic efficiency [121–123].In our experience with MSCs [69,124],like in other aforementioned reports(Fig.10),cadherin-based biomaterials significantly promote cell adhesion,survival,proliferation,and the maintenance of stemness in the early stage of cell culture.These features strongly support their practical application in cell enrichment and the xenogeneic agent-free stem cell culture.Moreover,the cell-cell interactions mediated by cadherins play an important role in the whole process of cell homing toward lesion sites and responding to the local microenvironment [125].The tissue-specific signals from different subtypes of cadherin not only provide a highly selective recognition property to the desired cells,but can also guide the paracrine and differentiation tendency of MSCs.It can be expected that more bionic tissue construction can be achieved by optimizing the combination of cadherin subtypes and the application method.

In recent years,the majority of research works have focused on the implementation of 3D culturing models such as MSC aggregate formation to provide whose native niche resembles in vivo characteristics[126].The expression of cell adhesion proteins of MSCs in 3D aggregates was significantly increased,particularly that of cadherins.Our study showed that,hE-cadherin-Fc,when introduced into MSC aggregates,further upregulated the expression of E-cadherin,stimulated the E-cadherin/β-catenin cellular membrane complex,and activated the phosphorylation of EGF receptors as well as the Akt and ERK1/2 signaling pathways in the MSCs.These results indicate that,in addition to the similar effects to natural cadherin signals,the stimulation of cadherin-based materials seems to simultaneously activate other signaling pathways,which strongly affect cell survival and secretion,and even direct differentiation in complex internal environments [60,77].Although the definite regulatory mechanisms of cadherin-based biomaterials on MSC behaviors in native multicellular structures remain unclear,these achieved gratifying results provide distinct advantages for the meticulous and modular control of cell-cell interaction in the tissue construction and MSC therapeutics.

6.Conclusions

Cadherins are one of the most widely studied biomaterials mimicking cell-cell interactions at the cell-matrix interface because of their special structure and irreplaceable biological function during embryonic development.Cadherin-based biomaterials are capable of guiding and even altering the cellular response to the stimulation of growth factors/morphogens,ECM,and mechanical signals in the extracellular microenvironment,thereby manipulating cell fate of stem cells.Further detailed experimental and mathematical analyses are required in order to deepen our understanding of(i)the characteristics of stem cells cultured with cadherin-based biomaterials in temporal and spatial manners and (ii) the key molecular mechanism responsible for the observed changes of cellular behavior.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig.9.Cadherin-based biomaterials modulate cellular matrix mechanosensing.A.Integrin and cadherin molecules modified on the surface of biomaterials modulate the cellular mechanical landscape.B and C.Immunofluorescence staining the nuc/cyt statistical analysis of YAP in differentiating hMSCs cultured for 1 week on P/H,R–P/H,and hVE-P/H hydrogels (white arrows indicate YAP entering the nucleus) ([19]).D.YAP/TAZ nuclear-to-cytoplasmic ratios across a range of substrate stiffnesses (n ＞ 31 cells per group).E.YAP/TAZ ratios after blocking cellular N-cadherin with a neutralizing antibody before seeding on 10 kPa substrates(n ＞23 cells per group) [12].The data are reported as the mean ± SD,n=3.＊P ＜0.05;＊＊P ＜0.01;＊＊＊P ＜0.001.

Fig.10.Cadherin-based biomaterials regulate MSC behaviors and affect their potential of therapeutic application [12,98,128–135].

Acknowledgements

The authors gratefully acknowledge financial support from the Youth Science Fund of the National Nature Science Foundation of China(Grant No.81703094),the National Key Research and Development Program of China (No.2016YFC1101304),the National Natural Science Foundation of China (Grant No.31771067),and the Nature Science Foundation of Tianjin (No.17JCZDJC37600).