Human E-cadherin-Fc Fusion Protein As Bioartificial Matrix Constructing Extracellular Microenvironment For Stem Cells

Extracellular matrix (ECM), neighboring cells and soluble factors are most important effectors of cell behavior and function. Among them, cell-matrix interaction plays a fundamental role in regulating cell adhesion, migration, proliferation, differentiation and the expression of specific functions. In recent years, there was some important progress in tissue engineering and regenerative medicine, which widened a new theory in various research fields. The recombinant fusion proteins, an engineered artificial ECM, which was based on gene engineering technology, has gained extensive attention because of its selectivity to specific cell lineages and tissue responses. In this study, human E-cadherin-Fc fusion protein was constructed and used as a cell-cell adhesion biomimicking matrix for human mesenchymal stem cells (hMSCs). It can not only be used in basic biological research, but also in tissue engineering and regenerative medicine.Firstly, a fusion protein consisting of human E-cadherin extracellular domain and the immunoglobulin G Fc region (hE-cadherin-Fc) was biosynthesized by gene engineering technology. The hE-cadherin-Fc fusion protein expressed and purified by the Free-Style MAX293Expression System was analyzed by Elisa and western blotting. Water contact angles (WCA) and Atomic Force Microscope (AFM) showed that the hE-cadherin-Fc was stably immobilized onto a polystyrene plate due to the hydrophobicity of the Fc domain, enhancing the surface wettability and topography. The hE-cadherin-Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs comparing with the tissue culture-treated plate (TC-PS) and the gelatin-coated plate. Furthermore, the expanded hMSCs on the hE-cadherin-Fc were positive for CD105, showing the hE-cadherin-Fc matrix could maintain the undifferentiation of hMSCs. Additionally, the expressions of E-cadherin and β-catenin in the hMSCs were improved on the hE-cadherin-Fc matrix, suggesting that the cell-cell adhesion junctions were substituted by the interactions between the hE-cadherin-Fc matrix and the hMSCs during the initial culture stage in the absence of cell-cell interactions.Secondly, we focused on the effects of the hE-cadherin-Fc matrix on the hepatic differentiation from hMSCs. After4weeks of directly differentiation on modified surfaces, hepatocyte-like cells were identified through cell morphology, RT-PCR, flow cytometry, immunofluorescence, Periodic acid-Shiff staining, indocyanine green (ICG) uptake and hepatocellular synthesis and metabolism functions by albumin/urea assays. Compared with the TC-PS and gelatin-coated surface, the expression of hepatic lineage surface markers CD117was higher on hE-cadherin-Fc surface. The mRNA expressions of hepatocyte-specific markers, such as ALB, CK18and HNF4, were performed by RT-PCR. The expression levels of these markers were found to be significantly upregulated during the differentiation on hE-cadherin-Fc surface. Similarly, the expression of ALB protein which was detected by immunofluorescence staining appeared to be higher than TC-PS and gelatin-coated surface. To confirm whether differentiated hepatocyte-like cells derived from hMSC were functionally competent, we examined the glycogen storage, ICG uptake, albumin secretion and urea production. It demonstrated that the differentiated cells expressed higher function on hE-cadherin-Fc-coated surface. These results show that the hE-cadherin-Fc may be a promising artificial extracellular matrix (ECM) for the hMSCs differentiation via the homophilic interaction of hE-cadherin and the synergy between cell adhesion molecular and growth factors.Lastly, we fabricated nanofibrous scaffolds and porous scaffolds, modified with hE-cadherin-Fc fusion protein and studied the synergistic effect of the scaffolds. The morphology of three-dimensional scaffolds which modified by hE-cadherin-Fc fusion protein was not changed by using scanning electron microscopy (SEM). Surface modification was investigated via monitoring the changes of the surface by X-ray photoelectron spectroscopy (XPS) and Elisa showed that the hE-cadherin-Fc was stably modified on three-dimensional scaffolds. WCA measurements confirmed that the hE-cadherin-Fc fusion protein could improve the surface properties of scaffolds. The hE-cadherin-Fc matrix markedly promoted the cell adhesion and proliferation of hMSCs on three-dimensional scaffolds. The cell morphology was evaluated by F-actin staining, and the cells had a more spreading morphology on hE-cadherin-Fc-coated scaffolds. The results demonstrated that the hE-cadherin-Fc improved the cell compatibility and biological functions of three-dimensional scaffolds.In summary, hE-cadherin-Fc was successfully prepared as a cell-cell adhesion biomimicking matrix which can be used for the surface modification of hydrophobic materials effectively. The hE-cadherin-Fc fusion protein matrix could enhance the adhesion and proliferation of the hMSCs, and improve the differentiation regulation of stem cells. The hE-cadherin-Fc was shown to be a promising artificial ECM for hMSCs.