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Adnan N.,Tokyo Institute of Technology | Mie M.,Tokyo Institute of Technology | Haque A.,University of California at Davis | Hossain S.,Biomaterials Center for Regenerative Medical Engineering | And 3 more authors.
Bioconjugate Chemistry | Year: 2016

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3β (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro. © 2016 American Chemical Society.


PubMed | Tokyo Institute of Technology, University of California at Davis and Biomaterials Center for Regenerative Medical Engineering
Type: Journal Article | Journal: Bioconjugate chemistry | Year: 2016

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3 (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.


PubMed | Tsinghua University, CAS Institute of Genetics and Developmental Biology, Chinese People's Liberation Army and Biomaterials Center for Regenerative Medical Engineering
Type: | Journal: International journal of nanomedicine | Year: 2015

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O--D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose-asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.


Miyagi H.,Tokyo Institute of Technology | Nag K.,Tokyo Institute of Technology | Nag K.,Biomaterials Center for Regenerative Medical Engineering | Sultana N.,Tokyo Institute of Technology | And 3 more authors.
Gene | Year: 2016

Zebrafish connexin 36.7 (cx36.7/ecx) has been identified as a key molecule in the early stages of heart development in this species. A defect in cx36.7 causes severe heart malformation due to the downregulation of nkx2.5 expression, a result which resembles congenital heart disease in humans. It has been shown that cx36.7 is expressed specifically in early developing heart cardiomyocytes. However, the regulatory mechanism for the cardiac-restricted expression of cx36.7 remains to be elucidated. In this study we isolated the 5'-flanking promoter region of the cx36.7 gene and characterized its promoter activity in zebrafish embryos. Deletion analysis showed that a 316-bp upstream region is essential for cardiac-restricted expression. This region contains four GATA elements, the proximal two of which are responsible for promoter activation in the embryonic heart and serve as binding sites for gata4. When gata4, gata5 and gata6 were simultaneously knocked down, the promoter activity was significantly decreased. Moreover, the deletion of the region between -. 316 and -. 133. bp led to EGFP expression in the embryonic trunk muscle. The distal two GATA and A/T-rich elements in this region act as repressors of promoter activity in skeletal muscle. These results suggest that cx36.7 expression is directed by cardiac promoter activation via the two proximal GATA elements as well as by skeletal muscle-specific promoter repression via the two distal GATA elements. © 2015 Elsevier B.V.


Xu K.,Nankai University | Shuai Q.,Nankai University | Li X.,Nankai University | Zhang Y.,Nankai University | And 7 more authors.
Biomacromolecules | Year: 2016

In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/β-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering. © 2016 American Chemical Society.


Zhang Y.,Nankai University | Mao H.,Nankai University | Mao H.,RIKEN | Qian M.,Nankai University | And 8 more authors.
Journal of Materials Chemistry B | Year: 2016

To effectively expand human mesenchymal stem cells (hMSCs) in vitro without affecting their innate biological properties, a fusion protein (hE-cad-Fc) consisting of a human E-cadherin extracellular domain and an immunoglobulin G Fc region was fabricated and used as a biomimetic matrix for MSC culture surface modification. The results showed that cells cultured on hE-cad-Fc-modified polystyrene surfaces exhibited improved proliferation and paracrine functions compared with cells cultured on unmodified and collagen-modified polystyrene surfaces. Meanwhile, surfaces modified with hE-cad-Fc effectively inhibited cell apoptosis even under the serum deprivation conditions. Additionally, the hE-cad-Fc not only up-regulated the expression of β-catenin in MSCs and stimulated the cellular membrane complex of E-cadherin/β-catenin, but also effectively activated the intracellular signals such as EGFR, AKT and ERK phosphorylation. Therefore, hE-cad-Fc appeared to be a promising candidate for biological surface modification and stem cell culture. © 2016 The Royal Society of Chemistry.


PubMed | Nankai University, University of Sichuan, Biomaterials Center for Regenerative Medical Engineering and Capital Medical University
Type: Journal Article | Journal: Advanced healthcare materials | Year: 2016

E-cadherin-modified poly(lactic-co-glycolic acid) (hE-cad-PLGA) microparticles were fabricated and then mediated the 3D cell aggregates of human mesenchymal stem cells (MSCs) on page 1949 by Jun Yang and co-workers. The hE-cad-Fc matrix and the PLGA microparticles synergistically regulate the proliferation and bioactive factors secretions of MSCs by activating EGFR, AKT and ERK1/2 signaling pathways. The hE-cad-PLGA microparticles offer a novel route to expand multipotent stem cell-based clinical applications.


PubMed | Nankai University, University of Sichuan and Biomaterials Center for Regenerative Medical Engineering
Type: Journal Article | Journal: Biomacromolecules | Year: 2016

In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering.


PubMed | University of Toyama and Biomaterials Center for Regenerative Medical Engineering
Type: | Journal: Journal of biomedical materials research. Part A | Year: 2016

The development of new three-dimensional (3D) cell culture system that maintains the physiologically relevant signals of hepatocytes is essential in drug discovery and tissue engineering research. Conventional two-dimensional (2D) culture yields cell growth, proliferation and differentiation. However, gene expression and signaling profiles can be different from in vivo environment. Here, we report the fabrication of a 3D culture system using an artificial scaffold and our custom-made inkjet 3D-bioprinter as a new strategy for studying liver-specific functions of hepatocytes. We built a 3D culture platform for hepatocytes-attachment and formation of cell monolayer by interacting the galactose chain of galactosylated alginate gel (GA-gel) with asialoglycoprotein receptor (ASGPR) of hepatocytes. The 3D geometrical arrangement of cells was controlled by using 3D bioprinter, and cell polarity was controlled with the galactosylated hydrogels. The fabricated GA-gel was able to successfully promote adhesion of hepatocytes. To observe liver-specific functions and to mimic hepatic cord, an additional parallel layer of hepatocytes was generated using two gel sheets. These results indicated that GA-gel biomimetic matrices can be used as a 3D culture system that could be effective for the engineering of liver tissues. This article is protected by copyright. All rights reserved.


PubMed | Nankai University, University of Sichuan, Biomaterials Center for Regenerative Medical Engineering and Capital Medical University
Type: Journal Article | Journal: Advanced healthcare materials | Year: 2016

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications.

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