Development of Phase-Separating Microfiber Network Hydrogels to Promote In Vitro Vascularization.

Engineered vascularized tissues in vitro exhibit the potential for transplantation therapy and disease modeling. Despite efforts to design hydrogels as cell culture platforms for in vitro vascularization, development of vascularized tissues recapitulating the natural structures and functions remains difficult due to a poor understanding of the relationships between the matrix microstructures and tube formation of endothelial cells. Herein, we developed microfiber network hydrogels with microporous structures by controlling the liquid-liquid phase separation (LLPS) of proteins and matrix structures in hydrogels. Extracellular matrix protein gelatin was modified with hydrogen-bonding moieties and mixed with hyaluronic acid sodium salt to form microfiber network structures. Gelatin gelation and hyaluronic acid sodium salt dissolution led to the formation of a microporous microfiber network hydrogel formation. Matrix structures of hydrogels were modified by controlling LLPS that affects endothelial cell tube formation. Vascularization was improved using laminin peptides and coculturing with mesenchymal stem cells. Overall, our approach exhibits the potential to induce in vitro vascularization for regenerative medicine and disease modeling applications.

The α2,8-sialyltransferase 6 (St8sia6) localizes in the ER and enhances the anchorage-independent cell growth in cancer.

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

Analysis of biochemical features of ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase (St8sia) 5 isoforms.

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.