2D-DIGE Proteomic Analysis of Mesenchymal Stem Cell Cultured on the Elasticity-tunable Hydrogels

Abstract

ABSTRACT. The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signalingrelated proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and β-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity.

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…A wide variety of cell types, such as human MSCs, 61,105,106 mouse ES cells, 107 fibroblasts, 18,108,109 and glioma cells, 110 have been reported to respond to the ECM elasticity within the range of in vivo tissue elastic modulus from 0.1 kPa in the brain to 100 kPa in pre-calcified bone. 111 This property of cells can be used to specify strength of the cytoplasm-ECM link (Fig.…”

Time-Dependent Migratory Behaviors in the Long-Term Studies of Fibroblast Durotaxis on a Hydrogel Substrate Fabricated with a Soft Band

“…Immunostaining of collagen was then performed as previously described. 30 The samples were fixed with 4% paraformaldehyde, washed 3 times with 1% BSA, and permeabilized and blocked with 10% donkey serum, 1% BSA, and 0.5% Triton-X 100 in PBS. 5 μg/mL of rabbit anticollagen I (primary antibody, ab34710, Abcam, Tokyo, Japan) and 1/1000-diluted anti-rabbit conjugated Alexa 488 (secondary antibody, Invitrogen, Carlsbad, CA) were then allowed to react with the gel samples overnight.…”

Redox gene expression of adipose-derived stem cells in response to soft hydrogel

“…The details of the primers used in this experiment are presented in the Table. 2.5. Immunofluorescence staining of class III betatubulin Immunofluorescence staining of the ADSCs on gels was performed as previously described (Kuboki et al, 2012). After being cultured for 1 week, the cells were incubated with primary antibody polyclonal antimouse, β3-tubulin (Sigma, Tokyo, Japan), followed by incubation with secondary antibodies (donkey antimouse conjugated with Alexa Fluor @ 488, Tokyo, Japan).…”