ABSTRACT
Mesenchymal stem cells (MSCs) represent one of the most promising stem cells for a number of degenerative conditions due to their multipotency, immunoprivileged properties, and easy expansion in vitro. However, the limited life span of primary MSCs during in vitro expansion greatly hampers their use in clinical applications and basic research. Immortalization of MSCs will overcome this problem and may provide a very useful tool with which to study MSC biology. Here we showed that silencing p53 expression with lentivirus-mediated small interfering RNA delayed the senescence by extended passage number, but was not sufficient to immortalize primary MSCs. However, combination of p53 knockdown and human telomerase reverse transcriptase (hTERT) overexpression was sufficient to immortalize MSCs. The effects of p53 knockdown and hTERT overexpression on MSCs, including proliferation, colony formation, and differentiation, were determined. The resultant immortal MSCs displayed similar surface antigen profile to primary MSCs and retained MSC differentiation potential. Gene expression profile showed high similarity between immortalized MSCs and primary MSCs. In addition, immortalization-associated genes were also identified. Our data suggested immortalization of MSCs related to upregulation of cell cycle regulator and DNA repair genes enabling them to bypass cell crisis and complete mitosis. This study provides a new cellular model for basic studies of MSCs and understanding of the molecular basis of MSC immortalization.
Subject(s)
Cell Transformation, Neoplastic , Gene Expression Regulation, Enzymologic , Mesenchymal Stem Cells/metabolism , Telomerase/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Chondrogenesis , Gene Knockdown Techniques , Humans , Lentivirus/genetics , Lentivirus/metabolism , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Telomerase/genetics , Time Factors , Transcriptome , Tumor Suppressor Protein p53/geneticsABSTRACT
In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.
