No abstract

Cell transplantation is a potential methodology for the treatment of Parkinson's disease. However, the therapeutic effect is limited by poor viability of transplanted cells. To overcome this problem, we hypothesized that a dual step approach, whereby providing an adhesive substrate for transplanted cells and, at the same time, by preventing the infiltration of activated microglia into the site of transplantation promotes the cell survival. To establish above conditions, attempts were made to prepare 3-D matrices using collagen hydrogels that incorporated integrin-binding polypeptides derived from laminin-1. Tandem combinations of laminin globular domains as well as a single globular domain 3 were prepared using recombinant DNA technology as a fusion with hexahistidine and bound to metal chelated surfaces to screen for the adhesion and proliferation of neural stem cells (NSCs). In addition, a small peptide derived from laminin γ1 chain was prepared and heterodimerized with the globular domain-containing chimeric proteins to evaluate for the enhancement of integrin-mediated cell adhesion. As a result, a heterodimer consisting of the globular domain 3 of the laminin α1 chain and the peptide from the laminin γ1 chain was selected as the best candidate among the polypeptides studied here for the incorporation into a collagen hydrogel. It was shown that the survival of NSCs was indeed promoted in the collagen hydrogel incorporating the heterodimer compared to the pure collagen hydrogel.

No abstract

To develop biomaterials that serve to improve the survival of neural cells transplanted into central nervous tissues, type I collagen-based hydrogels were prepared as a cell carrier. The hydrogels were modified with a laminin-derived peptide that is known to have an affinity for alpha3beta1 integrin, to transduce antiapoptotic signaling in embedded cells. For the modification of collagen, the peptide was fused to the N- or C-terminus, or both termini of a collagen-binding polypeptide domain by means of recombinant DNA technology. The chimeric proteins were characterized by polyacrylamide gel electrophoresis and circular dichroism spectroscopy, while binding of chimeric proteins to collagen-coated substrates was verified by surface plasmon resonance analysis under physiological conditions. Cell culture assays revealed that the adhesion of neurosphere-forming cells to collagen-coated polystyrene surfaces was significantly promoted by the incorporation of the chimeric proteins in a peptide-density dependent manner. The live/dead assays for cells cultured for 24 or 48 h in the hydrogels revealed that peptide incorporation improved the survival of cells embedded in collagen hydrogels. These results suggest that collagen hydrogel containing the laminin-derived peptide provides microenvironments suitable for the survival of neural cells.

Poor viability of cells transplanted into the brain has been the critical problem associated with stem cell-based therapy for Parkinson's disease. To overcome this problem, a collagen hydrogel incorporating an integrin-binding protein complex was prepared and used as a carrier for neural stem cells. The protein complex consisted of two polypeptides containing the G3 domain of a laminin α1 chain and the C-terminal oligopeptide of a laminin γ1 chain. These polypeptides were fused with α-helical segments which spontaneously formed a coiled-coil heterodimer and with the collagen-binding peptide that facilitated the binding of the heterodimer to collagen networks. In this study, neural stem cells stably expressing the enhanced green fluorescent protein (EGFP) were suspended in the hydrogel and transplanted into the striatum of healthy rats. The viability of transplanted cells was evaluated by histological analysis and quantitative reverse-transcriptase polymerase chain reaction for EGFP mRNA present in the tissue explants. Our results showed that the collagen hydrogel incorporating the integrin-binding protein complex serves to improve the viability of neural stem cells (NSCs) in the early stage after transplantation into the striatum.