Dynamics of host and graft after cell sheet transplantation: Basic study for the application of amyotrophic lateral sclerosis.

Stem cells offer great hope for the therapy of neurological disorders. Using a human artificial chromosome (HAC), we generated modified mesenchymal stem cells (MSCs), termed HAC-MSC that express 3 growth factors and 2 marker proteins including luciferase, and previously demonstrated that intrathecal administration of HAC-MSCs extended the lifespan in a mouse model of amyotrophic lateral sclerosis (ALS). However, donor cells disappeared rapidly after transplantation. To overcome this poor survival, we transplanted the HAC-MSCs as a sheet structure which retained the extracellular matrix. We investigated, here, whether cell sheet showed a longer survival than intrathecal administration. Also, the therapeutic effects on ALS model mice were examined. In vivo imaging showed that luciferase signals increased immediately after transplantation up to 7 days, and these signals were sustained for up to 14 days. In contrast, following intrathecal administration, signals were drastically decreased by day 3. Moreover, cell sheet transplantation successfully prolonged the survival of donor HAC-MSCs. Cell sheet transplantation increased the level of p-Akt at the graft area. Pathologically, none of the donor cells differentiated into neurons, astrocytes or microglial cells. When the cell sheet was transplanted into ALS model mice, there was an encouraging trend in the delayed onset of symptoms and increased lifespan. If each group was subdivided into rapid and slow progressors based on cut-off values for respective median survival, the survival of rapid progressors differed significantly between groups (treated vs. sham-operated = 145.4 ±â€¯1.4 vs. 139.2 ±â€¯1.2). The effect of HAC-MSC sheet transplantation still has a temporally narrow therapeutic window. Further improvement could be achieved by optimization of the transplantation conditions, e.g. co-transplantation of HAC-MSCs with endothelial progenitor cells.

Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.

BACKGROUND: Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. RESULTS: We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. CONCLUSIONS: These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.