Maturation of human iPS cell-derived dopamine neuron precursors in alginate-Ca(2+) hydrogel.

BACKGROUND: Pluripotent stem cells (embryonic stem/induced pluripotent stem cells) have been widely studied as a potential cell source for cell transplantation therapy of Parkinson's disease. However, some difficulties remain to be overcome. These include the need to prepare a large number of dopamine (DA) neurons for clinical use and to culture the cells for a long period to allow their functional maturation and the removal of undifferentiated cells. METHODS: In this study, aggregates of DA neuron precursors were enclosed in alginate-Ca(2+) microbeads, and the encapsulated aggregates were cultured for 25days to induce cell maturation. RESULTS: More than 60% of cells in the aggregates differentiated into tyrosine hydroxylase-positive DA neurons. The aggregates could release DA at the same level as aggregates maintained on culture dishes without encapsulation. In addition, by exposure to a citrate solution, the alginate-Ca(2+) gel layer could be easily removed from aggregates without damaging the DA neurons. When the aggregates were transplanted into rat brain, viable cells were found in the graft at one week post-transplantation, with cells extending neurites into the host tissue. CONCLUSIONS: Cell aggregates encapsulated in alginate-Ca(2+) beads successfully differentiated into mature DA neurons. GENERAL SIGNIFICANCE: The alginate-Ca(2+) microbead is suitable for maintaining DA precursor aggregates for a long period to allow their functional maturation.

A DNA hybridization system for labeling of neural stem cells with SPIO nanoparticles for MRI monitoring post-transplantation.

Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 µg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully detected by MRI in vitro as well in vivo. Cells transplanted into the rat brain striatum could be detected by MRI scanning up to 1 month post-transplantation.

Enhanced proliferation of neural stem cells in a collagen hydrogel incorporating engineered epidermal growth factor.

Neural stem cells (NSCs) have received much attention in cell-transplantation therapy for central nervous disorders such as Parkinson's disease. However, poor engraftment of transplanted cells limits the efficacy of the treatments. To overcome this problem, collagen-based hydrogels were designed in this study to provide microenvironments for embedded cells to survive and proliferate. Our approach was to incorporate epidermal growth factor (EGF), known as a mitogen for NSCs, into a collagen hydrogel. For the stable binding of EGF with collagen under mild conditions, EGF was fused with a collagen-binding polypeptide domain by recombinant DNA technology. A cell population containing NSCs was derived from the fetal rat brain and cultured in the composite hydrogels for 7 d followed by analysis for cell proliferation. It was shown that the number of living cells was significantly higher in hydrogels incorporating collagen-binding EGF. This effect is largely owing to the collagen-binding domain that serves to sustain presentation of EGF toward cells within the hydrogel. It is further revealed by gene expression analysis that cells proliferated in the EGF-incorporating collagen hydrogel contained subpopulations expressing the marker of stem cells, neurons, astrocytes, or oligodendrocytes.