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In CNS, transient global or severe focal ischemia leads to devastating irreversible cellular losses and functional impairments of the nervous tissue. Stem/progenitor cells have been considered the salvage therapy. However, besides decision on the cells source, stage of their development and proliferation, the limited survival of any implanted exogenous cells in the host tissue is one of the major obstacles for effective neurotransplantation. In preclinical animal experiment this problem gets even more complicated by necessity to use xenogeneic systems for initial testing of any human transplants and persistant lack of adequately humanized animal models. Other still unresolved questions which must be addressed are the routes, dosage and timing of cells delivery. Thus, the final answer must be at first approximated experimentally in animal models, then finally proved by case reports followed by clinical trials performed according to EBM rules. Here I will review the preclinical data gathered in our laboratory which led us toward the first, MRI monitored, clinical experiment on autologous, cord blood -derived progenitors transplantation. The neurally committed cells, prelabelled by the high signal of iron oxide nanoparticles (SPIO) were infused into the frontal horn of the lateral ventricle of 16 month old child with severe global cerebral ischemic injury. The dynamics of cell engraftment was visualized in time by MR imaging. Gradually decreased signal was noted over 4 month without any adverse side-effects. The child was followed up for next 6 month and his neurological status slightly but significantly improved. This is the first case study based on neurally –induced stem cells from the patient’s frozen cord blood and considered feasible, well tolerated and safe procedure which could be monitored by MRI after intraventricular cell transplantation.
Human umbilical cord blood (HUCB) has been established as a promising source of hematopoietic as well as various non- hematopoietic stem cell populations. This offers numerous advantages of HUCB stem/progenitor cells for therapies, however in vitro conditions that contribute to long term propagation of proliferating undifferentiated cells have not yet been established. Here we evaluate culture conditions promoting spheroid aggregates/neurospheres formation which, together with serum withdrawal and mitogenes treatments in strictly defined media, maintain population of HUCB progenitor cells in undifferentiated and dividing state exhibiting neurogenic potential in vitro. Our results indicate that formation and maintenance of three-dimensional aggregates enhanced by cell culture rotating motion, is crucial for high and prolonged expression of genes and proteins characteristic for cord blood stem cells and their further neural commitment.
The fate choice of neural progenitors could be dictated by local cellular environment of adult CNS. The aim of our study was to investigate the effect of hippocampal tissue on the differentiation and maturation of the oligodendrocyte NG2 precursors. Methods: The hippocampal slice culture was established from the brains of 7-day old rats. The NG2 precursors, obtained from a 12-day old mixed primary culture of neonatal rat hemispheres, were labeled with CMFDA and seeded on hippocampal slices. After 7ñ14 days in co-culture, the cells were stained with neural markers. Results: The NG2 cells differentiated predominantly into oligodendrocytes, presenting various stages of maturation: progenitors NG2+, O4+ and fi nally mature Galc-positive cells. However, except for a few cells with astrocyte-specifi c S100β staining, a considerable number of these cells differentiated into TUJ+ and MAP-2+ neurons. Moreover, a certain population of these cells preserved proliferative properties of primary precursors, as revealed by the Ki67 expression. Conclusions: Neuronal microenvironment provided by the culture of hippocampal slices is potent to induce neurogenesis from oligodendrocyte NG2+/PDGFRα+/CNP+ progenitors and promotes their differentiation not only into macroglia but also into neurons. It also sustains their proliferative capacity. The results indicate a crucial role of the local cellular environment in fatedecision of primary NG2+ multipotent neural progenitors, which may affect their behavior after transplantation into CNS. Supported by MSHE grant N40101832 /0296
Oligodendrocytes, the cells responsible for myelin formation and maintenance in CNS, are depleted in many acute and chronic conditions. The stem/progenitor cells stimulation or transplantation might be seriously considered as a long hopedfor therapeutic perspective. Better understanding of the mechanism(s) regulating the activation of the cell lineage from the endogenous progenitor reservoir might be helpful. Therefore an efficient source of donor cells for transplantation in humans is being craved for. In this study we show that the application of extracellular matrix component-laminin promotes oligogliogenesis from neural stem-like cells of human cord blood cells (HUCB-NSC). Although oligodendrocytes constitute a minor subpopulation of spontaneously differentiated HUCB-NSC, the manipulation of active compounds regulating the process of cell commitment results in a several fold increase in their number. Thus cells of the HUCB-NSC line could be considered as a potential source of glial cells, fulfilling the suitable candidate criteria for oligodendrocyte replacement therapy.
Brain infl ammation contributes to the propagation of neuropathological events that involves activation of astrocytes and microglia. It remains obscure how activated glial cells affect the survival and differentiation of neural stem cells (NSC). The aim of the study was to analyze neuronal commitment of Human Umbilical Cord Blood derived Neural Stem Cells (HUCB-NSC) cultured in the presence of normal and LPS- or TMT-activated glial cells. Methods: HUCB-NSC (5 × 104/cm2 ) were co-cultured with normal or LPS (0.1 μg/ ml) and TMT (1μM)-stimulated astrocytes and microglial cells isolated from neonatal rat brain for proliferation and cell phenotype assessment. Pro-infl ammatory cytokines were estimated (ELISA). Results: Normal rat astrocytes induce HUCB-NSC to differentiate mostly into neurones but microglia stimulate HUCB-NSC to differentiate into neurons as well as into astrocytes. LPS- and TMTinduced astrocytes diminish neurogenesis of HUCB-NSC and increase astrocyte differentiation in comparison to non-stimulated astrocytes. Microglia activation by LPS and TMT decreases HUCBNSC differentiation into neurons but enhances oligodendrogenesis compared to normal microglia. Stimulation of astrocytes and microglia by LPS and TMT declines HUCB-NSC proliferation cocultured with astrocytes or with microglia. The presence of IL-1β, IL-6, TNF-α and NO was observed in glia cell culture supernatants after LPS and TMT implementation. Conclusion: Activation of astrocytes and microglia induced by LPS and TMT attenuate pro-neural effect of non-stimulated (resting) glia and suppress proliferation of HUCB-NSC in vitro. The release of pro-infl ammatory cytokines and NO might be partly responsible for this effect. Supported by MSHE grant No 142/P01/2008/35
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