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      Neuronal hypoxia in vitro: Investigation of therapeutic principles of HUCB-MNC and CD133 + stem cells

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          Abstract

          Background

          The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood.

          Methods

          A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133 +, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133 -, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array.

          Results

          tMNC, CD133 + and surprisingly CD133 - reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% ± 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% ± 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133 - reduced apoptosis comparably. CD133 - produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133 + cells were not detectable in direct co-cultures.

          Conclusion

          Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.

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          Most cited references30

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          Neuronal replacement from endogenous precursors in the adult brain after stroke.

          In the adult brain, new neurons are continuously generated in the subventricular zone and dentate gyrus, but it is unknown whether these neurons can replace those lost following damage or disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts probably already formed before the insult, migrate into the severely damaged area of the striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the new neurons are functional and their formation can be stimulated, a novel therapeutic strategy might be developed for stroke in humans.
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            Intravenous administration of human umbilical cord blood reduces behavioral deficits after stroke in rats.

            Human umbilical cord blood cells (HUCBC) are rich in stem and progenitor cells. In this study we tested whether intravenously infused HUCBC enter brain, survive, differentiate, and improve neurological functional recovery after stroke in rats. In addition, we tested whether ischemic brain tissue extract selectively induces chemotaxis of HUCBC in vitro. Adult male Wistar rats were subjected to transient (2-hour) middle cerebral artery occlusion (MCAO). Experimental groups were as follows: group 1, MCAO alone (n=5); group 2, 3x10(6) HUCBC injected into tail vein at 24 hours after MCAO (n=6) (animals of groups 1 and 2 were killed at 14 days after MCAO); group 3, MCAO alone (n=5); group 4, MCAO injected with PBS at 1 day after stroke (n=8); and group 5, 3x10(6) HUCBC injected into tail vein at 7 days after MCAO (n=5). Rats of groups 3, 4, and 5 were killed at 35 days after MCAO. Behavioral tests (rotarod and Modified Neurological Severity Score [mNSS]) were performed. Immunohistochemical staining was used to identify cells derived from HUCBC. Chemotactic activity of ischemia brain tissue extracts toward HUCBC at different time points was evaluated in vitro. Treatment at 24 hours after MCAO with HUCBC significantly improved functional recovery, as evidenced by the rotarod test and mNSS (P<0.05). Treatment at 7 days after MCAO with HUCBC significantly improved function only on the mNSS (P<0.05). Some HUCBC were reactive for the astrocyte marker glial fibrillary acidic protein and the neuronal markers NeuN and microtubule-associated protein 2. In vitro, significant HUCBC migration activity was present at 24 hours after MCAO (P<0.01) compared with normal brain tissue. Intravenously administered HUCBC enter brain, survive, migrate, and improve functional recovery after stroke. HUCBC transplantation may provide a cell source to treat stroke.
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              The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.

              G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.
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                Author and article information

                Journal
                BMC Neurosci
                BMC Neuroscience
                BioMed Central
                1471-2202
                2008
                19 September 2008
                : 9
                : 91
                Affiliations
                [1 ]Fraunhofer-Institute for Cell Therapy and Immunology, Perlickstraße 1, 04103 Leipzig, Germany
                [2 ]University of Leipzig, Faculty of Veterinary Medicine, Department of Anatomy, Histology and Embryology, An den Tierkliniken 43, 04103 Leipzig, Germany
                [3 ]University of Leipzig, Institute of Medical Informatics, Statistics and Epidemiology, Haertelstrasse 16-18, 04107 Leipzig, Germany
                [4 ]University of Leipzig, Faculty of Biosciences, Pharmacy and Psychology, Institute of Biology II, Talstrasse 33, 04103 Leipzig, Germany
                [5 ]Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Strasse 55, 04103 Leipzig, Germany
                [6 ]University of Leipzig, Institute of Clinical Immunology and Transfusion Medicine, Johannisallee 30, 04103 Leipzig, Germany
                Article
                1471-2202-9-91
                10.1186/1471-2202-9-91
                2569042
                18803816
                d28a3bc9-dafc-4936-b109-13ec5b8b3501
                Copyright © 2008 Reich et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 June 2008
                : 19 September 2008
                Categories
                Research Article

                Neurosciences
                Neurosciences

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