Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia in vitro.

BACKGROUND: One of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB) cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O2< 1%) for 48 hours. Subsequently, HUCB mononuclear cells (MNC) were added to post-hypoxic neuronal cultures. These cultures were characterized regarding to the development of apoptosis and necrosis over three days. Based on this we investigated the therapeutic influence of HUCB MNC on the progression of apoptotic cell death. The impact of HUCB cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved. RESULTS: Hypoxic cultivation of neurons initially induced a rate of 26% +/- 13% of apoptosis. Hypoxia also caused an enhanced expression of Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Necrosis was only detected in low amounts. Within the next three days rate of apoptosis in untreated hypoxic cultures cumulated to 85% +/- 11% (p < or = 0.001). Specific cytokine (VEGF) patterns also suggest anti-apoptotic strategies of neuronal cells. Remarkably, the administration of MNC showed a noticeable reduction of apoptosis rates to levels of normoxic control cultures (7% +/- 3%; p < or = 0.001). In parallel, clustering of administered MNC next to axons and somata of neuronal cells was observed. Furthermore, MNC caused a pronounced increase of chemokines (CCL5; CCL3 and CXCL10). CONCLUSION: We established an in vitro model of neuronal hypoxia that affords the possibility to investigate both, apoptotic neuronal cell death and neuroprotective therapies. Here we employed the therapeutic model to study neuroprotective properties of HUCB cells. We hypothesize that the neuroprotective effect of MNC was due to anti-apoptotic mechanisms related to direct cell-cell contacts with injured neuronal cells and distinct changes in neuroprotective, inflammatory cytokines as well as to the upregulation of chemokines within the co-cultures.

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

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.

Assessment of neuroprotective effects of human umbilical cord blood mononuclear cell subpopulations in vitro and in vivo.

Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34(+)) or depleted (CD34(-)) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34(+) and CD34(-) cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34(+) cells provided transient protection for 2 days. The CD34(-) fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34(+) cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34(+) cells seeming to be particularly involved in the protective action of MNCs. CD34(+) cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression.

Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease.

Magnetic resonance imaging (MRI) of magnetically labeled stem cells has become a valuable tool in the understanding and evaluation of experimental stem cell-based therapies of degenerative central nervous system disorders. This comprehensive study assesses the impact of magnetic labeling of both human and rodent stem cell-containing populations on multiple biologic parameters as maintenance of stemness and oxidative stress levels. Cells were efficiently magnetically labeled with very small superparamagnetic iron oxide particles. Only under the condition of tailored labeling strategies can the impact of magnetic labeling on vitality, proliferation, pluripotency, and oxidative stress levels be minimized. In a rat model of Parkinson disease, magnetically labeled mouse embryonic stem cells were tracked by high-field MRI for 6 months. Significant interindividual differences concerning the spatial distribution of cells became evident. Histologically, transplanted green fluorescent protein-positive iron oxide-labeled cells were clearly identified. No significant increase in oxidative stress levels at the implantation site and no secondary uptake of magnetic label by host phagocytotic cells were observed. Our study strongly suggests that molecular MRI approaches must be carefully tailored to the respective cell population to exert minimal physiologic impact, ensuring the feasibility of this imaging approach for clinical applications.