Abstract
BackgroundOne 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.ResultsHypoxic 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 ≤ 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 ≤ 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).ConclusionWe 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.
Highlights
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
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
Hypoxia caused an enhanced expression of Caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP)
Summary
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. 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. Mild ischemia (CBF 11–20 cm3 × 100 g1 × min-1) leads to the activation of complex neurochemical cascades of cell death, mainly apoptosis In principle these apoptotic cascades are reversible and form an important aspect of the penumbra concept, which is the major target of therapeutic interventions [2,3]. Recent findings indicate that transplantation of external cell fractions could accompany established therapeutic procedures limited by narrow time windows [4], but the underlying processes are still rather unclear
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