Alberto Serrano

Influence of Oxygen Tension on Dopaminergic Differentiation of Human Fetal Stem Cells of Midbrain and Forebrain Origin

Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinsons disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1±0.5 and 17.1±0.4 (P<0.001); forebrain: 1.9±0.4 and 3.9±0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.

Group i metabotropic glutamate receptors: A potential target for regulation of proliferation and differentiation of an immortalized human neural stem cell line

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post‐natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain‐derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.

Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson’s disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson’s disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of β-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson’s disease.

Determining and mapping species sensitivity to trawling impacts: the BEnthos Sensitivity Index to Trawling Operations (BESITO)

Determining and mapping species sensitivity to trawling impacts: the BEnthos Sensitivity Index to Trawling Operations (BESITO) Jose M. González-Irusta*, Ana De la Torriente, Antonio Punzón, Marian Blanco, and Alberto Serrano Instituto Espa~nol de Oceanografı́a, Centro Oceanográfico de Santander, 39080 Santander, Spain *Corresponding author: tel: þ34 942 291 716; fax: þ34942275072; e-mail: gonzalezirusta@gmail.com.

Commented checklist of marine fishes from the Galicia Bank seamount (NW Spain)

A commented checklist containing 139 species of marine fishes recorded at the Galician Bank seamount is presented. The list is based on nine prospecting and research surveys carried out from 1980 to 2011 with different fishing gears. The ichthyofauna list is diversified in 2 superclasses, 3 classes, 20 orders, 62 families and 113 genera. The largest family is Macrouridae, with 9 species, followed by Moridae, Stomiidae and Sternoptychidae with 7 species each. The trachichthyd Hoplostethus mediterraneus and the morid Lepidion lepidion were the most abundant species. Biogeographically, the Atlantic group, with 113 species (81.3%) is the best represented, followed by the Lusitanian one with 17 species (12.2%). Data on species abundance, as number of individuals caught, size and depth are reported. Habitat, distribution and vulnerability status are commented. Moreover, biometric data and meristic counts are also reported for several species. The results obtained showing a high fish biodiversity and a sensible number of threatened species, strongly support the future declaration of the Galicia Bank as a Marine Protected Area.