The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O 2 , whereas exposing these cells to 21% oxygen prohibited long-term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain-derived precursors cultured in 3 versus 21% oxygen after 1-2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self-renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons. Keywords: microarray, neural precursors, neurospheres, oxygen, proliferation, telomerase. Stem cells are undifferentiated cells that retain the ability to divide and proliferate throughout postnatal life to provide precursor cells that can differentiate into specialized cells. Neural stem cells can give rise to all the cells of the CNS including glial and neuronal cells (Gage 2003). As a result of their ability to differentiate following transplantation, these cells represent a potentially valuable therapeutic tool for the treatment of genetic, traumatic, inflammatory or degenerative neurological disorders (Studer et al. 1998;Storch and Schwarz 2002;Conti et al. 2003). To enable long-term expansion, all precursor cells must maintain the capacity to self-renew and a positive balance of proliferation versus differentiation or cell death. Therefore, it is crucial to understand the mechanisms governing cell division, differentiation and senescence (Sharpless and DePinho 2004).Mammals have evolved complex circulatory systems to ensure that every cell receives sufficient oxygen for normal metabolic processes. Lack of oxygen is defined as a reduction in oxygen supply to the tissues to below physiological levels. Hypoxia can develop as a result of ischaemia, representing a pathological component of stroke and heart attack. In addition to being a consequence of the growth of a malignant tumour and so a potential marker, hypoxia also acts to promote tumour development (Brizel et al. 1996;Brat et al. 2004). Address correspondence and reprint requests to Javorina Milosevic, Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. E-mail: javorina.Milosevic@medizin.uni-leipzig.deAbbreviations used: ADA, adenosine deaminase; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2-deoxyuridine; DA, dopamine; DAPI, 6¢-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF...

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Here, we conditionally inactivated the hypoxia-responsive transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) in murine NPCs to determine its role in proliferation, survival, and dopaminergic differentiation in vitro as well as survival of murine dopaminergic neurons in vivo. HIF-1␣ conditional knock-out (HIF-1␣ CKO) mNPCs showed midbrain-specific impairment of survival and proliferation. Dopaminergic differentiation of HIF-1␣ CKO mNPCs in vitro was markedly reduced. Expression of vascular endothelial growth factor (VEGF) mRNA was reduced in HIF-1␣ CKO mNPCs, whereas erythropoietin signaling was not affected. Treatment of HIF-1␣ CKO mNPCs with 50 ng/ml VEGF partially recovered proliferation and dopaminergic differentiation in vitro. In substantia nigra (SN) of adult HIF-1␣ CKO mice, protein levels of dopaminergic marker molecules such as tyrosine hydroxylase (TH) and aldehyde dehydrogenase were reduced by 41 and 61%, respectively. The cell survival marker Bcl-2 was reduced by 58% while caspase-3 was activated. Nonbiased stereological cell counts of TH-positive neurons in SN of young adult HIF-1␣ CKO mice revealed a reduction of 31% compared with cre/wt mice (in which the wildtype Hif1a allele is expressed in parallel with the Cre recombinase allele). However, we found no impairment of striatal dopamine concentrations or locomotor behavior. In conclusion, HIF-1␣ seems to be a transcription factor relevant to the development and survival of substantia nigra dopaminergic neurons involving VEGF signaling.

Global gene expression profiling was performed using RNA from adult human hippocampus-derived neuroprogenitor cells (NPCs) and multipotent frontal cortical fetal NPCs compared with adult human mesenchymal stem cells (hMSCs) as a multipotent adult stem cell control, and adult human hippocampal tissue, to define a gene expression pattern that is specific for human NPCs. The results were compared with data from various databases. Hierarchical cluster analysis of all neuroectodermal cell/tissue types revealed a strong relationship of adult hippocampal NPCs with various white matter tissues, whereas fetal NPCs strongly correlate with fetal brain tissue. However, adult and fetal NPCs share the expression of a variety of genes known to be related to signal transduction, cell metabolism and neuroectodermal tissue. In contrast, adult NPCs and hMSCs overlap in the expression of genes mainly involved in extracellular matrix biology. We present for the first time a detailed transcriptome analysis of human adult NPCs suggesting a relationship between hippocampal NPCs and white matter-derived precursor cells. We further provide a framework for standardized comparative gene expression analysis of human brain-derived NPCs with other stem cell populations or differentiated tissues.

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 lM) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTPinduced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2¢,4¢-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 lM) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 lM) and PPADS (100 lM). In addition, UDP (1 lM) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5¢-O-(2-thiophosphate) (ADPbS); all 100 lM) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation. Keywords: extracellular signal-regulated kinase 1/2 pathway, neural stem/precursor cells, P2 receptors, tyrosine hydroxylase, UTP. Neural stem/precursor cells (NPCs) serve as a model of brain development, providing promise for currently incurable brain diseases and injuries. Based on their unique capacity to migrate throughout the brain and to target invading tumour cells, transplantation of NPCs offers a new

Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC) propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxia-inducible factor 1 alpha (HIF-1alpha) is rapidly degraded via the prolyl hydroxylase (PHD)/VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1alpha in NPCs and influence the transcription of HIF-regulated genes. Here, we investigate various hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine (DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl(2)) with respect to their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to stabilize HIF-1alpha, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl(2) was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497 enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both CoCl(2) and FG-4497 were protective to human dopaminergic neurons. Finally, exposure to hyperbaric oxygen (HBO) also stabilized HIF-1alpha in hmNPCs and induced neurogenesis in vitro. These findings suggest that several HIF stabilizing agents or conditions can rescue impaired neurons and promote neurogenesis in vitro.