In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH‐positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine‐β‐hydroxylase, a marker of noradrenergic neurons. PA6‐induced neurons were able to release dopamine and 3,4‐dihydroxphe‐hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+. When transplanted into 6‐hydroxydopamine–treated animals, hES‐derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH‐positive cells were present but in very small numbers compared with the high frequency of TH‐positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.

Adenosine A3 receptor (A3R) agonists have been shown to reduce cardiac and lung injury, but the protective roles of A3R agonists in the CNS are not well characterized. The protective effect of selective A3R agonist chloro-N(6)-(3-iodo-benzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) was first examined in primary cortical cultures. In cortical culture, Cl-IB-MECA pretreatment antagonized the hypoxia-mediated decrease in cell viability. In vivo, Cl-IB-MECA or vehicle was given intracerebroventricularly or intravenously to anesthetized rats. Animals were subjected to focal cerebral ischemia induced by transient middle cerebral artery (MCA) ligation. Intracerebroventricular or repeated intravenous administration (i.e., at 165 min and 15 min before MCA ligation) of Cl-IB-MECA did not alter blood pressure during ischemia but increased locomotor activity and decreased cerebral infarction 2 days after. In these animals, Cl-IB-MECA also reduced the density of TUNEL labeling in the lesioned cortex. The possibility of endogeneous neuroprotection was further examined in A3R knockout mice. After MCA ligation, an increase in cerebral infarction was found in the A3R knockouts compared with the A3R wild-type controls, suggesting that A3Rs are tonically activated during ischemia. Additionally, intracerebroventricular pretreatment with Cl-IB-MECA decreased the size of infarction in the wild-type controls, but not in the A3R knockout animals, suggesting that Cl-IB-MECA-induced protection was mediated through the A3 receptors. Collectively, these data suggest that Cl-IB-MECA reduced cerebral infarction through the activation of A3Rs and suppression of apoptosis.

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