Nodal, a member of the TGF-beta family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of beta-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of beta-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.

Silencing and variegated transgene expression are poorly understood problems that can interfere with gene function studies in human embryonic stem cells (hESCs). We show that transgene expression (enhanced green fluorescent protein [EGFP]) from random integration sites in hESCs is affected by variegation and silencing, with only half of hESCs expressing the transgene, which is gradually lost after withdrawal of selection and differentiation. We tested the hypothesis that a transgene integrated into the adenoassociated virus type 2 (AAV2) target region on chromosome 19, known as the AAVS1 locus, would maintain transgene expression in hESCs. When we used AAV2 technology to target the AAVS1 locus, 4.16% of hESC clones achieved AAVS1-targeted integration. Targeted clones expressed Oct-4, stage-specific embryonic antigen-3 (SSEA3), and Tra-1-60 and differentiated into all three primary germ layers. EGFP expression from the AAVS1 locus showed significantly reduced variegated expression when in selection, with 90% ؎ 4% of cells expressing EGFP compared with 57% ؎ 32% for randomly integrated controls, and reduced tendency to undergo silencing, with 86% ؎ 7% hESCs expressing EGFP 25 days after withdrawal of selection compared with 39% ؎ 31% for randomly integrated clones. In addition, quantitative polymerase chain reaction analysis of hESCs also indicated significantly higher levels of EGFP mRNA in AAVS1-targeted clones as compared with randomly integrated clones. Transgene expression from the AAVS1 locus was shown to be stable during hESC differentiation, with more than 90% of cells expressing EGFP after 15 days of differentiation, as compared with ϳ30% for randomly integrated clones. These results demonstrate the utility of transgene integration at the AAVS1 locus in hESCs and its potential clinical application. STEM CELLS 2008;26: 496 -504 Disclosure of potential conflicts of interest is found at the end of this article.

Summary:Purpose: Approved neural-stimulation therapies for epilepsy use prolonged intermittent stimulation paradigms with no ability to respond automatically to seizures.Methods: A responsive neurostimulator that can automatically analyze electrocortical potentials, detect electrographic seizures, and rapidly deliver targeted electrical stimuli to suppress them was evaluated in an open multicenter trial in 50 patients, 40 of whom received responsive cortical stimulation via subdural electrodes implanted for epilepsy surgery evaluations.Results: Four patients, ages 15 to 28 years, monitored at three institutions, with clinical and electrographic response to neurostimulation, are described. Electrographic seizures were altered and suppressed in these patients during trials of neurostimulation lasting ≤68 h, with no major side effects. In one patient, stimulation appeared also to improve the baseline EEG.Conclusions: Responsive cortical neurostimulation may be a safe and effective treatment for partial epilepsy. This information was derived from a small group of patients in an observation study. A double-blind, controlled Food and Drug Administration (FDA)-approved study of a permanently implanted responsive neurostimulation system to treat medically refractory partial seizures is under way.

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