Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.

Standard cell culture systems impose environmental oxygen (O 2 ) levels of 20%, whereas actual tissue O 2 levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O 2 environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O 2 and in lowered O 2 (3 Ϯ 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O 2 , yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O 2 compared with 18% in 20% O 2 . Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O 2 . Erythropoietin supplementation of 20% O 2 cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O 2 . These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O 2 , making this method an important advance in the ex vivo generation of specific neurons for brain repair. Key words: CNS precursors; CNS stem cells; dopaminergic neurons; erythropoietin; oxygen; Parkinson's diseaseCultured CNS stem cells have proved useful in defining the pathways that lead to generation of neurons and glia (McKay, 1997). These cells self-renew, and after mitogen withdrawal, differentiate into neurons, astrocytes and oligodendrocytes in predictable proportions (Johe et al., 1996;McKay, 1997). Single extrinsic factors can shift the fate of CNS stem cells toward specific cell lineages (Johe et al., 1996;Panchision et al., 1998). The potential therapeutic application of CNS stem cells in common degenerative and ischemic diseases has become a major focus of research. The generation of dopaminergic neurons from CNS precursors is of special interest given the promising results of fetal cell transplantation in patients with Parkinson's disease (Olanow et al., 1996; Piccini at al., 1999;Freeman et al., 2000).In clinical settings, gases are appreciated as primary variables in organ survival, with O 2 as the critical gas parameter. However, traditional CNS stem cell culture (as well as virtually all other ex vivo cell culture) is performed in nonphysiologically high O 2 . Standard tissue culture incubator conditions are 5% CO 2 and 95% air, which exposes cells to a 20% O 2 environment. In mammalian brain, interstitial tissue O 2 levels range from ϳ1 to 5% (Table 1). We tested the effects of culturing CNS progenitor cells in physiological "lowered" (3 Ϯ 2%) O 2 , comparing the cultures with those grown in the usual 20% O 2 . Our results indicate that oxygen lowere...

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Human brain organoid techniques have rapidly advanced to facilitate investigating human brain development and diseases. These efforts have largely focused on generating telencephalon due to its direct relevance in a variety of forebrain disorders. Despite its importance as a relay hub between cortex and peripheral tissues, the investigation of three-dimensional (3D) organoid models for the human thalamus has not been explored. Here, we describe a method to differentiate human embryonic stem cells (hESCs) to thalamic organoids (hThOs) that specifically recapitulate the development of thalamus. Single-cell RNA sequencing revealed a formation of distinct thalamic lineages, which diverge from telencephalic fate. Importantly, we developed a 3D system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids representing the developing thalamus or cortex.Our study provides a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain.(C) qPCR analysis for expressions of regional markers in developing hThOs, hMGEOs, and hCOs. Each data point represents expressions in pooled batch of 3-4 organoids, and 3 batches were collected for analysis. Mean ± SD is shown. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Immunostaining for MAP2 and thalamic marker TCF7L2 in day 41 hThO, hCO, and hMGEO. The scale bar represents 250 mm. (E) Immunostaining for thalamic and cortical progenitor marker PAX6, and cortical marker TBR1 in day 41 hThO, hCO, and hMGEO. The scale bar represents 250 mm. See also Figure S1.

Nonlinear wave propagation is ubiquitous in nature, appearing in chemical reaction kinetics, cardiac tissue dynamics, cortical spreading depression and slow wave sleep. The application of dynamical modelling has provided valuable insights into the mechanisms underlying such nonlinear wave phenomena in several domains. Wave propagation can also be perceived as sweeping waves of visibility that occur when the two eyes view radically different stimuli. Termed binocular rivalry, these fluctuating states of perceptual dominance and suppression are thought to provide a window into the neural dynamics that underlie conscious visual awareness. Here we introduce a technique to measure the speed of rivalry dominance waves propagating around a large, essentially one-dimensional annulus. When mapped onto visual cortex, propagation speed is independent of eccentricity. Propagation speed doubles when waves travel along continuous contours, thus demonstrating effects of collinear facilitation. A neural model with reciprocal inhibition between two layers of units provides a quantitative explanation of dominance wave propagation in terms of disinhibition. Dominance waves provide a new tool for investigating fundamental cortical dynamics.

Human embryonic stem (hES) cells, due to their capacity of multipotency and self-renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinson's disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co-cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed-1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell-derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl-induced depolarization and reuptake of DA. Finally, transplantation of hES-derived DA neurons into the striatum of hemi-parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine-induced rotation and step-adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES-derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cellderived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains.