Efforts to study the development and function of the human cerebral cortex in health and disease have been limited by the availability of model systems. Extrapolating from our understanding of rodent cortical development, we have developed a robust, multistep process for human cortical development from pluripotent stem cells: directed differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to cortical stem and progenitor cells, followed by an extended period of cortical neurogenesis, neuronal terminal differentiation to acquire mature electrophysiological properties, and functional excitatory synaptic network formation. We found that induction of cortical neuroepithelial stem cells from human ES cells and human iPS cells was dependent on retinoid signaling. Furthermore, human ES cell and iPS cell differentiation to cerebral cortex recapitulated in vivo development to generate all classes of cortical projection neurons in a fixed temporal order. This system enables functional studies of human cerebral cortex development and the generation of individual-specific cortical networks ex vivo for disease modeling and therapeutic purposes.

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.

Human cellular models of Alzheimer's disease (AD) pathogenesis would enable the investigation of candidate pathogenic mechanisms in AD and the testing and developing of new therapeutic strategies. We report the development of AD pathologies in cortical neurons generated from human induced pluripotent stem (iPS) cells derived from patients with Down syndrome. Adults with Down syndrome (caused by trisomy of chromosome 21) develop early-onset Alzheimer's disease, probably due to increased expression of the amyloid precursor protein (APP) encoded by a gene on chromosome 21. We found that cortical neurons generated from iPS cells and embryonic stem (ES) cells from Down syndrome patients developed Alzheimer's disease pathologies over months in culture, rather than years in vivo. The cortical neurons processed the transmembrane APP protein resulting in secretion of the pathogenic amyloid-β42 (Aβ42) peptide fragment. Aβ42 peptides formed insoluble intracellular and extracellular amyloid aggregates. Production of Aβ peptides was blocked by a gamma-secretase inhibitor. Finally, hyperphosphorylated tau protein, a pathological hallmark of AD, was found to be localized to cell bodies and dendrites in iPS cell-derived cortical neurons from Down syndrome patients, recapitulating later stages of the AD pathogenic process.

Mutations in SCN1A, the gene encoding the α subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks, suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary, our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons, and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation.

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.

We establish Anderson localization for general analytic k-frequency quasi-periodic operators on Z d for arbitrary k, d.

Alzheimer's disease (AD) is the most common form of dementia in the elderly; important risk factors are old age and inheritance of the apolipoprotein E4 (APOE4) allele. Changes in amyloid precursor protein (APP) binding, trafficking, and sorting may be important AD causative factors. Secretase-mediated APP cleavage produces neurotoxic amyloid-beta (Aβ) peptides, which form lethal deposits in the brain. In vivo and in vitro studies have implicated sortilin-related receptor (SORL1) as an important factor in APP trafficking and processing. Recent in vitro evidence has associated the APOE4 allele and alterations in the SORL1 pathway with AD development and progression. Here, we analyzed SORL1 expression in neural stem cells (NSCs) from AD patients carrying null, one, or two copies of the APOE4 allele. We show reduced SORL1 expression only in NSCs of a patient carrying two copies of APOE4 allele with increased Aβ/SORL1 localization along the degenerated neurites. Interestingly, SORL1 binding to APP was largely compromised; this could be almost completely reversed by γ-secretase (but not β-secretase) inhibitor treatment. These findings may yield new insights into the complex interplay of SORL1 and AD pathology and point to NSCs as a valuable tool to address unsolved AD-related questions in vitro.

This paper presents BCube, a new network architecture specifically designed for shipping-container based, modular data centers. At the core of the BCube architecture is its server-centric network structure, where servers with multiple network ports connect to multiple layers of COTS (commodity off-the-shelf) mini-switches. Servers act as not only end hosts, but also relay nodes for each other. BCube supports various bandwidth-intensive applications by speedingup one-to-one, one-to-several, and one-to-all traffic patterns, and by providing high network capacity for all-to-all traffic.BCube exhibits graceful performance degradation as the server and/or switch failure rate increases. This property is of special importance for shipping-container data centers, since once the container is sealed and operational, it becomes very difficult to repair or replace its components.Our implementation experiences show that BCube can be seamlessly integrated with the TCP/IP protocol stack and BCube packet forwarding can be efficiently implemented in both hardware and software. Experiments in our testbed demonstrate that BCube is fault tolerant and load balancing and it significantly accelerates representative bandwidthintensive applications.