Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca(2+) imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.

The affymetrix GeneChip platform: an overview.

The intent of this chapter is to provide the reader with a review of GeneChip technology and the complete system it represents, including its versatility, components, and the exciting applications that are enabled by this platform. The following aspects of the technology are reviewed: array design and manufacturing, target preparation, instrumentation, data analysis, and both current and future applications. There are key differentiators between Affymetrix' GeneChip technology and other microarray-based methods. The most distinguishing feature of GeneChip microarrays is that their manufacture is directed by photochemical synthesis. Because of this manufacturing technology, more than a million different probes can be synthesized on an array roughly the size of a thumbnail. These numbers allow the inclusion of multiple probes to interrogate the same target sequence, providing statistical rigor to data interpretation. Over the years the GeneChip platform has proven to be a reliable and robust system, enabling many new discoveries and breakthroughs to be made by the scientific community.

Monitoring eukaryotic gene expression using oligonucleotide microarrays.

An increasing number of biological and medical research questions depend on obtaining global views of gene expression. In this chapter, we will describe how oligonucleotide microarrays have been used to accomplish this goal. In particular, we will focus on the use of GeneChip arrays, which provide high levels of reproducibility, sensitivity, and specificity. Target preparation, hybridization, washing, signal detection, and data analysis will be described in detail. Additionally, we will discuss options for facilitating data sharing, including the creation of databases, and the use of internet tools that help users place their results in the context of data from public and proprietary databases. There is so much interest and innovation in the field of genomics that protocols are constantly evolving. This chapter should be used as a genomic profiling guide only. We urge readers to consult www.affymetrix.com for the most current products and protocols.

Microarrays in pharmacogenomics--advances and future promise.

With their ability to provide global views of genome sequence and gene activity, microarrays have emerged as key analytical tools in the field of pharmacogenomics. Vast amounts of data must be collected and analyzed to meet pharmacogenomics' ambitious goals, ranging from identifying markers that predict individuals' responses to therapy to discovering new drug targets. Microarrays will be instrumental to these efforts because they provide bountiful sources of gene expression and genotypic data. Attesting to their productivity, microarrays have been the central technology used in thousands of peer-reviewed publications and have also become important contributors to many databases including PharmGKB, the Cancer Microarray Database and the database of single nucleotide polymorphisms (dbSNP). Microarrays are also making more focused contributions, however, in helping pursue hypothesis-driven inquiries that extend or complement broad genomic surveys. In addition, their potential as clinical tools is being increasingly recognized. This review identifies some of the varied and changing needs of pharmacogenomics research and discusses the ways in which microarrays are tending to these demands. The technique's strongpoints and limitations are examined, as well as its future potential.

Microarrays and genetic epidemiology: a multipurpose tool for a multifaceted field.

The advent of molecular technologies that allow the collection and analysis of large amounts of genetic data is rapidly transforming the field of genetic epidemiology. Whether monitoring infectious outbreaks or identifying genotypic variations that underlie disease susceptibility, genetic epidemiology relies heavily on the analysis of multiple, independently derived results. By allowing the simultaneous monitoring of thousands of genetic or expression data points, microarrays are emerging as particularly powerful tools. Several recent reviews have described array manufacturing and the types of scientific questions that can exploit this technology, but few have addressed how the intended use of an array can dictate its design. This review will focus on this latter issue, with particular emphasis on the genetic epidemiology of infectious disease. The design of arrays for genotyping, expression profiling, and fingerprinting are presented, and examples of recent epidemiological studies are used to illustrate the applications' strong points and limitations. In addition to discussing arrays' ability to provide global views of gene identity or function, the review will describe design options for creating arrays that detect multiple genetic variations. It will also examine the reliability of array-generated fingerprints, assay accessibility, and possibilities for sharing and comparing data across studies. Although many challenges lie ahead, microarrays' multiple abilities appear uniquely poised to accelerate the advance of genetic epidemiology's multiple fronts.

Single nucleotide polymorphisms and their characterization with oligonucleotide microarrays.

Small variations in human DNA sequences influence our predisposition to disease and our response to medications. Many psychiatric diseases appear to be polygenic. Because our molecular understanding of the genetic etiology of neuropsychiatric disorders is very limited, the discovery and characterization of these variations is crucial in identifying disease genes. Furthermore, this knowledge may enable the customized selection of drug treatments based on an individual's genotype so as to maximize efficacy yet avoid adverse side effects. Examples of findings using conventional methods for determining genotypes, as well as new and future technologies for the characterization of single nucleotide polymorphisms, will be discussed, with a focus on psychiatric diseases and medications.