Thomas R. Kiehl

CORTECON: A Temporal Transcriptome Analysis of In Vitro Human Cerebral Cortex Development from Human Embryonic Stem Cells

Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.

Transcriptomic Responses to Sodium Chloride-Induced Osmotic Stress: A Study of Industrial Fed-Batch CHO Cell Cultures

The rapidly expanding market for monoclonal antibody and Fc‐fusion‐protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc‐fusion‐protein to hyperosmotic stress, a well‐known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132–145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine‐hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant‐protein productivity.

Development of antifouling surfaces to reduce bacterial attachment

It is well documented that bacterial adhesion to surfaces is mediated by the physical and chemical properties of the substrate, as well as the surface characteristics of the organism. Topographical features that limit cell–surface interactions have been shown to reduce surface colonization and biofilm formation. In this study, bacterial attachment to medically relevant materials was evaluated. Our data show that Escherichia coli attachment to glass, silicone, and titanium surfaces was most affected by the surface energy of these materials, as determined by water contact angle. The inherent roughness of the surface, however, was not correlated with cell attachment density. To study the effect of engineered surface roughness on bacterial attachment, topographical features, including arrays of holes and repeating lines/trenches, were formed from silicon wafers and then used as a template to imprint silicone-based polydimethylsiloxane (PDMS). Patterned silicone surfaces were then used in static and microfluidic flow-based experiments to evaluate cellular settlement and attachment. Cell attachment was observed to be strongly dependent upon the topographical features under both static and microfluidic flow conditions. The highest attachment density was observed on flat, un-patterned surfaces, while linear patterned surfaces showed greatly reduced cell attachment. Moreover, surfaces consisting of arrays of holes further reduced cell attachment as compared to linear patterns. These results demonstrate that the size, spacing, and shape of surface features play a significant role in cell–surface attachment and provide insight for the design of surfaces with antifouling properties.

Observations of cell size dynamics under osmotic stress

Cultured mammalian cells [e.g., murine hybridomas, Chinese hamster ovary (CHO) cells] used to produce therapeutic and diagnostic proteins often exhibit increased specific productivity under osmotic stress. This increase in specific productivity is accompanied by a number of physiological changes, including cell size variation. Investigating the cell size variation of hyperosmotically stressed cultures may reveal, in part, the basis for increased specific productivity as well as an understanding of some of the cellular defense responses that occur under hyperosmotic conditions. The regulation of cell volume is a critical function maintained in animal cells. Although these cells are highly permeable to water, they are significantly less permeable to ionic solutes. Appropriate cell–water content is actively maintained in these cells by regulation of ion and osmolyte balances. Transport appropriate to extracellular conditions, leading to accrual or release of these species, is activated in response to acute cell volume changes. Osmotically induced regulatory volume increases (RVI) and regulatory volume decreases (RVD) are known to occur under a variety of conditions. We observed the time evolution of size variation in populations of two CHO cell lines under hyperosmotic conditions. Observations were made using multiple instruments, multiple cell lines, and multiple cell culture conditions. Size variation of CHO A1 was gauged by flow cytometry using an LSRII® flow cytometer while CHO B0 cells were quantified using a Cedex® cell analyzer. Hyperosmotic stress had a dose‐dependent effect on the regulatory control of cell volume. Stressed cultures of CHO cells grown in suspension exhibited a shift in mean cell diameter. This shift in mean was not due to a change in the whole population, but rather to the emergence of distinct subpopulations of cells with larger cell diameters than those in the bulk of the population.

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.

Non-monotonic Changes in Progenitor Cell Behavior and Gene Expression during Aging of the Adult V-SVZ Neural Stem Cell Niche

Summary Neural stem cell activity in the ventricular-subventricular zone (V-SVZ) decreases with aging, thought to occur by a unidirectional decline. However, by analyzing the V-SVZ transcriptome of male mice at 2, 6, 18, and 22 months, we found that most of the genes that change significantly over time show a reversal of trend, with a maximum or minimum expression at 18 months. In vivo, MASH1+ progenitor cells decreased in number and proliferation between 2 and 18 months but increased between 18 and 22 months. Time-lapse lineage analysis of 944 V-SVZ cells showed that age-related declines in neurogenesis were recapitulated in vitro in clones. However, activated type B/type C cell clones divide slower at 2 to 18 months, then unexpectedly faster at 22 months, with impaired transition to type A neuroblasts. Our findings indicate that aging of the V-SVZ involves significant non-monotonic changes that are programmed within progenitor cells and are observable independent of the aging niche.

Evolving biochemical reaction networks with stochastic attributes

Biochemical networks display a wide range of behaviors. While many of these networks tend to operate in a steady-state regime, others exhibit distinctly stochastic behaviors. Fitting models to data from these systems challenges many of the linear and steady-state assumptions of typical modeling techniques. The genetic algorithm described herein seeks to generate networks which exhibit desired average/steady-state behaviors while minimizing or maximizing the standard deviation of those behaviors.

Molecular Response to Osmotic Shock

The cellular responses of cultured mammalian cells and non-mammalian organisms to changes in osmolarity are discussed. A number of common themes including activation of protein kinase cascades can be observed in a diverse group of organisms. A combination of physiological and transcriptional studies has been performed to identify regulatory factors and proteins that play a causal role in the cellular responses to osmotic changes. These factors may serve as targets for cellular engineering strategies to improve the productivity of cultured mammalian cells, particularly in response to osmotic shock

Big Data access and infrastructure for modern biology: case studies in data repository utility

Big Data is no longer solely the purview of big organizations with big resources. Todays routine tools and experimental methods can generate large slices of data. For example, high‐throughput sequencing can quickly interrogate biological systems for the expression levels of thousands of different RNAs, examine epigenetic marks throughout the genome, and detect differences in the genomes of individuals. Multichannel electrophysiology platforms produce gigabytes of data in just a few minutes of recording. Imaging systems generate videos capturing biological behaviors over the course of days. Thus, any researcher now has access to a veritable wealth of data. However, the ability of any given researcher to utilize that data is limited by her/his own resources and skills for downloading, storing, and analyzing the data. In this paper, we examine the necessary resources required to engage Big Data, survey the state of modern data analysis pipelines, present a few data repository case studies, and touch on current institutions and programs supporting the work that relies on Big Data.

Visualizing spike activity during neuronal network development.

We are interested in explaining neuronal network development through visualizations that summarize trends in large data. We utilized previously-recorded spiking patterns of embryonic rat cortex cells grown on multielectrode arrays [1]. We present results for batch 1 culture 3. Recordings were divided into 100 17.7 s intervals (the time required to sequentially stimulate each electrode at 0.3 s intervals). In our representation, each trail depicts an interval. The first 50 intervals recorded spontaneous activity (1-25 in red, 26-50 in pink); the last 50 intervals, activity in response to electrical stimulation (51-75 in green, 76-100 in blue). Each trail begins at the origin,