Eric Jervis

Support for the immortal strand hypothesis neural stem cells partition DNA asymmetrically in vitro

The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.

Cyclosporin A Has Direct Effects on Adult Neural Precursor Cells

Multipotent, self-renewing neural stem cells and their progeny [collectively referred to as neural precursor cells (NPCs)] represent a population of cells with great promise for CNS repair. To effectively harness their potential for therapeutic applications, the factors that regulate NPC behavior and/or fate must be well understood. The ability of immunomodulatory molecules to affect NPC behavior is of interest because of recent work elucidating the complex interactions between the immune system and nervous system. Herein, we examined the effects of cyclosporin A, a commonly used immunosuppressive molecule, on NPC proliferation kinetics, survival, and fate using in vitro assays at the population level and at the single-cell level. The use of pure populations of NPCs revealed a direct effect of cyclosporin A on cell survival, resulting in increased numbers and larger colonies, with no effect on proliferation kinetics. Cyclosporin A did not alter the differentiation profile of NPC colonies, indicating that it did not promote selective survival of a particular neural lineage. Additionally, we observed decreased cell–cell adhesions in developing cyclosporin A-treated NPC colonies. Consistent with the in vitro observations, in vivo administration of cyclosporin A to adult animals increased the numbers of NPCs within the neurogenic niche lining the lateral ventricles. Together, our findings establish that cyclosporin A has direct effects on NPCs both in vitro and in vivo, making it a promising candidate molecule for developing clinically relevant strategies to stimulate NPCs for brain repair.

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor.

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.

An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G1 phase assembly of pre‐replicative complexes (pre‐RC). Only the Orc1‐5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6‐YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre‐RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G1, Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

Probabilistic Model-Based Cell Tracking

The study of cell behavior is of crucial importance in drug and disease research. The fields of bioinformatics and biotechnology rely on the collection, processing, and analysis of huge numbers of biocellular images, including cell features such as cell size, shape, and motility. However manual methods of inferring these values are so onerous that automated methods of cell tracking and segmentation are in high demand. In this paper, a novel model-based cell tracker is designed to locate and track individual cells. The proposed cell tracker has been successfully applied to track hematopoietic stem cells (HSCs) based on identified cell locations and probabilistic data association.

Watershed deconvolution for cell segmentation

Cell segmentation and/or localization is the first stage of a (semi)automatic tracking system. We addressed the cell localization problem in our previous work where we characterized a typical blood stem cell in a microscopic image as an approximately circular object with dark interior and bright boundary. We also addressed the modelling of adjacent and dividing cells in our previous work as a deconvolution method to model individual blood stem cell as well as adjacent and dividing blood stem cells where an optimization algorithm was combined with a template matching method to segment cell regions and locate the cell centers. Our previous cell deconvolution method is capable of modelling different cell types with changes in the model parameters. However in cases where either a complex parameterized shape is needed to model a specific cell type, or in place of cell center localization, an exact cell segmentation is needed, this method will not be effective. In this paper we propose a method to achieve cell boundary segmentation. Considering cell segmentation as an inverse problem, we assume that cell centers are located in advance. Then, the cell segmentation will be solved by finding cell regions for optimal representation of cell centers while a template matching method is effectively employed to localize cell

A Statistical Thresholding Method for Cell Tracking

Tracking the motion of cells in culture is a task, which often still Is undertaken manually, and for which automated methods are strongly desirable. Researchers visually perform cell motion analysis, observe cell movements and cell shape changes for hours to discover when, where and how fast It moves, splits or dies. Hematopoletlc stem cells (HSCs) proliferate and differentiate to different blood cell types continuously during their lifetime, and are of substantial interest in gene therapy, cancer, and stem-cell research. In this paper a statistical method is introduced to track HSCs over time. A statistical thresholding method is combined with joint probabilistic data association in the proposed HSC tracker

Human ESC Colony Formation Is Dependent on Interplay Between Self-Renewing hESCs and Unique Precursors Responsible for Niche Generation

Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long‐term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self‐renewal is dependent on colony‐proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC‐derived cells, leading to clonal selection of colony reestablishing cells. hESC‐derived feeders co‐transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self‐renewing hESCs. Our findings demonstrate that hESC‐derived feeders arise from a distinct subpopulation of hESCs that respond to paracrine cues at the colony periphery that are required to sustain and establish clonal hESC self‐renewal.

True monolayer cell culture in a confined 3D microenvironment enables lineage informatics

There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo‐like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates “lineage informatics,” a lineage‐centric approach to cytomics.

Synthesis and evaluation of trans 3,4-cyclopropyl l-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.