Biomedical subjects

Rebecca Stewart

Publications and source records attributed to Rebecca Stewart.

13 recordsLinked to original sources

PubMed2006

The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis.

Understanding the molecular mechanism by which pluripotency is maintained in human embryonic stem cells (hESC) is important for the development of improved methods to derive, culture and differentiate these into cells of potential therapeutic use. Large-scale transcriptional comparison of the hES-NCL1 line derived from a day 8 embryo with H1 line derived from a day 5 embryo (WiCell Inc.) showed that only 0.52% of the transcripts analysed varied significantly between the two cell lines. This is within the variability range that has been reported when hESC derived from days 5-6 embryos have been compared with each other. This implies that transcriptional differences between the cell lines are likely to reflect their genetic profile rather than the embryonic stage from which they were derived. Bioinformatic analysis of expression changes observed when these cells were induced to differentiate as embryoid bodies suggested that quite a few of the downregulated genes were components of signal transduction networks. Subsequent analysis using western blotting, flow cytometry and antibody arrays implicated components of the PI3K/AKT kinase, MAPK/ERK and NFkappabeta pathways and confirmed that these components are decreased upon differentiation. Disruption of these pathways in isolation using specific inhibitors resulted in loss of pluripotency and/or loss of viability suggesting the importance of such signalling pathways in embryonic stem cell maintenance.

Cell Survival↗

PubMed2006

Mechanisms of self-renewal in human embryonic stem cells.

Embryonic stem cells (ESCs) are the pluripotent cell population derived from the inner cell mass of pre-implantation embryos and are characterised by prolonged self-renewal and the potential to differentiate into cells representing all three germ layers both in vitro and in vivo. Preservation of the undifferentiated status of the ESC population requires the maintenance of self-renewal whilst inhibiting differentiation and regulating senescence and apoptosis. In this review, we discuss the intrinsic and extrinsic factors associated with self-renewal process, together with possible signalling pathway interactions and mechanisms of regulation.

Biomarkers↗

PubMed2005

Human-serum matrix supports undifferentiated growth of human embryonic stem cells.

One of the most frequently used matrices for feeder-free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast-conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.

Animals↗

PubMed2005

Cloning and characterization of Xenopus beta2-microglobulin.

cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.

Amino Acid Sequence↗

PubMed2005

An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses. We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.

Animals↗

PubMed2004

Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture.

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

Adult↗

PubMed2003

Formation of neurospheres from human embryonal carcinoma stem cells.

Embryonal carcinoma (EC) stem cells derived from germ cell tumours are valuable tools for the study of embryogenesis and closely resemble embryonic stem cells. When human TERA2.cl.SP12 EC cells are exposed to retinoic acid and grown as adherent monolayers, approximately 10-15% of cells commit toward becoming neurons whilst the remainder of cells produce non-neuronal cell types. Using established protocols it is possible to isolate and purify neurons from these cultures but such a process takes several weeks and the numbers of neurons produced are relatively low. In this study, we describe the development of novel procedures to enhance neuronal productivity with dramatically increased efficiency, which will be of value for research purposes and drug discovery programmes.

Antigens, Surface↗

PubMed2003

Ontogeny of Xenopus NK cells in the absence of MHC class I antigens.

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at approximately 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3-4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3-4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRbeta V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.

Animals↗

PubMed2003

Manipulation of human pluripotent embryonal carcinoma stem cells and the development of neural subtypes.

There are few reliable cell systems available to study the process of human neural development. Embryonal carcinoma (EC) cells are pluripotent stem cells derived from teratocarcinomas and offer a robust culture system to research cell differentiation in a manner pertinent to embryogenesis. Here, we describe the recent development of a series of culture procedures that together can be used to induce the differentiation of human EC stem cells, resulting in the formation of either pure populations of differentiated neurons, populations of differentiated astrocytes, or populations of immature neuronal cell types. Cell-type-specific markers were used to examine the induction of EC stem cell differentiation by retinoic acid. In direct response to manipulation of the culture environment, the expression of cell type markers correlated with the differentiation and appearance of distinct neural cell types, including neurons and astrocytes. These experiments demonstrate that cultured human EC stem cells provide a robust model cell system capable of reproducibly forming neural subtypes for research purposes.

Astrocytes↗

PubMed2002

Identification and characterization of Xenopus CD8+ T cells expressing an NK cell-associated molecule.

Growing evidence suggests that some immune responses are mediated not only by conventional and distinct NK cells and CTL, but also by T cell subsets expressing NK receptors and NK cell-associated molecules. Consistent with our previously published finding that the mAb 1F8 identifies non-T/non-B cells in Xenopus that effect NK-like killing in vitro, we now report that in vivo treatment with this mAb impairs rejection of transplanted MHC class I-negative tumor cells. However, we also find that the NK cell-associated molecule recognized by mAb 1F8 is expressed by a minor population of CD8+ T cells, in which fully rearranged TCRbeta mRNA of at least three different V families can be identified, by contrast, 1F8+/CD8- (NK) cells lack such TCRbeta message. Additionally, the expression of the NK cell-associated molecule can be induced in vitro by a transient submitogenic stimulation of naïve CD8+ T cells with PMA and ionomycin. Such induced expression of 1F8 also occurs in alloantigen-activated CTL and is coincident with a down-regulation of MHC-specific cytotoxicity. Taken together, these new data suggest that regulation of CD8+ T cell activity involving NK cell-associated molecules is a general and evolutionarily ancient phenomenon.

Animals↗

PubMed2002

Non-neural adult stem cells: tools for brain repair?

Stem cells isolated from adult mammalian tissues may provide new approaches for the autologous treatment of disease and tissue repair. Although the potential of adult stem cells has received much attention, it has also recently been brought into question. This article reviews the recent work describing the ability of non-hematopoietic stem cells derived from adult bone marrow to form neural derivatives and their potential for brain repair. Earlier transplantation experiments imply that grafted adult stem cells can differentiate into neural derivatives. Recent reports suggest, however, that such findings may be misleading and grafted cells acquiring different identities may merely be explained by their fusion with host cells and not the result of radical changes to their program of cellular differentiation. Nonetheless, in vitro studies have shown that neural development by bone-marrow-derived stem cells also appears possible. Understanding the molecular mechanisms that specify the neural lineage will lead to the development of tools for the targeted production of neural cell types in vitro that may ultimately provide a source of material to treat specific neurological deficits.

Animals↗

PubMedDate not supplied

Command and control in emergency services operations: a social network analysis.

There is increasing interest in the use of social network analysis as a tool to study the performance of teams and organizations. In this paper, processes of command and control in the emergency services are explored from the perspective of social network theory. We report a set of network analyses (comprising visualization, a selection of mathematical metrics, and a discussion of procedures) based on the observation of six emergency service incidents: three fire service operations involving the treatment of hazardous chemicals, and three police operations involving immediate response to emergency calls. The findings are discussed in terms of our attempts to categorize the network structures against a set of extant command and control network archetypes and the relationship between those structures; comments on the qualities the networks display are put into the contexts of the incidents reported. We suggest that social network analysis may have a valuable part to play in the general study of command and control.

Efficiency, Organizational↗

PubMedDate not supplied

Understanding mentoring and preceptorship: clarifying the quagmire.

Mentoring! Preceptorship! These two terms are widely used within nursing. The vast literature on mentoring and preceptorship defines these concepts as discrete roles. However, confusion exists as to what defines mentorship when compared to preceptorship. It is apparent that these terms are being used interchangeably, despite the obvious and not so obvious differences portrayed in the literature. The authors therefore have questioned whether the roles of a mentor and preceptor are discrete and unique or can be integrated into one role? Furthermore, is one of these constructs more appropriate to new academics whilst the other is more appropriate for nurses in the clinical setting? This discussion paper is a journey into understanding preceptorship and mentoring and the value of sustaining a relationship with both.

Attitude of Health Personnel↗