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Biomedical subjects

James A Thomson

Publications and source records attributed to James A Thomson.

At least 19 recordsLinked to original sources
PubMed2007

Nanog and transcriptional networks in embryonic stem cell pluripotency.

Several extrinsic signals such as LIF, BMP and Wnt can support the self-renewal and pluripotency of embryonic stem (ES) cells through regulating the "pluripotent genes." A unique homeobox transcription factor, Nanog, is one of the key downstream effectors of these signals. Elevated level of Nanog can maintain the mouse ES cell self-renewal independent of LIF and enable human ES cell growth without feeder cells. In addition to the external signal pathways, intrinsic transcription factors such as FoxD3, P53 and Oct4 are also involved in regulating the expression of Nanog. Functionally, Nanog works together with other key pluripotent factors such as Oct4 and Sox2 to control a set of target genes that have important functions in ES cell pluripotency. These key factors form a regulatory network to support or limit each other's expression level, which maintains the properties of ES cells.

Amino Acid Sequence↗
PubMed2006

A severe de novo methylation of episomal vectors by human ES cells.

Episomal vectors can allow efficient genetic modification of cells and have the potential advantage of avoiding chromosomal position of integration effects. Here we explore the use of an Epstein-Barr virus-based episomal vector with human embryonic stem (ES) cells, and find high initial transfection rates, but a rapid loss of reporter gene expression. Similar to mouse ES cells, human ES cells express high levels of the de novo DNA methyltransferases, and we detected dramatic CpG methylation and minor non-CpG methylation on the episomes recovered from the human ES cells 7 days after the transfection, which was not present on the same episome recovered from 293 cells. Interestingly, the oriP region of the episomes was relatively excluded from this methylation. These findings define some of the limitations of using episomal vectors with human ES cells and offer a unique platform for analyzing epigenetic gene silencing in human ES cells.

Cells, Cultured↗
PubMed2006

Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.

During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile.

Antigens, CD34↗
PubMed2006

Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway.

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.

Animals↗
PubMed2006

Derivation of human embryonic stem cells in defined conditions.

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

Cell Culture Techniques↗
PubMed2006

Microarray analysis of stem cells and differentiation.

Microarrays have revolutionized molecular biology and enabled biologists to perform global analysis on the expression of tens of thousands of genes simultaneously. They have been widely used in gene discovery, biomarker determination, disease classification, and studies of gene regulation. Microarrays have been applied in stem cell research to identify major features or expression signatures that define stem cells and characterize their differentiation programs toward specific lineages. Here we provide a review of the microarray technology, including the introduction of array platforms, experimental designs, RNA isolation and amplification, array hybridization, and data analysis. We also detail examples that apply microarray technology to address several of the main questions in stem cell biology.

Animals↗
PubMed2005

Basic fibroblast growth factor support of human embryonic stem cell self-renewal.

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

Cell Culture Techniques↗
PubMed2005

Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients.

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.

ADP-ribosyl Cyclase 1↗
PubMed2005

Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion.

Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell-cell fusion. Using an OP9 coculture system, we produced CD45+ CD33+ myeloperoxidase+ myeloid precursors from an Oct4-enhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.

Animals↗
PubMed2005

Human ERas gene has an upstream premature polyadenylation signal that results in a truncated, noncoding transcript.

The ERas gene is expressed in mouse embryonic stem (ES) cells and promotes their in vitro proliferation and tumorigenicity. We analyzed the expression of the human ERas gene in human ES cells by reverse transcription-polymerase chain reaction (RT-PCR) and serial analysis of gene expression but could not detect a full-length coding transcript. Sequence analysis predicted a premature polyadenylation signal for the human ERas transcript, which we confirmed by 3' RACE analysis. By RT-PCR, we identified a truncated noncoding transcript in human ES cells that is downregulated during differentiation, suggesting conserved tissue specificity of the promoter region. Previous reports and expressed sequence tag databases indicate that orthologues of this gene are expressed in other mammals, including the mouse, dog, and cow, which suggests that it became a silenced pseudogene relatively recently in mammalian evolution. In addition to the premature polyadenylation site, both the human and chimpanzee ERas genes include typical Alu-S retrotransposon insertions that could also influence expression at this locus. The lack of ERas expression in human ES cells suggests that they could have significantly different tumorigenic properties than mouse ES cells.

Animals↗
PubMed2005

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.

Bone Morphogenetic Proteins↗
PubMed2005

Influence of virtual reality training on the roadside crossing judgments of child pedestrians.

The roadside crossing judgments of children aged 7, 9, and 11 years were assessed relative to controls before and after training with a computer-simulated traffic environment. Trained children crossed more quickly, and their estimated crossing times became better aligned with actual crossing times. They crossed more promptly, missed fewer safe opportunities to cross, accepted smaller traffic gaps without increasing the number of risky crossings, and showed better conceptual understanding of the factors to be considered when making crossing judgments. All age groups improved to the same extent, and there was no deterioration when children were retested 8 months later. The results are discussed in relation to theoretical arguments concerning the extent to which children's pedestrian judgments are amenable to training.

Accidents, Traffic↗
PubMed2005

A germ cell origin of embryonic stem cells?

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.

Animals↗
PubMed2005

The effects of adult guidance and peer discussion on the development of children's representations: evidence from the training of pedestrian skills.

It was hypothesized that practical training is effective in improving children's pedestrian skills because adult scaffolding and peer discussion during training specifically promote E3 level representation (linguistically-encoded, experientially-grounded, generalizable knowledge), as defined by Karmiloff-Smith's (1992) representational redescription (RR) model. Two studies were conducted to examine in detail the impact of this social input in the context of simulation-based training in roadside search skills. A group of 5-8-year-olds were pre-tested on ability to detect relevant road-crossing features. They then participated in four training sessions designed to promote attunement to these, under peer discussion versus adult guidance conditions (Study 1), and adult-child versus adult-group conditions (Study 2). Performance at post-test was compared with that of controls who underwent no training. Study 1 found that children in the adult guidance condition improved significantly more than those in the peer discussion or control conditions, and this improvement was directly attributable to appropriation of E3 level representations from adult dialogue. Study 2 found that progress was greater still when adult scaffolding was supplemented by peer discussion, with E3 level representation attributable to the children's exploration of conflicting ideas. The implications of these findings for the RR model and for practical road safety education are discussed.

Accident Prevention↗
PubMed2005

Differentiation of human embryonic stem cells occurs through symmetric cell division.

Embryonic (ES) stem cells can be expanded indefinitely, yet retain the ability to form all cell types of the body. Here we report that human ES cells differentiate exclusively by symmetric cell division in each of four distinct differentiation conditions examined. This suggests that, in some respects, ES cells more closely resemble precursor or transit amplifying cells rather than adult stem cells.

Cell Differentiation↗
PubMed2004

Crystal structure of human ABAD/HSD10 with a bound inhibitor: implications for design of Alzheimer's disease therapeutics.

The enzyme 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid beta-peptide-binding alcohol dehydrogenase (ABAD), has been implicated in the development of Alzheimer's disease. This protein, a member of the short-chain dehydrogenase/reductase family of enzymes, has been shown to bind beta-amyloid and to participate in beta-amyloid neurotoxicity. We have determined the crystal structure of human ABAD/HSD10 complexed with NAD(+) and an inhibitory small molecule. The inhibitor occupies the substrate-binding site and forms a covalent adduct with the NAD(+) cofactor. The crystal structure provides a basis for the design of potent, highly specific ABAD/HSD10 inhibitors with potential application in the treatment of Alzheimer's disease.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2004

Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

Antigens, CD34↗