Biomedical subjects

James A Thomson

Publications and source records attributed to James A Thomson.

31 records · Page 2Linked to original sources

PubMed2004

Arrays for the combinatorial exploration of cell adhesion.

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.

Animals↗

PubMed2003

Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells.

Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

Biocompatible Materials↗

PubMed2003

Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

Aneuploidy↗

PubMed2003

Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors.

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

Carcinoma, Embryonal↗

PubMed2003

Functional endothelial cells derived from rhesus monkey embryonic stem cells.

We have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell-derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin alpha v beta 3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.

Animals↗

PubMed2003

Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization.

Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. beta-Adrenergic stimulation with 1.0 micromol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology recorded from different cells, suggesting that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for IKr based on E-4031 induced prolongation of AP duration as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially therapeutic applications.

Action Potentials↗

PubMed2003

Homologous recombination in human embryonic stem cells.

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells are to fulfill their promise as a basic research tool. It also has important implications for ES cell-based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

DNA-Binding Proteins↗

PubMed2003

Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme.

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.

Amino Acid Sequence↗

PubMed2003

High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors.

Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.

Animals↗

PubMed2002

BMP4 initiates human embryonic stem cell differentiation to trophoblast.

The excitement and controversy surrounding the potential role of human embryonic stem (ES) cells in transplantation therapy have often overshadowed their potentially more important use as a basic research tool for understanding the development and function of human tissues. Human ES cells can proliferate without a known limit and can form advanced derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES cells can also form the extra-embryonic tissues that differentiate from the embryo before gastrulation. The use of human ES cells to derive early human trophoblast is particularly valuable, because it is difficult to obtain from other sources and is significantly different from mouse trophoblast. Here we show that bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor-beta (TGF-beta) superfamily, induces the differentiation of human ES cells to trophoblast. DNA microarray, RT-PCR, and immunoassay analyses demonstrate that the differentiated cells express a range of trophoblast markers and secrete placental hormones. When plated at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin (CG). These results underscore fundamental differences between human and mouse ES cells, which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for studying the differentiation and function of early human trophoblast and could provide a new understanding of some of the earliest differentiation events of human postimplantation development.

Bone Morphogenetic Protein 4↗

PubMed2002

Efficient method for expressing transgenes in nonhuman primate embryos using a stable episomal vector.

Transgenesis in the nonhuman primate can enhance the study of human biology by providing animal models for the study of primate-specific physiology, pathophysiology, and embryonic development. Progress with this technology has been hindered by the inherent inefficiency of transgenesis, transgene silencing, and practical restrictions on the production of sufficient pronuclear stage nonhuman primate zygotes. We have developed a novel technique using an Epstein Barr virus (EBV)-based episomal vector to produce rhesus monkey (Macaca mulatta) embryos expressing a transgene. Plasmid DNA containing the latent origin of replication, oriP, and Epstein Barr Nuclear Antigen-1 (EBNA-1) of EBV, as well as a CMV IE-enhanced green fluorescent protein (eGFP) expression cassette, was introduced into rhesus embryos by direct pronuclear microinjection. We detected eGFP in early cleavage stage embryos (4-8 cell) and throughout the duration of culture (day 8-9 blastocysts) by epifluorescent microscopy. A 50% transduction rate was obtained with the EBV-based vector. Microinjected embryos expressed eGFP and retained their developmental capacity as evidenced by development to the blastocyst stage. EBV-based vectors present a novel and efficient means of delivering transgenes for the study of the molecular control of primate embryonic development.

Animals↗

PubMed2002

Surface antigens of human embryonic stem cells: changes upon differentiation in culture.

We have analysed the surface antigen phenotype of a human embryonic stem (hES) cell line (H7) and the changes that occur upon differentiation induced by retinoic acid, hexamethylene bisacetamide and dimethylsulphoxide. The undifferentiated stem cells expressed Stage Specific Embryonic Antigen-3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-8 but not SSEA1. In these characteristics they closely resemble human embryonal carcinoma (EC) cells derived from testicular teratocarcinomas, and are distinct from murine EC and ES cells. The undifferentiated cells also expressed the liver/bone/kidney isozyme of alkaline phosphatase detected by antibody TRA-2-54, the class 1 major histocompatability antigens, HLA-ABC, and the human Thy1 antigen. Differentiation of hES cells was induced by retinoic acid, HMBA and DMSO with the appearance of various cell types including neurons and muscle cells. The surface antigens characteristically expressed by hES cells were down-regulated following induction of differentiation and other antigens appeared, notably several ganglioside glycolipids detected by antibodies VIN-IS-56 (GD3 and GD2), VIN-2PB-22 (GD2), A2B5 (GT3) and ME311 (9-O-acetyl-GD3). Whereas the expression of HLA was slightly down-regulated upon differentiation, its expression was strongly induced by interferon-y in both the undifferentiated and the differentiated cells, although the induction in the differentiated cultures was considerably stronger than in the stem cells. In all of these features the human ES cells, and their pattern of differentiation, resembled the pluripotent human EC cell line NTERA-2 although clearly the range of cells generated by the hES cells was considerably greater.

Acetamides↗

PubMedDate not supplied

Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.

How dopamine (DA) neuronal subtypes are specified remains unknown. In this study we show a robust generation of functional DA neurons from human embryonic stem cells (hESCs) through a specific sequence of application of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH). Treatment of hESC-derived Sox1+ neuroepithelial cells with FGF8 and SHH resulted in production of tyrosine hydroxylase (TH)-positive neurons that were mostly bipolar cells, coexpression with gamma-aminobutyric acid, and lack of midbrain marker engrailed 1 (En1) expression. However, FGF8 treatment of precursor cells before Sox1 expression led to the generation of a similar proportion of TH+ neurons characteristic of midbrain projection DA neurons with large cell bodies and complex processes and coexpression of En1. This suggests that one mechanism of generating neuronal subtypes is temporal availability of morphogens to a specific group of precursors. The in vitro-generated DA neurons were electrophysiologically active and released DA in an activity-dependent manner. They may thus provide a renewable source of functional human DA neurons for drug screening and development of sustainable therapeutics for disorders affecting the DA system.

Animals↗