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Peter W Andrews

Publications and source records attributed to Peter W Andrews.

At least 19 recordsLinked to original sources
PubMed2006

A prospective on stem cell research.

Stem cell research has stimulated considerable recent interest, but the concepts are old. Nevertheless, our understanding of the basic biology of different stem cell systems is poor. Many questions remain to be answered: How can we recognize stem cells? Are the underlying control mechanisms common to different types of stem cell, the so-called stemness concept, or is the control of self-renewal and commitment distinct in different stem cell types? What is the significance of differences in stem cells from different species? Do stem cells from somatic tissues really show plasticity with an ability to generate cells from distinct lineages, or are the observed examples consequences of experimental artifact, or rare events of no physiological significance? Do genetic mutations in the genes controlling stem cell self-renewal and differentiation lie at the heart of carcinogenesis? Answers to these and related questions now offer exciting future possibilities for both basic biology and medicine.

Adaptation, Biological↗
PubMed2006

Novel genomic aberrations in testicular germ cell tumors by array-CGH, and associated gene expression changes.

INTRODUCTION: Testicular germ cell tumors of adolescent and young adult men (TGCTs) generally have near triploid and complex karyotypes. The actual genes driving the tumorigenesis remain essentially to be identified. MATERIALS AND METHODS: To determine the detailed DNA copy number changes, and investigate their impact on gene expression levels, we performed an integrated microarray profiling of TGCT genomes and transcriptomes. We analyzed 17 TGCTs, three precursor lesions, and the embryonal carcinoma cell lines, NTERA2 and 2102Ep, by comparative genomic hybridization microarrays (array-CGH), and integrated the data with transcriptome profiles of the same samples. RESULTS: The gain of chromosome arm 12p was, as expected, the most common aberration, and we found CCND2, CD9, GAPD, GDF3, NANOG, and TEAD4 to be the therein most highly over-expressed genes. Additional frequent genomic aberrations revealed some shorter chromosomal segments, which are novel to TGCT, as well as known aberrations for which we here refined boundaries. These include gains from 7p15.2 and 21q22.2, and losses of 4p16.3 and 22q13.3. Integration of DNA copy number information to gene expression profiles identified that BRCC3, FOS, MLLT11, NES, and RAC1 may act as novel oncogenes in TGCT. Similarly, DDX26, ERCC5, FZD4, NME4, OPTN, and RB1 were both lost and under-expressed genes, and are thus putative TGCT suppressor genes. CONCLUSION: This first genome-wide integrated array-CGH and gene expression profiling of TGCT provides novel insights into the genome biology underlying testicular tumorigenesis.

Adolescent↗
PubMed2005

Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells.

Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression.

Adaptation, Physiological↗
PubMed2005

Differentiation of human embryonal carcinomas in vitro and in vivo reveals expression profiles relevant to normal development.

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Carcinoma in Situ↗
PubMed2005

The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells.

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.

Base Sequence↗
PubMed2004

Culture and characterization of human embryonic stem cells.

Human embryonic stem (ES) cells offer substantial opportunities for providing well-defined differentiated cells for drug discovery, toxicology, and regenerative medicine, but the development of efficient techniques for their large-scale culture under defined conditions, and for controlling and directing their differentiation, presents a substantial challenge. Markers for defining the undifferentiated cells are well established, based upon previous studies of embryonal carcinoma (EC) cells, their malignant counterparts from teratocarcinomas. These provide valuable tools for monitoring human ES cultures and their state of differentiation. However, current culture techniques are suboptimal and involve the use of poorly defined culture media and the use of feeder cells. Over time, the cells may also acquire karyotypic changes, reflecting genetic selection and adaptation to in vitro culture conditions. Nevertheless, progress is being made. Originally, human ES cells were derived and maintained in medium containing fetal calf serum. They are now widely cultured in a proprietary serum-free formulation (serum replacement from Invitrogen Corp., Carlsbad, CA), and recently we have derived a new human ES line in this medium without fetal calf serum. Human fibroblasts can also be used to replace mouse embryo fibroblasts as feeder cells. We have now found it possible to culture a subline of human ES cells on Matrigel, or purified collagen type IV, laminin, and fibronectin, without feeders or feeder-conditioned medium. These cells nevertheless retain the features of undifferentiated human ES cells, including a capacity for differentiation. Although these cells also carried karyotypic changes, further research focused upon understanding the mechanisms that control self-renewal, apoptosis, and commitment to differentiation will facilitate the development of defined culture conditions that minimize genetic change and optimize the maintenance of the undifferentiated stem cells.

Antigens, Surface↗
PubMed2004

Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells.

We have used RNA interference (RNAi) to downregulate beta2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not beta2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.

Antigens, Surface↗
PubMed2004

LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.

Murine embryonic stem (mES) cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF), and activation of signal transducer and activator of transcription 3 (STAT3) via LIF receptor (LIFR) signaling appears sufficient for maintenance of mES cell pluripotency. Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored. In this study, we show that the LIFRbeta and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs. Nevertheless, despite the functional activation of the LIF-STAT3 signaling pathway, human LIF is unable to maintain the pluripotent state of hESCs. Feeder-free culture conditions that maintain hESCs in an undifferentiated state do not show activation of STAT3, suggesting that distinct signaling mechanisms govern the self-renewal of hESCs.

Active Transport, Cell Nucleus↗
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

Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell.

Embryonal carcinoma (EC) cells, the pluripotent stem cells of teratocarcinomas, show many similar-ities to embryonic stem (ES) cells. Since EC cells are malignant but their terminally differentiated derivatives are not, understanding the molecular mechanisms that regulate their differentiation maybe of value for diagnostic and therapeutic purposes. We have examined the expression of multiple components of two developmentally important cell-cell signalling pathways, Wnt and Notch, in the pluripotent human EC cell line, NTERA2, and the human ES cell line, H7. Both pathways have well-documented roles in controlling neurogenesis, a process that occurs largely in response to retinoicacid (RA) treatment of NTERA2 cultures and spontaneously in H7 cultures. In NTERA2, many ofthe genes tested showed altered transcriptional regulation following treatment with RA. These include members of the frizzled gene family (FZDI, FZD3, FZD4, FZD5, FZD6), encoding receptors forWnt proteins, the Frizzled Related Protein family (SFRPI, SFRP2, FRZB, SFRP4), encoding solubleWnt antagonists and also ligands and receptors of the Notch pathway (Dlkl, Jaggedl; Notchl, Notch2, Notch3). Few differences were found in the repertoire of Wnt and Notch pathway genes expressed by NTERA2 EC cells and H7 ES cells. We present a model in which interactions between and regulation of Wnt and Notch signalling are important in maintaining EC/ES stem cells and also controlling their differentiation.

Carcinoma, Embryonal↗
PubMed2003

Reprogramming in inter-species embryonal carcinoma-somatic cell hybrids induces expression of pluripotency and differentiation markers.

Somatic cell reprogramming holds great promise for the development of novel cellular therapeutics. A number of sources of reprogramming potential have been identified, including oocytes, embryonic germ (EG) cells and embryonic stem (ES) cells. However, each of these sources of reprogramming factors is problematic, since they are either not freely available or have special growth requirements. Embryonal carcinoma (EC) cells are another source of pluripotent cells that, unlike ES and EG cells, do not usually require special growth conditions. Since they share many of the key characteristics of ES cells, such as pluripotency, EC cells may provide a readily amenable alternative source of reprogramming factors and could serve as a model for ES cells in this respect. Here we show that mouse EC cells can also function as donors of reprogramming factors. PEG-mediated fusion between murine EC cells (P19) and the cells of a human T-lymphoma cell line (CEM-GFP) resulted in inter-species hybrid colony formation. Colonies of hybrid cells displayed heterogeneity in cellular morphology as well as in their pattern of human gene expression. Expression of two human transcription factors characteristic of undifferentiated pluripotent stem cells, Oct-4 and Sox-2, was detected in the hybrid cells, demonstrating activation of endogenous human markers of pluripotency. Simultaneously, down-regulation of CD45, a marker present in lymphocytic cells, was observed in some hybrids. The detection of human specific markers of differentiation, such as nestin, lamininbeta1, and collagen IValpha1, indicates that fusion resulted in reprogramming of the human cells to reflect the differentiation potential of the murine EC partner.

Animals↗
PubMed2002

Multidimensional differential display via ion-pair reversed-phase denaturing high-performance liquid chromatography.

Experimental approaches are now available for the analysis of whole transcriptome expression in cells and tissues. Since the introduction of such methods for the investigation of differences in mRNA populations, they have been applied successfully to many areas of biology and medicine including development, differentiation, physiology, pharmacology, and carcinogenesis. Here we describe an improved and automated approach based on the differential mRNA display method developed by Liang and Pardee (P. Liang and A. B. Pardee, 1992, Science 257, 967-971). We report the use of ion-pair reversed-phase denaturing high-performance liquid chromatography (IP RP DHPLC), for the first time, to produce a "fingerprint," after amplification of the cDNA corresponding to the mRNA populations, from two or more of the samples that are to be compared. By overlaying the chromatograms produced from the amplification of different samples derived from the same set of oligodeoxynucleotide primers, those genes that are differentially expressed can be selected and subsequently cloned and sequenced rapidly to establish a profile of differentially expressed genes. In addition, validation of the data obtained is readily achieved by this method using IP RP DHPLC and quantitative RT-PCR. In this study total RNA was prepared from NTERA2 cells before and after differentiation induced by retinoic acid and was reverse-transcribed into cDNA prior to amplification to produce fluorescently tagged products. This methodology facilitates multiple rounds of interrogation of RT-PCR products and we tentatively refer to this approach as Multidimensional Differential Display.

Base Sequence↗
PubMed2002

From teratocarcinomas to embryonic stem cells.

The recent derivation of human embryonic stem (ES) cell lines, together with results suggesting an unexpected degree of plasticity in later, seemingly more restricted, stem cells (so-called adult stem cells), have combined to focus attention on new opportunities for regenerative medicine, as well as for understanding basic aspects of embryonic development and diseases such as cancer. Many of the ideas that are now discussed have a long history and much has been underpinned by the earlier studies of teratocarcinomas, and their embryonal carcinoma (EC) stem cells, which present a malignant surrogate for the normal stem cells of the early embryo. Nevertheless, although the potential of EC and ES cells to differentiate into a wide range of tissues is now well attested, little is understood of the key regulatory mechanisms that control their differentiation. Apart from the intrinsic biological interest in elucidating these mechanisms, a clear understanding of the molecular process involved will be essential if the clinical potential of these cells is to be realized. The recent observations of stem-cell plasticity suggest that perhaps our current concepts about the operation of cell regulatory pathways are inadequate, and that new approaches for analysing complex regulatory networks will be essential.

Animals↗