K. Sue O'Shea

L1 Retrotransposition in Human Neural Progenitor Cells

Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.

Embryonic stem cell models of development.

Pluripotent mouse embryonic stem (ES) cell lines have provided a means to analyze gene function in development via gene targeting. At the same time, they provide an opportunity to directly probe gene function by assessing the in vitro differentiation capacity of the ES cells themselves. In addition to providing direct data on lineage decisions not accessible in the complex three‐dimensional milieu of the early mouse embryo, controlled differentiation of ES into specific lineages may provide a source of cells for transplantation and gene therapy. Anat. Rec. (New Anat.): 257:32–41, 1999.

Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells

Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution. L1s can retrotranspose in the germline, during early development and in select somatic cells; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.

Laminin in lung development: effects of anti-laminin antibody in murine lung morphogenesis.

The pattern of deposition and the role of laminin, a major glycoprotein constituent of basement membranes, were investigated during lung morphogenesis in the fetal mouse. Lung primordia were removed from Day 13 embryos, right lower lobes were further dissected and placed in filter membrane assemblies. Explants were then cultured at the liquid-air interface for 3 days in the presence of anti-laminin, anti-thrombospondin (another extracellular matrix constituent), preimmune serum, laminin-neutralized anti-laminin, or medium alone. Cultures were monitored by (direct) phase-contrast microscopy, light microscopy, and immunofluorescence. We found that anti-laminin antibodies altered normal lung morphogenesis in a dose-dependent manner. The anti-laminin-treated explants presented a marked inhibition of branching morphogenesis and a distortion of the bronchial tree. A lower rate of growth was also observed in the explants exposed to this antibody. High concentrations of anti-thrombospondin antibodies, normal rabbit serum, or laminin-neutralized anti-laminin antibodies had no effect on lung morphogenesis. These results were not modified by culturing the explants in submersion culture or on Vitrogen 100-coated surfaces.

Glutamatergic Neuronal Differentiation of Mouse Embryonic Stem Cells after Transient Expression of Neurogenin 1 and Treatment with BDNF and GDNF: In Vitro and In Vivo Studies

Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50–75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.

State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish.

A meeting was convened so that users of three models for in vitro developmental toxicity (embryonic stem cells, whole embryo culture, and zebrafish) could share their experiences with each model, and explore the areas for improvement. We present a summary of this meeting and the recommendations of the group.

Coupled Global and Targeted Proteomics of Human Embryonic Stem Cells during Induced Differentiation

Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.

Differential Expression of the Carbonic Anhydrase Genes for CA VII (Car7) And CA-RP VIII (Car8) in Mouse Brain

The spatial expression patterns of the two α-carbonic anhydrase genes, CA VII and CA-RP VIII (called Car7 and Car8 in the mouse) were examined in the mouse brain by in situ hybridization. These two genes are the most highly conserved evolutionarily among the mammalian α-CAs. Both genes showed a similarly wide expression pattern in the brain. In the cerebrum, mRNA expression was detected in the pia, choroid plexus, and neurons of the cortical layer, thalamus, and medial habenulae. A high level of expression appeared in the pyramidal and granular cells of the hippocampus. In the cerebellum, both Car7 and Car8 were transcribed to different degrees in the Purkinje cells, and a lower expression level occured in the molecular and granular cell layers. Transcription signals for both genes were excluded from the white matter regions.

Neurodevelopmental origins of bipolar disorder: iPSC models.

Bipolar disorder (BP) is a chronic neuropsychiatric condition characterized by pathological fluctuations in mood from mania to depression. Adoption, twin and family studies have consistently identified a significant hereditary component to BP, yet there is no clear genetic event or consistent neuropathology. BP has been suggested to have a developmental origin, although this hypothesis has been difficult to test since there are no viable neurons or glial cells to analyze, and research has relied largely on postmortem brain, behavioral and imaging studies, or has examined proxy tissues including saliva, olfactory epithelium and blood cells. Neurodevelopmental factors, particularly pathways related to nervous system development, cell migration, extracellular matrix, H3K4 methylation, and calcium signaling have been identified in large gene expression and GWAS studies as altered in BP. Recent advances in stem cell biology, particularly the ability to reprogram adult somatic tissues to a pluripotent state, now make it possible to interrogate these pathways in viable cell models. A number of induced pluripotent stem cell (iPSC) lines from BP patient and healthy control (C) individuals have been derived in several laboratories, and their ability to form cortical neurons examined. Early studies suggest differences in activity, calcium signaling, blocks to neuronal differentiation, and changes in neuronal, and possibly glial, lineage specification. Initial observations suggest that differentiation of BP patient-derived neurons to dorsal telencephalic derivatives may be impaired, possibly due to alterations in WNT, Hedgehog or Nodal pathway signaling. These investigations strongly support a developmental contribution to BP and identify novel pathways, mechanisms and opportunities for improved treatments.