Diana Hernandez

Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: Identification of novel gene and pseudogene clusters

OBJECTIVES To date, six flavin-containing monooxygenase (FMO) genes have been identified in humans, FMOs 1, 2, 3, 4 and 6, which are located within a cluster on chromosome 1, and FMO5, which is located outside the cluster. The objectives were to review and update current knowledge of the structure and expression profiles of these genes and of their mouse counterparts and to determine, via a bioinformatics approach, whether other FMO genes are present in the human and mouse genomes. RESULTS AND CONCLUSIONS We have identified, for the first time, a mouse Fmo6 gene. In addition, we describe a novel human FMO gene cluster on chromosome 1, located 4 Mb telomeric of the original cluster. The novel cluster contains five genes, all of which exhibit characteristics of pseudogenes. We propose the names FMO 7P, 8P, 9P, 10P and 11P for these genes. We also describe a novel mouse gene cluster, located approximately 3.5 Mb distal of the original gene cluster on Chromosome 1. The novel mouse cluster contains three genes, all of which contain full-length open-reading frames and possess no obvious features characteristic of pseudogenes. One of the genes is apparently a functional orthologue of human FMO9P. We propose the names Fmo9, 12 and 13 for the novel mouse genes. Orthologues of these genes are also present in rat. Sequence comparisons and phylogenetic analyses indicate that the novel human and mouse gene clusters arose, not from duplications of the known gene cluster, but via a series of independent gene duplication events. The mammalian FMO gene family is thus more complex than previously realised.

Hypoxia Enhances the Generation of Retinal Progenitor Cells from Human Induced Pluripotent and Embryonic Stem Cells

The efficient differentiation of retinal cells from human pluripotent stem cells remains a major challenge for the development of successful and cost-effective cellular therapies for various forms of blindness. Current differentiation strategies rely on exposing pluripotent stem cells to soluble growth factors that play key roles during early development (such as DKK-1, Noggin, and IGF-1) at 20% oxygen (O(2)). This O(2) tension is, however, considerably higher than O(2) levels during organogenesis and may impair the differentiation process. In this study, we examined the effect of mimicking the physiological O(2) tension (2%) on the generation of retinal progenitor cells (RPCs) from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Both cell types were induced to differentiate into RPCs at 20% and 2% O(2). After 3 days in suspension culture as embryoid bodies (EBs), 2% O(2) caused the activation of hypoxia inducible factor responsive genes VEGF and LDHA and was accompanied by elevated expression levels of the early eye field genes Six3 and Lhx2. Twenty-one days after plating EBs in an adherent culture, we observed more RPCs co-expressing Pax6 and Chx10 at 2% O(2). Quantitative polymerase chain reaction analysis confirmed that lowering O(2) tension had caused a rise in the expression of both genes compared with 20% O(2). Our results indicate that mimicking physiological O(2) is a favorable condition for the efficient generation of RPCs from both hiPSCs and hESCs.

Feeder-Free Culture of Human Embryonic Stem Cells for Scalable Expansion in a Reproducible Manner

Human embryonic stem (hES) cells have the potential as starting materials for a wide variety of applications in cell therapy, drug discovery and development. However, the challenge is to produce large numbers of well-characterized hES cells that are pluripotent and of high quality. This is needed to be capable of producing future cell therapies that are safe, effective, and affordable for use in routine clinical practice. A major bottleneck is the present requirement for complex culturing regimes that are very labor intensive and unscalable. hES cells have traditionally been grown on feeder layers made from inactivated mouse or human embryonic fibroblasts, in medium containing serum and other nondefined factors. This makes conditions difficult to reproduce over multiple passages. With a view to simplifying culture conditions we have tested a novel proprietary good manufacturing practice-based system that circumvents the use of feeders completely. The system consists of a matrix and a formulated medium that, in combination, demonstrate a reliable and reproducible way to culture hES cells without the use of feeders. We have been able to grow hES cells (Shef3 and Shef6) for over 20 passages, in this system, without loss of pluripotency, capacity to differentiate, or acquisition of karyotypic abnormalities. Furthermore, we have demonstrated the feasibility of propagating hES cells at clonal dilutions from single cells using this system.

Deletion of the mouse fmo1 gene results in enhanced pharmacological behavioural responses to imipramine

Objectives Many drugs are the subject of multipathway oxidative metabolism catalyzed by one or more cytochromes P450 or flavin-containing monooxygenases (FMOs). This complicates assessment of the role of individual enzymes in metabolizing the drug and, hence, in understanding its pharmacogenetics. To define the role of FMOs in drug metabolism, we produced FMO-deficient mice. Methods An Fmo1(−/−), Fmo2(−/−), Fmo4(−/−) mouse line was produced by using chromosomal engineering and Cre-loxP technology. To assess the utility of the mutant mouse line, it was used to investigate the role of FMO in the metabolism of and response to the antidepressant imipramine, which has four major metabolites, three produced by cytochromes P450 and one, imipramine N-oxide, solely by FMO1. Results On treatment with imipramine, wild-type mice became sedated and produced imipramine N-oxide in the brain and other tissues. In contrast, knockout mice did not produce imipramine N-oxide, but showed exaggerated pharmacological behavioural responses, such as tremor and body spasm, and had a higher concentration of the parent compound imipramine in the serum and kidney and there was an increase in desipramine in the brain. Conclusion The absence of FMO1-mediated N-oxidation of imipramine results in enhanced central nervous system effects of the drug. The results provide insights into the metabolism of imipramine in the brain and may explain the basis of the adverse reactions to the drug seen in some patients. The knockout mouse line will provide a valuable resource for defining the role of FMO1 in the metabolism of drugs and other foreign chemicals.

The phenotype of a knockout mouse identifies flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic ageing

Graphical abstract

Precisely delivered nanomechanical forces induce blebbing in undifferentiated mouse embryonic stem cells

The aim of this study was to probe the morphological response of single mouse embryonic stem cells (mESC) to precisely delivered nanomechanical forces. Plating mESC as single cells gave rise to either round compact or flattened fibroblastic morphologies. The expression of OCT4 and Nanog was reduced in flattened cells, indicating that this population had begun to differentiate. Upon application of.> nN of force, using atomic force microscopy and simultaneous laser scanning confocal microscopy, round cells, but not flattened cells, were capable of forming mechanically induced blebs (miBlebs). Flattened cells appeared to have a more highly developed cytoskeleton than undifferentiated stem cells as characterized by the distribution of phospho-ezrin-radixin-moesin (pERM). Higher levels of pERM and an inability to form miBlebs in flattened cells imply that the earliest stages of embryonic stem cell differentiation are associated with the development of stronger mechanical links between the plasma membrane and the cytoskeleton

Characterization of the phenotype and functionality of corneal epithelial cells derived from mouse embryonic stem cells

AIMS To investigate the optimum conditions for the differentiation of a mouse embryonic stem cell line towards corneal epithelial cell fate. MATERIALS & METHODS The effect of conditioned media from both metabolically active (to produce lineage A) and growth-arrested limbal fibroblasts (lineage G) were compared with basal media (lineage N) in terms of morphology and marker expression, assessed by immunocytochemistry and reverse transcription PCR. Cultures were transplanted into a porcine ex vivo model to investigate their ability for wound healing and cornea repair. RESULTS Lineage N exhibited cobblestone morphology and expressed CK12 and p63α, while OCT4 and SSEA1 were downregulated. Post-transplantation, these cells were able to multilayer and heal after wounding while maintaining marker expression. CONCLUSION Lineages with corneal epithelial-like characteristics, which are derived from embryonic stem cells, have potential for use in the study of corneal wound healing and therapy.

Deletion of Genes From the Mouse Genome Using Cre/loxP Technology.

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmo1, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Microinjection of Targeted Embryonic Stem Cells and Establishment of Knockout Mouse Lines for Fmo Genes

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.