Melany Jackson

Severe Global DNA Hypomethylation Blocks Differentiation and Induces Histone Hyperacetylation in Embryonic Stem Cells

ABSTRACT It has been reported that DNA methyltransferase 1-deficient (Dnmt1−/−) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a−/−,3b−/−] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a−/−,3b−/−] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a−/−,3b−/−] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1−/− ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.

Psoriatic keratinocytes show reduced IRF-1 and STAT-1α activation in response to γ-IFN

Psoriasis is a chronic inflammatory dermatosis characterized by hyperproliferative keratinocytes (KC). The skin lesions are infiltrated by T cells, which secrete gamma interferon (γ‐IFN) and are believed to be necessary to maintain the psoriatic phenotype. In normal KC, γ‐IFN is a potent inhibitor of proliferation, but proliferation of KC persists in psoriatic plaques despite the presence of γ‐IFN. Immunostaining of interferon regulatory factor‐1 (IRF‐1) revealed that IRF‐1 was localized to the basal cells of the epidermis in normal and in nonlesional psoriatic skin, but was suprabasal or completely absent in lesional psoriatic skin. This finding led to the hypothesis that abnormal signaling in the γ‐IFN pathway may occur in psoriatic KC. To test this hypothesis, we measured activation of IRF‐1 and signal transducer and activator of transcription (STAT)‐1α transcription factors in KC after stimulation with γ‐IFN. Primary cultures of KC from normal and nonlesional psoriatic skin were stimulated with γ‐IFN and subsequent transcription factor activation was measured by electrophoretic mobility shift assay. Psoriatic KC showed a reduced induction of IRF‐1 and STAT‐1α activation after stimulation with γ‐IFN, compared with normal KC. Reduced activation of IRF‐1 and STAT‐1o in response to γ‐IFN indicates a fundamental defect in the growth and differentiation control of psoriatic KC in the absence of the influence of other cell types.—Jackson, M., Howie, S. E. M., Weller, R., Sabin, E., Hunter, J. A. A., McKenzie, R. C. Psoriatic keratinocytes show reduced IRF‐1 and STAT 1‐o activation in response to γ‐IFN. FASEB J. 13, 495–502 (1999)

A murine specific expansion of the Rhox cluster involved in embryonic stem cell biology is under natural selection

BackgroundThe rodent specific reproductive homeobox (Rhox) gene cluster on the X chromosome has been reported to contain twelve homeobox-containing genes, Rhox1-12.ResultsWe have identified a 40 kb genomic region within the Rhox cluster that is duplicated eight times in tandem resulting in the presence of eight paralogues of Rhox2 and Rhox3 and seven paralogues of Rhox4. Transcripts have been identified for the majority of these paralogues and all but three are predicted to produce full-length proteins with functional potential. We predict that there are a total of thirty-two Rhox genes at this genomic location, making it the most gene-rich homoeobox cluster identified in any species. From the 95% sequence similarity between the eight duplicated genomic regions and the synonymous substitution rate of the Rhox2, 3 and 4 paralogues we predict that the duplications occurred after divergence of mouse and rat and represent the youngest homoeobox cluster identified to date. Molecular evolutionary analysis reveals that this cluster is an actively evolving region with Rhox2 and 4 paralogues under diversifying selection and Rhox3 evolving neutrally. The biological importance of this duplication is emphasised by the identification of an important role for Rhox2 and Rhox4 in regulating the initial stages of embryonic stem (ES) cell differentiation.ConclusionThe gene rich Rhox cluster provides the mouse with significant biological novelty that we predict could provide a substrate for speciation. Moreover, this unique cluster may explain species differences in ES cell derivation and maintenance between mouse, rat and human.

Expression of nitric oxide synthase III (eNOS) mRNA by human skin cells: melanocytes but not keratinocytes express eNOS mRNA

Nitric oxide (NO) has been shown to have diverse physiological functions including vasodilation and antitumour and antimicrobial effects, and has a role as a neurotransmitter (reviewed in reference 1). NO is formed by the oxidation of L-arginine to L-citrulline which is catalysed by nitric oxide synthase (NOS) enzymes. Different isoforms of NOS exist which vary in intracellular location and are the products of three different genes. NOS I (nNOS or cNOS) is a constitutive isoform first identified in the peripheral and central nervous systems; NOS II (iNOS) is a calcium-independent inducible isoform usually absent in resting cells but can be induced by a variety of stimuli, and NOS III (eNOS, cNOS or ecNOS) is a second constitutive, calcium-dependent isoform present in endothelial cells.

HOXB4 Can Enhance the Differentiation of Embryonic Stem Cells by Modulating the Hematopoietic Niche

Hematopoietic differentiation of embryonic stem cells (ESCs) in vitro has been used as a model to study early hematopoietic development, and it is well documented that hematopoietic differentiation can be enhanced by overexpression of HOXB4. HOXB4 is expressed in hematopoietic progenitor cells (HPCs) where it promotes self‐renewal, but it is also expressed in the primitive streak of the gastrulating embryo. This led us to hypothesize that HOXB4 might modulate gene expression in prehematopoietic mesoderm and that this property might contribute to its prohematopoietic effect in differentiating ESCs. To test our hypothesis, we developed a conditionally activated HOXB4 expression system using the mutant estrogen receptor (ERT2) and showed that a pulse of HOXB4 prior to HPC emergence in differentiating ESCs led to an increase in hematopoietic differentiation. Expression profiling revealed an increase in the expression of genes associated with paraxial mesoderm that gives rise to the hematopoietic niche. Therefore, we considered that HOXB4 might modulate the formation of the hematopoietic niche as well as the production of hematopoietic cells per se. Cell mixing experiments supported this hypothesis demonstrating that HOXB4 activation can generate a paracrine as well as a cell autonomous effect on hematopoietic differentiation. We provide evidence to demonstrate that this activity is partly mediated by the secreted protein FRZB. STEM CELLS 2012; 30:150–160.

Expression of a novel homeobox gene Ehox in trophoblast stem cells and pharyngeal pouch endoderm

Ehox is an X‐linked paired like homeobox gene identified from a differentiating embryonic stem (ES) cell cDNA library and is expressed at low levels in the preimplantation blastocyst and in ES cells in vitro. In embryos at 6.5 days post coitum (dpc), Ehox expression was restricted to the extraembryonic ectoderm which correlates with high‐level expression in cultures of trophoblast stem cells. Extraembryonic expression becomes further restricted to the chorion and by 15.5 dpc Ehox is expressed in chorionic trophoblast of the labyrinth and spongiotrophoblast layers of the placenta. Ehox expression in the embryo proper first appears at 8.5 dpc in the anterior foregut endoderm and by 9.5 dpc is visible in pharyngeal pouches 2–4. By 10.5 dpc, Ehox expression becomes restricted to the ventral end of pouches 2 and 3. The data presented here is the first description of Ehox expression during embryogenesis and suggests a dual role for Ehox: (1) in trophoblast stem cells and compartments of the developing placenta, and (2) during development of the pharyngeal pouches, possibly delineating the area to become thymus. Development Dynamics 228:740–744, 2003.

Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

The culture of mouse embryonic stem cells and formation of embryoid bodies.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of the pre-implantation blastocyst. They have the capacity to undergo indefinite rounds of self-renewing cell division and differentiate into all the cell lineages of the developing embryo. In suspension culture, ES cells will differentiate into aggregates known as embryoid bodies in a manner similar to the early embryo. This culture system therefore provides a useful model to study the relatively inaccessible stages of mammalian development. We describe methods for the routine maintenance of mouse embryonic stem cells in culture, assays of stem cell self-renewal potential in monolayer culture and the generation of embryoid bodies to study differentiation pathways.

Induction of Hematopoietic Differentiation of Mouse Embryonic Stem Cells by an AGM-Derived Stromal Cell Line is Not Further Enhanced by Overexpression of HOXB4

Hematopoietic differentiation of embryonic stem (ES) cells can be enhanced by co-culture with stromal cells derived from hematopoietic tissues and by overexpression of the transcription factor HOXB4. In this study, we compare the hematopoietic inductive effects of stromal cell lines derived from different subregions of the embryonic aorta-gonad-mesonephros tissue with the commonly used OP9 stromal cell line and with HOXB4 activation. We show that stromal cell lines derived from the aorta and surrounding mesenchyme (AM) act at an earlier stage of the differentiation process compared with the commonly used OP9 stromal cells. AM stromal cells were able to promote the further differentiation of isolated brachyury-GFP(+) mesodermal cells into hematopoietic progenitors, whereas the OP9 stromal cells could not support the differentiation of these cells. Co-culture and analyses of individual embryoid bodies support the hypothesis that the AM stromal cell lines could enhance the de novo production of hematopoietic progenitors, lending support to the idea that AM stromal cells might act on prehematopoietic mesoderm. The induction level observed for AM stromal cells was comparable to HOXB4 activation, but no additive effect was observed when these 2 inductive strategies were combined. Addition of a γ-secretase inhibitor reduced the inductive effects of both the stromal cell line and HOXB4, providing clues to possible shared molecular mechanisms.

Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells.

Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion‐transmitted infections and complications can arise from immunological incompatibility. Clinically‐compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel‐like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC‐derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen‐inducible KLF1‐ERT2 expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic‐like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886–897