John D. Ansell

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.

Trophoblast giant cell differentiation in the mouse, expression of glucose phosphate isomerase (gpi-1) electrophoretic variants in transferred and chimeric embryos.

Abstract Trophoblast differentiation in the mouse embryo is characterized by the formation of giant cells whose nuclei contain as much as 500–1000 times the haploid amount of DNA. Three mechanisms which have been proposed to account for the increase in DNA include (1) endomitosis, (2) uptake of maternal nuclei or macromolecular DNA as the trophoblast cells invade the endometrium, and (3) the formation of syncytial trophoblast by cell fusion followed by the fusion of nuclei. We have used genetic variants of the dimeric isozyme glucose phosphate isomerase (GPI-1) to examine whether heterokarotypes are expressed in trophoblast tissue from transferred embryos (GPI-1A embryos to GPI-1B females) and chimeric embryos (GPI-1A↔GPI-1B). In the electrophoretic assay system we used, the GPI-1 phenotype of individual ectoplacental cones could be determined. We also established that different electrophoretic forms of GPI-1 could be discerned when one form comprises only 1% of the total GPI-1 activity of the sample. We found no evidence of heteropolymer (GPI-1AB) in electrophoretic assays of trophoblast from either the transferred or chimeric embryos. These data indicate that trophoblast cells do not functionally incorporate maternal DNA nor do they form syncytial heterokaryons by cell fusion. Our results are consistent with the proposal that DNA increases in trophoblast giant cells by endomitosis.

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.

Differentiating Embryonal Stem Cells Are a Rich Source of Haemopoietic Gene Products and Suggest Erythroid Preconditioning of Primitive Haemopoietic Stem Cells

The difficulties associated with studying molecular mechanisms important in hemopoietic stem cell (HSC) function such as the problems of purifying homogeneous stem cell populations, have prompted us to adapt the murine ES cell system as anin vitro model of HSC generation and function. We now report that careful analysis of the time course of HSC generation in differentiating ES cells allows them to be used as a source of known and novel hemopoietic gene products. We have generated a subtracted library using cDNA from ES cells collected just prior to and just following the emergence of HSCs. Analysis of this library shows it to be a rich source of known hemopoietic and hemopoietic related gene products with 44% of identifiable cDNAs falling into these camps. We have demonstrated the value of this system as a source of novel genes of relevance to HSC function by characterizing a novel membrane protein encoding cDNA that is preferentially expressed in primitive hemopoietic cells. Intriguingly, further analysis of the known components of the subtracted library is suggestive of erythroid preconditioning of the ES cell-derived HSC. We have used dot-blot andin situ analysis to indicate that this erythroid preconditioning is probably restricted to primitive but not definitive HSC.

Parental influences on X chromosome expression

Using mice that were mosaics for both Xce and phosphoglycerate kinase ( Pgk -1) alleles, we have established that the parental source of the Xce gene may affect the probability with which the X chromosome carrying it will remain active. This effect was seen in one allelic combination of Xce but not in another. The relationship between these effects and other phenomena of maternal ‘imprinting’ is discussed.

In vivo expansion of the endogenous B‐cell compartment stimulated by radiation and serial bone marrow transplantation induces B‐cell leukaemia in mice

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells. This B‐cell lineage is established during ontogeny and replenished by the process of self‐renewal. Spontaneous and induced leukaemias that frequently affect this lineage are thought to arise as a result of the frequent cell division required to maintain the population throughout adulthood and in response to repeated exposure to environmental antigens. In a series of bone marrow transplant (BMT) experiments performed in B6D2F1 mice, B‐cell leukaemia occurred in recipients of serially transplanted syngeneic bone marrow. This study was therefore designed to determine the frequency and phenotype of the observed leukaemia. Male donor cells were initially transplanted into lethally irradiated female hosts and secondary (2°) BMT was performed at 3 months. At 1, 2, 3 and 16 months following primary (1°) BMT, and when 2° BMT recipients developed leukaemia, animals were sacrificed and their tissues extensively examined. These analyses confirmed a host‐derived CD5+ transplantable B‐cell leukaemia that was initiated in 50% of 1° BMT recipients. With serial passage, the leukaemia became more aggressive and lost CD5 expression, suggesting transformation to a high‐grade leukaemia/lymphoma. This previously unreported observation suggests that the combination of radiation and subsequent serial transplantation induces a proliferative stress to the host B‐cell compartment that is causative in leukaemic transformation.

Hematopoietic Differentiation of Embryonal Stem Cells in vitro.

The hematopoietic system can be considered as a spectrum of differentiation and self renewal with at one extreme terminally differentiated cells such as erythrocytes or macrophages, and at the other, pluripotential hematopoietic stem cells (HSC), which can give rise to all the different lineages of the hematopoietic system, and due to their capacity for self renewal, persist throughout adult life (Spangrude et al. 1991). Introduction of limiting numbers of these cells into recipients whose own hematopoietic systems have been ablated (by, for example, irradiation) results in long-term reconstitution by the donor cells. These properties of HSC have make them tempting targets for gene manipulation, both for investigating the molecular and cellular biology of genes that are expressed in different lineages of the hematopoietic system and in somatic gene therapy. In the former case, gene manipulation of HSC would have particular benefit where the mutations, if transmitted through the germ line, would result in embryonic lethality