Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB

Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

Background: The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings: We report on the ability of STRO-1 + deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and a-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1 + DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) unde

Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex

Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect

The histone deacetylase inhibitors (HDACi) have demonstrated anticancer efficacy across a range of malignancies, most impressively in the hematological cancers. It is uncertain whether this clinical efficacy is attributable predominantly to their ability to induce apoptosis and differentiation in the cancer cell, or to their ability to prime the cell to other pro-death stimuli such as those from the immune system. HDACi-induced apoptosis occurs through altered expression of genes encoding proteins in both intrinsic and extrinsic apoptotic pathways; through effects on the proteasome/aggresome systems; through the production of reactive oxygen species, possibly by directly inducing DNA damage; and through alterations in the tumor microenvironment. In addition HDACi increase the immunogenicity of tumor cells and modulate cytokine signaling and potentially T-cell polarization in ways that may contribute the anti-cancer effect in vivo. Here, we provide an overview of current thinking on the mechanisms of HDACi activity, with attention given to the hematological malignancies as well as scientific observations arising from the clinical trials. We also focus on the immune effects of these agents

Oligodendrocytes: biology and pathology

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). They are the end product of a cell lineage which has to undergo a complex and precisely timed program of proliferation, migration, differentiation, and myelination to finally produce the insulating sheath of axons. Due to this complex differentiation program, and due to their unique metabolism/physiology, oligodendrocytes count among the most vulnerable cells of the CNS. In this review, we first describe the different steps eventually culminating in the formation of mature oligodendrocytes and myelin sheaths, as they were revealed by studies in rodents. We will then show differences and similarities of human oligodendrocyte development. Finally, we will lay out the different pathways leading to oligodendrocyte and myelin loss in human CNS diseases, and we will reveal the different principles leading to the restoration of myelin sheaths or to a failure to do so

20-Year Risks of Breast-Cancer Recurrence after Stopping Endocrine Therapy at 5 Years

The administration of endocrine therapy for 5 years substantially reduces recurrence rates during and after treatment in women with early-stage, estrogen-receptor (ER)-positive breast cancer. Extending such therapy beyond 5 years offers further protection but has additional side effects. Obtaining data on the absolute risk of subsequent distant recurrence if therapy stops at 5 years could help determine whether to extend treatment