Investigation of phosphoinositide 3-kinase dependent signalling in the regulation of embryonic stem cell fate

In order to harness the therapeutic potential of stem cells, a clear understanding of the factors and mechanisms governing their fate is required. Self-renewal, pluripotency and proliferation are important cellular functions for maintaining the stem cell state. The class IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses including cell migration, proliferation and survival. Moreover, this class of PI3Ks were previously reported to play a role in proliferation and maintenance of self-renewal in murine embryonic stem cells (mESCs). Here the activation of PI3Ks, the intracellular signalling, including cross-talk between other important pathways, and the roles of specific catalytic subunits of the PI3K class I family have been investigated. Despite inhibition of PI3Ks giving rise to differentiated cell types, early lineage commitment of mESCs was shown to be regulated by pathways not involving PI3Ks. Differentiation towards mesoderm, endoderm and ectoderm were detected upon broad selectivity inhibition of PI3Ks with LY294002. Cross-talk between PI3K and MAPK pathway signalling was highlighted as a possible mechanism for PI3Ks to regulate self-renewal. Inhibition of PI3Ks with LY294002 led to an enhancement in MAPK pathway activation. On further investigation, activation of MAPK pathway signalling by inducible expression of constitutively active Mek brought forth a minimal reduction in self-renewal. Furthermore, inhibition of p110β induced an enhancement in Erk phosphorylation akin to that induced by LY294002, implicating this isoform in regulating MAPK signalling under normal mESC culture conditions. Insulin was shown to activate PI3Ks in mESCs and could be inhibited by treatment with pharmacological inhibitors of the p110α catalytic subunit isoform. Further investigation into the role of p110α in mESCs revealed a role in cell proliferation and metabolic activity. However, pharmacological or siRNA-mediated interference of this isoform did not perturb self-renewal. In contrast, p110β was identified as having a predominant role in the maintenance of self-renewal of mESCs. Both specific pharmacological inhibition and siRNA targeted knockdown of p110β led to a marked loss in alkaline phosphatase staining and a reduction in Nanog and Rex1 expression, indicating a loss of self-renewal. Thus, independent roles for p110α and p110β in regulating mESC proliferation and self-renewal were found to be the result of coupling to different PI3K catalytic subunit isoforms. Interestingly, reducing proliferation by inhibition of p110α or mTOR led to a greater decline in mESC self-renewal when induced by inhibition of p110β. This demonstration of cross talk between the pathways that regulate proliferation and self-renewal suggests a priming effect where the rate of proliferation could sensitise mESCs to levels of p110β activation in order to regulate self-renewal and ultimately cell fate.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K-and GSK-3- dependent signalling

Abstract ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs. Control of ESC (embryonic stem cell) fate: an overview ESCs are derived from early pre-implantation embryos and, when cultured appropriately, can be maintained in a proliferative, self-renewing and pluripotent state almost indefinitely. Pluripotency is the ability to differentiate into all of the cells found in an adult organism, while self-renewal describes the generation of a daughter stem cell from its mother. In the case of ESCs, self-renewal occurs symmetrically, such that when an undifferentiated ESC divides and pluripotency is maintained, both its progeny will be undifferentiated Over the last 5-10 years, our understanding of the molecular components involved in maintaining pluripotency of mESCs (mouse ESCs) has increased dramatically, from a simple 'prelude' where STAT3 (signal transducer and activator of transcription 3) activation by LIF (leukaemia Key words: cell cycle, embryonic stem cell, glycogen synthase kinase 3 (GSK-3), phosphoinositide 3-kinase (PI3K), pluripotency, proliferation, self-renewal. Abbreviations used: CDK, cyclin-dependent kinase; ESC, embryonic stem cell; Esrrb, oestrogenrelated receptor β; GSK-3, glycogen synthase kinase-3; hESC, human ESC; LIF, leukaemia inhibitory factor; MEK, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase; mESC, mouse ESC; miRNA, microRNA; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; siRNA, short interfering RNA. 1 To whom correspondence should be addressed (email [email protected]). inhibitory factor) was all that seemed necessary, to a complex 'symphony' where extrinsic factors, intracellular signals, transcription factors, epigenetic regulators and miRNAs (microRNAs) have all been implicated The ESC cell cycle mESCs proliferate rapidly in culture and display unique cell-cycle kinetics, distinct from those of somatic cells, dividing approximately every 11-16 h and exhibiting a shortened G 1 -phase INK4a [13] and neither do mESCs arrest following DNA damag

Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells

Self-renewal of embryonic stem cells (ESCs) is essential for maintenance of pluripotency, which is defined as the ability to differentiate into any specialised cell type comprising the adult organism. Understanding the mechanisms that regulate ESC self-renewal and proliferation is required before ESCs can fulfil their potential in regenerative therapies, and murine ESCs (mESCs) have been widely used as a model. Members of the class-IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses, including cell migration, proliferation and survival. PI3Ks have been reported to regulate both proliferation and self-renewal of mESCs. Here we investigate the contribution of specific class-IA PI3K isoforms to the regulation of mESC fate using small-molecule inhibitors with selectivity for particular class-IA PI3K catalytic isoforms, and siRNA-mediated knockdown. Pharmacological inhibition or knockdown of p110beta promoted mESC differentiation, accompanied by a decrease in expression of Nanog. By comparison, pharmacological inhibition or siRNA-mediated knockdown of p110alpha had no effect on mESC self-renewal per se, but instead appeared to reduce proliferation, which was accompanied by inhibition of leukaemia inhibitory factor (LIF) and insulin-induced PI3K signalling. Our results suggest that PI3Ks contribute to the regulation of both mESC pluripotency and proliferation by differential coupling to selected p110 catalytic isoforms

Skeletal Regeneration: application of nanotopography and biomaterials for skeletal stem cell based bone repair

The application of selected skeletal progenitor cells and appropriate biomimetic microenvironments and nanotopographical surfaces offer the potential for innovative approaches to bone disease treatment and bone regeneration. Skeletal stem cells, commonly referred to as mesenchymal stem cells or human bone marrow stromal stem cells are multipotent progenitor cells with the ability to generate the stromal lineages of bone, cartilage, muscle, tendon, ligament and fat. This review will examine i) the application of innovative nanotopography surfaces that provide cues for human stem cell differentiation in the absence of chemical cues, ii) unique biomimetic microenvironments for skeletal tissue repair as well as iii) data from translational studies from the laboratory through to the clinic demonstrating the potential of skeletal cell based repair using impaction bone grafting as an exemplar. The development of protocols, tools and above all multidisciplinary approaches that integrate biomimetic materials, nanotopography, angiogenic, cell and clinical techniques for skeletal tissue regeneration for de novo tissue formation offers an opportunity to improve the quality of life of many

Changes in the methylation status of osteocalcin during fetal femur cell development.

<p><b>A.</b> Osteocalcin promoter methylation status in embryonic, fetal and adult cells. <b>B.</b> Osteocalcin promoter methylation status in STRO-1+ (skeletal stem cell containing) and STRO-1⁻ fractions of adult bone-marrow cells. <b>C</b>. Localisation of STRO-1 in a fetal bone from a 6 mm fetal sample. See higher magnification in <b>D–E</b>.</p

Using nanotopography and metabolomics to identify biochemical effectors of multipotency

It is emerging that mesenchymal stem cell (MSC) metabolic activity may be a key regulator of multipotency. The metabolome represents a "snapshot" of the stem cell phenotype, and therefore metabolic profiling could, through a systems biology approach, offer and highlight critical biochemical pathways for investigation. To date, however, it has remained difficult to undertake unbiased experiments to study MSC multipotency in the absence of strategies to retain multipotency without recourse to soluble factors that can add artifact to experiments. Here we apply a nanotopographical systems approach linked to metabolomics to regulate plasticity and demonstrate rapid metabolite reorganization, allowing rational selection of key biochemical targets of self-renewal (ERK1/2, LDL, and Jnk). We then show that these signaling effectors regulate functional multipotency

Changes in <i>DNMT1</i> expression in HFBCs and adult chondrocytes.

<p>Relative expression of <i>DNMT1</i> during chondrocyte evolution. Levels of DNMT1 were measured by qRT-PCR.</p