Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox)) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho)(x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.

The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small numbers of mitochondria present in hESC, thus it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilisation) and to show that hiPSC have similar antioxidant defences to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterised two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whilst the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show similar ROS stress defence mechanisms and mitochondrial biogenesis to hESC. Together our data suggest that during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defence mechanisms against accumulation of ROS as the hESC.

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence activated cell sorting (FACS) there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells but in contrast to species such as Mus musculus in which reporter constructs expressing GFP under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterisation of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that express GFP in a population of differentiating human ESC cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen 1 (SSEA-1) expressing cells in our previous publication.

It is well documented that adipogenic differentiation of the cell is associated with downregulation of Wnt/beta-catenin signalling. Using preadipocytes and dermal fibroblasts, we have found that activation of the adipogenic program was associated with marked changes in the expression of nuclear beta-catenin-interacting partners, emerin and lamins A/C, to influence expression and activation of peroxisome proliferators-activated receptors gamma (PPARgamma). In addition, silencing of protein expression with siRNA revealed that beta-catenin and emerin influenced each other's levels of expression and the onset of adipogenesis, suggesting that changes in the expression of nuclear lamina proteins were intimately linked to the stability of beta-catenin. By contrast, dermal fibroblasts, which are emerin null, demonstrated increased nuclear accumulation of stable beta-catenin and constant lamin expression. This was also associated with an unusual adipogenic capacity of the cells, with adipogenesis occurring in the presence of activated beta-catenin but declining upon silencing of the protein expression with siRNA. We propose that the process of adipogenesis is affected by a dynamic link between complexes of emerin and lamins A/C at the nuclear envelope and nucleocytoplasmic distribution of beta-catenin, to influence cellular plasticity and differentiation.

Of all the cell types that can be obtained from the differentiation of embryonic stem cells, primordial germ cells are arguably the most fascinating as they represent the in vitro completion of the reproductive cycle of the organism form which the embryonic stem cell line was derived. It is also possible to obtain these cells from embryos at an appropriate stage of development but this yields only small numbers which are not applicable to investigations of their epigenetic architecture. Considerable data have been generated from the differentiation of mouse embryonic stem cells to this cell type but despite the demonstration of their presence in human embryoid bodies, there has been little progress towards methods of producing human primordial germ cells in useful numbers. We present here a robust protocol to differentiate two human embryonic stem cell lines (H9 and hES-NCL1) that maximises the numbers of primordial germ cells that may be obtained using a simple FACS strategy for their isolation. These demonstrate high level expression of the germ cell specific VASA gene and show removal of parental imprints and chromatin modification changes that support their primordial germ cell identity.______________________________________________________________________________ Author contributions: K.T.: Collection and assembly of data, manuscript writing; S.P.A.: Collection and assembly of data, manuscript writing; A.G.: Collection and assembly of data; M.S.: Manuscript writing; M.L.: Conception and design, manuscript writing; L.A.: Conception and design, manuscript writing, financial support.

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of beta-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with beta-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, beta-catenin is restricted to the cytoplasm and beta-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerinDelta), dominantly stimulates beta-catenin activity and increases nuclear accumulation of beta-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of beta-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active beta-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.