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Pluripotent Stem Cells

The Expanding Talents of Oct4 and Sox2: Pluripotency Factors in Embryonic Stem Cells Regulate Differentiation into Germ Layers

From Cell
By Stuart P. Atkinson

While much is known about the relative contribution to pluripotency by key transcription factors such as Oct4, Nanog and Sox2, little is known about the role they may have, if any, in lineage-specific differentiation. Detailed transcriptional maps have been generated which show the integration of these factors into pluripotency networks (Chen et al, Lu et al, Marson et al, Wang et al) but how this map alters and how transcription factor function alters when a cell leaves the pluripotent state and becomes lineage restricted is unknown. This question has now begun to be answered in a Cell paper from the laboratory of Sharad Ramanathan at Harvard University, Cambridge, USA using directed differentiation of mouse embryonic stem cells (mESCs) as a model system to study the relative contributions of Oct4 and Sox2 to germ layer development and show that, in addition to their roles in pluripotency, they also act to regulate lineage choice (Thomson et al).

Methylation Dictates Fate? Cell fate potential of human pluripotent stem cells is encoded by histone modifications

From Cell Stem Cell
By Stuart P. Atkinson

Multiple studies over the last 5 years have shown us that human embryonic stem cells (hESCs) growing under self-renewing conditions in vitro are surprisingly heterogeneous with individual cells displaying dynamic phenotypes (Hayashi et al and Stewart et al) while others studies have demonstrated cell-to-cell variance in the levels of pluripotency-associated transcription factors (Chambers et al, Hayashi et al and Toyooka et al). This heterogeneity is most easily observed during directed differentiation of hESCs which generally generates a spectrum of differentiated cell types which impacts on the usefulness of such strategies for therapeutic use. The study by Hayashi et al begins to unravel the links between heterogeneous ESC states in mouse and specific chromatin modifications and now results from Hong et al from the lab of Mick Bhatia at the McMaster University, Hamilton, Canada, presented in Cell Stem Cell, reveal the identification of cells in hESC cultures that are lineage biased, through the use of specific surface markers, and link this lineage bias to specific chromatin modification patterns.

Differential Recruitment of Methyl CpG-Binding Domain Factors and DNA Methyltransferases by the Orphan Receptor Germ Cell Nuclear Factor Initiates the Repression and Silencing of Oct4

From the July Edition of Stem Cells
By Stuart P. Atkinson

Oct4 (or Pou5f1) is one of a few genes recognised as being vitally important for the pluripotent nature of embryonic stem cells (ESCs) and is commonly used for induced pluripotent stem cells (iPSC) generation. Its downregulation during differentiation is essential, and indeed it has been suggested that a failure to properly down-regulate Oct4 could have detrimental effects for the application of cell types differentiated from iPSCs (Zhao et al). Gcnf (also known as Nr6a1), an orphan nuclear receptor germ cell nuclear factor, has been previously linked to Oct4 repression through binding to specific sequences, and the loss of Gcnf in mouse ESCs (mESCs) leads to the failure of Oct4 repression during differentiation (Gu et al, 2005). While the epigenetic contribution to pluripotency and differentiation has been the focus of intense study, specific studies of the epigenetic mechanisms regulating Oct4 expression have been lacking, and so now Gu et al, 2011, from the lab of Austin J. Cooney at the Baylor College of Medicine, Houston, Texas in a study published in the July edition of Stem Cells, attempt to delineate the mechanisms by which Gcnf mediates DNA methylation and repression of Oct4.

Defining the Nature of Human Pluripotent Stem Cell Progeny

From Cell Research
By Stuart P. Atkinson

Multiple studies into the differentiation capabilities of pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have shown that they all have some potential to generate an array of clinically relevant cell types, even if this can vary widely across different hESC and hiPSC lines (Bock et al, Boulting et al, Feng et al and Osafune et al). Cell derivatives are often tested for specificity by a few generally well understood markers discovered through the study of natural development, but it remains to be seen if, on a global level, these derivatives are truly analogous to their natural counterparts and if their in vitro development follows what is observed in vivo. A recent study in Cell Research (Patterson et al) from the laboratory of William Lowry at the Department of Molecular, Cell and Developmental Biology, UCLA has addressed this point and overall demonstrates that hESCs and iPSCs can be differentiated towards specified derivatives that are transcriptionally similar to their developmental counterparts, but that these derivatives retain the expression of a group of genes known to play a role during very early embryonic development.

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

From Nature Biotechnology
By Stuart P. Atkinson

Human pluripotent cells types, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold considerable promise for cell-based therapeutics through their directed differentiation to desirable cell types. However, current differentiation protocols are generally very inefficient and the risk of teratoma formation by residual undifferentiated cells remaining amongst the differentiated products is a major concern (Ben-David and Benvenisty, Tang and Drukker). Early attempts at addressing this issue through suicide genes and chemotherapy had several major caveats (Cao et al, Schuldiner et al), while recent attempts have shown the efficacy of a cell death inducing antibody in undifferentiated hESC cultures, but not in targeting potentially teratoma forming cells in differentiating cultures (Tan et al, Choo et al). This encouraged researchers from the group of Micha Drukker at the Institute of Stem Cell Biology and Regenerative Medicine at the Stanford University School of Medicine, USA to find an antibody-mediated method of removing potentially teratoma forming residual cells from differentiated hESC cultures. This led to the discovery of a novel antibody, SSEA-5, which was shown to be highly specific to pluripotent cell types and, in combination with other specific antibodies, was able to efficiently deplete teratoma forming cells from differentiating cultures. The study by, Tang et al, was published as an advance online publication from Nature Biotechnology.

Human iPSCs Harbor Homoplasmic and Heteroplasmic Mitochondrial DNA Mutations while Maintaining hESC-Like Metabolic Reprogramming

From the September Edition of Stem Cells
By Stuart P. Atkinson

Studies into human induced pluripotent stem cells (hiPSCs) have recently begun to look at mutational changes which may occur during the reprogramming process (See Genetic Instability in Induced Pluripotent Stem Cells: One Step Forward in Understanding, Two Steps Back from the Clinic?) but mitochondrial genetics has not yet been studied in great detail. Mitochondrial kinetics are an important aspect of iPSC generation as cells undergo a metabolic switch, for example from oxidative phosphorylation in fibroblasts to glycolysis in pluripotent cells and may also be important in modelling mitochondrial mutation-associated diseases in iPSC. Therefore researchers from the laboratory of James Adjaye at the Max Planck Institute for Molecular Genetics, Berlin, used next generation sequencing technology to analyse the mitochondrial genome in iPSCs and found that although various mitochondrial DNA (mtDNA) point mutations arose, these did not affect the bioenergetic profile of the cells. This report is published in the September edition of Stem Cells by Prigione et al.

Complete Meiosis from Human Induced Pluripotent Stem Cells

From the August Edition of Stem Cells
By Stuart P. Atkinson

Multiple studies have shown that mouse and human embryonic stem cells (ESCs) can differentiate in vitro into primordial germ cells (PGCs) and oocyte- or sperm-like cells (Marques-Mari et al). Therapeutic use of these cells would require patient specificity, so generation of such cells from individualised induced pluripotent stem cells (iPSC) would be required; a feat which has yet to be reported. However, in a study published in the August Edition of Stem Cells, Eguizabal et al from the laboratory of Juan Carlos Izpisúa Belmonte at the Center for Regenerative Medicine in Barcelona, Spain the complete differentiation of human iPSCs to post-meiotic cells has now been demonstrated, perhaps leading the way towards the ultimate goal of patient specific gamete formation.

Radical Acceleration of Nuclear Reprogramming By Chromatin Remodeling with the Transactivation Domain of MyoD

From the September Edition of Stem Cells
By Stuart P. Atkinson

Generation of induced pluripotent stem cells (iPSCs) is both slow and inefficient; the route from somatic target cell generally takes a minimum of 4 weeks and only 1 in a thousand target cells being reprogrammed. The proper reconfiguration of the chromatin landscape is deemed a potential obstacle in the reprogramming process; so much so that small molecule inhibitors which promote a more open chromatin configuration are becoming common place in many reprogramming protocols (Huangfu, Maehr et al and Huangfu, Osafune et al). One potential problem with this approach is the lack of specific chromatin changes; instead these inhibitors promote global chromatin changes. Specific chromatin changes occur due to the specific recruitment of epigenetic regulators to specific loci by transcription factors; and so this suggests that currently used transcription factors in reprogramming (Oct4, Sox2, Klf4, Myc and Nanog) may have limited means to reconfigure chromatin. Myod1 is a master transcription factor for skeletal myogenesis and can directly convert one cell type into another, as exemplified by its ability to generate myotubes from pigmented retinal epithelial cells (Choi et al). This suggests that Myod1 has a more potent ability to recruit epigenetic modifiers leading to activation of suppressed genes embedded in closed chromatin. This hypothesis has been now been tested by the laboratory of from the Stem Cell Institute, University of Minnesota, USA and Laboratory of Animal Reproduction, Kinki University, Nara, Japan and is presented in the September Edition of Stem Cells (Hirai et al). Full-length mouse Oct4 (O) was fused with various fragments of mouse Myod1, excluding the basic helix-loop-helix (bHLH) domain to avoid activation of Myod1 target genes, and were co-transduced with a polycistronic retroviral vector encoding mouse Sox2, Klf4, and Myc (SKM) into MEFs derived from Oct4-GFP mice, allowing for the monitoring of the reprogramming process. Using this system, the authors demonstrate that that a specific chimeric Oct4-Myod1 protein can reprogram MEFs to an iPSC state more efficiently than OSKM.

New insight into how a somatic past shapes the future of human iPSCs

From Nature Cell Biology
Paper commentary by Carla Mellough

The differences between human embryonic stem cells (hESC) and their somatically derived counterparts, induced pluripotent stem cells (iPSC), have been under close scrutiny following the accumulation of various reports indicating greater disparity between the two cell types than originally envisaged (for example see iPSC don’t forget their origins). Conflicting results have been reported and remain unresolved, for example the transcriptional signature of iPSCs, although ascribed to partial memory retention of their somatic origin, does not always correlate with the differences in gene expression between iPSCs and hESCs. This has led to some of the biases being attributed to interlaboratory methodological variation. Functional disparity between differentiating hESCs and iPSCs has also been reported, with iPSC derivatives showing limited differentiation potential and early senescence and perhaps indicating that iPSCs do not hold a comparable clinical value to hESCs. This, alongside numerous reports highlighting the remarkable similarity of both cell types has resulted in some confusion regarding the applicability of iPSCs to translational research. For this reason, elucidation of the true likeness between iPSCs and hESCs has been somewhat limited. A recent study published in Nature Cell Biology from various centres at the University of California by Ohi et al.attempts to address these limitations by systematically comparing human iPSC lines derived from multiple somatic cells types and under the same methodology, in parallel.

Conversion of Sox17 into a pluripotency reprogramming factor by reengineering its association with Oct4 on DNA --

From the June 2011 Issue of Stem Cells
By Carla Mellough

The selective dimerization of transcription factors with binding partners can allow those factors lacking site-specific DNA binding capability to elicit transcriptional control. This type of synergistic action between the Sox and POU (Oct) family members of transcription factors is well known. Co-operation between various members facilitates numerous key regulatory roles, for example the well documented interaction between Sox2 and Oct4 in stem cells and more recently the cooperation of Sox17 with Oct4 for mesodermal development. Interestingly, both Sox2 and Sox17 interact with Oct4 during early development, yet both yield different effects and Sox17 cannot replace Sox2 as a pluripotency or reprogramming factor. An article published in the June issue of Stem Cells by Jauch et al., now begins to unravel the mechanisms underlying the competitive interactions between Sox2 and Sox17 for binding to Oct4.


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