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Pluripotent Stem Cells

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First SCNT-hESCs: Here’s the Science

“Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer”

Few papers have received more attention than the recent study from the laboratory of Shoukhrat Mitalipov at the Oregon Health & Science University, Beaverton, USA in Cell, describing the first derivation of human embryonic stem cells (hESCs) from cloned embryos (Tachibana et al).   Herein is a brief review on the science behind the study which describes the circumnavigation of persistent problems in this field and provides analysis of the resultant derived human nuclear transfer-ESCs (NT-ESCs).

iPSCs De-Liver

“Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells”

From Stem Cells Translational Medicine

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have long been postulated as a potential source for hepatocytes; for both regenerative medicine (transplantation and use in bioartificial liver devices) and for the pharmaceutical industry as an efficient means to assess toxicity of a range of drugs.   iPSCs are advantageous due to their patient and/or disease specificity and while hepatocyte production has been described previously (Rashid et al, Si-Tayeb et al, Song et al and Sullivan et al), their functionality has been suggested to be limited.   Now in a report in Stem Cells Translational Medicine, the group of Mark A. Zern at the University of California Davis Medical Center, California, USA describe the generation of relatively homogeneous hiPSC-derived hepatocytes (hiHs) which exhibit properties of metabolically functional hepatocytes (Ma and Duan et al).

Epigenetics of hESC Differentiation under the Microscope

“Epigenomic Analysis of Multilineage Differentiation of Human Embryonic Stem Cells” and “Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells”

In a collaborative study, published recently in Cell, James A. Thomson, Joseph R. Ecker and Bing Ren have defined the roles of specific epigenetic marks in the maintenance of lineage-specific gene expression patterns.   As access to early embryonic development in humans is prohibitive, this was studied by the differentiation of human embryonic stem cells (hESCs) into different progenitor types representing key lineages in the human embryo, and their systematic characterization with regards to DNA-me, chromatin modifications and mRNA expression.   This has found that promoters of genes involved in early developmental stages tend to be GC rich and modulated by histone modifications, while promoters of genes expressed at latter stages are GC poor and controlled by DNA-me (Xie et al).   Complementing this study in the same issue of Cell is a report from the laboratory of Alexander Meissner from the Harvard Stem Cell Institute, USA which also describes the study of epigenetic alterations during specification of hESCs confirming the findings of the previous study and identifies DNA-me and H3K4me1 as markers of pluripotency associated distal regulatory elements in hESCs (Gifford and Ziller et al).

Is hypoxic the new normoxic?

“Neural Precursor Cells Cultured at Physiologically Relevant Oxygen Tensions Have a Survival Advantage Following Transplantation”

From Stem Cells Translational Medicine

The common culture of cells under atmospheric (20%) as opposed to physiological oxygen levels (for example, 13% in oxygenated arterial blood, and 0.5-8% in brain tissue) is an important consideration for experimental outcome when using cultured cells for differentiation and transplantation experiments. Previous studies have indicated that the culture of cells under physiological oxygen levels or hypoxic preconditioning of cells prior to transplantation can yield improved survival, integration and differentiation of grafted cells. Nevertheless, the vast majority of researchers continue to culture cells under atmospheric oxygen tension. A recent study published in Stem Cells Translational Medicine by Stacpoole et al.1 from Robin Franklin’s group in Cambridge, has investigated the cellular stress that is created by dramatically dropping the oxygen tension of cells cultured under atmospheric conditions upon transplantation and the impact this has upon cellular integration and viability. Their results reveal that maintaining physiological levels of oxygen in vitro elicits clear benefits upon cellular function, and prompts us to reconsider this commonplace culture practise.

Telomerase and Stem Cells – An Epigenetic Link

"Short Telomeres in ESCs Lead to Unstable Differentiation"

The ability to maintain telomere length is one of the many attributes of embryonic stem cells (ESCs) (Huang et al). Comprising of reverse transcriptase (Tert) and RNA template (Terc), telomerase mediates the addition of new telomeric DNA to the end of chromosomes which is required for ESC function (Agarwal et al, Batista et al and Marion et al) and also for iPSC generation (Marion et al). Previous studies have found that ESCs from late generation Terc -/- mice have short telomeres and have a reduced teratoma formation ability (Huang et al); and now the group of Lea Harrington at the Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, The University of Edinburgh, Scotland have found that mice with critically short telomeres do not undergo stable differentiation, which the group have linked to alterations in pluripotency-associated gene expression and genome wide epigenetic alterations (Pucci et al).

Enhancing RPE-derivation from Pluripotent Stem Cells

"A Simple and Scalable Process for the Differentiation of Retinal Pigment Epithelium From Human Pluripotent Stem Cells" and "Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium"

The high prevalence of blindness caused by age-related macular degeneration (AMD) (Gehrs et al), due to damaged or dysfunctional retinal pigment epithelium (RPE) cells (Khandhadia et al) has led to the use of pluripotent stem cells to derive RPE for transplantation. Various studies have shown that RPE can be derived from human embryonic stem cells (hESCs) (Klimanskaya et al) and human induced pluripotent stem cells (hiPSCs) (Buchholz et al, Hirami et al, Meyer et al and Osakada et al) and a human clinical trial of hESC-RPE cell transplantation is currently under way (Schwartz et al). However, techniques used so far are problematic for large scale production of consistent high quality cells. Now in two studies published in Stem Cells Translational Medicine, advancements in differentiation protocols are presented. In the first study, researchers from the group of Donald J. Zack at the Johns Hopkins University School of Medicine Baltimore, Maryland, USA have described a less labour-intensive myosin inhibitor-mediated differentiation protocol which, after enrichment, leads to a highly pure population of cells which display many characteristics of native RPE cells (Maruotti et al). In the latter study researchers from the laboratories of Peter J. Coffey and Dennis O. Clegg at the Neuroscience Research Institute and the Center for the Study of Macular Degeneration at the University of California, Santa Barbara, USA describe their work into the modification of current protocols by the addition of retinal induction factors and other factors at specific times giving an increased efficiency of RPE derivation at earlier time points (Buchholz et al).

Modulation of the Cell Cycle Boosts Differentiation

"A simple tool to improve pluripotent stem cell differentiation"

Studies of pluripotent cells types have suggested that cell lines differ in their differentiation propensities (Bock et al and Osafune et al), meaning that only certain lines can be used to generate certain cells/tissues. However this contention has now been challenged by researchers from the laboratory of Douglas Melton at the Harvard Stem Cell Institute, Cambridge, Massachusetts, USA who have studied the altered cell cycle structure in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). These pluripotent cells types have an abbreviated G1 phase, during which the cell is prepared for DNA synthesis, and also have minimal checkpoint controls (Orford and Scadden) similar to what is understood about early development in vivo (Pardee and Weinberg). This suggested to the researchers that boosting cells in G1 phase could be important for directed differentiation in all pluripotent cell types and they have now shown that treatment with dimethylsulfoxide (DMSO) boosts the number of cells in G1, activates G1 checkpoints and improves directed differentiation into multiple lineages, removing any differentiation bias (Chetty et al).

Stable Pluripotent Intermediate Lines Established

Original article from STEM CELLS

 “Clonal Isolation of an Intermediate Pluripotent Stem Cell State”

Embryonic stem cells (ESCs) established from preimplantation embryos, and epiblast stem cells (EpiSCs) established from postimplantation embryos, represent two different cell types which have the ability to self-renew and are pluripotent (Brons et al, Evans and Kaufman and Tesar et al) but are sustained using different pathways. Recently observed heterogeneity in ESC cultures (Hayashi et al and Toyooka et al) suggests that some cells may exist in a metastable state, somewhere between an ESC and EpiSC state, although a stable cell line of this sort has never been established. Now, a study published in Stem Cells, Chang and Li from Imperial College London, United Kingdom and Chang Gung University, Taoyuan, Taiwan reports the successful establishment of clonal intermediate pluripotent lines from ESCs providing a means to better understand multiple pluripotent states.

Tracking Reprogramming Highlights Novel Hallmarks

Original article from STEM CELLS

“Dynamic Migration and Cell-Cell Interactions of Early Reprogramming Revealed by High-Resolution Time-Lapse Imaging”

The creation of induced pluripotent stem cells (iPSCs) from somatic cells is generally a long process with only a few rare cells undergoing the reprogramming process (Hanna et al). For this reason, analysis of the early stages of reprogramming is difficult and our knowledge about this stage remains scarce. Time-lapse microscopy has been used previously to define early events (Araki et al, Chan et al and Smith et al) but is hampered by problems; long imaging intervals, the identification of which cell to track, slow reprogramming kinetics and the nature of monitoring pluripotent colonies. Now researchers from the laboratory of Shangqin Guo at Yale University School of Medicine, Connecticut, USA have described a method to overcome these problems and through this have identified a novel two-cell intermediate which manifests before other reprogramming landmarks (Megyola et al).

Genome Stability Key to Efficient Reprogramming and Differentiation

Original article from STEM CELLS

“HPSC Models of Fanconi Anemia Deficiency Reveal an Important Role for Fanconi Anemia Proteins in Cellular Reprogramming and Survival of Hematopoietic Progenitors”

Fanconi anemia (FA) is caused by mutations in replication-dependant repair genes and while several mouse models with targeted deletions have been studied, (Cheng et al, Haneline et al 1998, Haneline et al 1999, and Wong et al) they do not faithfully recapitulate the human forms of disease. The generation of induced pluripotent stem cells (iPSCs) from somatic cells with FA gene mutations is a possible alternative method of studying FA disease progression (Müller et al and Raya et al), and now in a study published in Stem Cells, researchers from the group of Majlinda Lako at the Institute of Genetic Medicine, Newcastle University, UK, report on the derivation of iPSCs from FA patients and their hematopoietic differentiation (Yung and Tilgner et al).

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