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Pluripotent Stem Cells

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Early Reprogramming Factors Uncovered

“Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2”

The inner mechanics of the processes which take place during the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are gradually being delineated. One of the most important aspects of the reprogramming process is erasure of the somatic epigenetic pattern and the establishment of an embryonic stem cell (ESC)-like epigenetic pattern (Mikkelsen et al). Now in a study published in Nature, researchers from the laboratory of Asa Abeliovich at the Taub Institute for Aging, Columbia University, New York have identified factors important for the early and essential stage of reprogramming; poly(ADP-ribose)polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), which seem to have complementary roles in the establishment of early epigenetic marks that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming (Doege et al).

Towards the Production of Sperm from Human Pluripotent Cells

“Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells”

For adult and prepubescent patients who have been rendered sterile, no treatments to restore fertility are currently available. Multiple studies have however shown the ability of embryonic stem cells (ESCs) to be differentiated to primordial germ cells (PGCs), precursors of the spermatogenic lineage, and excitingly, a recent science paper has demonstrated that female ESCs and induced pluripotent stem cells (iPSCs) in mice can be induced into primordial germ cell-like cells (PGCLCs) which can then mature into germinal vesicle-stage oocytes and contribute to fertile offspring after in vitro maturation and fertilization (Hayashi et al). Recent studies have suggested that human pluripotent stem cells (hPSCs) can enter meiosis and, in some cases, produce haploid products (Eguizabal et al, Kee et al and Panula et al), but no definitive proof of the production of spermatogonia, haploid spermatocytes, or spermatids has been demonstrated. However, in a report published in Cell Reports, researchers from the laboratory of Gerald P. Schatten from the University of Pittsburgh School of Medicine have developed an in vitro method that gives rise to both adult-type spermatogonia and cells which resemble post-meiotic round spermatids (Easley et al).

Mature airway epithelia formation breakthrough for Cystic Fibrosis

“Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein”

Previous attempts to differentiate human pluripotent cells into lung epithelia have generated mixed results; studies in human embryonic stem cells (hESCs) have produced cells that express distal airway epithelial phenotypes at low efficiency (Van Haute et al, Samadikuchaksaraei et al and Wang et al) while studies in human induced pluripotent stem cells (hiPSCs) generated lung endoderm progenitors but not mature proximal and distal lung epithelial cells (Mou et al). However, in a study published recently in Nature Biotechnology, researchers from the laboratories of James Ellis and Janet Rossant at the Department of Molecular Genetics, University of Toronto, Canada have now developed a method to generate functional proximal conducting airway epithelia express­ing the cystic fibrosis transmembrane conductance regulator gene (CFTR) from human pluripotent stem cells (hESCs and hiPSCs). Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in CFTR and the researchers also demonstrate that the treatment of CF patient iPSC–derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation results in enhanced plasma membrane localization of mature CFTR protein (Wong et al).

Another Chapter in the Story of ESCs and ABCG2

Original article from STEM CELLS

“Regulation and Expression of the ATP-Binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells”

ATP-binding cassette sub-family G member 2 (ABCG2) is known to function as a multidrug efflux pump and plays a role in resistance to chemotherapeutic agents in cancer cells. While it has been implicated as the cause of the “side population” in adult stem cells, its role in human embryonic stem cells (hESCs) remains unclear. Two competing groups have reported seemingly contradictory findings; one reports that ABCG2 mRNA and protein are present at a high level in two hESC lines (HUES1 and HUES9) (Apati et al and Sarkadi et al), while another has suggested that ABCG2 mRNA and protein is absent in three hESC lines (HUES1, H9 and CT2) (Zeng et al). These results therefore suggest that the role ABCG2 in hESCs merits another detailed investigation, which has now been attempted in a recent report in Stem Cells from the laboratory of Michael M. Gottesman at the National Cancer Institute at the National Institutes of Health. Their analysis found ABCG2 mRNA to be expressed in all hESC lines while protein levels were low or undetectable; suggesting a putative role for miRNA-mediated regulation of ABCG2 expression (Padmanabhan and Chen et al).

Variability Necessitates More iPSCs

Original article from STEM CELLS Translational Medicine

“Variability in the Generation of Induced Pluripotent Stem Cells - Importance for Disease Modeling”

One of the most interesting uses of induced pluripotent stem cells (iPSCs), alongside potential immunologically matched cell replacement therapy, is the modelling of complex human diseases (Vitale et al, 2011). However, diseases with undefined genetic components such as idiopathic Parkinson disease (Soldner et al) raise questions about the effectiveness of iPSCs as disease models. However comparisons between multiple patients and controls to identify disease-specific attributes that are independent of individual differences may overcome this problem. Such variability includes both genetic and epigenetic mechanisms and many laboratories are moving to attempt to maximise the efficiency and efficacy of iPSC generation and standardise any production protocols in order to minimise any identified variations so that disease-associated changes can be observed more clearly (Boulting et al and Saha and Jaenisch). Now, in a study published in the September edition of Stem Cell Translational Medicine researchers from the laboratory of Alan Mackay-Sim have derived 18 human iPSC lines from 8 individuals in order to determine the variability in the generation, selection, and characterization of these iPSCs (Vitale et al, 2012), finding low inter-individual and interclonal variability but also reprogramming instability.

Changing Methylation Accompanies Changing Culture

“Epigenetic stability, adaptability, and reversibility in human embryonic stem cells”

Human embryonic stem cells (hESCs) are known to adapt during extended periods of ­­in vitro culture, a process potentially detrimental to later differentiation ability. However, the mode of initiation of transcriptional changes and the potential for reversibility of adaptation mediated by culture conditions is relatively unknown. DNA methylation is a potential mechanism behind transcriptional changes during culture adaptation through the reinforcement of environmental cues (Bird 2002 and Riggs 1989), but the recently observed plasticity of DNA methylation (Gong and Zhu 2011 and Riggs and Xiong 2004) suggests that it may also allow for the reversal of observed transcriptional changes. Researchers from the laboratory of Arthur D. Riggs have now analysed hESCs transcriptional and DNA methylation status during passaging and transfer between different growth conditions, finding that DNA methylation patterns appear to be remarkably stable but that a few regions of differential methylation are significant and often irreversible (Tompkins et al).

Targeting Small Molecules Hits the Spot for Reprogramming

“A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts”

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is known to involve the genome-wide remodelling of chromatin and it has been suggested that small molecules which can affect the chromatin landscape could be used to reactivate the endogenous pluripotency network in place of transcription factor transduction (Onder et al and Hirai et al). Researchers from the laboratory of Hiroshi Sugiyama at Kyoto University, Japan have previously shown that the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) can be conjugated with selective DNA-binding hairpin pyrrole-imidazole polyamides (PIPs) (Ohtsuki et al), allowing the upregulation of pluripotency-associated factors through the targeted increase in permissive histone modifications at the promoter regions of Oct4 and Nanog in mouse embryonic fibroblasts (MEFs) (Pandian et al, ChemBioChem, 2011). However, the induction values observed were very low (Pandian et al, ChemBioChem, 2011 and Pandian et al, Bioorg Med Chem, 2012) and so the laboratory sought to synthesise new SAHA-PIPs with improved recognition ability (Pandian et al, Sci Reps, 2012).

Epigenetic Showdown: SCNT vs. Normal Development

“Mouse ooplasm confers context-specific reprogramming capacity”

The mammalian oocyte mediates one of the largest periods of DNA methylation dynamics; global demethylation of the paternal genome, a process required for the establish­ment of totipotency and developmental competence (Reik et al 2011). Somatic cell nuclear transfer (SCNT) attempts to recapitulate this artificially by using enucleated oocytes to reprogram a somatic cell nucleus, but only achieves this with very low efficiency, likely compromised by the retention of uncharacterised somatic epigenetic modifications (Rideout et al and Wakayama et al).   Now, researchers from the laboratory of Alexander Meissner have reported the results from their study of genome-scale DNA methylation patterns after SCNT and their comparison to observed dynamics during normal fertilization, and identify specific targets for DNA demethylation during SCNT and identify some unique limitations (Chan et al).

Source of ESC mutations Uncovered?

Original article from STEM CELLS

“Deficient DNA Damage Response and Cell Cycle Checkpoints Lead to Accumulation of Point Mutations in hESCs”

The long term culture of human embryonic stem cells (hESCs) is known to have some detrimental effects, such as the acquirement of an abnormal karyotype, increased copy number variations, loss of heterozygosity, increased rate of proliferation and resistance to apoptosis (Draper et al, Baker et al and Narva et al). These changes are all indicative of the gain of a tumourigenetic phenotype, so it is not surprising that culture-adapted hESCs form teratocarcinomas when transferred into severe combined immunodeficiency (SCID) mice, while non-adapted hESCs lead to  teratoma formation (Solter, Blum and Benvinisty and Andrews). How long term culture leads to this effect is relatively unknown, although it is thought that uncontrolled cell cycle checkpoints and abnormal DNA damage response and repair are among the mechanisms contributing to hESC adaptation and major karyotypic changes (Rodriguez-Jiminez et al, Mantel et al and Momcilovic et al). In a study described in Stem Cells, researchers from the laboratories of Peter W. Andrews and Thierry Nouspikel at the University of Sheffield, United Kingdom have now analyzed one mode of DNA damage repair; Nucleotide Excision Repair (NER) with respect to the DNA damage response and cell cycle checkpoints in hESCs and have found that point mutations result from a combination of defects in the DNA damage signaling pathway which leads to incomplete arrest at cell cycle checkpoints, the rapid proliferation rate of hESCs and insufficient NER activity (Hyka-Nouspikel et al).

All the Self-Renewal and None of the Tumourigenesis!

“Modulating Glypican4 Suppresses Tumorigenicity of ESCs While Preserving Self-Renewal and Pluripotency”

September 2012 — Stem cell self-renewal and differentiation are highly complex processes involving the integration of a variety of signals, both external and internal, such as soluble cytokines, growth factors, and extracellular matrix proteins which converge onto complex networks of transcription factors which mediate fate choice (He et al, Morrison and Spradling and Ying et al).   However, how extracellular signaling cues are accurately perceived by stem cells to ensure specific outcomes is poorly understood.   Now, in a study published in Stem Cells, researchers from the group of Rosanna Dono at the Developmental Biology Institute of Marseilles (IBDML), France have shown that the cell surface protein Gpc4, a member of the heparan sulfate proteoglycan (HSPG) superfamily, is required for mouse embryonic stem cell (mESC) self renewal and neural stem cell (NSC) maintenance, functioning through the modulation of Wnt/β-catenin signaling and, additionally, that the impairment of Gpc4 disrupts teratoma formation of mESCs but not pluripotency (Fico et al).

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