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Copyright of Revista Espacios is the property of Talleres de Impresos Oma and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The article presents an essay on the involvement of biotechnology in cultural and political controversy and the book "Brave New World," by Aldous Huxley. Topics discussed include the debate over the great embryonic stem cell (ESCR), the issuance of the report "Human Cloning and Human Dignity" and the development of genetic engineering.

Progress has recently been made toward the production of human skeletal muscle cells from induced pluripotent stem (iPS) cells. However, the functional and ultrastructural characterization, which is crucial for disease modeling and drug discovery, remains to be documented. We show, for the first time to our knowledge, that the electrophysiological properties of human iPS-derived skeletal myocytes are strictly similar to those of their embryonic stem (ES) cell counterparts, and both are typical of aneural mammalian skeletal muscle. In both cell types, intracellular calcium signaling that links membrane depolarization to contraction occurs in the absence of extracellular Ca2+, a unique feature of skeletal muscle. Detailed analysis of the Ca2+ signal revealed diverse kinetics of the rising phase, and hence various rates in the release of Ca2+ from the sarcoplasmic reticulum. This was mirrored by ultrastructural evidence of Ca2+ release units, which varied in location, shape, and size. Thus, the excitation contraction coupling machinery of both iPS- and ES-derived skeletal myocytes was functional and specific, but did not reach full maturity in culture. This is in contrast with the myofibrillar network, which displayed the same organization as in adult skeletal muscle. Overall, the present study validates the human iPS-based skeletal myocyte model in comparison with the embryonic system, and provides the functional and ultrastructural basis for its application to human skeletal muscle diseases. [ABSTRACT FROM AUTHOR]

Background Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. Methods Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. Results CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. Conclusions Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the transcriptional profile and role of WNT3A signalling in maintaining buffalo embryonic stem (ES) cells in a pluripotent state and in the induction of their differentiation. ES cells were derived from embryos produced by in vitro fertilisation (iESC), parthenogenesis (pESC) and hand-made cloning (cESC). The expression of WNT3A, its receptors and intermediate signalling pathways were found to be conserved in ES cells derived from the three different sources. WNT3A was expressed in ES cells but not in embryoid bodies derived from iESC or in buffalo fetal fibroblast cells. It was revealed by real-time polymerase chain reaction analysis that following supplementation of culture medium with WNT3A (100, 200 or 400 ng mL[sup -1]) a significant increase (P < 0.05) was observed in the expression level of β-CATENIN, which indicated the activation of the canonical WNT pathway. WNT3A, in combination with exogenous fibroblast growth factor-2 and leukaemia inhibitory factor, induced proliferation of undifferentiated ES cells. Differentiation studies showed that WNT3A caused formation of scaffold-like structures and inhibition of differentiation into neuron-like cells. In conclusion, the WNT3A signalling pathway is necessary both for maintaining undifferentiated buffalo ES cells as well as for directing their differentiation. In our study, we investigated the transcriptional profile and role of WNT3A signaling in maintaining the pluripotency of buffalo embryonic stem (ES) cells as well as in inducing their differentiation. We concluded that WNT3A, in combination with exogenous FGF-2 and LIF, induced proliferation of undifferentiated ES cells, while differentiation studies revealed its role in causing formation of scaffold-like structures and preventing differentiation into neuron-like cells. Thus, WNT3A signalling pathway is necessary both for maintaining pluripotency and for directed differentiation of buffalo ES cells. [ABSTRACT FROM AUTHOR]

To elucidate detailed functional mechanisms of key fate-determining transcription factors (eg, Nurr1, Pitx3, and Lmx1a) and their functional interplay for midbrain dopamine (mDA) neurons, we developed highly efficient gain-of-function system by transducing the neural progenitors (NPs) derived from embryonic stem cells (ESCs) with retroviral vectors, allowing the analysis of downstream molecular and cellular effects. Overexpression of each factors, Nurr1, Pitx3, and Lmx1a robustly promoted the dopaminergic differentiation of ESC-NP cells exposed to sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8). In addition, each of these factors directly interacts with potential binding sites within the tyrosine hydroxylase (TH) gene and activated its promoter activity. Interestingly, however, overexpression of Nurr1, but not of Pitx3 or Lmx1a, generated a significant number of nonneuronal TH-positive cells. In line with this, Pitx3 and Lmx1a, but not Nurr1, induced expression of the Ngn2 gene, which is critical for neurogenesis. We also observed that Pitx3 directly bound to its potential binding sites within the Ngn2 gene and the pan-neuronal marker β-tubulin III gene, suggesting that Pitx3 contributes to mDA neurogenesis by directly regulating these genes. Taken together, our data demonstrate that key mDA regulators ( Nurr1, Pitx3, and Lmx1a) play overlapping as well as distinct roles during neurogenesis and neurotransmitter phenotype determination of mDA neurons. [ABSTRACT FROM AUTHOR]

Cardiac cell replacement therapy by using human embryonic stem cell (hESC) derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA) and FK506, as well as rapamycin and a selective inhibitor of calcineurin-binding downstream nuclear factor of activated T-cell (NFAT) transcription factor VIVIT on the proliferative activity, function, and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506, and rapamycin all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC with the mitotic marker Ki67+ by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the proapoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mammalian target of rapamycin (mTOR) pathways in hESC-CM survival (using apoptotic marker caspase-3), whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation, while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressant therapy would be compatible with stem cell transplantation, but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation. [ABSTRACT FROM AUTHOR]

Lefty expression has been recognized as a stemness marker because Lefty is enriched both in undifferentiated embryonic stem cells (ESCs) and in blastocysts. Here, we examined the function of Lefty1 and Lefty2 in the maintenance of self-renewal and pluripotency of mouse ESCs (mESCs). Suppression of Lefty1 or Lefty2 expression in mESCs did not alter the self-renewal properties of mESCs under nondifferentiating conditions, but suppression of these genes did affect Smad2 phosphorylation and differentiation. Lefty1 knockdown mESCs showed enhanced phosphorylation of Smad2 and increased differentiation potential, whereas Lefty2 knockdown mESCs exhibited reduced phosphorylation of Smad2 and enhanced self-renewal in the presence of a differentiation signal. In vivo, teratomas developed from Lefty2 knockdown mESCs contained massive expansions of immature neuroepithelium, a marker of malignant teratomas. Taken together, these results suggest that optimal expression of Lefty1 and Lefty2 is critical for the balanced differentiation of mESCs into three germ layers. [ABSTRACT FROM AUTHOR]

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system. [ABSTRACT FROM AUTHOR]

Copyright of Dirasat: Shar'ia & Law Sciences is the property of University of Jordan and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The developments arising from human IVF are remarkable. Embryos were studied for developmental patterns that have consequences for viability and fertility. Growing human blastocysts in vitro allowed further exploration of the differentiation of primitive embryonic cells, leading to the discovery of human embryonic stem cells (ESC). The availability of perhaps unlimited numbers of human ESC could inform the study of differentiation and also provide cells for therapies in human regenerative medicine. The developments in cell biology have been impressive, including the discovery of induced pluripotent stem cells - adult cells transduced by specific transcription factors to behave like human ESC. Key regulators of development such as activators or inhibitors of lineage progression have also been explored, particularly the fibroblast growth factor, Wnt and transforming growth factor β signalling pathways and miRNA. Such regulators can be utilized in algorithms to predict how cells differentiate in vitro. Using multistep differentiation protocols, many different cell types can be formed and matured into functionally effective cells, some of which are already in translational research for clinical applications. Possible future developments include destruction of cancer stem cells, reversal of type I diabetes, restoration of vision, repair of motor function, cure for HIV/AIDS and heart muscle regeneration. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo. They hold a huge promise for cell therapy with their self-renewing ability and pluripotency, which is known as the potential to differentiate into all cell types originating from three embryonic germ layers. However, their unique pluripotent feature could not be utilised for therapeutic purposes due to the ethical and legal problems during derivation. Recently, it was shown that the cells from adult tissues could be reverted into embryonic state, thereby restoring their pluripotent feature. This has strenghtened the possiblity of directed differentition of the reprogrammed somatic cells into the desired cell types in vitro and their use in regenerative medicine. Although these cells were termed as induced pluripotent cells, the mechanism of pluripotency has yet to be understood. Still, induced pluripotent stem cell technology is considered to be significant by proposing novel approaches in disease modelling, drug screening and cell therapy. Besides their self-renewing ability and their potential to differentiate into all cell types in a human body, they arouse a great interest in scientific world by being far from the ethical concerns regarding their embryonic counterparts and their unique feature of being patient-specific in prospective cell therapies. In this review, induced pluripotent stem cell technology and its role in cell-based therapies from past to present will be discussed. [ABSTRACT FROM AUTHOR]

The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state. [ABSTRACT FROM AUTHOR]

In 2011, courts in both the United States and European Union handed down decisions related to human embryonic stem cell (hESC) research. In both cases, the definition of research was challenged – but the two courts reached different opinions. In the US case, Sherley v. Sebelius, research was defined as a specific project. The US District Court of Appeals did not link research utilizing existing hESC lines to the act of destroying a human embryo in order to create the line, which is not eligible for federal funding. In contrast, the Court of Justice of the European Union in the Brüstle v. Greenpeace case determined inventions related to hESCs were unpatentable since they resulted from research that involved the destruction of human embryos. In this article, we will compare and contrast these two court cases, the politics related to the rulings, and their impacts. We find that these cases significantly impacted current research and have the potential to negatively impact future stem cell research and development. However, the long-term effects of the cases remain to be seen, and there is a chance that these cases could actually strengthen this area of science. Ultimately, we feel that stem cell polices must be straightforward and supported by the public to prevent courts and judges from making decisions on science, which are disruptive to the progression of research. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2004.10.006 Byline: Calvin Simerly (a)(b), Christopher Navara (a)(b), Sang Hwan Hyun (a)(b), Byeong Chun Lee (c), Sung Keun Kang (c), Saverio Capuano (a)(b), Gabriella Gosman (a)(b), Tanja Dominko, Kowit-Yu Chong (a)(b), Duane Compton (d), Woo Suk Hwang (c), Gerald Schatten (a)(b) Keywords: Embryogenesis; Blastocyst; Meiotic spindle extraction; Nuclear transfer; Stem cells; Molecular motors; NuMA Abstract: Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for 'therapeutic cloning,' though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or 'fertilization failures' might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming. Author Affiliation: (a) Department of Obstetrics-Gynecology-Reproductive Sciences, Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA (b) Department of Cell Biology-Physiology, Pittsburgh Development Center, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA (c) Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea (d) Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA Article History: Received 13 August 2004; Revised 28 September 2004; Accepted 12 October 2004 Article Note: (footnote) [star] In accordance with the International Society for Stem Cell Research's (ISSCR) Nomenclature Statement (September 2, 2004), 'nuclear transfer (NT)' is used instead of 'therapeutic cloning' and 'NT stem cells' or NTSC describe stem cells derived from NT embryos. The terms 'reproductive cloning' and 'therapeutic cloning' are used herein as well for clarity.

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling, which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs, suggesting that they may influence self-renewal. To test this hypothesis, we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation, whereas SPRY4 KD enhanced survival. In both cases, after KD the cells were able to differentiate into cells of the three germ layers, although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage, explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs [ABSTRACT FROM AUTHOR]

Highlights: [•] Electrochemical cell is fabricated that enables culturing and monitoring of embryonic stem cell. [•] The electrochemical signal is analyzed between undifferentiated embryonic stem cell and differentiated one. [•] The proposed technique can be applied to real time embryonic stem cell monitoring for stem cell therapy. [Copyright &y& Elsevier]

Safety is the foremost issue in all human cell therapies, but human induced pluripotent stem cells (iPSCs) currently lack a useful safety indicator. Studies in chimeric mice have demonstrated that certain lines of iPSCs are tumorigenic; however a similar screen has not been developed for human iPSCs. Here, we show that in vitro cartilage tissue engineering is an excellent tool for screening human iPSC lines for tumorigenic potential. Although all human embryonic stem cells (ESCs) and most iPSC lines tested formed cartilage safely, certain human iPSCs displayed a pro-oncogenic state, as indicated by the presence of secretory tumors during cartilage differentiation in vitro.We observed five abnormal iPSC clones amoungst 21 lines derived from five different reprogramming methods using three cellular origins. We conclude that in vitro cartilage tissue engineering is a useful approach to identify abnormal human iPSC lines. [ABSTRACT FROM AUTHOR]

Oct4 is the gatekeeper of stem cell pluripotency, but recent evidences also support Oct4 as a key regulator of germ layer formation and lineage commitment. How Oct4 contributes to lineage commitment is not well understood. We identified a Tcf/Lef-Oct4 composite site in the promoter of the cardiac mesoderm gene Mesp1, with a nucleotide sequence identical to the previously established Sox2-Oct4 composite site. This Tcf/Lef-Oct4 composite site mediated synergistic activation of the Mesp1 promoter by Oct4 and canonical Wnt signaling. Transcription ternary complexes were formed with Oct4 and Wnt terminal components, Lef1. Point mutations on the Tcf/Lef-Oct4 composite site impaired Oct4 and Lef1 binding and Mesp1-β-gal transgene reporter expression during mouse embryogenesis. In ZHBTc4 murine embryonic stem cells, the loss of Oct4 during differentiation impaired Mesp1 expression and the development of the cardiac program. This Tcf/Lef-Oct4 composite site appears to be a unique nodal point regulatory element that may drive pluripotency via Sox2-Oct4 and switch on lineage-related genes through Oct4's recruitment of Tcf/Lef factors. S TEM C ells 2013;31:1213-1217 [ABSTRACT FROM AUTHOR]

Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a smallmolecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage- specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancerBRD4binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells [ABSTRACT FROM AUTHOR]

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. [ABSTRACT FROM AUTHOR]

Recent developments in stem cell research suggest that it may be time to reconsider the current focus of stem cell induction strategies. During the previous five years, approximately, the induction of pluripotency in somatic cells, i.e., the generation of so-called 'induced pluripotent stem cells' (iPSCs), has become the focus of ongoing research in many stem cell laboratories, because this technology promises to overcome limitations (both technical and ethical) seen in the production and use of embryonic stem cells (ESCs). A rapidly increasing number of publications suggest, however, that it is now possible to choose instead other, alternative ways of generating stem and progenitor cells bypassing pluripotency. These new strategies may offer important advantages with respect to ethics, as well as to safety considerations. The present communication discusses why these strategies may provide possibilities for an escape from the dilemma presented by pluripotent stem cells (self-organization potential, cloning by tetraploid complementation, patenting problems and tumor formation risk). [ABSTRACT FROM AUTHOR]

The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested. [ABSTRACT FROM AUTHOR]

Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs. S tem C ells 2012;30:1852-1862 [ABSTRACT FROM AUTHOR]

This article reports on two qualitative studies of tissue banks in the United Kingdom: the onCore UK Cancer Tissue Bank and the UK Stem Cell Bank. I use the language of Waldby and Mitchell's tissue economies to interrogate the shared ground between the two institutions as collators, moral guardians and distributors of human tissue. The article articulates the key ontological and ethical differences between cancer tissue taken directly from patients and embryonic stem cell material taken from 'spare' IVF embryos donated by couples undergoing treatment. In this context, a key focus of the article is the spatial and temporal formations both constraining and produced by each bank to demonstrate the ways in which both institutions are engaged in bringing stable modes of exchange to socially complex human tissue, to both control temporal multiplicity and tame geographical spread. I take seriously the spaces and places of each bank, and the movement and flows of tissue through them to demonstrate how tissue banks operate as 'moral refineries' supplying healthcare research. [ABSTRACT FROM AUTHOR]

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly l-lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo. [ABSTRACT FROM AUTHOR]

Analysis of gene expression to define molecular mechanisms and pathways involved in human embryonic stem cells (hESCs) proliferation and differentiations has allowed for further deciphering of the self-renewal and pluripotency characteristics of hESC. Proteins associated with hESCs were discovered through isobaric tags for relative and absolute quantification (iTRAQ). Undifferentiated hESCs and hESCs in different stages of spontaneous differentiation by embryoid body (EB) formation were analyzed. Using the iTRAQ approach, we identified 156 differentially expressed proteins involved in cell proliferation, apoptosis, transcription, translation, mRNA processing, and protein synthesis. Proteins involved in nucleic acid binding, protein synthesis, and integrin signaling were downregulated during differentiation, whereas cytoskeleton proteins were upregulated. The present findings added insight to our understanding of the mechanisms involved in hESC proliferation and differentiation. [ABSTRACT FROM AUTHOR]

In this paper we propose a firing statistics based neuronal network burst detection algorithm for neuronal networks exhibiting highly variable action potential dynamics. Electrical activity of neuronal networks is generally analyzed by the occurrences of spikes and bursts both in time and space. Commonly accepted analysis tools employ burst detection algorithms based on predefined criteria. However, maturing neuronal networks, such as those originating from human embryonic stem cells (hESC), exhibit highly variable network structure and time-varying dynamics. To explore the developing burst/spike activities of such networks, we propose a burst detection algorithm which utilizes the firing statistics based on interspike interval (ISI) histograms. Moreover, the algorithm calculates interspike interval thresholds for burst spikes as well as for pre-burst spikes and burst tails by evaluating the cumulative moving average and skewness of the ISI histogram. Because of the adaptive nature of the proposed algorithm, its analysis power is not limited by the type of neuronal cell network at hand. We demonstrate the functionality of our algorithm with two different types of microelectrode array (MEA) data recorded from spontaneously active hESC-derived neuronal cell networks. The same data was also analyzed by two commonly employed burst detection algorithms and the differences in burst detection results are illustrated. The results demonstrate that our method is both adaptive to the firing statistics of the network and yields successful burst detection from the data. In conclusion, the proposed method is a potential tool for analyzing of hESC-derived neuronal cell networks and thus can be utilized in studies aiming to understand the development and functioning of human neuronal networks and as an analysis tool for in vitro drug screening and neurotoxicity assays. [ABSTRACT FROM AUTHOR]

The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lensspecific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLECderived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology [ABSTRACT FROM AUTHOR]

Abstract: Ever since the technique of coaxing ordinary skin cells into becoming pluripotent stem cells (iPSCs) has been developed, which have the potential to become any cell or tissue in the body, efforts were made to improve the approach because some major challenges. Increasing evidence suggests that several microRNAs (miRNAs) are involved in early embryonic development and embryonic stem cell formation, known as embryonic stem cell (ESC)-specific miRNAs, particularly the miR-302 family. We summarized here a novel approach to generate iPSCs by using miR-302 and its related miRNAs such as miR-367. The development of this miR-302/367-mediated iPSC (termed mirPSC) may provide tools to deal with the obstacles facing some current iPSC reprogramming methods. The mechanism by which miR-302/367 induce iPSC reprogramming is proposed. [Copyright &y& Elsevier]

Consensus about the toxicity of silver nanoparticles (Ag-NPs) has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs), which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (∼20nm and ∼100 nm) in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles. [ABSTRACT FROM AUTHOR]

Background: Human embryonic stem cell derived cardiomyocytes (hESC-CMs) hold high potential for basic and applied cardiovascular research. The development of a reliable simulation platform able to mimic the functional properties of hESC-CMs would be of considerable value to perform preliminary test complementing in vitro experimentations. Methods: We developed the first computational model of hESC-CM action potential by integrating our original electrophysiological recordings of transient-outward,funny, and sodium-calcium exchanger currents and data derived from literature on sodium, calcium and potassium currents in hESC-CMs. Results: The model is able to reproduce basal electrophysiological properties of hESC-CMs at 15-40 days of differentiation (Early stage). Moreover, the model reproduces the modifications occurring through the transition from Early to Late developmental stage (50-110, days of differentiation). After simulated blockade of ionic channels and pumps of the sarcoplasmic reticulum, Ca2+ transient amplitude was decreased by 12% and 33% in Early and Late stage, respectively, suggesting a growing contribution of a functional reticulum during maturation. Finally, as a proof of concept, we tested the effects induced by prototypical channel blockers, namely E4031 and nickel, and their qualitative reproduction by the model. Conclusions: This study provides a novel modelling tool that may serve useful to investigate physiological properties of hESC-CMs. [ABSTRACT FROM AUTHOR]

To celebrate 30 years of peer-reviewed publication of cutting edge stem cell research in Stem Cells, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this, we will present our views of the 10 most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments would have occurred in recent years, but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body [Naturliche Schopfungsgeschichte, 1868; Berlin: Georg Reimer] but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960s did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960s-1970s and the first embryonic stem cell in 1981, such adult or tissue-specific stem cells were the only known class. The first issue of Stem Cells was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently, induced pluripotent stem cells (iPSCs) have been developed, and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only in Stem Cells but also in other journals over the last 5 years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation. S TEM C ELLS 2012;30:2-9 [ABSTRACT FROM AUTHOR]

The state of a cell is defined by the genes it transcribes and the epigenetic landscape that regulates their expression. Pluripotent cells have markedly different epigenetic signatures when compared with differentiated cells. Permissive chromatin, high occurrence of bivalent domains, and low levels of heterochromatin allow pluripotent cells to react to distinctive stimuli and undergo changes of cell state by differentiating into various tissues. Differentiated cells can be reprogrammed by a set of transcription factors to induced pluripotent stem cells (iPSC) that convert their transcriptional and epigenetic state to pluripotency and thus closely resemble embryonic stem cells (ESC). However, questions remain on whether the epigenetic reprogramming is complete or if there are some recurring iPSC specific aberrations that impede their full pluripotency potential. For this reason, iPSC need to be closely compared with ESC, which is used as a golden standard for in vitro pluripotency. Transcribed genes, epigenetic landscape, differentiation potential, and mutational load show small but distinctive dissimilarities between these two cell types. S TEM C ELLS 2012;30:33-41 [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illumina's Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV-/low methylation, EBV-/high methylation, and EBV+/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV+ tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV+ and EBV-/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g. AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV-/high-methylation genes and commonly methylated gastric cancer genes P = 2 x 10-15 and 2 x 10-34, respectively), but not among EBV+ tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV+-associated de novo methylation. When recombinant EBV was introduced into the EBV-/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV+ gastric cancers showed distinct methylation patterns likely attributable to EBV infection. [ABSTRACT FROM AUTHOR]

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG. [ABSTRACT FROM AUTHOR]

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms. [ABSTRACT FROM AUTHOR]

Sox2 is a key component of the transcription factor network that maintains the pluripotent state of embryonic stem cells (ESCs). Sox2 is regulated by multiple post-translational modifications, including ubiquitination, sumoylation, acetylation and phosphorylation. Here we report that Sox2 is in association with and methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase that plays a pivotal role in ESCs. We found that CARM1 facilitates Sox2-mediated transactivation and directly methylates Sox2 at arginine 113. This methylation event enhances Sox2 self-association. Furthermore, the physiological retention of Sox2 on chromatin restricts the Sox2 methylation level. Our study reveals the direct regulation of Sox2 by CARM1 that sheds lights on how arginine methylation signals are integrated into the pluripotent transcription factor network. [ABSTRACT FROM AUTHOR]

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 μN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the b-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research. [ABSTRACT FROM AUTHOR]

δ-Catenin is an Armadillo protein of the p120-Catenin sub- family capable of modulating cadherin stability, small GTPase activity, and nuclear transcription. From yeast two- hybrid screening of a human embryonic stem cell cDNA library, we identified δ-Catenin as a potential interacting partner of the caspase-3 protease, which plays essential roles in apoptotic as well as non-apoptotic processes. Interaction of δ-Catenin with caspase-3 was confirmed using cleavage assays conducted in vitro, in Xenopus apoptotic extracts, and in cell line chemically induced contexts. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo repeat 6 of δ-Catenin, was identified through pep- tide sequencing. Cleavage thus generates an amino-terminal (residues 1-816) and carboxyl-terminal (residues 817-1314) fragment, each containing about half of the central Armadillo domain. We found that cleavage of δ-Catenin both abolishes its association with cadherins and impairs its ability to modulate small GTPases. Interestingly, 817-1314 possesses a conserved putative nuclear localization signal that may facilitate the nuclear targeting of δ-Catenin in defined contexts. To probe for novel nuclear roles of δ-Catenin, we performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating interaction with an uncharacterized KRAB family zinc finger protein, ZIFCAT. Our results indicate that ZIFCAT is nuclear and suggest that it may associate with DNA as a transcriptional repressor. We further determined that other p120 subfamily catenins are similarly cleaved by caspase-3 and likewise bind ZIFCAT. Our findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120-Catenin subfamily members, facilitating the coordinate modulation of cadherins, small GTPases, and nuclear functions. [ABSTRACT FROM AUTHOR]

In embryonic stem cells (ESCs), Wnt-responsive development-related genes are silenced to maintain pluripotency and their expression is activated during differentiation. Acetylation of histones by histone acetyltransferases stimulates transcription, whereas deacetylation of histones by HDACs is correlated with transcriptional repression. Although Wnt-mediated gene transcription has been intimately linked to the acetylation or deacetylation of histones, how Wnt signaling regulates this type of histone modification is poorly understood. Here, we report that Smek, a regulatory subunit of protein phosphatase 4 (PP4) complex, plays an important role in histone deacetylation and silencing of the Wnt-responsive gene, brachyury, in ESCs. Smek mediates recruitment of PP4c and HDAC1 to the Tcf/Lef binding site of the brachyury promoter and inhibits brachyury expression in ESCs. Activation of Wnt signaling during differentiation causes disruption of the Smek/PP4c/HDAC1 complex, resulting in an increase in histones H3 and H4 acetylation at the brachyury gene locus. These results suggest that the Smek-containing PP4 complex represses transcription of Wnt-responsive development-related genes through histone deacetylation, and that this complex is essential for ESC pluripotency maintenance. [ABSTRACT FROM AUTHOR]

Pluripotency and self-renewal are the hallmarks of embryonic stem cells. This state is maintained by a network of transcription factors and is influenced by specific signalling pathways. Current evidence indicates that multiple pluripotent states can exist in vitro. Here we review the recent advances in studying the transcriptional regulatory networks that define pluripotency, and elaborate on how manipulation of signalling pathways can modulate pluripotent states to varying degrees. [ABSTRACT FROM AUTHOR]

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derivedhematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo. [ABSTRACT FROM AUTHOR]

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4. [ABSTRACT FROM AUTHOR]