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The online space has continued to be a platform for not only private and mundane discussions, but also a tribune for voicing critical political and national opinions. Nigerians and the international community have employed online media, as well as other media platforms to articulate their thoughts on the claims which favoured the possibility of the demise of the immediate past president of Nigeria, President Muhammadu Buhari, after a prolonged illness in the year 2007 and the ploy of a Jubril of Sudan clone in his place. This study examined the pragmatic strategies employed in the online articles on the national dilemma caused by the Buhari/Jubril claims and refutals, in response to the lacuna in the literature on such analytical investigations on the subject. An online article titled, "Buhari: The real, the fake and the dead" authored by a Nigerian writer, Tunde Odesola retrieved from the online page of The Punch Newspaper on December 3, 2018, served as data for the study. The article was analysed using insights from the Stance Theory and the Pragmatics in general. The analysis revealed eight pragmatic strategies utilised by the writer, namely Biblical allusion, evaluative stance and positioning, epistemic stance and evocation of sarcasm, metaphors, and derogative labelling as anti-Jubrilist positioning, epistemic stance, and evocation of (dis)alignment, antithetical evocations, evaluative stance and berating, evaluative stance and justifying. These result in a pragmatic reconstruction of the readers' views on the issue. [ABSTRACT FROM AUTHOR]

Ethical and theological debates concerning human cloning are getting more intense as cloning technology continues to develop. This has led to debates originating from both secular ethics and Christian theological perspectives. This paper aims to coordinate the debate according to these two perspectives. It will draw from the positions of Christian theology, the position of the United Nations Universal Declaration of Human Rights, and the positions of scientific authorities. It will conclude by stressing the importance of regulating new technologies in a manner which recognizes the shared ethico-theological concerns for human dignity, rights, freedoms, and the moral growth of humanity. [ABSTRACT FROM AUTHOR]

Answering climate change challenges, the paper proposes an intelligent decision support system (DSS) for the management of green-blue infrastructure (GBI). Addressing the gaps identified in other studies, the designed DSS incorporates four key elements: 1/interdisciplinary collaboration among all stakeholders 2/inclusion of practical operation and maintenance activities, 3/main components of distributed DSS, with practical examples of use, 4/consideration of conditions specific to the location. The multi-layered DSS architecture can be implemented as a unified platform that provides a comprehensive, customizable, and flexible framework based on AI tools, big data and analytics, edge computing, cloud, and mobile, IIoT, and biometric system tools. The use of cobots and digital clones alongside humans results in the implementation of hybrid human-machine units. DSS for GBI increases decision-making capacity and can serve as a foundation for the implementation of similar systems by governments and local communities to build sustainable and resilient communities. [ABSTRACT FROM AUTHOR]

Many proteins form complexes that function in reaction pathways. Therefore, to understand protein function, it is necessary to reconstitute complexes and pathways in vitro. However, it is not straightforward to achieve full activity in reconstituted systems. To address this problem, we present a yeast system named "in saccharo" analysis, which uses budding yeast for simultaneous expression and analysis of many kinds of non-host proteins, such as human proteins. For this purpose, vectors that can accommodate many genes are required. Here, we describe the construction of a chromosome vector by insertion of unique barcode sequences (BCs) into the ribosomal RNA gene repeat (rDNA). Each unit of the rDNA has a BC that is used as the target for integration of an external gene. Because rDNA is naturally capable of maintaining many repetitive copies, the vector is expected to retain the numerous external genes that may be required for reconstitution of functional protein complexes and reaction pathways. Consistent with this prediction, we were able to clone three human genes that form the RNA silencing pathway, which has no functional equivalent in budding yeast, and to demonstrate functionality in this in saccharo analysis system. [ABSTRACT FROM AUTHOR]

The article offers Parliamentary news brifs from Australia and discusses the Mitochondrial Donation Law Reform (Maeve's Law) Bill 2021 that amends Prohibition of Human Cloning for Reproduction Act 2002 and Freedom of Information Act 1982 and Prohibition of Human Cloning for Reproduction Act 2002. It offers views of several legislators including Julian Leeser, Kevin Andrews, and Matthew Canavan on the bill.

Background : The unique phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. The potential of this phenomenon has already been demonstrated in animal models. In the present study, the translational potential of several human endothelium-specific antibodies for their use in the endocapt-based approach was analysed. Methods : The binding of different endothelium-surface specific monoclonal antibody clones was analysed in human tissue and in endothelial cells using image-based immunofluorescence and the determination of half-maximal effective concentration (EC 50). The potential of endocapt-based locoregional application of antibodies or antibody-coated liposomes was analysed ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumour models. Results: Eight out of ten antibody clones were assigned to the group of "fast binding antibodies". Different antibody clones showed various binding kinetics to the same endothelial marker whereas the binding kinetics of single antibody clones was independent from the tissue type. Anti-CD49e, anti-CD31, anti-CD34 and anti-CD102 antibodies showed the lowest EC 50 of antibody binding concentration and constant results in EC 50 determination of antibody binding to cells and human tissue. Experimental studies using anti-mouse CD49e antibody and coated immunoliposomes confirmed their effective capture by endothelial cells in vitro and in vivo by which fluorescent liver segment labelling was achieved. Conclusions: Our findings identify the high potential of several human antibody clones, especially anti-CD49e, -CD31, -CD34 and -CD102, for endocapt technology. We also propose important translational implications of these antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. Toxicological studies are indispensable for further translational development of new antibodies for endocapt. The phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. This phenomenon broadly prevents systemic circulation of the antibody or antibody-drug conjugates. In the present study, our findings identify several human antibody clones promising for endothelial capture technology. This study provided the experimental demonstration of liver segment labelling ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumor models. In addition, this study proposed the important translational implications of selected antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. [ABSTRACT FROM AUTHOR]

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 (DMP1), sclerostin (SOST), and E11/gp38/podoplanin (E11). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein (DSPP). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration. [ABSTRACT FROM AUTHOR]

This paper explores the representation of cannibalism in David Mitchell’s novel, Cloud Atlas, and delineates two levels on which it appears; the first context portrays cannibalistic tribes, while the second one displays an industry of producing and recycling human clones as fast food. In addition, this analysis focuses on the confrontation of what it means to be civilized or savage, and on the emerging social issues revolving around consumer culture, as the juxtaposition of consumer society and the figure of the cannibal challenges the concept of consumer society by questioning if it is civilised at all or not. [ABSTRACT FROM AUTHOR]

This article reports and analyses the method and findings from a 3-year interdisciplinary project investigating how the medium of law can support understanding of socio-scientific issues. Law represents one of the most important means by which society decides and communicates its values. Activities mirroring legal processes therefore have significant potential to inform, inspire and involve school students in exploring the conceptual, social and ethical issues relating to developments in biomedical science. This article focusses on an intervention-style study in which UK-based 16- to 17-year-old students role played a Supreme Court moot, developed by modifying a domestic appeal case concerned with whether the contemporary legislation covered the creation of cloned human embryos. We draw attention to how the science of cloning has been slightly misunderstood by the courts and in science materials provided to UK school students. We argue that moot-centred engagement activities offer great potential for science communication among post-16 students and, despite the limitations of the judicial process for addressing complex socio-scientific issues, such role plays aid development of scientific and sociolegal understanding, as well as enhancing students’ self-confidence and argumentation skills. [ABSTRACT FROM AUTHOR]

Cloning technology has been used successfully in the past fifteen years for over twenty different species to produce embryonic stem cells or for reproductive purposes. The application of this technology for human cloning, however, has been a scientific challenge until very recently. In May of 2013, a seminal paper was published in the journal Cell that described innovative cloning technology used to create human embryonic stem cell lines from healthy individuals and from patients with genetic diseases (Tachibana et al. 2013). These scientists were not interested in using this technology for human reproduction to clone people. Nonetheless, this technology is transforming theoretical human reproductive cloning into a potential reality. The possibilities enabled by this recent breakthrough in cloning technology raises significant and challenging halakhic issues. I shall discuss the scientific basis of this technology and raise halakhic questions that emerge from this technology that our rabbinic authorities will have to adjudicate. [ABSTRACT FROM AUTHOR]

This article discusses writer C.J. Cherryh's novels on creation and portrayal of genetically-engineered human clones called Azi, and states that azi cannot survive in future as a new humanity as they lose their sense of creativity owing to tampering of their Autonomic Nervous System responses.

In the present paper I discuss the fantasy of human cloning and the clone as a myth of the "culture of the copy" society. I follow the idea that science fiction is, from an ethical point of view, the literary genre most concerned with shaping possible, often unsettling worlds and contexts, while also debating the main differences between the concept of "clone" and the unsettling category of the "double" in the realm of literature. [ABSTRACT FROM AUTHOR]

Abstract: In addition to orchid mycorrhizal fungi (OrMF), the roots of orchids harbour plant fungal endophytes termed root-associated fungi (RAF). In the present study, the endangered photosynthetic orchid Pseudorchis albida was screened for OrMF and RAF using culture-dependent (isolations from root sections and pelotons) and culture-independent (cloning from root sections) techniques. The efficiency of the different approaches for detecting the fungi and the effect of the sampling season (summer or autumn) were evaluated. In total, 66 distinct OTUs of mycorrhizal and non-mycorrhizal fungi were found, which, to our knowledge, is the highest diversity of RAF that has yet been detected in a single orchid species. The OrMF community was dominated by Tulasnella species, which were mainly detected by isolation from pelotons or cloning from root sections. The roots and tubers showed higher mycorrhizal colonization in summer, corroborating the frequent reports of Tulasnella from pelotons in this season. In contrast, two helotialean fungi, Varicosporium elodeae and Leohumicola sp., the latter of which was repeatedly isolated from pelotons, were significantly more abundant in the autumn. [Copyright &y& Elsevier]

The article considers the ethical aspects of human cloning. It presents the most important arguments against human cloning which can be found in Kazuo Ishiguro's novel Never Let Me Go. Author of the article examines the issue of human cloning as presented in the arguments from instrumentalisation and the internal life of clones and from limiting the freedom of the individual and making him/her unhappy. Then author considers an argument that appeals to the right of a person to be a unique individual, and an argument that appeals to the idea that a person has the right to an open future. Later, the author tries to analyze other arguments such as: psychological distress and the desintegration of personal identity. The author also considers the argument from the natural right to family relationships, and reformulates argument from the wisdom of repugnance. In her interpretation this argument becomes an argument from the wisdom of compassion. [ABSTRACT FROM AUTHOR]

This study has a twofold objective: firstly, it aims to examine the main types of argument that have been formulated against human cloning, to identify their presuppositions and to evaluate their strength; secondly, it aims to argue that the most important objections against human cloning are philosophical and religious, in particular the objection that human cloning represents a radical form of disenchantment or an abuse of rationality. The birth of a cloned mammal, a sheep named Dolly, which was announced on 23 February 23 1997, began a wide public debate on the morality of human cloning. I shall try to show that this debate should not be limited only to ethical and legal considerations, since it has more profound ramifications. The science of cloning is rooted in a certain metaphysical background, which, discreetly, but firmly, accompanies its spectacular promises, and which has profound consequences on our metaphysical and religious beliefs. This background needs to be openly discussed and evaluated. Moreover, it warrants the special attention of philosophers and religious thinkers alike. [ABSTRACT FROM AUTHOR]

Many Christian scholars, if not all of them, consider Genesis to be foundational texts of the Bible and the spring for all the other doctrines of the Scripture. Therefore, I'm considering the attempt to search and find arguments for cell therapy ethical issues in the fundamental text of Genesis as a challenging and educative task. Moreover, this could be the first step in analyzing the relationships between Christian religions and bioethics, in terms of finding reasonable decisions for ethical challenges, raised by the current biomedical research. As for many other dilemmas of humanity, we have to recall the text of Genesis for analyzing the goodness or evilness of our actions in translational medicine, even though that is not the only way to get a reasonable ethical decision. My contribution is an essay that is trying to correlate the Genesis lessons with the needed arguments in deciding what could be good and what could be evil in the stem cell research, according to the religious convictions. The biggest challenges of biomedical research for Christian religions were due to the human cloning issue, made possible by the somatic cell nuclear transfer, but those challenges update the older debates on birth control pill, technologically assisted reproduction, or gene therapy. Issues related to in vitro fertilization, gene enhancement and gene therapy, human cell cloning, embryonic stem cell using, and chimera cell obtaining for research are being considered and related to the putative arguments extracted from the book of Genesis, describing the origins. As a matter of fact, I may conclude that the single way to reach a reasonable ethical decision in our society is to intersect ethics, science and theology and to engage large debates involving scientists, theologians, civil society representatives, ethicists (experts in applied ethics) and moral philosophers, having the two latest professionals as referees. [ABSTRACT FROM AUTHOR]

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TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Leftyl and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs. [ABSTRACT FROM AUTHOR]

The announcement of the birth of Dolly raised many ethical questions about cloning and its implications. This genetic technology raised questions in all scientific dimensions, that come out either in favour or against the various applications of cloning. Aim: The aims of this study were to present the moral and theological questions that arise from the application of cloning in humans, and to analyze the Greek legislation regarding reproductive and therapeutic cloning. Material and Method: A literature review, based on both review and research literature, was conducted covering the period 1952-2010, derived from the MEDLINE, SCOPUS and ???????? databases, using as key words: Reproductive cloning, Bioethical, Therapeutic cloning, Legislation, Orthodox Theology. Results: There is considerable ethical debate about whether or not human cloning should be used. The main argument against human reproductive cloning is that it is an offence to human dignity. On the other hand, those in favour of cloning strongly believe that it would be the most effective method of assisted reproduction in cases where no other method can be used. In contrast to human reproductive cloning, which is rejected by the majority of scientists, therapeutic cloning is expected to give new momentum to the treatment of a variety of diseases. Firstly, the use of stem cells will help to treat conditions such as the degenerative diseases of the nervous system. Subsequently, at a later stage, given its complexity and following long-term laboratory research, cloning could help to produce tissues or complete organs for transplantation. The Orthodox religion is totally opposed to cloning, claiming that experiments on embryos constitute an insult on the sanctity of human life. The Greek legislation prohibits reproductive cloning, but it considers the application of therapeutic cloning to be legal. Conclusions: Reproductive cloning is considered morally and scientifically unacceptable, because it is believed to offend the human dignity and to disturb the genetic diversity of humans and of nature as a whole. Further research is needed on therapeutic cloning using germinal cells, which could be particularly important in the area of transplantation medicine. [ABSTRACT FROM AUTHOR]

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, pro- grammed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies, their viability is significantly reduced. Here, we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actinmyosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. [ABSTRACT FROM AUTHOR]

The article focuses on the issues involving human reproductive cloning using induced pluripotent stem cells (iPSCs) and tetraploid complementation, and the associated legislation and professional guidelines. It discusses the debate over human reproductive cloning, asserting that individuals who cannot have genetically related children should have the liberty to choose cloning. The authors propose that policies on human reproductive cloning should be reviewed to ban other methods of cloning.

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in KrUppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of KIf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal. [ABSTRACT FROM AUTHOR]

Abstract: WNT and bone morphogenetic protein (BMP) signaling are known to stimulate hemogenesis from pluripotent embryonic stem (ES) cells. However, osteochondrogenic mesoderm was generated effectively when BMP signaling is kept to a low level, while WNT signaling was strongly activated. When mesoderm specification from ES cells was exogenous factor dependent, WNT3a addition supported the generation of cardiomyogenic cells expressing lateral plate/extraembryonic mesoderm genes, and this process involved endogenous BMP activities. Exogenous BMP4 showed a similar effect that depended on endogenous WNT activities. However, neither factor induced robust chondrogenic activity. In support, ES cell differentiation in the presence of either WNT3a or BMP4 was associated with elevated levels of both Bmp and Wnt mRNAs, which appeared to provide sufficient levels of active BMPs and WNTs to promote the nonchondrogenic mesoderm specification. The osteochondrogenic mesoderm expressed PDGFRα, which also expressed genes that mark somite and rostral presomitic mesoderm. A strong WNT signaling was required for generating the mesodermal progeny, while approximately 50- to 100-fold lower concentration of WNT3a was sufficient for specifying axial mes(end)oderm. Thus, depending on the dose and cofactor (BMP), WNT signaling stimulates the generation of different biological activities and specification of different types of mesodermal progeny from ES cells. [Copyright &y& Elsevier]

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases. [ABSTRACT FROM AUTHOR]

Abstract: The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces, which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable, continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks, thus opening the prospect of propagation in controlled bioreactors. [Copyright &y& Elsevier]

Abstract: In mice, during early embryonic development UTF1 (undifferentiated embryonic cell transcription factor 1) is expressed in the inner cell mass of blastocysts and in adult animals expression is restricted to the gonads. (Embryonic) Cells expressing UTF1 are generally considered pluripotent, meaning they can differentiate into all cell types of the adult body. In mouse it was shown that UTF1 is tightly associated with chromatin and that it is required for proper differentiation of embryonic carcinoma and embryonic stem cells. In this study we functionally characterized the human UTF1 protein. We show with localization, subnuclear fractionation, and strip-FRAP analyses that human UTF1 is a tightly DNA-associated protein with transcriptional repressor activity. Our data identify human UTF1 as a pluripotency-associated chromatin component with core histone-like characteristics. [Copyright &y& Elsevier]

Abstract: Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications. [Copyright &y& Elsevier]

The article explains the processes and components involved in human cloning. It describes the three types of cloning technologies, recombinant deoxyribonucleic acid (DNA) technology, reproductive cloning, and therapeutic cloning. The uses of these technologies include learning about related methods such as gene therapy, repopulating endangered animals, and producing whole organs from certain cells. The process of therapeutic cloning to make organs for transplants is also tackled. The American Medical Association and scientists are against human cloning.

Abstract: Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-μm-diameter colonies give rise to mesoderm, while small 200-μm colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages. [Copyright &y& Elsevier]

Abstract: ESTOOLS, a project funded by the European Commission (FP6), gathers expertise on human embryonic stem cells in 10 countries of the European Research Area. The ESTOOLS outreach program uses Art extensively as the only universal cross-cultural and cross-religion means of communication. The Smile of a Stem Cell photo exhibition, a major component of this program, aims to fill a missing link between public dissemination of science and science-illiterate citizens. Scientists are also engaged to stand at a distance from their work and observe it with an outsider’s perspective, which enhances their competency to communicate science. The photo exhibition, by its situation upstream of scientific education, makes itself open to interest and enthusiasm among a public with no prerequired scientific knowledge or abilities. [Copyright &y& Elsevier]

Abstract: Generating surrogate insulin-producing cells from embryonic stem cells (ESCs) through in vitro replication of successive steps during pancreatic development has been challenging . Here we describe a novel reproducible protocol to establish homogeneous and scalable insulin-producing cell lines from mouse (m) ESCs via differentiation of the previously described lineage-restricted clonal mESC-derived E-RoSH cells. Unlike their parental mESCs, E-RoSH cells expressed high levels of mesodermal and endodermal genes. Nutrient depletion in the presence of nicotinamide inhibited proliferation of E-RoSH cells and induced differentiation into heterogeneous cultures comprising vascular-like structures that produced detectable levels of insulin and C-peptide in an equimolar ratio. Limiting dilution of these cultures resulted in the isolation of eight independent insulin-producing cell lines in five experiments. All these lines were cloned and shown to be amenable to repeated cycles of freeze and thaw and to replicate for months with a doubling time of 3–4 days. Under such conditions, the cultured cells exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic β cells, including storage of an equimolar ratio of insulin and C-peptide in granules and release of the contents of these organelles through a glucose-sensitive machinery. After transplantation, these cells reversed hyperglycemia in streptozotocin-treated SCID mice and did not form teratomas. [Copyright &y& Elsevier]

Abstract: We have previously described the derivation of insulin-producing cell lines from mouse embryonic stem cells (mESCs) by differentiation of an intermediate lineage-restricted E-RoSH cell line through nutrient depletion in the presence of nicotinamide followed by limiting dilution. Here we investigated whether insulin-producing cell lines could be similarly derived directly from mouse embryo cells or tissues. Using a similar approach, we generated the RoSH2.K and MEPI-1 to -14 insulin-producing cell lines from the 5.5-dpc embryo-derived E-RoSH-analogous RoSH2 cell line and a 6.0-dpc mouse embryo culture, respectively. Insulin content was ∼8 μg/106 MEPI-1 cells and 0.5 μg/106 RoSH2.K cells. Like insulin-producing mESC-derived ERoSHK cell lines, both MEPI and RoSH2.K lines were amenable to repeated cycles of freeze and thaw, replicated for months with a doubling time of 3–4 days, and exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic β-cells, including storage and release of insulin and C-peptide in an equimolar ratio. Transplantation of these cells also reversed hyperglycemia in streptozotocin-treated SCID mice and did not induce teratoma. Like ERoSHK cells, both RoSH2.K and MEPI-1 cells also induced hypoglycemia in the mice. Therefore, our protocol is robust and could reproducibly generate insulin-producing cell lines from different embryonic cell sources. [Copyright &y& Elsevier]

The article focuses on the issue of human cloning. It describes the processes involved in therapeutic human cloning and stresses on the need of comprehensive human cloning legislation. It mentions various international and national laws regulating human cloning and discusses the future of legislation related to human cloning in the U.S.

Abstract: Proteomic profiling of human embryonic stem cells (hESC) can identify cell fate determination and self-renewal biomarkers. Employing Fourier transform LC-ESI-MS/MS and MS3 mass spectrometry, we obtained a membrane proteomic signature overlapping between hESC cultured on mouse embryonic fibroblast (MEF) feeders and those grown under MEF-free culture conditions. We identified 444 transmembrane or membrane-associated proteins, of which 157 were common between both culture conditions. Functional annotation revealed CD antigens (10%), adhesion proteins (4%), proliferation-associated proteins (4%), receptors (41%), transport proteins (21%), structural proteins (5%), and proteins with miscellaneous functions (15%). In addition, 15 CD antigens and a number of surface marker molecules not previously observed in hESC at a proteome level, e.g., Nodal modulator 1, CD222, transgelin-2, and CD81, were identified. In conclusion, we describe the first membrane proteome profile of hESC that is independent of culture conditions. These data can be used to define the phenotype of hESC. [Copyright &y& Elsevier]

Abstract: The derivation of human embryonic stem cells (hESCs) brought cell therapy-based regenerative medicine significantly closer to clinical application. However, expansion of undifferentiated cells and their directed differentiation in vitro have proven difficult to control. This is mainly because of a lack of knowledge of the intracellular signaling events that direct these complex processes. Additionally, extracellular factors, either secreted by feeder cells that support self-renewal and maintain pluripotency or present in serum supplementing proprietary culture media, that influence hESC behavior are largely unknown. Xeno-free media that effectively support long-term hESC self-renewal and differentiation to specific types of specialized cells are only slowly becoming available. Microarray-based transcriptome analyses have produced valuable gene expression profiles of hESCs and indicated changes in transcription that occur during differentiation. However, proteins are the actual effectors of these events and changes in their levels do not always match changes in their corresponding mRNA. Furthermore, information on posttranslational modifications that influence the activity of pivotal proteins is still largely missing. Over the years, mass spectrometry has experienced major breakthroughs in high-throughput identification of proteins and posttranslational modifications in cells under different conditions. Mass spectrometry-based proteomic techniques are being applied with increasing frequency to analyze hESCs, as well as media conditioned by feeder cells, and have generated proteome profiles that not only support, but also complement, existing microarray data. In this review, the various proteomic studies on hESCs and feeder cells are discussed. In a meta-analysis, comparison of published data sets distinguished 32 intracellular proteins and 16 plasma membrane proteins that are present in multiple hESC lines but not in differentiated cells, which were therefore likely to include proteins important for hESCs. In addition, 13 and 24 proteins, respectively, were commonly found in different feeder cell lines of mouse and human origin, some of which may be extracellular signaling molecules that play a key role in the undifferentiated propagation of hESCs. These findings underscore the power of mass spectrometry-based techniques to identify novel proteins associated with hESCs by studying these cells in an unbiased, discovery-oriented manner on a proteome-wide scale. [Copyright &y& Elsevier]

Hematopoietic stem cells derived from human embryonic stem cells (hESCs) could provide a therapeutic alternative to bone marrow transplants, but the efficiency of currently available derivation protocols is low. In this study, we investigated whether coculture with monolayers of cells derived from mouse AGM and fetal liver, or with stromal cell lines derived from these tissues, can enhance hESC hematopoietic differentiation. We found that under such conditions hESC-derived differentiating cells formed early hematopoietic progenitors, with a peak at day 18-21 of differentiation that corresponded to the highest CD34 expression. These hESC-derived hematopoietic cells were capable of primary and secondary hematopoietic engraftment into immunocompromised mice at substantially higher levels than described previously. Transcriptional and functional analysis identified TGF-β1 and TGF-β3 as positive enhancers of hESC hematopoietic differentiation that can further stimulate this process when added to the culture. Overall, our findings represent significant progress toward the goal of deriving functional hematopoietic stem cells from hESCs. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) are considered one promising new approach to generate a transplantable cell source for the treatment of liver diseases. Because traditional methods, such as the initial formation of embryoid body in the presence of serum result in all three germ layer derivatives, strategies have been utilized that favor cell-specific differentiation to generate more uniformity. Here, we have presented the use of a multistep protocol with growth factors in a serum-free adherent culture configuration to mediate the hepatocyte differentiation of human ESCs (hESCs). The differentiated cells exhibited characteristic hepatocyte morphology, ultrastructure, and expressed hepatic-related genes as shown by reverse transcription-polymerase chain reaction and displayed antibody detectable expression of markers specific for hepatic maturation. These hepatocyte-like cells also demonstrated evidence of albumin and α-fetoprotein secretion, glycogen storage, urea production, uptake of low-density lipoprotein, and indocyanine green. Therefore, we propose that the hepatocyte-like cells derived from hESCs by the present method may provide a useful model for the studies of key events during early liver development and a potential source of drug screening and transplantable cells for cell-replacement therapies. [ABSTRACT FROM AUTHOR]

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process.Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers. [ABSTRACT FROM AUTHOR]

Background & Aims: Krüppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. Methods: Heterozygous Klf5 knockout (Klf5+/− ) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5+/− mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt. Results: Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5+/− mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5+/− animals as compared with WT littermates. Conclusion: Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection. [Copyright &y& Elsevier]

Accessibility of human oocytes for research poses a serious ethical challenge to society. This fact categorically holds true when pursuing some of the most promising areas of research, such as somatic cell nuclear transfer and embryonic stem cell studies. One approach to overcoming this limitation is to use an oocyte from one species and a somatic cell from another. Recently, several attempts to capture the promises of this approach have met with varying success, ranging from establishing human embryonic stem cells to obtaining live offspring in animals. This review focuses on the challenges and opportunities presented by the formidable task of overcoming biological differences among species. [ABSTRACT FROM AUTHOR]

The article presents a comment on the generation of six clinical-grade human embryonic stem cell (hESCs) lines. The edict for producing clinically compliant hESCs requires adherence to global ethical standards for egg procurement and embryo donation, conformity to regulations controlling clinical-grade cell and tissue product development, and compliance with current good tissue. Six clinical-grade hESC lines were generated with the adherence to regulations.

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is highly variable. So, developing generic differentiation protocols and their empirical testing on a range of independently derived hESC lines pose a daunting challenge due to considerable diversity in culture methods practiced between lines. Maintenance of BG01V and ReliCell®hES1 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual passaging. We assessed cardiac differentiation from both the cell lines via embryoid body (EB) formation. Subsequent culture in low fetal bovine serum (5%)-containing medium produced spontaneously contracting EBs, in the presence of bone morphogenetic protein-2 (BMP-2; 25 ng/ml). Derived cardiomyocytes expressed cardiac genes and proteins and responded to functional assays. Further, the activation of the Smad signaling machinery evoked by BMP-2 has been confirmed through inhibitor studies. Therefore, in our hands, the same differentiation conditions functioned in two independently derived hESC lines. Similar studies in other lines may facilitate development of universal protocols. The present data may also provide valuable insights for testing of other factors that might promote cardiomyocyte differentiation in low-serum formulations. [ABSTRACT FROM AUTHOR]

The article presents abstracts of medical research. They include "Enhancing Mammalian Regeneration," "Nuclear Cloning, Embryonic Stem Cells and Cell Therapy: Promise, Problems, Reality," "Chromatin and nuclear re-organisation during differentiation and development," and "Cell-type Specific Genome Organization in Euchromatic & Heterochromatic Neighborhoods."