Treatment of an Ectopic Pregnancy: An Ethical Reanalysis.

There is considerable lack of clarity on the medical facts surrounding management of ectopic pregnancy. In particular, it is not widely appreciated that by the time an ectopic pregnancy is diagnosed, in most cases, there is no viable fetus (i.e., the fetus has already died). Moreover, there is very little ethical guidance from the medical profession regarding the emotionally difficult decision to terminate a wanted pregnancy when the life of the mother is at risk. The best articulated positions on this topic come from religious groups, based on the principle of double effect. Yet the application of this reasoning to termination of an ectopic pregnancy is inconsistent with the medical facts in many cases. To resolve these inconsistencies, while still providing a robust ethical context for resolving such difficult situations, we propose clear guidelines for determining when a viable fetus is present in ectopic pregnancy and clarify the moral object in ectopic pregnancy management. Summary: This paper explores the ethical framework for clinical decision making in the case of ectopic pregnancies. Focusing on the disordered union of mother and unborn child clarifies the object and purpose of the actions used to separate the mother and fetus in order to save the life of both, or at least one. Since over 90% of tubal ectopic pregnancies present as embryos who have already died, these cases present no ethical dilemma. This paper proposes a modification of currently used criteria for determining the viability of ectopic pregnancies and calls for further research.

Determination of Death: A Scientific Perspective on Biological Integration.

Human life is operationally defined by the onset and cessation of organismal function. At postnatal stages of life, organismal integration critically and uniquely requires a functioning brain. In this article, a distinction is drawn between integrated and coordinated biologic activities. While communication between cells can provide a coordinated biologic response to specific signals, it does not support the integrated function that is characteristic of a living human being. Determining the loss of integrated function can be complicated by medical interventions (i.e., "life support") that uncouple elements of the natural biologic hierarchy underlying our intuitive understanding of death. Such medical interventions can allow living human beings who are no longer able to function in an integrated manner to be maintained in a living state. In contrast, medical intervention can also allow the cells and tissues of an individual who has died to be maintained in a living state. To distinguish between a living human being and living human cells, two criteria are proposed: either the persistence of any form of brain function or the persistence of autonomous integration of vital functions. Either of these criteria is sufficient to determine a human being is alive.

Symposium on the Definition of Death: Summary Statement.

This statement summarizes the conclusions of the Symposium on the Definition of Death, held at The Catholic University of America in June 2014. After providing the background and context for contemporary debates about brain death and describing the aims of the symposium, the statement notes points of unanimous and broad agreement among the participants, and highlights areas for further study.

2-O-sulfotransferase regulates Wnt signaling, cell adhesion and cell cycle during zebrafish epiboly.

O-sulfotransferases modify heparan sulfate proteoglycans (HSPGs) by catalyzing the transfer of a sulfate to a specific position on heparan sulfate glycosaminoglycan (GAG) chains. Although the roles of specific HSPG modifications have been described in cell culture and invertebrates, little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. Here, we report that 2-O-sulfotransferase (2-OST) is an essential component of canonical Wnt signaling in zebrafish development. 2-OST-deficient embryos have reduced GAG chain sulfation and are refractory to exogenous Wnt8 overexpression. Embryos in which maternally encoded 2-OST is knocked down have normal activation of several zygotic mesoderm, endoderm and ectoderm patterning genes, but have decreased deep cell adhesion and fail to initiate epiboly, which can be rescued by re-expression of 2-OST protein. Reduced cell adhesion and altered cell cycle regulation in 2-OST-deficient embryos are associated with decreased ß-catenin and E-cadherin protein levels at cell junctions, and these defects can be rescued by reactivation of the intracellular Wnt pathway, utilizing stabilized ß-catenin or dominant-negative Gsk3, but not by overexpression of Wnt8 ligand. Together, these results indicate that 2-OST functions within the Wnt pathway, downstream of Wnt ligand signaling and upstream of Gsk3ß and ß-catenin intracellular localization and function.

Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia.

The molecular mechanisms responsible for specifying the dorsal-ventral pattern of neuronal identities in dorsal root ganglia (DRG) are unclear. Here we demonstrate that Sonic hedgehog (Shh) contributes to patterning early DRG cells. In vitro, Shh increases both proliferation and programmed cell death (PCD). Increasing Shh in vivo enhances PCD in dorsal DRG, while inducing greater proliferation ventrally. In such animals, markers characteristic of ventral sensory neurons are expanded to more dorsal positions. Conversely, reducing Shh function results in decreased proliferation of progenitors in the ventral region and decreased expression of the ventral marker trkC. Later arising trkA(+) afferents make significant pathfinding errors in animals with reduced Shh function, suggesting that accurate navigation of later arising growth cones requires either Shh itself or early arising, Shh-dependent afferents. These results indicate that Shh can regulate both cell number and the distribution of cell types in DRG, thereby playing an important role in the specification, patterning and pathfinding of sensory neurons.

Regulatory issues for personalized pluripotent cells.

The development of personalized pluripotent stem cells for research and for possible therapies holds out great hope for patients. However, such cells will face significant technical and regulatory challenges before they can be used as therapeutic reagents. Here we consider two possible sources of personalized pluripotent stem cells: embryonic stem cells derived from nuclear transfer (NT-ESCs) and induced pluripotent stem cells (iPSCs) derived from direct reprogramming of adult somatic cells. Both sources of personalized pluripotent stem cells face unique regulatory hurdles that are in some ways significantly higher than those facing stem cells derived from embryos produced by fertilization (ESCs). However, the outstanding long-term potential of iPSCs and their relative freedom from the ethical concerns raised by both ESCs and NT-ESCs makes direct reprogramming an exceptionally promising approach to advancing research and providing therapies in the field of regenerative medicine.

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Alternative sources of pluripotent stem cells: scientific solutions to an ethical dilemma.

Stem cell researchers in the United States have faced a quagmire of uncertainty due to multiple factors: the ethical divide over the use of embryos for research, the lack of clarity in federal guidelines governing this research, the restrictive patent situation surrounding the generation of new human embryonic stem (HES) cell lines; and the limits on types of research eligible for federal funding. In this commentary, we describe how recent advances in derivation of hES cell-like lines may allow at least some of these uncertainties to be resolved. More importantly, we suggest that the derivation of hES cell-like lines by morally acceptable methods would not only avoid the corrosive effects of a protracted ethical debate over stem cell research, but would also allow U.S. researchers to access federal funds and compete on a more level international playing field.

Adaptation of sensory neurons to hyalectin and decorin proteoglycans.

Proteoglycans are abundantly expressed in the pathways of developing and regenerating neurons, yet the responses of neurons to specific proteoglycans are not well characterized. We have shown previously that one chondroitin sulfate proteoglycan (CSPG), aggrecan, is potently inhibitory to sensory axon extension in short-term assays and that over time, embryonic neurons adapt to aggrecan-mediated inhibition through the transcriptional upregulation of integrin expression (Condic et al., 1999). Here, we have compared the response of embryonic sensory neurons to structurally distinct CSPGs that belong to either the hyalectin (or lectican) family of large, aggregating proteoglycans or the decorin (or small leucine-rich proteoglycan) family of smaller proteoglycans. Both of these structurally diverse proteoglycan families are expressed in developing embryos and inhibit outgrowth of embryonic sensory neurons in short-term cultures. These results document a previously uncharacterized inhibitory function for the decorin-family proteoglycan biglycan. Interestingly, embryonic neurons adapt to these diverse proteoglycans over time. Adaptation is associated with upregulation of select integrin alpha subunits in a proteoglycan-specific manner. Overexpression of specific integrin alpha subunits improves neuronal regeneration on some but not all decorin-family CSPGs, suggesting that neurons adapt to inhibition mediated by closely related proteoglycans using distinct mechanisms. Our findings indicate that CSPGs with diverse core proteins and distinct numbers of chondroitin sulfate substitution sites mediate a similar response in sensory neurons, suggesting that increased integrin expression may be an effective means of promoting axonal regeneration in the presence of diverse inhibitory proteoglycan species in vivo.

The Role of Maternal-Effect Genes in Mammalian Development: Are Mammalian Embryos Really an Exception?

The essential contribution of multiple maternal factors to early mammalian development is rapidly altering the view that mammals have a unique pattern of development compared to other species. Currently, over 60 maternal-effect mutations have been described in mammalian systems, including critical determinants of pluripotency. This data, combined with the evidence for lineage bias and differential gene expression in early blastomeres, strongly suggests that mammalian development is to some extent mosaic from the four-cell stage onward.

Sensory neuron subtypes have unique substratum preference and receptor expression before target innervation.

The factors controlling the specification and subsequent differentiation of sensory neurons are poorly understood. Data from embryological manipulations suggest that either sensory neuron fates are specified by the targets they encounter or sensory neurons are considerably more "plastic" with respect to specification than are neurons of the CNS. The prevailing view that sensory neurons are specified late in development is not consistent, however, with the directed outgrowth of sensory neurons to their targets and the characteristic spatial distribution of sensory neuron fates within the peripheral ganglia. To address when in development different classes of sensory neurons can first be distinguished, we investigated the interactions of early dorsal root ganglia neurons with the extracellular matrix before neurite outgrowth to targets. We found that subclasses of sensory neurons in early dorsal root ganglia show different patterns of neurite outgrowth and integrin expression that are predictive of their fates. In the absence of neurotrophins, presumptive proprioceptive neurons extend neurites robustly on both laminin and fibronectin, whereas presumptive cutaneous neurons show a strong preference for laminin. Cutaneous afferents that have innervated targets show a similar strong preference for laminin and show higher levels of integrin alpha7beta1 than do proprioceptive neurons. Finally, presumptive proprioceptive neurons express fibronectin receptors, integrin alpha3beta1, alpha4beta1, and alpha5beta1, at higher levels than do presumptive cutaneous neurons. Our results indicate that subtypes of sensory neurons have unique patterns of neurite outgrowth and receptor expression before target innervation.

Characterization of Netrin-1, Neogenin and cUNC-5H3 expression during chick dorsal root ganglia development.

The molecular mechanisms that control the axon pathfinding of different subtypes of dorsal root ganglia (DRG) neurons are poorly understood. To address whether Netrin-1-Neogenin/UNC-5 signaling contributes to sensory axon pathfinding, we cloned chick UNC5 homolog 3 (cUNC-5H3) and characterized the spatial, cellular and temporal expression patterns of Netrin-1, Neogenin and cUNC-5H3 in cutaneous and proprioceptive DRG neurons. We have found that Netrin-1 is only expressed by late-arising cutaneous neurons. In contrast, cUNC-5H3 expression is restricted to proprioceptive neurons. Neogenin is expressed in all DRG neurons. Our results indicate that the pathfinding molecules, Netrin-1 and cUNC-5H3 are differentially expressed by early cutaneous and proprioceptive neurons.

Totipotency: what it is and what it is not.

There is surprising confusion surrounding the concept of biological totipotency, both within the scientific community and in society at large. Increasingly, ethical objections to scientific research have both practical and political implications. Ethical controversy surrounding an area of research can have a chilling effect on investors and industry, which in turn slows the development of novel medical therapies. In this context, clarifying precisely what is meant by "totipotency" and how it is experimentally determined will both avoid unnecessary controversy and potentially reduce inappropriate barriers to research. Here, the concept of totipotency is discussed, and the confusions surrounding this term in the scientific and nonscientific literature are considered. A new term, "plenipotent," is proposed to resolve this confusion. The requirement for specific, oocyte-derived cytoplasm as a component of totipotency is outlined. Finally, the implications of twinning for our understanding of totipotency are discussed.

Genome-wide analysis reveals the unique stem cell identity of human amniocytes.

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage.

Alternative sources of pluripotent stem cells: ethical and scientific issues revisited.

Stem cell researchers in the United States continue to face an uncertain future, because of the changing federal guidelines governing this research, the restrictive patent situation surrounding the generation of new human embryonic stem cell lines, and the ethical divide over the use of embryos for research. In this commentary, we describe how recent advances in the derivation of induced pluripotent stem cells and the isolation of germ-line-derived pluripotent stem cells resolve a number of these uncertainties. The availability of patient-matched, pluripotent stem cells that can be obtained by ethically acceptable means provides important advantages for stem cell researchers, by both avoiding protracted ethical debates and giving U.S. researchers full access to federal funding. Thus, ethically uncompromised stem cells, such as those derived by direct reprogramming or from germ-cell precursors, are likely to yield important advances in stem cell research and move the field rapidly toward clinical applications.