Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase

The biomedical application of human embryonic stem (hES) cells will increasingly depend on the availability of technologies for highly controlled genetic modification. In mouse genetics, conditional mutagenesis using site-specific recombinases has become an invaluable tool for gain- and loss-of-function studies. Here we report highly efficient Cre-mediated recombination of a chromosomally integrated loxP-modified allele in hES cells and hES cell-derived neural precursors by protein transduction. Recombinant modified Cre recombinase protein translocates into the cytoplasm and nucleus of hES cells and subsequently induces recombination in virtually 100% of the cells. Cre-transduced hES cells maintain the expression of pluripotency markers as well as the capability of differentiating into derivatives of all three germ layers in vitro and in vivo. We expect this technology to provide an important technical basis for analyzing complex genetic networks underlying human development as well as generating highly purified, transplantable hES cell-derived cells for regenerative medicine.     

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.     

Ethics and synthetic gametes

The recent in vitro derivation of gamete-like cells from mouse embryonic stem (mES) cells is a major breakthrough and lays down several challenges, both for the further scientific investigation and for the bioethical and biolegal discourse. We refer here to these cells as gamete-like (sperm-like or oocyte-like, respectively), because at present there is still no evidence that these cells behave fully like bona fide sperm or oocytes, lacking the fundamental proof, i.e. combination with a normally derived gamete of the opposite sex to yield a normal individual. However, the results published so far do show that these cells share some defining features of gametes. We discuss these results in the light of the bioethical and legal questions that are likely to arise would the same process become possible with human embryonic stem (hES) cells.     

Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera

Despite a long and valuable history, human-animal chimera research has often been questioned. Among the moral issues raised by chimeras is the concept that integration of human cells into anatomical locations such as the brain might endow animals with "human-like" capacities including self-awareness. We present a justification for one type of human-animal chimera experiment: the evaluation of hES cell developmental potency via teratoma formation in immunodeficient mice. We argue that this experiment raises no significant moral concerns and should be the jurisdiction of animal care and use committees and exempt from formal review by the stem cell research oversight process.     

Neurotrophins mediate human embryonic stem cell survival

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.     

Morphological variation within Acyrthosiphon pisum and in ability to transmit broad bean severe chlorosis virus

The EL and WARF biotypes of Acyrthosiphon pisum (Harris), which were originally isolated on the basis of their differential efficiency in transmitting pea enation mosaic virus (PEMV), were studied morphologically, and a key based on observed morphological differences is presented. The two biotypes also were compared as vectors of broad bean severe chlorosis virus (BBSCV). The EL biotype was more efficient than that of WARF in transmitting BBSCV; 1st-stage nymphs of EL and WARF were more efficient vectors of BBSCV than their counterpart adults.

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Zusammenfassung Die EL und WARF Biotypen von Acyrthosiphon pisum, die ursprünglich nach ihrer Übertragungsfähigkeit für das Scharfe Adernmosaik der Erbse (PEMV) isoliert worden waren, wurden morphologisch studiert und ein Bestimmungsschlüssel ausgearbeitet. Die beiden Biotypen wurden auch als Vektoren des Schweren Chlorosevirus der Ackerbohne (BBSCV) verglichen. Der EL Biotyp war leistungsfähiger in der Übertragung von BBSCV als der WARF Typ. Erstlarven von EL und WARF waren wirksamere Vektoren von BBSCV als die entsprechenden Adulttiere.

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Michigan Agricultural Experiment Station Journal Article Number 7540.     

Discovering the Neural Nature of Moral Cognition? Empirical,Theoretical, and Practical Challenges in Bioethical Research with Electroencephalography (EEG)

In this article we critically review the neural mechanisms of moral cognition that have recently been studied via electroencephalography (EEG). Such studies promise to shed new light on traditional moral questions by helping us to understand how effective moral cognition is embodied in the brain. It has been argued that conflicting normative ethical theories require different cognitive features and can, accordingly, in a broadly conceived naturalistic attempt, be associated with different brain processes that are rooted in different brain networks and regions. This potentially morally relevant brain activity has been empirically investigated through EEG-based studies on moral cognition. From neuroscientific evidence gathered in these studies, a variety of normative conclusions have been drawn and bioethical applications have been suggested. We discuss methodological and theoretical merits and demerits of the attempt to use EEG techniques in a morally significant way, point to legal challenges and policy implications, indicate the potential to reveal biomarkers of psychopathological conditions, and consider issues that might inform future bioethical work.     

Comparative characteristics of three human embryonic stem cell lines

Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.     

International stem cell registries

Rapid advances in stem cell research have led to the derivation of hundreds of human embryonic stem (hES) cell lines in centers throughout the world, as well as the development of new technologies for producing pluripotent stem cells. These cell lines have unique characteristics and were derived using a variety of ethical guidelines. Stem cell registries have been developed in order to collect, organize, and disseminate cell line-specific information. In this review, we describe the current state of the field by providing an overview of the unique qualities and mandates of the three major stem cell registries: the European hES Cell Registry, the Registry of hES Cell Line Provenance developed by the International Society for Stem Cell Research, and the International Stem Cell Registry of hES and induced pluripotent stem cell lines established at the University of Massachusetts Medical School. While each registry has its own unique mandate and features, there is some overlap in the goals and information provided. This review discusses the challenges and prospects for an integrated approach in which all three registries effectively collaborate to minimize duplication and facilitate information exchange within the stem cell community.     

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4, and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers.     

Embryonic stem cells in science and medicine,part II: Law,ethics, and the continuing need for dialogue

Just as our first article, “Embryonic Stem Cells in Science and Medicine: An Invitation for Dialogue,” in the December 2007 issue of Gender Medicine went to press, two groups of researchers had just announced that adult human somatic cells had been reprogrammed to behave like pluripotent stem cells, and that the reprogrammed cells were able to differentiate into cell types of the 3 germ layers in vitro and in a mouse model. A third group has since done so. Because the reprogrammed cells were not embryonic in origin, the announcements were heralded as “stunning” and “leaps forward,” because, it was argued, the ability to generate stem cells, without destroying embryos in the process, would avoid the difficult ethical questions raised by human embryonic stem (hES) cell research. This article addresses the most recent announcements and briefly retraces the relevant history so that we may consider whether the moral, ethical, and social issues do in fact disappear as a result of these new advancements. We conclude that, despite the hoopla, little has changed. If indeed there were ethical issues surrounding hES cell research, they remain—and remain as urgent to address and resolve as they had been previously. Lastly, we argue that the medical and scientific communities continue to do themselves a disservice by failing to create a cohesive governing body to address and make concrete recommendations concerning the moral, ethical, and related social issues affecting their communities.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell-derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell-derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell-derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.     

Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines

The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.     

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.     

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho‐associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow‐freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post‐thaw culture, the presence of 10 μM ROCK inhibitor (Y‐27632) and 1 μM pifithrin‐μ together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder‐dependent or feeder‐independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Stem cell medicine encounters the immune system

Recent progress in deriving human embryonic stem (hES) cells and defining their capacity to differentiate has inspired hope that they could become a source of replacement cells for damaged or diseased tissues. We review the immunological barriers to transplanting hES cells and consider several potential solutions, including stem-cell banking, modification of the immunogenicity of donor cells and induction of tolerance to the graft. We evaluate the probable efficacy of these approaches with a view to facilitating the use of hES cells in clinical practice.