In Vivo Tumorigenicity of the 20q11.21 Amplicon in an Engraftment Model of hPSCs and Differentiated Liver Cells.

Human pluripotent stem cells (hPSCs) are a promising source of somatic cells for clinical applications and disease modelling. However, during culture they accumulate genetic aberrations such as amplification of 20q11.21 which occurs in approximately 20% of extensively cultured hPSC lines and confers a BCL2L1-mediated survival advantage. During the production of the large number of cells required for transplantation and therapy these aberrations may become unavoidable which has important safety implications for therapies and may also impact upon disease modelling. Presently, these risks are poorly understood; whilst it is apparent that large-scale genetic aberrations can pose an oncogenic risk, the risks associated with smaller, more insidious changes have not been fully explored. In this report, the effects of engraftment of human embryonic stem cells (hESCs) and hESC-derived hepatocyte-like cells (HLCs) with and without amplification of the 20q11.21 minimal amplicon and isochromosome 20q (i20q) in SCID-beige mice are presented. The cells were tracked in vivo using a luminescent reporter over a period of approximately four months. Intrasplenic injection of hESCs showed greater engraftment potential and the formation of more severely disruptive lesions in the liver and spleen of animals injected with cells containing 20q11.21 compared with i20q and wild type. HLCs with 20q11.21 engrafted more successfully and formed more severely disruptive lesions than wild type cells or cells with i20q. These results reinforce the notion that karyotyping of therapeutic hPSC is required for transplant, and suggest that screening for known common aberrations is necessary. Further work to identify commonly arising genetic aberrations should be performed and routine screening for hPSCs intended for therapeutic use should be used.

MAIA, Fc receptor-like 3, supersedes JUNO as IZUMO1 receptor during human fertilization.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor-like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Artificial refuges for wildlife conservation: what is the state of the science?

Artificial refuges are human-made structures that aim to create safe places for animals to breed, hibernate, or take shelter in lieu of natural refuges. Artificial refuges are used across the globe to mitigate the impacts of a variety of threats on wildlife, such as habitat loss and degradation. However, there is little understanding of the science underpinning artificial refuges, and what comprises best practice for artificial refuge design and implementation for wildlife conservation. We address this gap by undertaking a systematic review of the current state of artificial refuge research for the conservation of wildlife. We identified 224 studies of artificial refuges being implemented in the field to conserve wildlife species. The current literature on artificial refuges is dominated by studies of arboreal species, primarily birds and bats. Threatening processes addressed by artificial refuges were biological resource use (26%), invasive or problematic species (20%), and agriculture (15%), yet few studies examined artificial refuges specifically for threatened (Vulnerable, Endangered, or Critically Endangered) species (7%). Studies often reported the characteristics of artificial refuges (i.e. refuge size, construction materials; 87%) and surrounding vegetation (35%), but fewer studies measured the thermal properties of artificial refuges (18%), predator activity (17%), or food availability (3%). Almost all studies measured occupancy of the artificial refuges by target species (98%), and over half measured breeding activity (54%), whereas fewer included more detailed measures of fitness, such as breeding productivity (34%) or animal body condition (4%). Evaluating the benefits and impacts of artificial refuges requires sound experimental design, but only 39% of studies compared artificial refuges to experimental controls, and only 10% of studies used a before-after-control-impact (BACI) design. As a consequence, few studies of artificial refuges can determine their overall effect on individuals or populations. We outline a series of key steps in the design, implementation, and monitoring of artificial refuges that are required to avoid perverse outcomes and maximise the chances of achieving conservation objectives. This review highlights a clear need for increased rigour in studies of artificial refuges if they are to play an important role in wildlife conservation.

GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells.

BACKGROUND: A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. METHODS: We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank. RESULTS: Here, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. CONCLUSIONS: Overall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In-Vitro-Derived Neural Crest.

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.

Phytoestrogen intake and other dietary risk factors for low motile sperm count and poor sperm morphology.

BACKGROUND: Few potentially modifiable risk factors of male infertility have been identified, and while different diets and food groups have been associated with male infertility, evidence linking dietary factors including phytoestrogens and semen quality is limited and contradictory. OBJECTIVES: To study the associations between phytoestrogen intake and other dietary factors and semen quality. MATERIALS AND METHODS: A case-referent study was undertaken of the male partners, of couples attempting conception with unprotected intercourse for 12 months or more without success, recruited from 14 UK assisted reproduction clinics. A total of 1907 participants completed occupational, lifestyle and dietary questionnaires before semen quality (concentration, motility and morphology) were assessed. Food intake was estimated by a 65-item food frequency questionnaire (FFQ) covering the 12 months prior to recruitment. Analyses of dietary risk factors for low motile sperm concentration (MSC: <4.8 × 106 /mL) and poor sperm morphology (PM: <4% normal morphology) used unconditional logistic regression, accounting for clustering of subjects within the clinics, first without, and then with, adjustment for confounders associated with that outcome. RESULTS: High consumption of daidzein (≥13.74 µg/d), a phytoestrogen found in soy products, was a protective factor for MSC with an odds ratio (95%CI) of 0.58 (0.42-0.82) after adjustment for clustering and potential confounding. Dietary risk factors for PM after similar adjustment showed that drinking whole milk (OR 0.67, 95%CI 0.47-0.96) and eating red meat were protective with an OR 0.67 (0.46-0.99) for eating red meat >3 times/wk. DISCUSSION: In this case-referent study of men attending an infertility clinic for fertility diagnosis, we have identified that low MSC is inversely associated with daidzein intake. In contrast, daidzein intake was not associated with PM but eating red milk and drinking whole milk were protective. CONCLUSIONS: Dietary factors associated with semen quality were identified, suggesting that male fertility might be improved by dietary changes.

Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.

1,3-Dinitrobenze-Induced Genotoxicity Through Altering Nuclear Integrity of Diploid and Polyploidy Germ Cells.

1,3-Dinitrobenzene (mDNB) is a widely used intermediate in commercial products and causes testicular injury. However, genotoxic effects upon low-level exposure are poorly understood. The present study evaluated the effects of very low-chronic doses of mDNB on sperm nuclear integrity. Male hamsters were treated with 1.5 mg/kg/d/4 wks (group A), 1.5 mg/kg/mDNB/d/week/4 weeks (group B), 1.0 mg/kg/mDNB/3 d/wk/4 wks (group C), or polyethylene glycol 600 (control). Nuclear integrity of distal cauda epididymal sperm was determined using the sperm chromatin structure assay and acridine orange staining (AOS). The germ cell nuclear integrity was assessed by the comet assay. Testicular histopathology was conducted to evaluate the sensitive stages. The comet assay revealed denatured nuclear DNA in group A (in diploid and polyploid cells from weeks 2-5); respectively at week 4 and weeks 3 to 4 in groups B and C. According to AOS, only group A animals exhibited denatured sperm DNA (weeks 1 and 3). The effective sperm count declined from weeks 1 to 6. Mean sperm DNA denaturation extent, percentage cells outside the main population, and standard deviation indicated altered sperm nuclear integrity in group A. Same animals exhibited progressive disruption of the Sertoli cells, while groups B and C exhibited damages on germ cells. The results suggest that mDNB affects sperm nuclear integrity at very low chronic doses targeting cell-specific testicular damage.

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.

Animal movements in fire-prone landscapes.

Movement is a trait of fundamental importance in ecosystems subject to frequent disturbances, such as fire-prone ecosystems. Despite this, the role of movement in facilitating responses to fire has received little attention. Herein, we consider how animal movement interacts with fire history to shape species distributions. We consider how fire affects movement between habitat patches of differing fire histories that occur across a range of spatial and temporal scales, from daily foraging bouts to infrequent dispersal events, and annual migrations. We review animal movements in response to the immediate and abrupt impacts of fire, and the longer-term successional changes that fires set in train. We discuss how the novel threats of altered fire regimes, landscape fragmentation, and invasive species result in suboptimal movements that drive populations downwards. We then outline the types of data needed to study animal movements in relation to fire and novel threats, to hasten the integration of movement ecology and fire ecology. We conclude by outlining a research agenda for the integration of movement ecology and fire ecology by identifying key research questions that emerge from our synthesis of animal movements in fire-prone ecosystems.

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report.

BACKGROUND: Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. OBJECTIVE: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. MATERIALS AND METHODS: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. RESULTS: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. CONCLUSION: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.

Preservation and stability of cell therapy products: recommendations from an expert workshop.

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.

Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine.

Comparability: manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14-15 September 2015.

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this 'may be difficult for cell-based medicinal products'. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

Mucin-Inspired Thermoresponsive Synthetic Hydrogels Induce Stasis in Human Pluripotent Stem Cells and Human Embryos.

Human pluripotent stem cells (hPSCs; both embryonic and induced pluripotent) rapidly proliferate in adherent culture to maintain their undifferentiated state. However, for mammals exhibiting delayed gestation (diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent under these conditions. Here we report cellular stasis for both hPSC colonies and preimplantation embryos immersed in a wholly synthetic thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) [PGMA55-PHPMA135] diblock copolymer worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained PSC viability and pluripotency in the quiescent G0 state without passaging for at least 14 days. Similarly, gel-coated human embryos remain in a state of suspended animation (diapause) for up to 8 days. The discovery of a cryptic cell arrest mechanism for both hPSCs and embryos suggests an important connection between the cellular mechanisms that evoke embryonic diapause and pluripotency. Moreover, such synthetic worm gels offer considerable utility for the short-term (weeks) storage of either pluripotent stem cells or human embryos without cryopreservation.

Expression of Syncytin 1 (HERV-W), in the preimplantation human blastocyst, embryonic stem cells and trophoblast cells derived in vitro.

STUDY QUESTION: As Syncytin 1 (human endogenous retrovirus (HERV-W)) is crucial for human embryo placentation is it expressed during preimplantation embryo development? SUMMARY ANSWER: Syncytin 1 was expressed mainly in trophoblast cells of the blastocyst particularly in cells underlying the inner cell mass (ICM). WHAT IS KNOWN ALREADY: Syncytin 1 (along with HERV-FRD or Syncytin 2) is expressed in first-trimester placenta and required for cell-cell fusion to enable formation of syncytiotrophoblast and effective placentation. STUDY DESIGN, SIZE AND DURATION: Preimplantation human embryos donated for research were cultured in vitro and protein expression of Syncytin 1 at the blastocyst stage of development investigated. Comparisons were made with protein (Syncytin 1) and mRNA (Syncytin 1 and 2) expression in human embryonic stem cells (hESCs) undergoing differentiation to trophoblast-like cells in vitro. In total, 10 blastocysts (×3 or 4 replicates) were analysed and 4 hESC lines. The study was terminated after consistent observations of embryos were made. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated embryos were thawed and cultured to blastocyst, fixed with 4% w/v paraformaldehyde. Syncytin 1 protein expression was determined by immunofluorescent localisation and confocal microscopy. Additionally, hESCs were differentiated to trophoblast-like cells in standard and conditioned culture medium with growth factors (bone morphogenetic protein 4 (BMP4) or fibroblast growth factor 4 (FGF4) and assessed for mRNA (Syncytin 1 and 2) by quantitative polymerase chain reaction (qPCR) and protein expression by immunolocalization and western blot. MAIN RESULTS AND ROLE OF CHANCE: Syncytin 1 was expressed in cytoplasm and on the cell surface of some trophoblast cells, and consistently the trophectoderm underlying the ICM of the blastocyst. There was weak but consistent expression of Syncytin 1 in cells on the periphery of the ICM also displaying pluripotency antibody marker (Tra-1-60). Three-dimensional reconstruction of confocal slice data provided good visualization of expression. The time course of expression of Syncytin 1 was replicated in hESCs differentiated in vitro confirming the embryo observations and providing statistically significant differences in protein and mRNA level (P= 0.002) and (P< 0.05), respectively. LIMITATION, REASONS FOR CAUTION: Culture of a limited number of embryos to blastocyst in vitro may not replicate the range and quality of development in situ. Probes (antibodies, PCR) were tested for specificity, but might have non-specific reactions. WIDER IMPLICATIONS OF FINDINGS: Syncytin expression is a prerequisite for embryo implantation and placentation. Understanding when expression first occurs during embryo development may be informative for understanding conditions of abnormal gestations such as pre-clampsia. STUDY FUNDING/COMPETING INTERESTS: The study was supported partly by an ERASMUS training grant and grant G0801059 from the Medical Research Council, U.K. There were no competing interests.

Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples.

Stopping the past from intruding the present: Social anxiety disorder and proactive interference.

It has recently been suggested that social anxiety disorder (SAD) entails a deficit in downregulating unwanted (even non-threatening) memories. In the present study we test this hypothesis by comparing a sample of young adults diagnosed with SAD and healthy controls in their ability to resist proactive interference in a working memory task. Where participants performed similarly in the control condition of the memory task, participants with SAD were more susceptible to interference in the experimental condition than the healthy controls. This finding is in line with previous studies that show anxiety to be associated with impoverished executive control and, specifically, suggests that SAD entails a reduced ability to get rid of interfering memories. Clinical implications are discussed.

Pulmonary Embolism Following Outpatient Vasectomy.

Venous thromboembolic events have several known major risk factors such as prolonged immobilization or major surgery. Pulmonary embolism has rarely been reported after an outpatient vasectomy was completed. We present the rare case of a healthy 32-year-old Caucasian male with no known risk factors who presented with pleuritic chest pain 26 days after his outpatient vasectomy was performed. Subsequently, he was found to have a pulmonary embolism as per radiological imaging. We explore the association between outpatient vasectomies and venous thromboembolic events. A review of the literature is also included.