Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

Autophagic response to cell culture stress in pluripotent stem cells.

Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function.

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.

Occupation exposures and sperm morphology: a case-referent analysis of a multi-centre study.

OBJECTIVE: We examined occupational exposures and sperm morphology to establish whether exposures implicated differed from those affecting motile sperm concentration. METHODS: Computer aided sperm morphometric assessment was undertaken on morphology slides obtained as part of a multi-centre study in 1999-2002 of occupational factors in male infertility. Men attending 14 fertility clinics across the UK were recruited and gave a semen sample. Before results of the semen analysis were known, the men completed detailed questionnaires about their employment and lifestyle. Occupational exposures were assessed by occupational hygienists. Data were analysed using an unmatched case-referent design, allowing for clustering and for confounders. Three case definitions were used: poor morphology (normal morphology <4%), low motile sperm count (MSC) (<4.8×10(6)) and either condition. RESULTS: Morphology results were available for 1861/2011 men employed at the time of recruitment. Of these 1861, 296 (15.9%) had poor morphology; of the 2011with sperm count, 453 (22.5%) had low MSC; 654/1981 (33.0%) had either condition. Poor morphology, adjusted for confounding, was related to self-reported lifetime exposure to lead (OR=1.33; 95% CI 1.00 to 1.75). Low MSC was also related to self-reported lead and to hygienist-assessed glycol ether exposure. Self-reported use of paint stripper (OR=1.47; 95% CI 1.07 to 2.03) and lead, but not glycol ether, were significantly related to the combined case definition. CONCLUSIONS: While this study did not identify any occupational exposure uniquely related to sperm morphology, the capacity of the study to detect risk was increased by including morphology with sperm concentration and motility.

Age differences in the principal temporo-spatial components of EEG activity during a proactive interference task.

Proactive interference (PI) is the disruptive effect of no longer relevant information on current working memory (WM) processing. PI effects in EEG data have been previously found to be altered in healthy aging, although it remains unclear the extent to which such changes reflect delayed or different brain mechanisms employed to overcome PI. Hence, we had twenty-six young (18-34 years) and sixteen old (53-68 years) healthy adults complete a Recent Probes task while EEG was recorded. Compared to young adults, old adults were slower, less accurate and less able to discriminate when they last saw a given stimulus, but PI effects on reaction time were greater in the former, likely due to a general difficulty that old adults had in the task. Temporo-spatial principal component analysis of the EEG data showed young and older adults to differ in terms of temporal and spatial characteristics of brain activity associated with resolving PI. YA showed a factor indicative of a medial frontal negativity (MFN) that showed greater amplitude in low compared to high PI trials. OA, in contrast, showed a late positive component (LPC), although similarly with larger amplitude in low compared to high PI trials. The modulation of the MFN component in YA may reflect the recruitment of cognitive control to overcome PI. The modulation of the LPC in OA may represent the detection of conflict between familiarity and context recollection during PI.

Variation in mean human sperm length is linked with semen characteristics.

STUDY QUESTION: Are there any links between the length measurements of sperm components (head, midpiece, flagellum, total sperm length and the flagellum:head ratio) and data obtained during semen analysis? SUMMARY ANSWER: Both the mean measurement and the variation in the lengths of sperm components are related to characteristics of semen. WHAT IS KNOWN ALREADY: Studies in non-human species have shown that sperm morphology (size and shape) is associated with testes productivity and the consistency of sperm manufacture. However, no study to date has investigated whether there are relationships between the size and consistency of human sperm components, and measures of semen characteristics, including sperm numbers and how well they swim. STUDY DESIGN, SIZE AND DURATION: A retrospective laboratory study of the semen provided by 103 randomly selected men from a 500-man cohort who enrolled into the study between April and December 2006. PARTICIPANTS AND SETTING: Men attending Sheffield Teaching Hospital NHS Foundation Trust for semen analysis as part of investigations for infertility and whose ejaculates were found to contain sperm. MAIN RESULTS AND THE ROLE OF CHANCE: The mean flagellum length and the mean total sperm length were positively associated with semen characteristics measured manually, but were not associated with the sperm swimming speed measured by computer-aided sperm analysis. Ejaculates with a lower variation in the length of sperm components contained sperm that were more likely to be motile. The mean sperm length components accounted for up to 9% of the variance in semen characteristics, while the coefficient of variation accounted for up to 21%. LIMITATIONS AND REASONS FOR CAUTION: The sperm examined were obtained from men undergoing fertility investigations and so these results may not reflect men in the general population. WIDER IMPLICATIONS OF THE FINDINGS: Sperm length measurements may provide a useful insight into testis function and the efficiency of spermatogenesis. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by funding from the University of Sheffield. The authors declare no conflicts of interest.

A small molecule modulator of prion protein increases human mesenchymal stem cell lifespan, ex vivo expansion, and engraftment to bone marrow in NOD/SCID mice.

Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine.

Chlorination by-products in tap water and semen quality in England and Wales.

OBJECTIVES: Disinfection by-products (DBPs) have been associated with adverse semen outcomes in laboratory animals, although the evidence for trihalomethanes (THMs) is limited. Three small epidemiological studies found little evidence for an association between DBPs and adverse semen outcomes in humans. Using data from a large case-referent study (Chemicals and Pregnancy Study, Chaps-UK), we investigated the association between total THM (TTHM), chloroform and total brominated THMs and sperm concentration, percent motile sperm and motile sperm concentration (MSC). METHODS: Chaps-UK recruited men from 13 fertility clinics in nine urban centres across England and Wales between 1999 and 2002. We linked modelled THM concentrations in water zones to semen quality data for 642 cases (men with low MSC) and 926 referents (other men investigated for infertility), based on the men's residence during semen sampling. We assessed risk of low MSC in relation to DBP exposure using continuous THM concentrations. A secondary analysis investigated continuous outcomes (MSC, sperm concentration and percent motile sperm). RESULTS: In the case-referent analysis there was little evidence of elevated risk associated with chloroform, total brominated THM or TTHM concentration after adjustment (OR per 10 µg/L TTHM 1.01; 95% CI 0.91 to 1.12). Similarly, there was no significant effect of THMs on the continuous outcomes. CONCLUSIONS: In the largest study to date on DBPs in public water supplies, and semen quality we found that concentrations of THMs were not associated with poor semen quality. Large-scale investigation of other DBPs (eg, haloacetic acids) and other semen quality parameters (eg, sperm morphology and/or sperm DNA integrity) is recommended.

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.

ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

The first definitive cell fate decision in development occurs at the blastocyst stage with establishment of the trophoblast and embryonic cell lineages. In the mouse, lineage commitment is achieved by epigenetic regulation of a critical gatekeeper gene, the transcription factor Elf5, that reinforces placental cell fate and is necessary for trophoblast stem (TS) cell self-renewal. In humans, however, the epigenetic lineage boundary seems to be less stringent since human embryonic stem (ES) cells, unlike their murine counterparts, harbour some potential to differentiate into trophoblast derivatives. Here, we show that ELF5 is expressed in the human placenta in villous cytotrophoblast cells but not in post-mitotic syncytiotrophoblast and invasive extravillous cytotrophoblast cells. ELF5 establishes a circuit of mutually interacting transcription factors with CDX2 and EOMES, and the highly proliferative ELF5(+)/CDX2(+) double-positive subset of cytotrophoblast cells demarcates a putative TS cell compartment in the early human placenta. In contrast to placental trophoblast, however, ELF5 is hypermethylated and largely repressed in human ES cells and derived trophoblast cell lines, as well as in induced pluripotent stem cells and murine epiblast stem cells. Thus, these cells exhibit an embryonic lineage-specific epigenetic signature and do not undergo an epigenetic reprogramming to reflect the trophoblast lineage at key loci such as ELF5. Our identification of the trophoblast-specific transcriptional circuit established by ELF5 will be instrumental to derive human TS cell lines that truly reflect early placental trophoblast and that will be most beneficial to gain insights into the aetiology of common pregnancy complications, including intra-uterine growth restriction and pre-eclampsia.

Mitochondrial haplotype does not influence sperm motility in a UK population of men.

BACKGROUND: Sperm motility is regulated by mitochondrial enzymes that are partially encoded by mitochondrial DNA (mtDNA). MtDNA has therefore been suggested as a putative genetic marker of male fertility. However, recent studies in different populations have identified both significant and non-significant associations between mtDNA variation and sperm motility. Here, we tested whether mtDNA variation was associated with sperm motility in a large cohort of men from the UK, to test the robustness of previous studies and the reliability of mtDNA as a marker of poor sperm motility. METHODS: A total of 463 men attending for semen analysis as part of infertility investigations were recruited from a UK laboratory. Sperm motility was measured using both computer-assisted sperm analysis and traditional manual measurements. MtDNA haplogroup and haplotype were determined in 357 and 298 men, respectively, using single nucleotide polymorphism (SNP) markers throughout the mtDNA genome, and compared with sperm motility data. The linkage between the SNP markers, and possible associations between individual SNPs and motility, were also investigated. RESULTS: We found no statistical association between haplogroup or haplotype and sperm motility, regardless of how it was measured (P > 0.05 in all cases). Moreover, individual SNPs which were in linkage disequilibrium and dispersed across the mitochondrial genome, and therefore sensitive to mtDNA variation, were not predictive of sperm motility. CONCLUSIONS: Mitochondrial haplotype is unlikely to be a reliable genetic marker of male factor infertility.

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.

Generation of T cells from human embryonic stem cell-derived hematopoietic zones.

Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34(high)CD43(low) population, further substantiating the notion that hESC-derived CD34(high)CD43(low) cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34(+)CD7(+) T/NK committed, CD7(+)CD4(+)CD8(-) immature single positive, CD4(+)CD8(+) double positive, and finally CD3(+)CD1(-)CD27(+) mature T cell stages. TCRalphabeta(+) and TCRgammadelta(+) T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.

Pinacidil enhances survival of cryopreserved human embryonic stem cells.

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.

Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 microg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

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.

Specific knockdown of OCT4 in human embryonic stem cells by inducible short hairpin RNA interference.

Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen beta2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods. STEM CELLS 2009;27:776-782.

Human fetal auditory stem cells can be expanded in vitro and differentiate into functional auditory neurons and hair cell-like cells.

In the quest to develop the tools necessary for a cell-based therapy for deafness, a critical step is to identify a suitable stem cell population. Moreover, the lack of a self-renovating model system for the study of cell fate determination in the human cochlea has impaired our understanding of the molecular events involved in normal human auditory development. We describe here the identification and isolation of a population of SOX2+OCT4+ human auditory stem cells from 9-week-old to 11-week-old fetal cochleae (hFASCs). These cells underwent long-term expansion in vitro and retained their capacity to differentiate into sensory hair cells and neurons, whose functional and electrophysiological properties closely resembled their in vivo counterparts during development. hFASCs, and the differentiating protocols defined here, could be used to study developing human cochlear neurons and hair cells, as models for drug screening and toxicity and may facilitate the development of cell-based therapies for deafness.