[Show abstract][Hide abstract] ABSTRACT: The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson's, Huntington's, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.
Full-text · Article · Dec 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Sep 2015 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Aims: The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled 111Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS. hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.
Full-text · Article · Oct 2014 · EuroIntervention: journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology
[Show abstract][Hide abstract] ABSTRACT: 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) (<4.8×106) 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.
No preview · Article · Sep 2014 · Occupational and Environmental Medicine
[Show abstract][Hide abstract] ABSTRACT: Common methods for the generation of human embryonic-derived neural stem cells (hNSCs) result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders). Additionally, sufficient expansion of resulting hNSCs for scaling out or up in a clinically relevant time frame has proven to be difficult. Here, we report a strategy that produces hNSCs in completely “Xeno-Free” culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro. Critically, we have also confirmed neural lineage specificity upon sorted hNSC transplantation into the immunodeficient NOD-scid mouse brain. The future use or adaptation of these protocols has the potential to better facilitate the advancement of pre-clinical strategies from the bench to the bedside.
Full-text · Article · Sep 2014 · Stem Cell Research
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION
Are common lifestyle factors associated with poor sperm morphology?
Common lifestyle choices make little contribution to the risk of poor sperm morphology.
WHAT IS KNOWN ALREADY
Although many studies have claimed that men's lifestyle can affect sperm morphology, the evidence is weak with studies often underpowered and poorly controlled.
STUDY DESIGN, SIZE, DURATION
Unmatched case-referent study with 318 cases and 1652 referents. Cases had poor sperm morphology (
[Show abstract][Hide abstract] ABSTRACT: The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells.
No preview · Article · Jun 2014 · Heart (British Cardiac Society)
[Show abstract][Hide abstract] ABSTRACT: Differential methylation between the two alleles of a gene has been observed at 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 bisulphite 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 identifying 21 novel loci harbouring 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 to identifying primary DMRs and reveal the extent of reprograming during pre-implantation 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.
[Show abstract][Hide abstract] ABSTRACT: 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).
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).
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.
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.
Full-text · Article · Jun 2013 · Occupational and environmental medicine
[Show abstract][Hide abstract] ABSTRACT: Recent studies investigating possible causes of male subfertility have largely focused on how lifestyle or environmental factors impact on the process of spermatogenesis. Markedly, fewer studies have investigated those risk factors that result in reduced sperm quality, such as poor sperm motility. The speed at which sperm swim is a major predictor of fertility and is extremely variable in human populations. It has been hypothesized that offspring sex may be adaptively manipulated to maximize the offspring's reproductive fitness (e.g., parents with genes for good male fertility traits, such as high sperm speed, would produce primarily sons and fewer daughters because the offspring will inherit advantageous male fertility genes). Conversely, parents with poor male fertility genes would produce primarily daughters. We tested whether there was an association between how fast a man's sperm swam and the sex bias of his siblings in a sample of men attending clinic for fertility investigations with their partner and with a wide range of semen characteristics, including sperm speed. We found that the sex bias of a man's siblings is associated with his sperm speed; men with female-biased siblings had significantly slower sperm (judged using computer-assisted sperm analysis (CASA)) than men from male-biased sibships. This observation suggests family composition is an important factor that needs to be considered in future epidemiological and clinical studies of human fertility.Asian Journal of Andrology advance online publication, 3 December 2012; doi:10.1038/aja.2012.109.
Preview · Article · Dec 2012 · Asian Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: To circumvent difficulties of isolating pure populations of cancer stem cells for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of five embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumour, to their non-malignant caricature, specifically six human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within two publically available data sets of primary EC tumours and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by RT-PCR-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both germ cell tumour and ES cell biology, and in general to the concept of cancer stem cells.
No preview · Article · Nov 2012 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: STUDY QUESTION
Are common lifestyle factors associated with low-motile sperm concentration (MSC)?
Common lifestyle choices make little contribution to the risk of low MSC.
WHAT IS KNOWN AND WHAT THIS PAPER ADDS
Reviews of male subfertility often highlight how aspects of men's adult lifestyle can significantly increase their risk of subfertility but the strength of supporting evidence is weak. In this study, although low MSC was associated with a history of testicular surgery, being in manual work, not wearing loose underwear and black ethnicity, no relation was found to consumption of alcohol, use of tobacco or recreational drugs or high body mass index (BMI). These results suggest that delaying assisted conception to make changes to lifestyle is unlikely to enhance conception.
Unmatched case-referent study with 939 cases and 1310 referents. Cases had a low-MSC relative to the time since last ejaculation (
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Mitochondrial haplotype is unlikely to be a reliable genetic marker of male factor infertility.
[Show abstract][Hide abstract] ABSTRACT: Human embryo implantation in situ remains a ‘blackbox’ as it is virtually inaccessible to investigation. The finding that pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem cells generate trophoblast has created a new paradigm for studying the very earliest stages of human development in vitro. The phenotype of trophoblast cells generated from pluripotent stem cells depends on the specific culture conditions and displays considerable variation with some uncertainty. These derived cells offer an important new route to investigate the earliest stages of human trophoblast differentiation and in co-culture with endometrial cells an in vitro model of embryo invasiveness at implantation.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.
Full-text · Article · Nov 2011 · Nature Biotechnology