[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.
EuroIntervention: journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology 10/2014; 10(6):709-716. · 3.76 Impact Factor
[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.
[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.
[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). 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.
Occupational and environmental medicine 06/2013; · 3.23 Impact Factor
[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.
Asian Journal of Andrology 12/2012; · 2.14 Impact Factor
[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.
Stem cells and development 11/2012; · 4.15 Impact Factor
[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: 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.
Human Reproduction 01/2012; 27(3):641-51. · 4.59 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.
[Show abstract][Hide abstract] ABSTRACT: The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state', yet it appears that this categorization may be an oversimplification, because a number of sub-states appear to exist within this state. To increase the resolution of the undifferentiated state, we have generated eight novel monoclonal antibodies, all capable of recognizing undifferentiated human ES and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment.
International Journal of Andrology 06/2011; 34(4 Pt 2):e175-87; discussion e187-8. · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal human embryo implantation leads to poor foetal development and miscarriage, or pre-eclampsia. Ethical and practical considerations concerning implantation limit its investigation, and it is often difficult to extrapolate findings in laboratory animals when implantation processes show diverse species differences. Therefore, it is important to develop new in vitro models to study the earliest events of human implantation. The aim of this study was to derive trophoblast cell lines from human embryonic stem cells (hESCs) by a robust protocol and co-culture of these cells with an established endometrial cell culture system to validate a model of trophoblast invasion at implantation.
Derivation of trophoblast cell lines from hESC lines was established by spontaneous and induced differentiation of embryoid bodies and by initial measurement of hCGβ secretion by enzyme-linked immunosorbent assay and their phenotype investigated using gene- and protein-expression markers. Vesicles formed from an aggregating trophoblast were co-cultured with decidualized human endometrial stromal cells in hypoxic (2% oxygen) and normoxic (20% oxygen) environments.
Derived villous cytotrophoblast cell (CTB) lines further differentiated to invasive, extra-villous CTBs. Eventually, cells lost their proliferative capacity, with some lines acquiring karyotypic changes, such as a gain in the X chromosome. Cell-invasion assays confirmed that the extra-villous CTBs were invasive and erosion of the endometrial stromal layer occurred, particularly under hypoxic conditions in vitro.
Trophoblast cell lines derived from hESCs differentiate and adapt in vitro and can be used as a model to study the mechanisms of early trophoblast invasion.
Human Reproduction 02/2011; 26(2):398-406. · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterise the properties of these cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterised extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSCs exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.
Epigenetics: official journal of the DNA Methylation Society 01/2011; 6(1):52-62. · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.
The International journal of developmental biology 01/2011; 55(2):175-80. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS) cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.
In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT) and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES) cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine) and cultured in human embryonic stem cell (ES) medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.
Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.
PLoS ONE 12/2010; 5(12):e14397. · 3.53 Impact Factor