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

Abstract: 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

Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD

A systematic review of non-invasive modalities used to identify women with anal incontinence symptoms after childbirth

© 2018, The International Urogynecological Association. Introduction and hypothesis: Anal incontinence following childbirth is prevalent and has a significant impact upon quality of life (QoL). Currently, there is no standard assessment for women after childbirth to identify these symptoms. This systematic review aimed to identify non-invasive modalities used to identify women with anal incontinence following childbirth and assess response and reporting rates of anal incontinence for these modalities. Methods: Ovid Medline, Allied and Complementary Medicine Database (AMED), Cumulative Index of Nursing and Allied Health Literature (CINAHL), Cochrane Collaboration, EMBASE and Web of Science databases were searched for studies using non-invasive modalities published from January 1966 to May 2018 to identify women with anal incontinence following childbirth. Study data including type of modality, response rates and reported prevalence of anal incontinence were extracted and critically appraised. Results: One hundred and nine studies were included from 1602 screened articles. Three types of non-invasive modalities were identified: validated questionnaires/symptom scales (n = 36 studies using 15 different instruments), non-validated questionnaires (n = 50 studies) and patient interviews (n = 23 studies). Mean response rates were 92% up to 6 weeks after childbirth. Non-personalised assessment modalities (validated and non-validated questionnaires) were associated with reporting of higher rates of anal incontinence compared with patient interview at all periods of follow-up after childbirth, which was statistically significant between 6 weeks and 1 year after childbirth (p < 0.05). Conclusions: This systematic review confirms that questionnaires can be used effectively after childbirth to identify women with anal incontinence. Given the methodological limitations associated with non-validated questionnaires, assessing all women following childbirth for pelvic-floor symptomatology, including anal incontinence, using validated questionnaires should be considered

Oxygen controlled processing of pluripotent stem cells

A major challenge facing the development of effective cell therapies is the efficient differentiation of pluripotent stem cells (PSCs) into pure populations. This is caused, in part, by the heterogeneous presence of functionally distinct subpopulations in undifferentiated PSCs. These can exhibit variable developmental potential, suggesting that there will be a heterogeneous response to differentiation cues, and a low yield of the target cell type. The importance of recapitulating the in vivo stem cell niche during stem cell process development is now widely acknowledged, and thus, manipulation of microenvironmental factors will be invaluable in engineering optimal in vitro conditions for cell culture. Lowering oxygen tension to physiological levels can affect both the expansion and differentiation stages. However, to date, there are no studies investigating the consequences of culturing PSCs under hypoxic conditions on subsequent lineage commitment at ambient oxygen levels. Mouse Embryonic Stem Cells (mESCs) were passaged three times at 2% O2 before allowing cells to spontaneously differentiate as embryoid bodies (EBs) in normoxic (20% O2) conditions. Maintenance of mESCs under hypoxia was associated with a population shift away from a Rex1-positive ICM-like (Inner Cell Mass-like) state, to a primed epiblast-like state exhibiting a significant increase in the expression of early differentiation markers FGF5 and Eomes. Conversely, expression of these committed markers was decreased in human induced pluripotent stem cells (hiPSCs) following the same hypoxic step, and was instead associated with a significant increase in Rex1 expression. Hypoxic preconditioning primed mESCs for their subsequent differentiation into mesodermal and endodermal lineages, as confirmed by increased gene expression of Eomes, Goosecoid, Brachyury, AFP, Sox17, FoxA2, and protein expression of Brachyury, Eomes, Sox17, FoxA2, relative to normoxic cultures. The effects extended to the subsequent formation of more mature mesodermal lineages. A significant upregulation of cardiomyocyte marker Nkx2.5 was also observed, and critically a decrease in the number of contaminant pluripotent cells after 12 days using a directed cardiomyocyte protocol. However, the impact of hypoxic preconditioning was to preferentially prime human cells for ectodermal lineage commitment during subsequent EB differentiation, with significant upregulation of Nestin and β3-tubulin. The research discussed in this thesis demonstrates the importance of oxygen-tension control during cell maintenance on the subsequent differentiation of both mouse and human PSCs, and highlights the differential effects. The reported results have indicated that further to focusing on the differentiation stage itself, it will be critical to consider the prior maintenance of PSCs to fully optimise future stem cell processes. Furthermore, the work has highlighted the importance of considering each stage of bioprocess development individually, to best recapitulate the transient conditions in vivo

The method of educational assessment affects children\u27s neural processing and performance: behavioural and fMRI Evidence

Standardised educational assessments are now widespread, yet their development has given comparatively more consideration to what to assess than how to optimally assess students\u27 competencies. Existing evidence from behavioural studies with children and neuroscience studies with adults suggest that the method of assessment may affect neural processing and performance, but current evidence remains limited. To investigate the impact of assessment methods on neural processing and performance in young children, we used functional magnetic resonance imaging to identify and quantify the neural correlates during performance across a range of current approaches to standardised spelling assessment. Results indicated that children\u27s test performance declined as the cognitive load of assessment method increased. Activation of neural nodes associated with working memory further suggests that this performance decline may be a consequence of a higher cognitive load, rather than the complexity of the content. These findings provide insights into principles of assessment (re)design, to ensure assessment results are an accurate reflection of students\u27 true levels of competency

Stem cell-derived retinal pigment epithelium transplantation for treatment of retinal disease

Age-related macular degeneration remains the most common cause of blindness in the western world, severely comprising patients' and carers' quality of life and presenting a great cost to the healthcare system. As the disease progresses, the retinal pigmented epithelium (RPE) layer at the back of the eye degenerates, contributing to a series of events resulting in visual impairment. The easy accessibility of the eye has allowed for in-depth study of disease progression in patients, while in vivo studies have facilitated investigations into healthy and diseased RPE. Consequently, a number of research groups are examining different approaches for the replacement of RPE cells in age-related macular degeneration (AMD) patients. This chapter examines some of these initial proof-of-principle studies and goes on to review the use of pluripotent stem cells as a source for RPE replacement in a number of current AMD clinical trials. Finally, we consider just some of the regulatory and manufacturing challenges presented in taking a promising AMD treatment from the research bench into clinical trials in patients, and how to mitigate potential risks early in process development