Towards manufactured red blood cells for the treatment of inherited anemia

Patients with inherited anemia and hemoglobinopathies (such as sickle cell disease and β-thalassemia) are treated with red blood cell (RBC) transfusions to alleviate their symptoms. Some of these patients may have rare blood group types or go on to develop alloimmune reactions, which can make it difficult to source compatible blood in the donor population. Laboratory-grown RBC represent a particularly attractive alternative which could satisfy an unmet clinical need. The challenge, however, is to produce - from a limited number of stem cells - the 2x1012 RBC required for a standard adult therapeutic dose. Encouraging progress has been made in RBC production from adult stem cells under good manufacturing practice. In 2011, the Douay group conducted a successful proof-of-principle mini-transfusion of autologous manufactured RBC in a single volunteer. In the UK, a trial is planned to assess whether manufactured RBC are equivalent to RBC produced naturally in donors, by testing an allogeneic mini-dose of laboratory-grown manufactured RBC in multiple volunteers. This review discusses recent progress in the erythroid culture field as well as opportunities for further scaling up of manufactured RBC production for transfusion practice

Polyurethane scaffolds seeded with CD34⁺ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors

We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34+ cells exhibit a remarkable reproducibility of egress, with an increased output when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34+ cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36+GPA−/low). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded “CD34+ cell” population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression

© 2018 The Authors. Published under the terms of the CC BY 4.0 license Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and invivo. Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis.Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance

Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds

Macrophage Reprogramming with Anti-miR223-Loaded Artificial Protocells Enhances In Vivo Cancer Therapeutic Potential

Several immune cell‐expressed miRNAs (miRs) are associated with altered prognostic outcome in cancer patients, suggesting that they may be potential targets for development of cancer therapies. Here, translucent zebrafish (Danio rerio) is utilized to demonstrate that genetic knockout or knockdown of one such miR, microRNA‐223 (miR223), globally or specifically in leukocytes, does indeed lead to reduced cancer progression. As a first step toward potential translation to a clinical therapy, a novel strategy is described for reprogramming neutrophils and macrophages utilizing miniature artificial protocells (PCs) to deliver anti‐miRs against the anti‐inflammatory miR223. Using genetic and live imaging approaches, it is shown that phagocytic uptake of anti‐miR223‐loaded PCs by leukocytes in zebrafish (and by human macrophages in vitro) effectively prolongs their pro‐inflammatory state by blocking the suppression of pro‐inflammatory cytokines, which, in turn, drives altered immune cell‐cancer cell interactions and ultimately leads to a reduced cancer burden by driving reduced proliferation and increased cell death of tumor cells. This PC cargo delivery strategy for reprogramming leukocytes toward beneficial phenotypes has implications also for treating other systemic or local immune‐mediated pathologies

Importance of Anatomical Efficacy for Disease Control in Neovascular AMD: An Expert Opinion

BACKGROUND: Neovascular age-related macular degeneration (nAMD) presents a significant treatment burden for patients, carers and medical retina services. However, significant debate remains regarding how best to manage nAMD when assessing disease activity by optical coherence tomography (OCT), and particularly the significance of different types of fluid and how the understanding of anatomical efficacy can influence treatment strategies. This article provides opinion on the practical implications of anatomical efficacy and significance of fluid in the management of nAMD and proposes recommendations for healthcare professionals (HCPs) to improve understanding and promote best practice to achieve disease control. METHODS: An evidence-based review was performed and an expert panel debate from the Retina Outcomes Group (ROG), a forum of retinal specialists, provided insights and recommendations on the definition, role and practical implications of anatomical efficacy and the significance of fluid at the macula in the management of nAMD. RESULTS: The ROG has developed recommendations for achieving disease control through a zero-tolerance approach to the presence of fluid in nAMD as patients who avoid fluctuations in fluid at the macula have better visual outcomes. Recommendations cover five key areas: service protocol, training, regimen, multidisciplinary teams and engagement. This approach facilitates more standardised protocol-based treatment strategies. CONCLUSIONS: Targeting a fluid-free macula and aiming for disease control are essential to improve outcomes. As new therapies and technologies become available, drying the macula and maintaining disease control will become even more achievable. The outlined recommendations aim to promote best practice among HCPs and medical retina services to improve patient outcomes

The use of a pre-operative scoring system for the prediction of phacoemulsification case difficulty and the selection of appropriate cases to be performed by trainees

Background: To establish whether a previously validated scoring system (Habib) for the prediction of risk or likelihood of posterior capsule rupture during phacoemulsification surgery could be used to: 1. Predict the difficulty of a phacoemulsification case, and 2. Select appropriate phacoemulsification cases for trainees. Methods: The study sample was consecutive phacoemulsification cases undertaken by senior surgeons at a single ophthalmic unit over a three-week period (170 cases). Each case was scored using a potential difficulty scoring system. Immediately post-operatively, each case was given two scores by the operating surgeon (who was masked with regard to the potential complication score). The first score indicated the perceived difficulty of the case, and the second score, the degree of experience that they thought a trainee would require in order to have performed the same case without complication. Results: Using Cuzick's non-parametric test for trend, there was evidence for a trend of increasing perceived difficulty with increasing potential difficulty score (p = 0.05), and of increasing experience required with increasing potential difficulty score (p < 0.001) Conclusion: The authors advocate that Habib's potential difficulty scoring system can be used to inform the surgeon of the likely difficulty of a phacoemulsification case and to aid selection of appropriate cases for trainees prior to surgery

Third generation artificial pitch quality in commercial football centers

Small sided football is the most popular and fastest growing area of adult football in the UK with an estimated 1.5m adults playing every week. The sport's popularity has led to an influx of commercial football centers offering organized 5, 6 and 7-a-side leagues on third generation artificial pitches. The range of quality and maintenance of these pitches is not fully understood despite the established links between surface quality, player performance and injury. Currently researchers and manufacturers use national governing body standards as guidelines for quality; however, many commercial centers are not approved by governing bodies and therefore are not obliged to meet these criteria. In this paper we characterize the quality of 23 pitches at five, UK based, commercial football centers using portable, low cost methods including; the FIFA rotational resistance test, the Clegg Impact Hammer and an infill depth probe. This paper describes the range of qualities observed, alongside maintenance procedures and usage statistics. To the authors knowledge this is the first study that characterizes commercial football center pitches. Twenty-two of the 23 pitches met the FIFA 1 star guidelines for rotational resistance (25 - 50 Nm), however, mean Clegg Impact Hammer readings are high (208 G), suggesting surface compliance in commercial centers falls outside FIFA standards. Within pitch variance was common at all centers and was an order of magnitude higher in some pitch comparisons. These findings have two practical implications; 1) pitch quality and maintenance at commercial centers is highly variable across and within pitches, 2) the harder surfaces and the high levels of variability found in commercial football centers suggests that players require footwear researched and designed specifically for these conditions