Forward programming of human pluripotent stem cells to a megakaryocyte-erythrocyte bi-potent progenitor population: an in vitro system for the production of platelets and red blood cells for transfusion medicine.

There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.British Heart Foundation funded PhD

Biomechanical Dependence of SARS-CoV-2 Infections

Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, that is, the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.This work was supported by the Leukemia and Lymphoma Society grant, the UK Medical Research Council (Roger Pedersen), the National Institute for Health Research (NIHR; RP-PG-0310-1002; Willem Ouwehand and Cedric Ghevaert) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. Cedric Ghevaert is supported by the British Heart Foundation (FS/09/039); Marloes Tijssen is supported by the European Hematology Association (Research fellowship) and the British Heart Foundation (PG/13/77/30375). Catherine Hobbs was supported by the National Health Service Blood and Transplant. Matthew Trotter was supported by a Medical Research Council Centre grant (MRC Centre for Stem Cell Biology and Regenerative Medicine); since participation in the work described, Matthew Trotter has become an employee of Celgene Research SLU, part of Celgene Corporation. Nicole Soranzo's research and Sanger Institute affiliates are supported by the Wellcome Trust (WT098051 and WT091310), the EU FP7 (Epigenesys 257082 and Blueprint HEAL TH-F5-2011-282510). The Cambridge Biomedical Centre (BRC) hIPSCs core facility is funded by the NIHR.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/ncomms1120

Wild waterfowl migration and domestic duck density shape the epidemiology of highly pathogenic H5N8 influenza in the Republic of Korea

Highly pathogenic avian influenza (HPAI) viruses threaten human and animal health yet their emergence is poorly understood, partly because sampling of the HPAI Asian-origin H5N1 lineage immediately after its identification in 1996 was comparatively sparse. The discovery of a novel H5N8 virus in 2013 provides a new opportunity to investigate HPAI emergence in greater detail. Here we investigate the origin and transmission of H5N8 in the Republic of Korea, the second country to report the new strain. We reconstruct viral spread using phylogeographic methods and interpret the results in the context of ecological data on poultry density, overwintering wild bird numbers, and bird migration patterns. Our results indicate that wild waterfowl migration and domestic duck density were important to H5N8 epidemiology. Specifically, we infer that H5N8 entered the Republic of Korea via Jeonbuk province, then spread rapidly among western provinces where densities of overwintering waterfowl and domestic ducks are higher, yet rarely persisted in eastern regions. The common ancestor of H5N8 in the Republic of Korea was estimated to have arrived during the peak of inward migration of overwintering birds. Recent virus isolations likely represent re-introductions via bird migration from an as-yet unsampled reservoir. Based on the limited data from outside the Republic of Korea, our data suggest that H5N8 may have entered Europe at least twice, and Asia at least three times from this reservoir, most likely carried by wild migrating birds

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

PANC Study (Pancreatitis: A National Cohort Study): national cohort study examining the first 30 days from presentation of acute pancreatitis in the UK

Abstract Background Acute pancreatitis is a common, yet complex, emergency surgical presentation. Multiple guidelines exist and management can vary significantly. The aim of this first UK, multicentre, prospective cohort study was to assess the variation in management of acute pancreatitis to guide resource planning and optimize treatment. Methods All patients aged greater than or equal to 18 years presenting with acute pancreatitis, as per the Atlanta criteria, from March to April 2021 were eligible for inclusion and followed up for 30 days. Anonymized data were uploaded to a secure electronic database in line with local governance approvals. Results A total of 113 hospitals contributed data on 2580 patients, with an equal sex distribution and a mean age of 57 years. The aetiology was gallstones in 50.6 per cent, with idiopathic the next most common (22.4 per cent). In addition to the 7.6 per cent with a diagnosis of chronic pancreatitis, 20.1 per cent of patients had a previous episode of acute pancreatitis. One in 20 patients were classed as having severe pancreatitis, as per the Atlanta criteria. The overall mortality rate was 2.3 per cent at 30 days, but rose to one in three in the severe group. Predictors of death included male sex, increased age, and frailty; previous acute pancreatitis and gallstones as aetiologies were protective. Smoking status and body mass index did not affect death. Conclusion Most patients presenting with acute pancreatitis have a mild, self-limiting disease. Rates of patients with idiopathic pancreatitis are high. Recurrent attacks of pancreatitis are common, but are likely to have reduced risk of death on subsequent admissions. </jats:sec

An interview with A. Koneti Rao: Editor of Platelets (1990-2018)

Professor A. Koneti Rao has made many critical contributions to the field of platelet research for over forty years. He joined the editorial board of Platelets as a Principal Editor in 1989 before the start of the journal and the appointment of Stan Heptinstall, who was Editor-in-Chief for 25 years. Professor Rao retired from the editorial board in 2018. This article is based on an interview with Professor Rao that took place prior to the Platelets Editorial Board meeting and lunch in 2019 during the ISTH Congress in Melbourne. Professor Rao was presented with a plaque in recognition of his service to the journal. The article is a reflection on Professor Rao’s personal life and his career in science, along with his views on the past and future of Platelets. Professor Rao continues to serve as a referee for the journal