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Background: Predicting hospital length of stay (LoS) for patients with COVID-19 infection is essential to ensure that adequate bed capacity can be provided without unnecessarily restricting care for patients with other conditions. Here, we demonstrate the utility of three complementary methods for predicting LoS using UK national- and hospital-level data.Method: On a national scale, relevant patients were identified from the COVID-19 Hospitalisation in England Surveillance System (CHESS) reports. An Accelerated Failure Time (AFT) survival model and a truncation corrected method (TC), both with underlying Weibull distributions, were fitted to the data to estimate LoS from hospital admission date to an outcome (death or discharge) and from hospital admission date to Intensive Care Unit (ICU) admission date. In a second approach we fit a multi-state (MS) survival model to data directly from the Manchester University NHS Foundation Trust (MFT). We develop a planning tool that uses LoS estimates from these models to predict bed occupancy.Results: All methods produced similar overall estimates of LoS for overall hospital stay, given a patient is not admitted to ICU (8.4, 9.1 and 8.0 days for AFT, TC and MS, respectively). Estimates differ more significantly between the local and national level when considering ICU. National estimates for ICU LoS from AFT and TC were 12.4 and 13.4 days, whereas in local data the MS method produced estimates of 18.9 days.Conclusions: Given the complexity and partiality of different data sources and the rapidly evolving nature of the COVID-19 pandemic, it is most appropriate to use multiple analysis methods on multiple datasets. The AFT method accounts for censored cases, but does not allow for simultaneous consideration of different outcomes. The TC method does not include censored cases, instead correcting for truncation in the data, but does consider these different outcomes. The MS method can model complex pathways to different outcomes whilst accounting for censoring, but cannot handle non-random case missingness. Overall, we conclude that data-driven modelling approaches of LoS using these methods is useful in epidemic planning and management, and should be considered for widespread adoption throughout healthcare systems internationally where similar data resources exist. [ABSTRACT FROM AUTHOR]

Insulin is routinely used to manage hyperglycaemia in organ donors and during the peri‐transplant period in islet transplant recipients. However, it is unknown whether donor insulin use (DIU) predicts beta‐cell dysfunction after islet transplantation. We reviewed data from the UK Transplant Registry and the UK Islet Transplant Consortium; all first‐time transplants during 2008‐2016 were included. Linear regression models determined associations between DIU, median and coefficient of variation (CV) peri‐transplant glucose levels and 3‐month islet graft function. In 91 islet cell transplant recipients, DIU was associated with lower islet function assessed by BETA‐2 scores (β [SE] ‐3.5 [1.5], P =.02), higher 3‐month post‐transplant HbA1c levels (5.4 [2.6] mmol/mol, P =.04) and lower fasting C‐peptide levels (−107.9 [46.1] pmol/l, P =.02). Glucose at 10 512 time points was recorded during the first 5 days peri‐transplant: the median (IQR) daily glucose level was 7.9 (7.0‐8.9) mmol/L and glucose CV was 28% (21%‐35%). Neither median glucose levels nor glucose CV predicted outcomes post‐transplantation. Data on DIU predicts beta‐cell dysfunction 3 months after islet transplantation and could help improve donor selection and transplant outcomes. [ABSTRACT FROM AUTHOR]

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency. [ABSTRACT FROM AUTHOR]

Diffuse congenital hyperinsulinism in infancy (CHI-D) arises from mutations inactivating the KATP channel; however, the phenotype is difficult to explain from electrophysiology alone. Here we studied wider abnormalities in the β-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared with controls. PAX4 and ARX expression was decreased. A tendency toward increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas, and implied immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. In CHI-D, increased proliferation was most elevated in duct (5- to 11-fold) and acinar (7- to 47-fold) lineages. Increased β-cell proliferation observed in some cases was offset by an increase in apoptosis; this is in keeping with no difference in INSULIN expression or surface area stained for insulin between CHI-D and control pancreas. However, nuclear localization of CDK6 and P27 was markedly enhanced in CHI-D β-cells compared with cytoplasmic localization in control cells. These combined data support normal β-cell mass in CHI-D, but with G1/S molecules positioned in favor of cell cycle progression. New molecular abnormalities in δ-cells and marked proliferative increases in other pancreatic lineages indicate CHI-D is not solely a β-cell disorder. [ABSTRACT FROM AUTHOR]

Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), IxB kinase-α (IKKα), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease. [ABSTRACT FROM AUTHOR]

Knowledge of human pancreas development underpins our interpretation and exploitation of human pluripotent stem cell (PSC) differentiation toward a β-cell fate. However, almost no information exists on the early events of human pancreatic specification in the distal foregut, bud formation, and early development. Here, we have studied the expression profiles of key lineage-specific markers to understand differentiation and morphogenetic events during human pancreas development. The notochord was adjacent to the dorsal foregut endoderm during the fourth week of development before pancreatic duodenal homeobox-1 detection. In contrast to the published data from mouse embryos, during human pancreas development, we detected only a single-phase of Neurogenin 3 (NEUROG3) expression and endocrine differentiation from approximately 8 weeks, before which Nirenberg and Kim homeobox 2.2 (NKX2.2) was not observed in the pancreatic progenitor cell population. In addition to revealing a number of disparities in timing between human and mouse development, these data, directly assembled from human tissue, allow combinations of transcription factors to define sequential stages and differentiating pancreatic cell types. The data are anticipated to provide a useful reference point for stem cell researchers looking to differentiate human PSCs in vitro toward the pancreatic β-cell so as to model human development or enable drug discovery and potential cell therapy. [ABSTRACT FROM AUTHOR]

Amongst the different types of adverse drug reactions, drug-induced liver injury is the most prominent cause of patient morbidity and mortality. However, the current available hepatic model systems developed for evaluating safety have limited utility and relevance as they do not fully recapitulate a fully functional hepatocyte, and do not sufficiently represent the genetic polymorphisms present in the population. The rapidly advancing research in stem cells raises the possibility of using human pluripotent stem cells in bridging this gap. The generation of human induced pluripotent stem cells via reprogramming of mature human somatic cells may also allow for disease modelling in vitro for the purposes of assessing drug safety and toxicology. This would also allow for better understanding of disease processes and thus facilitate in the potential identification of novel therapeutic targets. This review will focus on the current state of effort to derive hepatocytes from human pluripotent stem cells for potential use in hepatotoxicity evaluation and aims to provide an insight as to where the future of the field may lie. [ABSTRACT FROM AUTHOR]

The molecular basis of type 2 diabetes predisposition at most established susceptibility loci remains poorly understood. KCNQ1 maps within the 11p15.5 imprinted domain, a region with an established role in congenital growth phenotypes. Variants intronic to KCNQ1 influence diabetes susceptibility when maternally inherited. By use of quantitative PCR and pyrosequencing of human adult islet and fetal pancreas samples, we investigated the imprinting status of regional transcripts and aimed to determine whether type 2 diabetes risk alleles influence regional DNA methylation and gene expression. The results demonstrate that gene expression patterns differ by developmental stage. CDKN1C showed monoallelic expression in both adult and fetal tissue, whereas PHLDA2, SLC22A18, and SLC22A18AS were biallelically expressed in both tissues. Temporal changes in imprinting were observed for KCNQ1 and KCNQ1OT1, with monoallelic expression in fetal tissues and biallelic expression in adult samples. Genotype at the type 2 diabetes risk variant rs2237895 influenced methylation levels of regulatory sequence in fetal pancreas but without demonstrable effects on gene expression. We demonstrate that CDKN1C, KCNQ1, and KCNQ1OT1 are most likely to mediate diabetes susceptibility at the KCNQ1 locus and identify temporal differences in imprinting status and methylation effects, suggesting that diabetes risk effects may be mediated in early development. [ABSTRACT FROM AUTHOR]

The transcription factor SOX9 is crucial for multiple aspects of development. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder concordant with the expression profile of SOX9 during embryogenesis. The mechanistic understanding of development has revealed roles for SOX9 in regulating cartilage extracellular matrix (ECM) production and cell proliferation, among others. More recently, it transpires that SOX9 becomes expressed and induces destructive ECM components in organ fibrosis and related disorders. Although commonly absent from the parent cell type, SOX9 is expressed in a wide range of cancers, where it regulates cell proliferation. These data have potential diagnostic, prognostic and therapeutic relevance, suggesting that disease mechanisms might result from re-expressing this developmental transcription factor in ectopic locations. [ABSTRACT FROM AUTHOR]

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm-Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney. [ABSTRACT FROM AUTHOR]

Abstract: The cranial base is essential for integrated craniofacial development and growth. It develops as a cartilaginous template that is replaced by bone through the process of endochondral ossification. Here, we describe a novel and specific role for the homeoprotein Six2 in the growth and elongation of the cranial base. Six2-null newborn mice display premature fusion of the bones in the cranial base. Chondrocyte differentiation is abnormal in the Six2-null cranial base, with reduced proliferation and increased terminal differentiation. Gain-of-function experiments indicate that Six2 promotes cartilage development and growth in other body areas and appears therefore to control general regulators of chondrocyte differentiation. Our data indicate that the main factors restricting Six2 function to the cranial base are tissue-specific transcription of the gene and compensatory effects of other Six family members. The comparable expression during human embryogenesis and the high protein conservation from mouse to human implicate SIX2 loss-of-function as a potential congenital cause of anterior cranial base defects in humans. [ABSTRACT FROM AUTHOR]

Dehydroepiandrosterone (DHEA) sulfotransferase, known as SULT2A1, converts the androgen precursor DHEA to its inactive sulfate ester, DHEAS, thereby preventing the conversion of DHEA to an active androgen. SULT2A1 requires 3′-phosphoadenosine-5′-phosphosulfate (PAPS) for catalytic activity. We have identified compound heterozygous mutations in the gene encoding human PAPS synthase 2 (PAPSS2) in a girl with premature pubarche, hyperandrogenic anovulation, very low DHEAS levels, and increased androgen levels. In vitro coincubation of human SULT2A1 and wild-type or mutant PAPSS2 proteins confirmed the inactivating nature of the mutations. These observations indicate that PAPSS2 deficiency is a monogenic adrenocortical cause of androgen excess. [ABSTRACT FROM AUTHOR]

Appropriate temporospatial expression of the transcription factor SOX9 is important for normal development of a wide range of organs. Here, we show that when SOX9 is expressed ectopically, target genes become expressed that are associated with disease. Histone deacetylase inhibitors in clinical trials for cancer therapy induced SOX9 expression via enhanced recruitment of nuclear factor Y (NF-Y) to CCAAT elements in the SOX9 proximal promoter. The effect of histone deacetylase inhibitors could be elicited in cells that normally lack SOX9, such as hepatocytes. In human fetal hepatocytes, this aberrant induction of SOX9 protein caused ectopic expression of COL2A1 and COMP1 that encode extracellular matrix (ECM) components normally associated with chondrogenesis. Previously, ectopic expression of this "chondrogenic" profile has been implicated in vascular calcification. More broadly, inappropriate ECM deposition is a hallmark of fibrosis. We demonstrated that induction of SOX9 expression also occurred during activation of fibrogenic cells from the adult liver when the transcription factor was responsible for expression of the major component of fibrotic ECM, type 1 collagen. These combined data identify new aspects in the regulation of SOX9 expression. They support a role for SOX9 beyond normal development as a transcriptional regulator in the pathology of fibrosis. [ABSTRACT FROM AUTHOR]

In humans, sexual differentiation of the external genitalia is established at 7-12 weeks post conception (wpc). During this period, maintaining the appropriate intrauterine hormone environment is critical. In contrast to other species, this regulation extends to the human fetal adrenal cortex, as evidenced by the virilization that is associated with various forms of congenital adrenal hyperplasia. The mechanism underlying these clinical findings has remained elusive. Here we show that the human fetal adrenal cortex synthesized cortisol much earlier than previously documented, an effect associated with transient expression of the orphan nuclear receptor nerve growth factor IB-like (NGFI-B) and its regulatory target, the steroidogenic enzyme type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). This cortisol biosynthesis was maximal at 8-9 wpc under the regulation of ACTH. Negative feedback was apparent at the anterior pituitary corticotrophs. ACTH also stimulated the adrenal gland to secrete androstenedione and testosterone. In concert, these data promote a distinctive mechanism for normal human development whereby cortisol production, determined by transient NGFI-B and HSD3B2 expression, provides feedback at the anterior pituitary to modulate androgen biosynthesis and safeguard normal female sexual differentiation. [ABSTRACT FROM AUTHOR]

Identifying genes linked to extreme phenotypes in humans has the potential to highlight biological processes not shared with all other mammals. Here, we report the identification of homozygous loss-of-function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein family, a large and rapidly evolving group of epigenetic silencers which target transposable elements. We show that loss of ZNF808 in vitro results in aberrant activation of regulatory potential contained in the primate-specific transposable elements it represses during early pancreas development. This leads to inappropriate specification of cell fate with induction of genes associated with liver identity. Our results highlight the essential role of ZNF808 in pancreatic development in humans and the contribution of primate-specific regions of the human genome to congenital developmental disease., (© 2023. The Author(s).)

Myofibroblasts are responsible for scarring during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state and increasing profibrotic phenotype. Here, we show mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. Integrating chromatin accessibility profiles with RNA expression provides insight into the transcription network underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive regulation of PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, loss of PAK1-dependent signaling impairs the mechanoadaptive response in vitro and dramatically improves fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1-mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms as potential therapeutic targets in fibrosis., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Cystic Fibrosis related liver disease (CFLD) is the 3rd largest cause of death in Cystic Fibrosis (CF). As advances in pulmonary therapies have increased life-expectancy, CFLD has become more prevalent. Current guidelines may underdiagnose liver fibrosis, particularly in its early stages. Newer modalities for the assessment of fibrosis may provide a more accurate assessment. FibroScan is validated in assessing fibrosis for several aetiologies including alcohol and fatty liver, the CFLD cohort have an entirely different phenotype so the cut off values are not transferrable. We appraised fibrosis assessment tools to improve diagnosis of CFLD., Methods: A prospective cohort (n = 114) of patients from the Manchester Adult Cystic Fibrosis Centre, UK were identified at annual assessment. Demographic data including co-morbidity, CFTR genotyping, biochemistry and imaging were used alongside current guidelines to group into CFLD and CF without evidence of liver disease. All patients underwent liver stiffness measurement (LSM) and assessment of serum-based fibrosis biomarker panels. A new diagnostic criterion was created and validated in a second, independent cohort., Results: 12 of 114 patient classified as CFLD according to the European Cystic Fibrosis Society best practice guidelines. No specific risk factors for development of CFLD were identified. Liver enzymes were elevated in patients with CFLD. Serum biomarker panels did not improve diagnostic criteria. LSM accurately predicted CFLD. A new diagnostic criterion was proposed and validated in a separate cohort, accurately predicating CFLD in 10 of 32 patients (31 %)., Conclusion: We present a cohort of patients with CF assessed for the presence of liver fibrosis using blood biomarkers and LSM based platforms. We propose a new, simplified diagnostic criteria, capable of accurately predicting liver disease in patients with CF.Clinical trials number: NCT04277819., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

Background: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea., Methods: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2-7 months after hospital discharge and a later time point 10-14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107)., Findings: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4-6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5-8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (-19%; 95% CI -20 to -16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18-39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27-41% of this effect., Interpretation: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition., Funding: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council., Competing Interests: Declaration of interests IDS declares a statistical editor honoraria role with Thorax. TP declares support from the National Institute for Health Research (NIHR) Leicester Biomedical Research Centre (BRC) to complete the work. ALH declares an editor-in-chief role for Computer Physics Communications. RA declares speaker fees and travel support from Boehringer Ingelheim. CEB declares their institute was awarded a grant from the UK Research and Innovation (UKRI)/NIHR and institutional support from NIHR Nottingham BRC to complete this work; the author reports grants from Nottingham Hospitals Charity and Nottingham University Hospitals Research and Innovation Department. TC declares support from the NIHR South London and Maudsley NHS Foundation Trust, King's College London BRC to complete the work; the author reports grants from Guy's and St Thomas' Charity, NIHR, and UKRI; the author has published self-help books on chronic fatigue for which she receives royalties; the author has received ad-hoc payments for workshops carried out in long-term conditions; the author declares travel and accommodation support; the author is part of the scientific committee of the British Association for Behavioural and Cognitive Psychotherapies (BABCP) and she is on the expert advisory panel for COVID-19 Rapid Guidelines. JDC declares grants from AstraZeneca, Novartis, Boehringer Ingelheim, Genentech, Gilead Sciences, Insmed, GlaxoSmithKline, and Grifols; the author reports consulting fees from AstraZeneca, Insmed, Boehringer Ingelheim, Janssen, Antabio, Chiesi, Novartis, Pfizer, Zambon, GlaxoSmithKline, and Grifols. GD declares support from the NIHR Imperial BRC, Royal Brompton and Harefield NGS Foundation Trust, Royal Brompton & Harefield Hospitals Charity, Imperial College Healthcare NHS Trust, and the Medical Research Council (MRC) to complete this work; the author reports grants from Genentech, AstraZeneca, British Lung Foundation (BLF) Early Cohort, GlaxoSmithKline, Novartis, Chiesi, and Boehringer Ingelheim; the author has a published chapter in a textbook for which he receives payment; the author declares a role in the advisory boards for AstraZeneca and Novartis and as honorarium as deputy editor of the American Journal of Respiratory and Critical Care Medicine (AJRCCM). L-PH declares grants from NIHR Oxford BRC. MGJ declares grants from the Royal Society, MRC, BLF, Boehringer Ingelheim, and the Asthma, Allergy and Inflammatory Research Charity. BR declares support from the British Heart Foundation (BHF) Oxford Centre of Research Excellence (CRE) to complete this work. SLR-J declares support for her institute from UKRI to complete the work; the author reports grants from UKRI, The European and Developing Countries Clinical Trials Partnership (EDCTP), MRC, Rosetrees Trust, and the Global Challenges Research Fund (GCRF); the author reports an honorarium for chapter contribution from the Federation of European Academies of Medicine; the author is Data and Safety Monitoring Board (DSMB) Chair for a Bexsero trial funded by Wellcome Trust; the author declares an editor role for AIDS journal. AVR declares support from NIHR Leicester BRC to complete the work. MS declares support for his institute from MRC/Department of Health and Social Care (DHSC) to complete the work. ASi declares support for her institute from UKRI/NIHR to complete the work. DGW declares an advanced Fellowship grant from NIHR; the author receives an honorarium from bioMérieux to present. TY declares support from the NIHR Leicester BRC to complete the work. RGJ declares grants from AstraZeneca, Biogen, Galecto, GlaxoSmithKline, Nordic Biosciences, RedX, Pliant; the author received consulting fees from AstraZeneca, Brainomix, Bristol Myers Squibb, Chiesi, Cohbar, Daewoong, GlaxoSmithKline, Veracyte, Resolution Therapeutics, and Pliant; the author has received payments from Boehringer Ingelheim, Chiesi, Roche, patientMpower, and AstraZeneca; the author was paid for expert testimony by Pinsent Masons LLP; the author has participated on a DSMB for Boehringer Ingelheim, Galapagos, and Vicore; the author has held a leadership role at NuMedii and is President of Action for Pulmonary Fibrosis. SJS declares grants from NIHR, Wellcome Doctoral Training Programme (DTP), the Human Tissue Authority, NIHR DHSC/UKRI COVID-19 Rapid Response Initiative, NIHR Global Research Group, Actegy Limited, and as an NIHR Senior Investigator; the author has presented for GlaxoSmithKline, Ministry of Justice, CIPLA, and Sherbourne Gibbs; the author is on the National Institute for Health and Care Excellence (NICE) Expert Advisor Panel for Long COVID and was on the Wales Long COVID Advisory Board; the author is the American Thoracic Society (ATS) Pulmonary Rehabilitation Assembly Chair, Clinical Lead Royal Society of Physicians (RSP) Pulmonary Rehabilitation Accreditation Scheme, and Clinical Lead Nation Asthma and COPD Audit Programme (NACAP) for Pulmonary Rehabilitation. WD-CM declares grants for his institution from NIHR, National Health Service (NHS) Accelerated Access Collaborative, and BLF; the author is the honorary President of the Association for Respiratory Technology and Physiology but receives no payment. CEBr declares support from UKRI/DHSC and NIHR Leicester BRC to complete the work; the author reports grants from GlaxoSmithKline, AstraZeneca, Sanofi, CI, Chiesi, Novartis, Roche, Genentech, Mologic, and 4DPharma; the author received consulting fees paid to his institution from GlaxoSmithKline, AstraZeneca, Sanofi, BI, Chiesi, Novartis, Roche, Genentech, Mologic, 4DPharma, and TEVA. LVW declares support from UKRI, GlaxoSmithKline/Asthma + Lung UK, and NIHR to complete this work; the author receives grants from Orion Pharma, GlaxoSmithKline, Genentech, and AstraZeneca; the author received consulting fees paid to her institution from Galapagos and Boehringer Ingelheim; the author received support for attending a meeting from Genentech. JCP received consulting fees from Istesso and The Limbic; the author participated on a DSMB for Vicore. AART declares a fellowship grant from BHF and a grant from NIHR to his institution; the author received an honorarium for lectures from Janssen-Cilag Ltd; the author received support for attending meetings from Janssen-Cilag Ltd. AH declares support from UKRI, NIHR, and NIHR Manchester BRC to complete the work; the author is Chair NIHR Translational Research Collaboration (unpaid). PLM declares a grant for his institution from AstraZeneca; the author received consulting fees from Hoffmann-La Roche, Boehringer Ingelheim, AstraZeneca, Trevi, and Qureight; the author received speaker fees from Boehringer Ingelheim and Hoffmann-La Roche. RAE declares support from UKRI/MRC to complete the work; the author reports a grant from NIHR/Wolfson Foundation; the author received consulting fees from AstraZeneca for Long COVID; the author received a speaker fee from Boehringer Ingelheim for a lecture on Long COVID; the author received support to attend BTS conference virtually from Chiesi; the author is the European Respiratory Society Group 01.02 Pulmonary Rehabilitation Secretary (unpaid). JFB declares support to his institute from an MRC Transition Fellowship, Asthma + Lung UK, NIHR Manchester BRC, and UKRI; the author reports grants paid to his institution from the Small Business Research Initiative Home Spirometer and the National Institute of Academic Anaesthesia; the author has received support for attending meetings from TEVA and Therakos; the author is a committee member of the Royal Society of Medicine (RSM). All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted Dl CO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.

Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation., (© 2023. The Author(s).)

Chronic liver disease (CLD) is an ignored epidemic. Premature mortality is considerable and in the United Kingdom (UK) liver disease is in the top three for inequitable healthcare alongside heart and respiratory disease. Fifty percentage of patients with CLD are first diagnosed with cirrhosis after an emergency presentation translating to poorer patient outcomes. Traditional models of care have been based in secondary care when the need is at community level. Investigating patients for disease based on their risk factors at a population level in the community will identify its presence early when there is potential reversibility. Innovation is needed in three broad areas to improve clinical care in this area: better access to diagnostics within the community, integrating diagnostics across primary and secondary care and utilizing digital healthcare to enhance patient care. In this article, we describe how the Integrated Diagnostics for Early Detection of Liver Disease (ID-LIVER) project, funded by UK Research and Innovation, is developing solutions in Greater Manchester to approach the issue of diagnosis of liver disease at a population level. The ambition is to build on innovative pathways previously established in Nottingham by bringing together NHS organizations, academic partners and commercial organizations. The motivation is to co-create and implement a commercial solution that integrates multimodal diagnostics via cutting edge data science to drive growth and disrupt the currently inadequate model. The ambitious vision is for this to be widely adopted for early diagnosis and stratification of liver disease at a population level within the NHS., Competing Interests: ING and JM have received investigator led funding research from Gilead Sciences. Gilead had no intellectual input into the study concept or design, interpretation of the results or editing the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bennett, Purssell, Street, Piper Hanley, Morling, Hanley, Athwal and Guha.)

Introduction: The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD)., Methods and Analysis: The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment., Ethics and Dissemination: All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals., Conclusion: This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Aims/hypothesis: Approximately 50% of organ donors develop hyperglycaemia in intensive care, which is managed with insulin therapy. We aimed to determine the relationships between donor insulin use (DIU) and graft failure in pancreas transplantation., Methods: UK Transplant Registry organ donor data were linked with national data from the UK solid pancreas transplant programme. All pancreas transplants performed between 2004 and 2016 with complete follow-up data were included. Logistic regression models determined associations between DIU and causes of graft failure within 3 months. Area under the receiver operating characteristic curve (aROC) and net reclassification improvement (NRI) assessed the added value of DIU as a predictor of graft failure., Results: In 2168 pancreas transplant recipients, 1112 (51%) donors were insulin-treated. DIU was associated with a higher risk of graft loss from isolated islet failure: OR (95% CI), 1.79 (1.05, 3.07), p = 0.03, and this relationship was duration/dose dependent. DIU was also associated with a higher risk of graft loss from anastomotic leak (2.72 [1.07, 6.92], p = 0.04) and a lower risk of graft loss from thrombosis (0.62 [0.39, 0.96], p = 0.03), although duration/dose-dependent relationships were only identified in pancreas transplant alone/pancreas after kidney transplant recipients with grafts failing due to thrombosis (0.86 [0.74, 0.99], p = 0.03). The relationships between donor insulin characteristics and isolated islet failure remained significant after adjusting for potential confounders: DIU 1.75 (1.02, 2.99), p = 0.04; duration 1.08 (1.01, 1.16), p = 0.03. In multivariable analyses, donor insulin characteristics remained significant predictors of lower risk of graft thrombosis in pancreas transplant alone/pancreas after kidney transplant recipients: DIU, 0.34 (0.13, 0.90), p = 0.03; insulin duration/dose, 0.02 (0.001, 0.85), p = 0.04. When data on insulin were added to models predicting isolated islet failure, a significant improvement in discrimination and risk reclassification was observed in all models: no DIU aROC 0.56; DIU aROC 0.57, p = 0.86; NRI 0.28, p < 0.00001; insulin duration aROC 0.60, p = 0.47; NRI 0.35, p < 0.00001., Conclusions/interpretation: DIU predicts graft survival in pancreas transplant recipients. This assessment could help improve donor selection and thereby improve patient and graft outcomes.

Background: Posterior urethral valves (PUVs) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair kidney development., Objective: To identify genetic variants associated with kidney injury in patients with obstructive uropathy., Design Setting and Participants: We included 487 patients born in 1981 or later who underwent pyeloplasty or valve resection before 18 yr of age in the discovery phase, 102 PUV patients in a first replication phase, and 102 in a second replication phase., Outcome Measurements and Statistical Analysis: Signs of kidney injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m 2 , high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning at <45%. We used χ 2 tests to calculate p values and odds ratios for >600 000 single-nucleotide polymorphisms (SNPs) in the discovery sample comparing patients with and without signs of kidney injury within 5 yr after surgery. We performed stratified analyses for PUV and UPJO and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples for the associated SNPs, and RNA and protein expression analyses for the associated gene in fetal tissues., Results and Limitations: Despite the small and nonhomogeneous sample, we observed suggestive associations for six SNPs in three loci, of which rs6874819 in the CDH12 gene was the most clear ( p  = 7.5 × 10 -7 ). This SNP also seemed to be associated with time to kidney injury in the PUV discovery and replication samples. RNA expression analyses showed clear CDH12 expression in fetal kidneys, which was confirmed by protein immunolocalization., Conclusions: This study identified CDH12 as a candidate gene for kidney injury in PUV., Patient Summary: We found that variants of the CDH12 gene increase the risk of kidney injury in patients with extra flaps of tissue in the urethra (posterior urethral valves). This is the first report on this gene in this context. Our study provides interesting new information about the pathways involved and important leads for further research for this condition., (© 2021 The Author(s).)

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721)., (© 2016 by the American Association for the Study of Liver Diseases.)

Genome-wide association studies implicate the CYP17A1 gene in human blood pressure regulation although the causative polymorphisms are as yet unknown. We sought to identify common polymorphisms likely to explain this association. We sequenced the CYP17A1 locus in 60 normotensive individuals and observed 24 previously identified single-nucleotide polymorphisms with minor allele frequency >0.05. From these, we selected, for further studies, 7 polymorphisms located ≤ 2 kb upstream of the CYP17A1 transcription start site. In vitro reporter gene assays identified 3 of these (rs138009835, rs2150927, and rs2486758) as having significant functional effects. We then analyzed the association between the 7 polymorphisms and the urinary steroid metabolites in a hypertensive cohort (n=232). Significant associations included that of rs138009835 with aldosterone metabolite excretion; rs2150927 associated with the ratio of tetrahydrodeoxycorticosterone to tetrahydrodeoxycortisol, which we used as an index of 17α-hydroxylation. Linkage analysis showed rs138009835 to be the only 1 of the 7 polymorphisms in strong linkage disequilibrium with the blood pressure-associated polymorphisms identified in the previous studies. In conclusion, we have identified, characterized, and investigated common polymorphisms at the CYP17A1 locus that have functional effects on gene transcription in vitro and associate with corticosteroid phenotype in vivo. Of these, rs138009835--which we associate with changes in aldosterone level--is in strong linkage disequilibrium with polymorphisms linked by genome-wide association studies to blood pressure regulation. This finding clearly has implications for the development of high blood pressure in a large proportion of the population and justifies further investigation of rs138009835 and its effects., (© 2016 The Authors.)

Background & Aims: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes., Methods: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes., Results: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics., Conclusions: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.

Unlabelled: Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise, with differentiated hepatocyte-like cells currently considered to be "fetal-like" in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation., Conclusion: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein "toolkit" for determining human hepatocyte maturity in cultured cells., (Copyright © 2013 by the American Association for the Study of Liver Diseases.)

Unlabelled: Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression., Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background: Asthma pathogenesis involves gene and environmental interactions. A disintegrin and metalloprotease 33 (ADAM33)/Adam33 is a susceptibility gene for asthma and bronchial hyperresponsiveness in human beings and mice. ADAM33 is almost exclusively expressed in mesenchymal cells, including mesenchymal progenitors in developing lungs., Objective: Because maternal allergy is a risk factor for asthma, we hypothesized that an allergic environment affects ADAM33/Adam33 expression during human and mouse lung development., Methods: Human embryonic/fetal lung (HEL) tissues were collected from first-trimester terminations of pregnancy. These were processed immediately or used for explant culture +/- IL-13. MF1 mice or ovalbumin-sensitized A/J mice (Bronchial hyperresponsivness (Bhr)1/Adam33 locus-positive) were time-mated and challenged with ovalbumin (A/J mice only) during pregnancy. Lungs were harvested at different times during gestation and post partum. ADAM33/Adam33 expression was analyzed by using reverse transcriptase quantitative polymerase chain reaction and Western blotting., Results: ADAM33 mRNA was detectable in HELs in the pseudoglandular stage of development and showed a significant increase from 7 to 9 weeks postconception. IL-13 significantly suppressed ADAM33 mRNA in HEL explants. In developing murine lungs, Adam33 mRNA and protein expression increased significantly in the early pseudoglandular stage and showed another large increase post partum. In A/J mice, maternal allergy significantly suppressed Adam33 mRNA in lungs of newborn pups, whereas processed Adam33 protein increased and several smaller isoforms were detected., Conclusion: Adam33/Adam33 shows 2 significant increments in expression during lung morphogenesis, suggesting important developmental regulation. The ability of maternal allergy or exogenous IL-13 to suppress Adam33/ADAM33 mRNA but enhance Adam33 processing suggests a gene-environment interaction that may be relevant for asthma pathogenesis.

Human sexual differentiation is a critical process whereby a strict dimorphism is established that enables future reproductive success as phenotypic males and females. Significant components of this differentiation pathway unfold during the first three months of gestation when they are sensitive to disruption by abnormal hormonal influences. Excessive exposure of female development to androgens in conditions such as congenital adrenal hyperplasia causes virilization. However, recently we have suggested that female development normally takes place in the presence of low, yet significant, levels of androgen, implying a need for strict regulation to avoid virilization and the potential for a biological role of androgens in females that has not been fully elucidated. Here, we review androgen-dependent male differentiation of the external genitalia in humans, and link this to current understanding of female development and steroidogenesis in the developing adrenal cortex.

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations.

Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.