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The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM + population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles. The liver parenchyma consists of several cell types, but the origin of this tissue in humans is unclear. Here, the authors perform single cell RNA sequencing of human fetal and adult liver to identify a hepatobiliary hybrid progenitor population of cells, which have a similar gene signature to mouse oval cells.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFβ signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne. Although acne vulgaris is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here the authors show that GATA6 is involved in maintaining homeostasis of the upper pilosebaceous unit of human skin and may contribute to acne pathogenesis.

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC‐Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC‐Heps generated using a chemically defined four‐step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)‐diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune‐privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP‐compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. Stem Cells Translational Medicine 2019;8:124&14

We have previously shown that E-cadherin regulates the naive pluripotent state of mouse embryonic stem cells (mESCs) by enabling LIF-dependent STAT3 phosphorylation, with E-cadherin null mESCs exhibiting over 3000 gene transcript alterations and a switch to Activin/Nodal-dependent pluripotency. However, elucidation of the exact mechanisms associated with E-cadherin function in mESCs is compounded by the difficulty in delineating the structural and signalling functions of this protein. Here we show that mESCs treated with the E-cadherin neutralising antibody DECMA-1 or the E-cadherin binding peptide H-SWELYYPLRANL-NH 2 (Epep) exhibit discrete profiles for pluripotent transcripts and NANOG protein expression, demonstrating that the type of E-cadherin inhibitor employed dictates the cellular phenotype of mESCs. Alanine scanning mutation of Epep revealed residues critical for Tbx3, Klf4 and Esrrb transcript repression, cell-cell contact abrogation, cell survival in suspension, STAT3 phosphorylation and water solubility. STAT3 phosphorylation was found to be independent of loss of cell-cell contact and Activin/Nodal-dependent pluripotency and a peptide is described that enhances STAT3 phosphorylation and Nanog transcript and protein expression in mESCs. These peptides represent a useful resource for deciphering the structural and signalling functions of E-cadherin and demonstrate that complete absence of E-cadherin protein is likely required for hierarchical signalling pathway alterations in mESCs.

Although human dermis contains distinct fibroblast subpopulations, the functional heterogeneity of fibroblast lines from different donors is under-appreciated. We identified one commercially sourced fibroblast line (c64a) that failed to express α-smooth muscle actin (α-SMA), a marker linked to fibroblast contractility, even when treated with transforming growth factor-β1 (TGF-β1). Gene expression profiling identified insulin-like growth factor 1 (IGF1) as being expressed more highly, and Asporin (ASPN) and Wnt family member 4 (WNT4) expressed at lower levels, in c64a fibroblasts compared to three fibroblast lines that had been generated in-house, independent of TGF-β1 treatment. TGF-β1 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) in c64a cells to a greater extent than in the other lines. The c64a gene expression profile did not correspond to any dermal fibroblast subpopulation identified by single-cell RNAseq of freshly isolated human skin cells. In skin reconstitution assays, c64a fibroblasts did not support epidermal stratification as effectively as other lines tested. In fibroblast lines generated in-house, shRNA-mediated knockdown of IGF1 increased α-SMA expression without affecting epidermal stratification. Conversely, WNT4 knockdown had no consistent effect on α-SMA expression, but increased the ability of fibroblasts to support epidermal stratification. Thus, by comparing the properties of different lines of cultured dermal fibroblasts, we have identified IGF1 and WNT4 as candidate mediators of two distinct dermal functions: myofibroblast formation and epidermal maintenance.

IntroductionPrimary sclerosing cholangitis (PSC) is a chronic inflammatory condition of the bile ducts leading to fibrosis and end stage liver disease. A lack of robust non-invasive biomarkers has been hindering disease monitoring and development of optimal therapies. We have previously noted that the high levels of faecal calprotectin (fcal) seen in PSC-IBD patients belie the mild or quiescent intestinal inflammation. An unsupervised proteomics study identified biliary calprotectin as a potential biomarker. Here, we test the hypothesis that fcal is a marker of biliary injury in PSC.MethodsWe analysed paired endoscopic activity data (UCEIS) and fcal results of patients with PSC-IBD (n=20) or UC (n=20) who underwent colitis surveillance in the context of a colitis surveillance pilot study. Relevant clinical data was recorded prospectively. Recruiting consecutive patients attending for ERCP (n=6) allowed for the concomitant testing of biliary and faecal calprotectin.ResultsAs expected, fcal strongly correlated with severity of mucosal injury (UCEIS) in UC [r=0.82, 95% CI(0.58, 0.92), p<0.0001]. However, the correlation was weaker in PSC-IBD [r=0.59, 95% CI(0.19, 0.82), p=0.006]. Moreover, in patients with PSC-IBD and quiescent colitis (UCEIS: 0–1) fcal concentration was significantly higher in comparison to UC patients with comparable endoscopic activity [279 ug/g (10, 1560) vs. 30 (10, 161), p=0.015)]. A trend towards abnormal liver biochemistry was seen in those PSC-IBD with higher fcal [ALP: 250IU/L (113, 561) vs. 83 (59, 170), p=0.06, GGT: 351 U/L (117, 1014) vs. 51 (29, 153) p=0.02, AST: 53 U/L (26, 85) vs. 37 (22, 43), p=ns]. UC patients with quiescent colitis and fcal >150 had a higher risk of colitis relapse in 12 months [HR=7.6, 95% CI(1.8, 33.6)] in comparison to those with fcal <150. However, in patients with PSC-IBD and quiescent colitis a fcal >150 was associated instead with a higher risk of cholangitis associated complications (need for antibiotics or stent insertion), HR=6.5, 95% CI(1.3, 33.9). Strikingly, biliary calprotectin concentration showed a strong correlation with fcal concentration (r=0.90, p=0.04). Interestingly, immunostaining of biliary brushings for calprotectin demonstrated positive staining in cholangiocytes as well as neutrophils and macrophages.ConclusionIn patients with PSC-IBD and quiescent colitis the identification of a raised fcal is likely to herald complications of inflammation in the bile ducts rather than the colon. In this setting, fcal may be a valuable prognostic biomarker of cholangitis. Additionally, our data suggest that in PSC, the source of raised fcal may also be the damaged biliary epithelium.

Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients. Competing Interest Statement The authors have declared no competing interest.

The Ward group have previously demonstrated that E-cadherin regulates the naïve pluripotent state of mouse Embryonic Stem (mES) cells via activation of the LIF/STAT3 pathway. Loss of E-cadherin in mES cells leads to a 'primed' pluripotent phenotype characterized by LIF-independent self-renewal through ACTIVIN/NODAL signalling, similar to mouse Epiblast Stem (EpiS) and human (h)ES cells. However, the exact mechanism of E-cadherin's regulation of pluripotency in both mouse and human ES cells has remained elusive. In this study we describe a novel network modelling approach of transcriptomic analysis using gene expression changes shared between three independently derived E-cadherin negative cell lines (collectively termed ESDeltaEcad cells). Our models predicted regulation of pluripotency pathways via the co-activator protein EP300. We show that E-cadherin regulates the cell localisation and binding of EP300 to NANOG-OCT4-SOX2 DNA clusters. EP300 also functions to phosphorylate SMAD2 in the absence of E-cadherin and to inhibit pluripotency-associated transcripts in Ecad-/- mES cells. Using comparative network analysis of E-cadherin antagonism in mouse and human transcriptomes we have identified that E-cadherin plays a major role in regulating the transcriptional circuitry between naive and primed ES cell states. We show that this is likely to be mediated at least partly by positive activation of NCOA3 and ESRRB. Differential transcript expression in Ecad-/- mES cells correlates with loss of ESRRB protein expression and altered cellular localisation of NCOA3, both regulators of naive pluripotency. We have gone on to show through peptide antagonism that while E-cadherin is an important upstream regulator of genes critical in defining the naïve pluripotent state, this action is not necessarily dependent on the loss of cell-cell contact associated with E-cadherin inhibition. We present the first data that E-cadherin regulates naïve mES cell pluripotency by regulation of EP300, ESRRB and NCOA3 activity. Furthermore we have shown E-cadherin to inhibit the naïve transcriptional phenotype in hES cells. Peptides that target E-cadherin also provide a valuable tool for controlling the transition between naive and pluripotent states in mouse and human ES cells.

Off-grid solar applications offer undeniable benefits: they are a safer, healthier, and cheaper alternative to kerosene or candles and also can lead to the direct and indirect creation of jobs. However, the rapidly increasing rate of off-grid solar system production highlights the need to create a system where those products and the resources that compose them are kept in use for as long as possible: a circular economy of off-grid solar applications. This chapter explores the key drivers for adoption, business models currently being applied, and market penetration of off-grid solar applications and the opportunities created by these innovative models. The chapter then goes on to explore the end-of-life challenges for these products and the proactive approaches that are being taken to ensure that these valuable products, components, and materials are safely kept in use for longer. It builds on the results and insights of an ongoing pilot project financially supported by CDC Plus funding provided by UK Aid.

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D PEG-DA fabricated inverted colloid crystal (ICC) scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications.