Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Endothelial Network Formation.

UNLABELLED: Vascular smooth muscle cells (SMCs) from distinct anatomic locations derive from different embryonic origins. Here we investigated the respective potential of different embryonic origin-specific SMCs derived from human embryonic stem cells (hESCs) to support endothelial network formation in vitro. SMCs of three distinct embryological origins were derived from an mStrawberry-expressing hESC line and were cocultured with green fluorescent protein-expressing human umbilical vein endothelial cells (HUVECs) to investigate the effects of distinct SMC subtypes on endothelial network formation. Quantitative analysis demonstrated that lateral mesoderm (LM)-derived SMCs best supported HUVEC network complexity and survival in three-dimensional coculture in Matrigel. The effects of the LM-derived SMCs on HUVECs were at least in part paracrine in nature. A TaqMan array was performed to identify the possible mediators responsible for the differential effects of the SMC lineages, and a microarray was used to determine lineage-specific angiogenesis gene signatures. Midkine (MDK) was identified as one important mediator for the enhanced vasculogenic potency of LM-derived SMCs. The functional effects of MDK on endothelial network formation were then determined by small interfering RNA-mediated knockdown in SMCs, which resulted in impaired network complexity and survival of LM-derived SMC cocultures. The present study is the first to show that SMCs from distinct embryonic origins differ in their ability to support HUVEC network formation. LM-derived SMCs best supported endothelial cell network complexity and survival in vitro, in part through increased expression of MDK. A lineage-specific approach might be beneficial for vascular tissue engineering and therapeutic revascularization. SIGNIFICANCE: Mural cells are essential for the stabilization and maturation of new endothelial cell networks. However, relatively little is known of the effect of the developmental origins of mural cells on their signaling to endothelial cells and how this affects vessel development. The present study demonstrated that human smooth muscle cells (SMCs) from distinct embryonic origins differ in their ability to support endothelial network formation. Lateral mesoderm-derived SMCs best support endothelial cell network complexity and survival in vitro, in part through increased expression of midkine. A lineage-specific approach might be beneficial for vascular tissue engineering and therapeutic revascularization.This work was supported by the British Heart Foundation (BHF), the UK Medical Research Council (MRC) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding.This is the author accepted manuscript. The final version is available from AlphaMed Press via http://dx.doi.org/10.5966/sctm.2015-028

SARS-CoV-2 Infects Human Pluripotent Stem Cell-Derived Cardiomyocytes, Impairing Electrical and Mechanical Function

COVID-19 patients often develop severe cardiovascular complications, but it remains unclear if these are caused directly by viral infection or are secondary to a systemic response. Here, we examine the cardiac tropism of SARS-CoV-2 in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and smooth muscle cells (hPSC-SMCs). We find that that SARS-CoV-2 selectively infects hPSC-CMs through the viral receptor ACE2, whereas in hPSC-SMCs there is minimal viral entry or replication. After entry into cardiomyocytes, SARS-CoV-2 is assembled in lysosome-like vesicles and egresses via bulk exocytosis. The viral transcripts become a large fraction of cellular mRNA while host gene expression shifts from oxidative to glycolytic metabolism and upregulates chromatin modification and RNA splicing pathways. Most importantly, viral infection of hPSC-CMs progressively impairs both their electrophysiological and contractile function, and causes widespread cell death. These data support the hypothesis that COVID-19-related cardiac symptoms can result from a direct cardiotoxic effect of SARS-CoV-2

Cardiovascular ACE2 receptor expression in patients undergoing heart transplantation

Abstract: Aims: Membrane‐bound angiotensin‐converting enzyme (ACE)2 is the main cellular access point for SARS‐CoV‐2, but its expression and the effect of ACE inhibition have not been assessed quantitatively in patients with heart failure. The aim of this study was to characterize membrane‐bound ACE2 expression in the myocardium and myocardial vasculature in patients undergoing heart transplantation and to assess the effect of pharmacological ACE inhibition. Methods and results: Left ventricular (LV) tissue was obtained from 36 explanted human hearts from patients undergoing heart transplantation. Immunohistochemical staining with antibodies directed against ACE2 co‐registered with cardiac troponin T (cTnT) and α‐smooth muscle cell actin (SMA) was performed across the entire cohort. ACE2 receptor expression was quantitatively assessed throughout the myocardium and vasculature. ACE2 was consistently expressed throughout the LV myocardium (28.3% ± 22.2% of cardiomyocytes). ACE2 expression was also detected in small calibre blood vessels (range, 2–9 μm), albeit at quantitatively much lower levels (5% ± 9% of blood vessels). There was no significant difference in ACE2 expression between patients receiving ACE inhibitors prior to transplantation and ACE inhibitor‐negative controls (P > 0.05). ACE2 expression did not differ significantly between the different diagnostic groups as the underlying reason for heart transplantation (ANOVA > 0.05). N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) (R2 = 0.37, P = 0.0006) and pulmonary capillary wedge pressure (PCWP) (R2 = 0.13, P = 0.043) assessed by right heart catheterization were significantly correlated with greater ACE2 expression in cardiomyocytes. Conclusions: These data provide a comprehensive characterization of membrane‐bound cardiac ACE2 expression in patients with heart failure with no demonstrable effect exerted by ACE inhibitors

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium

Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration.

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them a promising adjuvant therapeutic for cardiac repair.: This work was supported by the British Heart Foundation (BHF; Grants NH/11/1/28922, G1000847, FS/13/29/30024 and FS/18/46/33663), Oxford-Cambridge Centre for Regenerative Medicine (RM/13/3/30159), the UK Medical Research Council (MRC) and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding (SS), as well as National Institutes of Health Grants P01HL094374, P01GM081619, R01HL12836 and a grant from the Fondation Leducq Transatlantic Network of Excellence (CEM). J.B. was supported by a Cambridge National Institute for Health Research Biomedical Research Centre Cardiovascular Clinical Research Fellowship and subsequently, by a BHF Studentship (Grant FS/13/65/30441). DI received a University of Cambridge Commonwealth Scholarship. LG is supported by BHF Award RM/l3/3/30159 and LPO is funded by a Wellcome Trust Fellowship (203568/Z/16/Z). NF was supported by BHF grants RG/13/14/30314. NL was supported by the Biotechnology and Biological Sciences Research Council (Institute Strategic Programmes BBS/E/B/000C0419 and BBS/E/B/000C0434). SS and MB were supported by the British Heart Foundation Centre for Cardiovascular Research Excellence. Core support was provided by the Wellcome-MRC Cambridge Stem Cell Institute (203151/Z/16/Z), The authors thank Osiris for provision of the primary mesenchymal stem cells (59

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids

Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro

The Role of Human Embryonic Stem Cell-derived Epicardium in Myocardial Graft Development

BH

Proinflammatory cytokines driving cardiotoxicity in Covid-19.

AIMS: Cardiac involvement is common in patients hospitalised with COVID-19 and correlates with an adverse disease trajectory. While cardiac injury has been largely attributed to direct viral cytotoxicity, serum-induced cardiotoxicity secondary to serological hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. METHODS AND RESULTS: To investigate serological drivers of cardiotoxicity in COVID-19 we have established a robust bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell (hESC)-derived cardiomyocytes. We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability independent of viral transduction, an effect that was also seen in acute respiratory distress syndrome (ARDS). Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-β, IL-10, CRP and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partly rescued the phenotype and preserved cardiomyocyte viability and function. Bulk RNA sequencing of serum-treated cardiomyocytes elucidated specific pathways involved in the COVID-19 response impacting cardiomyocyte viability, structure and function. The observed effects of serum-induced cytotoxicity were cell-type selective as serum exposure did not adversely affect microvascular endothelial cell viability but resulted in endothelial activation and a procoagulant state. CONCLUSION: These results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and characterise the downstream activated pathways in human cardiomyocytes. The serum signature of patients with severe disease indicates possible targets for therapeutic intervention

Epicardially secreted fibronectin drives cardiomyocyte maturation in 3D-engineered heart tissues.

Ischemic heart failure is due to irreversible loss of cardiomyocytes. Preclinical studies showed that human pluripotent stem cell (hPSC)-derived cardiomyocytes could remuscularize infarcted hearts and improve cardiac function. However, these cardiomyocytes remained immature. Incorporating hPSC-derived epicardial cells has been shown to improve cardiomyocyte maturation, but the exact mechanisms are unknown. We posited epicardial fibronectin (FN1) as a mediator of epicardial-cardiomyocyte crosstalk and assessed its role in driving hPSC-derived cardiomyocyte maturation in 3D-engineered heart tissues (3D-EHTs). We found that the loss of FN1 with peptide inhibition F(pUR4), CRISPR-Cas9-mediated FN1 knockout, or tetracycline-inducible FN1 knockdown in 3D-EHTs resulted in immature cardiomyocytes with decreased contractile function, and inefficient Ca2+ handling. Conversely, when we supplemented 3D-EHTs with recombinant human FN1, we could recover hPSC-derived cardiomyocyte maturation. Finally, our RNA-sequencing analyses found FN1 within a wider paracrine network of epicardial-cardiomyocyte crosstalk, thus solidifying FN1 as a key driver of hPSC-derived cardiomyocyte maturation in 3D-EHTs