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Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic small vessel disease caused by variants in theNOTCH3gene. Patients with CADASIL experience recurrent strokes, developing into cognitive defect and vascular dementia. CADASIL is a late-onset vascular condition, but migraine and brain MRI lesions appear in CADASIL patients as early as their teens and twenties, suggesting an abnormal neurovascular interaction at the neurovascular unit (NVU) where microvessels meet the brain parenchyma. To understand the molecular mechanisms of CADASIL, we established induced pluripotent stem cell (iPSC) models from CADASIL patients and differentiated the iPSCs into the major NVU cell types including brain microvascular endothelial-like cells (BMECs), vascular mural cells (MCs), astrocytes and cortical projection neurons. We then built anin vitroNVU model by co-culturing different neurovascular cell types in Transwells and evaluated the blood brain barrier (BBB) function by measuring transendothelial electrical resistance (TEER). Results showed that, while the wild-type MCs, astrocytes and neurons could all independently and significantly enhance TEER values of the iPSC-BMECs, such capability of MCs from iPSCs of CADASIL patients was significantly impeded. Additionally, the barrier function of the BMECs from CADASIL iPSCs was significantly impaired, accompanied with disorganised tight junctions in iPSC-BMECs, which could not be effectively rescued by the wild-type MCs, astrocytes and neurons. Our findings provide new insight into early disease pathologies on the neurovascular interaction and BBB function at the molecular and cellular levels for CADASIL, which helps inform future therapeutic development.

Hepatocyte nuclear factor 1B(HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. HeterozygousHNF1Bmutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygousHNF1Bmutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family withHNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.

Kabuki syndrome (KS) is a rare multisystem disorder, characterized by intellectual disability, growth delay, and distinctive craniofacial features. It is mostly caused by de novo mutations of KMT2D, which is responsible for histone H3lysine 4 mono-methylation (H3K4me1) that marks active and poised enhancers. We assessed the impact of KMT2D mutations on chromatin and transcriptional regulation in a cohort of multiple KS1 tissues, including primary patient samples and disease-relevant lineages, namely cortical neurons (iN), neural crest stem cells (NCSC), and mesenchymal cells (MC). In parallel, we generated an isogenic line derived from human embryonic stem cells (hESC) for the stepwise characterization of neural precursors and mature neurons. We found that transcriptional dysregulation was particularly pronounced in cortical neurons and widely affected synapse activity pathways. This was consistent with highly specific alterations of spontaneous network-bursts patterns evidenced by Micro-electrode-array (MEA)-based neural network. Profiling of H3K4me1 unveiled the almost complete uncoupling between this chromatin mark and the effects on transcription, which is instead reflected by defects in H3K27ac. Finally, we identified the direct targets of KMT2D in mature cortical neurons, uncovering TEAD2 as the main mediator of KMT2D haploinsufficiency. Our results uncover the multi-tissue architecture of KS1 dysregulation and define a unique electrical phenotype and its molecular underpinnings for the cortical neuronal lineage.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the spent medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient media for 48 hours to replicate an early, undirected dissolution of pluripotency.We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of 4 E8-against 4 E6-enriched biomarkers provides 16 ratio abundances which are each robustly diagnostic for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to recovery assays.

BackgroundHeterozygous variants in the cardiac ryanodine receptor gene (RYR2) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Most pathogenic RYR2 variants are missense variants which result in a gain of function, causing ryanodine receptors to be increasingly sensitive to activation by calcium, have an increased open probability and an increased propensity to develop calcium waves. However, some RYR2 variants can lead to arrhythmias by a loss of function mechanism.ObjectiveTo understand the mechanism by which a novel nonsense variant in RYR2 p.(Arg4790Ter) leads to ventricular arrhythmias.MethodsHuman induced pluripotent stem cells (hiPSCs) harbouring the novel nonsense variant in RYR2 were differentiated into cardiomyocytes (RYR2-hiPSC-CMs) and molecular and calcium handling properties were studied.ResultsRYR2-hiPSC-CMs displayed significant calcium handling abnormalities at baseline and following treatment with isoproterenol. Treatment with carvedilol and nebivolol resulted in a significant reduction in calcium handling abnormalities in the RYR2-hiPSC-CMs. Expression of the mutant RYR2 allele was confirmed at the mRNA level and partial silencing of the mutant allele resulted in a reduction in calcium handling abnormalities at baseline.ConclusionThe nonsense variant behaves similarly to other gain of function variants in RYR2. Carvedilol and nebivolol may be suitable treatments for patients with gain of function RYR2 variants.

At implantation, trophoblast derived from the trophectoderm of the blastocyst-stage embryo invades the endometrium to establish pregnancy. To understand how embryos breach the endometrial epithelium, we modelled human implantation using blastocysts or trophoblast stem cell spheroids cultured with endometrial epithelial cells (EEC). Blastocyst invasion of the EEC layer was initiated by multinuclear syncytiotrophoblast. Spheroids also invaded the epithelium with syncytiotrophoblast, and EEC induced upregulation of syncytiotrophoblast markers. Modelling implantation in silico using blastocyst and EEC transcriptomes revealed gene networks that exhibited greater connectivity and organisation in trophectoderm of the polar region of the embryonic axis. However, gene ontologies and machine learning suggested that EEC drives syncytiotrophoblast differentiation in polar and mural trophectoderm. This is the first evidence for endometrial epithelium-induced trophectoderm differentiation to invasive syncytiotrophoblast as the cellular mechanism of embryonic breaching of the endometrium in humans, with implications for reproductive medicine and our understanding of human embryonic development.

Epigenetic modification is a key driver of differentiation and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the ACAN –10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating association of ARID5B with SIRT1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, whilst altering ECM composition and suppressing GAG content during cartilage development. These results suggest that SIRT1 activity can be beneficial to cartilage development and matrix protein synthesis but tailored by addition of other positive GAG mediators.

Bone Morphogenetic Proteins (BMPs) are members of the Transforming Growth Factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise perturbation of the BMP signalling pathway. Here, we develop a novel optogenetic BMP signalling system (optoBMP) that enables rapid induction of the canonical BMP signalling pathway through illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signalling pathway through variable light stimulation. Optogenetic control of BMP signalling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

Human embryonic stem cells (hESCs) derived from the pluripotent Inner cell mass (ICM) of the blastocyst are fundamental tools for understanding human development, yet are not identical to their tissue of origin. To investigate this divergence we compared the transcriptomes of genetically paired ICM and trophectoderm (TE) samples with three hESC lines: MAN1, HUES3 and HUES7 at similar passage. We generated inferred interactome networks using transcriptomic data unique to the ICM or TE, and defined a hierarchy of modules (highly connected regions with shared function). We compared network properties and the modular hierarchy and show that the three hESCs had limited overlap with ICM specific transcriptome (6%-12%). However this overlap was enriched for network properties related to transcriptional activity in ICM (p=0.016); greatest in MAN1 compared to HUES3 (p=0.048) or HUES7 (p=0.012). The hierarchy of modules in the ICM interactome contained a greater proportion of MAN1 specific gene expression (46%) compared to HUES3 (28%) and HUES7 (25%) (p=9.0×10−4).These findings show that traditional methods based on transcriptome overlap are not sufficient to identify divergence of hESCs from ICM. Our approach also provides a valuable approach to the quantification of differences between hESC lines.And Manchester Academic Health Sciences Centre