William G. Bernard

Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening. HIGHLIGHTED ARTICLE: Epicardial cells and their derivatives are generated from human pluripotent stem cells, an approach that has potential applications in the field of regenerative medicine.

An iPSC-derived vascular model of Marfan syndrome identifies key mediators of smooth muscle cell death

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in FBN1, which encodes the extracellular matrix protein fibrillin-1. To investigate the pathogenesis of aortic aneurysms in MFS, we generated a vascular model derived from human induced pluripotent stem cells (MFS-hiPSCs). Our MFS-hiPSC-derived smooth muscle cells (SMCs) recapitulated the pathology seen in Marfan aortas, including defects in fibrillin-1 accumulation, extracellular matrix degradation, transforming growth factor-β (TGF-β) signaling, contraction and apoptosis; abnormalities were corrected by CRISPR-based editing of the FBN1 mutation. TGF-β inhibition rescued abnormalities in fibrillin-1 accumulation and matrix metalloproteinase expression. However, only the noncanonical p38 pathway regulated SMC apoptosis, a pathological mechanism also governed by Krüppel-like factor 4 (KLF4). This model has enabled us to dissect the molecular mechanisms of MFS, identify novel targets for treatment (such as p38 and KLF4) and provided an innovative human platform for the testing of new drugs.

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Histamine and prostaglandin E2 (PGE2), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. Highlighted article: Novel optimized inducible knockdown and knockout platforms are developed and used to assess gene function in human pluripotent stem cells and their differentiated progeny.

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

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.

Temporal and Embryonic Lineage-Dependent Regulation of Human Vascular SMC Development by NOTCH3

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8–9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner.

25 Embryological origin of human smooth muscle cells influences their ability to support vasculogenesis

Background We hypothesised that the distinct embryonic origins of vascular smooth muscle cells (SMCs) at different anatomic locations, contribute to differences in vascular development and disease. Here we investigated the respective vasculogeneic potential of different embryonic-origin specific SMC derived from human embryonic stem cells (hESCs) to support vasculogenesis in vitro. Methods and results SMCs of three distinct embryonic origins were derived from an mStrawberry-expressing hESC line and were co-cultured with GFP-expressing human umbilical vein endothelial cells (HUVEC) to investigate the effects of distinct SMC subtypes on endothelial network formation. As demonstrated by quantitative data, LM-derived SMCs best supported HUVEC network survival in 3D co-culture in matrigel. In addition, LM-SMCs facilitated more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. The effects of the LM-SMCs on HUVECs were at least in part paracrine in nature. A TaqMan Array was performed to identify possible mediators responsible for the differential effects of the SMC lineages and a microarray to determine lineage-specific angiogenesis gene signatures. Midkine (MDK) was identified as one important mediator for the enhanced vasculogenic effect of LM-SMCs. Subsequent siRNA-mediated knockdown of MDK in SMCs resulted in a decrease of total HUVEC network area and number of branch points in LM-derived SMC co-cultures. Conclusion SMCs from distinct embryonic origins differ in their ability to support HUVEC network formation. LM-derived SMCs best support vasculogenesis in vitro, in part through increased expression of MDK.

191 NOTCH3 REGULATES HUMAN VASCULAR SMOOTH MUSCLE CELL DEVELOPMENT AND FUNCTIONALITY IN A LINEAGE SPECIFIC MANNER: IMPLICATIONS IN THE PATHOLOGY OF CADASIL

Introduction CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is characterised by degeneration of the smooth muscle cells (SMC) within the cerebrovasculature, resulting in recurrent subcortical ischemic episodes and vascular dementia. Mutations of the single-pass transmembrane protein Notch3 have been described as the causative component of CADASIL. Whilst Notch3 is expressed in all vascular SMCs, the major pathology observed in CADASIL is primarily located within the cerebrovasculature, with other vascular beds remaining predominantly unaffected. SMCs have diverse embryological origins, and we hypothesise that these origins may regulate the physiology and regional disease pre-disposition within this cell type. Methods To address this anomaly, we have utilised our recently developed protocol which allows the generation of SMCs, from human embryonic stem cells (hESC), through multiple developmental lineage intermediates: lateral-plate (LM) and paraxial mesoderm (PM), and neuroectoderm (NE). By integrating either siRNA mediated knockdown of Notch3 at critical time points, or the generation of stably expressing Notch3 shRNA hESC lines, we are beginning to address the role of Notch3 in human SMC development and pathology. Results We observe that expression levels of components of the notch signalling pathway vary strikingly between LM-, PM- and NE-derived populations during differentiation, with a distinct peak in Notch3 expression observed early in the development of NE-derived populations. Notch3 knockdown, through either siRNA or shRNA approaches, inhibits the differentiation of NE-derived SMCs as observed by markedly impaired expression of SMC-specific markers, whilst LM- and PM-derived population show an additional lineage specific phenotype. Conclusions/Implications These results provide further insights into the pathology observed in CADASIL patients, with NE-derived populations showing Notch3 dependence for differentiation, and maturation. We believe that this in vitro lineage-specific Notch3 knockdown phenotype may translate to the pathophysiology observed during disease. To further elucidate this, we aim to unravel the mechanisms through which Notch3 signalling regulates these lineages, utilising both next generation sequencing to identify novel pathways through which Notch3 may regulate SMC biology, and through the generation of induced pluripotent stem cells (iPSC) from CADASIL patients. These approaches may lead to the identification of novel therapeutic targets in the management of CADASIL.