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

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.This work was supported by the British Heart Foundation (BHF) [NH/11/1/28922], by the UK Medical Research Council and BHF [G1000847 to S.S. and R.A.P.), and by Cambridge Hospitals National Institute for Health Research Biomedical Research Centre funding (S.S. and R.A.P.). D.I. is on a University of Cambridge Commonwealth Scholarship. S.S. and F.S. are supported by the BHF [FS/13/29/ 30024]. L.G. is supported by the BHF [RM/l3/3/30159]. W.G.B. and V.L.M. are supported by BHF PhD studentships [FS/11/77/29327 and FS/10/48/28674].This is the final published version. It first appeared at http://dev.biologists.org/content/early/2015/03/25/dev.119271.abstract

A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration.

The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-β inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/- HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/- HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/- NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro

Process development and scale-up for gene circuit engineered CAR-NK cell manufacturing

Allogeneic Natural Killer (NK) cell therapy has shown promise in recent years for treating cancer in patients without inducing graft versus host disease and with potential for off-the-shelf administration. Senti Bio is using gene circuits to introduce logic-gating and regulated expression of payloads into next-generation CAR-NK cell therapies to broaden the therapeutic indications and improved efficacy in liquid and solid tumors. Key process development objectives for gene circuits include the ability to efficiently and stably transduce multi-gene constructs into primary NK cells while retaining cell expandability and anti-cancer function. Here, we describe a scalable GMP-ready manufacturing process for generating clinically relevant numbers of CAR-NK cells, and we demonstrate its potential applicability to our product pipeline. To achieve a batch size target of \u3e10^11 NK cells, we aimed to develop a process to start with ~25*10^6 isolated NK cells, achieve \u3e40% CAR+ transduction, and obtain \u3e5,600-fold expansion over 21 days. Enrichment of adult apheresis material from 12 healthy donors via CD3 depletion and CD56 selection yielded an average of ~3*10^8 NK cells, which were cryopreserved for later use. Upon thaw, NK cells were activated using proprietary irradiated gene-modified feeder cells and expanded in a closed system 1L G-Rex chamber. Seven days later, NK cells were transduced with retroviral vectors using closed system procedures, resulting in up to 80% CAR+ population. Gene circuits were tested across multiple retroviral vector delivery systems, and successful constructs were developed into producer cell lines (HEK293) using various single cell cloning techniques with the goal of generating stable, high titer vector producer clones. Primary NK cell transduction efficiency was optimized by testing a range of MOI, comparing different vector addition and spinoculation vessels, and the effect of GMP-compatible transduction enhancers. Transduced NK cells were expanded further in multiple closed system G-Rex culture vessels for a total process time (initial NK thaw to CAR-NK harvest) of approximately 21 days. Different expansion methods were assessed including different irradiated modified cell lines and feeder-free NK expansion technologies achieving ~10,000-fold expansion in the 1L vessels. At cell harvest, the cell suspension was volume-reduced, harvested and formulated into cryopreservation medium using an automated cell processing system, yielding ~4*10^9 cells per liter of culture. Formulated cells were filled in vials and stored in liquid nitrogen vapor phase. Functional assessment was performed via both in vitro and in vivo studies, demonstrating significant CAR-specific cancer cell killing compared to non-transduced NK cells. We also evaluated multiple donors for transduction efficiency, growth characteristics, cancer cell killing specificity, scalability, immunomodulatory function, single cell transcriptomics and distribution and kinetics in vivo to determine desirable attributes for manufacturing. This CAR-NK manufacturing process is expected to be suitable for translation to GMP clinical manufacturing in support of Senti Bio’s internal allogeneic CAR-NK cell pipeline

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

Embryonic origins of human vascular smooth muscle cells: implications for in vitro modeling and clinical application.

Vascular smooth muscle cells (SMCs) arise from multiple origins during development, raising the possibility that differences in embryological origins between SMCs could contribute to site-specific localization of vascular diseases. In this review, we first examine the developmental pathways and embryological origins of vascular SMCs and then discuss in vitro strategies for deriving SMCs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We then review in detail the potential for vascular disease modeling using iPSC-derived SMCs and consider the pathological implications of heterogeneous embryonic origins. Finally, we touch upon the role of human ESC-derived SMCs in therapeutic revascularization and the challenges remaining before regenerative medicine using ESC- or iPSC-derived cells comes of age

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

This is the final published version. It first appeared at http://dev.biologists.org/content/early/2015/03/25/dev.119271.abstract.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 TGF?1, 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.This work was supported by the British Heart Foundation (BHF) [NH/11/1/28922], by\ud the UK Medical Research Council and BHF [G1000847 to S.S. and R.A.P.), and by\ud Cambridge Hospitals National Institute for Health Research Biomedical Research\ud Centre funding (S.S. and R.A.P.). D.I. is on a University of Cambridge\ud Commonwealth Scholarship. S.S. and F.S. are supported by the BHF [FS/13/29/\ud 30024]. L.G. is supported by the BHF [RM/l3/3/30159]. W.G.B. and V.L.M. are\ud supported by BHF PhD studentships [FS/11/77/29327 and FS/10/48/28674]

Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk.

Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies, efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Recent studies have investigated the disease mechanism of CAD associated gene SMAD3, a central transcription factor (TF) in the TGFβ pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cellular phenotype, promoting expression of differentiation marker genes while inhibiting proliferation. RNA sequencing and chromatin immunoprecipitation sequencing studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and atherosclerosis processes in this cell type. HCASMC phenotype, and gene expression patterns promoted by SMAD3 were noted to have opposing direction of effect compared to another CAD associated TF, TCF21. At sites of SMAD3 and TCF21 colocalization on DNA, SMAD3 binding was inversely correlated with TCF21 binding, due in part to TCF21 locally blocking chromatin accessibility at the SMAD3 binding site. Further, TCF21 was able to directly inhibit SMAD3 activation of gene expression in transfection reporter gene studies. In contrast to TCF21 which is protective toward CAD, SMAD3 expression in HCASMC was shown to be directly correlated with disease risk. We propose that the pro-differentiation action of SMAD3 inhibits dedifferentiation that is required for HCASMC to expand and stabilize disease plaque as they respond to vascular stresses, counteracting the protective dedifferentiating activity of TCF21 and promoting disease risk

<i>TCF21</i> and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

<div><p>Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor <i>TCF21</i> as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of <i>TCF21</i> expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of <i>AHR</i>, its heterodimerization partner <i>ARNT</i>, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including <i>IL1A</i>, <i>MMP1</i>, and <i>CYP1A1</i>. TCF21 was shown to bind in <i>AHR</i>, <i>ARNT</i> and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.</p></div

A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration

The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-β inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/- HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/- HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/- NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro