Human pluripotent stem cell-derived epicardial progenitors can differentiate to endocardial-like endothelial cells.

During heart development, epicardial progenitors contribute various cardiac lineages including smooth muscle cells, cardiac fibroblasts, and endothelial cells. However, their specific contribution to the human endothelium has not yet been resolved, at least in part due to the inability to expand and maintain human primary or pluripotent stem cell (hPSC)-derived epicardial cells. Here we first generated CDH5-2A-eGFP knock-in hPSC lines and differentiated them into self-renewing WT1+ epicardial cells, which gave rise to endothelial cells upon VEGF treatment in vitro. In addition, we found that the percentage of endothelial cells correlated with WT1 expression in a WT1-2A-eGFP reporter line. The resulting endothelial cells displayed many endocardium-like endothelial cell properties, including high expression levels of endocardial-specific markers, nutrient transporters and well-organized tight junctions. These findings suggest that human epicardial progenitors may have the capacity to form endocardial endothelium during development and have implications for heart regeneration and cardiac tissue engineering

Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells.

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications

Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions.

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies

Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions.

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-β inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications

DDEL-05DIFFERENTIAL BLOOD-BRAIN BARRIER (BBB) PERMEABILITY OF ALKYLPHOSPHOCHOLINE (APC) ANALOGS ANALYZED USING AN IN VITRO PLURIPOTENT STEM CELL-DERIVED BRAIN MICROVASCULAR ENDOTHELIAL CELL SYSTEM

Photograph of a scene during the restoration of Guthrie's Historic Downtown District

Destroying pathogen-tumor symbionts synergizing with catalytic therapy of colorectal cancer by biomimetic protein-supported single-atom nanozyme

Abstract The crucial role of intratumoral bacteria in the progression of cancer has been gradually recognized with the development of sequencing technology. Several intratumoral bacteria which have been identified as pathogens of cancer that induce progression, metastasis, and poor outcome of cancer, while tumor vascular networks and immunosuppressive microenvironment provide shelters for pathogens localization. Thus, the mutually-beneficial interplay between pathogens and tumors, named “pathogen-tumor symbionts”, is probably a potential therapeutic site for tumor treatment. Herein, we proposed a destroying pathogen-tumor symbionts strategy that kills intratumoral pathogens, F. nucleatum, to break the symbiont and synergize to kill colorectal cancer (CRC) cells. This strategy was achieved by a groundbreaking protein-supported copper single-atom nanozyme (BSA-Cu SAN) which was inspired by the structures of native enzymes that are based on protein, with metal elements as the active center. BSA-Cu SAN can exert catalytic therapy by generating reactive oxygen species (ROS) and depleting GSH. The in vitro and in vivo experiments demonstrate that BSA-Cu SAN passively targets tumor sites and efficiently scavenges F. nucleatum in situ to destroy pathogen-tumor symbionts. As a result, ROS resistance of CRC through elevated autophagy mediated by F. nucleatum was relieved, contributing to apoptosis of cancer cells induced by intracellular redox imbalance generated by BSA-Cu SAN. Particularly, BSA-Cu SAN experiences renal clearance, avoiding long-term systemic toxicity. This work provides a feasible paradigm for destroying pathogen-tumor symbionts to block intratumoral pathogens interplay with CRC for antitumor therapy and an optimized trail for the SAN catalytic therapy by the clearable protein-supported SAN

Neuronal activity regulates blood-brain barrier efflux transport through endothelial circadian genes

The blood vessels in the central nervous system (CNS) have a series of unique properties, termed the blood-brain barrier (BBB), which stringently regulate the entry of molecules into the brain, thus maintaining proper brain homeostasis. We sought to understand whether neuronal activity could regulate BBB properties. Using both chemogenetics and a volitional behavior paradigm, we identified a core set of brain endothelial genes whose expression is regulated by neuronal activity. In particular, neuronal activity regulates BBB efflux transporter expression and function, which is critical for excluding many small lipophilic molecules from the brain parenchyma. Furthermore, we found that neuronal activity regulates the expression of circadian clock genes within brain endothelial cells, which in turn mediate the activity-dependent control of BBB efflux transport. These results have important clinical implications for CNS drug delivery and clearance of CNS waste products, including Aβ, and for understanding how neuronal activity can modulate diurnal processes