The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

The blood-brain barrier is a dynamic and highly organized structure that strictly regulates the molecules allowed to cross the brain vasculature into the central nervous system. The blood-brain barrier pathology has been associated with a number of central nervous system diseases, including vascular malformations, stroke/vascular dementia, Alzheimer's disease, multiple sclerosis, and various neurological tumors including glioblastoma multiforme. There is a compelling need for representative models of this critical interface. Current research relies heavily on animal models (mostly mice) or on two-dimensional (2D) in vitro models, neither of which fully capture the complexities of the human blood-brain barrier. Physiological differences between humans and mice make translation to the clinic problematic, while monolayer cultures cannot capture the inherently three-dimensional (3D) nature of the blood-brain barrier, which includes close association of the abluminal side of the endothelium with astrocyte foot-processes and pericytes. Here we discuss the central nervous system diseases associated with blood-brain barrier pathology, recent advances in the development of novel 3D blood-brain barrier -on-a-chip systems that better mimic the physiological complexity and structure of human blood-brain barrier, and provide an outlook on how these blood-brain barrier-on-a-chip systems can be used for central nervous system disease modeling. Impact statement The field of microphysiological systems is rapidly evolving as new technologies are introduced and our understanding of organ physiology develops. In this review, we focus on Blood-Brain Barrier (BBB) models, with a particular emphasis on how they relate to neurological disorders such as Alzheimer's disease, multiple sclerosis, stroke, cancer, and vascular malformations. We emphasize the importance of capturing the three-dimensional nature of the brain and the unique architecture of the BBB - something that until recently had not been well modeled by in vitro systems. Our hope is that this review will provide a launch pad for new ideas and methodologies that can provide us with truly physiological BBB models capable of yielding new insights into the function of this critical interface.

Different approaches have investigated the effects of different extracellular matrices (ECMs) and threedimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM.

No abstract

Neural stem cells (NSCs) give rise to all the major cell types in the brain, including neurons, oligodendrocytes, and astrocytes. However, the intracellular signaling pathways that govern brain NSC proliferation and differentiation have been incompletely characterized to date. Since some neurodevelopmental brain disorders (Costello syndrome and Noonan syndrome) are caused by germline activating mutations in the RAS genes, Ras small GTPases are likely critical regulators of brain NSC function. In the mammalian brain, Ras exists as three distinct molecules (H-Ras, K-Ras, and N-Ras), each with different subcellular localizations, downstream signaling effectors, and biological effects. Leveraging a novel series of conditional-activated Ras molecule-expressing genetically engineered mouse strains, we demonstrate that activated K-Ras, but not H-Ras or N-Ras, expression increases brain NSC growth in a Raf-dependent, but Mek-independent, manner. Moreover, we show that activated K-Ras regulation of brain NSC proliferation requires Raf binding and suppression of retinoblastoma (Rb) function. Collectively, these observations establish tissue-specific differences in activated Ras molecule regulation of brain cell growth that operate through a noncanonical mechanism.