There is widespread recognition of the complementary value o

There is widespread recognition of the complementary value of hiPSCs in disease modeling. Indeed, a number of studies have revealed mutant HTT-related molecular and cellular abnormalities using human pluripotent stem cells and differentiated cells, e.g., alterations in transcriptomes, proteomes, ATM-p53 and TGF-β signaling, and monoamine oxidase activity (Mattis and Svendsen, 2015; Ooi et al., 2015). Cellular abnormalities include enhanced lysosomal activity in HD hiPSCs, altered neuronal glutamate signaling and calcium homeostasis, reduced mitochondrial length and function, reduced neurite length in GABAergic and MSNs, impaired neuronal brain-derived neurotrophic factor (BDNF)-vesicular transport, and reduced viability in response to a number of cellular stress paradigms including BDNF withdrawal, H2O2 treatment, and inhibition of autophagy (Mattis and Svendsen, 2015). While many of these pathological features had been previously identified in animal models of HD, some human-specific discoveries are starting to emerge from studies in hiPSCs (Ruzo et al., 2015). Ultimately the use of isogenic control lines will facilitate such efforts, and will help authenticate mutant HTT-specific effects.
Experimental Procedures
Author Contributions
Acknowledgments We thank Qiyu Chen for technical assistance, Sumanty Tohari for whole-exome sequencing, and Kerry McLaughlin for editorial assistance. The work was partly funded by a Strategic Positioning Fund for Genetic Orphan Diseases (SPF2012/005) and a Joint Council Office Project grant (1431AFG122) from the Agency for Science, Technology and Research (Singapore), and a Tier 1 grant R-172-000-297-112 from the Ministry of Education (Singapore) to M.A.P. F.G. and D.L. are supported by the Singapore Immunology Network (SIgN) core funding. C.R. is supported by the A∗STAR Research Attachment Program (ARAP) and A.Z. is supported by the A∗STAR Singapore International Graduate Award (SINGA). We thank the NINDS iPSC Repository for the HD and control iPSC lines (ND36997 and ND36999).
Introduction The blood-brain barrier (BBB) is composed of specialized Amyloid Beta-Peptide (1-40) endothelial cells (BECs) that are surrounded by pericytes, astrocytes, and neurons. These neurovascular units form intracellular tight junctions between BECs, which limit the passive diffusion of molecules into the CNS. BECs are enriched with nutrient transporters, such as glucose transporters, amino acid transporters, and fatty acid transporters, for efficient uptake into the brain from the blood. On the other hand, the enrichment of polarized efflux transporters, such as P-glycoprotein (PGP), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) in BECs protects the brain from toxic factors and pathogens. Because of drug efflux by the BBB, the delivery of therapeutic drugs into the brain to treat CNS diseases has been a major challenge. The BBB is also associated with brain disease, as its impairment correlates with neurodegenerative diseases including Alzheimer\'s disease and Parkinson\'s disease (Desai et al., 2007; Saito and Ihara, 2014). These reasons have spurred researchers to establish a BBB model for analyzing the dysfunction of neurovascular units and drug permeability in vitro. Current in vitro BBB models use brain microvessels and astrocytes isolated from non-human species such as pig, rat, or mouse (Deli et al., 2005). However, the characteristics and functions of the BBB from these species differ from that in humans (Aday et al., 2016; Hoshi et al., 2013; Syvänen et al., 2009; Warren et al., 2009). Therefore, a human-derived BBB model is needed for pre-clinical drug screening or research about human BBB physiology and pathology. Although human primary brain microvessels isolated from the brain specimens of tumors or epilepsy patients can be used, they have low availability and reproductivity (Cecchelli et al., 2007). Immortalized human brain microvessels have also been considered, but the resulting BBB models do not form strong barrier properties due to discontinuous tight junctions (Weksler et al., 2005). Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) have been used to prepare human in vitro BBB models, but these cells must be co-cultured with primary rat astrocytes or C6 rat glioma cells for maturation of the BBB (Lippmann et al., 2012; Minami et al., 2015). In the present work, we sought to induce all four BBB components, ECs, pericytes, neurons, and astrocytes, from hiPSCs, which are an unlimited human source and recapitulate development, in order to create a reproducible and robust human BBB model.