In our protocol we developed a hiPSC differentiation system
In our protocol, we developed a hiPSC differentiation system to produce ECs that depends on cyclic AMP (cAMP). cAMP is crucial for enhancing the EC differentiation and arterial specification of mouse embryonic stem dofetilide (Yamamizu et al., 2009, 2010, 2012a, 2012b). To generate BECs, we co-cultured the ECs with pericytes, neurons, and astrocytes, which were also derived from hiPSCs. Culturally induced BECs (ciBECs) showed properties consistent of the BBB, including an enrichment of BBB-specific transporters, good barrier function, and the efflux of drugs. This method allowed us to investigate the mechanisms of BBB formation, which led to our discovery that ciBEC specification occurs through Notch signaling via Dll1 in neurons. Our model should be useful for the study of BBB development and homeostasis, and also for the discovery of CNS drugs that bypass the BBB.
Discussion Understanding how the BBB operates is crucial for drug development, as it impedes chemical molecules from reaching the CNS. Current non-primate BBB models fail to adequately recapitulate human BBB function because of species-specific differences (Aday et al., 2016; Hoshi et al., 2013; Syvänen et al., 2009; Warren et al., 2009). We therefore considered human iPSCs as a resource for creating a reproducible and robust human BBB model. Here, we generated hiPSC-derived BBB that expressed specific transporters and receptors and showed low permeability properties, suggesting that it could be used as a platform for initial drug screening before in vivo clinical tests. Importantly, using simple co-culturing systems with hiPSC-derived ECs, pericytes, neurons, and astrocytes, we revealed a molecular mechanism for ciBEC specification. In particular, this technology allows us to investigate cell-cell interactions, which led to the discovery that Dll1 expression by neurons is crucial for activating the Notch signaling that leads to ciBEC specificity. Notch is a single-pass transmembrane receptor known for its function in controlling cell-fate decisions and creating boundaries through cell-cell communication. Notch signaling in ECs is known to specify arterial ECs (Yamamizu et al., 2010). Dll1, a Notch ligand in neurons, functions in neural differentiation and maintenance (Grandbarbe et al., 2003; Kageyama et al., 2008). Furthermore, it communicates with radial glial cells for the differentiation to astrocytes (Namihira et al., 2009). Our results showed that Dll1 was highly expressed in mature neurons (Figure S6A), and we demonstrated that it interacted with ECs for ciBEC specification (Figure 4C). Some reports have shown that the Wnt canonical pathway is crucial for proper neurovascular development. For example, the simultaneous ablation of Wnt7a/7b is embryonic lethal for neurovascular phenotypes (Daneman et al., 2009; Stenman et al., 2008). Our results showed that Wnt7a was expressed in neurons and Wnt7b was expressed in pericytes and astrocytes (Figure S6B), but treatment with Wnt inhibitors in our co-culture system did not cause significant differences in the expression of BBB-specific transporters (Figure 3A). Moreover, we found that Wnt7a was highly expressed in early-stage neural progenitors (Figure S6C), indicating that Wnt7a may be necessary for early vascularization in the brain. Overall, our technology could help elucidate the underlying molecular machinery for finely regulated BBB formation, which should offer new therapeutic strategies to repair the BBB following stroke or other neurological impairments. The brain vasculature sprouts into the neuronal tube and elongates toward the ventricular zone to form vascular networks that are surrounded by neuroepithelial cells, radial glia, neuroblasts, and neurons at around embryonic day 9.5 (E9.5) in mouse embryo (Engelhardt, 2003). The vascular permeability decreases at E16 as it interacts with BECs, neurons, and radial glial cells (Risau et al., 1986). Our ciBEC specification process seems to involve Notch signaling in response to neuron-derived Dll1 (Figure 4C), which may recapitulate the in vivo microenvironment for BBB development. We found that mature barrier characteristics of the BBB were induced by crosstalk between iPSC-derived astrocytes (Figure 5C). Several astrocytic factors for BBB maturation, including transforming growth factor, glial cell-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), interleukin-6, angiopoietin-1 (Ang1), and hydrocortisone, are capable of glial-mediated barrier induction (Abbott, 2002; Lee et al., 2003). Indeed, hiPSC-derived astrocytes highly expressed GDNF, bFGF, EGF (epidermal growth factor), and Ang1 (Figure S6D). Further analyses are needed to identify the BBB maturation factors from hiPSC-derived astrocytes. In addition, previous anatomical examination of the brain microvasculature showed that the endfeet of astrocytes form a lacework of fine lamellae to closely support the outer surfaces of the endothelium (Kacem et al., 1998). Although we successfully used Transwell dishes, which separate the apical and basolateral sides, to analyze drug kinetics, the construction of a three-dimensional BBB model from all four cell populations will expedite mature BBB formation and BBB physiology analysis.