To better understand morphogenesis of the human brain, three- dimensional organoid culture systems with human pluripotent stem cells, termed (partial) cerebral organoids, have been extensively studied in recent years. Multidiscipline approaches such as genetic engineering, microfluidics, and tissue engineering have been used to promote cerebral organoids maturation and create functional cerebral organoids. For example, a microfabricated device was used to spatially control the direction of axon extension from the cerebral organoids and motor nerve organoids. However, further replication of in vivo system may be required. Axon bundles in vivo are composed of nerves and adjacent blood vessels, and interactions between nerve and blood vessels play important roles in morphogenesis of the brain. In this study, we fabricated a microdevice to test neuro-vascular interactions in organoids prepared with human induced pluripotent stem cell (hiPSC).

　201B7 hiPSC line was provided by Riken Cell Bank. We differentiated hiPSCs to cerebral neurons, vascular endothelial cells, and pericytes, respectively. In the preparation of cerebral organoids, hiPSCs were seeded in a low-adhesive culture plate to form spheroids and cultured in differentiation medium for 25 days. Neural differentiation was assessed by RT-PCR and immunohistochemistry. Since the length of axons in the central nervous system is of several mm in length, a microdevice with a few mm-long microfluidic channel was fabricated using photolithography. Cerebral organoids at 25 days were placed into the microdevice. The next day, endothelial cells differentiated from hiPSCs for 6 days were seeded into the microdevice (Figure 1A). The formation of an axon fascicle and vascular sprouts were evaluated by immunohistochemical analysis.
In the differentiation culture, expression of pluripotent stem cell marker, OCT3/4, decreased, and cortical markers, TBR1 and CTIP2, increased over time. Immunohistochemical analysis revealed that forebrain neural lineage marker FOXG1-positive and Tuj1-positive cells were abundant in the cerebral organoids at 50 days of culture (Figure 1B). After 30 days of differentiation culture, pericytes and endothelial cells were visualized with PDGFRβ and CD31, respectively (Figure 1C). When the two cerebral organoids were placed at both ends of the microchannel and the endothelial cells were seeded around them, axons spontaneously grew from the organoids, and eventually connected between the two organoids. The axon bundle tissues were then maintained over several weeks (Figure 1D). Additionally, endothelial cells were seen to penetrate the cerebral organoid and axon bundle tissues.
This model will be a fundamental platform for better understanding neurogenesis via neural-vascular interactions and for finding new cerebral nerve drugs.