Human cerebral organoids derived from induced pluripotent stem cells (iPSCs) provide novel tools for recapitulating the cytoarchitecture of human brain and for studying biological mechanisms of neurological disorders. However, the heterotypic interactions of neurovascular units, composed of neurons, pericytes, astrocytes, and brain microvascular endothelial cells, in brain-like tissues are less investigated, in addition, most cortical organoids lack a microglia component, the resident immune cells in the brain. The first objective of this study is to investigate the impacts of neurovascular interactions on the brain regional patterning in cortical spheroids and organoids derived from human iPSCs. The second objective is to engineer brain-region-specific organoids from hiPSCs incorporated with isogenic microglia-like cells in order to enhance brain-like microenvironments. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting mesenchymal stem cells (MSCs). Single hybrid spheroids were constructed at different iNPC: iEC: MSC ratios of 4:2:0, 3:2:1 2:2:2, and 1:2:3 in low-attachment 96-well plates, which can be promoted using Geltrex and hyaluronic acid hydrogels. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1. In addition, tri-cultured spheroids promoted the expression of TBR1 (deep cortical layer VI) and Nkx2.1 (ventral cells), and matrix remodeling genes, MMP2 and MMP3, as well as Notch-1, indicating the crucial role of matrix remodeling and cell-cell communications on cortical spheroid and organoid patterning. Moreover, tri-culture system elevated blood-brain barrier gene expression (e.g., GLUT-1), CD31 and tight junction protein ZO-1 expression. In the last part of this study, microglia-like cells were derived from hiPSCs using stage-wise growth factor induction into mesoderm. The derived microglia-like cells expressed several phenotypic markers, including CD11b, IBA-1, CX3CR1, and P2RY12, and phagocytosed micron-size super-paramagnetic iron oxide particles. Microglia-like cells were able to upregulate pro-inflammatory genes (TNF-α) and secrete anti-inflammatory cytokines (i.e., VEGF, TGF-β1, and PGE2) when stimulated with amyloid β 42 oligomers, lipopolysaccharides, or dexamethasone. Dorsal cortical (higher expression of TBR1 and PAX6) and ventral (higher expression of Nkx2.1 and Prox-1) spheroids/organoids were derived from the same hiPSC line, which displayed action potentials and synaptic activities. Co-culturing the isogenic microglia-like cells with the dorsal or ventral organoids showed differential migration ability, intracellular Ca2+ transients imaging, and the response to pro-inflammatory stimuli (ventral group had higher TNF-α expression). The whole study should advance our understanding of cellular interplay of the neurovascular unit in diseased human brain and the effects of microglia on brain tissue function and establish a transformative approach to modulate cellular microenvironment during neurogenesis toward the goal of treating various neurological disorders.
