Abstract
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. The objective of this study is to investigate the impacts of neural spheroids and vascular spheroids interactions on the regional brain-like tissue patterning in cortical spheroids derived from human iPSCs. Hybrid neurovascular spheroids were constructed by fusion of human iPSC-derived cortical neural progenitor cell (iNPC) spheroids, endothelial cell (iEC) spheroids, and the supporting human 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. The incorporation of MSCs upregulated the secretion levels of cytokines VEGF-A, PGE2, and TGF-β1 in hybrid spheroid system. In addition, tri-cultured spheroids had high levels 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 ZO1 expression. Treatment with AMD3100, a CXCR4 antagonist, showed the immobilization of MSCs during spheroid fusion, indicating a CXCR4-dependent manner of hMSC migration and homing. This forebrain-like model has potential applications in understanding heterotypic cell-cell interactions and novel drug screening in diseased human brain.
Highlights
Www.nature.com/scientificreports administration and nutrient transport[13,20]
For iPSC-derived cortical neural progenitor cell (iNPC)-iEC-mesenchymal stem cells (MSCs) spheroids, Human MSC (hMSC) were added into the well of day 14 iNPC-iEC hybrid spheroids and cultured for another 7 days or MSCs and iEC spheroids were added together to the wells containing iNPC spheroids (Fig. 1B–D). hMSCs were integrated with the iNPC-iEC hybrid spheroids and migrated toward the spheroid center
Due to the higher squared aspect ratio, iNPC-iECMSC spheroids were mainly used in the following experiments
Summary
Www.nature.com/scientificreports administration and nutrient transport[13,20]. Functional BBB models require the interactions of brain microvascular endothelial cells (ECs), astrocytes, neurons, and pericytes, which can be realized using hiPSC-derived cells[21,22,23,24]. Mesenchymal stem cell (MSC)-driven condensation has been observed in organ buds formation based on hiPSC-derived cells for multiple tissue types including kidney, intestine, brain, and heart etc., in the presence of MSCs19. The objective of this study is to investigate heterotypic neural-vascular-mesenchymal interactions in cortical organoids in vitro through tri-culture of iNPCs, hiPSC-derived ECs (iECs), and human MSCs. The long-term goal is to fabricate next-generation of brain organoids with additional cellular components from hiPSCs for disease modeling, drug screening, and possibly cell therapy. This study used a simple approach to assemble hiPSC-derived vascular spheroids with hiPSC-derived cortical spheroids in the presence of human MSCs. The cellular localization, fusion kinetics, cytokine secretion and gene expression of brain regional markers, cell-cell interactions, extracellular matrix remodeling proteins, and BBB functional proteins are characterized. This study provides understanding of differential effects of heterotypic cell-cell interactions on hiPSC-based organoid engineering which to date are poorly understood
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