Pro-inflammatory cytokine stimulation of human cortical spheroids containing neuroectoderm-derived and mesoderm-derived cell types

Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)functioning. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. In this study, we developed a five-months culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) comprising not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations. We then set out to explore the effects of stimulating our hCSs for three time periods with the cytokines TNFa and IL-1ß. We use transcriptome-wide RNA-sequencing (RNA-seq) analysis to show that the major process induced by TNFa as well as by IL-1ß is neuroinflammation. Central in this process are endothelial cells, microglia and astrocytes, and activation of the NF?B and STAT pathways. In addition to this equiva-lent impact of TNFa and IL-1ß, we find that each of the two cytokines has specific and stimulation-time-dependent effects on hCS gene expression, and that IL-1ß exhibits a faster self-inhibitory feedback response to dampen neuroinflammation than TNFa. Thus, our protocol provides an inducible 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms. Overall design: We analyzed the pooled total RNA of 4 spheroids (0h, control condition for the TNFa- or IL-1ß-stimulation conditions), pooled total RNA of 5 spheroids (4h TNFa stimulation), pooled total RNA of 5 spheroids (12h TNFa stimulation), pooled total RNA of 5 spheroids (36h TNFa stimulation), pooled total RNA of 5 spheroids (4h IL-1ß stimulation), pooled total RNA of 5 spheroids (12h IL-1ß), pooled total RNA of 5 spheroids (36h IL-1ß stimulation). Furthermore, we analyzed pooled total RNA of 4 spheroids (0h_2, control condition for the LPS+TNFa+IL-1ß stimulation conditions) and pooled total RNA of 5 spheroids (24h LPS- + 24h TNFa+IL-1ß stimulation).