(183ab) Engineering Human Brain Assembloids to Investigate Forebrain and Choroid Plexus Interactions Under BMP4 and Wnt Signaling Modulation

Brain organoids, the three-dimensional models derived from human induced pluripotent stem cells (iPSCs), self-organize and are cued towards differentiating into neural tissue that captures aspects of brain development and function. Differentiation of iPSCs to forebrain generally involves using exogenous signals to mimic the development of the human neocortex while choroid plexus (ChP) organoids aim to replicate the cerebrospinal fluid (CSF) producing properties as well as barrier functions of the choroid plexus region in the brain. However, research on forebrain-ChP organoid interactions remains limited, leaving a critical knowledge gap in understanding how these structures influence each other in healthy and diseased conditions. Particularly, research related to neurological disorders such as stroke and Alzheimer’s disease, characterized by cortical disruption and altered CSF, could benefit from more precise modeling of the brain environment.

In this study forebrain and ChP organoids were differentiated from human iPSCs and introduced as the assembloids based on maturation time. Forebrain (Fb) cultures at day 16 of differentiation were assembled with day 12 ChP group at approximately 1:1 ratio in each well (x5/group). Based on established differentiation protocol, the ChP spheroids (i.e., premature organoids) are treated with CHIR99021 (CHIR) and BMP4 during day 10-15. To assess the effects of these small molecules on the Fb-ChP co-culture or assembloids, one group continued to receive the factors after assembly while the other group did not. The culture was assessed using imaging and gene analysis, which were performed after about a week. The results indicated that ChP-associated markers (TTR, AQP1, and CLIC6) were downregulated with the addition of CHIR and BMP4 while TBR1, the marker associated with deep cortical neuron development in the forebrain, was upregulated. Markers for inflammatory response, exosome biogenesis, as well as cell-cell interaction were assessed, and in some cases significant differences were found between the two groups. The upregulation of CXCR4, TNFα and NOTCH1 with the addition of BMP4 and CHIR suggests an increase in cell migration, stress, cell-cell interactions, and retention of progenitor state qualities, indicating increased susceptibility in change of cell fate. The addition of forebrain organoids may influence the development of ChPs towards more anterior-like structures (as indicated by the increase in TBR1), rather than more posterior as would happen in ChP only cultures. To help visualize the interaction of ChP with forebrain cells, ChP organoids (on day 19 of differentiation) were labelled with CellTracker™ red before introduction to day 19 forebrain group. Image analysis was performed for more than a week. This study provides insights for the maturation of different brain region specific organoids as assembloids at different stages of development, as well as the complexity of interactions of small molecules in co-culture compared to single-type organoids. The established physiologically relevant brain tissue models can be applied for drug screening towards the treatment of neurological disorders.