Figure 1.

Fusion kinetics of iNPC spheroids and hMSCs with iEC spheroids to construct hybrid spheroids. (A) (i) Schematic illustration of endothelial cell (iEC) spheroid derivation from hiPSCs. (ii) Schematic illustration of generating hybrid spheroids from iNPCs. hMSCs were added to iNPCs for iNPC-MSC-iEC spheroids (method A), before iEC transfer. Or hMSCs were added to the well containing iNPC and iEC spheroids for iNPC-iEC-MSC spheroids (method B). hMSCs were labeled with CellTracker Red. (B) Phase contrast images of iNPC-iEC-MSC spheroids morphology at day 1, 3, 5, and 7 (total day 15–21). iEC spheroids and MSCs were added to the preformed iNPC aggregates. Scale bar: 400 μm. (C) (i) Schematic illustration of calculation of squared aspect ratio of contact length between two aggregates over maximum diameter $\left(\frac{Lneck}{Lmax}\right){}^2$. The aggregation kinetics were evaluated by (ii) the squared aspect ratio over 7 days and (iii) the inter-sphere angle formed by the two aggregates. (D) Overlay of phase contrast images (iNPCs and iECs) with fluorescent images (hMSCs) of hybrid spheroids (i) with or (ii) without ROCKi Y27632 (10 μM) when MSCs were added to the culture one week after fusion of iNPC spheroids and iEC spheroids. Scale bar: 400 μm. (E) The aggregation kinetics were analyzed in (Ei) and (Eii), respectively. *Indicates p < 0.05 for the different test conditions.