Fig. 2

Characterization of mLOs consisting of hPSC-derived HE, ECs, and HscLCs. A Schematic protocol for self-aggregation and differentiation of mLOs. mLOs were generated and differentiated with or without HscLCs for 16 days in DM (created with BioRender.com). B Phase-contrast images of mLOs generated with [mLO(+ HSC)] or without [mLO(-HSC)] HscLCs on the indicated days of differentiation. Enlarged images showing the periphery of organoids at day 16 are shown separately in C Scale bar, 200 μm. D Violin plot showing the size distributions of mLO(+ HSC) and mLO(-HSC) on the indicated days of differentiation. Each black dot represents a single organoid. E Whole-mount immunofluorescence images of NG2 (pericytes), CD31 (ECs), and HNF1β (HE) in mLOs at the end of self-aggregation (day 5). High-magnification images of the boxed areas (CD31) are shown separately in the right panels. Scale bar, 200 μm. F Representative immunostaining image showing close association between CD31 + ECs and αSMA + HscLCs in mLO(+ HSC) on day 16. Scale bar, 50 μm. G Immunofluorescence images of cleaved caspase-3 and EdU in cryo-sectioned mLOs after 16 days of differentiation. mLOs were pre-incubated with EdU for 6 h before fixation. Scale bar, 200 μm. Quantification of immunoreactive signals is shown in H. Each black dot represents the percent positive area of the immunoreactive signal in each mLO. **p < 0.01 and ***p < 0.001 by unpaired t tests. I Whole-mount immunofluorescence images of HNF4α (HLCs), CD31, and E-cadherin (hepatic epithelium) in mLOs after differentiation (day 16). The insets show high-magnification images of E-cadherin staining. Scale bar, 200 μm. J Quantification of immunoreactive signals in (I). Each black dot represents the percent positive area of the immunoreactive signal in each mLO. ****p < 0.0001 by unpaired t test