Placenta-derived factors contribute to human iPSC-liver organoid growth

Abstract

Organoids derived from human induced pluripotent stem cells (hiPSC) are potentially applicable for regenerative medicine. However, the applications have been hampered by limited organoid size and function as a consequence of a lack of progenitor expansion. Here, we report the recapitulation of progenitor expansion in hiPSC-liver organoids based on the analysis of mouse development. Visualization of blood perfusion and oxygen levels in mouse embryos reveals a transient hypoxic environment during hepatoblast expansion, despite active blood flow. During this specific stage, the placenta expresses various growth factors. Human and mouse placenta-liver interaction analysis identifies various placenta-derived factors. Among them, IL1α efficiently induces the growth in hiPSC-liver organoids as well as mouse fetal livers following progenitor expansion under hypoxia. Furthermore, subsequent oxygenation demonstrates that progenitors expanded by IL1α contribute to hiPSC-liver organoid size and function. Taken together, we demonstrate that treatment with the placenta-derived factor under hypoxia is a crucial human organoid culture technique that efficiently induces progenitor expansion.

Fig. 1. Localized perfusion promotes the growth of the liver ventral lobe between E10.5 and E11.5.

A Illustration of the method for visualizing placenta-derived perfused vessels using CD31 antibodies (Ab). B Confirmation of intravital CD31 Ab perfusion (gray). Scale bar = 1 mm. C Confirmation of CD31 Ab perfusion into the liver tissue sections. Images indicate perfused CD31 (gray) and HNF4α (magenta). Scale bar = 500 μm or 100 μm. D Whole-mount imaging of perfused CD31 (gray) and HNF4α (magenta) immunostaining at each stage. Perfused CD31 in the liver was visualized (green) using Imaris. Scale bar = 300 μm. E Visualization of the perfused blood vessels in the ventral and dorsal lobes of the liver (blue or gray). Scale bar = 200 μm. F Quantification of perfused blood vessels in the dorsal and ventral lobes of the liver at E10.5. Error bars are represented as SEM; Ventral (n = 3), Dorsal (n = 3), two-tailed Student’s t-test, Ventral vs. Dorsal (p-value = 0.0227). G Quantification of perfused blood vessels in the dorsal and ventral lobes of the liver at E11.5. Error bars are represented as SEM; Ventral (n = 3), Dorsal (n = 3), two-tailed Student’s t-test, Ventral vs. Dorsal (p-value = 0.4640). H Quantification of the volume of the dorsal and ventral lobes of the liver at E10.5 and E11.5. Error bars are represented as SEM; Ventral (n = 3), Dorsal (n = 3), One-way ANOVA with Tukey’s test, E10.5 ventral vs. E11.5 ventral (p-value = 0.001), E11.5 ventral versus E11.5 dorsal (p-value = 0.004). I Ventral liver growth between E10.5 and E11.5 followed by hepatoblast expansion induced by placenta-derived blood perfusion. Source data are provided as a Source Data file.

Fig. 2. Hepatoblast expansion induced by placenta-derived blood perfusion under hypoxia.

A Images of perfused CD31 and HNF4α/EdU immunostaining in each stage. Scale bar = 50 μm. B Quantification of hepatoblast expansion by counting the number of HNF4α⁺EdU⁺ cells at each stage. Error bars are represented as SEM; E9.5 (n = 3), E10.5 Ventral (n = 5), E10.5 Dorsal (n = 4), E11.5 Ventral (n = 3), E11.5 Dorsal (n = 3), One-way ANOVA with Tukey’s test, E10.5 Ventral vs. E10.5 Dorsal (p-value = 0.002). E11.5 Ventral vs. E11.5 Dorsal (p-value = 0.9467). C Oxygen level in the liver at each stage. Pimonidazole indicated hypoxic regions (green). Scale bar = 50 μm. D Quantification of pimonidazole positive regions in the liver. Error bars are represented as SEM; E9.5 (n = 4), E10.5 Ventral (n = 3), E10.5 Dorsal (n = 3), E11.5 Ventral (n = 4), E11.5 Dorsal (n = 4), One-way ANOVA with Tukey’s test, E10.5 ventral versus E11.5 ventral (p-value < 0.0001). E10.5 dorsal versus E11.5 dorsal (p-value < 0.0001). E Microarray data of growth factor-related gene expression in placenta or liver. F Putative spatiotemporal interactions between liver and placenta. Placenta-derived blood containing factors perfuses into the ventral lobe of the liver. Source data are provided as a Source Data file.

Fig. 3. Placenta-derived IL1α promotes hepatoblast expansion at E10.5.

A Schematic overview of placenta-liver interaction analysis. B KEGG pathway analysis using human and mouse placenta and liver microarray data. C Extraction of enriched genes in humans and mice, as shown in the Venn diagram. D Ligands and receptors with matched signaling using CellTalkDB. E Effect of placental ligands on fetal liver culture. Whole-mount staining three days after culture. P: Parenchyma. Scale bar = 500 μm or 100 μm. F Liver area calculated using macro-images during culture. Error bars are represented as SEM; Control (n = 8), FGF2 (n = 8), IL1α (n = 10), IL13 (n = 7), InhBB (n = 9), PDGFAB (n = 8), One-way ANOVA with Tukey’s test, IL1α versus Control (p-value = 0.0196). Liver volume calculated using whole-mount images. Error bars are represented as SEM; Control (n = 5), FGF2 (n = 5), IL1α (n = 4), IL13 (n = 3), InhBB (n =  5), PDGFAB (n = 4), two-tailed Mann–Whitney U-test, IL1α versus Control (p-value = 0.0159). Hepatoblast expansion calculated using whole-mount images. Error bars are represented as SEM; Control (n = 5), FGF2 (n = 5), IL1α (n = 4), IL13 (n = 3), InhBB (n = 5), PDGFAB (n = 4), two-tailed Mann–Whitney U-test, IL1α versus Control (p-value = 0.0159), InhBB versus Control (p-value = 0.0159). G IL1α production from isolated each tissue. Error bars are represented as SEM; Placenta (n = 6), Liver+STM (n = 4), Heart (n = 3), Fetus (n = 3), One-way ANOVA with Tukey’s test, Placenta versus each group (p-value < 0.0001). H Examination of IL1rap expression in the ventral and dorsal lobes of the liver using MOSTA. Error bars are represented as SEM; Ventral (n = 11), Dorsal (n = 6), two-tailed Mann–Whitney U-test, Ventral versus Dorsal (p-value = 0.0063). Scale bar = 500 μm or 100 μm. I Examination of IL1rap expression in the ventral and dorsal lobes of the liver using immunostaining. Scale bar = 100 μm. J E11.5 whole liver after IL1 receptor antagonist (IL1RA) treatment at E10.5. Scale bar = 100 μm. K Ratio of ventral/dorsal liver lobe after IL1RA treatment. Error bars are represented as SEM; Vehicle (n = 5), IL1RA (n = 6), two-tailed Mann–Whitney U-test, IL1RA versus Vehicle (p-value = 0.0043). L Hepatoblast expansion after IL1RA treatment. Error bars are represented as SEM; Vehicle (n = 5), IL1RA (n = 6), two-tailed Student’s t-test, IL1RA versus Vehicle (p-value = 0.0314). M Hepatoblast expansion occurs in the ventral liver lobe after perfusion with placenta-derived IL1α at E10.5. Source data are provided as a Source Data file.

Fig. 4. IL1α promotes hiPSC-derived liver organoid growth.

A Schematic overview of hiPSC-liver organoid culture and subsequent analysis. B Effect of IL1α on hiPSC-liver organoid culture. KO (Kusabira–Orange) indicates hiPSC-hepatoblasts (KO-HE). Whole-mount staining three days after organoid culture. Scale bar = 500 μm or 100 μm. Quantification of liver volume using the HNF4α⁺ regions. Box plots show the minimum value to maximum value. Error bars are represented as SEM; Control (n = 4), IL1α (n = 4), two-tailed Mann–Whitney U-test, IL1α versus Control (p-value = 0.0286). C Colony assay using single or small cluster of cells derived from organoids with or without IL1α. D Ratio of HE colony areas with and without IL1α pre-treatment. Error bars are represented as SEM; Control small colonies (n = 3), IL1α small colonies (n = 4), Control large colonies (n = 3), IL1α large colonies (n = 4), One-way ANOVA with Tukey’s test, IL1α small colonies versus Control small colonies (p-value = 0.3600). IL1α large colonies versus Control large colonies (p-value = 0.0019). E The expression of ALB⁺CK19⁺ in IL1α pre-treated HE colony. Scale bar =  10 μm. F Expression of IL1 receptor related genes using single cell RNA-seq data set (Camp JG et al. Nature 2017). G Ingenuity Pathway Analysis (IPA) in organoid using RNAseq data. H Effects of inhibitors against each pathway estimated by IPA on HE expansion for 7 days. Box plots show the minimum value to maximum value. Error bars are represented as SEM; Control (n = 4), ZINC00784494 (n = 4), Linsitinib (n = 4), Compstatin (n = 4), CCR6 antagonist (n = 4), One-way ANOVA with Tukey’s test, CCR6 antagonist versus Control (p-value = 0.0219). Scale bar = 500 μm. I Gene expression of SAA1, CCR6 and TLR2 on HE, EC and MC. Immunostaining of CCR6 and TLR2. Scale bar = 10 μm. J CCL20 secretion from hiPSC-liver organoid after SAA1 treatment. Error bars are represented as SEM; Control (n = 4), SAA1 6 μg/mL (n = 4), SAA1 20 μg/mL (n = 4), One-way ANOVA with Tukey’s test, SAA1 20 μg/mL versus Control (p-value < 0.0001). K Estimated cell-cell interaction within hiPSC-liver organoid after IL1α treatment. Hepatoblast expands through IL1α-induced SAA1-TLR2-CCL20-CCR6 signaling. Source data are provided as a Source Data file.

Fig. 5. Oxygen enhances IL1α-induced hiPSC-liver organoid growth.

A Culture protocol of hiPSC-liver organoid based on the analysis of liver development. B Effect of IL1α and oxygen (Oxy) on hiPSC-liver organoid. KO-HE was observed in 3D on day 14. Ki67 (green) and HNF4α (red) immunostaining using hiPSC-liver organoid tissue sections. Scale bar = 500 μm or 100 μm. C Quantification of hepatoblast proliferation indicated by HNF4α⁺Ki67⁺ on day 14. Box plots show the minimum value to maximum value. Error bars are represented as SEM; Control (n = 3), IL1α (n = 4), Oxy (n = 4), IL1α/Oxy (n = 4), One-way ANOVA with Tukey’s test, IL1α/Oxy versus Control (p-value = 0.0008), IL1α/Oxy versus IL1α (p-value = 0.0072), IL1α/Oxy versus Oxy (p-value = 0.0005). Quantification of hiPSC-liver organoid volume on day 14. Error bars are represented as SEM; Control (n = 6), IL1α (n = 6), Oxy (n = 4), IL1α/Oxy (n = 6), One-way ANOVA with Tukey’s test, IL1α/Oxy versus Control (p-value = 0.0002), IL1α/Oxy versus IL1α (p-value = 0.0010), IL1α/Oxy versus Oxy (p-value = 0.0437). D Quantification of hALB secretion from hiPSC-liver organoids over time. Box plots are shown the minimum value to maximum value. Error bars are represented as SEM; IL1α day1 (n = 4), IL1α/Oxy day1 (n = 4), IL1α day7 (n = 4), IL1α/Oxy day7 (n = 4), IL1α day14 (n = 4), IL1α/Oxy day14 (n = 4), two-tailed Student’s t-test, IL1α/Oxy day14 versus IL1α day14 (p-value = 0.0485). E Gene expressions of hepatocyte and hepatoblast markers on HE sorted from IL1α/Oxy-treated hiPSC-liver organoid. Source data are provided as a Source Data file.