Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 8;17(3):584-598.
doi: 10.1016/j.stemcr.2022.01.003. Epub 2022 Feb 3.

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes

Claudia Raggi  1 , Marie-Agnès M'Callum  2 , Quang Toan Pham  2 , Perrine Gaub  3 , Silvia Selleri  2 , Nissan Vida Baratang  4 , Chenicka Lyn Mangahas  2 , Gaël Cagnone  5 , Bruno Reversade  6 , Jean-Sébastien Joyal  7 , Massimiliano Paganelli  8
Affiliations

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes

Claudia Raggi et al. Stem Cell Reports. .

Abstract

Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.

Keywords: differentiation; disease modeling; drug discovery; hepatoblasts; hepatocyte-like cells; human; induced pluripotent stem cells; liver development; liver organoids; pluripotent stem cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Differentiation prototocol and PSC-derived definitive endoderm and posterior foregut (A) Description of our new differentiation protocol. (B–F) PSC-derived endoderm (DE). (B) Expression of EOMES, FOXA2, and SOX17 genes over the first 5 days of differentiation (qRT-PCR, expressed as log10 mean fold change relative to PSCs ± SEM, n = 9, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (C) Expression of endoderm-enriched genes in PSC-derived cells after 5 days of differentiation compared with undifferentiated PSCs (qRT-PCR, expressed as log10 mean fold change relative to DE ± SEM; n = 3 for PSCs, n = 6 for DEs, ∗p < 0.05, ∗∗p < 0.01; see also Figure S2A). (D) Expression of DE markers at the end of day 5 of differentiation compared with undifferentiated PSCs (immunofluorescence, representative images, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining in included images, scale bar, 200 μm). (E) Expression of DE markers is highly homogeneous in PSC-derived DE cells (flow cytometry, n ≥ 4, mean ± SEM). (F) FOXA2/CXCR4/SOX17 triple-positive cells constitute >90% of PSC-derived DEs (flow cytometry, n = 4; left: mean ± SEM, center and right: representative experiment). (G and H) PSC-derived ventral posterior foregut (VPFG): expression of HHEX, PROX1, HNF4A, AFP, and ALB genes in PSC-derived cells (qRT-PCR expressed as log10 mean fold change ± SEM, relative to: G, undifferentiated PSCs and H, PSC-derived DEs; n = 3 for PSCs, n ≥ 3 for DE, n ≥ 5 for VPFG; ∗p < 0.05, ∗∗∗p < 0.001; see also Figures S2E and S2F).
Figure 2
Figure 2
PSC-derived liver bud (LB) (A) PSC-derived cells after 15 days of differentiation achieve a gene expression profile consistent with hepatoblasts in the forming LB (qRT-PCR, expressed as log10 mean fold change relative to VPFG ± SEM; n = 3 for VPFG, n = 8 for DE, ∗∗p < 0.01; see also Figure S2G). (B) Expression of hepatoblast-enriched AFP, albumin, CK19, EpCAM, and ZO-1 in PSC-derived LB cells (immunofluorescence, representative images with DAPI nuclear staining, scale bars, 200 μm, left, and 50 μm, right). (C) Expression of hepatoblast-enriched TBX3 increases in LB cells and decreases upon further maturation into HLCs (qRT-PCR, expressed as fold change relative to DE ± SEM; n = 8, ∗p < 0.05). (D) LB cells show high homogeneity for EpCAM expression, while expression of a marker of pluripotency is negligible and comparable to background noise (flow cytometry; TRA-1-60: n = 6 for PSCs, n = 3 for LB, mean ± SEM; EpCAM: n = 5, mean ± SEM, representative experiment). (E) LB cells replicate actively (left panels), whereas HLCs reach a quiescent state (Ki67-albumin fluorescence co-staining, representative images, DAPI nuclear staining in included images, scale bars, 200 μm, left panels of each group, and 50 μm, right panels). (F) The differentiation protocol allows for the expansion of LB cells (automated cell count, n = 6 for PSCs and LB, n = 3 for DE, n = 4 for HLCs; mean ± SEM, ∗∗p < 0.01).
Figure 3
Figure 3
Characterization of PSC-derived HLCs (A) Representative morphology at the end of the differentiation protocol (phase contrast; arrowheads showing binucleated hepatocytes; scale bars, 200 μm, left, 100 μm, center, and 50 μm, right). (B) Expression of liver-specific genes compared with LB cells (qRT-PCR, expressed as log10 mean fold change relative to LB ± SEM; n = 7, ∗p < 0.05, ∗∗∗p < 0.001; see also Figure S3C). (C) Expression of liver-specific genes compared with adult PHHs (qRT-PCR, expressed as log10 mean fold change relative to PHH ± SEM; n = 9 for PHHs, n = 10 for HLCs; ∗∗p < 0.01, ∗∗∗p < 0.001, not significant [ns] p ≥ 0.05; see also Figure S3D). (D) CYP3A4 expression and induction upon supplementation with 20 μM rifampicin for 72 h (qRT-PCR, mean fold change relative to baseline ± SEM; n = 9, ∗∗∗p < 0.001). (E) Expression of liver-specific genes LXR, CAR, and PXR in HLCs compared with LB cells (qRT-PCR, log10 mean fold change relative to LB ± SEM; n = 9 for LXR and CAR, n = 5 for PXR, ∗∗∗∗p < 0.0001). (F) Effect of different maturation conditions between days 16 and 30 on the expression of liver-specific genes by HLCs (qRT-PCR, mean fold change relative to the protocol shown in Figure 1A ± SEM; n = 3; p > 0.05 for all conditions; “shorter” corresponds to 3 days per step instead of 5). (G) Expression of AFP, albumin, CK19, CK18, E-cadherin, and EpCAM in PSC-derived HLCs (immunofluorescence, representative images, DAPI nuclear staining; yellow arrowheads showing binucleated hepatocytes; scale bar, 50 μm). (H) Percentage of HLCs expressing AFP, albumin, CK19, EpCAM, and CK18 (immunofluorescence, representative images, DAPI nuclear staining; scale bar, 200 μm; bottom: percentage of positive cells compared with DAPI-positive nuclei, n = 9, mean ± SEM). (I) HLCs were highly homogeneous for albumin expression (flow cytometry; n = 6, mean ± SEM; p > 0.05 when HLCs were compared with LB cells; see also Figures S3E and S3F).
Figure 4
Figure 4
Liver-specific functions performed by HLCs (A and B) Cyp3A4 activity performed by HLCs in comparison to PHHs and HepG2 cells (A; relative light units per million cells; n = 6 HLCs, n = 7 PHHs, n = 5 PSCs, n = 3 HepG2 cells) and its induction upon supplementation with 20 μM rifampicin for 72 h (B; n = 9; mean ± SEM, ∗p < 0.05, ∗∗∗p < 0.001). (C) Albumin secretion in HLCs at days 25 and 30 compared with PHHs (ELISA, n = 4 for HLCs and PHHs, mean ± SEM). (D) Urea production by HLCs compared with PHHs and HepG2 cells (ELISA, n = 3 for HLCs, PSCs, and HepG2 cells, n = 12 for PHHs, mean ± SEM). (E and F) Hepatotoxicity of acetaminophen (APAP) and amiodarone (AMIO) assessed through the measurement of oxygen consumption rate on HLCs (O, oligomycin; F, FCCP; R/A, Rotenone/Antimycin A; n = 12, mean ± SEM).
Figure 5
Figure 5
Comparison of our HLCs with cells obtained with previously described protocols, PHHs, and adult liver samples (A) Heatmap showing the expression of the top 46 liver-enriched genes in HLCs obtained with our protocol compared with LB cells, HLCs obtained with a protocol not acting on Wnt and TGFβ signaling pathways beyond the DE stage (HLC-2; see Figure S3G), HLCs obtained with other previously described protocols (1: Warren et al., 2017), hepatocytes derived by transdifferentiation (hiHeps; 2: Gao et al., 2017), and PHHs (RNA-seq, unsupervised clustering; 2: Gao et al., 2017; list of genes from the Human Protein Atlas; Uhlén et al., 2015). Transcriptome profile through the differentiation stages and comparison of the HLCs with adult liver samples is shown in Figures S4A and S4B. (B) Our HLCs are more similar to freshly isolated adult PHHs than most HLCs obtained with previously described protocols (1: Warren et al., 2017; 2: Gao et al., 2017; 3: Li et al., 2017; 4: Touboul et al., 2016; 5: Xie et al., 2019; 6: Fu et al., 2019; 7: Kim et al., 2019). 2D representation of Uniform Manifold Approximation and Projection (UMAP)-based dimensionality reduction of the top 17 principal components obtained analyzing the 3,000 most variable genes across datasets (Dorrity et al., 2020). (C) Liver-specific genes are overexpressed in HLCs obtained with our new protocol compared with HLC-2 (Figure S3G; qRT-PCR, log10 mean fold change relative to HLC-2 ± SEM, n = 9, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (D) Volcano plot showing the most variable genes in our HLCs compared with HLC-2 (n = 2). (E) Representative sample of liver-enriched genes overexpressed in HLCs obtained with the new protocol compared with HLC-2 (differential gene expression; >3-fold change, p adjusted < 0.05; n = 2). (F) Semantic similarity-based scatterplot of Gene Ontology terms showing families of genes that are overexpressed in HLCs compared with HLC-2 (REVIGO tree map, n = 2, p adjusted < 0.05). (G) HLCs obtained with our new protocol show significantly more Cyp3A4 activity than HLC-2 (relative light units per million cells; n = 6 HLCs, n = 4 HLC-2; mean ± SEM, ∗∗p < 0.01). (H) Our HLCs secrete more albumin per million cells than HLC-2 (ELISA, n = 4 for HLCs, n = 10 for HLC-2; mean ± SEM, ∗∗p < 0.01). (I) The new differentiation protocol allows for generating HLCs with significantly better efficiency (automated cell count, n = 4; mean ± SEM, ∗p < 0.05). See also Figures S4C and S4D.
Figure 6
Figure 6
Complex, syngeneic liver organoids generated with bipotent PSC-derived hepatoblasts (A) Schematic representation and macroscopic appearance organoids generated with syngeneic, PSC-derived LB cells (hepatoblasts) and mesenchymal and endothelial progenitor cells (MPCs and EPCs, respectively; characterization in Figure S5). (B) Liver-specific functions performed by the organoids: cyp3A4 activity at day 7 (relative light units per million cells compared with PHHs and HLCs; n = 6 HLCs, n = 7 PHHs, n = 3 organoids; mean ± SEM); albumin secretion at day 7 and 10 compared with PHHs and HLCs (ELISA, n = 4 for HLCs and PHHs, n = 3 organoids day 7, n = 6 organoids day 10; mean ± SEM, ∗p < 0.05, ∗∗∗∗p < 0.0001). (C) Histological appearance of liver organoids at day 7 showing cords of small hepatocytes at the center, and bile ducts surrounded by extracellular matrix in the outer layer of the structure (H&E staining, representative images, scale bar, 500 μm). (D and E) Mature bile ducts within the organoids: a single layer of CK19-positive, SOX9-positive cholangiocytes delimiting a lumen surrounded and by the extracellular matrix (D: H&E staining, representative image, scale bar, 100 μm; E, immunofluorescence, scale bar, 50 μm). (F) Within the organoids, LB cells differentiate into CK18-positive, CK19-negative, BSEP-positive hepatocytes mostly located at the center of the organoids (white dashed line) and CK19- and CK18-positive cholangiocytes forming bile ducts (white arrowheads; immunofluorescence, scale bars, 20 μm, top left and right, 50 μm, top center, and 200 μm, bottom; additional staining in Figure S6). (G) Within the organoids, hepatocytes reach a state of quiescence (Ki67-negative). Cholangiocytes form bile ducts at different stages of maturation, with immature ducts still showing cell replication and mature ones (arrowhead) being Ki67-negative (immunofluorescence, representative images, scale bar, 200 μm).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Asai A., Aihara E., Watson C., Mourya R., Mizuochi T., Shivakumar P., Phelan K., Mayhew C., Helmrath M., Takebe T., et al. Paracrine signals regulate human liver organoid maturation from iPSC. Development. 2017;144:dev.142794. - PMC - PubMed
    1. Baxter M., Withey S., Harrison S., Segeritz C.-P., Zhang F., Atkinson-Dell R., Rowe C., Gerrard D.T., Sison-Young R., Jenkins R., et al. Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes. J. Hepatol. 2015;62:581–589. - PMC - PubMed
    1. Berasain C., Avila M.A. Regulation of hepatocyte identity and quiescence. Cell. Mol. Life Sci. 2015;72:3831–3851. - PMC - PubMed
    1. Berger D.R., Ware B.R., Davidson M.D., Allsup S.R., Khetani S.R. Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro. Hepatology. 2015;61:1370–1381. - PubMed
    1. den Braver-Sewradj S.P., den Braver M.W., Vermeulen N.P.E., Commandeur J.N.M., Richert L., Vos J.C. Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer. Toxicol. Vitro. 2016;33:71–79. - PubMed

Publication types

Grants and funding