Generation of human liver organoids from pluripotent stem cell-derived hepatic endoderms

Abstract

Background: The use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.

Methods: The peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.

Results: CD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Keywords: Differentiation; Hepatic endoderm; Hepatocyte; Liver organoid; Matrigel; Three-dimensional culture; Albumin; Hepatocyte; Human induced pluripotent stem cell.

Figure 1. Hepatic differentiation potential of the HuiPSC MUi019 line.

(A) Schematic diagram of hepatic cell differentiation in the 2D culture system. The protocol consisted of four stepwise phases: endoderm, hepatic endoderm, hepatoblast, and hepatocyte. Cells were cultured under 5% or 20% O₂. (B–E) Representative microscopic bright-field images of differentiated iPS cells after exposure to different sets of cytokines (20X objective lens). (F) Microscopic image of 25-day-old differentiated cells at higher magnification. The dotted line indicates the area where polygonal cells having large nuclei were observed. (G–K) Gene expression profile of cells at different stages of culture. Relative expression levels were calculated using the 2^−ΔΔCT method. Data are the mean ± SD (n = 3) and statistically analyzed using Student’s t test. (L–P) Confocal microscopic observation of albumin (red color) and CD81 (green color). Scale bar = 50 mm. (Q) Percentage of albumin-expressing cells observed using immunofluorescence and confocal microscopy. (R) Glycogen storage in the 25-day differentiated cells from E. Representative image shown was obtained with a 100X objective lens. Light microscopic and confocal images are representative of three independent experiments.

Figure 2. Organoid formation potential of the hepatic endoderm, hepatoblast, and hepatocyte phases.

(A) Schematic diagram of the methods used for assessing organoid formation ability. (B) Hepatic endoderm (day 10), hepatoblasts (day 15), and hepatocytes (day 20) were cultured in semisolid Matrigel (pink color). Liquid medium (light orange) was added on top of the Matrigel. (C–E) Ability of hepatic endoderm, hepatoblasts, and hepatocytes to form an organoid in the Matrigel-based 3D culture was assessed at day 5 of the 3D culture. Images shown were obtained with a 40× objective lens. (F–G) Two representative images of the organoid in the Matrigel-based 3D culture at day 7. (H–I) Two representative images of the organoid in the Matrigel-based 3D culture at day 10. Three independent experiments were performed and representative microscopic images are shown.

Figure 3. Morphology of hepatic endoderm-derived organoids (HEOs).

(A–C) Morphology of the HEOs at days 12, 14, and 17 postculture in Matrigel (4X objective lens). (D–F) Representative images of single HEOs are shown at high magnification (40X). (G) The outer layer of cells is shown. (H) Zoomed-in image of the HEOs showing the cells with a polygonal shape and large nuclei (arrowheads). (I–LL) Confocal images of a day 12- and 17-derived organoid showing the expression of hepatic nuclear factor 4 (HNF4), hepatocyte-specific albumin (ALB) and α-fetoprotein (AFP), cytochrome P450 4A3 (CYP4A3), and CD81, an important receptor of Plasmodium falciparum sporozoites. (MM–PP) Co-localization of CD81 and ALB in the 17-day HEO. The confocal images of organoid show the expression of CD81 (green) and hepatocyte-specific albumin (red). (QQ–TT) Zoomed-in images reveal the albumin- and CD81-expressing cells. (UU–XX) Cells stained with 2nd antibody specific to IgG of rabbit and mouse (unstained samples) served as background control. Four independent experiments were performed and representative images are shown. (YY) Total amount of human albumin secreted within 48 h in the culture medium of the 25- and 40-day 2D culture and the 17- and 60-day 3D culture. Total amount (ng) of human albumin in each experiments were calculated based on number of cells. Individuals represent independent experiments. Data are the mean ± SD (n =3–4) and statistically analyzed using Student’s t test. Culture medium of undifferentiated HuiPSCs was used for comparison. (ZZ–EEE) Hepatocyte functions (glycogen and lipid storage, and metabolism of indocyanine) of a day 12- and 17-derived organoids. Four independent experiments were performed and representative images are shown.

Figure 4. Minimal concentration of Matrigel needed to maintain the 3D structure of the organoid.

(A) Schematic diagram showing a method used to determine the minimum concentration of Matrigel in which the 3D structure of the organoid could be maintained. (B–E) Microscopic images of the liver organoid. The 17-day-old organoids were split to obtain 25%, 12.5%, and 6.25% Matrigel in RPMI/B27, which were supplemented with HGF and oncostatin M, and cultured for 5 days. All images were captured using a 20X objective lens. Three independent experiments were performed and representative images are shown.

Figure 5. Long-term maintenance of the hepatic endoderm-derived organoid.

(A) Morphology of the HEOs at days 60 postculture in Matrigel. Representative images of a well of 96-well plate show the HEOs after a week of sub-culture. (B) At higher magnification, the outer layer of cells remain single. (C) Focused image of the HEOs showing the cells with a polygonal shape and large nuclei (white arrowhead) as well as binucleated cell (black arrowhead), characteristics of hepatic cells. (D–L) Confocal images of the day 60-derived HEOs showing the expression of hepatocyte-specific albumin (ALB), cytochrome P450 3A4 (CYP4A3) and CD81. (M–U) Zoomed-in confocal images shown in (D). (V–X) Hepatocyte functions (glycogen and lipid storage, and metabolism of indocyanine) of the day 60-derived organoids. Four independent experiments were performed and representative images are shown. (Y–AA) Confocal images of the day 60-derived HEOs showing the expression of EpCAM. (BB–DD) Confocal images of the day 60-derived HEOs showing the expression of CD31.