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

Ethical approval/declaration

The study research protocol for the use of MUi019 was approved by the Mahidol University Central Institutional Review Board (COA no. 2016/038.2803) and the Human Research Protection Unit, Faculty of Medicine Siriraj Hospital, Mahidol University (COA no. Si459/2019).

Culture of human iPS cells (Si-Tayeb et al., 2010)

HuiPS cells (MUi019 line) (Tangprasittipap et al., 2017) were cultured on a cell culture plate coated with 2 µg/mL Matrigel (Growth Factor Reduced; Corning, BD Bioscience). The culture medium for iPS cells consisted of Dulbecco’s modified Eagle medium (DMEM) F12, 100 µM penicillin/streptomycin (Invitrogen), L-glutamine, 100 ng/mL fibroblast growth factor 2 (FGF2 or bFGF; Invitrogen), two ng/mL TGFβ1, 64 µg/mL L-ascorbic acid, and 1X insulin/transferrin/selenium. Cells were passaged every 5–6 days using 0.5 mM EDTA (Invitrogen) as the cell dissociation solution.

Generation of hepatic endoderm, hepatoblasts, and hepatocytes from human iPS cells (Si-Tayeb et al., 2010)

HuiPS cells were cultured as mentioned above for 5–6 days. Hepatic differentiation consisted of four sequential phases. For the first phase, the culture medium consisted of 100 ng/mL activin A (R&D Systems) in RPMI plus 1x B27 (RPMI/B27) medium (Invitrogen) under ambient oxygen/5% CO₂ for 5 days. For the second phase, the culture medium contained 20 ng/mL bone morphogenetic protein 4 (BMP4, Peprotech) and 10 ng/mL basic FGF (Invitrogen) in RPMI/B27 medium under 4% O₂/5% CO₂ for 5 days. For the third phase, the culture medium consisted of 20 ng/mL hepatic growth factor (HGF, Peprotech) in RPMI/B27 medium under 4% O₂/5% CO₂ for 5 days. For the final phase, the cells were cultured with 20 ng/mL oncostatin-M (R&D Systems) in RPMI/B27 medium for 5 days and then maintained in ambient oxygen/5% CO₂.

Gene expression analysis

RNA was extracted, and cDNA was prepared using a cDNA synthesis kit (Biotechrabbit). The expression of pluripotency-related, endodermal and hepatic genes was assessed using real-time conventional PCR. Primer sets were obtained from previous reports (Schwartz et al., 2002; Takahashi et al., 2007; Chen et al., 2012; Irie et al., 2015; Aguila et al., 2014) and are shown in Table 1. Luna^® Universal qPCR Master Mix (New England BioLabs) was used, and the primer concentration was 1 µM for each primer. The thermal cycles were as follows: initial denaturation at 95 °C for 3 min, 30 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s, followed by a final extension at 72 °C for 1 min. Transcript of human actin beta (ACTB) served as internal control to normalize gene expression. Threshold cycles (CT) of each samples were compared with a reference sample using the 2^−ΔΔCT method (Rao et al., 2013) and is shown as relative expression. Gene expression analyses were from three independent experiments, and each run of real-time PCR were carried out in triplicate.

Immunofluorescence

The cells or organoids were attached to a glass slide and air-dried. The cells were fixed with 1% paraformaldehyde in PBS at room temperature for 30 min and then permeabilized with 0.05% Triton X-100 in PBS for 15 min. After washing with PBS, the cells were exposed to 3% BSA in PBS for 15 min. Rabbit polyclonal anti-human HNF4 (1:100 from Sigma-Aldrich), mouse monoclonal anti-human α-fetoprotein antibody (1:500, Sigma-Aldrich), rabbit polyclonal anti-human albumin (1:100, Sigma-Aldrich), rabbit polyclonal anti-human CYP3A43 antibody (1:100, Abcam), mouse monoclonal anti-human EpCAM antibody (1:100, GeneTex), rabbit polyclonal anti-human CD31 antibody (1:20, Abcam) and mouse anti-human CD81 (1:200, Abcam) antibodies diluted in 1% BSA were applied onto the glass slide and incubated overnight. After washing with PBS, the cells were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies (Invitrogen). The DAPI-containing medium was mounted on the glass slide and covered with a glass coverslip. Images of the cells were visualized using a confocal microscope (ECLIPSE Ti-Clsi4 Laser Unit, Nikkon).

Generation of liver organoids from the hepatic endoderm

HuiPSC-derived hepatic endoderm was collected from a well of a 24-well plate using cell dissociation buffer (Gibco, LifeTechnologies). The cells were then suspended in a culture medium consisting of 40 ng/mL HGF and 20 ng/mL oncostatin M in RPMI/B27. The cell suspension was immersed in ice for 15 min and immediately mixed with an equal volume of Matrigel. The final mixture contained 50% Matrigel, 20 ng/mL HGF (Peprotech), and 10 ng/mL oncostatin M. After solidification of the Matrigel at 37 °C for 15 min, RPMI/B27 medium containing 20 ng/mL HGF and 10 ng/mL oncostatin M was added on top of the solid Matrigel. The cells were cultured under 4% O₂/5% CO₂ at 37 °C. The covering medium was renewed every 48 h. From day 10 onward of 3D culture, the medium on top of Matrigel was changed to the HCM™ Hepatocyte Culture Medium BulletKit™ (herein called HCM) containing HBM™ Basal medium and HCM™ SingleQuots Supplements, which include transferrin, ascorbic acid, human endothelial growth factor, insulin, hydrocortisone, fatty acid-free BSA and Gentamicin sulfate-Amphotericin. To sub-culture, the plate was place on ice for 10 min. The cold HCM was then added into a well and gently mixed by pipetting to liquefy the Matrigel. A part of liquefied Matrigel was suspended in the cold HCM. An equal volume of the organoid mixture was then mixed with Matrigel and plated into a 96-well plate. After solidification of the Matrigel at 37 °C for 15 min, the HCM was added on top. The HCM was changed every 48 h.

Enzyme-linked immunosorbent assay (ELISA) for human albumin

Amount of human albumin secreted in culture medium was assessed using the Human Albumin/Serum albumin ELISA kit (Millipore) following manufacturer’s instruction. The hepatocyte culture medium was used as background control. Triplicates were performed for each independent experiments.

Glycogen accumulation

The cells were treated with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS. Cells treated with 1 mg/mL diastase in PBS (Sigma) were used as the negative control. The cells were then incubated with periodic acid for 5 min, washed with distilled water, and incubated with freshly prepared Schiff’s solution for 15 min. Finally, the cells were rinsed, and the nuclei were stained with hematoxylin. After rinse with water, cells were incubated with Bluing reagent for 30 s followed by incubation with Light Green solution for two min. The glass slide was immersed in absolute alcohol to dehydrate and air-dried.

Lipid stain using Oil Red O

Lipid stored in cells was visualized using fat-soluble Oil Red O following a manufacture’ instruction (Oil Red O Stain Kit, Abcam). Cells were attached onto a glass slide using cytocentrifugation. After air dry, the glass slide was placed in propylene glycol for 2 min and then incubated with Oil Red O solution for 6 min. The glass slide was immersed in 85% propylene glycol in distilled water for 1 min and rinsed twice with distilled water. The cells were then stained with Hematoxylin for 1–2 min and rinse thoroughly in tap water. Then, the glass slide was rinsed with two changes of distilled water and air-dried.

Metabolism of indocyanine

Indocyanine green (ICG) is an organic anion, non-toxic and exclusively eliminated by hepatocytes and has been useful as a marker of hepatocyte. The ICG was added into culture medium at 1 mg/mL and incubated at 37 °C for 60 min. After medium removal, cells were washed with PBS to remove excess extracellular indocyanine.

Statistical analysis

Data are presented as means ± standard deviation (SD). Differences were statistically evaluated using Student t test. P-values <0.05 were considered statistically significant.

Hepatic differentiation potential of the HuiPS MUi019 cell line

The potential of the peripheral blood CD34+ hematopoietic progenitor cell-derived MUi019 cell line for endodermal differentiation has been demonstrated in vivo (Tangprasittipap et al., 2017); however, its capability for hepatic differentiation in vitro is unknown. Here, we first assessed the capability of the HuiPSC MUi019 cell line (Tangprasittipap et al., 2017) for hepatic differentiation. Hierarchically differentiated cells, namely, definitive endoderm, hepatic endoderm (HE), hepatoblasts, and hepatocytes, were prepared according to a method described in a previous report (Si-Tayeb et al., 2010). Upon exposure to four different sets of cytokines (Fig. 1A), the morphologies of the differentiated cells tended to change in a stepwise fashion (Figs. 1B–1E). On day 25 of differentiation, groups of cells having the same morphology appeared in many focal areas of the cell culture plate (Figs. 1E–1F). Higher magnification observation revealed polygonal cells having large nuclei, which is a main characteristic of hepatocytes (Fig. 1F).

To characterize the hepatic cells, mRNA transcripts indicating each cell type were examined using real-time PCR. Here, octamer-binding transcription factor 4 (OCT4) identified an undifferentiated pluripotent stem cells, forkhead box A2 (FOXA2) and sex determining region Y box 17 (SOX17) indicated definitive endoderm. The hepatic endoderm expresses FOXA2. Alpha-fetoprotein (AFP) and albumin (ALB) indicated hepatoblast and mature hepatocytes, respectively. Based on their transcript profile in Figs. 1G–1K, mRNA level of OCT4 decreased as the HuiPSC differentiated into hepatocytes (Fig. 1G). By contrast, that of SOX17 and FOXA2 up-regulated in the 10-day 2D culture, in which the HuiPSCs specified to hepatic endoderm, respectively; however, level of both transcripts gradually decreased in the 15-day 2D culture during differentiation of hepatic endoderm into hepatoblasts (Figs. 1H and 1I, respectively). Level of AFP transcript was detectable at day 10 until day 25 (Fig. 1J), while that of ALB transcript could be detected at day 25 of 2D culture (Fig. 1K). Collectively, the 25-day-old cells exhibited characteristics of endoderm (SOX17 and FOXA2), hepatoblasts (AFP), and hepatocyte (ALB) cells. In Figs. 1L–1Q, confocal microscopic observations confirmed the presence of intracellularly synthesized albumin (Fig. 1L), a specific marker of hepatic cells, and CD81 (Fig. 1M), an important receptor of the Plasmodium falciparum sporozoite (Silvie et al., 2003) and hepatitis C virus entry (Bruening et al., 2018). Coexistence of albumin and CD81 was also observed (arrowheads in Fig. 1O). Cells stained with 2nd antibody was served as background control (Fig. 1P). Representative confocal image revealed 5.9 ± 1.3% of the 25-day-old 2D cultured cells expressing albumin (Fig. 1Q). The HuiPSC-derived hepatocytes were able to store glycogen (Fig. 1R). Taken together, the results indicated that the HuiPS MUi019 cell line was capable of hepatic cell differentiation in vitro.

HuiPSC-derived hepatic endoderm spontaneously formed organoids, but the hepatoblasts and hepatocytes could not

To determine which cell type is capable of forming an organoid, we collected HE, hepatoblasts, and hepatocytes at days 10, 15, and 20 of cell differentiation (Fig. 2A) and cultured them in a 3D setting (50% Matrigel prepared in RPMI supplemented with 1% B27, HGF, and oncostatin M) (Fig. 2B). The top of the solidified Matrigel was covered with the same medium described above without Matrigel. On day 5 of the 3D culture, a small, irregularly shaped structure appeared in the culture of the HEs (Fig. 2C), while neither hepatoblasts nor hepatocytes were observed (Figs. 2D and 2E, respectively). Therefore, 2D-cultured, 10-day-old HE was used further for organoid formation. On day 7 of the 3D culture, irregularly shaped cell clumps composed of an outer layer and inner mass could be clearly observed under a microscope. The outer layer had a protruding, spike-like structure (Figs. 2F–2G). By day 10 of the 3D culture, the sizes of cell clumps increased, while their pikes disappeared, and the outer layer became smooth (Figs. 2H–2I). At this time point, the organoid showed the characteristics of a spherical, hollow shape, hereafter called a hepatic endoderm organoid or HEO. From day 10 onward of the 3D culture, the covering medium was changed to the HCM for hepatocyte maturation.

Characterization of the HuiPS-derived HEOs

On day 12 of the 3D culture, the HEO had space at the center surrounded by a layer of cells, which are characteristics typical of a blastocyst (Fig. 3A). The sizes of the HEOs varied and increased from day 12 onward (Figs. 3B–3C). Despite their increasing sizes, there was no fusion of adjacent HEOs. Instead, the outer layer of the HEO was bent (Figs. 3B and 3C, arrowheads). Microscopic observation of the HEOs on days 12 and 14 revealed a thin, single-layer structure (Figs. 3D and 3E, respectively). By day 17, dome-shaped cells appeared (Fig. 3F, arrowheads). Higher magnification observation of the 17-day HEO yielded microscopic images showing a very thin layer (Fig. 3G). When focusing on the cells, polygonal cells could be observed (arrowheads in Fig. 3H).

To characterize the 17-day-old HEOs, gene and protein expression analyses were performed. Although the HEOs did express the pluripotency-related OCT4 (Fig. 1G) and the endoderm-specific transcription factor SOX17 (Fig. 1H); however, the level was much lower than the 2D culture. By contrast, the 17-day-old HEOs expressed hepatic transcription factor FOXA2 (Fig. 1I), hepatoblast-specific factor AFP (Fig. 1J), and hepatocyte-specific ALB (Fig. 1K) at higher level compaired to the 25-day-old 2D-cultured hepatocyte, thus implying the greater maturation of the 3D culture. Then, we evaluated the expression of hepatocyte-related proteins in a time course manner using immunofluorescence and confocal microscopy. The confocal images revealed expression of hepatic nuclear factor 4 (HNF4) at both day 12 (Figs. 3I–3K) and 17 (Figs. 3L–3N) of the 3D culture. Moreover, the confocal images also confirmed that the ALB transcripts were translated into albumin (Figs. 3O–3T). As shown in Figs. 3U–3Z, the HEOs expressed AFP at day 12 and 17 of the 3D culture. Although CYP4A3 was not observed at the 12-day-old HEOs, CYP4A3 was subsequently detected at day 17 of the 3D culture (Figs. 3AA–3FF). To ensure that HEO could be applied as a model of infectious diseases, we examined the expression of CD81, an important receptor for Plasmodium falciparum sporozoites and hepatitis C virus. As shown in Figs. 3GG–3LL, the 12-day- and 17-day-old HEOs expressed CD81. Localization of albumin and CD81 was performed. Albumin was expressed in an asymmetric pattern (Figs. 3MM–3PP); however, the expression of CD81 overlapped with that of albumin in some areas of the HEOs (Fig. 3PP). Higher magnification images revealed the coexpression of albumin and CD81 (Figs. 3QQ–3TT). Based on cell counting, higher percentage of ALB+ cells was observed in the HEOs (bar graph in Fig. 1Q). All confocal images were compared with the HEOs stained with the 2nd antibody (Figs. 3UU–3XX). In comparable to immunofluorescence, the 17-day-old HEOs secreted albumin into the culture medium at 1,077 ± 176 ng per 10⁶ examined cells within 48 h of culture (Fig. 3YY). By contrast, among three independent experiments, the 25-day-old 2D cultured hepatic cells secreted albumin into the culture medium at 44.9 ± 53 ng per 10⁶ cells within 48 h of culture. Apart from the immunofluorescence, both day 12 and 17-derived HEOs were capable of storing glycogen (Figs. 3ZZ and 3AAA) and lipid (Figs. 3BBB–3CCC) and metabolizing indocyanine (Figs. 3DDD–3EEE) in a time-course manner. Collectively, the data of gene and protein expression imply a full maturation of the HEOs within 17 days of 3D culture, which is equivalent to the 27-day period.

Minimal concentration of Matrigel required for maintaining the 3D structure of the HEOs

Given the high cost of Matrigel, we next determined the minimum concentration of Matrigel in which the 3D structure of the organoid remained unchanged. The 17-day-old organoids were split by dilution with the medium to obtain 25%, 12.5%, and 6.25% Matrigel prepared in RPMI/B27 supplemented with HGF and oncostatin M (Fig. 4A). Regardless of the Matrigel concentration, the HEOs could maintain their spherical structure, and their outer layer remained intact (Figs. 4B–E). In our experience, using Matrigel at a concentration lower than 6.25% leads to difficulty in changing the upper liquid medium during long-term culture.

Long-term culture of the HEOs

We further evaluated whether the HEOs could be maintained hepatocyte characteristics in a long-term culture. The HEOs were sub-cultured weekly and the culture continued for 60 days in total. Representative images of a well of 96-well plate show morphology and size of the 60-day-old HEOs under a microscope after a week of sub-culture (Fig. 5A). At higher magnification, the HEOs remain single layer of cells (Fig. 5B) having a polygonal shape plus large nuclei as well as binucleated cell (respective white and black arrowhead in Fig. 5C), characteristics of hepatic cells. Confocal images of the day 60-derived HEOs showing the expression of hepatocyte-specific ALB (Figs. 5D–5F), CYP4A3 (Figs. 5G–5I) and CD81 (Figs. 5J–5L). To clearly demonstrate, higher magnifications of the images are shown in Figs. 5M–5U. In agreement with immunofluorescence, the 60-day-old HEOs secreted albumin at 1,106 ± 77 ng per 10⁶ cells within 48 h, a similar level to the 17-day-old HEOs (Fig. 3YY). By contrast, when we maintained the 2D culture of the HuiPSC-derived hepatic cells for 40 days, lower amount of albumin could be detected relative to that of the day 25-derived hepatic cells (Fig. 3YY), implying loss of albumin synthesis an in vitro culture. Moreover, the day 60-derived HEOs were still able to storing glycogen (Fig. 5V) and lipid (Fig. 5W), and metabolizing indocyanine (Fig. 5X) as well as expressing hepatic stem/progenitor marker EpCM (Figs. 5Y–5AA) but not endothelial marker CD31 (Figs. 5BB–5DD). Taken together, the HEOs remained characteristics of hepatic cells after long-term culture in a Matrigel.