Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids

Abstract

Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs).

Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation.

Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia.

Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

Keywords: HLCs; bile acids; bile canaliculi network; detoxification; hepatic mature functions; hiPSC-derived hepatocytes; hiPSCs; liver organoids; metabolism; self-assembling.

Figure 1

Generation of iHep-Orgs from hiPSCs. (A) Schematic representation of the differentiation protocol and brightfield images of undifferentiated hiPSCs, differentiated endoderm (day 5), hepatoblasts (day 11), and self-organized iHep-Orgs after 24 h of assembling (day 12) and over time (days 18, 25, 28, 38). Scale bars for brightfield images = 100 µm. Scale bars for iHep-Orgs = 500 µm. Scale bars for magnifications = 100 µm. (B) Evolution of diameter and DNA content of iHep-Orgs over time (measures carried out on 35 and 200 organoids, respectively). Graph represents mean values ± SD (n = 16). (Ca) Viability assay of iHep-Orgs at day 38 of differentiation. Alive cells’ nuclei are stained in blue; dead cells’ nuclei are stained in green. Scale bars = 100 µm. Images are the Z-projections obtained by combining multiple images taken at different focal distances (z-stacking); (Cb) H&E staining on section of a representative iHep-Org. Scale bar = 100 µm. (D) RT-PCR analysis of AFP, ALB, CYP3A7, and CYP3A4 gene expression in iHep-Orgs over time and PHH-Orgs at day 8 of culture. (E) AFP, ALB, and CYP3A4 gene expression in iHep-Orgs over time (blue bars). PHHs have been used as control (brown bars). Quantification is relative to the expression level in fetal human hepatocytes (GS 20 weeks). Histograms represents mean ± SD (n = 16). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05.

Figure 2

Immunofluorescence staining of iHep-Orgs at the final stage of the differentiation. (A) ELISA quantification of AFP and ALB secretion of iHep-Orgs (blue bars) and PHH-Orgs (brown bars). Histograms represent mean ± SD (n = 16). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05 (B) Whole iHep-Orgs immunostained for HNF4α, ALB, CYP3A4, ZO-1, CK-8, BSEP, and MDR3. Nuclei are stained with DAPI. Scale bars = 200 µm. (C) Sections (7 µm thick) of iHep-Orgs immunostained for CK-19, EPCAM, AFP, ALB, HNF1α, ASGR, CX32, BSEP, MDR1, MDR3, CYP3A4, UGT1A1, G6Pase, mitochondria, coagulation factor IX (FIX). Nuclei are stained with DAPI. Scale bars = 100 µm.

Figure 3

Bile acid secretion and bile canaliculi network analysis of iHep-Orgs. (A) Left panel: bile acid production and secretion by iHep-Orgs in culture supernatants at day 20 (light blue bars) and 38 (blue bars) of culture. Histograms represent mean ± SD (n = 3). CA = cholic acid; DCA = deoxycholic acid; CDCA = chenodeoxycholic acid; GCA = glycocholic acid; GDCA = glycodeoxycholic acid; GCDCA = glycochenodeoxycholic acid; TCA = Taurocholic acid; TDCA = taurodeoxycholic Acid; TCDCA = taurochenodeoxycholic acid. Right panel: total bile acid production and secretion by iHep-Orgs (blue bars) and PHH-Orgs (brown bars) over time. Histograms represent mean ± SD (n = 3). (B) Live imaging of the bile canaliculi network formed by excretion of the fluorescent probe DCFA in iHep-Org at day 38. Scale bar = 100 µm. (C) Immunofluorescence staining for the apical membrane transporter BSEP in iHep-Org sections (75 µm) at day 38. Scale bar = 75 µm. (D) Left panel: 3D reconstruction of the BC network from the apical membrane transporter BSEP staining in iHep-Org sections (75 µm) at day 38. Right panel: 3D reconstruction of a segment (ROI 1) of the BC network (rotation in x axis). (E) Quantification and length measurements of bile canaliculi recorded on the surface (light blue bars) and in the core (blue bars) of the iHep-Orgs after DCFA treatment and BSEP staining. Three iHep-Orgs from each of 6 independent experiments were analyzed.

Figure 4

Activity of phase I and II metabolisms of iHep-Orgs. (A) CYP1A1, CYP1A2, CYP2B6, CYP3A7 and CYP3A4 activities measured in iHep-Orgs without (light bars) and after induction (blue bars). Histograms represent mean ± SD (n = 3). (B) CYP1A2 (EROD) and CYP3A4 (BROD) specific activities of iHep-Orgs (blue shade bars) and PHH-Orgs (brown shade bars). Histograms represent mean ± SD (n = 8). (C) UGT1A1 activity of iHep-Orgs (blue bars) and PHH-Orgs (brown bars). Graph represents mean ± SD (n = 6). (D) ADH activity of iHep-Orgs (blue shade bars) and PHH-Orgs (brown shade bars). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05.

Figure 5

Metabolism in iHep-Orgs. (A) Scheme depicting the glucose metabolism analysis in iHep-Orgs. (a,b) Periodic acid–Schiff staining of glycogen in iHep-Orgs under hyperglycemic and hypoglycemic conditions, respectively. Scale bar = 50 µm. (c) Representation of the gluconeogenesis process from pyruvate in hepatocytes. Graph of glucose quantification after glycogenolysis (bottom left) and gluconeogenesis (bottom right) in iHep-Orgs (blue bars) and PHH-Orgs (brown). (B) Lipid metabolism in iHep-Orgs. Oil Red O’ staining of lipid droplets in (a) iHep-Orgs cultured in normal conditions and (b) iHep-Orgs cultured in presence of high lipid concentration.

Figure 6

Detoxification abilities of iHep-Orgs in moderate pathological conditions. (A) Lactate detoxification and (B) urea synthesis of iHep-Orgs (blue bars) and PHH-Orgs (brown bars). Graphs represent mean ± SD (n = 8). *** indicate p < 0.001; ** indicate p < 0.01.