Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

Abstract

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1.

Figure 1

(a) Schematic representation of the 4-stage differentiation protocol and the major regulatory factors administered at each stage: the differentiation protocol recapitulates the stages of ontogenic development of liver; starting from the undifferentiated human induced pluripotent stem cells (hiPSCs), the cells undergo differentiation to Definitive Endoderm (DE), followed by Foregut Endoderm (FE) from where the Hepatic Progenitor Cells (HPCs) or Hepatoblasts arise. The final maturation step leads to mature Hepatocyte-Like Cells (HLCs). (b) The hiPSC-EBs differentiated with both WIF-1 and DKK-1 exhibited greater expressions of hepatocyte-specific markers relative to the ones differentiated without the WIF-1 and DKK-1. Data presented as mean ± SD (n = 3). (c) The hiPSC-EBs differentiated without WIF-1 and DKK-1 showed greater expressions of cholangiocyte-specific markers relative to the ones differentiated with both WIF-1 and DKK-1. Data presented as mean ± SD (n = 3).

Figure 2

(a) Stage-specific protein expressions of hiPSC-EBs during the differentiation process. SOX17 and FOXA2 for the definitive endoderm stage; HHEX and GATA4 for the foregut endoderm; AFP and HNF-4α for the hepatic progenitor cells; ALBUMIN and CK-18 for the mature HLCs. DAPI stains for cell nuclei. Scale bar 100 μm. (b) Stage-specific gene expression analysis by Real-Time PCR. The relative quantities of stage-specific genes were measured at the mRNA level to follow the progression of the differentiation process. Sox17 as the definitive endoderm marker; Gata4 as the foregut endoderm marker; HNF-4α as the hepatic progenitor cells marker; Albumin was used to determine the final maturation for the hepatocyte-like cells (HLCs). Undifferentiated cells were used as negative control. (c) quantitative RT-PCR displayed the presence of mRNA for AFP, five P450 isoforms (Cyp3A4, Cyp2C9, Cyp3A7, Cyp1B1, and Cyp2B6), Albumin, and CK18 in the terminally differentiated hiPSC-EB-HLCs with and without inhibitors. Gene expression for the condition with inhibitors was greater compared with the one without inhibitors for any gene tested; (d,e) Following the differentiation program, terminally differentiated hiPSC-EB-HLCs expressed mature hepatocyte-specific markers, as evidenced by co-staining of ALBUMIN and HNF-1α, and ALBUMIN and C-MET. Scale bar 100 μm. (f) FACS analysis for albumin positive cells showed a higher percentage of albumin producing cells in the condition with inhibitors compared with the one without inhibitors (80% vs 68%).

Figure 3. Secretion of hepatic proteins by hiPSC-EB-HLCs.

The conditioned medium from hiPSC-EB-HLCs was collected 48 hours following the completion of the differentiation process for both conditions with and without inhibitors. (a) Albumin, (b) Alpha Fetoprotein (AFP) and (c) fibrinogen were detected in the medium and (d) intracellular Urea was detected. The difference in secretion between the conditions with inhibitors was statistically significant with respect to the condition without inhibitors. Undifferentiated hiPSCs were used as negative control. The results are representative of at least three independent experiments. Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

The resultant hiPSC-EB-HLCs displayed functional activities typical of mature primary hepatocytes, such as (a) Acetylated low-density lipoprotein (DiI-ac-LDL) uptake in red; (b) Indocyanine green (ICG - Cardiogreen) uptake; (c) ICG release after 6 hours; (d) glycogen storage indicated by PAS staining; and (e) cytoplasmic accumulation of neutral triglycerides and lipids indicated by Oil-Red O staining for both conditions with and without inhibitors. Undifferentiated hiPSCs were used as negative control. Scale bar 100 μm.

Figure 5

(a) Ammonium metabolism assay over a 24-hour period for both conditions with and without inhibitors; Cytochrome P450 (CYP450) induction analysis comparing the two experimental conditions with and without inhibitors. Several CYP enzymes were evaluated through incubation of the cells with different inducers: (b) Phenobarbital for the CYP2B6, (c) Rifampicin for the CYP3A4 and (d) Omeprazole for the CYP1A2 for a period of 72 hours. DMSO was used as control to test the basal activity of different CYP450. Data presented as mean ± SD (n = 3). *p < 0.05; ***p < 0.001.

Figure 6. In vivo functionality of the hiPSC-EB-HLCs in a rat model of acute liver failure induced by D-Galactosamine.

(a) Serum level of alanine aminotransferase (ALT). The mean values of ALT prior to liver injury was 53 U/L; after injury was significantly higher at 3781 U/L; and at 2 weeks was 78 U/L following transplantation of hiPSC-EB-HLCs treated with the two inhibitors, and 364 U/L for the hiPSC-EB-HLCs without inhibitors; (b) The Kaplan-Meier survivals were determined for 12 days after cell transplantation; (c) Histological examination of the liver sections of the survived animals at 14 days after hiPSC-EB-HLCs transplantation showed intense positive staining for human albumin; 20x and 40X. (e) Immunofluorescence of the rat liver after transplantation showed the co-staining of several human hepatic proteins such as HNF-3β, Albumin and C-MET; (f) Immunofluorescence of human liver was used as positive control displaying staining of all three human hepatic proteins.