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. 2019 Jun 20;9(1):8920.
doi: 10.1038/s41598-019-45514-3.

Generation of Fully Functional Hepatocyte-Like Organoids From Human Induced Pluripotent Stem Cells Mixed With Endothelial Cells

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Free PMC article

Generation of Fully Functional Hepatocyte-Like Organoids From Human Induced Pluripotent Stem Cells Mixed With Endothelial Cells

Giuseppe Pettinato et al. Sci Rep. .
Free PMC article

Abstract

Despite advances in stem cell research, cell transplantation therapy for liver failure is impeded by a shortage of human primary hepatocytes (HPH), along with current differentiation protocol limitations. Several studies have examined the concept of co-culture of human induced pluripotent cells (hiPSCs) with various types of supporting non-parenchymal cells to attain a higher differentiation yield and to improve hepatocyte-like cell functions both in vitro and in vivo. Co-culturing hiPSCs with human endothelial cells (hECs) is a relatively new technique that requires more detailed studies. Using our 3D human embryoid bodies (hEBs) formation technology, we interlaced Human Adipose Microvascular Endothelial Cells (HAMEC) with hiPSCs, leading to a higher differentiation yield and notable improvements across a wide range of hepatic functions. We conducted a comprehensive gene and protein secretion analysis of our HLCs coagulation factors profile, showing promising results in comparison with HPH. Furthermore, a stage-specific glycomic analysis revealed that the differentiated hepatocyte-like clusters (HLCs) resemble the glycan features of a mature tissue rather than cells in culture. We tested our HLCs in animal models, where the presence of HAMEC in the clusters showed a consistently better performance compared to the hiPSCs only group in regard to persistent albumin secretion post-transplantation.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Real-time PCR of the coagulation cascade and fibrinolysis genes. The main factors involved in the coagulation cascade, from both the intrinsic, extrinsic and common pathway, as well as some factors involved in the fibrinolysis, were tested. In most of the cases, in both hiPSC-EB-HLCs and hiPSC-EB + EC-HLCs we observed a gene expression that was statistically higher than the one observed in human primary hepatocyte (HPH) and human neonatal hepatocyte (HNH). For some genes, such as FXII, FIX, FVIII, FV, Fibrinogen, FXIIIb, and VWF the expression of our differentiated hiPSC-EB + EC-HLCs was significantly higher than the hiPSC-EB-HLCs. HPH was used as a positive control for mature phenotype, while HNH was used as a control for immature genotype. Results for the differentiated hiPSCs for both conditions were normalized GAPDG and to undifferentiated hiPSCs (experimental negative control). Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2
Figure 2
Quantification analysis of secreted coagulation factors. We collected the conditioned medium for 6 days every other day for both primary hepatocyte, hiPSC only and plus endothelial cells (HAMEC), and we analyzed the conditioned media to study the secretion of several proteins involved in the coagulation cascade. (al) As expected, primary hepatocyte showed higher level of coagulation factors secretion when compared with our experimental conditions in the beginning, however, starting from day four, they start to lose functions, where our experimental conditions showed a better performance in most of the factors studied, and in certain cases even higher than the HPH themselves. In most of the cases hiPSC-EB + EC-HLCs performed better than hiPSC-EB-HLCs. (m) Factor Xa generation assay. The presence of endothelial cells in our differentiated clusters led to a higher affinity of the factor Xa generation enzyme to the substrate as indicated from the lower Km when comparing the hiPSC only with the hiPSCs plus endothelial cells. As positive control HPH, and HAMEC in monolayer culture and HAMEC in 3D culture (dashed line), were used. (n) Results for the thrombin generation assay showed that the microparticles released from hiPSC-EB + EC-HLCs amplified thrombin generation when compared with the condition without HAMEC.
Figure 3
Figure 3
Tissue-specific gene expression analysis by Real-Time PCR. The relative quantities of tissue-specific genes were measured at the mRNA level to assess the final maturation of the terminally differentiated HLCs for both experimental conditions. The results showed an overall trend of higher expression of all markers for the hiPSC-EB + EC-HLCs when compared to the hiPSC-EB-HLCs, with statistically significant higher expression for the albumin, HNF-4α, HNF-4β, CYP1A2, CYP2B6 and UGT1A3. Almost for all the genes tested, both hiPSC-EB-HLCs and hiPSC-EB + EC-HLCs showed a gene expression that was statistically higher than the one observed in human primary hepatocyte (HPH) and human neonatal hepatocyte (HNH). HPH was used as a positive control for mature phenotype, while HNH was used as a control for immature genotype. Results for the differentiated hiPSCs for both conditions were normalized to undifferentiated hiPSCs (experimental negative control). Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
Tissue-specific marker analysis through immunofluorescence and FACS analysis. Following the differentiation program, terminally differentiated hiPSC-EB + EC-HLCs expressed mature hepatocyte-specific markers, as evidenced by the presence co-staining of (a) ALBUMIN and Alpha-1 Anti-Trypsin (A1AT), (b) ALBUMIN and ALT, (c) ALBUMIN and AST (d) ALBUMIN and CD31, (e) ALBUMIN and HNF-3β. Scale bar 100 µm. (f) FACS analysis for albumin positive cells showed a greater percentage of albumin positive cells in the condition with endothelial compared with the one without (86% vs 59%). (g) Magnified (60X) detail of ALBUMIN/CD31 positive cells showing the rosettes organization of CD31 positive cells interspersed with ALBUMIN positive cells. Scale bar 50 µm.
Figure 5
Figure 5
Secretion pattern of several hepatic proteins by hiPSC-EB-HLCs. Conditioned media from hiPSC-EB-HLCs were collected after 48 hours from the completion of the differentiation protocol for both conditions with and without endothelial cells. (a) Albumin, (b) fibrinogen and (c) Alpha Fetoprotein (AFP) were detected in the medium and (d) intracellular Urea was detected. Differences in secretion between the conditions with endothelial cells were statistically significant with respect to the condition without endothelial cells for the Albumin and AFP. There was not statistically significant difference between the two experimental conditions for the Fibrinogen and Urea intracellular concentration. Undifferentiated hiPSCs were used as negative control, and human primary hepatocyte as positive 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; Detoxification property analysis of the differentiated HLCs. (e) The ammonium metabolism assay conducted on a period over 24-hour for both conditions with and without endothelial cells showed a higher ability of ammonium clearance for the hiPSC-EB + EC-HLCs (about 45% from the first hour) when compared with hiPSC-EB-HLCs (about 20% from the first hour); (f) Phase II detoxification analysis through resorufin conjugation assay: The results showed a higher formation rate for the condition hiPSC-EB + EC-HLCs compared with the hiPSC-EB-HLCs, reaching similar level of the HPH used as positive control. (gn) Cytochrome P450 (CYP450) induction analysis: Several CYP enzymes were assessed through incubation of the differentiated HLCs with specific inducers: Omeprazole for the (g) CYP1A1, and (h) CYP1A2; Rifampicin for the (i) CYP3A4, and (l) CYP3A7; and Phenobarbital for the (m) CYP2B6, and (n) CYP2C9 for a period of 72 hours. DMSO was used as control to test the basal activity of the different CYP450. Data presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
Ability of the differentiated HLCs to store different metabolites. The resultant hiPSC-EB-HLCs and hiPSC-EB + EC-HLCs showed hepatocyte functional activities, such as (a) Indocyanine green (ICG - Cardiogreen) uptake; (b) ICG release after 6 hours; (c) cytoplasmic accumulation of neutral triglycerides and lipids indicated by Oil-Red O staining; (d) glycogen storage indicated by PAS staining; and (e) Acetylated low-density lipoprotein (DiI-ac-LDL) uptake in red. Both conditions with and without endothelial cells showed a similar ability for all the metabolites assessed. Undifferentiated hiPSCs were used as negative control. Scale bar 100 μm.
Figure 7
Figure 7
Mass spectrometry N- and O-glycan analyses of stage-specific hiPSCs differentiation. (a) Heat map representation of the relative abundance of the N-glycans identified in undifferentiated hEBs (Undiff. hEBs), hiPSCs Stage 1, hiPSCs Stage 2, and hiPSCs Stage 3, with (+) or without (−) co-culture of HAMEC. N-glycans with a relative abundance of less than 0.2% in all cell conditions are not represented here. (b) Heat map representation of the relative abundance of the O-glycans identified in Undiff. hEBs, hiPSCs Stage 1, hiPSCs Stage 2,and hiPSCs Stage 3, + or − co-culture of HAMEC. (c) Heat map representation of the relative abundance of the O-glycans identified HPH, HNH, Undiff. hiPSCs and hiPSC Stage 4 + or − co-culture HAMEC. (d). Heat map representation of the relative abundance of the N-glycans identified in human primary hepatocytes (HPH), human neonatal hepatocytes (HNH), undifferentiated hiPSCs (Undiff. hiPSCs) and hiPSC Stage 4 with (+) or without (−) co-culture HAMEC. N-glycans with a relative abundance of less than 0.2% in all cell conditions are not represented here.
Figure 8
Figure 8
In vivo transplantation of hiPSC-EB-HLCs with and without HAMEC in a D-Galactosamine induced acute liver failure rat model. (a) Kaplan–Meier survival curve of 10- to 14-week-old control animals. 90% (8 of 9) of the rats that acquired liver injury with values of alanine aminotransferase (ALT) > 3,000 U/L at 1 day after injection died within 3-days, compared with a 3-day mortality (2 of 5) in those with an ALT < 3,000 U/L; (b) Kaplan–Meier survival plot of animals after HLCs transplantation with and without HAMEC, showed a similar survival rate between the two experimental conditions; (c–f) Representative liver and spleen sections with immunohistochemical staining. Background staining with hematoxylin-eosin. (c) Image of rat liver section transplanted with hiPSC-EB + EC-HLCs (20X left panel and 40X right panel). Double immunostaining was performed using a non-rat cross-reactive antibody to human albumin and non-rat cross-reactive antibody to human EC marker platelet EC adhesion molecule (PECAM)-1. Black arrows indicate presence of intracellular human albumin but there was not presence of PECAM-1. (c) Image of a rat spleen section transplanted with hiPSC-EB + EC-HLCs (20X left panel and 40X right panel). Double immunostaining was performed using a non-rat cross-reactive antibody to human albumin and non-rat cross-reactive antibody to human EC marker platelet EC adhesion molecule (PECAM)-1. Black arrows indicate human PECAM-1 positive ECs within the spleen but not the presence of human albumin. (e) Liver section from a control rat that was transplanted with medium only (negative control). Section was stained for human albumin and human PECAM-1 (10X on left panel and 20X on right panel). The sections show the absence of staining for both antibodies, indicating the specificity for human species of the antibodies used. (f) Normal human liver section (positive control) stained for human albumin (10X on left panel and 20X on right panel). Black arrows indicate presence of human albumin both intracellularly and extracellularly. Scale bar 2.5 µm. (gi) Representative liver and spleen sections with immunofluorescence staining. (g) Representative image of liver section from a rat transplanted with hiPSC-EB + EC-HLCs. Immunofluorescence staining with non-rat cross-reactive antibodies to hepatocyte markers human HNF-3β, human albumin, and human C-MET displayed the presence of all 3 markers. (h) Representative image of a spleen section from a rat transplanted with hiPSC-EB + EC-HLCs. Immunofluorescence staining with non-rat cross reactive antibodies to PECAM-1, human albumin and human C-MET display presence of human PECAM-1 but no presence of human albumin or C-MET. (i) Normal human liver section (positive control) stained for human HNF-3b, human albumin and human C-MET. Image displays presences of all 3 markers. Nuclear staining with DAPI. Scale bar represents 100 µm.

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