Fig 1: (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-4a 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-4a 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-1a, 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%).
Fig 2: Comparison of qRT-PCR and newly developed TLN assay.(A) Comparable qRT-PCR (200 μl plasma) and TLN (20 μl plasma) assay results on GAPDH levels in plasma EVs. (B) Results of different AFP molecular beacons detecting different locations of AFP mRNA fragments and no significant difference was found among different AFP molecular beacons (P>0.5).
Fig 3: 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. (g–n) 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.
Fig 4: Median expression levels of EV AFP and GPC-3 mRNA are significantly higher in HCC patients than in healthy controls.(A) Representative TLN-TIRF images (80 × 80 μm) (100X) of EV AFP and GPC-3 mRNA expression in a HCC patient and a healthy donor plasma samples. (B) Dot charts of EV AFP and GPC-3 mRNA expression in HCC patient and healthy donor plasma samples. (C) Scatter plots of plasma AFP protein vs. EV GPC-3 mRNA, EV AFP mRNA vs. EV GPC-3 mRNA, and plasma AFP protein vs. EV GPC-3 mRNA between HCC patients and healthy donors.
Fig 5: GP73 promotes AFP secretion.A GP73 overexpression decreases the protein level of intracellular AFP and increases the level of extracellular AFP. The protein levels of GP73 and AFP in cell lysate and supernatant were measured by western blotting in HepG2 and PLC cells transfected with PCDNA3-GP73 or PCDNA3 (as a control). β-actin or GST was respectively used as an intracellular or extracellular loading control. The mRNA level of AFP in cell lysate was measured by Real-time PCR. B GP73 knockout increases the protein level of intracellular AFP and decreases the level of extracellular AFP. The protein levels of GP73 and AFP in cell lysate and supernatant were measured by western blotting in HepG2 and PLC cells mediated GP73 knockout by CRISPR/Cas9 system. β-actin or GST was respectively used as an intracellular or extracellular loading control. The mRNA level of AFP in cell lysate was measured by Real-time PCR. C GP73 overexpression increases the secretion level of AFP. Stable transfected HepG2 and PLC cells overexpressed GP73. Cell culture supernatant was collected and AFP level was measured by ELISA. D GP73 knockout decreases the secretion level of AFP. The expression of GP73 in HepG2 and PLC cells were knockout by CRISPR/Cas9 system. Cell culture supernatant was collected and AFP level was measured by ELISA. Error bars represent S.D. *p < 0.05, **p < 0.01.
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