Exosomes From Intestinal Epithelial Cells Promote Hepatic Differentiation of Liver Progenitor Cells in Gut-Liver-on-a-Chip Models

Abstract

Hepatic progenitor cells (HPCs) are frequently overactivated, and their differentiation into hepatocytes is impaired in advanced liver diseases. To explore the effects of intestinal epithelial cells and their exosomes on the hepatic differentiation of HPCs, co-culture systems of Caco-2/HepaRG cell lines and intestine/HPC organoids are established in a novel gut-liver-on-a-chip. Exosomes derived from intestinal organoids are administered to mice with carbon tetrachloride (CCL4)-induced liver fibrosis. The results showed that the co-culture of HPCs and intestinal epithelial cells promoted the hepatic differentiation of HPCs, mediated by exosomes derived from intestinal epithelial cells. Treatment with exosomes derived from intestinal organoids ameliorated liver fibrosis in a mouse model of CCL4-induced liver fibrosis. A cluster of miRNAs, miR-371-373, is identified within the exosomes of the intestinal epithelial cells, which target RPS6KA2 to modulate hepatic differentiation. This findings demonstrate that exosomes from intestinal epithelial cells promote the hepatic differentiation of HPCs. Exosomes from intestinal organoids may be a novel therapeutic strategy for the treatment of advanced liver diseases.

Keywords: exosomes; hepatic progenitor cells; intestinal epithelial cells; liver fibrosis; organ‐on‐a‐chip.

Figure 1

Establishment of an integrated gut‐liver‐on‐a‐chip with cell lines and organoids. a) The schema of the gut‐liver‐on‐a‐chip displays the upper left gut chamber (green), vessel‐like medium channel (red), and right bilateral liver chambers (blue). The bright field images of each area of the chip are shown below the schema, including the gut chamber (left), vessel‐like medium channel (middle), and liver chamber (right). Scale bar = 100 µm. b) Schematic diagram of the chip structure. c) Diagram of the assembled microfluidic device. d) Bright‐field images of intestinal epithelial cells (Caco‐2) and hepatic progenitor cells (HepaRG) cultured on the chip on Day 6. Scale bar = 100 µm. e) Left panel: Caco‐2 cells in the gut chamber were co‐stained with antibodies against E‐CAD (red), L‐FABP (green), and DAPI (blue) to show the nuclei. Right panel: HepaRG cells in the liver chamber were stained with antibodies against KRT18 (green). Scale bar = 100 µm. f) The Caco‐2 cells that grew near the inlet and outlet of the gut chamber were immunostained with antibodies against Villin, KRT20, LGR5, MUC2 (green), E‐CAD (red), and DAPI (blue) to show nuclei. Left panels: low magnification; right panels: high magnification. Scale bar = 100 µm. g) Full field of view: mini‐gut (Caco‐2 cells) on the chip cultured for 4 days, with four continuous bright‐field images. Scale bar = 100 µm. h) Images of Caco‐2 cells cultured for five days in the gut chamber. Immunostaining for intestinal cell markers: SOX9+ crypt stem cells (green), MUC2+ goblet cells (green), lysozyme+ Paneth cells (green), and L‐FABP+ enterocytes (green) were co‐stained with E‐CAD (red) and DAPI (blue). The rightmost images show enlarged views of the selected areas in the corresponding pictures immediately to the left. Scale bar = 100 µm. i) HepaRG cells cultured on the chip for 12 days were co‐stained with antibodies against AAT (green), KRT18 (red), and DAPI (blue). Scale bar = 100 µm. j) Bright‐field images of Int‐Orgs on the chip. Upper left panel and upper right panel: Int‐Orgs at the stage of maintenance perfusion I (Day 2) and high‐magnification field. Middle left and middle right panels: Int‐Orgs at stage of maintenance perfusion II (Day 6) and high‐magnification field. Lower left panel: Int‐Orgs after differentiation for 5 days. Lower right panel: Int‐Orgs after differentiation for 7 days. Scale bar = 100 µm. k) Bright‐field images of HPC‐Orgs on chip. Upper left panel: HPC‐Orgs on Day 3. Middle left and middle right panels: HPC‐Orgs at the stage of maintenance perfusion (Day 5 and Day 7). Lower left and lower right panels: Hep‐Orgs at differentiation stage II (Day 10) and high‐magnification field. Lower right corner: Hep‐Orgs at differentiation stage II (Day 10) with the morphological and structural details of the organoid cells in high magnification field. Scale bar = 100 µm. l) Immunofluorescence images of Int‐Orgs (differentiated) on the chip: Villin, MUC2, L‐FABP, lysozyme, and KRT‐20 (green), E‐CAD (red), and DAPI (blue). The three panels in the top right are magnified images corresponding to the square in the adjacent panels on the left. Scale bar = 100 µm. m) Immunofluorescence images of Hep‐Orgs on the chip: KRT‐18 (green), BSEP (red), and DAPI (blue). Scale bar = 100 µm.

Figure 2

Enhanced hepatic differentiation of HPCs (HepaRG/HPC‐Orgs) cocultured with intestinal cells (Caco‐2/Int‐Orgs) on the chip. a) Caco‐2 and HepaRG cells were cocultured on the chip for 5 days. Immunostaining was used to compare the protein levels between the RG‐M (HepaRG monoculture) and RG‐C (HepaRG cocultured with Caco‐2) groups on the chip. Red: KRT‐18, green: ALB, yellow: HNF4A, blue: DAPI. Scale bar = 100 µm. Right‐side charts show the fluorescence intensity of the labelled proteins in the left‐side panel (n = 3, mean ± SD, Student's t‐tests). ^* p < 0.05. b) Immunofluorescence staining of the markers of hepatic differentiation in mono‐ and cocultured Hep‐Orgs on the chip. Hep‐Orgs on chip‐M: Hep‐Orgs monocultured on chip at differentiation stage II (Day 5); Hep‐Orgs on chip‐C: Hep‐Orgs cocultured with Int‐Orgs on the chip at differentiation stage II (Day 5). ALB (green), KRT‐18 (red), HNF4A (red), EPCAM (red), E‐CAD (red or green), and DAPI (blue) are shown in the left panel. Scale bar = 100 µm. The right‐side charts show the fluorescence intensity of the detected proteins (n = 3, mean ± SD, Student's t‐tests). ^* p < 0.05. c) Expression of genes related to hepatic differentiation and stemness in Hep‐Orgs cultured in Transwell plates (3D) and the chip was measured by qRT‒PCR. A: Hep‐Orgs‐M‐3D, Hep‐Orgs monocultured in the Transwell 3D system. B: Hep‐Orgs‐C‐3D, Hep‐Orgs cocultured with Int‐Orgs in the Transwell 3D system. C: Hep‐Orgs‐M‐chip, Hep‐Orgs monocultured on chip. D: Hep‐Orgs‐C‐chip, Hep‐Orgs cocultured with Int‐Orgs on chip; 3: after two‐stage differentiation for 3 days; 6: after two‐stage differentiation for 6 days. The heatmap displays the log2 (fold change) value of the genes. Correlation analysis was conducted according to the gene expression of each group (n = 3). d) Expression of genes related to hepatic differentiation and stemness in HepaRG cells in Transwell (3D) and the chip was detected by qRT‐PCR. A: RG‐M‐3D, HepaRG monocultured in Transwell 3D system; B: RG‐C‐3D, HepaRG cocultured with Caco‐2 cells in Transwell 3D system; C: RG‐M‐chip, HepaRG monocultured on chip; D: RG‐C‐chip, HepaRG cocultured with Caco‐2 cells on chip. 1, 2, and 3 represent the day(s) of coculture. The heat map displays the log2 (fold change) value of the genes. Correlation analysis was conducted according to the gene expression trend of each group (n = 3). e,f) Measurement of CYP metabolites: DXO (CYP2D6), 6βOH‐TEST (CYP3A4), and APAP (CYP1A2) in HepaRG cells (e) and Hep‐Orgs (f) mono‐ and coculture groups on chip within 8 days (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05. g) ELISA data of the levels of secreted ALB and AAT in the HepaRG cells monocultured and cocultured in the Transwell‐3D system and chip in 6 days (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05 in RG‐Caco‐2 3D(static) versus RG 3D(static), ^# p < 0.05 in RG‐Caco‐2 chip(perfused) versus RG chip(perfused). h) ELISA data of the levels of secreted ALB and AAT in Hep‐Orgs monocultured and cocultured in the Transwell‐3D system and chip in 6 days (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05 in Hep‐Int‐Orgs 3D(static) versus Hep‐Orgs 3D(static), ^# p < 0.05 in Hep‐Int‐Orgs chip(perfused) versus Hep‐Orgs chip(perfused). i) Urea synthesis analysis of HepaRG cells monocultured and cocultured in Transwell and chip detected by colorimetry within 6 days (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05 in RG‐Caco‐2 3D(static) versus RG 3D(static), ^# p < 0.05 in RG‐Caco‐2 chip(perfused) versus RG chip(perfused). j) Urea synthesis analysis of Hep‐Orgs monocultured and cocultured in Transwell and chip, detected by colorimetry within 6 days (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05 in Hep‐Int‐Orgs 3D(static) versus Hep‐Orgs 3D(static), ^# p < 0.05 in Hep‐Int‐Orgs chip(perfused) versus Hep‐Orgs chip(perfused).

Figure 3

The effects of intestinal EXOs inhibition on the hepatic differentiation of HPCs (HepaRG/Hep‐Orgs). a) Bright‐field images of Hep‐Orgs on the chip at the differentiation stage II (Day 1). Upper panels and lower panels: Hep‐Orgs on chip under monoculture (left), coculture (middle), and coculture treated with GW4869 (right) at differentiation stage II (Day 1), and high‐magnification images corresponding to the squares. Scale bar = 100 µm. The column chart on the right shows the proportion of the area of tight junction of Hep‐Orgs in panel (a) (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. b) Bright‐field images of Hep‐Orgs on the chip at the differentiation stage II (Day 6). Upper panels and lower panels: Hep‐Orgs on chip under monoculture (left), coculture (middle), and coculture treated with GW4869 (right) at differentiation stage II (Day 6) and high‐magnification images corresponding to the squares. Scale bar = 100 µm. The column chart on the right shows the proportion of the area of tight junction of Hep‐Orgs in panel (b) (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. c) The levels of secreted proteins (ALB, AAT) of Hep‐Orgs on chip were detected by ELISA in monoculture (control), coculture, and coculture treated with GW4869 during the 6 days of the differentiation stage II (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 for Hep‐Int‐Orgs(chip) versus Hep‐Orgs(chip), #p < 0.05 for Hep‐Int‐Orgs‐GW4869(chip) versus Hep‐Orgs(chip). d) Levels of secreted proteins (ALB, AAT) in HepaRG cells on the chip detected by ELISA in monoculture (control), coculture, and coculture treated with GW4869 during the 6 days of the differentiation stage II (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 for RG‐Caco‐2(chip) versus RG(chip), #p < 0.05 in RG‐Caco‐2‐GW4869(chip) versus RG(chip). e) The levels of secreted proteins (ALB, AAT) in monocultured, cocultured, and EXO‐treated HepaRG cells on the chip were detected by ELISA during 6 days of differentiation (stage II) (n = 3, mean ± SD, Two‐way ANOVA). ^* p < 0.05 in RG‐Caco‐2(chip) versus RG(chip), # p < 0.05 for RG‐EXO (Caco‐2)‐5 µg mL⁻¹ (chip) versus RG(chip). f) The levels of secreted proteins (ALB, AAT) of Hep‐Orgs on the chip were detected by ELISA in monoculture, coculture, and EXO‐treated groups during the 6 day differentiation stage II (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 in Hep‐Int‐Orgs(chip) versus Hep‐Orgs(chip), #p < 0.05 for Hep‐Orgs‐EXO(Int‐Orgs)‐10 µg mL⁻¹ (chip) versus Hep‐Orgs(chip). g) Urea synthesis of Hep‐Orgs in the different groups was measured by colorimetry within 6 days of differentiation II (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 in Hep‐Int‐Orgs(chip) versus Hep‐Orgs(chip), #p < 0.05 in Hep‐Orgs‐EXO(Int‐Orgs)‐10 µg mL⁻¹ (chip) versus Hep‐Orgs(chip). h) HepaRG cells in monoculture (RG‐M), EXO‐treated (RG‐EXOs), and coculture (RG‐C) groups were cultured on chips for three, five, and nine days. The corresponding high‐magnification images of the squares are shown on the right side. Scale bar = 100 µm. The line graph on the right presents the size variations of the HepaRG cell spheroids in panel (h) (n = 3, mean ± SD, One‐way ANOVA). ^* p <0.05, RG‐EXOs versus RG‐M; # p <0.05, RG‐C versus RG‐M. i) Expression of hepatic cell markers, including ALB (green), KRT18 (red), and HNF4A (yellow), in monocultured and EXO‐treated HepaRG cells on the chip, detected by immunostaining on day 5. Scale bar = 100 µm. The charts show the fluorescence intensity of the proteins in each group (n = 3, mean ± SD, Student's t‐tests). ^* p < 0.05.

Figure 4

Changes in hepatic function and morphology of Hep‐Orgs cultured in the coculture system and EXO‐treated system. a) The expression of ALB (green), KRT18 (red), and HNF4A (red) in hepatocytes, as well as EPCAM (red), in the monocultured and EXO‐treated Hep‐Orgs on the chip on Day 5 of the differentiation stage II detected by immunostaining. E‐CAD, red or green; DAPI, blue. Scale bar = 100 µm. The bar charts show the fluorescence intensity of detected proteins (n = 3, mean ± SD, Student's t‐tests). ^* p < 0.05. b) Scatter plots showing the expression of ALB (FITC), KRT18 (APC), HNF4A (FITC), and AAT (APC) in HepaRG on‐chip (undif)‐M, HepaRG on‐chip (dif)‐M, HepaRG on‐chip (dif)‐C, and HepaRG on‐chip (dif)‐EXOs groups after 5 days, as detected by flow cytometry. M, monoculture; C, coculture with Caco‐2 cells; EXOs, treatment with EXOs from Caco‐2 cells. Histograms show the differences in the expression of these indices among the groups (n = 3). c) The scatter plots showed the expression of ALB (FITC), KRT18 (APC), HNF4A (FITC), and AAT (APC) in HPC‐Orgs on the chip (undif)‐M, Hep‐Orgs on the chip (dif)‐M, Hep‐Orgs on the chip (dif)‐C, and Hep‐Orgs on the chip (dif)‐EXOs groups after 6 days, detected by Flow cytometry. M = monoculture; C = cocultured with Int‐Orgs; EXOs = treated with EXOs of Int‐Orgs. The histograms show the difference in the expression of these indexes among the groups (n = 3). d) The first column panel: TEM images of HPC‐Orgs on the chip. N = Nucleus, Nu = Nucleoli, Mit = Mitochondria, Tj = Tight junction, Mv = Microvilli; The second column panel: TEM image of Hep‐Orgs on the chip. Lys = lysosome, Bc = bile canaliculi, RER = rough endoplasmic reticulum, Gly = glycogen storage. The red arrow points to the bile canaliculi. The third column panel: TEM images of Hep‐Orgs cocultured with Int‐Orgs on the chip at the differentiation stage II (Day 5). Mvb = Multivesicle bodies. The red arrow points to the bile canaliculi. The fourth column panel: TEM image of EXO‐treated Hep‐Orgs on the chip. Po = Peroxisome, Mvs = Microvesicles. The red arrow points to the bile canaliculi. Scale bar = 2 µm. e) Upper panels: bright‐field images of Hep‐Orgs on the chip under monoculture (left), coculture (right), and EXO‐treated (middle) conditions at the differentiation stage II (Day 6). Lower panels: The corresponding high‐magnification images of the squares in the upper panels and the cells framed by a red box in the panels are magnified in the lower right corner (200×). Scale bar = 100 µm. f) PAS staining of Hep‐Orgs on the chip under monoculture (left), coculture (right), and EXO‐treated (middle) conditions at the differentiation II stage (Day 6). Scale bar = 100 µm. The column chart on the right shows the proportion of the PAS‐stained area in panel (h) (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, ns: not significant.

Figure 5

Treatment of EXOs from Int‐Orgs improved the hepatic fibrosis in mice. a) The macrograph, HE staining, MASSON trichrome staining, and α‐SMA staining images of CCL4‐induced hepatic fibrosis in mice at 6 weeks and 9 weeks in the control groups (Oil+PBS), modelling groups (CCL4+PBS), and treatment groups (CCL4+EXOs). Scale bar = 100 µm. b) The histogram shows the positive area (%) of Masson trichrome staining of the different groups (6w‐left, 9w‐right) (n = 6, mean ± SD, One‐way ANOVA). ^* p < 0.05. c) The histogram shows the positive area (%) of α‐SMA staining of the different groups (6w‐left, 9w‐right) (n = 6, mean ± SD, One‐way ANOVA). ^* p < 0.05. d) Schematic diagram of the procedures of animal experiments. e) Distribution of EXOs in vivo (day 1, day 3, day 5, and day 7 after EXOs injection). f) The ratios of liver weight to body weight of 6w (upper) and 9w (lower) CCL4‐induced mice (n = 6, mean ± SD, One‐way ANOVA). ^* p < 0.05. g) The scatter plots showed the expression of ALB (APC), KRT18 (FITC), and HNF4A (FITC) in C57(9w): Oil + PBS, C57(9w): CCL4 + PBS, and C57(9w): CCL4 + EXOs groups detected by Flow cytometry. The histograms show the difference in the expression of these indexes among the groups (n = 6). h) Serum levels of T‐BIL, D‐BIL, ALT, AST, BUN, ALB, and AAT in control groups (Oil+PBS), modelling groups (CCL4+PBS), and treatment groups (CCL4+EXOs) (n = 6, mean ± SD, One‐way ANOVA). ^* p < 0.05.

Figure 6

Treatment of EXOs from Int‐Orgs increased the differentiation and functional expression of hepatocytes in mice with hepatic fibrosis. a) IHC staining images show the expression of ALB, KRT18, AAT, and AFP of mice liver at 6 weeks (upper) and 9 weeks (down) in the control groups (Oil+PBS), modelling groups (CCL4+PBS), and treatment groups (CCL4+EXOs). Scale bar = 100 µm. b) Immunofluorescence staining images show the expression of ALB and KRT19 of mice liver at 9 weeks in the control groups (Oil+PBS), modelling groups (CCL4+PBS), and treatment groups (CCL4+EXOs). ALB, red; KRT19, green; DAPI, blue. Scale bar = 100 µm. c) The column chart (left) shows the fluorescence intensity of ALB in panel (b). The column chart (right) shows the positive expression area of KRT19 in panel (b) (n = 6, mean ± SD, One‐way ANOVA). ^* p < 0.05. d) Immunofluorescence staining images show the expression of HNF4A and KRT19 in human liver in normal liver groups and cirrhotic liver groups. Scale bar = 100 µm. The column chart on the right presents the proportion of the HNF4A/KRT19 dual‐positive area in panel (d) (n = 10, mean ± SD, Student's t‐tests). ^* p < 0.05.

Figure 7

Regulatory effects of the miR‐371‐373 cluster on the differentiation of HPCs a) The expression of hepatic cell markers in HepaRG cells (cultured in Transwell) transfected with miR‐371‐373 cluster mimics and inhibitors detected by immunostaining on Day 3 after transfection. RG‐NC: HepaRG cells transfected with NC; RG‐371‐M: HepaRG cells transfected with miR‐371a‐5p mimics; RG‐371‐I: HepaRG cells transfected with miR‐371a‐5p inhibitor; RG‐372‐M: HepaRG cells transfected with miR‐372‐3p mimics; RG‐372‐I: HepaRG cells transfected with miR‐372‐3p inhibitor; RG‐373‐M: HepaRG cells transfected with miR‐373‐3p mimics; RG‐373‐I: HepaRG cells transfected with miR‐373‐3p inhibitor; ALB (red), HNF4A (red), KRT18 (green), and DAPI (blue). Scale bar = 100 µm. The charts show the fluorescence intensity of ALB (upper), HNF4A (middle), and KRT18 (bottom) (n = 3, mean ± SD, One‐way ANOVA). ^*p<0.05. b) The expression of hepatic cell markers in HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐371‐373 cluster inhibitors) was detected by immunostaining on Day 3 after coculture. RG‐NC: HepaRG cells transfected with NC; RG‐Caco‐2‐NC: HepaRG cells cocultured with Caco‐2 cells (transfected with NC); RG‐Caco‐2‐iNC: HepaRG cells cocultured with Caco‐2 cells (transfected with iNC); RG‐Caco‐2‐371‐I: HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐371a‐5p inhibitor); RG‐Caco‐2‐372‐I: HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐372‐3p inhibitor); RG‐Caco‐2‐373‐I: HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐373‐3p inhibitor); ALB (green), HNF4A (red), KRT18 (red), and DAPI (blue). Scale bar = 100 µm. The charts show the fluorescence intensity analysis of ALB (left), HNF4A (middle), and KRT18 (right) (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. c) The expression levels of genes related to hepatic differentiation and stemness in HepaRG cells (cultured in Transwell), transfected with miR‐371‐373 cluster mimics and inhibitors measured by qRT‒PCR. The heatmap shows the log2(fold change) value of the genes (n = 3). d) The expression levels of genes related to hepatic differentiation and stemness in HepaRG cells cocultured with Caco‐2 cells (transfected with the miR‐371‐373 cluster inhibitors) were measured by qRT‒PCR. The heatmap shows the log2(fold change) value of the genes (n = 3). e) The effects of miR‐371‐373 cluster mimics on ALB expression in HepaRG cells on the chip were detected by immunostaining on Day 3 after transfection. ALB (red) and DAPI (blue). Scale bar = 100 µm. f) ALB expression in HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐371‐373 cluster inhibitors) on the chip was detected by immunostaining on Day 3 after coculture. ALB (red) and DAPI (blue). Scale bar = 100 µm. g) The upper and lower panels display the fluorescence intensity analysis of ALB in panel (e) and panel (f), respectively (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with RG‐NC (negative control). h) The secreted ALB levels of HepaRG cells transfected with the mimics or inhibitor of miR‐371a‐5p (upper panel)/372‐3p (middle panel)/373‐3p (lower panel) on the chip were measured by ELISA during 6 days after transfection (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 for RG‐miR‐371 mimics versus RG‐NC, #p < 0.05 for RG‐miR‐371 inhibitor versus RG‐iNC (upper panel); ^* p < 0.05 for RG‐miR‐372 mimics versus RG‐NC, #p < 0.05 for RG‐miR‐372 inhibitor versus RG‐iNC (middle panel); ^* p < 0.05 for RG‐miR‐373 mimics versus RG‐NC, #p < 0.05 for RG‐miR‐373 inhibitor versus RG‐iNC (lower panel). i) Secreted ALB levels in HepaRG cells cocultured with Caco‐2 cells (transfected with miR‐371a‐5p (left panel)/372‐3p (middle panel)/373‐3p (right panel) inhibitor) on the chip measured by ELISA within 6 days after transfection (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 for RG‐Caco‐2‐miR‐371 inhibitor versus RG‐Caco‐2‐iNC (left panel); ^*p<0.05 for RG‐Caco‐2‐miR‐372 inhibitor versus RG‐Caco‐2‐iNC (middle panel); ^* p < 0.05 for RG‐Caco‐2‐miR‐373 inhibitor versus RG‐Caco‐2‐iNC (right panel). j) Secreted ALB levels in HepaRG cells treated with EXOs from Caco‐2 cells (transfected with miR‐371a‐5p (left panel)/372‐3p (middle panel)/373‐3p (right panel) inhibitor) on the chip measured by ELISA during 6 days after transfection (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05 for RG‐EXO‐miR‐371 inhibitor versus RG‐EXO‐iNC; ^*p<0.05 for RG‐EXO‐miR‐372 inhibitor versus RG‐EXO‐iNC; ^*p<0.05 for RG‐EXO‐miR‐373 inhibitor versus RG‐EXO‐iNC.

Figure 8

Validation of the miR‐371‐373 cluster target gene RPS6KA2 a) Dual‐luciferase reporter assays were performed to test putative binding sites between RPS6KA2 3′ UTR and miR‐371a‐5p/372‐3p/373‐3p. The wide type (WT) sequence and mutant (MUT) sequence containing the binding site were inserted into the luciferase reporter vector. Luciferase activity indicated the molecular interactions between miR‐371a‐5p/372‐3p/373‐3p mimics and RPS6KA2 mRNA, as shown in the chart on the right (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with NC. b) The expression of RPS6KA2 (red) and the downstream protein p‐ATF4 (red) and DAPI (blue) in HepaRG cells with and without EXO treatment (on the chip), visualized by immunostaining on Day 5. Scale bar = 100 µm. The charts display the fluorescence intensity of RPS6KA2 and p‐ATF4 (n = 3, mean ± SEM, Student's t‐tests). ^* p < 0.05. c) Immunofluorescence staining visualizing the expression of functional proteins (ALB, AAT (green)), the target protein RPS6KA2 (red), the downstream protein p‐ATF4 (red), and DAPI (blue) in both monocultured and cocultured HepaRG cells on the chip on Day 5. Scale bar = 100 µm. The charts show fluorescence intensity of the detected proteins (n = 3, mean ± SD, Student's t‐tests). ^*p<0.05. d) The expression of RPS6KA2 (red) in HepaRG cells transfected with miR‐371‐373 cluster mimics was visualized by immunostaining on the chip on Day 3 after transfection. Scale bar = 100 µm. e) The expression of RPS6KA2 (red) in HepaRG cells cocultured with Caco‐2 cells (transfected with the miR‐371‐373 cluster inhibitors) was detected by immunostaining on the chip on Day 3 after coculture. Scale bar = 100 µm. f) The charts show fluorescence intensity of RPS6KA2 in Figure 8d (left panel) and e (right panel) (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with RG‐NC. g) Comparison of RPS6KA2 expression levels in HepaRG cells transfected with miR‐371‐373 cluster mimics or inhibitors (on chips) measured by qRT‒PCR (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. h) Gene expression levels of RPS6KA2 in HepaRG cells cocultured with Caco‐2 cells (transfected with the miR‐371‐373 cluster inhibitors) on chips measured by qRT‒PCR (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. i) Monocultured, cocultured, and EXO‐treated Hep‐Orgs were extracted from the chip, and immunostaining images of RPS6KA2 (in red) in the paraffin sections of the Hep‐Orgs were captured using a confocal microscope on Day 5. E‐CAD was labelled in green and DAPI in blue. Scale bar = 100 µm. The chart shows the fluorescence intensity analysis of RPS6KA2 (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with Hep‐Orgs‐M. j) Gene expression levels of RPS6KA2 in monocultured, cocultured, and EXO‐treated Hep‐Orgs (on chips) measured by qRT‒PCR (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with Hep‐Orgs‐M.

Figure 9

Verification of the function of RPS6KA2 on hepatic differentiation. a) The knockdown efficiency of RPS6KA2 siRNAs in HepaRG cells was detected by qRT‐PCR (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with NC. b) Expression of genes related to hepatic function and stemness in HepaRG cells (transfected with NC, RPS6KA2 siRNA‐1, RPS6KA2 sgRNA‐1, and RPS6KA2 siRNA‐1 + sgRNA‐1) detected by qRT‐PCR at day 3 after transfection (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with NC. c) Expression of ALB, KRT18 (green), HNF4A (yellow), and RPS6KA2 (red) in HepaRG cells (transfected with NC and RPS6KA2 siRNA‐1) detected by immunostaining at day 3 after transfection. DAPI (blue) shows the nuclei. Scale bar = 100 µm. The charts display the fluorescence intensity of proteins (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05. d) Flow chart of transcription activation screening using sgRNA‐dCas9 SAM (synergistic activation mediator system). Blast, blasticidin; Hygro, hygromycin; Puro, puromycin. e) Schematic illustration of the three‐component SAM system, including RPS6KA2‐sgRNA, dCas9‐VP64, and MS2‐P65‐HSF1. f) The overexpression efficiency of RPS6KA2 sgRNAs in HepaRG cells was detected by qRT‐PCR (n = 3, mean ± SD, One‐way ANOVA). ^* p < 0.05, compared with NC. g) The level of RPS6KA2 expression in HepaRG cells was evaluated using qRT‐PCR following co‐transfection of siRNA‐1 and sgRNA‐1 or following transfection of sgRNA‐1(n = 3, mean ± SD, One‐way ANOVA). ^* p <0.05, compared with NC.