Volume 136, Issue 3, Pages 1000-1011 (March 2009) Sall4 Regulates Cell Fate Decision in Fetal Hepatic Stem/Progenitor Cells  Tsunekazu Oikawa, Akihide Kamiya, Sei Kakinuma, Mikio Zeniya, Ryuichi Nishinakamura, Hisao Tajiri, Hiromitsu Nakauchi  Gastroenterology  Volume 136, Issue 3, Pages 1000-1011 (March 2009) DOI: 10.1053/j.gastro.2008.11.018 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Expression of Sall4 during liver development. (A) Expression of human SALL4A and SALL4B mRNAs in 3 HCC cell lines (Huh2.2, Huh7, and HepG2). An acute myeloid leukemia cell line, Kasumi-1, was used as a positive control. Samples were normalized by copy numbers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with equal copies applied as templates. (B) Sorting gates for Dlk+CD45−Ter119− hepatoblasts and hematopoietic cells. Cells were dissociated from E14.5 livers and stained with FITC-conjugated anti-Dlk antibody and allophycocyanin-conjugated anti-CD45 and -Ter119 antibodies. (C) Expression of Sall4 splicing variants in hepatoblasts and hematopoietic cells derived from E11.5, 12.5, and 14.5 livers. E12.5 whole embryo (E12.5WE) was used as a positive control. (D) Production of Sall4 protein in Dlk+ hepatoblasts. Cells were sorted by MACS and cultured for 2, 4, and 6 days in hepatocyte culture medium. Western blot analyses revealed that Sall4 protein was increased after 2 to 6 days of culture in the absence of the hepatic maturation factors OSM and DMSO. Black arrow shows the main band of Sall4 protein in ES cells. Gray arrow shows the main band of Sall4 protein in hepatoblasts. (E) In-droplet staining of Sall4 in hepatoblasts. Using FACS, Dlk+CD45−Ter119− cells derived from E11.5 or 13.5 livers were directly sorted into a droplet of PBS on glass slide and incubated with anti-Sall4 antibody. Expression of Sall4 (red) was visualized with Alexa546-conjugated antibody. Nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI) (scale bars, 10 μm). Control staining with isotype-matched IgG as a primary antibody yielded negative results. Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 The effects of Sall4a overexpression on differentiation of hepatoblasts into mature hepatocytes. (A) Protein expression of Sall4 in culture derived from hepatoblasts infected with Sall4a-expressing virus. Cells were sorted using MACS and infected with mock- or Sall4a-expressing virus. After 2, 4, and 6 days of culture without OSM and DMSO, cells were collected, and Sall4 protein was detected using Western blot analyses. Actin was used as an internal control. (B) Morphologic changes in Dlk+ hepatoblasts infected with Sall4a-expressing virus in cultures supplemented with the hepatic maturation factors OSM and Matrigel. Hepatoblasts infected with mock- or Sall4a-expressing viruses were cultured with differentiation into mature hepatocytes induced by OSM and Matrigel. Many mature hepatocytes, with clear round nuclei and highly condensed and granulated cytoplasm, were detected in cultures infected with mock virus but not in those infected with Sall4a-overexpressing virus (scale bar, 25 μm). (C) Expression of hepatocyte marker genes in cultures supplemented with hepatic maturation factors. After 7 days of culture, total RNAs were extracted from hepatoblasts infected with mock- or Sall4a-expressing viruses. Expression of immature hepatocyte (α-fetoprotein [AFP]), hepatocyte (Alb), mature hepatocyte markers (TAT, TO, CPS, and G6Pase) and of a transcription factor regulating hepatic maturation (HNF4α) was evaluated using semiquantitative and quantitative RT-PCR (left and right panels, respectively). (D) Expression of cholangiocyte marker genes in cultures supplemented with hepatic maturation factors. Expression of cholangiocyte markers (CK19, CK7, and integrinβ4), Notch signaling molecules (Notch1, Notch2, and Jagged1), and transcription factors regulating cholangiocytic differentiation (HNF6 and HNF1β) were detected using semiquantitative and quantitative RT-PCR. (C and D) Samples were normalized by copy numbers of GAPDH, with equal copies applied as templates. Data from quantitative RT-PCR are mean ± SD of 3 separate experiments (triplicate samples, *P < .05). Adult whole liver was used as a positive control. Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Overexpression of Sall4a induced differentiation of hepatoblasts into bile duct structures. (A) Representative views of branching structures in collagen gel-embedding cultures derived from hepatoblasts. Hepatoblasts infected with mock- or Sall4a-expressing viruses were cultured in collagen gel in the presence of insulin, epidermal growth factor, HGF, and TNF-α for 7 days. Many large branching structures (consisting of >100 cells) were observed in Sall4a-overexpressing culture (scale bars, 100 μm). (B) Expression of cholangiocyte marker genes in collagen gel-embedding culture. For a normal culture control, cells were infected with mock-expressing viruses and cultured for 4 days. For embedding culture, cells infected with mock- or Sall4a-expressing viruses were cultured for 4 days and subjected to collagen gel-embedding culture for 7 days. Cells were collected, and total RNA was purified. Expression of cholangiocyte markers (CK19 and CK7) and Sall4a was detected using RT-PCR. (C) Expression of CK19 by branching structures in collagen gel-embedding culture. Hepatoblasts were subjected to collagen gel-embedding culture for 7 days and stained with anti-CK19 antibody. Expression of CK19 (red) was detected using Alexa546-conjugated antibody. Nuclei (blue) were stained with DAPI (scale bars, 50 μm). (D) Overexpression of Sall4a increased numbers of branching structures derived from hepatoblasts. Small (S, consisting of 10–49 cells), medium (M, 50–99 cells), and large (L, >100 cells) branching structures were counted after 7 days of embedding culture. Data are expressed as mean ± SD of 3 separate experiments (triplicate samples, *P < .05). Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Signaling pathways regulating bile duct formation induced by Sall4a. (A and B) Hepatoblasts infected with mock- or Sall4a-expressing viruses were subjected to collagen gel-embedding culture in the presence of LY294002 (A, a PI-3K inhibitor) and U0126 (B, a MEK1 inhibitor). Small (S, consisting of 10–49 cells), medium (M, 50–99 cells), and large (L, >100 cells) branching structures were counted after 7 days of embedding culture. Data are expressed as mean ± SD of 3 separate experiments (triplicate samples, *P < .05). (C) Dlk+ hepatoblasts infected with mock- or Sall4a-expressing viruses were cultured for 4 days in normal culture conditions. Expression and phosphorylation of molecules in the PI-3K and MAPK signaling pathways were analyzed using Western blots. Actin was used as an internal control. Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effect of knockdown of Sall4 expression using shRNA on differentiation of hepatoblasts into bile ductal structures. (A) Hepatoblasts transduced with control shRNA or with shRNAs against Sall4 were subjected to collagen gel-embedding culture in the presence of epidermal growth factor, HGF, and TNF-α for 4 days (scale bars, 100 μm). (B) Numbers of branching structures cultured in A. Small (S, consisting of 10–49 cells), medium (M, 50–99 cells), and large (L, >100 cells) branching structures were counted after 7 days of embedding culture. Data are expressed as mean ± SD of 3 separate experiments (triplicate samples, *P < .05). (C) Cell survival of Dlk+ hepatoblasts infected with shRNA-expressing lentiviruses. Hepatoblasts infected with control- and Sall4 shRNA-expressing viruses were cultured for 7 days. Apoptotic cells were detected using TUNEL assays. Cells infected with lentiviruses were detected by expression of EGFP (scale bars, 100 μm). Apoptosis induced by the addition of β-cyclodextrin provided positive control cells. Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Transplantation of mock- or Sall4a-expressing hepatoblasts using a bile duct disruption model. KSN mice were treated with DAPM. Dlk+ hepatoblasts infected with mock- or Sall4a-expressing viruses were cultured for 3 days. Aliquots of 1 × 106 hepatoblasts were transplanted 2 days after administration of DAPM. (A) Representative views of recipient livers. Liver tissues were harvested 4 weeks after transplantation. Transplanted cells derived from mock- or Sall4a-transduced hepatoblasts were detected by EGFP expression. Using serial sections, expression of Alb (red) and CK19 (green) was visualized with Alexa546- and Alexa488-conjugated antibodies. Arrowheads show bile ducts derived from EGFP-expressing donor hepatoblasts. Arrows show bile ducts derived from recipient cells (scale bars, 100 μm). (B) EGFP+ cells derived from transplanted hepatoblats were detected in cryosections of liver tissues of recipients transplanted with mock- or Sall4a-overexpressing hepatoblasts but not in cryosections of DAPM-injured liver tissues without transplantation (No transplantation). Using the same sections, expression of CK19 (red) was visualized with Alexa546-conjugated antibody (scale bars, 25 μm). (C) Percentages of portal triad bile ducts containing EGFP-expressing transplanted cells. Three sections per mouse (recipients of mock- or Sall4a-overexpressing hepatoblasts; n = 3 each) were chosen randomly, and 50 portal triads, again chosen randomly, were assessed per liver. Data are expressed as mean ± SD (*P < .05). Gastroenterology 2009 136, 1000-1011DOI: (10.1053/j.gastro.2008.11.018) Copyright © 2009 AGA Institute Terms and Conditions