Supplementary Fig. 1. Isolation and in vitro expansion of EpCAM + cholangiocytes. For collagenase perfusion, enzyme solution was injected from the

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1 Supplementary Fig. 1. Isolation and in vitro expansion of EpCAM + cholangiocytes. For collagenase perfusion, enzyme solution was injected from the portal vein for digesting adult livers, whereas it was from the right ventricle for neonatal livers. Cholangiocytes were isolated from undigested tissues after collagenase perfusion. They were further purified by MACS based on the expression of EpCAM. After expanding cells on type I collagen gel, they were used for immunostaining, FACS analysis, and culture.

2 EpCAM+ (%) Number of Cyst/5000 cells st 2nd 3rd 4tn Passage st 2nd 3rd 4th Passage MG coat MG gel Collagen coat Collagen gel Supplementary Fig. 2. Adult cholangiocytes keep expression of EpCAM and the ability to form cysts during the serial culture on type I collagen gel. EpCAM + cells isolated from 6W mouse livers were cultured on dishes coated with type I collagen, type I collagen gel, a thin layer of Matrigel, and Matrigel. Cells were replated every 7 days. Cholangiocytic characteristics were examined by analyzing expression of EpCAM by FACS and the ability to form cysts in three dimensional culture.

3 CK19 Albumin Merge 50mm EpCAM HNF1 Merge EpCAM Supplemental Fig. 3. Human cholangiocytes proliferate on type I collagen gel. Human cholangiocytes were purified by MACS based on the expression of EpCAM. Cholangiocyte markers including CK19, EpCAM, and HNF1 are expressed in human cholangiocytes after 7 days of culture. EpCAM expression is also shown by FACS analysis.

4 Supplementary Fig. 4. Neonatal cholangiocytes express cholangiocyte markers during culture on collagen gel. EpCAM + neonatal cholangiocytes were isolated from 1W mouse livers and cultured on Col-I gel for a week. Cells were replated onto Col-I gel and incubated for 2 days before fixation. Neonatal cholangiocytes kept cholagiocyte markers, whereas they did not express a hepatocyte marker, HNF4a during culture on Col-I gel.

5 Confluent OSM + MG Confluent OSM + MG Confluent OSM + MG Confluent OSM + MG CK7/GAPDH EpCAM/GAPDH 10 P= P= Neonate Adult Neonate Adult Supplementary Fig.5. Downregulation of CK7 and EpCAM in neonatal cholangocytes during hepatocytic differentiation. Epression of CK7 and EpCAM were downregulated in neonatal but not in adult cholangiocytes during hepatocytic differentiation. Two sided student s test was performed. Bars represent SEM.

6 Supplementary Fig. 6. A g-secretase inhibitor increases albumin expression in adult cholangiocytes. A. Effect of DAPT on Hes1 expression. DAPT, a g-secretase inhibitor and a potent inhibitor for the Notch signaling pathway, slightly downregulated Hes1, a major target of the pathway. B. Effect of DAPT on expression of hepatocyte markers. DAPT increased expression of albumin in adult cholangiocytes.

7 Supplementary Fig. 7. Expression of Grhl2 protein in adult and neonatal cholangiocytes. Expression of Grhl2 was examined before and after inducing hepatocytic differentiation. Grhl2 was expressed in adult cholangiocytes even after the treatment of OSM and MG, whereas it was remarkably downregulated in neonatal cells.

8 Supplementary Fig. 8. Downregulation of Grhl2 slightly increases expression of hepatocyte markers in adult cholangiocytes. A. Design of three sirna on Grhl2 gene. Three stealth RNAi against Grhl2 were purchased from Life Technologies Inc. B and C. Downregulation of Grhl2 by sirna. HPPL expressing Grhl2 were introduced with negative control or Grhl2-targeted stealth RNAi. Sequence 3 most efficiently downregulated Grhl2. D and E. Downregulation of Grhl2 in adult cholangiocytes. F and G. Experssion of hepatocyte markers. Downregulation of Grhl2 slightly increased albumin mrna detected by PCR. Expression of CPSI and TAT were also slightly upregulated by downregulation of Grhl2.

9 TER (Ω cm 2 ) 300 Neonate Adult Incubation time (days) Supplementary Fig. 9. Change of TER during culture of neonatal and adult cholangiocytes. Neonatal and adult cholangiocytes expanded on Col-I gel were replated onto culture inserts coated with Col-I gel. Average TER values of 3 wells are shown in the graph. Bars represent SD.

10 Neonate Adult Supplementary Fig. 10. Appearance of neonatal gallbladder. Neonatal gallbladder (GB) (arrow in the left panel) is small, colorless, and transparent as compared with adult one (arrow in the right panel). Neonatal GB appears to only contain a small amount of bile.

11 Supplemental Fig. 11. Methylation status of CpG islands in the proximal promoters of C/EBPa and HNF4a. Cytosines in both promoters are more methylated in cholangiocytes than hepatocytes. However, there are no clear difference between 1W and 6W cholangiocytes. Genomic DNAs isolated from EpCAM + cholangiocytes and ICAM + hepatocytes were bisulfonated by using a kit. The regions containing CpG islands were amplified by PCR and PCR products were inserted into a puc119 cloning vector. DNA sequences were determined to know which CpGs are methylated.

12 Conf OSM+MG Conf OSM+MG Conf OSM+MG Conf OSM+MG HNF4/GAPDH C/EBPa/GAPDH Control Grhl2 Control Grhl2 Supplementary Fig. 12. Grhl2 does not inhibit induction of C/EBPa mrna in neonatal cholangiocytes. Expressions of HNF4a and C/EBPa were upregulated during hepatocytic differentiation of neonatal cholangiocytes. Grhl2 inhibited induction of HNF4a mrna but not C/EBPa.

13 Supplementary Fig. 13. Induction of hepatocytic differentiation by overexpression of C/EBPa and downregulation of Grhl2. A. Effect of insulin concentration on hepatocytic differentiation. As it was reported that C/EBPa was more active in the presence of a low concentration of insulin (Sekine et al. EMBO J. 2007, 26, 3067), we found that 0.1 insulin/transferrin/selenium (ITS) was better for induction of CPSI than 1xITS. B and C. Effect of C/EBPa and Grhl2 on hepatocytic differentiation. In the presence of sirna against Grhl2 and DAPT, C/EBPa further promotes expression of hepatocytic markers. Some cells expressed albumin and CPSI proteins.

14 1 Table S1. Primers used for PCR Gene name Sequence Albumin Sense 5 -GAA AGC CCA CTG TCT TAG TG-3 Antisense 5 -GGG TGT AGC GAA CTA GAA TG-3 CK19 Sense 5 -GTC CTA CAG ATT GAC AAT GC-3 Antisense 5 -CAC GCT CTG GAT CTG TGA CAG-3 C/EBPα Sense 5 -AAA CAA CGC AAC GTG GAG A-3 Antisense 5 -GCG GTC ATT GTC ACT GGT C-3 CPSI Sense 5 -ACT GAG AGA TGC TGA CCC TA-3 Antisense 5 -CCT GGA AAT TGG TGA GGA GA-3 Cyp1a2 Sense 5 -CCCTGCCCTTCAGTGGTACA-3 Antisense 5 -AAGCTGTAGAGGTCTGGTCG-3 Cyp2b10 Sense 5 -GTTGAGCCAACCTTCAAGGAA-3 Antisense 5 -AAGAGCTCAAACATCTGGCTG-3 Cyp2d10 Sense 5 -GATCCCAAGGTGTGGTCCTT-3 Antisense 5 -GCAGGAGTATGGGGAACATA-3 FoxA1 Sense 5 -GAACAGCTACTACGCGGACA-3 Antisense 5 -CGG AGT TCA TGT TGC TGA CA-3 GAPDH Sense 5 -ACC ACA GTC CAT GCC ATC AC-3 Antisense 5 -TCC ACC ACC CTG TTG CTG TA-3 HNF4α Sense 5 -CAG CAA TGG ACA GAT GTG TGA-3 Antisense 5 -TGG TGA TGG CTG TGG AGT C-3 PEPCK Sense 5 -TTGATGCCCAAGGCAACTTA-3 Antisense 5 -ACGGCCACCAAAGATGATAC-3 TAT Sense 5 -GAG GAG TGT GAC AAA TAG GC-3 Antisense 5 -AGA GGA CAC TCC TGT GTC AG-3 Tdo2 Sense 5 -TGAGTAAAGGTGAACGACGAC-3 Antisense 5 -AGCCGACTGAGAATCCTGTA-3.

15 2 Table S2. Primary antibodies Antigen Company Host animal Dilution Albumin Bethyl laboratory goat 1:1000 C/EBPα SantaCruz Biotechnology Inc. rabbit 1:200 CPSI Santa Cruz Biotechnology Inc. goat 1:200 Cytokeratin 19 Tanimizu et al rabbit 1:2000 EpCAM BD Pharmingen rabbit 1:500 Grhl2 Sigma-Ardrich rabbit 1:1000 HNF1β Santa Cruz rabbit 1:200 HNF4α SantaCruz Biotechnology Inc. rabbit 1:200 Osteopontin R&D goat 1:400 Sox9 Millipore rabbit 1:500