Lentiviral Vector

RU5

SIN

PBSdGag

RRE

PPTSFFV PRE*

IRES

puro, EGFP, dTomato

RSV

Foxa2, Hnf4a, Cebpa

Lentiviral Vector used:

SIN: Self-Inactivating 3' Long Terminal Repeat (LTR); PRE: post-transcriptional regulatory elementIRES: EMCV internal ribosome entry siteSFFV: strong internal promoter of SFFV (Spleeen focus forming virus)PPT: central polypurine tractRRE: Rev responsive elementdGag: portion of the HIV-1 gag gene with a closed reading framePBS: primer binding siteRU5: 5'-HIV-1 repeat and U5-region of the LTRRSV: rous sarcoma virus promoter

Constructs in Iacob et al.:

Foxa2 IRES puro, plasmid/vector pL-S-Foxa2-I-puro Hnf4α IRES EGFP, plasmid/vector pL-S-Hnf4a-I-egfpC/ebpα IRES dTomato, plasmid/vector pL-S-Cebpa-I-dTomcontrols:IRES puro, plasmid/vector p:-S-I-puroIRES EGFP, plasmid/vector pL-S-I-egfpIRES dTomato, plasmid/vector pL-S-I-dTom

Suppl. Figure 1

Suppl. Table 1

PCR Primer used:

Gene Sequence (5'-3' direction) Acc. No.

Gapdh CCTGCACCACCAACTGCTTA NM_008084.2TCAATGAGCCCCTTCCACAATG

Foxa2 GTCCATTTTGTGGGGCTGAT NM_010446.1CACCATTACGCCTTCAACCA

Hnf4a TGCCAACCTCAATTCATCCA NM_008261.2GCTCGAGGCTCCGTAGTGTT

Cebpa AAGAAGTCGGTGGACAAGAACAG NM_007678.3GTTGCGTTGTTTGGCTTTATCTC

Alb CTCAGGTGTCAACCCCAA NM_009654.2TCCACACAAGGCAGTCTC

Aat AGGTTAGCCCAGATCCACT NM_009243.3ATTGTTGAAGATTCGGGTGA

Tat ATGGTGGGAATTGAGATGGA NM_146214.2TGCCTTCAGCACAGTGGTAG

Tdo2 AGGGAACAAAATCCATGACG NM_019911.2TTCCAGAACCGAGAACTGCT

G6pc TCTGTCCCGGATCTACCTTG NM_008061.3GTAGAATCCAAGCGCGAAAC

Cldn1 CGTGGTGTTGGGTAAGAGGT NM_016674.3AGGTCTGGCGACATTAGTGG

Cps1 CCATCCCATAATTCCTGCTG NM_001080809.1TCTCCAGTGGAAGCCATCTC

Gys2 TCCACCAGTCCCTTACTACCC NM_145572.2ACTGGGGATACCCATCACAG

Fah CGGGCCGGAGCCAGAAAAC NM_010176.4ACCATTCCCCAGGTCTATG

Cyp2a5 GACCGAATGAAGATGCCCTA NM_007812.4CGAAACTTGGTGTCCTTGGT

Cyp3a11 CAGCTTGGTGCTCCTCTACC NM_007818.3TCAAACAACCCCCATGTTTT

Nr1i2 (Pxr) GACCTGCCTATTGAGGACCA NM_010936.2TTCTGGAAGCCACCATTAGG

Apoa1 GTGGCTCTGGTCTTCCTGAC NM_009692.2ACGGTTGAACCCAGAGTGTC

Apoa4 GCATCTAGCCCAGGAAACTG NM_007468.2ATGTATGGGGTCAGCTGGAG

Apoc3 ACATGGAACAAGCCTCCAAG NM_023114.3TGGTTGGTCCTCAGGGTTAG

Krt18 CGAGGCACTCAAGGAAGAAC NM_010664.1CTTGGTGGTGACAACTGTGG

Krt19 CTCGGATTGAGGAGCTGAAC NM_008471.2TCACGCTCTGGATCTGTGAC

Primer for cloning (restriction sites underlined)

Foxa2_for GCGGATCCAATGCTGGGAGCCGTGAAGATFoxa2_rev GCGGATCCTTAGGATGAGTTCATAATAGHnf4a_for GCGGATCCAGAATGCGACTCTCTAAAACHnf4a_rev GCAGATCTCTAGATGGCTTCTTGCTTGGCebpa_for GCGGATCCAATGGAGTCGGCCGACTTCTACebpa_rev GCGGATCCTCACGCGCAGTTGCCCATG

Suppl. Figure 2

A

B

C

100x, 18 days100x, 6 days 100x, 30 days

100x, 4 days400x, 24h 400x, 48hK

rt18

/ G

APD

H

0

2

4

6

8

10

12

14

16

Krt

19 /

GA

PDH

0,0

0,5

1,0

1,5

2,0

2,5

pHc day

1

pHc day

0

ALDPC-MM

ALDPC

pHc day

1

pHc day

0

ALDPC-MM

ALDPC

100x, Collagen I 100x, Matrigel

Suppl. Figure 2: Further Characterisation of murine ALDPC.A) Emergence of ALDPC in long term tissue culture an collagen I-coated plates. B) Bipotentiality of ALDPC: The cholangiocyte differentiation potential visualizes with the formation of bile duct-like structures after 6 days of culture on Matrigel. C) ALDPC express both Keratins characteristic for hepatocytes (Krt18) and cholangiocytes (Krt19). mRNA-expression levels normalized for GAPDH, pHc: freshly isolated primary hepatocytes, ALDPC-MM: ALDPC in Maintenance medium, ALDPC: ALDPC in liver differentiation medium (LD). Mean values (N=3) and standard deviation are shown.

Suppl. Figure 3. Transcription factor transduced murine embryonic fibroblasts NIH3T3.Using the same protocol as described for the generation of ALDPC-F-H-C (sections 4.5. and 2.3), triple transduced NIH3T3 cells were established to preliminarily investigate the potential of the three transcription factors Foxa2, Hnf4α and C/ebpα to transdifferentiate non-hepatic cell sources towards a hepatocyte-like phenotype.Shown are non-transduced or triple transduced (FoxA2 + Hnf4α + C/ebpα) NIH3T3 cells in liver differentiation medium (LD) at different time-points: A) Non transduced at day 7; B) Triple transduced, day 2; C) Triple transduced, day 5 and D) Triple transduced at day 7. B-D: right panel: green and orange cells express eGFP, thus Hnf4α; middle panel: red cells express dTomato, thus C/ebpα.PAS staining (glycogen synthesis, to compare with Figure 7):E) PAS staining of non transduced NIH3T3 fibroblasts after 7 days of culture using LD medium. F) PASstaining of triple transduced NIH3T3 fibroblasts at day 7: PAS positive, binucleated epitheliod-shaped cells are visible.

Suppl. Figure 3

Suppl. Figure 4. RT-qPCR analysis for mRNA expression levels of liver specific genes in iPS-derived hepatocytes (iPS-hep) and triple transcription factor transduced NIH3T3 murine embryonic fibroblasts (NIH3T3-FHC).Values of ALDPC (ALDPC, 7 days in LD Medium) and ALDPC-FHC (triple transduced ALDPC) are copied from Figure 3 for comparison.ALDPC, ALDPC in liver differentiation medium (LD); iPS-hep – murine induced pluripotent stem cellderived hepatocyte-like cells obtained according to protocol described in detail in reference [3]; ALDPC-FHC, ALDPC transduced with FoxA2, Hnf4α and C/ebpα in LD at day7; NIH3T3-FHC,NIH3T3 fibroblasts transduced with FoxA2, Hnf4α and C/ebpα, according to the same protocol used for ALDPCs transduction in LD at day7. The data show mRNA levels normalized to Gapdh as the internal standard and presented as mean of three independent experiments and standard deviation (SD). n.d.: not detected.Methods: iPS-heps were obtained according to a recently published protocol [3]. Briefly, the hepatic differentiation protocol took 28 days and the following cytokines (R&D Systems) were added sequentially: Activin-A (100 ng/ml) and Wnt3a (50 ng/ml) from day 0 to day 6, followed by BMP4 (bone morphogenetic protein 4, 50 ng/ml) and FGF2 (fibroblast growth factor 2, 10 ng/ml) (day 7 - 10), then acidic FGF (50 ng/ml), FGF4 (10 ng/ml) and FGF8b (25 ng/ml) from day 11 to 14. Finally, from day 15 to 28 HGF (hepatocyte growth factor, 20 ng/ml) and follistatin (100 ng/ml) were supplemented. NIH-3T3 fibroblasts transduced with the three transcription factors were generated using the same protocol as described for transduction of ALDPC in section 4.5. RNA extractions and RT-qPCR were performed as described in section 4.6. Suppl. Figure 4

Alb

ALDPC

Alb/

Gap

dh

0,1

1

10

100

iPS-hep

ALDPC-FHC

NIH3T3-FHC

0,0026 0,1

1

10

100

1000

Aat/G

apdh

Aat

ALDPC iPS-hep

ALDPC-FHC

NIH3T3-FHC

0,08

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

Tdo2

/Gap

dh

ALDPC iPS-hep

ALDPC-FHC

NIH3T3-FHC

Tdo2

0,00

0,01

0,02

0,03

0,04

0,05

0,06

0,07

G6p

/Gap

dh

G6p

ALDPC iPS-hep

ALDPC-FHC

NIH3T3-FHC

0,00

0,02

0,04

0,06

0,08

Tat/G

apdh

ALDPC iPS-hep

ALDPC-FHC

NIH3T3-FHC

n.d. n.d.

n.d. n.d.

Tat

0,00

0,05

0,10

0,15

0,20

0,25

Cld

n1/G

apdh

Cldn1

n.d.0,0002ALDPC iPS

-hepALDPC-FHC

NIH3T3-FHC

0,04