Supplementary Material and Methods
The isolation of liver oval cells , liver small cells ,liver stellate cells
Human liver
oval cells were isolated through Thy-1/EpCAM/CD29/CD73/CD44/CD90 MicroBead
(Miltenyi technic,USA) from human liver cancer tissues and its adjacent noncancerous
liver tissues; Hepatic stellate cells were sorted by Fluorescence Activated Cell Sorting
(Beckman) using anti-CD133,anti-Oct4 from human liver cancer tissues and its
adjacent noncancerous liver tissues; Small hepatocyte like progenitor cells (liver small
cells) (Alb+、CK8+、CK18+ 、transferring+) were isolated
from human liver cancer
tissues and its adjacent noncancerous liver tissues by laser capture microdissection
(LCM) (Applied Biosystems® ArcturusXT™ LCM, Life Technologies).
Cell lines
Human liver cell line HELE, Fetal liver cell line L02 Human hepatoma
cell lines ,Huh7,Hep3B,HepG2,SMMC7721,QGY7701 were obtained from the Cell
Bank of Chinese Academy of Sciences (Shanghai, China).These cell lines were
maintained in Dulbecco’s modified Eagle medium (Gibco BRL Life Technologies)
supplemented with 10% heat-inactivated (56ºC, 30 minutes) fetal bovine serum
(sigma) in a humidified atmosphere of 5% CO2 incubator at 37ºC.
Embryonic stem (ES) cells differentiate into hepatocyte-like cell in vitro
Human
ES cell line MEL-2 could efficiently generate definitive endoderm (DE) tissue by
treating the the modified cultures with high concentrations of the TGFβ family ligand
activin A(100 ng/ml, R&D, Minneapolis) for 5 days. A number of groups have
generated hepatoblosts using this DE tissue as a starting material, plating the DE on
matrix (e.g. collagen) to mimic the hepatic ECM and then added FGF4(100
ng/ml,R&D) and BMP(100 ng/ml ,R&D, Minneapolis) to mimic hepatic induction for
5 days (induced hepatoblasts). This is followed by some combination of insulin,
transferrin, selenite (ITS,5μg/ml, R&D, Minneapolis), HGF(20ng/ml, R&D,
Minneapolis),OSM(10ng/ml, R&D, Minneapolis),aFGF(50ng/ml, R&D, Minneapolis)
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and Dexamethasone(10-7M, R&D, Minneapolis) to expand the hepatoblast population
and to promote hepatic maturation for 10 days( induced hepatocyte-like cells)(S1).
Hepatoblasts isolation Liver cells were prepared from mouse E14.5 liver. The cells
resuspended in Dulbecco's modified Eagle's medium (DMEM) were cultured in the
same conditions used for the primary culture of fetal hepatic cells, in which hepatic
differentiation is induced by oncostatin M (OSM). Alternatively, after 5 days of
incubation, cells were overlaid with Matrigel (Becton Dickinson) and incubated for an
additional 5 days).
Pancreas stem cells differentiate into hepatocyte-like cell in vitro Cells were
isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were
characterized for the absence and presence of pancreatic and hepatic markers. In vitro
expanded HFP were treated with fibroblast growth factor 2 (FGF2) and
dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in
various passages were further studied for their capacity to be functional in hepatic
parenchyma . Amylase- and EPCAM-positive-enriched cells isolated from HFP and
treated with FGF2 and DX lost expression of pancreatic markers and gained a liver
phenotype.
Blood stem cells differentiate into hepatocyte-like cell in vitro Mesenchymal stem
cells (MSCs) were isolated from human umbilical cord blood (UCB) ,human bone
marrow (BM) and human menstrual blood (MenSC) were cultured under the
pro-hepatogenic condition. After three weeks of incubation in hepatogenic
differentiation medium containing hepatocyte growth factor (HGF), fibroblast growth
factor-4 (FGF-4), and oncostain M (OSM), these derived MSCs are able to
differentiate into hepatocyte-like cells(HLC)in which expression of a variety of
hepatic lineage specific markers [Thy-1, c-Kit, Flt-3, albumin (ALB), α-fetoprotein
(AFP),
cytokeratin18/19,cytochrome
P450
2
1A1/3A4
(CYP1A1/3A4).albumin,
alpha-fetoprotein, and cytokeratin-18 and 19 etc) was analyzed by flow
cytometry(Beckman), RT-PCR, Western blot, and immunofluorescence. Differentiated
cuboidal HLCs were observed and the functionality of mature hepatocyte cells were
further assessed in vitro , such as urea synthesis, glycogen storage, and indocyanine
green (ICG) uptake, the ability to incorporate DiI-acetylated low-density lipoprotein
(DiI-Ac-LDL)
(S2).
Blood and fetal tissue collection was approved by the Research
Ethics Committee in compliance with national guidelines regarding the use of fetal
tissue for research purposes. All patienfs gave written informed consent for collection
and use of human tissues.
Adipose-deived stem cells differentiate into hepatocyte-like cell in vitro Human
Adipose-Derived Stem Cells( ADSCs) were isolated from human lipoaspirate tissue
and cryopreserved from primary cultures. ADSCs were passaged
and expanded
once after isolation(Low passage)in MesenPRO RS™ Medium(2%serum) that
reduces ADSC doubling times using STEMPRO® Human Adipose-Derived Stem
Cell (ADSC) Kit(LONZA). After three weeks of incubation in hepatogenic
differentiation medium containing hepatocyte growth factor (HGF), fibroblast growth
factor-4 (FGF-4), and oncostain M (OSM), these derived ADSCs are able to
differentiate into hepatocyte-like cells(HLC).
Western Blotting
The logarithmically growing cells were washed twice with
ice-cold phosphate-buffered saline (PBS, Hyclone Lab. INC) and lysed in a lysis
buffer (50 mmol/L Tris-HCl PH8.0, 150mmol/L NaCl, 1%Nonidet P-40, 5mmol/L
ethylenediaminetetraacetic acid [pH8.0], 1mmol/L phenylmethyl sulfonyl fluoride, 2
µg/mL aprotenine, and 2 µg/mL leupeptine, 2 µg/mL pepstatine and when assay
phosphorylation, add 50mmol/l sodium fluoride ,25 mmol/l glycerophosphate,or
1mmol/L Na3VO4). Cells lysates were centrifuged at 12,000g for 20 minutes at 4°C
after sonication on ice , and supernatants were separated. Protein concentration was
measured using a Bio-Rad protein assay assay kit( Bio-Rad laboratories,Inc). After
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being boiled for 10 minutes in the presence of 2-mercaptoethanol, samples containing
cells or tissue lysate proteins were separated on a 10%
sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a
nitrocellulose membranes (Invitrogen, Carlsbad, CA,USA). To visualize the
transferring efficiency, membranes were stained with Ponceau S (Sigma), destained
with Milli-Q water, and then blocked in 10% dry milk-TBST (20mM Tris-HCl [PH
7.6], 127mM NaCl, 0.1% Tween 20) for 1 h at 37°C. Following three washes in
Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with 0.2 µg/ml of
antibody(appropriate dilution) overnight at 4°C. Following three washes, membranes
were then incubated with secondary antibody for 60 min at 37°C or 4°C overnight in
TBST. Signals were visualized by enhanced chemiluminescence plus kit(GE
Healthcare) or ODYSSEY infrared imaging system(LI-COR). Signals were visualized
by ODYSSEY infrared imaging system(LI-COR, Lincoln, Nebraska USA). Standard
Western blotting procedures were used with the following antibodies: rabbit
polyclonal anti-Foxa2, mouse monoclonal anti-Sox17, mouse monoclonal anti-AFP,
mouse monoclonal anti-Biotin, bvmouse monoclonal anti-Histone, mouse monoclonal
anti-Albumin, mouse monoclonal anti-RNA polII, mouse monoclonal anti-HNF4α,
mouse monoclonal anti-HGF, polyclonal anti-β-catenin, polyclonal anti-CTCF,
mouse monoclonal anti-β-actin were purchased from Santa Cruz, Biotech, and rabbit
polyclonal anti- rabbit polyclonal anti-H3K27me3 were purchased from cell signaling
technology from Abcam. IRDye 680LT /IRDye 800CW secondary antibodies were
purchased from LI-COR scientific company. All other reagents and compounds were
analytical grades (Sigma,Promega,etc).
Reverse-Transcriptase Polymerase Chain Reaction
Total RNA was purified
using Trizol (Invitrogen) according to manufacturer's instructions . cDNA was
prepared by using oligonucleotide (dT)18 and a SuperScript First-Strand Synthesis
System (Invitrogen).The PCR amplification kit (TaKaRa) were adopted according to
the manufacturer's instructions. H19 cDNA was amplified using the upstream primer
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(5’-attgcgcagcaaggaggctg-3’) and the down stream primer(5’-cctccctcctgagagctcat-3’
)(synthesized by Shenggong ,Shanghai,China) under the PCR reaction conditions
performed in 36 cycles with each cycle consisting of a denaturation step (94℃ for 30
seconds, and 3 minutes for the first cycle only), an annealing step (58℃ for 30
seconds) and an elongation step (72℃ for 30 seconds, 10 minutes for the last cycle
only
).
CUDR
cDNA
was
amplified
(CUDR/P1:5’-atgagtcccatcatctctcca-3’)
and
using
the
the
down
upstream
stream
primer
primer(;
CUDR/P2:5’-taatgtaggtggcgatgagtt-3’)(synthesized by Shenggong ,Shanghai,China)
under the PCR reaction conditions performed in 36 cycles with each cycle consisting
of a denaturation step (94℃ for 30 seconds, and 3 minutes for the first cycle only), an
annealing step (55℃ for 30 seconds) and an elongation step (72℃ for 45 seconds, 10
minutes for the last cycle only ).β-actin served as a internal control for the efficiency
of
the
RT-PCR(β-actin
primer:
P1:5’-gggaaatcgtgcgtgacatt-3’;P2:5’-ctcaggaggagcaatgatct-3’).
PCR products were
analyzed by 1.0% agarose gel electrophoresis and visualized by ethidium bromide
staining using Image imaging system (Baygene).
Dual Luciferase Reporter Assay Cells (1x105/well of a six-well plate) were
transiently transfected with 1 µg of luciferase construct
(promega) or indicated plasmids
with the use of
and 0.1 µg of pRL-tk
the LipofectiamineTM 2000
(Invitrogen) . After incubation for 36 h, the cells were harvested with Passive Lysis
Buffer (Promega), and luciferase activities of cell extracts were measured with the use
of the Dual luciferase assay system (Promega) according to manufacturer's
instructions. luciferase activity was measured and normalized for transfection
efficiency with Renilla luciferase activity.Transfection was performed with at least
three different batches of each reporter plasmid.
Nuclear Run on assay Nuclear run-on was performed by supplying biotin-probe to
nuclei, and labeled transcripts were bound to streptavidin-coated streptavidin-agarose
Resin(S3). The cells are chilled, and the membranes are permeabilized or lysed. The
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nuclei are then incubated for a short time at 37 °C in the presence of nucleoside
triphosphates (NTPs) and biotin labeled probe. The number of nascent transcripts on
the gene at the time of chilling is thought to be proportional to the frequency of
transcription initiation. To determine the relative number of nascent transcripts in each
sample, the biotin labeled RNA is purified and hybridized to a membrane containing
immobilized DNA from the gene of interest. The amount of biotin activity that
hybridizes to the membrane is approximately proportional to the number of nascent
transcripts.
DNA methylation analysis The CpG Amplification Kit contains primers that can be
used for analysis of DNA samples by MSP. Prior to performing PCR with the primer
sets provided in CpG Amplification Kits (Millipore), one microgram of purified DNA
must undergo bisulfate modification with the CpGenome™ Fast DNA Modification.
The amount of reagents required in each reaction is:10X Universal PCR buffer 2.5
TaKaRa™
Taq or "hot start" enzyme (5 U/μL) 0.2 μL (1 Unit),dH2O 16.8 μL,Template DNA (50
ng/μL) 2.0 μL,TOTAL VOLUME 25.0 μL.Place tubes in the thermocycler block, and
perform PCR under the following conditions: Denature:for Taq Polymerase 95°C / 5
minutes. Then, perform 35-40 cycles of the following conditions: denature 95°C / 45
seconds,anneal 55°C / 45 seconds,extend 72°C / 60 seconds. After the completion of
PCR, add an appropriate amount of loading dye to the sample and analyze 10 μL of the
reaction on a 2% agarose.
DNA pull down
Cells were lysed by sonication in HKMG buffer (10 mM HEPES,
PH7.9, 100 mM KCl, 5 mM MgCl2, 100% glycerol, 1 mM DTT, and 0.5% NP40)
containing protease inhibitors for the preparation of nuclear exact. Equal amount of
cell nuclear extracts were precleared with Streptavidin-agarose Resin (Thermo) for 1
hours,
and
then
were
double-stranded-oligonucleotides
incubated
(synthesized
6
with
1μg
by
biotinylated
Shenggong
company,Shanghai,China), and together with 10μg poly(dI-dC) at 4°C for 24 hours.
DNA-bound proteins were collected with the incubation with streptavidin-agarose
Resin for 1 hour with gently shaking to prevent precipitation in solution. Following
five times washings of the resin bound complex with 0.5-1.0 ml of binding buffer, the
samples were boiled and subjected to SDS-PAGE and Western blotting analysis.
Chromatin immunoprecipitation
Cells were cross-linked with 1% (v/v)
formaldehyde (Sigma) for 10 min at room temperature and stopped with 125 mM
glycine for 5 min. Crossed-linked cells were washed with phosphate-buffered saline,
resuspended in lysis buffer, and sonicated for 8-10 min in a SONICS to generate DNA
fragments with an average size of 500 bp-1000bp. Chromatin extracts were diluted
5-fold with dilution buffer, pre-cleared with Protein-A/G-Sepharose beads (Sigma),
and immunoprecipitated with specific antibody on Protein-A/G-Sepharose beads.
After washing, elution and de-cross-linking, the ChIP DNA was detected by either
traditional PCR (35 cycles) and PCR products were run on a 1.5% agarose gel.
Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP)
(3C-ChIP)
described
Antibody-specific immunoprecipitated chromatin was obtained as
above
for
ChIP
assays.
Chromatin
still
bound
to
the
antibody-Protein-A/G-Sepharose beads were resuspended in 500 μl of 1.2× restriction
enzyme buffer at 37 °C for 1 h. 7.5 μl of 20% SDS was added, the mixture was
incubated for 1 h, followed by addition of 50 μl of 20% Triton X-100, and then
incubation for an additional 1 h. Samples were then incubated with 400 units of
selected restriction enzyme at 37 °C overnight. After digestion, 40 μl of 20% SDS was
added to the digested Chromatin, and the mixture was incubated at 65 °C for 10 min.
6.125 ml of 1.15× ligation buffer and 375 μl of 20% Triton X-100 was added, the
mixture was incubated at 37 °C for 1 h, and then 2000 units of T4 DNA ligase was
added at 16 °C for a 4-h incubation. Samples were then de-cross-linked at 65 °C
overnight followed by phenol-chloroform extraction and ethanol precipitation. After
7
purification, the ChIP-3C material was detected for long range interaction with
specific primers. PCR products were amplified with AccuPrime Tag High Fidelity
DNA Polymerase (Invitrogen) and PCR products were run on a 1.5% agarose gel.
BrdU staining
Cells were cultured for 24 hour before treatment with 10μl BrdU
(Roche) for 4 hours. Immunofluorescent staining with an anti-BrdU antibody was
performed according to the manufacturer’s instructions (Becton Dickinson). BrdU
positive cells from ten random chosen fields of at least three independent samples
were counted.
Wound healing assay
Cells were cultured to >90% confluence in 10 cm dishes.
The cells were rinsed with PBS and starved in low serum media (1.5 ml; 0.5% - 0.1%
serum in DMEM) overnight. A sterile 200 μl pipette tip was used to scratch wounds
through the cells. The cells were then rinsed gently with PBS. Photographs were
taken using phase contrast at 10X at 0 and 24 hours (the media were changed after
each measurement).
Statistical analysis The significant differences between mean values obtained from at
least three independent experiments. Each value was presented as mean±standard
error of the mean (SEM) unless otherwise noted, with a minimum of three replicates.
The results were evaluated by SPSS20.0 statistical soft (SPSS Inc Chicago, IL) and
Student’s t-test was used for comparisons, with P<0.05 considered significant.
Supplementary Reference
S1.Shiraki
N,
Umeda
K,
Sakashita
N,
Takeya
M,
Kume
K,
Kume
S.(2008)Differentiation of mouse and human embryonic stem cells into hepatic
lineages. Genes Cells 13(7):731-46
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S2.Hong SH, Gang EJ, Jeong JA, Ahn C, Hwang SH, Yang IH, Park HK, Han H, Kim
H.(2005) In vitro differentiation of human umbilical cord blood-derived mesenchymal
stem cells into hepatocyte-like cells. Biochem Biophys Res Commun 330(4):1153-61
S3.Patrone G, Puppo F, Cusano R, Scaranari M, Ceccherini I, Puliti A, Ravazzolo
R.(2002)Nuclear run-on assay using biotin labeling, magnetic bead capture and
analysis by fluorescence-based RT-PCR. Biotechniques 29(5):1012-4, 1016-7
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