Supplemental Information
Supplemental Material and Methods
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from human ESCs, iPSCs, their derivatives, HepG2 cells, and
primary hepatocytes (CellzDirect, Austin, TX) using an RNeasy Plus Mini kit (Qiagen,
Hilden, Germany).
Human fetal (22–40 weeks old) and adult (51-years-old) liver total
RNA were purchased from Clontech Laboratories, Inc. (Mountain View, CA).
cDNA
was synthesized using 500 ng of total RNA with a Superscript VILO cDNA synthesis kit
(Invitrogen).
Semi-quantitative RT-PCR was performed with the use of TaKaRa Ex Taq
Hot Start Version (TaKaRa, Shiga, Japan).
Real-time PCR was performed with Taqman
gene expression assays (Applied Biosystems, Foster City, CA) using an ABI PRISM 7700
Sequence Detector (Applied Biosystems).
Relative quantification was performed against
a standard curve and the values were normalized against the input determined for the
housekeeping gene, GAPDH.
The primer sequences used in this study are described in
Table S1.
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Immunostaining
The cells were fixed under the conditions shown in Table S2 (ref. 8 in manuscript).
After
blocking with PBS containing 1% bovine serum albumin (BSA), the cells were incubated
with primary antibody at 4°C overnight, followed by incubation with a secondary antibody
that was labeled with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1 h at room
temperature.
All the antibodies and conditions are listed in Table S2.
experiments were performed without primary antibody.
The control
The immunopositive cells were
counted in at least 8 random chosen fields.
Flow cytometry
Monolayer cultures were dissociated and single-cell suspensions were fixed (0.4% PFA) for
30 minutes at 4 ℃ .
For CXCR4 straining, cells were stained with biotinylated
anti-CXCR4 (R&D) antibody in PBS containing 1% FCS, followed by incubation with
streptavidin APC (Molecular Probes).
FACS analysis was performed using a FACS
LSRFortessa (Becton, Dickinson and Company, Franklin Lakes, NJ).
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LacZ Assay
The human ESC- and iPSC-derived definitive endoderms were transduced with Ad-LacZ at
3,000 VP/cell for 1.5 h.
At 24 h after incubation, X-galactosidase (Gal) staining was
performed as described previously (ref. 26 in manuscript).
Assay for cytochrome P450 3A4 activity
To measure cytochrome P450 3A4 activity, we performed Lytic assays by using a
P450-GloTM CYP3A4 Assay Kit (Promega, Madison, WI).
For the cytochrome P450
3A4 activity assay, Ad-HEX-transduced cells and non-transduced cells as well as HepG2
cells were treated with rifampicin, which is the substrate for CYP3A4, at a final
concentration of 25 μM or DMSO for 72 h.
We measured the fluorescence with a
luminometer (Lumat LB 9507; Berthold, Tokyo, Japan) according to the manufacturer’s
instructions.
HepG2 cells were cultured as per the instructions.
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