SUPPLEMENTAL MATERIALS AND METHODS · value determined by TOFMS. The metabolic pathway map was provided using public-domain software, VANTED: Visualization and Analysis of Networks

SUPPLEMENTAL MATERIALS AND METHODS

Animals

For experiments using amino acid depleted diets, mice were mated and vaginal plugs identified

the following morning. Plugged females were naturally fed BCAAs or valine depleted diets from

gestational day 8 and harvested at day 13 to dissect fetal liver tissue for assessing HPC

frequency.

For preparation of fetal liver cells, the tissues were acquired from fetal mice at embryonic day

(E) 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, 19.5. The livers from E9.5, 10.5, 11.5, 13.5 and 15.5 were

dissociated by treating them with DMEM/F12 (Life technologies, USA) containing 0.2% trypsin

(Life technologies) and 5% fetal bovine serum (FBS: MP Biomedicals, USA) by shaking at 37°C

for 15 minutes. The livers from E17.5 and 19.5 were dissociated by treating with 0.1%

collagenase solution. After pipetting and being washed, cells were triturated and passed through

40 μm nylon meshes to obtain a single-cell suspension.

Cell culture

For mouse cell culture, E11.5, 13.5 and 15.5 mouse fetal liver cells were seeded on laminin-

coated 6-well plates (BD, USA) at a density of 5.0 x 103 cells/cm2. Our standard culture medium

for the primary mouse HPCs was described previously (Suzuki et al., 2000). To optimize HPCs

culture condition, BCAAs, L-isoleucine (Wako, Japan), L-leucine (Wako), and L-valine (Wako)

or commercial non-essential amino acids solution (Life technologies) or essential amino acids

solution (Life technologies) were added at various concentrations, from 0.4 mM to 4.0 mM, into

the standard culture medium and the same amount of PBS was added as control. After the

optimization, we used 4.0 mM concentration of each BCAAs.

Development 144: doi:10.1242/dev.143032: Supplementary information

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Undifferentiated human iPSCs were maintained without feeder cells in mTeSR1 medium

(STEMCELL Technologies). For differentiation, iPSCs were seeded onto a Matrigel (Growth

factor reduced; Corning) coated dish, and the medium was replaced with RPMI1640 medium

(Wako) containing 1% B-27 supplement minus insulin (Gibco) and Human activin A (100

ng/mL; R&D Systems) for 6 days. The cells were further treated with RPMI1640 containing 1%

B27 supplement (Gibco), bFGF (10 ng/mL; R&D systems) and BMP4 (20 ng/mL; R&D

systems) for 3 days. After 9 days of differentiation, generated cells, which were comparable to

HPCs, were passaged to laminin coated 6-well plates (5 x 104 cells per well) and then cultured in

RPMI1640 containing 1% B27 supplement with addition of BCAAs for 6 days.

Human umbilical vein endothelial cells (HUVECs: Lonza) and human mesenchymal stem cells

(MSCs: Lonza) were maintained in endothelial growth medium (EGM: Lonza) or MSC growth

medium (Lonza). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.

Staining and image analysis

For immunostaining, the tissue was cryoprotected with Tissue-Tek (Sakura Finetechnical) and

then frozen with liquid nitrogen. Frozen tissue samples were sliced (5 um) and placed on MAS-

coated slides (Matsunami, Osaka, Japan) for immunostaining.

The cultured cells were washed with PBS. Tissue sections and cultured cells were fixed for 10

minutes at −30°C in acetone/methanol. Next, the sections were incubated for 2 hours in a

blocking solution [10% goat serum (Sigma–Aldrich) in PBS] and were incubated with primary

antibodies overnight at 4°C. The sections were then rinsed 3 times in PBS containing 0.05%

Tween 20 (PBST) and subsequently incubated with an appropriate secondary antibody for 1

hour. After a final rinse with PBST (thrice), the slides were encapsulated with fluorescent


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protective agent containing DAPI (Vecton), cover-slipped, and examined using an LSM510 laser

scanning microscope (Carl Zeiss). The primary antibodies are shown in supplemental table S1.

For flow cytometry analysis, Isolated E13.5 mouse liver single cells were suspended in FACS

buffer (3% FBS in PBS). The samples were then stained with antibodies against Dlk1, CD45 and

TER119 for 30 min at room temperature, followed by staining with Alexa 647-conjugated and

APC-Cy7-conjugated secondary antibodies. The sample were analyzed on a FACSAria II (BD)

to obtain the fluorescence intensity values. The detail of primary antibodies is shown in

supplemental table S1

Transcriptome and metabolome analysis

Isolation of non-hematopoietic liver parenchymal cells was performed as below. Fetal liver cells

were incubated with biotin-conjugated anti-TER119 (BD Biosciences) and biotin-conjugated

anti-CD45 (BD Biosciences) antibodies on ice for 30 min. After wash, cells were reacted with

Streptavidin Particles Plus (BD Biosciences) on ice for 30 min. The reacted sample was added

into the 2mL IMag buffer (PBS containing 0.5% BSA and 2mM EDTA), and TER119+/CD45+

hematopoietic cells were removed by a Cell Separation Magnet (BD Biosciences). Total RNA of

fetal mouse TER119- CD45- liver cells and cultured HPCs was extracted using TRIzol reagent

according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA using

the high capacity cDNA reverse Transcription Kit (Applied Biosystems, CA, USA). Quantitative

RT-PCR was performed with the Roche Universal Probe system and the Eagle Taq Master Mix

(Roche Applied Science, Germany).

The relative quantification of gene expression was carried out according to the delta-delta Ct

(threshold cycle) method. Gapdh and Eukaryotic 18S rRNA was chosen as a reference

endogenous control. Mouse and human-specific primer sequences (forward and reverse) are


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listed in supplemental table S2. The following PCR conditions were used: 50°C for 2 min, 95°C

for 10 min, 95°C for 15 s, and 60°C for 1 min for a total of 45 cycles.

For microarray analysis, total RNA of fetal liver excluded hematopoietic cells was isolated using

TRIzol Reagent (Life Technologies, CA, USA). For quality control of total RNA samples,

Agilent 2100 Bioanalyzer was used. Expression profiling was obtained with Whole Mouse

Genome 4x44K v2 OligoDNA Microarray Kit (Agilent Technologies) according to

manufacturer’s instruction. Hybridization signals were scanned and processed by 75% percentile

shift normalization using GeneSpring GX11.5.1 software.

For Metabolome analysis, collected mouse liver tissues from several time points of

developmental process were analyzed by Human Metabolome Technologies (Japan). Mouse

liver sample and methanol including 50 μM of internal standard material were put into the tube

on ice to fracture by crushing machine. Chloroform and water were added to the sample and

centrifuged to isolate the aqueous layer. After ultrafiltration of isolated layer, pellet was re-

suspended in water. Acquisition of metabolome profiles was performed as described previously

(Takebe et al., 2013). CE-TOFMS was carried out using an Agilent CE Capillary Electrophoresis

System equipped with an Agilent 6210 Time of Flight mass spectrometer, Agilent 1100 isocratic

HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit

(Agilent Technologies, Waldbronn, Germany). The system was controlled by Agilent G2201AA

ChemStation software version B.03.01 for CE (Agilent Technologies, Waldbronn, Germany).

Cationic metabolites were analysed with a fused silica capillary (50 μm i.d. × 80 cm total

length), with Cation Buffer Solution (Human Metabolome Technologies) as the electrolyte. The

sample was injected at a pressure of 50 mbar for 10 sec (approximately 10 nl). The applied


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voltage was set at 27 kV. Electrospray ionisation-mass spectrometry (ESI-MS) was conducted in

the positive ion mode, and the capillary voltage was set at 4,000 V. The spectrometer was

scanned from m/z 50 to 1,000. Other conditions were as in the cation analysis(Soga and Heiger,

2000). Anionic metabolites were analysed with a fused silica capillary (50 μm i.d. × 80 cm total

length), with Anion Buffer Solution (Human Metabolome Technologies) as the electrolyte. The

sample was injected at a pressure of 50 mbar for 25 sec (approximately 25 nl). The applied

voltage was set at 30 kV. ESI-MS was conducted in the negative ion mode, and the capillary

voltage was set at 3,500 V. The spectrometer was scanned from m/z 50 to 1,000. Other

conditions were as in the anion analysis (Soga et al., 2007).

Raw data obtained by CE-TOFMS were processed with the automatic integration software,

MasterHands (Sugimoto et al., 2010). Peak information including m/z, migration time (MT) and

area was obtained. Each peak was aligned according to similar migration time on CE and m/z

value determined by TOFMS. The metabolic pathway map was provided using public-domain

software, VANTED: Visualization and Analysis of Networks containing Experimental Data

(Junker et al., 2006).


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Supplemental Table S1. Antibodies used in this study

Target protein Source Clone Catalog number

Bcat1 Abcam 3F5 Ab195663

Cytokeratin 8/18 Progen GP11

Albumin Genetex GTX19196

Cytokeratin 7 Dako OV-TL 12/30 M7018

Dlk1 R&D 1168B FAB8634G

Cd45 BD Biosciences 30-F11 557659

Ter119 BD Biosciences TER-119 560609

FOXA2 Millipore 07-633

SOX17 Abcam 3B10 Ab84990

HNF4A Santa Cruz C-19 Sc-6556

Supplemental Table S2. The quantitative reverse transcriptase PCR primers used in this

study.

Gene Forward Reverse

mBcat1 CTGGATAAACGAAGACGGAGA CACTCCTGGGAGAATGATGC mBcat2 GTCGGTGACTGCAAGTTGG CTCAGTGAGCTGGTGGTCTG mAlb CTTTGCAATGGATGCTCTCTT TTCTCCTTCACACCATCAAGC mKrt7 GGAGATGGCCAACCACAG GGCCTGGAGTGTCTCAAACTT hBCAT1 GATGTTTGGCTCTGGTACAGC GGACCATTCTCCATAGTTGGAA hALB GTGAGGTTGCTCATCGGTTT GAGCAAAGGCAATCAACACC hRBP4 CCAGAAGCGCAGAAGATTG TTTCTTTCTGATCTGCCATCG hASGR1 GCTGGAGAAACAGCAGAAGG CGCAGGTCAGACACGAACT hNANOG GAGATGCCTCACACGGAGAC AGGGCTGTCCTGAATAAGCA hFOXA2 CGTTCCGGGTCTGAACTG ACCGCTCCCAGCATACTTT hSOX17 ACGCCGAGTTGAGCAAGA TCTGCCTCCTCCACGAAG hAFP TGTACTGCAGAGATAAGTTTAGCTGAC TCCTTGTAAGTGGCTTCTTGAAC

hSOX7 TTCCTCACCAGCCAGGTC ATTTGCGGGAAGTTGCTCTA


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Supplemental Table S3. Metabolic process related gene expressions, which were up-

regulated in early stage of liver development (E9.5-15.5)

slope correlation slope correlation

Bcat1 -1.091427822 -0.927018574 Sms -0.286414887 -0.992034695

Ankrd1 -0.833494415 -0.876968076 Smarcd3 -0.278771529 -0.769421765

Elovl4 -0.833034423 -0.86991936 Pla2g4a -0.268174693 -0.701431955

Dbh -0.665281065 -0.72222807 Prodh -0.259766971 -0.729608008

Pcsk1 -0.622647231 -0.718138862 Tpo -0.244371904 -0.765737863

Pkm2 -0.595858717 -0.982677091 Nfya -0.244295853 -0.927280009

Tph1 -0.592365033 -0.872990804 Far1 -0.236848492 -0.988611136

Pla2g5 -0.570729627 -0.892082398 Dio2 -0.226840919 -0.88789484

Tead2 -0.53954806 -0.908668122 Ggps1 -0.220490958 -0.931414327

Arg2 -0.487726378 -0.735185899 Inppl1 -0.217876486 -0.814929418

Mboat2 -0.483728485 -0.928016441 Pla2g4d -0.215631742 -0.76020232

Trib3 -0.470797005 -0.86163102 Pla2g3 -0.207887267 -0.821641561

Prkd1 -0.459071821 -0.8994456 Lpcat1 -0.205041627 -0.868569513

Pik3r3 -0.454330529 -0.881939649 Agpat4 -0.199931197 -0.927996936

Eno3 -0.439509586 -0.877321159 Acsl6 -0.193361525 -0.990841759

Pik3r5 -0.404633478 -0.868151235 Cds1 -0.189529624 -0.820927752

Galc -0.391202416 -0.915787535 Pla2g1b -0.188348874 -0.802382837

Slc5a5 -0.349110546 -0.823723766 Mtap -0.186201477 -0.884661602

Pomc -0.342575048 -0.930281415 Med31 -0.184697453 -0.922510829

Grhl1 -0.338718654 -0.796224432 Pdpr -0.181372981 -0.911206194

Lpcat2 -0.334245105 -0.859360363 Psmc5 -0.179750201 -0.981813238

Slc44a5 -0.32971173 -0.743798989 Gys1 -0.178707453 -0.861743491

Gpd2 -0.329524922 -0.924589224 Slc6a8 -0.175280142 -0.762118092

Ckb -0.328707765 -0.916806664 Med30 -0.173174541 -0.949020825

Pdk3 -0.31033641 -0.965036038 Tead3 -0.171602613 -0.741704543


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SUPPLEMENTAL FIGURES

Supplemental Figure 1. Bcat1 expression during mouse liver development (A) The

immunofluorescent analysis of Bcat1 in E11.5, 13.5, 15.5 and 8w mouse fetal and adult liver

tissue. Red: Bcat1; Green: Ck8/18; Blue: DAPI

8WE11.5 E13.5 E15.5

Bcat1/Ck8/18/DAPIA


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Supplemental Figure 2. The effect of valine depleted diets in the mouse liver development

(A) The image of E13.5 mouse embryo and liver after feeding valine depleted diet to maternal

mouse from E8.5. (B) whole weight of each embryo, fetal liver weight ant the ratio of liver

weight against body weight. (data represent the mean ± SD, n = 11 for control and valine

depleted diet, Mann-Whitney’s U test: *P<0.05, **P < 0.001)

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Supplemental Figure 3. Comparison of BCAAs concentration for murine hepatic

progenitor cell expansion. (A) The change of ratio for large colony including more than 90 cells

after 6 days culture in medium supplemented various concentration of BCAAs (data represent

the mean ± SD, n = 12, Mann-Whitney’s U test toward control: *P<0.05)


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Supplemental Figure 4. Valine treatment on human iPSC derived HPC. (A) The image

scanned by INCell analyzer 2000 to quantize the size of iPSC derived LB in Figure 4C. The area

surrounded by yellow line was recognized as each LBs and measured. Left: LBs cultured in

Amino acid free medium, Middle: LBs cultured in basal medium as control, Right: LBs cultured

in medium supplemented by 4 mM of l-valine for amino acid free medium. (B) The ratio of cell

number change by BCAAs treatment on HUVECs and hMSCs. No significant change was

observed after BCAA supplemented culture. (C) Stage specific BCAT1 and BCAT2 expression

in various stages of human iPSC differentiation into hepatocyte like cells. Bottom panel show the

diagram of the differentiation methods for hepatocyte like cells. Stage1: at day 6 of induction,

Stage2: at day 9 of induction, Stage3: at day 13 of induction, Stage4: at day 21 of induction.

FLT: human fetal liver tissue (13 week and 40 week of gestation), ALT: human adult liver tissue.

(data represent the mean ± SD, n = 3). (D) Validation of iPSC derived cell at stage 2 by

immunostaining of HPC markers. In bottom left panel; red: HNF4A; blue: DAPI. In bottom right

panel; red: FOXA2; green: SOX17, blue: DAPI (E) Stage specific gene expressions were

confirmed by q-RT-PCR after BCAAs supplemented culture. Re-plated stage2 cells were

cultured in BCAAs supplemented medium for 6 days. Numbers above column indicated the

actual value which could not show in represent Y-axis range. (data represent the mean ± SD, n =

3).


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SUPPLEMENTAL REFERENCES

Soga, T. and Heiger, D. N. (2000). Amino acid analysis by capillary electrophoresis electrospray ionization mass

spectrometry. Anal Chem 72, 1236-1241.

Soga, T., Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu, Y. and Tomita, M. (2007). Analysis of nucleotides by

pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique. J Chromatogr

A 1159, 125-133.

Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T. and Tomita, M. (2010). Capillary electrophoresis mass

spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles.

Metabolomics 6, 78-95.

Suzuki, A., Zheng, Y., Kondo, R., Kusakabe, M., Takada, Y., Fukao, K., Nakauchi, H. and Taniguchi, H.

(2000). Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse

liver. Hepatology 32, 1230-1239.

Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang, R. R., Ueno, Y., Zheng, Y. W.,

Koike, N., et al. (2013). Vascularized and functional human liver from an iPSC-derived organ bud

transplant. Nature 499, 481-484.


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Documents

SUPPLEMENTAL MATERIALS AND METHODS · value determined by TOFMS. The metabolic pathway map was provided using public-domain software, VANTED: Visualization and Analysis of Networks