A small molecule that directs differentiation of human embryonic stem cells

into the pancreatic lineage Supplementary Material

Shuibing Chen , Malgorzata Borowiak , Julia L. Fox, René Maehr, Kenji Osafune, Lance

Davidow, Kelvin Lam, Lee F. Peng, Stuart L. Schreiber, Lee L. Rubin, Douglas Melton

Supplemental Figure 1. Analysis of the HUES 9-E cells after dissociation. The HUES 9-E cells

(after 9 day treatment with Wnt3a, Activin A, FGF10, CYC and RA) were split and plated into

384-well plate. After overnight incubation, the cells were stained with SOX17 and FOXA2

antibodies.

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplem

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Nature Chemical Biology: doi:10.1038/nchembio.154

b.

Name Number Chemical Structure

Boldine 10

NHO

O

OOH

Cedrelone 11

Dimethisoquin

hydrochloride

12

Nature Chemical Biology: doi:10.1038/nchembio.154

Ethopropazine hydrochloride 13

Harmin hydrochloride 14

Prieurianin 15

O OO

O

O

O

O

OO

OOH

O

O

OH

O

O

Rotenone 16

Strophanthidin 17

Nature Chemical Biology: doi:10.1038/nchembio.154

Terconazole 18

Trimeprazine tartrate 19

Supplemental Figure 3. Data analysis of the primary screen. (a) Data of primary screen. Each dot

represents one compound at one concentration. 5,000 compounds were tested at three

concentrations: 10 μM, 1 μM and 100 nM. The x-axis is the 5,000 compounds with 3

concentrations of each.  The y-axis is the percentage of cells positively stained by the Pdx1

antibody. Primary hits (above the green line) were designated as compounds that induced Pdx1

in more than 7% of all cells, which is 4 times higher than the average. Subsequent tests

confirmed eleven compounds that increase both the number and percentage of Pdx1+ cells. The

eleven compounds were labeled with different colors. The other dots above the green line are the

compounds that only increase the percentage not the number of Pdx1+ cells because of

compound toxicity. (b) Chemical Structure of other hit compounds.

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 4. ILV’s effect on HUES 9-E cells. After 9 days differentiation, the HUES

9-E cells were treated with 1 µM ILV for 4 days and then stained with Pdx1 antibody. The

number of Pdx1+ cells was analyzed with the Opera high content screening system

(PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 5. The proliferation ability of Pdx1+ cells under different treatments. The

HUES 9-E cells were treated with 300 nM ILV or 50 ng/ml FGF10 for 4 days and then stained

with Pdx1 antibody. No treatment was used as a control. In controls, 29.2±4.5% of Pdx1- cells

expressing Ki67, and only 0.8±0.2% Pdx1+ cells express Ki67. In FGF10-treated condition,

8.8±1.4% Pdx1+ cells express Ki67. In ILV-treated cells, 0.1±0.1% of Pdx1+ cells express Ki67.

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 6. ILV’s effect on HUES 8-E cells. After 9 days differentiation, the HUES

8-E cells were treated with 300 nM ILV, 50 ng/ml FGF10 or 300 nM ILV+50 ng/ml FGF10 for 4

days and then stained with Pdx1 antibody. The number of Pdx1+ cells was analyzed withthe

Opera high content screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 7. The effect of ILV on HUES-E population derived from HUES 2 and 4.

After 9 days treatment with Wnt, Activin, FGF10, KAAD-CYC and RA (same as HUES 8 and

9), the HUES-E cells derived form HUES 2 and 4 were treated with 300 nM ILV, or 300 nM

ILV+50 ng/ml FGF10 for 4 days and then stained with Pdx1 antibody. DMSO treatment and day

1 were used as controls. The percentage and number of Pdx1+ cells were analyzed with an Opera

high content screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 8. ILV also promotes pancreatic differentiation of mESCs. mESCs (AV3)

were treated with 1000 ng/ml recombinant mouse Nodal for 5 days to produce definitive

endoderm and then treated with 300 nM ILV for additional 6 days. The cells were analyzed by

immunocytochemistry with (a, b) Pdx1, Sox9 and Nkx6.1 antibodies and (c) qRT-PCR. DMSO

treatment alone is the control. Hnf6: Hepatocyte nuclear factor-6; Ptf1a: pancreas specific

transcription factor, 1a; Foxa2: forkhead box A2; Nkx2.2: NK2 transcription factor related, locus

2; Cdcp1: CUB-domain-containing protein 1.

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 9. ILV directs the specification of GTE population toward the pancreatic

lineage. DE population, without Pdx1+ cells, was derived from HUES 8 after 1 day treatment

with 25 ng/ml Wnt 3a and 100 ng/ml Activin A for 1 day and 100 ng/ml Activin A for additional

2 days. This DE population (without Pdx1+ cells) was treated with 300 nM ILV or 300 nM

ILV+50 ng/ml FGF10 for additional 4 days. The number of Pdx1+ cells was analyzed with an

Opera high content screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 10. ILV functions through PKC activation. PKC agonists mimic ILV’s

effect and PKC antagonists block ILV’s effect. HUES 8-E cells were treated with 500 nM TPB

or 14 nM PMA in the absence of ILV and 1 µM BISI, 10 µM Gö 6983 or 4 µM Gö 6976 in the

presence of 300 nM ILV for 4 days and stained with Pdx1 antibody. The number of Pdx1+ cells

was analyzed with an Opera high content screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 11. PKC agonists mimic ILV’s effect. (a) PKC agonists synergize with

FGF10. HUES 8-E cells were treated with 500 nM TPB or 14 nM PMA in the presence of 50

ng/ml FGF10 for 4 days and stained with Pdx1 antibody. (b) PKC agonists mimic ILV’s effect

on HUES-DE cells (without RA treatment). HUES 8-DE cells were treated with 500 nM TPB or

14 nM PMA in the absence or presence of 50 ng/ml FGF10 for 4 days and stained with Pdx1

antibody. The percentage and number of Pdx1+ cells were analyzed with the Opera high content

screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 12. Effect of ILV and RA on HUES 8-E cells. (a) Effect of ILV and RA on

HUES 8-E cells in different conditions. HUES 8-E cells were treated with 300 nM ILV or 2 µM

RA in the absence and presence of 0.25 µM KAAD-CYC, 50 ng/ml FGF10 and 50 ng/ml

FGF10+ 0.25 µM KAAD-CYC for 4 days and stained with Pdx1 antibody. The percentage and

number of Pdx1+ cells were analyzed withthe Opera high content screening system

(PerkinElmer). (b) The combined effect of ILV and RA. HUES 8-E cells were treated with 100

nM or 300 nM ILV in the absence and presence of 2 µM RA for 4 days and stained with Pdx1

antibody. The percentage and number of Pdx1+ cells were counted by the Opera high content

screening system (PerkinElmer).

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Figure 13. The characterization of ILV. 1H NMR (500 Mhz, d-DMSO): δ 6.99 (s,

1H), 6.89 (d, J=8.1 Hz, 1H), 6.85 (t, J=8.1 Hz, 1H) , 6.81(d, J=8.1 Hz, 1H), 6.38 (d, J=11.6 Hz,

1H), 4.27 (d, J=11.5 Hz, 1H), 3.94 (m, 1H), 3.35 (m, 2H), 2.98 (m, 2H), 2.75 (s, 3H), 2.45 (m,

1H), 0.89 (d, J=6.7 Hz, 3H), 0.48 (d, J=6.7 Hz, 3H). HRMS (m/z): [M+] calcd. For C17H23N3O2,

302.18630; found: 302. 18727.

Nature Chemical Biology: doi:10.1038/nchembio.154

Nature Chemical Biology: doi:10.1038/nchembio.154

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Methods.

mESC culture and differentiation.

mESCs (AV3) are routinely cultured on irradiated MEF feeder cells in DMEM

(Invitrogen) supplemented with 15% FBS (Hyclone), 2 mM L-Glu, 1.1 mM 2-mercaptoethanol,

1 mM nonessential amino acids, 1×PS and 103 unit LIF (Chemicon). Cells are passaged at the

ratio of 1:12 every 3 days by using 0.25% trypsin-EDTA (Invitrogen). To generate the definitive

endoderm population, mESCs were plated on gelatin-coated surface at 2500 cell/cm2 after

depletion of MEF feeder cells. Then, the cells were treated with 1000 ng/ml recombinant mouse

Nodal (R&D systems) in A-RPMI supplemented with 1×L-Glu, 1×L-PS and 0.2% FBS for 5

days. To test the small molecules, the definitive endoderm population was treated with 300 nM

ILV in DMEM supplemented with 1×L-Glu, 1×PS, 1× B27 for 6 days.

For the quantification of Pdx1+ cells, at least 10 images for each treatment were taken

using an Olympus IX70 fluorescent microscope. Total cell number was quantified based on

DAPI nuclear staining and Pdx1+ cells were quantified using Metamorph software (Molecular

Devices). The percentage of Pdx1+ out of the total cell number is shown. Quantification for each

treatment was done for four independent experiments.

Quantitative RT-PCR.

Total RNAs were extracted with the RNeasy Mini Kit (Qiagen) and reverse-transcribed

using SuperScript III RT-PCR system (Invitrogen) according to the manufacturer's protocol. One

μl of cDNA sample was PCR amplified with QuantiFast SYBR Green PCR Kit (Qiagen) and

analyzed with DNA engine Opticon2 (MJ Research). The data was normalized using GAPDH as

a control. Primers are listed in supplemental table 1.

Nature Chemical Biology: doi:10.1038/nchembio.154

Supplemental Tabel 1. Primers for qPCR.

h-Pdx1-F 5'-CCTTTCCCATGGATGAAGTC-3'

h-Pdx1-R 5'-CGAACTCCTTCTCCAGCTCTA-3'

h-HNF6-F 5'-GTGTTGCCTCTATCCTTCCCAT-3'

h-HNF6-R 5'-CGCTCCGCTTAGCAGCAT-3'

h-FOXA2-F 5'-ATTGCTGGTCGTTTGTTGTG-3'

h-FOXA2-R 5'-TACGTGTTCATGCCGTTCAT-3'

h-SOX9-F 5'-GCCACGGAGCAGACGCAC-3'

h-SOX9-R 5'-GCGCCTGCTGCTTGGACA-3'

h-PROX-F 5'-AAAGCAAAGCTCATGTTTTTTTATACC-3'

h-PROX-R 5'-GTAAAACTCACGGAAATTGCTAAACC-3'

h-HB9-F 5'-TCCACCGCGGGCATGATCCT-3'

h-HB9-R 5'-GCGCTTGGGCCGCGACAGCTA-3'

h-NKX6.1-F 5'-TCAACAGCTGCGTGATTTTC-3'

h-NKX6.1-R 5'-CCAAGAAGAAGCAGGACTCG-3'

h-NGN3-F 5'-CCCTCTACTCCCCAGTCTCC-3'

h-NGN3-R 5'-CCTTACCCTTAGCACCCACA-3'

h-NKX2.2-F 5'-ATGTAAACGTTCTGACAACT-3'

h-NKX2.2-R 5'-TTCCATATTTGAGAAATGTTTGC-3'

h-PTF1A-F 5'-CATAGAGAACGAACCACCCTTTGAG-3'

h-PTF1A-R 5'-GCACGGAGTTTCCTGGACAGAGTTC-3'

h-insulin-F 5'-GAGGCCATCAAGCACCATCAC-3'

h-insulin-R 5'-GGCTGCGTCTAGTTGCAGTA-3'

h-amylase-F 5'-CTGACAACTTCAAAGCAAA-3'

h-amylase-R 5'-TACAGCATCCACATAAATACGA-3'

h-somatostatin-F 5'-GATGCTGTCCTGCCGCCTCC-3'

h-somatostatin-R 5'-TGCCATAGCCGGGTTTGA-3'

h-glucagon-F 5'-AGGCAGACCCACTCAGTGA-3'

h-glucagon-R 5'-AACAATGGCGACCTCTTCTG-3'

h-SOX7-F 5'-AGAAGGAGGACAGGGGTGAG-3'

h-SOX7-R 5'-TGAGGACGAGAAGAAGGTCTG-3'

Nature Chemical Biology: doi:10.1038/nchembio.154

h-CDX2-F 5'-GGAACCTGTGCGAGTGGATG-3'

h-CDX2-R 5'-AGGTGGTGGGGCTTGCGGGGGCG-3'

h-CES2-F 5'-AATCCCAGCTATTGGGAAGGA-3'

h-CES2-R 5'-CTGGCTGGTCGGTCTCAAAC-3'

h-FABP2-F 5'-GATAAACTAAAAGCATAGGCTGCATATG-3'

h-FABP2-R 5'-TCAAAATCAGAATGGCAATTATCTCT-3'

h-AFP-F 5'-CTTTGGGCTGCTCGCTATGA-3'

h-AFP-R 5'-TGGCTTGGAAAGTTCGGGTC-3'

h-Albumin-F 5'-ACA GAATCCTTGGTGAACAGGCGA-3'

h-Albumin-R 5'-TCAGCCTTGCAGCACTTCTCTACA-3'

h-BRAX1-F 5'-AAACGCTTCGAGAAGCAGAA-3'

h-BRAX1-R 5'-TCCACTTCATCCTCCGATTC-3'

h-SOX2-F 5'-TTACCTCTTCCTCCCACTCCAG-3'

h-SOX2-R 5'-GGGTTTTCTCCATGCTGTTTCT-3'

h-Troponin T-F 5'-GGCAGCGGAAGAGGATGCTGAA-3'

h-Troponin T-R 5'-GAGGCACCAAGTTGGGCATGAACGA-3'

h-ISL1-F 5'-GATCTATGTCACTCTGCAAGG-3'

h-ISL1-R 5'-TACAACCACCATTTCACTG-3'

h-Desmin-F 5'-CCAACAAGAACAACGACG-3'

h-Desmin-R 5'-TGGTATGGACCTCAGAACC-3'

h-MYOD-F 5'-AGCACTACAGCGGCGACT-3'

h-MYOD-R 5'-GCGACTCAGAAGGCACGTC-3'

h-Nestin-F 5'-GAAACTCAAGCACCAC-3'

h-Nestin-R 5'-TTTTAAACTCCAGCCATCC-3'

h-SOX1-F 5'-CTCACTTTCCTCCGCGTTGCTTCC-3'

h-SOX1-R 5'-TGCCCTGGTCTTTGTCCTTCATCC-3'

h-GAPDH-F 5'-GTTGTCTCCTGCGACTTCA-3'

h-GAPDH-R 5'-TGGTCCAGGGTTTCTTACTC-3'

m-Actb-F 5'-CCAACCGTGAAAAGATGACC-3'

m-Actb-R 5'-CCATCACAATGCCTGTGGTA-3'

m-GAPDH-F 5'-ACCCAGAAGACTGTCGATGG-3'

Nature Chemical Biology: doi:10.1038/nchembio.154

m-GAPDH-F 5'-TTCAGCTCTGGGATGACCTT-3'

m-Ipf1-F 5'-GAAATCCACCAAAGCTCACG-3'

m-Ipf1-R 5'-GAATTCCTTCTCCAGCTCCA-3'

m-Cdcp1-F 5'-TGTGGGTGAATGTGGAGAAA-3'

m-Cdcp1-R 5'-GCTTCCAGGAGAAGTCATGC-3'

m-Ptf1a-F 5'-AACCAGGCCCAGAAGGTTAT-3'

m-Ptf1a-R 5'-AAAGAGAGTGCCCTGCAAGA-3'

m-Hnf6-F 5'-AGATCAATACCAAAGAGGTGGCG-3'

m-Hnf6-R 5'-TTGGTACAAGTGCTCGATGAGG-3'

m-FoxA2-F 5'-GACATACCGACGCAGCTACA-3'

m-FoxA2-R 5'-GGCACCTTGAGAAAGCAGTC-3'

m-Nkx2.2-F 5'-ATCGCTCTCCCCTTTGAACT-3'

m-Nkx2.2-R 5'-TAACGTTGGGATGGTTTGGT-3'

Nature Chemical Biology: doi:10.1038/nchembio.154