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
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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
or after d
KAAD-C
and overn
mental Figure
dissociation.
CYC and RA
night incuba
e 2. There is
The HUES
A) before dis
ation) stained
no detectab
9-E cells (af
ssociation a
d with Pdx1
ble difference
fter 9-day tre
and the HUE
antibody (%
e in the perc
eatment with
ES 9-E cells
% staining sh
centage of P
h Wnt 3a, A
after dissoc
hown).
dx1+ cells b
Activin A, FG
ciation (after
before
GF10,
r split
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