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-3 -2 -1 0 8 16 hES cell platingviral infectionpuro selectionhES cell plating passage and analysis analysis Day

Medium ES DMEM/F12/Neurobasal/N2/B27

Factors SB431542

Drosomophrin

PAX6 SOX1

H1+SOX2-shRNA GAPDH

GAPDH PAX6

PAX6 SOX2

SOX2

D0

D16 55KD

55KD 35KD 35KD

35KD 35KD 1# 2# 3# 4#

PAX6 (NPC differentiation at day 16)

% of Max

89.0 37.7 41.7 35.7 42.8

0 20 40 60 80 100

Percentage of PAX6+ cells (%)

**

Supplementary Figure 1

b c

d

Virus, SOX2-shRNAs

shctrl

H1+SOX2-shRNA 1# 2# 3# 4#

shctrl

g

Neural stem cell genes

H1+SOX2-shRNA

shctrl 1# 2# 3# 4#

e

50 μm

SOX2/NESTIN DAPIOCT4 DAPI

H1+SOX2-shRNA (day 16)

shctrl 1# 2# 3# 4#

0.0 0.1 0.2 0.3 0.4 SOX2

*

** H1+SOX2

-shRNA 1#

2#3#

4#

shctrl

Relative expression to GAPDH

NANOG

0.00 0.02 0.04 0.06 0.08 0.10 OCT4

0.0000 0.0005 0.0010 0.0015 0.0020

Relative expression to GAPDH

Day 0 Day 8 Day 16

Day 0 Day 8 Day 16 Day 0 Day 8 Day 16

0.00 0.05 0.10 0.15 0.20 0.25

OTX2 FOXG1 ZIC1 PAX3 KRT18 VEGFA

**

** **

**

Relative expression to GAPDH at day16

Meso-endoderm genes

f

**

H1+SOX2 -shRNA 1#2#

3#4#

shctrl

a

Day 0

Day 8

Merge

50 μm

Wild Type cells

SOX2 EZH2 DAPI

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