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Combined small molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors

Stuart M. Chambers, Yuchen Qi, Yvonne Mica, Gabsang Lee, Xin-Jun Zhang, Lei Niu, James Bilsland, Lishuang Cao, Edward Stevens, Paul Whiting, Song-Hai Shi, Lorenz Studer.

Supplementary Figures:

Figure S1 - LSB3i screen description

Figure S2 - LSB3i differentiation scheme.

Figure S3 - Cell cycle analysis of LSB and LSB3i

Figure S4 – Protein expression of NTRK2 and NTRK3

Figure S5 – LSB3i treated iPSC clone C72 rapidly acquires a nociceptor phenotype.

Figure S6 – NTRK1 FACS sorting enriches for hiPSC-derived LSB3i neurons

Figure S7 – qRT-PCR validation of SOX10::GFP BAC cell line.

Figure S8 – SOX10::GFP expression for all combinations of 3i factors.

Figure S9 – SOX10 and neuronal _3-tubulin upon passage of LSB3i cells.

Figure S10 – Cell progeny from cells SOX10+ at day 15.

Figure S11 – Microarray gene expression for mechanoreceptor and proprioceptor markers.

Figure S12 – Varying CHIR exposure indicates its requirement in both SOX10 expression and neuronogenesis

Figure S13 - A single action potential recorded following current injection of 75 pA.

Figure S14 - Photomontage of calcium flux images of LSB3i induced hESC derived neurons.

Figure S15 – LSB3i Differentiation model.

- Supplementary Tables:

Supplementary Table 1

Supplementary Table 2

Nature Biotechnology: doi:10.1038/nbt.2249

Day: 0 1 2 4 6 8 10

KSR

LDN-193189, SB431542 (LSB)

3 5 7 9

CHIR99021 (C)

DAPT (D)

SU5402 (S)

Cyclopamine (Cy)

N2

Day 1 - S, DLSB3i (Day 2 -

S, D, C)Day 3 - S, D

Day 3 - S, D, C

Day 3 - Cy, S, D, C

Day 6 - S, D, C

Day 0 - S, D, C

Day 2 - S

Day 8 - S, DDay 7 - Cy,

S, D, C

Merge

TUJ1

PAX6

DAPI

LSB from Day 0for all conditions

3i Added On Day: 1 2* 3 4 5 6 7 8

*LSB3i

PA

X6/

TU

J1P

AX

6/T

UJ1

a

b

c

Figure S1 - SM Chambers, et al.

Figure S1 – LSB3i screen description. (a) Cyclopamine, SU5402, DAPT, and CHIR99021 were added on different days in approximately 400 different combinations. (b) Cells were fixed on day 10 and examined for the loss of PAX6 and acquisition of �3- tubulin (TUJ1) by immunofluorescence (examples shown). (c) Day 2 was deter-mined to be the optimal day for addition of SU5402, DAPT, and CHIR99021 (3i).


Day: 0 1 2 4 6 8 10

KSR

LDN-193189, SB431542 (LSB)

3 5 7 9

CHIR99021, DAPT, SU5402 (3i)

N2


Figure S2 – LSB3i differentiation scheme. In the context of dual-SMAD inhibition using LDN-193189 and SB431542 (LSB), optimal neuronal differentiation was observed when CHIR99021, SU5402 and DAPT (3i) were added at day two of differentiation. Starting on day 4, N2 media was added in increasing 25% increments replacing KSR.


Cell Cycle (FACS)

2 3 5 7 9 110

20

40

60

80

100G1/G0 LSB

G1/G0 LSB3i

S/G2/M LSB

S/G2/M LSB3i

Day of Differentiation

% In

Sta

ge o

f Cel

l Cyc

le


Figure S3 – Cell cycle analysis of LSB and LSB3i. By day 7 of differentiation, LSB3i treatment slows cell proliferation as measured by FACS.


TUJ1 , NTRK3TUJ1 , NTRK2

0 102

103

104

105

0

20

40

60

80

100

% o

f Max

0 102

103

104

105

0

20

40

60

80

100

% o

f Max

Neg. controlNTRK3

Neg. controlNTRK2


Figure S4 – Protein expression of NTRK2 and NTRK3. Little or no expression of NTRK2 and NTRK3 could be detected in LSB3i cells by immunofluorescence or FACS.


TUJ1/BRN3A

C72

TUJ1/ISL1 TUJ1/RUNX1TUJ1/RET

C72 C72 C72


Figure S5 – LSB3i treated iPSC clone C72 rapidly acquires a nociceptor phenotype. TUJ1 positive neurons from LSB3i treated iPSC clone C72 expressISL1, BRN3A, RET, and RUNX1.


C14

C72

NTRK1 Negative NTRK1 Positive

TUJ1/Nestin


Figure S6 – NTRK1 FACS sorting enriches for hiPSC-derived LSB3i neurons. NTRK1 sorting on day 10 of differentiation can enrich for TUJ1 (green) positive neurons and remove nestin (red) positive progenitor cells.


SSEA4+

Sox10

:GFP+

HNK1+ in

P1

0.001

0.01

0.1

1

10

100

SSEA4+

Sox10

:GFP

HNK1+ in

P1

0.0001

0.001

0.01

0.1

1

10

SSEA4+

Sox10

:GFP

HNK1+ in

P1

1.0 10-01

1.0 1000

1.0 1001

1.0 1002

1.0 1003

1.0 1004

1.0 1005

1.0 1006

1.0 1007

SOX10 p75 Ap2b

Rel

ativ

e E

xpre

ssio

n (

qRT-

PC

R)


Figure S7 – qRT-PCR validation of SOX10::GFP BAC cell line. Compared to hPSCs sorted for SSEA4 and a previous method to enrich for neural crest stem cells by sorting for HNK1+ cells from neural cultures22, GFP+ cells sorted using the SOX10::GFP BAC are greatly enriched for the neural crest genes SOX10, p75, and AP2B measured by qRT-PCR. GFP+ cells under LSB3i were also negative for markers of other SOX10+ cell types (data not shown) such as oligodendrocytes (OLIG2) or otic placode precursors (SIX and FOXG1) confirming neural crest identity of the cells.



SOX10::GFP

4 6 8 100

10

20

30

40

50SUCHIRDAPTSU/CHIRSU/DAPTDAPT/CHIR3i

Day of Differentiation

% G

FP

Figure S8 – SOX10::GFP expression for all combinations of 3i factors. Using the SOX10::GFP BAC hESC line, we examined the level of expression of SOX10 under different combinations of 3i factors to gain further mechanistic insight. Both 3i and SU/CHIR treatments displayed the fastest onset of SOX10 expression, suggesting both are critical for neural crest induction and DAPT acts primarily at the later stages of differ-entiation. When CHIR or DAPT/CHIR was added, moderate, yet delayed, levels of SOX10 was observed. In the absence of CHIR (DAPT, SU, SU/DAPT) little SOX10 expression was observed.


TUJ1 / SOX10::GFP

TUJ1 / SOX10

SOX10 / DAPIc

a

b


Figure S9 – SOX10 and neuronal �3-tubulin upon passage of LSB3i cells. Co-expression of markers neuronal �3-tubulin with SOX10 is never observed by (a) SOX10::GFP or (b) SOX10 antibody staining. (c) When passaged SOX10 expression decreases to < 5%.


SMA GFP (SOX10), TUJ1


Figure S10 – Cell progeny from cells SOX10+ at day 15. SOX10::GFP cells sorted at late stages of LSB3i (day 15). Upon culturing the SOX10::GFP posi-tive cells, neural crest progeny are observed such as smooth muscle cells marked by smooth muscle alpha actin (SMA), neurons expressing neuronal �3-tubulin (TUJ1), and putative Schwann cells expressing SOX10 in close prox-imity to neurons.


NTRK2

2 3 5 7 9 150.0

0.5

1.0

1.5

2.0

2.5

Time (Days)

NTRK3

2 3 5 7 9 151.0

1.2

1.4

1.6

1.8

Time (Days)

ETV1 (Proprioceptor)

2 3 5 7 9 151.0

1.2

1.4

1.6

1.8

Time (Days)

MAFA (Mechanoreceptor)

2 3 5 7 9 150.8

1.0

1.2

1.4

1.6

Time (Days)

RUNX3

2 3 5 7 9 151.0

1.2

1.4

1.6

1.8

Time (Days)

LSBLSB 3i

PVALB (proprioceptor)

2 3 5 7 9 150.8

1.0

1.2

1.4

1.6

1.8

2.0

Time (Days)


Figure S11 – Microarray gene expression for mechanoreceptor and proprioceptor markers. Minimal expression changes (< 2-fold) were found for markers of mechanorecep-tors and proprioceptors including NTRK2, NTRK3, RUNX3, PVALB1, MAFA, and ETV1 when examined by microarray. Expression differences are normalized array values (log2).


TU

J1,B

RN

3AS

OX

10Day: 0 1 2 4 6 8 10

KSR

LDN-193189, SB431542 (LSB)

3 5 7 9

DAPT, SU5402

N2

12 14

CHIR99021

LSBLSB 3iC 2-4

LSB 3iC 2-8

LSB 3iC 2-14


LSB3i

CHIR 2-

4

LSB3i

CHIR 2-

8

LSB3i

CHIR 2-

14-4

-2

0

2

4

6

PAX6SOX10

Nor

mal

ized

Gen

e E

xpre

ssio

n (L

og2)

LSB3i

CHIR 2-

4

LSB3i

CHIR 2-

8

LSB3i

CHIR 2-

14-5

0

5

10

15

NTRK1NTRK2NTRK3

Nor

mal

ized

Gen

e E

xpre

ssio

n (L

og2)

LSB3i

CHIR 2-

4

LSB3i

CHIR 2-

8

LSB3i

CHIR 2-

140

5

10

15

ASCL1ISL1BRN3A

Nor

mal

ized

Gen

e E

xpre

ssio

n (L

og2)

Figure S12 – Varying CHIR exposure indicates its requirement in both SOX10 expression and neuronogenesis. The length of time CHIR exposure was varied from 2 days (Days 2-4) up to 12 days (Days 2-14) and the resulting differentiations were examined for markers of neural crest and autonomic and sensory neuron populations. When CHIR is added for 2 days, SOX10 expression is robust and comparable to longer exposure. When CHIR is added for 6 days, the majority of the cells express �3-tubulin (TUJ1) and BRN3A indicating prolonged CHIR exposure promotes sensory neurogenesis. Continued exposure of CHIR further biases the cell fates towards nociceptors and away from other peripheral sensory neurons on the basis of NTRK1,2, and 3 gene expression measured by Real-Time PCR.



0 400 800

-60-30

030

0 400 800

-60

-30

0

30

0 400 800

-60

-30

0

30

0 400 800

-60

-30

0

30

Vol

tage

(mV

)

time (ms)

control 500 nM A-803467

Vol

tage

(mV

)

time (ms)

500 nM TTX

Vol

tage

(mV

)

time (ms)

Vol

tage

(mV

)

time (ms)

wash

Figure S13 - A single action potential recorded following current injection of 75 pA. There was no significant effect upon application of 500 nM A-803467. Subsequent application of 500 nM TTX blocked the action potential with full recovery after wash.



Figure S14 - Photomontage of calcium flux images of LSB3i induced hESC derived neurons. Top panel shows the response to ,β Methylene ATP and inhibition by A- 317491. Images on the left hand side are basal images prior to addition of agonist or vehicle. Images on the right hand side are post treatment. α,β Methylene ATP induced an increase in calcium flux, which was blocked by the antagonist. The lower panel shows examples of calcium flux induced by capsaicin. Capsaicin induced a response in cell bodies in relatively few neurons (arrow in lower images); in images where no cell body response was detected, responses in neurites were frequently observed (upper panel, arrows).


SU5402, DAPT

NotchFGF

SB431542

TGF-�

Pluripotent Stem Cells(OCT4, NANOG)

TrophectodermMesendoderm

Non-neural EctodermLDN-193189SB431542

SMAD

Sensory Neuron(ISL1, BRN3A, RUNX1,

RET, NTRK1, PRPH)

Neural Crest Cells(SOX10, NGN1)

CHIR99021, SU5402

CHIR99021(WNT)

Days 2-4 Days 4-10


Mature Nociceptors(ISL1, BRN3A, SCN9A,

SCN10A, P2RX3, TRPM8, TRPV1, TAC1, VGLUT2)

Days 10-15

Neurotrophins(bNGF, GDNF, BDNF)

Figure S15 – LSB3i Differentiation model. Early LSB inhibits trophectoderm, mesen-doderm, and non-neural ectoderm cell fates yielding neuroectoderm. CHIR99021, SU5402 and DAPT induce and accelerate neural crest stem cell identity by day 8 and promote rapid differentiation of the neural crest stem cells to nociceptors expressing peptidergic markers by day 10.


Phases Genes

Neurectoderm PAX6, OTX2, DLK1, DKK1, CUZD1

Neural Crest SOX10, MSX1, ID2, AP2B, ETS1, FOXD3

Neuron NGN1, DCX, TUBB3, SYT4, STMN2, INA, GAP43, ISL1, POU4F1

Nociceptor TAC1, VGLUT2, SLC15A3

Table S1 - SM Chambers, et al.


Marker Determined By Maximum Percent Day of Expression Cell FateSOX10 BAC GFP, Antibody 80% Day 6-‐14 Neural Crest

Neuronal β3-‐Tubulin (TUJ1) Antibody (FACS and IF) 75% After Day 10 NeuronNestin Antibody (FACS and IF) 25% Day 10 Neural ProgenitorNTRK1 Antibody (FACS) 60% Day 10 Nociceptor

BRN3A of TUJ1 neurons Antibody (IF) > 95% After Day 11 Sensory NeuronISL1 of TUJ1 neurons Antibody (IF) > 95% After Day 11 Sensory Neuron

RUNX1 (early) Antibody (IF) > 80% Day 8 NociceptorRET Antibody (IF) > 80% Day 14 Nociceptor

Functional SCN10A Electrophysiology 20% After Day 21 NociceptorTTX-‐R inhibited by A-‐803467 Electrophysiology 90% After Day 21 Nociceptor

Functional P2RX3 Calcium flux 50% After Day 21 NociceptorFunctional TPVR1 Calcium flux 1-‐2% After Day 21 Nociceptor

Table S2 - SM Chambers, et al.


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