Inhibition of JAK/STAT signaling stimulates adult ... of JAK/STAT signaling stimulates adult satellite cell function ... FACS profile of digests from Pax7-ZsGreen mice at adolescent,

Price et al., (2014)

1

Inhibition of JAK/STAT signaling stimulates adult satellite cell function

Feodor D. Price, Julia von Maltzahn, C. Florian Bentzinger, Nicolas A. Dumont, Hang Yin,

Natasha C. Chang, David H. Wilson, Jérôme Frenette, and Michael A. Rudnicki

SUPPLEMENTARY INFORMATION

Supplementary Figures 1-9

Supplementary Table 1

Nature Medicine: doi:10.1038/nm.3655


2

Supplementary Figure 1

3 weeks 3 months 18 months

3 w

eeks

3 m

onth

s18

month

s

********

*******

1wk

Embryonic Postnatal

Co

nception

3 weeks

(Adolescent)

3 months

(Young Adult)

18 months

(Late Adult)

1 y

r

Log ZsGreen

Lo

g P

E

3 months - WT 3 weeks ZsGreen

104

103

102

101

100

100 101 102 103 104

104

103

102

101

100

100 101 102 103 104

104

103

102

101

100

100 101 102 103 104

104

103

102

101

100

100 101 102 103 104

0.08% 1.39%

3 months ZsGreen

0.90%

18 months ZsGreen

0.52%


a

b

***

******

c d

ß1 CXCR4 CD34 Pdgfra Mcad

***

0

20

40

60

80

100

120

Zsgre

en

+ c

ells

expre

ssin

g a

giv

en c

ell

surf

ace

mark

er

(%)

Supplementary Figure 1.

Tota

l # o

f Z

sG

reen

+ c

ells

/ m

ouse

hin

dlim

b m

uscle

dig

est

0

50000

100000

150000

200000

250000

Supplementary Figure 1 Satellite cells decrease in number and alter their cell

surface complement with age. (a) Isolation time points for satellite cells. 3 weeks

(Adolescent), 3–4 months (Young Adult) and 18 ± 2 months (Late Adult). (b)

FACS profile of digests from Pax7-ZsGreen mice at adolescent, young adult and

late adult stages. (c) Total number of ZsGreen+ cells per hind limb muscle digest

relative to age stage. (d) Cell surface profile of ZsGreen+

cells collected from

different age groups and analyzed with common satellite cell surface markers. * p

< 0.05 **, p < 0.01, *** p < 0.001. Abbreviations: PE, phycoerythrin; FSC,

forward scatter; SSC, side scatter; CXCR4, C-X-C chemokine receptor type 4;

Mcad, Muscle-cadherin; PDGFRα, platelet derived growth factor receptor α.



3


Saline


Zs

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en

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Zsg

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n P

ax7 D

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AP

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Pa

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Pa

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Dystr

op

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Lam

inin

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Dy

str

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La

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AP

I

Dy

str

op

hin

DA

PI

c

b

d

e


Saline

3 months

3 weeks

3 weeks

a

Zs

gre

en

Supplementary Figure 2 Adolescent satellite cells display improved regenerative capacity

relative to young adult satellite cells following transplantation. (a) Saline staining controls for

dystrophin (green) and laminin (red) on mdx TA muscle from 6–8 week old mice. (b)

Immunofluorescent staining of adolescent donor satellite for dystrophin (green) and laminin

(red) following transplantation into mdx mice. Arrowheads designate donor-derived

dystrophin expressing myofibers. (c) Saline staining controls for ZsGreen (green) and Pax7

(red) on mdx TA muscle from 6–8 week old mice. (d) Immunofluorescent staining of

adolescent donor satellite for ZsGreen (green) and Pax7 (red) following transplantation into

mdx mice. Arrows designate donor derived ZsGreen– and Pax7-expressing satellite cells.

Scale bars denote 100 m. (e) High magnificiation image depicting ZsGreen expressing donor

derived satellite cells following transplant of young adult satellite cells into mdx TA muscle.

Scale bar denotes 10 m. Arrows designate donor derived ZsGreen– and Pax7-expressing

satellite cells.



4


a b

dc


Invers

e p

-valu

e (

Log) GO terms enriched in

3 week Satellite cells

1.0E+00

1.0E+08

1.0E+16

1.0E+24

1.0E+32

1.0E+40

1.0E+48

1.0E+56

1.0E+64

Ce

ll C

ycle

Cell

Div

isio

n

Mitosis

DN

A P

ackag

ing

DN

A R

epa

ir

Chro

matin

Org

an

iza

tio

n

Ce

llula

r R

esp

onse t

o S

tre

ss

Str

iate

d M

uscle

Cell

Develo

pm

en

t

Cyto

ske

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n O

rgan

iza

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n

Re

gu

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n o

f M

icro

tub

ule

C

yto

ske

leto

n O

rgan

iza

tio

n

Invers

e p

-valu

e (

Log) GO terms enriched in

18 month Satellite cells

1.0E+00

1.0E+02

1.0E+04

1.0E+06

1.0E+08

1.0E+10

1.0E+12

Imm

une R

espo

nse

Vascula

ture

De

ve

lopm

ent

Inflam

mato

ry R

espo

nse

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spo

nse

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id

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ula

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ation R

ed

uction

Ossific

atio

n

Reg

ula

tio

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f A

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tosis

Po

sitiv

e R

eg

ula

tio

n o

f T

ranscrip

tio

n


Myobla

st

3 w

eeks

3 m

on

ths

18

mo

nth

s

Myoblast

3 weeks

3 months

18 months

fe

5 5.5 6

pI

p-Stat3

18

mo

nth

s3

mo

nth

s3

we

ek

s

1.5

2.0

Sig

nal

2.5

3.0

3.5

4.0

1.0

0.5

0.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 10 20 30 40 50 60 80

Glyoxylate and dicarboxylate

metabolism

Citrate cycle TCA cycle

Starch and sucrose metabolism

P53 signaling pathway

Galactose metabolism

N glycan biosynthesis

No

rma

lize

d E

nri

ch

men

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co

re

(NE

S)

for

3w

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Ce

ll

Tra

nscri

pto

me

s

KEGG Signaling Pathway

1

1

1

1

0.932 0.892 0.891

0.975 0.975

0.985

Supplementary Figure 3 Gene expression analysis reveals intrinsic differences between

skeletal muscle myoblasts and satellite cells of varying ages. (a,b) Pearson correlation

analysis and 3D component analysis identify a distinct difference between myoblasts and all

satellite cell samples irrespective of age. (c) GO term enrichment for genes >2 fold up

regulated in adolescent satellite cells. (d) GO term enrichment for genes >2 fold up regulated

in late adult satellite cells. (f) Representative chemiluminescent signal profiles of phospho-

Stat3 (p-Stat3) from freshly sorted satellite cell lysates isolated from adolescent, young adult

and late adult mice (n=3 for each age group).



5


Supplementary Figure 4 Age related transcriptional changes in myogenic, satellite cell,

quiescence, cell cycle and extracellular matrix markers. Log2 transformed RMA values from 5

d and 2 d differentiated myotubes, proliferating myoblasts along with adolescent, young adult

and late adult satellite cells were used to generate global ratio heat maps. Red indicates genes

that have high log2 expression values while blue represents those with low log2 expression

values. Data represents pooled replicates for adolescent (n=5), young adult (n=8), aged (n=8)

satellite cell samples along with 5 d myotubes (n=3), 2 day myotubes (n=3), and myoblasts

samples (n=3).



6


Supplementary Figure 5 Satellite cells on single EDL fibers decrease with age. (a)

Representative images of EDL fibers following 0 hours in culture (T0) from adolescent, young

adult and late adult mice. Pax7 (green) and DAPI (blue). Scale bars denote 100 m. (b)

Quantification of average satellite cell numbers per fiber from adolescent, young adult and late

adult EDL fibers at T0. Data represents biological triplicate samples expressed as the mean ±

SEM of 30 fibers per condition at 0 h. ** p < 0.01, *** p < 0.001. (c) Ex vivo treatment of single

muscle fibers isolated from EDL muscle with either a Jak2 inhibitor (Tyrphostin AG 490) or a

Stat3 inhibitor (5, 15 DPP) resulted in an increase in the number of satellite cells/fiber following

72 h in culture. Pax7 (green), MyoD (red) and DAPI (blue). Scale bar denotes 10um. (d)

Quantification of planar satellite cell divisions after 42 h following siRNA treatment with either

siStat3 or siJak2 on myofibers isolated from 18 month old mice. (e) Quantification of planar cell

divisions after 42 h following JAK/STAT inhibitor treatment on myofibers isolated from 18

month old mice. Data expressed as the mean ± SEM of 15 fibers per condition (n=4) at 42 h. ** p

< 0.01, *** p < 0.001.



7


*** ***

Price et al., (2014)Supplementary Figure 6.a

0

0.2

0.4

0.6

0.8

1

siSCR siJAK2

Rela

tive

Fo

ld C

ha

ng

e

in J

ak2

Exp

ressio

n

0

0.2

0.4

0.6

0.8

1

siSCR siSTAT3

Re

lative F

old

Ch

an

ge

in S

tat3

Expre

ssio

n

Supplementary Figure 6 siRNA knock down validation of Stat3 and Jak2. (a) QPCR validation

of siStat3 and siJak2 in mouse primary myoblasts. Data are presented as the mean ± SEM of 2

biological replicates normalized to GAPDH. *** p < 0.001.



8


c

d

TyrAG 4905,15 DPPVehicle

b

ZsG

reen

Pax7

DA

PI

% Z

sg

reen p

ositiv

e s

ate

llite

cells

NS

NS

0

5

10

15

20

25

30

Re

-in

jury

Inju

ry

Re

-in

jury

Inju

ry

CTX

injury

FACS isolation

and transplantation

CTX

re-injury

Assess self-renewal capacity

21 Days 21 Days 48 h


3 months 18 months

aSupplementary Figure 7.

NS NS

3 months of age

Age of donor cells transplanted into Bl/6 mice

18 months of age

Host

sa

telli

te c

ells

pe

r are

a (

%)

Host sate

llite

cells

per

are

a (

%)

0

20

40

60

80

100

120

140

0 20 40 60 80

100 120 140 160 180 200

Supplementary Figure 7 Ex vivo treatment with JAK/STAT inhibitors promotes satellite cells

expansion and self-renewal. (a) ZsGreen (green), Pax7 (red) and DAPI (blue) staining in mdx TA

muscle 3 weeks following transplantation of 10,000 donor ZsGreen+ satellite cells. Arrows

designate Pax7+/ZsGreen

+ nuclei. Scale bar denotes 100 m. (b) Quantification of host satellite

cells following the transplantation of young adult and late adult satellite cells ex vivo treated with

JAK/STAT inhibitors. NS designates values that are not significant. (c) Experimental schematic

for re-injury of transplanted young adult and late adult satellite cells. (d) Quantification of donor

derived ZsGreen+ satellite cells from young adult or late adult mice after 21 d of regeneration,

subsequent re-injury and an additional 21 d of regeneration. Data represent the mean ± SEM

conducted in biological triplicate. NS designates values that are not significant.



9


Supplementary Figure 8 Regenerating young adult or late adult TA muscle displays fewer

developmental myosin heavy chain (devMyHC) expressing fibers and a greater number of

Pax7+/MyoD

– satellite cells following treatment with JAK/STAT inhibitors. (a) Regenerating TA

muscle following direct IM injection of JAK/STAT inhibiters. Laminin (red), Pax7 (green) and

DAPI (blue). Scale bar denotes 100um. (b) Quantification of devMyHC+ fibers following 7 days

of regeneration in the presence of small molecule inhibitors of JAK/STAT signaling in either

young adult or late adult TA muscle. Data represent the mean ± SEM conducted in biological

triplicate (n=3). * p < 0.05, ** p < 0.01. (c) Quantification of satellite cells expressing Pax7

and/or MyoD following treatment with JAK/STAT signaling inhibitors in regenerating young

adult TA muscle 7 days after injury with CTX. MyoD (green), Pax7 (red) and DAPI (blue). Scale

bar denotes 100 m. (d) Quantification of Pax7+/MyoD

– satellite cells in regenerating young

adult and late adult TA muscle following treatment with JAK/STAT signaling inhibitors. Data

represent the mean ± SEM conducted in biological triplicate. * p < 0.05, ** p < 0.01.



10


TyrA

G 4

90

5,1

5 D

PP

+

TyrA

G 4

90

5,1

5 D

PP

Ve

hic

leU

nin

jure

d

a b

c0

5

10

15

20

25

30

35

40

45 A

vg C

d11b e

xpre

ssin

g c

ells

per

fie

ld

TyrAG 490

5,15 DPP

5,15 DPP+TyrAG 490

Vehicle

Supplementary Figure 9. Price et al., (2014)

Supplementary Figure 9 Late adult skeletal muscle displays a decrease in fibrosis and an

amelioration in muscle architecture following treatment with JAK/STAT signalling inhibitors. (a)

Average number of Cd11b cells per field following treatment with JAK/STAT signalling

inhibitors. (b) Sirius Red connective tissue stain in adult TA muscle following treatment with

JAK/STAT signalling inhibitors. Scale bar denotes 100um. (c) Hematoxylin and Eosin stain in

late adult TA muscle following treatment with JAK/STAT signalling inhibitors. Scale bar denotes

100um.



11

SUPPLEMENTARY TABLES

Supplementary Table 1 Gene expression analysis of older adult and geriatric satellite cells. (a)

GSEA motif enrichment in genes up regulated in aged satellite cells relative to adolescent

satellite cells. The normalized enrichment score (NES) is the primary statistic for examining

gene set enrichment results. By normalizing the enrichment score, GSEA accounts for

differences in gene set size and in correlations between gene sets and the expression dataset;

therefore, the normalized enrichment scores (NES) can be used to compare analysis results

across gene sets. Motifs associated with JAK/STAT signaling highlighted in blue. (b) Log2 fold

changes observed from microarray data between young adult and geriatric adult satellite cells

(Sousa-Victor et al. 2014 Nature). Genes associated with JAK/STAT signaling as well as

common Satellite cell and myogenic markers are displayed

(a) GSEA motif enrichment

NAME NES

HOXA4_Q2 1.4983312

ATF1_Q6 1.4656311

ATF3_Q6 1.4572374

FREAC4_01 1.4357421

PAX3_B 1.3992219

AP1_Q4_01 1.3987931

YNTTTNNNANGCARM_UNKNOWN 1.3984052

TTGCWCAAY_CEBPB_02 1.3891907



12

NFKAPPAB_01 1.3882123

NFKB_C 1.3881786

IRF2_01 1.3841386

GATA3_01 1.3798792

PAX8_01 1.3777196

OCT1_01 1.374703

LMO2COM_02 1.3714612

FXR_IR1_Q6 1.3699574

NFKB_Q6 1.369385

MYOGENIN_Q6 1.3535694

TGTYNNNNNRGCARM_UNKNOWN 1.3479842

NFKB_Q6_01 1.345761

EN1_01 1.3368281

GGGNNTTTCC_NFKB_Q6_01 1.3365896

SREBP1_02 1.3349676

ARP1_01 1.331565

AP1_C 1.329848

PXR_Q2 1.3287233

AAAYWAACM_HFH4_01 1.3222685

CEBPDELTA_Q6 1.3195753

AP1_Q6_01 1.3114696

CREBP1_Q2 1.3109635

CHX10_01 1.3049413

NFKAPPAB65_01 1.3032994

GCCNNNWTAAR_UNKNOWN 1.3000053

FOXJ2_02 1.2978952

YTAATTAA_LHX3_01 1.2976207

PAX8_B 1.2967724



13

AREB6_02 1.2902988

OCT1_02 1.2874292

ATF4_Q2 1.2851044

CEBP_Q2_01 1.28358

DBP_Q6 1.2817761

GGARNTKYCCA_UNKNOWN 1.2813796

STTTCRNTTT_IRF_Q6 1.2811085

CDPCR1_01 1.2790228

AR_Q2 1.2774649

IK2_01 1.2760376

HNF3B_01 1.2759829

LFA1_Q6 1.2714353

CREB_Q2 1.2659374

AP1_01 1.2652273

TAXCREB_01 1.2614539

AP1FJ_Q2 1.2588943

SRF_01 1.2537456

ATF_B 1.2503577

BACH2_01 1.2498549

STAT3_02 1.2484115

MZF1_01 1.2451184

T3R_Q6 1.2405772

FOXO4_01 1.2395285

SMAD4_Q6 1.2346556

POU1F1_Q6 1.2320962

FOXO1_02 1.2313038

CDPCR3HD_01 1.2303405

STAT5A_02 1.2291081



14

E12_Q6 1.2258477

TAL1BETAE47_01 1.2252668

TTAYRTAA_E4BP4_01 1.2251939

TFIII_Q6 1.224314

GNCF_01 1.2223188

AFP1_Q6 1.2214636

GFI1_01 1.2196105

ICSBP_Q6 1.215643

PBX1_01 1.2144768

PITX2_Q2 1.2133756

IK3_01 1.2075506

STAT5A_01 1.2064092

P300_01 1.2050723

VDR_Q3 1.2038164

IRF_Q6 1.2020484

PR_01 1.2014045

HNF3ALPHA_Q6 1.2002699



15

(b) Genes upregulated in geriatric satellite cells

P Value Log2 Fold Change Gene Symbol Description

(Young Adult vs Geriatric)

3.68E-03 -2.05247553 Myc myelocytomatosis oncogene (Myc), transcript variant 1, mRNA [NM_010849]

1.80E-02 -1.26492727 Vegfc vascular endothelial growth factor C (Vegfc), mRNA [NM_009506]

2.75E-02 -1.50489876 Pim1 proviral integration site 1 (Pim1), mRNA [NM_008842]

5.26E-04 -1.71281319 Bcl2 B-cell leukemia/lymphoma 2 (Bcl2), transcript variant 2, mRNA [NM_177410]

2.25E-03 -2.2512006 Bcl3 B-cell leukemia/lymphoma 3 (Bcl3), mRNA [NM_033601]

Absent Bcl6

1.45E-04 -2.14700878 Socs3 suppressor of cytokine signaling 3 (Socs3), mRNA [NM_007707]

1.36E-03 -2.23564161 Cebpa CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa), mRNA [NM_007678]

5.30E-03 -1.9677176 Cebpb CCAAT/enhancer binding protein (C/EBP), beta (Cebpb), mRNA [NM_009883]

5.39E-04 -1.97629476 Cebpd CCAAT/enhancer binding protein (C/EBP), delta (Cebpd), mRNA [NM_007679]

1.59E-04 -1.36519849 Jun Jun oncogene (Jun), mRNA [NM_010591]

7.89E-05 -2.10010602 Junb Jun-B oncogene (Junb), mRNA [NM_008416]

1.20E-04 -1.91883008 Jund Jun proto-oncogene related gene d (Jund), mRNA [NM_010592]

1.07E-03 -2.23269444 Fos FBJ osteosarcoma oncogene (Fos), mRNA [NM_010234]

1.18E-04 -1.57665627 Fosb FBJ osteosarcoma oncogene B (Fosb), mRNA [NM_008036]

1.89E-03 -1.97642273 Fosl2 fos-like antigen 2 (Fosl2), mRNA [NM_008037]

1.48E-03 -2.33884355 Egfr epidermal growth factor receptor (Egfr), transcript variant 1, mRNA [NM_207655]

5.00E-03 -1.11561855 Ar androgen receptor (Ar), mRNA [NM_013476]

Absent Pdgfra

2.50E-02 1.06482196 Pdgfrb platelet derived growth factor receptor, beta polypeptide (Pdgfrb), mRNA [NM_001146268]

1.87E-03 -1.05718032 Il15ra interleukin 15 receptor, alpha chain (Il15ra), transcript variant 1, mRNA [NM_008358]

6.54E-04 -2.82838612 Il17ra interleukin 17 receptor A (Il17ra), mRNA [NM_008359]

8.23E-03 -1.82475843 Il18 interleukin 18 (Il18), mRNA [NM_008360]

4.04E-02 -0.92452345 Il1r1 interleukin 1 receptor, type I (Il1r1), transcript variant 1, mRNA [NM_008362]

7.03E-03 -1.82705842 Il6st (gp130) interleukin 6 signal transducer (Il6st), mRNA [NM_010560]

4.78E-03 -1.38311088 Jak1 Janus kinase 1 (Jak1), mRNA [NM_146145]

1.85E-02 -1.12451392 Jak2 Janus kinase 2 (Jak2), transcript variant 1, mRNA [NM_008413]

3.66E-04 -1.75841306 Stat3 signal transducer and activator of transcription 3 (Stat3), transcript variant 3, mRNA [NM_011486]

3.09E-04 -2.201832 Stat5a signal transducer and activator of transcription 5A (Stat5a), transcript variant 1, mRNA [NM_011488]

5.67E-03 -1.80404438 Stat5b signal transducer and activator of transcription 5B (Stat5b), transcript variant 1, mRNA [NM_011489]

4.99E-03 -1.39478092 Stat6 signal transducer and activator of transcription 6 (Stat6), mRNA [NM_009284]

2.98E-04 2.84930079 Ccnb1 cyclin B1 (Ccnb1), mRNA [NM_172301]

1.63E-03 -2.71772068 Cd34 CD34 antigen (Cd34), transcript variant 2, mRNA [NM_133654]

3.56E-02 -1.23379025 Cxcr4 chemokine (C-X-C motif) receptor 4 (Cxcr4), mRNA [NM_009911]

2.69E-04 -2.09815709 Myf5 myogenic factor 5 (Myf5), mRNA [NM_008656]

8.49E-03 -0.86672614 Myod1 myogenic differentiation 1 (Myod1), mRNA [NM_010866]

6.02E-03 -1.25250692 Myog myogenin (Myog), mRNA [NM_031189]

2.28E-02 -0.99747603 Notch1 Notch gene homolog 1 (Drosophila) (Notch1), mRNA [NM_008714]



8.45E-04 -1.64980344 Pax7 paired box gene 7 (Pax7), mRNA [NM_011039]

6.82E-03 -1.76436267 Spry1 sprouty homolog 1 (Drosophila) (Spry1), mRNA [NM_011896]


Inhibition of JAK/STAT signaling stimulates adult ... of JAK/STAT signaling stimulates adult satellite cell function ... FACS profile of digests from Pax7-ZsGreen mice at adolescent,

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