Figure S1

Osteocyte

Chondrocyte

Adipocyte

bm-MSC

DIFFERENTIATION and VALIDATION

+/- AZA

+/- Cytokine

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Molecular characterization

3. Immunophenotyping

4. in vitro differentiation

A

B

Ost

eocy

teA

dipo

cyte

Cho

ndro

cyte

Oil Red O

Alcian Blue

Figure S2

Alizarin Red S

Enric

hmen

t sco

re (E

S)

Osteocyte vs bm-MSC

Chondrocyte vs bm-MSC

Adipocyte vs bm-MSC

p < 0.01

p < 0.01

p < 0.01

C

Osteocyte

Chondrocyte

Adipocyte

bm-MSC

DIFFERENTIATION and VALIDATION

+/- AZA

+/- Cytokine

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Molecular characterization

3. Immunophenotyping

4. in vitro differentiation

RUNX2/DAPI

SOX9/DAPI

PPARG/DAPI

Time (Days)

Cel

l Num

ber (

log1

0)

cCFU-F (PDGF-AB)cCFU-F (AZA+PDGF-AB)aCFU-F (Pdgf-AB)aCFU-F (AZA+PDGF-AB)

bm-MSCoCFU-F (PDGF-AB)oCFU-F (AZA+PDGF-AB)

Figure S3

C AdipocyteOsteocyte ChondrocyteB MC SC LC

MC LCSC

0 8 16 24 32 40 48 56 64 72 80 88 96 104

112

120

0

5

10

15

20

25

D

Osteocyte

Chondrocyte

Adipocyte

BM-MSC

DIFFERENTIATION and VALIDATION

+/- AZA

+/- Cytokine

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Immunophenotyping

3. in vitro differentiation

4. Molecular characterization

A

bm-M

SC

0

20

40

60

80

100

120

140

UT

AZA

A+P

-AB

P-A

BA

+bF bF

A+H HA

+I IA

+V V

1ry

Col

ony

/ 500

0 C

ells

UT

AZA

A+P

-AB

P-A

BA

+bF bF

A+H HA

+I IA

+V V UT

AZA

A+P

-AB

P-A

BA

+bF bF

A+H HA

+I IA

+V V

MC SC LC0

102030405060

2ry

Col

ony

%

MC SC LC0

102030405060

MC SC LC0

102030405060

MC SC LC0

102030405060

BM-MSC

+ AzaPDGF-AB

+ AzaPDGF-AB

+ AzaPDGF-AB

2ry

Col

ony

%

2ry

Col

ony

%

2ry

Col

ony

%

3X 3X 3X

Figure S4

B

bm-MSC

oCFU

-FaC

FU-F

bm-MSC bm-MSC

cCFU

-F

bm-MSC

bm-MSC

Ost

eocy

teC

hond

rocy

teA

dipo

cyte

bm-MSC

C200

100

0

-200-200

-100

-100 0 100 2001st Principal Component (30.6682%)

2nd

Prin

cipa

l Com

pone

nt (2

6.12

74%

)

bm-MSCOsteocyte

oCFU-F

Chondrocyte

cCFU-F

Adipocyte

aCFU-F

Osteocyte

Chondrocyte

Adipocyte

BM-MSC

DIFFERENTIATION and VALIDATION

+ AZA

+ PDGF-AB

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Molecular characterization

3. Immunophenotyping

4. in vitro differentiation

A

Figure S5

IgG

bm-MSC

AdipocyteOsteocyte Chondrocyte

SCA1

aCFU-FoCFU-F cCFU-F

IgG AdipocyteOsteocyte Chondrocyte

CD90.2

bm-MSC aCFU-FoCFU-F cCFU-F

IgG AdipocyteOsteocyte Chondrocyte

bm-MSC aCFU-FoCFU-F cCFU-F

CD105

CD166

bm-MSC aCFU-FoCFU-F cCFU-F

IgG AdipocyteOsteocyte Chondrocyte

0.0740.031

0.069 0.1

82.6 88.579.2 76.3

0 0.110 0

80.8 70.869.8 67.5

0 0 0.026 0.32

99.3 99.698.6 98.6

0 0 0.0150.034

98.1 97.596.7 96.5

Osteocyte

Chondrocyte

Adipocyte

BM-MSC

DIFFERENTIATION and VALIDATION

+AZA

+ PDGF-AB

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Molecular characterization

3. Immunophenotyping

4. in vitro differentiation

A

B

Figure S6

D Alizarin Red S Oil Red O Alcian Blue

Osteocyte

Chondrocyte

Adipocyte

BM-MSC

DIFFERENTIATION and VALIDATION

+ AZA

+ PDGF-AB

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

1. CFU-F activity

2. Molecular characterization

3. Immunophenotyping

4. in vitro differentiation

A

oCFU-F

cCFU-F

aCFU-F

A

B

Ost

eocy

te

D

Adi

pocy

teC

hond

rocy

te

Oil Red O

Alcian Blue

UT AZAPDGF-AB AZA+PDGF-AB

Ost

eocy

teA

dipo

cyte

Cho

ndro

cyte

C

AdipocyteOsteocyte Chondrocyte

Col

ony

/ 500

0 ce

lls

MC

LCSC

bm-M

SC UT

AZA

PDG

F-AB

AZA

+PD

GF-

AB UT

AZA

PDG

F-AB

AZA

+PD

GF-

AB UT

AZA

PDG

F-AB

AZA

+PD

GF-

AB

0

20

40

60

80

100

120

140

160Alizarin Red

Figure S7

Osteocyte

Chondrocyte

Adipocyte

BM-MSC

DIFFERENTIATION and VALIDATION

+/- AZA

+/- PDGF-AB

days20 4 6 8 10 121 3 5 97 11

CONVERSION

oCFU-F

cCFU-F

aCFU-F

In Vitro CHARACTERIZATION

CFU-F activity

Figure S8

B

OC AZA PDGF-AB AZA +PDGF-AB

02468

105060708090

100

SG0/G1

G2/M

Perc

enta

ge (%

)

Treatment

******

***

0

10

20

30

40

50

60

70

80

90

100

UT

AZA

PDG

F-AB

AZA

+

PD

GF-

AB

Treatment

Colo

ny /

5000

cel

lsMC

LCSC

C

A

0 8 16 24 32 40 48 56 64 72 80 88 96 1040

5

10

15

20 AZA

UTbmMSC

PDGF-ABPDGF-AB+AZA

Days

Cel

l Num

ber (

log1

0 )

Figure S9

MCLC SC

0 20 40 60 80 100 0 20 40 60 80 100

LC X MC

0 20 40 60 80 100

LC

LC X MC

0 20 40 60 80 100

LC

0 20 40 60 80 100

LC

0 20 40 60 80 100 X

LC X 0 2 4 6 8 10

MC

LC

0 20 40 60 80 100XLC

0 20 40 60 80 100XLC

0 20 40 60 80 100

1ry

2ry

3ry

4ry

Colony / 5000 cellsColony / 5000 cells X- No colony formation

0 20 40 60 80 100

0 20 40 60 80 100

0 20 40 60 80 100

0 20 40 60 80 100

(%)

(%)

(%)

bm-MSC oCFU-F

Figure S10

E

VE-CAD/ac-LDL/DAPIEn

doth

eliu

m

CAV1 / DAPI vWF / DAPI

Osteocytebm-MSCoCFU-F

NKX2-5 / DAPI CX43 / DAPI

Car

diom

yocy

te

MEF2C / DAPI GATA4 / DAPI

aSMA / SRF / DAPI

Smoo

th m

uscl

e

MYOCD / SRF / DAPI

PDGFRB / CD31 / DAPI

Peric

yte

/ End

othe

lium

bm-MSC

D ALBUMIN/HNF-4a/DAPI

Hep

atoc

yte

TUJ1 / DAPI

GFAP / DAPI

O4 / DAPI

Neu

ral

F

A B C

NKX

2-5

CX4

3

GAT

A4

MEF

2C

0

20

40

60

80

100

% o

f Pos

itive

Cel

ls

Osteocytebm-MSCoCFU-F

aSM

A

SRF

MYO

CD

0

20

40

60

80

100

% o

f Pos

itive

Cel

ls

Osteocytebm-MSCoCFU-F

TUJ1

GFA

P O40

10

20

30

40

50

60

PDGFRB / CD31 / DAPI

Osteocytebm-MSCoCFU-F

% o

f Pos

itive

Cel

ls

HNF4

a

ALBU

MIN

0

20

40

60

80

100

% o

f Pos

itive

Cel

ls

Osteocytebm-MSCoCFU-F

VE-CAD/eNOS/DAPI

% o

f Pos

itive

Cel

ls

VE-CAD/DAPI

VE-C

AD

eNO

S

CAV

1

vWF

0

20

40

60

80

100

Figure S11

MYH11/b2-MG-GFP/DAPI

Smoo

th m

uscl

e

Neu

rona

l

TUJ1 / b2-MG-GFP/ DAPI

Epith

eliu

mN-CAD / b2-MG-GFP/ DAPI

Gut

epi

thel

ium

b2-MG-GFP/ DAPI

CALPONIN1/b2-MG-GFP

RUNX2/b2-MG-GFP/DAPI

Car

diom

yocy

te

b-ACTININ / b2-MG-GFP/ DAPI

NESTIN / b2-MG-GFP/ DAPI

Skel

etal

mus

cle

Car

tilag

e

PPARG / b2-MG-GFP/ DAPI

Adip

ose

tissu

e

CD31 / b2-MG-GFP/ DAPI

BV

SOX9 / b2-MG-GFP/ DAPI

Bone

Bloo

d ve

ssel

Figure S12

0

10

20

30

40

50

60

70

80

90

100

110

120

130

Col

ony

/ 500

0 ce

llsMC

LCSC

oCFU-FOC

bm-M

SC OC OC

oCFU-F

oCFU-F

Rag1C56BL/6 QS-Swiss

A

**

N.S

Figure S13

Subcutaneous fat

Single CellSuspension

After 7-10Days

i

Adipocytes (d12)

C

Pdgf

ra-n

GFP

FSC-A

Day 00 0

Day 1 Day 6 Day 8 Day 10 Day 120 14 25 22 9 7.4

Ad-MSCGFP+

GFP-

Adipoc

yte

(Pdgfra

-nGFP-)

aCFU-FGFP+

GFP-

Col

ony

/ 500

0 ce

lls

MC

LCSC

D

0

10

20

30

40

50

60

70

80

90

100 E

Alizarin Red S Oil Red O Alcian Blue

Adip

ocyt

e

Ost

eocy

te

Cho

ndro

cyte

Pdgfra-nGFP

adipocytes(Pdgfra-nGFP-)

adipocytesstromal cells

ADIPOCYTES ISOLATIONA CONVERSIONB

ii +/- AZA

+/-PDGF-AB

days20 4 6 8 10 121 3 5 97 11

Ad-MSC (d12)

IN VITRO CHARACTERIZATION

1X

PDGF-AB + AZAUntreated controls (Media only)

***

HumanSubcutaneous

fat

Single CellSuspension

After 10 Days

adipocytesadipocytesstromal cells

ADIPOCYTES ISOLATIONA CONVERSIONB

+/- AZA

+/- PDGF-AB

days20 5 10 15 251

i 1X

Day 0 Day 15 Day 25

ChondrocyteOsteocyte

Adipocyte

IN VITRO CHARACTERIZATIONC D E

0 10 20 30 40 50 60 70 80 90 100

110

120

130

1400

2468

1012141618202224

Adipo MSCPDGF-AB + AZAPDGF-AB AZA Untreated

Cel

l Num

ber (

log1

0)

Days

PDGF-AB + AZA

Adip

oM

SC

Unt

reat

ed

PDG

F-AB AZ

A

PDG

F-AB

+AZA

0

10

20

30

40

Larg

e C

olon

y / 5

000

cells

Figure S14

PD

GFR

A

6.2

Figure S15

Sca1- CD31-

Dmp1eYFP

long bones Dmp1-eYFP

Osteocytes

ISOLATION

Rag1 Mice

L4

L5

L6TP

TP

CSCS

HELISTATCollagen

Sponge (CS)

A

+/-AZA

+/- PDGF-AB

days20 4 6 8 10 12

CONVERSIONB TRANSPLANTATIONC

Dmp1-eYFP/PDGFRA/DAPI Dmp1-eYFP/VIMENTIN/DAPIbm-MSC/PDGFRA/DAPI

IN VIVO CHARACTERIZATION

Dmp1-eYFP/PDGFRA/DAPI

DTreated OsteocytesUntreated Osteocytes BM-MSC

12 Weeks 12 Weeks 12 Weeks

b2-MG-GFP / VIMENTIN / DAPIA

Figure S16

Unt

reat

ed

AZA

+ PD

GF-

AB

Skel

etal

mus

cle

b2-MG-GFP / PDGFRA / DAPI

b2-MG-GFP / aSMA / DAPI

ii

iii

Bloo

d ve

ssel

Merged

b2-MG-GFP/DAPIVE-CAD

Bone

b2-MG-GFP / RUNX2 / DAPI

b2-MG-GFP/DESMIN/DAPI b2-MG-GFP / DAPIM-CADHERIN

b2-MG-GFP / CD166 / DAPI

i

iii

iv

v

Unt

reat

edU

ntre

ated

Unt

reat

ed

AZA

+ PD

GF-

ABAZ

A +

PDG

F-AB

AZA

+ PD

GF-

AB

b2-MG-GFP/CD146/DAPI

Bloo

d ve

ssel

Ost

eocl

ast

b2-MG-GFP/CATHEPSIN K/DAPI

B12 Weeks

Figure S17

A

R+SR+S

+

Osteoc

yte0

20

40

60

80

100

R+S+

bm-M

SC R+S+

oCFU-F

Day 3Day 5

CD

4 T-

Cel

l Pro

lifer

atio

n (%

)

N.SN.S

**** ****

***

****

N.S

0

20

40

60

80

100

CD

8 T-

Cel

l Pro

lifer

atio

n (%

)

R+SR+S

+

Osteoc

yte R+S+

bm-M

SC R+S+

oCFU-F

Day 3Day 5

N.S

N.S

**** ****

***

****

N.S

*

*

i iiG

ranz

yme

B

Cell Trace Violet

R only R + S R + S + bm-MSCR + S + Osteocytes R + S + oCFU-FB

A

bm-M

SC 0 50 1000

10

20

30

40

50

60

70

80

90

PDGF-BB (ng/ml)+ 10μM AZA

MC

LCSC

Col

ony

/ 500

0 ce

lls

bm-M

SC 0 50 1000

10

20

30

40

50

60

70

80

90

PDGF-AA (ng/ml)+ 10μM AZA

MC

LCSC

Col

ony

/ 500

0 ce

lls

Figure S18

i ii B CFU-FPA+P oCFU-F

Time (Days)

Cell

Num

ber (

log1

0)

0 8 16 24 32 40 48 56 64 72 800

5

10

15

20

25bm-MSCPDGF-AAAZA + PDGF-AA

Time (Days)

Cell

Num

ber (

log1

0)

oCFU-F

0 8 16 24 32 40 48 56 64 72 80 88 96 104

112

120

0

5

10

15

20

25

Figure S19

oCFU-F vs OsteocyteA

Figure S20

CD

51

PDGFRA

99

1

94

1

4

1

93 2.5

3 1.5

89 6

3 2

0

0

Untreated 48h AZA 48h PDGF-AB 48h AZA+PDGF-AB

CD

51

Pdgfrb

A943.4

0.4 2

9.4 88

0.4 2.1

23 52

1.8 2.7

57 38

2 3.3

Untreated 48h AZA 48h Pdgf-AB 48h AZA+Pdgf-AB

B

Ost

eocy

teAZ

A+PD

GF-

ABO

steo

cyte

263 bp

Klf4

CpG island

Sox2

CpG island21270 bp 227 bp

Ost

eocy

te

Methylated CpG

Unmethylated CpGTSS

AZA+

PDG

F-AB

Ost

eocy

tePou5f1(Oct4)

278 bp

Ost

eocy

teMyc

CpG island21373 bp 229 bp

Ost

eocy

te

A B

C

D

AZA+

PDG

F-AB

Ost

eocy

teAZ

A+PD

GF-

ABO

steo

cyte

Figure S212% 1%

OCT4/NANOG/DAPINANOG/DAPIOCT4/DAPI

ES C

ells

Trea

ted

Ost

eocy

te

E

Table S1

In vitrocharacteristics

In vivocharacteristics

1. Micro, small and large colony forming ability: Yes Yes

2. Long-term self renewal: Yes Yes

3. MSC-like immunophenotype: Yes Yes

4. MSC-like molecular signature: Yes No

5. Trans-germ layer multipotency: Yes Yes

6. Immune modulation Yes Yes

MSC iMS cells

1. Tumorigenesis: No No

2. In vivo plasticity (ES cell co-culture transplant under the kidney capsule): No Yes

3. Contribution to tissue regeneration and repair: No Yes

1

SUPPLEMENTAL FIGURE LEGENDS 1

FIGURE S1: Overview of experiments (Fig S1-S6) used to screen cytokines. Step 1: 2

Differentiation and validation; bmMSCs were differentiated into osteocytes, chondrocytes 3

and adipocytes and their cell identities verified. Step 2: Conversion; each cell type was then 4

cultured with various cytokines with or without AZA. Step 3: in vitro characterization; 5

Characteristics of treated cells were evaluated to establish their proliferative capacity, 6

variation in transcriptomics, cell surface phenotypes and multipotency. 7

8

FIGURE S2: (A) Schematic outline of Step 1: Differentiation and validation; bmMSCs were 9

differentiated into bone, cartilage and fat cells and their cell identities verified by (B) 10

histology; histochemistry and immunohistochemistry for lineage determining transcription 11

factors and (C) transcriptomics; GSEA plots showing enrichment of genes associated with 12

target cell types. 13

14

FIGURE S3: (A) Schematic outline of cell conversion and characterisation of CFU-F 15

activity. Following conversion, cells are passaged in MSC medium alone. (B) Images of 16

crystal violet stained CFU-Fs scored as micro (MC- 5-24 cells; < 2mM), small (SC- ≥ 25 17

cells; 2-4mM) or large (LC- >4mM) colonies as described in Chong et al Cell Stem Cell 18

2011. (C) Yield of primary (1ry) CFU-Fs (number based on size) for each cytokine (+/- 19

AZA). The insets show secondary (2ry) colony forming potential of re-plated single cells 20

from micro, small and large colonies generated from bmMSCs or PDGF-AB/AZA treated 21

osteocytes (oCFU-F), chondrocytes (cCFU-F) or adipocytes (aCFU-F). (D) Growth of oCFU-22

F, cCFU-F and aCFU-Fs derived from osteocytes, chondrocytes and adipocytes respectively 23

and treated with PDGF-AB alone and in combination with AZA. bm-MSC; bone marrow 24

derived mesenchymal stem cells, CFU-F; colony forming unit-fibroblast, oCFU-F; CFU-Fs 25

2

generated from osteocytes treated with cytokine/AZA, cCFU-F; CFU-Fs generated from 1

chondrocytes treated with cytokine/AZA, aCFU-F; CFU-Fs generated from adipocytes 2

treated with cytokine/AZA, AZA; 5-azacytidine, bFGF; basic fibroblast growth factor, HGF; 3

hepatocyte growth factor, IGF1; Insulin like growth factor 1, VEGF; vascular endothelial 4

growth factor, UT; untreated. Standard Deviation bars = SD between independent 5

experiments. 6

7

FIGURE S4: (A) Schematic outline of cell conversion and characterisation of transcriptomic 8

variation. (B) Global gene expression profiles of in vitro differentiated osteocytes, 9

chondrocytes and adipocytes maintained for 12 days in culture with and without PDGF-AB 10

and AZA are shown compared with control primary bmMSCs, which were also maintained in 11

culture for 12 days. Selected lineage specific genes are marked with arrows. (C) Principal 12

component analysis showing the spatial relationship between individual transcriptomes of in 13

vitro differentiated osteocytes, chondrocytes and adipocytes maintained for 12 days in culture 14

with and without PDGF-AB and AZA are shown compared with control primary bm-MSCs, 15

which were also maintained in culture for 12 days. bm-MSC; bone marrow derived 16

mesenchymal stem cells, CFU-F; colony forming unit-fibroblast, oCFU-F; CFU-Fs generated 17

from osteocytes treated with PDGF-AB/AZA, cCFU-F; CFU-Fs generated from 18

chondrocytes treated with PDGF-AB/AZA, aCFU-F; CFU-Fs generated from adipocytes 19

treated with PDGF-AB/AZA, AZA; 5-azacitidine. Standard Deviation bars = SD between 20

independent experiments. 21

22

FIGURE S5: (A) Schematic outline of cell conversion and characterisation of surface 23

immunophenotypes. (B) Flow cytometry profiles of in vitro generated osteocytes, 24

chondrocytes and adipocytes maintained in MSC medium alone or medium supplemented 25

3

with PDGF-AB and AZA. The MSC markers, SCA-1, CD90.2, CD105 or CD166 are 1

expressed on oCFU-F, cCFU-F and aCFU-Fs at levels and frequencies comparable with that 2

expressed on freshly isolated bm-MSCs. bm-MSC; bone marrow derived mesenchymal stem 3

cells, CFU-F; colony forming unit-fibroblast, oCFU-F; CFU-Fs generated from osteocytes 4

treated with PDGF-AB/AZA, cCFU-F; CFU-Fs generated from chondrocytes treated with 5

PDGF-AB/AZA, aCFU-F; CFU-Fs generated from adipocytes treated with PDGF-AB/AZA. 6

7

FIGURE S6: (A) Schematic outline of cell conversion and assessment of their tri-lineage 8

differentiation potential. (B) PDGF-AB/AZA treated bone (oCFU-F), cartilage (cCFU-F) and 9

fat (aCFU-F) cells show in vitro tri-lineage differentiation potential. 10

11

FIGURE S7: Independent verification of cell conversion (Y.Y and W.R.W) (A) Schematic 12

outline as in Fig S1. (B) Step 1; validation of in vitro generated osteocytes (alizarin red), 13

chondrocytes (alcian blue) and adipocytes (oil red O) from bm-MSCs. (C) Crystal violet 14

stained CFU-F colonies generated by treating in vitro generated osteocytes, chondrocytes and 15

adipocytes with PDGF-AB/AZA or PDGF-AB alone or AZA alone or MSC medium alone. 16

(D) Colony number based on size. MC; micro colony, SC; small colony, LC; large colony. 17

18

FIGURE S8: (A) Quantification of oCFU-F colonies (based on size) from osteocytes 19

cultured in media alone, media + AZA, media + PDGF-AB and media + PDGF-AB+AZA. 20

(B) Long-term growth curves of osteocytes in media alone (UT), bm-MSCs, osteocytes in 21

control media (media + AZA, media + PDGF-AB) and osteocytes in media + PDGF-22

AB+AZA. (C) Cell cycle analysis of osteocytes (OC) at day 12 cultured with no additives, 23

with AZA alone, with Pdgf-AB alone and with both AZA and PDGF-AB. ***; P< 0.001. 24

25

4

FIGURE S9: Clonogenic trees of serial replating of single micro and large colonies from 1

bmMSCs (left) and oCFU-Fs (right), showing type and number of colonies that are generated 2

from each. Following conversion oCFU-F cells are passaged in MSC medium alone. MC; 3

micro-colony, SC; small colony, LC; large colony, bm-MSCs; bone marrow derived 4

mesenchymal stem cells, oCFU-F; CFU-Fs generated from osteocytes treated with PDGF-5

AB/AZA. 6

7

FIGURE S10: Confocal images of oCFU-Fs generated from PDGF-AB/AZA treated 8

primary osteocytes (harvested from Pdgfrα-H2B: GFP mice) and differentiated into 9

mesodermal, ectodermal and endodermal derivatives. Histograms to the right of each panel of 10

images show the comparative efficiency of differentiation achieved with untreated 11

osteocytes, control bmMSCs and PDGF-AB/AZA treated osteocytes (oCFU-Fs). (A) 12

Endothelial cells (VE-Cadherin (VE-CAD), endothelial nitric oxide synthase (eNOS) 13

acetylated low-density lipoprotein (ac-LDL) uptake, Caviolin 1(CAV1) and von Willebrand 14

factor (vWF)). (B) Matrigel tube formation using bmMSCs (upper panel; endothelial cells 15

only) and oCFU-Fs (lower panel; endothelial cells and pericytes). (C) Cardiomyocytes 16

(NKX2.5, CONNEXIN-43 (CX43), Myocyte-specific enhancer factor 2C (MEF2C) and 17

GATA4). (D) Smooth muscle (α Smooth muscle actin (aSMA), Serum response factor 18

(SRF), Calponin, Myosin 11 (MYH11), and Myocardin (MYOCD)). (E) Neuro-ectoderm, 19

neurons (neuron specific class III β-tubulin (TUJ1)), astrocytes (glial fibrillary acidic protein 20

(GFAP)) and Oligodendrocytes (O4). (F) Endodermal derivative; hepatocytes (ALBUMIN 21

and hepatocyte nuclear factor 4 alpha (HNF-4a)). Standard Deviation bars = SD between 22

independent experiments. Scale bar = 20µm. 23

24

5

FIGURE S11: oCFU-Fs generated from PDGF-AB/AZA treated primary osteocytes (derived 1

from ubiquitous GFP (β2-microglobulin-GFP) mice) were mixed 3:1 with mES cells and 2

implanted under the kidney capsules of Rag1 deficient mice. Tissue sections from teratomas 3

harvested at six weeks and stained with anti-GFP-biotin/Streptavidin-HRP or anti-GFP/Alexa 4

Fluor 488 with lineage specific markers (N-CADHERIN; epithelium, TUJ1; neuronal, 5

MYH11 and CALPININ; smooth muscle, NESTIN; regenerating skeletal muscle, CD31; 6

endothelium, b-ACTININ; cardiomyocytes, RUNX2; Bone, SOX9; Cartilage and PPARG; 7

adipocytes. Contributions from reprogrammed cells stain either brown (anti-GFP/HRP) or 8

fluorescent green (β2-MG-GFP). CFU-F; colony forming unit-fibroblast, oCFU-F; PDGF-9

AB/AZA treated osteocytes cultured in MSC medium, DAPI; 4', 6-Diamidino-2-10

Phenylindole, Dihydrochloride nuclear stain, β2-MG-GFP; β2 microglobulin-GFP. Scale bar 11

= 20µm. 12

13

FIGURE S12: Comparison of CFU-Fs generated from primary osteocytes harvested from 14

long bones of C57/BL6, Q(S) and Rag1 mice of comparable sex and age. 15

16

FIGURE S13: (A) Schematic outline of steps followed to reprogram subcutaneous 17

unilocular mature adipocytes harvested from Pdgfra-nGFP mice. The insets show Oil red O 18

staining of unilocular mature adipocytes (i; low and ii; high magnification) attached to a petri 19

dish. (B) Schematic outline of treatment and time points at which cells were harvested for 20

flowcytometry. (C) Flow cytometry analysis of freshly isolated Pdgfra-nGFP- adipocytes 21

showing progressive acquisition Pdgfra-nGFP (stromal marker). Control untreated 22

adipocytes and adipocyte derived MSCs maintained in MSC medium without PDGF-23

AB/AZA are shown at the extreme right. (D) CFU-Fs derived from sorted Ad-MSCs, 24

adipocytes and aCFU-Fs scored based on colony size. (E) aCFU-Fs can be differentiated into 25

6

osteocytes (alizarin Red), adipocytes (oil Red O) and chondrocytes (alcian blue). Ad-MSCs; 1

adipocyte derived MSCs, aCFU-F; CFU-Fs generated from PDGF-AB/AZA treated 2

adipocytes. Standard Deviation bars = SD between independent experiments. 3

4

FIGURE S14: (A) Schematic outline of steps followed to harvest and isolate human 5

subcutaneous mature adipocytes. The insets show Oil red O staining of unilocular mature 6

adipocytes attached to a petri dish. (B) Schematic outline of treatments at selected time points 7

used to evaluate the morphology adipocytes in reprogramming media (PDGF-AB+AZA). (C) 8

Large colony forming units-fibroblast activity of adipocyte-MSCs, untreated and AZA or 9

PDGF-AB or PDGF-AB+AZA treated adipocytes. (D) Growth curves of adipocyte-MSCs, 10

untreated and AZA or PDGF-AB or PDGF-AB+AZA treated adipocytes that were harvested 11

at P0 and perpetuated in culture. (E) PDGF-AB+AZA treated adipocytes can be differentiated 12

into osteocytes (alizarin Red), adipocytes (oil Red O) and chondrocytes (alcian blue). Ad-13

MSCs; adipocyte derived MSCs. Standard Deviation bars = SD between independent 14

experiments. Scale bar = 30µm. 15

16

FIGURE S15: (A) Isolation of osteocytes from Dmp1-eYFP mice. The inset shows a bright 17

field image of isolated osteocytes in culture. (B) Schematic outline of the treatment applied to 18

osteocytes harvested from (A). (C) Schematic representation of the posterior-lateral inter-19

transverse lumber fusion model used for the implantation of Helistat collagen sponges 20

containing PDGF-AB/AZA treated Dmp1-eYFP+ osteocytes. (D) Confocal images of grafts 21

harvested at 12 weeks show activation of stromal markers (PDGFRA and VIMENTIN) in 22

Dmp1-eYFP cells that were treated with PDGF-AB/AZA. Grafts with untreated Dmp1-eYFP 23

osteocytes or control bmMSCs harvested from these mice show no activation at 12 weeks. 24

7

Scale bar = 20µm. CS; collagen sponge, TP; transverse process, bm-MSC; MSCs harvested 1

from bone marrow. 2

3

FIGURE S16: Confocal images of untreated and PDGF-AB/AZA treated bone fragments 4

harvested at 12 weeks. (A) GFP+ cells are abundant at the surface of treated fragments and 5

co-express stromal markers (VIMENTIN, aSMA, PDGFRA, CD166). (B) Confocal images 6

of tissues surrounding treated bone fragments showing donor cell contribution to bone cells 7

(RUNX2 and BMP2), skeletal muscle (DESMIN and M-CADHERIN) and blood vessel 8

(CD146 and VE-CADHERIN) and osteoclasts (CATHEPSIN-K). Standard Deviation bars = 9

SD between independent experiments. Scale bar = 20µm. 10

11

FIGURE S17: (A) Mixed lymphocyte reaction assays showing in vitro immune suppression 12

of (i) CD4 and (ii) CD8 T-cell alloreactivity. (B) Granzyme B production in cytotoxic T 13

lymphocytes in the presence of osteocytes, bmMSCs, and oCFU-Fs. bmMSCs; primary 14

MSCs from bone marrow, oCFU-F; CFU-Fs generated by treating primary osteocytes with 15

PDGF-AB/AZA, R; responder T cells, S; stimulator splenocytes. Standard Deviation bars = 16

SD between independent experiments. 17

18

FIGURE S18: CFU-F colony type and number from (i) control bone marrow MSCs (ii) 19

osteocytes cultured with AZA (2 days) + PDGF-AA (12 days) and (iii) and osteocytes 20

cultured with AZA (2 days) + PDGF-BB (12 days). (B) Growth curves of oCFU-Fs generated 21

from osteocytes exposed to PDGF-AA+/-AZA. Without AZA, PDGF-AA generates few 22

colonies that fail to propagate. With serial replating, AZA + PDGF-AA treated oCFU-Fs lose 23

their proliferative vigour. 24

25

8

FIGURE S19: Ingenuity pathway analysis of gene expression profiles of PDGF-AB/AZA 1

treated osteocytes and untreated osteocytes focussing on changes relevant to mediators of the 2

PDGF receptor signaling pathway. Red; up-regulated and Green; down-regulated. 3

4

FIGURE S20: Surface expression of (A) PDGFRB and (B) PDGFRA on primary osteocytes 5

cultured for 48 hrs in media alone or supplemented with AZA and/or PDGF-AB. 6

7

FIGURE S21: Allelic bisulfite sequencing to show CpG methylation in primary osteocytes 8

before and after PDGF-AB/AZA treatment at the promoters of (A) Oct4, (B) cMyc, (C) Klf4 9

and (D) Sox2 pluripotency genes. (E) OCT4 and NANOG expression in mouse embryonic 10

stem cell and PDGF-AB+AZA treated osteocytes. Scale bar = 20µm. 11

12

TABLE S1: Core features of MSCs and iMS cells. 13

14

Movie S1: Live cell imaging of SCA1-/CD31-/PDGFRA-GFP-/CD51+ osteocytes harvested 15

from Pdgfra-nGFP mice. The cells were treated with PDGF-AB (8 days)/AZA (2 days) and 16

imaged over 8 days. GFP is expressed when PDGFRA- osteocytes transform into PDGFRA+ 17

(MSC cell marker) cells. 18

19

Movie S2: Beating cardiomyocytes generated from primary osteocytes that were first 20

converted into iMS cells using PDGF-AB/AZA and then differentiated into cardiomyocytes 21

as detailed in methods. 22

23

Movie S3: Endothelial tubes formed in cytokine-enriched matrigel (see methods) from 24

PDGF-AB/AZA treated primary osteocytes. Z- stack 3D reconstruction of tube-like 25

9

structures stained with anti-CD31 (endothelial cells; green) and anti-PDGFRB (pericytes; 1

red). 2

3

Movie S4: Live cell imaging of primary mature adipocytes harvested from Pdgfra-nGFP 4

mice. The cells were treated with PDGF-AB (8 days)/AZA (2 days) and imaged over 8 days. 5

GFP is expressed when Pdgfra-ve adipocytes transform into PDGFRA+ (MSC cell marker) 6

cells. 7

8

9

10

SUPPLEMENTAL MATERIALS 1

Table S2: Mouse Strains 2

Common

Name

International

nomenclature

Knock-In

(KI),Knock-

Out (KO)

or

Transgenic

(Tg)

Developmental

Lineage

Genetic

Background

Reference

C57

Black

C57BL/6J Wild type N/A C57BL/6J

Pdgfra-

nGFP

Pdgfratm11(EGFP)Sor KI N/A C57BL/6J Hamilton

et al., 2003

DMP1-

Cre

R26eYFP

Tg(Dmp1-

cre)1Jqfe GtROSA)26Sor

Tg Osteocyte C57BL/6J Stern et al.,

2012

Rag1-/- Rag1tm1Mom KO N/A FVB/NJ Mombaerts

et al., 1992

GreenTg C57BL/6Tg(UBC-

GFP)30Scha/J

Tg N/A C57BL/6J Schaefer et

al., 2001

3

11

Table S3: PCR primers for the RT-PCR, DNA methylation and NOMe-Seq assays. 1

Assay Gene Sequence (5’-3’) DNA

Strand

RT-PCR

Oct4

(NM_013633.3)

TCTTTCCACCAGGCCCCCG

GCTC

Forward

TGCGGGCGGACATGGGGA

GATCC

Reverse

Nanog

(NM_028016.2)

AGGACAGGTTTCAGAAGCA

GA

Forward

CCATTGCTAGTCTTCAACC

ACTG

Reverse

Sox2

(NM_011443.3)

TAGAGCTAGACTCCGGGCG

ATGA

Forward

TTGCCTTAAACAAGACCAC

GAAA

Reverse

Klf4

(NM_010637.3)

GCGAACTCACACAGGCGA

GAAACC

Forward

TCGCTTCCTCTTCCTCCGAC

ACA

Reverse

cMyc

(NM_001177353.1)

TGACCTAACTCGAGGAGGA

GCTGGAATC

Forward

AAGTTTGAGGCAGTTAAAA

TTATGGCTGAAGC

Reverse

Rex1 ACGAGTGGCAGTTTCTTCT Forward

12

(NM_009556.3) TGGGA

TATGACTCACTTCCAGGGG

GCACT

Reverse

βActin

(NM_007393.3)

CCTAAGGCCAACCGTGAAA

AG

Forward

TCTTCATGGTGCTAGGAGC

CA

Reverse

NOMe-Seq Runx2

(NM_001145920)

TTTTGYTTTTTAGAGGYTTA

ATTTTATAGGAG

Forward

TATTCCTRCATAAACTATA

ATTAAARCACTCACTA

Reverse

CTAAAAAAAATTTRCACCR

CACTTATAATTCTA

Reverse

Nested

Bisulfite

sequencing

Pou5f1 (Oct4)

(NM_013633)

GAYGTTTTTAATTTTYGTTT

GGAAGATATAG

Forward

YAACATAAAAAAATCCCCA

ATACCTCTA

Reverse

Nanog

(NM_028016)

GTTTATGGTGGATTTTGYA

GGTGGGATTAAT

Forward

TCTTCRAAAACTAAATTCC

TTACCARCCTCTA

Reverse

CTTACCARCCTCTATRCAA

ARCATCTCAA

Reverse

Nested

Bisulfite Klf4 (NM_010637) YGYGGAGTTTGTTTATTTA Forward

13

1

2

sequencing GTTATTATGGT

CRCRAAATACRAAATCCTA

AAAACTATAC

Reverse

Sox2 (Region 1)

(NM_011443)

GYGTTTTATTTATTTTTATG

TATTTAAGAGAGAGT

Forward

AATAAACAACCATCCATAT

AATAAAAACTATCAA

Reverse

Sox2 (Region 2)

(NM_011443)

AGAAGTTTGGAGTTYGAGG

TTTAAGT

Forward

TTCAACTCCRTCTCCATCAT

ATTATACATA

Reverse

Myc (Region 1)

(NM_010849

GAATAATYGTATAGAAAG

GGAAAGGATTAG

Forward

CRAAAAACTTCTTTTATAC

TACRACTCAA

Reverse

Myc (Region 2)

(NM_010849)

TTGGAAGAGTYGTGTGTGT

AGAGT

Forward

CAACTCRAAAAACTCTTTT

CAAAAAAACTAATC

Reverse

14

Table S4: Identification of genes differently expressed during bmCFU-F differentiation 1

into osteocytes, chondrocytes and adipocytes. 2

Osteocyte

Gene Name Fold Change CORE ENRICHMENT

NPY 5.8 Yes

BMP8A 4.5 Yes

SPARCL1 2.1 Yes

COL22A1 1.8 Yes

TBX3 1.8 Yes

ANGPT2 1.7 Yes

BMP4 1.6 Yes

BMP3 1.5 Yes

DKK1 1.3 Yes

COL4A1 1.3 Yes

FGF1 1.3 Yes

RB1 1.2 Yes

COL4A2 1.2 Yes

BMP2 1.2 Yes

GAL 1.2 Yes

MSC 1.2 Yes

COL15A1 1.1 Yes

NOTCH3 1.1 Yes

COL18A1 1.1 Yes

RELN 1.1 Yes

15

MEPE 1.1 No

CAV1 1.0 No

TINAGL 1.0 No

FGF23 -1.0 No

NRARP -1.1 No

RUNX2 -1.1 No

ARSJ -1.1 No

DMP1 -1.1 No

LAMA5 -1.1 No

SOST -1.2 No

SOX11 -1.3 No

MYCL1 -1.4 No

DLK1 -1.4 No

PHEX -1.4 No

1

Chondrocyte

Gene Name Fold Change CORE ENRICHMENT

IBSP 56.2 Yes

COMP 16.8 Yes

BMP8A 10.4 Yes

CD48 6 Yes

MMP13 5.9 Yes

COL6A3 5.5 Yes

IGFBP6 4.7 Yes

16

CILP2 4 Yes

COL2A1 3.5 Yes

DCN 3.3 Yes

HAPLN1 3.2 Yes

CXCL16 2.6 Yes

TRPV4 2.6 Yes

FGF21 2.3 Yes

EMP2 2 Yes

SOX11 1.9 Yes

LEPRE1 1.9 Yes

FOXA3 1.4 No

CDKN1A 1.4 No

SOX9 1.3 No

KLF4 1.1 No

AVIL 1.1 No

FGFR4 -1.1 No

CUGBP1 -1.1 No

GDF5 -1.4 No

COL6A1 -1.5 No

CYR61 -1.9 No

CXCL15 -2.3 No

KLF2 -2.4 No

ESM1 -3.2 No

1

17

Adipocyte

Gene Name Fold Change CORE ENRICHMENT

C3 405.3 Yes

FABP5 50.7 Yes

ACSL1 39.3 Yes

LIPE 22.8 Yes

AGT 17.6 Yes

ABHD5 13.4 Yes

SCP2 11.9 Yes

SLC1A5 11.3 Yes

CRAT 11.3 Yes

CEBPA 10.4 Yes

POR 9.3 Yes

MGST3 9.3 Yes

CBR3 9.2 Yes

BRP44 8.8 Yes

LPIN1 7.8 Yes

PIM3 6.8 Yes

SLC5A6 6.7 Yes

PEX11A 6.3 Yes

FABP4 5.6 Yes

CYCS 5.1 Yes

HIST1H2BC 5 Yes

PPA1 5 Yes

18

ORMDL3 4.9 Yes

MKNK2 4.8 Yes

TALDO1 4.6 Yes

CPT2 4.5 Yes

TOB1 4.4 Yes

GNPAT 4.3 Yes

DNAJB9 4.2 Yes

SORBS1 4.2 Yes

RREB1 4 Yes

CISD1 4 Yes

ACO2 3.9 Yes

TYSND1 3.8 Yes

IFNGR1 3.7 Yes

TMEM97 3.6 Yes

NRIP1 3.6 Yes

DBI 3.5 Yes

AK2 3.4 Yes

PPARG 3.3 Yes

GPHN 3.2 Yes

BCL2L13 3.1 Yes

COQ9 3 Yes

ALDOA 3 Yes

PTGES2 2.8 Yes

PRDX3 2.7 Yes

19

CYC1 2.7 Yes

MAPK6 2.7 Yes

SDHD 2.7 Yes

SOD2 2.7 Yes

NDUFB10 2.6 Yes

MRPL12 2.5 Yes

IVD 2.4 Yes

MRPS34 2.2 Yes

PC 2.2 Yes

JAGN1 2.2 Yes

GRPEL1 2.2 Yes

PPIF 2.1 Yes

MDH2 2 Yes

MRPL20 1.9 Yes

ECHS1 1.9 Yes

MRPL34 1.8 Yes

TIMM9 1.8 Yes

MRPS2 1.8 Yes

LRRC59 1.8 Yes

NDUFAB1 1.8 Yes

COX6A1 1.7 Yes

ZNRF2 1.7 Yes

BCAP31 1.7 Yes

NDUFA8 1.7 Yes

20

RASD1 1.6 Yes

AIFM1 1.5 Yes

ATL2 1.5 Yes

COX17 1.4 Yes

MRPS26 1.4 Yes

TIMM23 1.4 Yes

ETFB 1.4 Yes

SLMO2 1.4 Yes

CLOCK 1.4 Yes

TIMM17A 1.4 Yes

MRPL15 1.4 Yes

PHB2 1.3 Yes

ESRRA 1.3 Yes

TOMM40 1.3 No

GYS1 1.3 No

PSMA1 1.2 No

PTCD3 1.2 No

ADAM12 1.2 No

ACADM 1.2 No

LMAN2 1.2 No

TBL2 1.1 No

PSMA5 1 No

PHB -1 No

SIPA1 -1.1 No

21

EXTL3 -1.1 No

PRPS1 -1.3 No

PDIA6 -1.3 No

MTX2 -1.4 No

FDX1 -1.6 No

PTRF -2.4 No

IGF2 -9.1 No

1

22

Table S5: Antibodies and Inhibitors 1

Antibody or Inhibitor Detection or Assay Manufacturer

Sca1-PECy7 FACS Biolegend

Pdgfra-APC FACS Biolegend

CD31-PE FACS Becton and Dickinson

CD31-APC FACS Biolegend

CD51-PE FACS Biolegend

CD45-eFluor FACS eBioscience

CD45-FITC FACS Becton and Dickinson

Mouse Lineage Panel FACS BD Pharmigen

Anti-GFP-Alex-488 IF Invitrogen

Anti-GFP-Alex-647 IF Invitrogen

αSarcomaric actin-Cy3 IF Sigme

αSarcomaric actinin IF Sigma

Connexin 43 IF Becton and Dickinson

Nkx2-5 IF Santa Cruz Biotechnology

Gata4 IF Santa Cruz Biotechnology

Mef2c IF Santa Cruz Biotechnology

CD31 IF Becton and Dickinson

VE-Cadherin IF Santa Cruz Biotechnology

eNOS IF Abcam

vWF IF Abcam

Caveolin 1 IF Becton and Dickinson

AcLDL-Alexa Fluor 488 IF Molecular probes

23

Serum response factor IF Becton and Dickinson

Myh11 IF Santa Cruz Biotechnology

Calponin IF Abcam

Myocd IF Santa Cruz Biotechnology

Pdgfrb IF Biolegend

Pdgfra IF Biolegend

CD166 IF eBioscience

Albumin IF Santa Cruz Biotechnology

Hnf4α IF Santa Cruz Biotechnology

Tuj1 IF Santa Cruz Biotechnology

Gfap IF Millipore

Oligodendrocyte O4 IF Millipore

Anti-GFP-biotin IH/IF Invitrogen

Anti-GFP IF/WB Invitrogen

HRP IH Invitrogen

Nestin IF Millipore

Bmp2 IF Santa Cruz Biotechnology

Cathepsin K IF Santa Cruz Biotechnology

Runx2 IF Santa Cruz Biotechnology

Sox9 IF Millipore

CD146 IF Biolegend

Desmin IF Abcam

M-Cadherin IF Santa Cruz Biotechnology

Pparγ IF Abcam

24

Stat3 WB Santa Cruz Biotechnology

p-Stat3 WB Millipore

p-Jnk WB Millipore

cJun WB Abcam

p-cJun WB Millipore

βActin WB Santa Cruz Biotechnology

Pdgfra (APA5) IF/Inhibitor Becton and Dickinson

AG1296 Inhibitor Calbiochem

LY294002 Inhibitor Calbiochem

Wortmannin Inhibitor Calbiochem

Jak Inhibitor I Inhibitor Calbiochem

Jak2 Inhibitor II Inhibitor Calbiochem

Jnk Inhibitor I (L) Form Inhibitor Calbiochem

Stat3 Inhibitor V Stattic Inhibitor Calbiochem

Stat3 Inhibitor Peptide Inhibitor Calbiochem

1

2

25

SUPPLEMENTAL METHODS 1

Mice 2

Mouse strains are listed in Table S2. Mice were housed in the Biological Resource Centre at 3

the Lowy Cancer Research Centre, University of New South Wales. All experiments 4

involving mice were approved by the University of New South Wales animal ethics 5

committee. 6

7

BM-MSC isolation and ex vivo expansion 8

BM-MSCs were isolated from wild-type C57BL/6, Pdgfra-nGFP or Dmp1-eYFP mice. The 9

mice were sacrificed at 8-16 weeks of age. Tibias and femurs were removed, cleaned of 10

excess soft tissues and transferred to ice cold PBS (Invitrogen, Carlsbad, CA). Bone marrow 11

was flushed out with 2% fetal calf serum (FCS) in PBS and bones were thoroughly crushed 12

using a mortar and pestle. Bone fragments were transferred into collagenase Type II (263 13

U/ml; Worthington Biosciences, Lakewood, NJ) and placed on a shaker at 37˚C for 20 min. 14

The supernatant was passed through a 40 µm filter into a fresh tube and inactivated with 15

100% FCS. Cells were washed twice in 2% FCS in PBS and plated in αMEM (Invitrogen, 16

Carlsbad, CA) with 20% FCS, and penicillin/ streptomycin/ glutamine (P/S/G, Invitrogen)(1) 17

and cultured in the incubator at 37˚C, 5% CO2 for 72 h. At the end of 72 h cells were washed 18

in PBS to remove non-adherent cells and cultures were continued in fresh medium. Cells 19

were passaged on reaching 80% confluence. After passaging cells, they were placed back in 20

tissue culture flasks (T75) with αMEM + 20% FCS + P/S/G for bulk passaging. Cells were 21

routinely cryopreserved in 10% DMSO and 90% culture medium. 22

23

26

Gene expression and epigenetic analyses 1

All primers are listed in Table S3. Total RNA (>200 nt) was prepared from cells using 2

Qiagen's RNeasy mini kit (Qiagen, Germany) as per manufacturer's instructions. High-quality 3

RNA (average RIN > 9) was profiled using Illumina's Mouse WG-6 v2.0 BeadArray. 4

Illumina BeadStudio was used to extract raw data as well as background subtracted and 5

quantile normalized expression levels. Array data has been deposited in the Gene Expression 6

Omnibus under accession GSE59282. The Combat algorithm (Johnson et al, 2007) and 7

Partek Genomics Suite (v 6.6) were used for downstream processing, including batch effect 8

removal, identification of differentially expressed genes (FDR <=0.01) and hierarchical 9

clustering (average linkage method with data shifted to mean zero and standard deviation 10

one). Principal component analysis was performed using Matlab (v 2012b), as described 11

previously(2), and correlation plots were created in R (v. 3.0.0) using the ggplot2 12

(H.Wickham. ggplot2: elegant graphics for data analysis. Springer New York, 2009) package. 13

Pathway analyses of differentially expressed genes was performed using IPA 14

(Ingenuity® Systems, www.ingenuity.com) and gene set enrichment analysis (GSEA) was 15

performed using the standalone application of GSEA and ran against gene sets available from 16

the c1 and c2 annotations of the MSig database (3). Gene expression was integrated with 17

publicly available datasets (See Table S4), measured on the same array platform (Illumina's 18

Mouse WG-6 v2.0 BeadArray), available form GEO and ArrayExpress under the following 19

accessions, E-GEOD-21516 (ESC, EpiSC), GSE31738 (MSC) and E-GEOD-36484 (NSC, 20

ESC). In brief, raw expression files were processed and after merging all arrays 18,436 21

probes were common to all experiments. The resulting dataset was quantile normalized, batch 22

effects were removed and expression values scaled to the same mean and standard deviation 23

across samples. PCA was performed on whole array data as described previously (2) and the 24

first two principal components (PCs) were plotted. 25

27

1

Primary osteocyte isolation from Pdgfra-GFP long bones and culture 2

Osteocytes were isolated from long bones of either wild-type C57BL/6 or Pdgfra-nGFP 3

mice. The mice were sacrificed at 8-16 weeks of age (n=12). Tibias and femurs were 4

removed, cleaned of soft tissues and transferred to ice cold PBS. Bone marrow was flushed 5

out with 2% FCS in PBS and bones were crushed and cut into ~200 μm- 400 μm pieces using 6

a mortar and pestle and scissors. Bone fragments were transferred into 5 ml of collagenase 7

(Type II, 2mg/ml) in PBS and placed on a water bath shaker at 37˚C for 30 min twice. 8

Supernatant was collected and collagenase was inactivated with 10 ml of 2% FCS and 5 mM 9

EDTA in PBS. Cells were collected through 40 μm cell strainer. Cells were stained for 10

SCA1, CD51, CD31, Lin/CD45 and PDGFRA. These cells were FACS sorted for SCA1-11

/CD31-/Pdgfra-nGFP-/PDGFRA-(Protein)- /CD51+ osteoblasts. FACS sorted osteoblasts and 12

bone fragments were cultured independently in complete culture medium (DMEM + P/S/G + 13

100 ug/ml of ascorbate + 10% FCS)(4). Cells were cultured at 370C and 5% CO2 in the 14

incubator for 8-10 days. Osteoblasts (SCA1-/CD31-/PDGFRA-/CD51+) derived from culturing 15

bone fragments were also FACS sorted before culturing in the reprogramming medium. 16

17

Primary osteocyte isolation from Dmp1-eYFP long bones and culture 18

Primary osteocytes were isolated from 8-16 weeks (n=7) old Dmp1-eYFP mice derived long 19

bones (femurs and tibia) using a previously described method (5). Cell suspensions resulting 20

from the primary isolation procedure and resulting bone fragments were cultured on type-I rat 21

tail collagen-coated six-well plates at a density of 25,000 cells per cm2 and 20-30 bone 22

fragments per well respectively in osteocyte culture medium (αMEM + 5% FCS + P/S/G). 23

28

Cells were maintained at 370C and 5% CO2 in the incubator for 7 days. Cells were cultured 1

from suspension and the outgrowths of cells from bone fragments were FACS sorted for 2

SCA1-/CD31-/ PDGFRA-/Dmp1-eYFP+ and cultured in osteocyte culture media for 3 days 3

before replaced with the reprogramming medium. 4

5

Primary mature adipocyte isolation from Pdgfra-nGFP subcutaneous fat and culture 6

Primary mature subcutaneous adipocytes were isolated from 8-16 weeks old Pdgfra-nGFP 7

mice (n=6) adipose tissue using a previously described method (6). After enzymatic dispersal 8

of cells, two different methods of differential plating were employed to ensure the purity of 9

mature adipocytes in culture (Fig S13A). The first method exploits the extended time needed 10

for unilocular adipocytes to attach to the cell culture dish surface. The second method of 11

differential plating involved cultures that were firmly adhered to the dish. Primary adipocytes 12

were cultured in adipocyte medium (DMEM-HG+ 10% FCS + P/S/G). Cells were cultured at 13

370C and 5% CO2 in the incubator for 8-10 days before exposing to reprogramming agents. 14

15

Isolation and culture of Primary human subcutaneous adipocytes and adipose tissue 16

MSCs 17

Subcutaneous fat was harvested with consent from patients undergoing surgery for 18

degenerative disc disease with approval from the Prince of Wales Hospital human research 19

ethics committee. A previously described method was used with modifications (6). 20

Subcutaneous fat tissues were first cleaned of excess soft tissues and gently minced using a 21

scalpel. Minced fat tissue was then transferred into Collagenase Type I (0.2%) and placed on 22

a shaker at 37°C for 40 min. The well-digested and homogenised suspension was then passed 23

29

through a 70µm filter into a fresh tube and inactivated with 100% FCS. The suspension was 1

then centrifuged at 300g at 4°C for 5 min. allowing its separation into 3 distinct layers. 2

Primary adipocytes from the uppermost fatty layer were plated in 35mm dishes and cultured 3

in adipocyte medium (6) (Fig S14A). The cell culture dishes were set in as shown in figure 4

S14A to allow unilocular adipocytes to attach to the cell culture dish surface. After discarding 5

the middle layer, adipose tissue MSCs (Ad-MSCs) pelleted in the stromal fraction were 6

cultured in T25 flasks in MSC media. Adipocytes were cultured at 37˚C and 5% CO2 in the 7

incubator for 10 days before exposing to reprogramming agents. Primary adipocytes were 8

exposed to MSC media containing 10μM 5’Azacytidine + 200ng/ml human recombinant 9

PDGF-AB for 2 days and 200ng/ml human recombinant PDGF-AB for a further 23 days. 10

11Cellular reprogramming and inhibitor studies 12

In vitro differentiated osteocytes, chondrocyte, adipocytes and long-bone derived osteocytes 13

were first cultured in MSC medium (αMEM + 20% FCS + P/S/G) with or without 10 μM 14

5’Azacytidine (Tocris Biosciences) and with or without cytokine (50 or 100 ng/ml PDGF-AA 15

or PDGF-BB or PDGF-AB, 10 ng/ml bFGF, 20 ng/ml HGF, 10 ng/ml IGF-1 and 10 ng/ml 16

VEGF (all from R&D Systems)) for 2 days and then cultured in MSC media with or without 17

cytokine for 10 days. In order to investigate the reprogramming cell signalling pathways, 18

inhibitors were added to the reprogramming cocktail from day 1 and kept for 12 days. Media 19

was refreshed every 3-4 days. At the end of day 12 cells were harvested for downstream 20

analysis. Inhibitors are listed in Table S5. 21

In vivo imaging 22

Changes in cellular morphology and GFP expression in individual osteocytes (n=4) and 23

adipocytes (n=3) derived from Pdgfra-GFP mice and change in cellular morphology and 24

acquisition of proliferative capacity in Dmp1-eYFP+ osteocytes was established by live 25

30

imaging using an IncuCyte microscope (Essen Bioscience) with 10x phase objective and a 1

Nikon Ti-E microscope with a 20x phase objective (0.45 NA). Images were captured every 2

60 and 30 mins respectively for 8 days. 12 bit images were acquired with a 1280x1024 pixel 3

array. 4

5

CFU-F long-term growth and serial clonogenicity 6

MSCs, oCFU-Fs, cCFU-Fs and aCFU-Fs were expanded in bulk culture after plating 10,000 7

cells per T75 flask. Resulting cells were split every 8 days. MSC and oCFU-F colonies were 8

isolated individually using cloning cylinders (‘O” rings) (Sigma-Aldrich) and MSC clones 9

(micro, small and large) and Osteocyte oCFU-F clones (micro, small and large) were pooled 10

individually as micro (MC), small (SC) and large (LC) colonies and cultured first in T25 11

flask for 12 days and then from passage 2 onwards in T75 flask for 12 days. Cumulative cells 12

numbers were calculated and plotted (log10 scale). Micro (n=46), small (n=22) and large 13

(n=19) colony’s 2ry, 3ry and 4ry colony formation was evaluated by plating single cell from 14

individual colonies into 96 well plates. 15

16

In Vitro Lineage Differentiation 17

Antibodies are listed in Table S5. 18

Osteogenic differentiation: Osteogenic differentiation was promoted by culturing cells in 19

either 6 well plate or in 4 chamber slide containing Dulbecco’s Minimum Essential Medium-20

low glucose (DMEM-LG) (Invitrogen, Carlsbad, CA), 10% FCS, 100 μg/ml penicillin and 21

250 ng/ml streptomycin, 200 mM L-Glutamine and 0.1μM dexamethasone (Sigma-Aldrich), 22

31

10 mM β-glycerophosphate. (Sigma-Aldrich), 200 μM L-ascorbic acid 2-phosphate (Sigma-1

Aldrich) for 21 days. The cells were stained with alizarin Red and anti-RUNX2 antibody. 2

Chondrogenic differentiation: 2.5 x 105cells to 1 X 105 were plated either in 6 well plate or 4 3

well chamber slide contained serum free Dulbecco’s Minimum Essential Medium high 4

glucose (DMEM-HG), 100 μg/ml penicillin and 250 ng/ml streptomysin, 200 mM L-5

Glutamine, 50 μg/ml insulin-transferrinselenious (ITS) acid mix (BD Biosciences), 2mM L-6

ascorbic acid 2-phosphate (Sigma-Aldrich), 1mM sodium pyruvate, 0.1 μM dexamethasone 7

(Sigma-Aldrich), and 10 ng/ml transforming growth factor β3 (TGF-β3; R and D Systems). 8

Medium was changed every 4 days for 28 days. Differentiated cells were stained for sulfated 9

proteoglycans with 1% alcian blue and anti-SOX9 antibody. 10

Adipogenic Differentiation: Cells were cultured in DMEM-HG containing 10% FCS, 100 11

μg/ml penicillin and 250 ng/ml streptomysin, 200 mM L-Glutamine and 0.5 μM 1methyl-3-12

isobutyl methylxantine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 10 μg 13

insulin (Sigma-Aldrich), 200 μM indomethacin (Sigma -Aldrich). Cells were cultured for 7-14

10 days. The cells were fixed and stained with Oil Red O or anti-PPARG antiboby. 15

Smooth muscle differentiation: Smooth Muscle differentiation was promoted by culturing 16

the cells in the presence of 50 ng/ml Platelet derived growth factor BB (PDGF-BB) (R and D 17

Systems) made up with 5% FCS in DMEM-HG and 100 μg/ml penicillin and 250 ng/ml 18

streptomycin and 200 mM L-Glutamine. The cells were induced for 14 days with media 19

changed every 3-4 days. The cells were stained for smooth muscle myosin heavy chain 20

(MYH1), Myocardin (MYOCD), Serum response factor (SRF) and calponin. 21

Endothelial differentiation: Endothelial cell differentiation was promoted by culturing the 22

cells in 5% FCS in Iscove’s modified Dulbecco’s Medium (Invitrogen, Carlsbad, CA) 23

containing 10 ng/ml bFGF and 10 ng/ml vascular endothelial growth factor (VEGF), (RND 24

32

Systems; 493-MV), 100 μg/ml penicillin and 250 ng/ml streptomycin and 200 mM L-1

Glutamine. Cells were stained for CD31, VE-Cadherin (VE-Cad), Caviolin-1 (Cav1), von-2

willebrand’s factor (vWF) and endothelial nitric oxide synthase (eNOS). For low-density 3

lipoprotein (LDL) uptake, acetylated apoprotein-LDL (AcLDL-Alexa Fluor 488- Molecular 4

probes) at final concentration of 10 ug/ml was added to endothelial differentiation assays at 5

the end of day 14. Then cells were cultured for a further 24 h. At the end of day 15 cells were 6

fixed and uptake was assessed by fluorescence yield. For matrigel assay, CFU-Fs, oCFU-F 7

and osteoblasts were plated on the chamber slides containing matigel and cultured for 7 days. 8

At the end of day 7 tubes were fixed and stained for CD31 and PDGFRB expression. 9

Cardiomyocyte differentiation: To promote cardiomyocyte differentiation, cells were first 10

cultured in 2% matrigel coated chamber slides or glass bottom petri dishes in normal MSC 11

medium for approximately 4-5 days. Then cells were differentiated towards cardiomyocytes 12

in cardiomyogenic differentiation medium consisting of DMEM-LG : Medium 199 (4:1), 13

1.0 mg/ml bovine insulin, 0.55 mg/ml human transferrin, 0.5 μg/ml sodium selenite, 14

50 mg/ml bovine serum albumin, and 0.47 μg/ml linoleic acid, 10−4 M ascorbate phosphate, 15

10-9 M dexamethasone, 100 μg/ml penicillin and 250 ng/ml streptomycin, 200 mM L-16

Glutamine and 10% FCS with 1ng/ml recombinant human neuregulin 1β2 for 14-21 days. 17

Fresh medium was changed every 3 days. The cells were stained for cardiac α-sarcomeric 18

actinin, Connexin 43, GATA4, Mef2c and NKX 2-5. Beating cardiomyocytes images were 19

acquired on a Nikon Ti-E microsope with a 20x phase objective (0.45 NA). 1000 frames were 20

acquired continuously with a 52 ms frame rate. 12 bit images were acquired with a 21

1280x1024 pixel array. For cell contraction frequency, we created customized software that 22

used normalized cross-correlation to track the displacement of a user specified region over 23

consecutive frames. Peak detection was performed on pixel displacement values to identify 24

33

the occurrence of a contraction, which was subsequently used to calculate the beating 1

frequency. 2

Neuronal differentiation: When the cells reach at 80% confluence, culture media was 3

switched to DMEM-HG media containing 100 μg/ml penicillin, 250 ng/ml streptomycin, 200 4

mM L-Glutamine and 1 mM β-mercaptoethanol. Media was changed every 3-4 days and 5

cultured for 8-10 days. Neural differentiation was confirmed by expression of Glial fibrillary 6

Acidic Protein (Gfap), Neuron specific beta III Tubulin (Tuj1), Oligodendrocyte marker O4. 7

Hepatocyte differentiation: At 80% cell confluence, culture media was switched to serum 8

free DMEM-HG containing 100 μg/ml penicillin, 250 ng/ml streptomycin, 200 mM L-9

Glutamine, 20 ng/mL EGF (R and D Systems) and 10 ng/mL of bFGF (R and D Systems) to 10

inhibit cell proliferation for 2 days. After conditioning the cells, differentiation medium was 11

added consisting of DMEM-HG supplemented with 20 ng/mL of HGF (R and D Sydtems) 12

and 10 ng/mL of bFGF for 7 days. The cells were then cultured in DMEM-HG supplemented 13

with 20 ng/mL OSM, 1 µmol/L dexamethasone, 10 µL/mL ITS premix and 100 μg/ml 14

penicillin and 250 ng/ml streptomycin for 14 days. Media was changed every 7 days. Hepatic 15

differentiation was assessed by immunofluorescence staining for albumin (Alb) and 16

hepatocyte nuclear factor 4 alpha (HNF4a). 17

18

Teratoma formation 19

Rag1 Mice were anaesthetized and the kidney was exposed through an incision on the dorsal 20

lumbar region. 1 x 106 cells in 20 ul of PBS containing 30% matrigel were injected under 21

kidney capsule using a fine needle (26G). Rag1 mice were injected (under the kidney 22

capsule) with mouse HM1 embryonic stem (ES) cells (N=2) or MSCs (N=3), osteocytes 23

34

(N=3) and oCFU-Fs (N=3) from β2-microglobulin-GFP mice either alone or as a mixture of 1

mESCs and MSCs (N=3), osteocytes (N=3) or oCFU-Fs (N=3) (mESCs: cells; 1:3). Mice 2

were sacrificed 4-6 weeks after injection. Tumour tissues were fixed in 4% PFA for 48 hrs. 3

Half of the tissue collected from each mouse was embedded in OCT embedding medium 4

while the other half was processed with paraffin embedding. GFP expression was revealed by 5

using immunohistochemistry with biotin conjugated anti-GFP antibody with secondary HRP 6

staining or by immunofluorescence staining as described below with confocal microscope. 7

8

Mixed lymphocyte reaction (MLR) 9

BmMSCs, osteocytes, and oCFU-Fs were plated at 3000 cells per well in a U-bottom 96-well 10

plate (Greiner, Kremsmunster, Austria) in complete alpha-MEM (αΜΕΜ, 10% FCS, β-11

mercaptoethanol, HEPES, P/S/G) and irradiated at 2000 cGy (X-RAD 320 Biologic 12

Irradiator, Precision X-Ray) prior to addition of stimulator splenocytes and responder T cells. 13

Stimulator splenocytes were prepared from MHC-mismatched BALB/c (H-2d) mice and 14

inactivated by incubating with mitomycin C (Sigma; 25µg/ml) at 37°C + 5% CO2 for 1 hour. 15

Following incubation, BALB/c splenocytes were washed twice with RPMI-1640 + 10% FCS. 16

Responder T cells were isolated from C57BL/6 (CD45.1 congenic, H-2b) mice using a Pan T 17

cell Isolation Kit II (Miltenyi Biotec, Gladbach, Germany). The purity of responding T cells 18

was >95%. Responder T cells were labeled with Cell Trace Violet (Life Technologies, 19

Invitrogen) for assessment of T cell proliferation. For the MLR, 3x105 responder cells and 20

3x105 stimulator cells were seeded in a 96-well round bottom plate and T cell proliferation 21

assessed by flow cytometry after 5 days. T cell proliferation in the MLR was determined 22

based on the dilution of the proliferation dye Cell Trace Violet assessed by flow cytometry. 23

Cells were stained with fluorochrome labeled antibodies against CD45.1 (1:200 dilution) to 24

35

label responder T cells. For the detection of intracellular granzyme B in responder T cells, 1

cells were incubated for 2 hours at 4°C in Fixation/Permeabilization solution (eBiosciences) 2

and labeled with anti-Granzyme B (1:50, eBiosciences) for 30 minutes at 4°C. Cells were 3

resuspended in PBS+5%BSA+NaN3 prior to analysis on a FACSCantoII flow cytometry with 4

FACSDiva software (both BD Biosciences). Data analysis was performed using FlowJo 5

(TreeStar Inc.; Ashland Oregon USA). Light scatter gating was performed on all samples to 6

include live lymphocytes and exclude doublet cells, dead cells and debris unless specified 7

otherwise. 8

9

Immuno-histochemistry: 10

Cells were washed with PBS (Invitrogen) for 10 minutes. The cells were then fixed with 4% 11

paraformaldehyde (ProsciTec) in PBS (w/v) for 15-20 minutes and then permeabilized with 12

0.03% Tween-20 in PBS (v/v) for 15 minutes at room temperature (RT). The cells were 13

washed once with PBS and then blocked with 10% donkey serum (v/v) in PBS for 1 h. The 14

cells were subsequently incubated overnight at 4°C with the primary antibodies in 2% bovine 15

serum albumin (BSA) (w/v) in PBS, stained accordingly with secondary antibodies in 2% 16

BSA and incubated for one hour at 4°C. Nuclear staining was done with DAPI. Slides were 17

mounted with Prolong-gold mounting medium (Invitrogen). Slides were analysed using either 18

L780 LSM Zeiss confocal microscope or Leica SP5 CW STED confocal microscope. 3D 19

rendering was performed using Imaris software in order to provide improved spacial 20

information in Z- direction. In this research we created 3D isosurface renderings from 21

confocal z-stacks of MSCs and oCFU-Fs cultured in matrigel for 7 days. Oil red O, alizarin 22

red and alcian blue staining for adipocytes, osteocytes and chondrocytes respectively 23

analysed by Nikon light microscope. Antibodies are itemised under Supplemental Materials 24

36

1

Immuno-blotting and densitometry 2

Cell pellets were lysed in non-denaturing lysis buffer contains Complete Protease inhibitor 3

cocktail (Roche) and PhosStop (Roche), followed by brief sonication using Bioruptor 4

(Diagenode). The supernatant of the whole-cell extracts were loaded onto NuPage 4-12% 5

Bis-Tri Gel (Life Technology) and transferred using iBlot Gel Transfer Kit (Nitrocellulose, 6

Life Technology) according to the manufacture instructions. Densitometry was performed 7

using GE ImageQuant TL Software Version 7.0. Antibodies are itemised under Supplemental 8

Materials 9

Allelic bisulphite sequencing. Allelic bisulphite sequencing was performed as described 10

previously (7). Briefly this involved sodium bisulfite modification using the EZ DNA 11

methylation Gold Kit (Zymo Research). Analysed regions were amplified from 40 ng of 12

bisulfite treated DNA using the primers listed in supplementary materials. PCR products 13

were cloned by ligation and transformation using the TOPO TA Cloning kit (Invitrogen). 14

Individual molecules were isolated from transformed colonies by colony PCR before 15

sequencing using BigDye Terminator v3.1 (ABI). All primers are listed in Table S3 16

Nucleosome occupancy and methylome sequencing (NOMe-Seq). NOMe-Seq was 17

performed as described previously(8). This involved treatment of intact nuclei with 200 U 18

GpC methyltransferase M.CviPl for 15 min at 37 oC followed by termination of the reaction 19

with an equal volume of 20 mM Tris HCl pH 7.9, 600 mM NaCl, 1 % (w/v) SDS and 10 mM 20

EDTA. Extracted DNA was bisulfite converted and amplified using primers listed in 21

supplementary materials M.CviPI enzyme methylates accessible DNA at GpC sites, whereas 22

nucleosome bound DNA is inaccessible and remains refractory to GpC methylation. PCR 23

amplicons were cloned and individual molecules sequenced as described above. Regions of 24

37

M.CviPI inaccessibility of ≥150bp were identified as nucleosome occupied. All primers are 1

listed in Table S3. 2

3

Posterior-lateral inter-transverse lumber fusion model 4

Long bones (femurs and tibias) were harvested from b2-microglobulin-GFP mice, soft tissues 5

were carefully removed and bone marrow was flushed out with cold 2% FCS in PBS in an 6

aseptic manner. Then bones were washed twice with cold PBS and crushed using a mortar 7

and pestle then cut into ~200μm - 400μm pieces using scissors. These bone fragments were 8

transferred to 5 ml of collagenase type II (2 mg/ml) in PBS and placed on a water bath shaker 9

at 37˚C for 30 min. This procedure was done three times. Supernatant was removed and the 10

bone fragments were cultured in a) MSC media only for 12 days, b) MSC media + 10uM 11

AZA for 2 days then in MSC media for 10 days, c) MSC media + 100 ng/ml PDGF-AB for 12

12 days and d) MSC media + 10uM AZA + 100 ng/ml PDGF-AB for 2 days, then MSC 13

media + 100 ng/ml PDGF-AB for 10 days. Sca1-/ CD31-/ PDGFRA-/ Dmp1-eYFP+ primary 14

osteocytes also harvested from Dmp1-eYFP mice derived long bones as describe earlier and 15

subject to culture in a) MSC media only for 12 days, b) MSC media + 10uM AZA for 2 days 16

then in MSC media for 10 days, c) MSC media + 100 ng/ml PDGF -AB for 12 days and d) 17

MSC media + 10uM AZA + 100 ng/ml PDGF-AB for 2 days, then MSC media + 100 ng/ml 18

PDGF-AB for 10 days. At the end of day 12 bone fragments or Dmp1-eYFP+ Osteocytes 19

(loaded onto a Helistat collagen sponge) were surgically implanted under anaesthesia into 20

posterior-lateral lumbar spine region (L4-L5) bilaterally on Rag1 mice (bone fragments 21

transplanted n= 30 and Dmp1-eYFP osteocytes transplanted n= 14)(9). A single orthopaedic 22

surgeon performed all the surgical procedures. At the end of 6 and 12 weeks mice were 23

euthanized and analysed by micro-computed tomography (micro CT). Then the spine from 24

38

the thoracic to caudal vertebral region was removed as whole, including pelvis. After the 1

animal was sacrificed, the spine-allograft complex was harvested; care was taken not to 2

disturb the spinal fusion region. The specimens were fixed in 4% PFA for 48 hours. After 48 3

hours of fixation in 4% PFA, spines were decalcified in EDTA and either embedded in OCT 4

or paraffin for immunofluorescence and histology analysis. 5

Micro CT: The lumbar spine-allograft complex was scanned using micro-CT (Siemens 6

Inveon Micro-CT System, Siemens Medical Solutions, Erlangen, Germany). Image analysis 7

software (Inveon Research Workplace [IRW], Siemens Medical Solutions, Knoxville, 8

Tennessee) was used for visualization and analysis of the spinal fusion. 9

Histology: After micro CT, samples were decalcified in 14% EDTA solution for routine 10

paraffin histology and cryosection for immunofluorescence imaging. The spine-allograft 11

complexes were sectioned sagittally at 5μm thickness. Histology was done using Harris 12

hematoxylin and eosin. Histology was qualitatively assessed for integrity, quality, graft 13

viability, degree of bone integration, new bone/cartilage formation, presence of fibrous tissue 14

interface layer, cellular activity, and Sharpey fibers in a blinded fashion by three independent 15

observers and scored for activity. 16

17

Data analysis 18

Unless otherwise stated, all experiments were performed with at least three technical and 19

three biological replicates. Analysis of results from flow cytometry used FlowJo software 20

(TreeStar, Oregon, USA). Comparison of means was analysed using t-test and standard 21

deviation was used as the measure of spread unless indicated otherwise. GraphPad Prism 22

version 6 was used for statistical analysis. 23

39

SUPPLEMENTAL REFERENCES 1

1. Friedenstein AJ, Chailakhjan RK, & Lalykina KS (1970) The development of 2fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. 3Cell Tissue Kinet 3(4):393-403. 4

2. Diffner E, et al. (2013) Activity of a heptad of transcription factors is associated with 5stem cell programs and clinical outcome in acute myeloid leukemia. Blood 6121(12):2289-2300. 7

3. Subramanian A, et al. (2005) Gene set enrichment analysis: a knowledge-based 8approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 9102(43):15545-15550. 10

4. Bakker AD & Klein-Nulend J (2012) Osteoblast isolation from murine calvaria and 11long bones. Methods Mol Biol 816:19-29. 12

5. Stern AR, et al. (2012) Isolation and culture of primary osteocytes from the long 13bones of skeletally mature and aged mice. BioTechniques 52(6):361-373. 14

6. Fernyhough ME, et al. (2004) Primary adipocyte culture: adipocyte purification 15methods may lead to a new understanding of adipose tissue growth and development. 16Cytotechnology 46(2-3):163-172. 17

7. Hesson LB & Ward RL (2013) Discrimination of pseudogene and parental gene DNA 18methylation using allelic bisulphite sequencing. Methods Mol Biol. . 19

8. Hesson LB, et al. (2013) Reassembly of nucleosomes at the MLH1 promoter initiates 20resilencing following decitabine exposure. PLoS Genetics. 21

9. Rao RD, Bagaria VB, & Cooley BC (2007) Posterolateral intertransverse lumbar 22fusion in a mouse model: surgical anatomy and operative technique. Spine J 7(1):61-2367. 24

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