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
25