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Supplemental Figure Legend
Supplemental Figure 1. Flow assisted cell sorting (FACS) of plastic adherent bone
marrow-derived human mesenchymal stem cells (hMSC) demonstrated a population
highly enriched (>99%) for CD90, CD105, CD133 while devoid of (>99%) CD 34 and
CD45.
Supplemental Figure 2. TGFβ superfamily members induce cytosolic Nkx2.5 in human
mesenchymal stem cells (hMSC). A, Immunostaining of hMSC treated for 5 days with
TGFβ (lower left), Activin-A (upper right), and BMP (lower right) demonstrated increased
Nkx2.5 expression versus naïve controls (upper left). B, The inductive effect by TGFβ
superfamily members was verified through quantitative PCR of Nkx2.5 mRNA levels
normalized to Tubulin.
Supplemental Figure 3. Retinoic Acid enhancement of TGFβ effect in embryonic stem
cells was recapitulated in hMSC. A, Touch-up PCR approach highlighted Nkx2.5
expression enhancement in retinoic acid treated versus untreated embryonic stem cells
in the presence of TGFβ. B, This effect was reproduced in hMSC for Nkx2.5 and MEF2C
as demonstrated on quantitative PCR normalized to tubulin.
Supplemental Figure 4. Recombinant factors needed to be applied in cocktail form to
achieve nuclear translocation. Nuclear translocation of Nkx2.5, MEF2C and Gata-4
quantified following individual or combined addition of TGFβ, BMP-4, Activin-A, IGF-1,
FGF-2, IL-6, α-Thrombin and retinoic acid (n=6 patients, excluding patients 3 and 9).
Supplemental Figure 5. IGF-1 and FGF-2 treatment is associated with AKT
phosphorilation. A, IGF-1 or FGF-2 treatment of embryonic stem cells resulted in
phosphorylation and nuclear translocation of AKT. B, IGF-1 and FGF-2 induced AKT
phosphorylation is preserved in hMSC.
Supplemental Figure 6. Cocktail induced nuclear translocation of cardiac transcription
factors was dependent on IGF-1 and FGF. A, Translocation of MEF2C into the nucleus
following stimulation with the complete cardiogenic cocktail was lost with removal of IGF-
1 and FGF-2, B. C, AKT dependence was demonstrated with loss of nuclear
translocation following addition of the AKT-P inhibitor SR13668.
Supplemental Figure 7. Guidance of patient-derived hMSC results in cardiopoietic
maturation. A, Cardiopoietic guidance of non-reparative patient (Pt.) hMSC resulted in
nuclear MEF2C expression indicative of cardiogenic commitment. B, hMSC derived
cardiomyocytes at day 10, 15 and 20 after decrease in platelet lysate concentration
(Troponin-I, top; α-actinin, bottom; and rare smooth myocyte, right) demonstrated an
increase in the number of mature mitochondria versus naïve hMSC, C.
Supplemental Figure 8. Cardiopoietic hMSC demonstrate capacity for cardiac
differentiation and engraftment. A, Gross evaluation of hearts 20 months after therapy
reveals a limited scar within the anterior all distal to the LAD ligation. B, Low
magnification (10X) immunohistological evaluation reveals a larger scar area in naïve
treated versus cardiopoietic treated (CP-hMSC) hearts with evidence of engrafted
human cells within the healed scar as demonstrated by human troponin stained
myocytes co-expressing ventricular myosin light chain (MLC2v). C, High magnification
(63X) evaluation demonstrates cardiomyocytes expressing human troponin co-localized
with α-actinin embedded within the anterior wall of the murine heart in CP-hMSC treated
hearts.
Supplemental Figure 9. Cardiopoietic hMSC demonstrate de novo cardiogenesis and
induce expression of endogenous stem cells, with rare evidence of fusion noted. A,
Rarely, human nuclei, were noted to be fused to murine nuclei on species specific
genomic probing (left) as quantified by confocal histogram evaluation (right). B,
Quantification of Sca-1 staining cells was found to be significantly upregulated in
cariopoietic hMSC treated hearts, when compared to naïve. C, human lamin (green) and
Sca-1 (yellow) staining reveals high abundance of non-human, murine Sca-1 cells in
cardiopoietic hMSC treated hearts.
Supplemental Figure 10. Cardiopoietic (CP) hMSC safety is determined by pathological
examination and electrocardiography. A, Infarcted hearts treated with patient derived
hMSC demonstrated heart muscle repair with no evidence of aberrant growth or tumor
formation. Examples of hMSC derived from four distinct patients (Pt) and transplanted
into four infarcted mouse hearts. B, Cardiopoietic hMSC treated mice revealed no
evidence of ventricular ectopy on Holter-type continuous ECG evaluation. C, Histological
evaluation of murine brain, lung, kidney, spleen and liver revealed no evidence of
abnormal cellular growth or tumorigenesis 6 months after cell delivery.
Supplemental Movies. (Microscopy) Calcium transients were appreciated in hMSC
derived cardiomyocytes under electrical current stimulation. (Echocardiography) Efficacy
of cardiopoietic (CP) hMSC is demonstrated by echocardiography at 1-year follow-up.
Short and Long axis imaging of naïve stem cell treated hearts revealed a fibrotic and
hypokinetic anterior wall most evident on apical M-Mode evaluation (Pt 11 Naïve
Movies). In contrast, CP hMSC treated hearts revealed a robust contractile profile
throughout the anterior wall reflecting a sustained benefit from guided stem cell therapy
(Pt 11 CP Movies).
Supplemental Figure 1
10 0 10 1 10 2 10 3 10 4APC-Cy7-A
99.62%
CD
34
CD45
A
100 101 102 103 104APC-Cy7-A
99.45%
CD45
CD
133
B
100 101 102 103 10PerCp-Cy5'5-A
99.14%
CD
105
CD90
C
Naïve
TGFβ BMP-4
Activin-A
A
B
Naïve TGFβ Activin BMP0
1
1.5
2
2.5
3
Nkx
2.5
mR
NA
Ex
pres
sion
(AU
)
10μm
DAPINkx2.5
Supplemental Figure 2
Nkx2.5
Tubulin
- + + +
mRNA expression in Embryonic stem cells
Supplemental Figure 3
Retinoic Acid - - + +
A
B
Nkx2.5 MEF2C Nkx2.5 MEF2C0
1
1.5
2
2.5
3
mR
NA
exp
ress
ion
in h
MSC
(AU
)Retinoic Acid
+ + + +TGFβ
TGFβ + + + +
20406080
100
Nuclear N
kx2.5 (%)
20406080
Nuclear M
ef2C (%
)100
100
20406080
0
Nuclear G
ata-4 (%)
0
0
Supplemental Figure 4
TGFβ
-1
BM
P-4
Act
ivin
-A
IGF-
1
FGF-
2
IL-6
Thro
mbi
n
Ret
inoi
c A
cid
Coc
ktai
l
Supplemental Figure 5
Phospho-AktSer473
DAPI
A
B(-) IGF-1
(+) IGF-1 (30 min)
Human Mesenchymal Stem Cell
20μm
30μm
Murine Embryonic Stem Cell
20μm
(+) IGF-1 (30 min)
30μm
(+) IGF-1(15 min)
(-) IGF-1
Phospho-AktSer473
DAPI
10μm
(+) FGF-2(15 min)
(-) FGF-2
30μm
20μm20μm
(+) FGF-2 (30 min)
Phospho-AktSer473
DAPI
10μm
(-) FGF-2
(+) FGF-2 (30 min)
10μm
Cocktail (+)IGF-1 (-)FGF-2(-)
Cocktail (+)SR13668 (+)
50μm
20μm
20μm
Cocktail (+)MEF2CMEF2CDAPIDAPI
Supplemental Figure 6
A
B
C
A Pt.1 Pt.4 Pt.5 Pt.6
Pt.7 Pt.11Pt.8 Pt.10
20μm
50μm
Supplemental Figure 7
MEF2CMEF2CDAPIDAPI 20μm50μm 20μm
50μm50μm50μm
B
02468
101214161820
Total Immature Mature
Num
ber o
f mito
chon
dria
Naive hMSCCP hMSC
n=5
C
D10
h-TroponinDAPI
D15 D20
D15
α-actininDAPI
D10 D20
D20
Sm M ActinDAPI
Supplemental Figure 8
C α−Actininh-Troponin
DAPI
Naïve hMSC CP hMSC50μm
A CP hMSC
20 months 20 months
CP hMSC
2mm
CP hMSC
Scar
h-TroponinMLC2vh-TroponinMLC2v
B
Scar
20 MonthsNaïve hMSC
20 Months
Site of ligation
500μm
Supplemental Figure 9
A
Ant
erio
r wal
lSc
a-1
posi
tive
cell
per f
ield
0
5
10
15
20
25
30∗
Naïve CP
Sca-1
h-Lamin
h-laminDAPI
Sca-1α-Actinin
CP hMSCC
250
00 2 4 6 8 10 12 14 16 18 20 22
B
HumanMouseDAPI
Brain
Kidney Alveolar spaceAlveolar space
LungLung
Spleen
Liver
Pt -1 Pt -4
Pt -12Pt -7
hMSC Treated murine hearts demonstrate no tumor formationA
B
C
Supplemental Figure 10
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