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