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T E C H N I C A L R E P O R T S

a 20

b 6,000

c

–6,000

d 2.0

*

*†‡

†‡

AWT diastole (mm)

1.6

15

*

Max dP/dt (mmHg/s)

*

Min dP/dt (mmHg/s)

LVEDP (mmHg)

4,000

*

–4,000

*

*

1.2

*†‡§

Sham

Untreated

10

DFB

*†‡

0.8

*

*

MSC

2,000

–2,000

5

0.4

* *

*

0

0

0

0

http://www.nature.com/naturemedicine

Sham

ntreated

DFB

MSC

Sham

ntreated

DFB

MSC

Sham

ntreated

DFB

MSC

Baseline

After treatment

U

U

U

e

11

*§

f

50

g

2,000

*§

h

Coronary ligation

Transplantation

100

10

*

*§

40

(pg/ml)

1,500

80

* *

MSC group

9

*†‡

(mm)

(n = 20)

30

level

60

8

*†‡

1,000

*

*§

LVDD

20

*

Plasma ANP

40

7

*

Untreated group

500

* *

*†‡

6

§

10

* *

*

20

(n = 20)

5

0

0

0

Baseline

After treatment

Baseline

After treatment

Baseline

After treatment

0

1

2

3

4

5

6

7

8

Figure 5 Cardiac structure and function afterPercentageFS

transplantation of monolayered MSCs. (a–c) Hemodynamic parametersSurvivalrate(percent)

Time (weeks)

obtained by catheterization. LVEDP, left

Group

ventricle end-diastolic pressure. (d–f) Echocardiographic findings. AWT, anterior wall thickness; LVDD, left ventricle end-diastolic dimension; FS, fractional

shortening. (g) Plasma atrial natriuretic peptide (ANP) level. Baseline represents measurements 4 weeks after coronary ligation; ’after treatment’ represents

measurements taken 4 weeks after transplantation (8 weeks after coronary ligation). Data are mean ± s.e.m. *P o 0.05 versus sham group; wP o 0.05

versus untreated group; zP o 0.05 versus DFB group; yP o 0.05 versus baseline. (h) Survival of rats with chronic heart failure with or without monolayered

Publishing

MSC transplantation. The Kaplan-Meier survival curve demonstrates an 8-week survival rate of 65% for the MSC group versus 45% for the untreated group.

Survival rate after transplantation was significantly higher in the MSC group than in the untreated group (100% versus 71% 4-week survival rate after

transplantation, log-rank test, P o 0.05).

Nature

conditions at 37 1C and becomes reversibly hydrophilic below 32 1C. Therefore,

COMPETING INTERESTS STATEMENT

cultured cells that adhere to the dish surface spontaneously detach from the

The authors declare competing financial interests (see the Nature Medicine website

grafted surface without enzymatic digestion.

for details).

2006

Preparation of monolayered cell grafts. We suspended MSCs at the third or

Published online at http://www.nature.com/naturemedicine/

©

fourth

passage from adipose

tissue or DFBs at the second passage by

Reprints and permissions information is available online at http://npg.nature.com/

trypsinization, and plated the cell suspension containing 3 ml of complete

reprintsandpermissions/

medium onto a 60-mm temperature-responsive dish at 5 105 cells per dish

(MSCs) or 8 105 cells per dish (DFBs) and cultured cells at 37 1C. After 3 d of

1.

Liu, J. et al. Autologous stem cell transplantation for myocardial repair. Am. J. Physiol.

culture, confluently cultured MSCs or DFBs on the temperature-responsive

Heart Circ. Physiol. 287, H501–H511 (2004).

dishes were incubated at 20 1C. By 40 min, both MSCs and DFBs detached

2.

Reinlib, L. & Field, L. Cell transplantation as future therapy for cardiovascular disease?:

spontaneously and floated up into the medium as monolayered cell grafts.

A workshop of the National Heart, Lung, and Blood Institute. Circulation 101, E182–

Immediately after detachment, we gently aspirated the monolayered cell grafts

E187 (2000).

3. Schuster, M.D. et al. Myocardial neovascularization by bone marrow angioblasts results

using a 1,000 ml pipette tip and transferred them onto an elastic plastic sheet.

in cardiomyocyte regeneration. Am. J. Physiol. Heart Circ. Physiol. 287, H525–H532

Statistical analysis. Numerical values are expressed as mean ± s.e.m. There are

(2004).

4.

Kocher, A.A. et al. Neovascularization of ischemic myocardium by human bone-

four groups of continuous variables in this study. Therefore, for multiple

marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling

comparisons of more than two groups, we performed one-way analysis of

and improves cardiac function. Nat. Med. 7, 430–436 (2001).

variance (ANOVA). If the ANOVA was significant, we used the Newman-Keul

5. Bel, A. et al. Transplantation of autologous fresh bone marrow into infarcted myocar-

dium: a word of caution. Circulation 108, II247–II252 (2003).

procedure as a post hoc test. For repeated measurement such as echocardio-

6.

Yamada, N. et al. Thermo-responsive polymeric surface: control of attachment and

graphic parameters, we performed two-way repeated ANOVA with the

detachment of cultured cells. Makromol. Chem. Rapid Commun. 11, 571–576

Newman-Keul test. Comparisons of parameters between two groups were made

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by unpaired Student t-test. A value of P o 0.05 was considered significant.

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Note: Supplementary information is available on the Nature Medicine website.

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8.

Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-

dimensional cell sheet manipulation technique and temperature-responsive cell

ACKNOWLEDGMENTS

culture surfaces. Circ. Res. 90, e40–e48 (2002).

We thank J.I. Hoffman for his statistical advice. We thank T. Iwase, T. Ito, S.

9.

Hirose, M., Kwon, O.H., Yamato, M., Kikuchi, A. & Okano, T. Creation of designed

Murakami, N. Sakata and Y. Isono for their technical support. We thank Y. Tsuboi

shape cell sheets that are noninvasively harvested and moved onto another surface.

Biomacromolecules 1, 377–381 (2000).

and H. Sonoda for their assistance with microscopic analysis of monolayered cell

10. Kushida, A. et al. Decrease in culture temperature releases monolayer endothelial cell

grafts. We also thank Y. Sawa for his suggestions on this study. This work was

sheets together with deposited fibronectin matrix from temperature-responsive culture

supported by research grants for Cardiovascular Disease (16C-6) and Human

surfaces. J. Biomed. Mater. Res. 45, 355–362 (1999).

Genome Tissue Engineering 005 and 009 from the Japanese Ministry of Health,

11. Herreros, J. et al. Autologous intramyocardial injection of cultured skeletal muscle-

Labor and Welfare, and the Program for Promotion of Fundamental Studies in

derived stem cells in patients with non-acute myocardial infarction. Eur. Heart J. 24,

Health Science of the Japanese National Institute of Biomedical Innovation.

2012–2020 (2003).

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VOLUME 12 [ NUMBER 4 [

APRIL 2006

NATURE MEDICINE