Supplementary Figure 1. Implants derived from human embryoid body preparations contain non-cardiac structures. In early studies, infarcted hearts

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1 a Supplementary Figure 1. Implants derived from human emryoid ody preparations ontain non-ardia strutures. In early studies, infarted hearts reeived ell preparations of low ardia purity (<20% ardiomyoytes), otained y Peroll enrihment of spontaneously differentiating emryoid ody outgrowths. Most reipients of this ruder ell preparation, delivered without the pro-survival oktail, showed no surviving graft ells y four weeks posttransplantation. However, a suset of hearts (6 of 34) did show small human grafts within the infart sar that ontained oth ardia and non-ardia elements. Panel a shows a representative field from one suh heart after in situ hyridization with the human-speifi panentromeri proe (rown hromagen indiates human nulei). Although this graft did ontain small lumps of myoardium (see Panel, whih shows an adjaent setion that has een immunostained with a β-myosin heavy hain antiody, rown hromagen), it was largely omprised of non-ardia tissue elements, inluding disorganized vessels and rare epitheliumlined ysts (see Panel, immunostained with a pan-ytokeratins antiody, rown). Note that immunostains in panels & were performed on adjaent setions to that shown in panel a, and the fields depited orrespond to the upper and lower oxed areas in panel a. Sale ar = 20 mirons.

2 Supplementary Figure 2. Effiient ardiogenesis via direted differentiation. As is illustrated y this example, serial treatment of hescs with ativin A and BMP-4 results in preparations with extensive ardia differentiation. With eah grafting experiment, a small suset of the Peroll-enrihed ells to e implanted were ultured, fixed, and immunostained for β-myosin heavy hain (rown). Panels depit suh a preparation using ells ultured prior to (left) and following (right) enrihment y Peroll gradient entrifugation. Numers indiate the perentage of β-myosin heavy hain positive ells in the depited ell preparation.

3 a d Supplementary Figure 3. Proliferation of human myoardial implants. To determine whether the graft ardiomyoytes ontinue to proliferate in vivo, engrafted animals were pulsed with the thymidine analogue BrdU one hour prior to sarifie. Panel a shows a representative luster of β-myosin heavy hain positive (red hromagen) graft ardiomyoytes, a suset of whih show inorporation of BrdU (rown nulear signal, see lower oxed area and magnified image in panel ). Overall, a mean of 1.4± 0.2% of the graft ells were doule-positive for β- myosin heavy hain and BrdU. Futhermore, oasional graft ardiomyoytes showed mitoti figures (note oxed area in the upper right-hand orner and magnified image in panel ). Together, this evidene demonstrates sustained ell yle ativity as late as four weeks after transplantation into the infarted heart. Panel d shows a β-myosin heavy hain positive graft that inludes two human ardiomyoytes in mitosis (arrows). Sale ars = 25 mirons.

4 Supplementary Information Supplementary Figure 4. Host mirovasulature within human ardia implants. The human ardia implants ontained numerous mirovessels lined y host-derived endothelial ells. This representative field, whih ontains oth host and graft myoardium, has een immunostained with the rat-speifi endothelial ell marker, RECA-1 (rown). By ontrast, human, graft-derived endothelial ells (identified with a human-speifi antiody against CD31/PECAM and Ulex europaeus) were rare and infrequently formed vasular strutures (data not shown). Sale ar = 100 mirons.

5 Supplementary Figure 5. M-Mode ehoardiograms. Upper panel. Representative ontrol heart reeiving PSC-only. The left ventriular avity is dilated and the anterior wall shows akinesis. Left arrow indiates left ventriular end-systoli dimension (LVESD); right arrow indiates left ventriular end-diastoli dimension (LVEDD). Vertial sale ar = 5 mm; horizontal sale ar = 250 mse. Lower panel. Representative heart reeiving hesc-derived ardiomyoytes in PSC (Cells+PSC). There is redued left ventriular dilation relative to the aove PSC-only ontrol. The anterior wall moves inward during systole, indiating preservation of wall motion. Left arrow indiates left ventriular end-systoli dimension (LVESD); right arrow indiates left ventriular end-diastoli dimension (LVEDD). Vertial sale ar = 5 mm; horizontal sale ar = 250 mse.

6 Supplementary Tale 1. Histologi, ehoardiographi, and MRI parameters. Parameter Cardios + PSC PSC-Only SFM-Only Non-ardia ells Non-infarted Histologi Infart area (% LV area) 10.6 ± ± ± ± 1.5 N/A Ehoardiographi LVEDD, 48 hours (mm) 6.9 ± ± ± ± ± 0.1 LVESD, 48 hours (mm) 4.8 ± ± ± ± ± 0.1 Frational shortening, 48 hours (%) 28.1 ± ± ± ± ± 1 Heart rate, 48 hours (pm) 394 ± ± ± ± ± 12 LVEDD, 4 weeks (mm) 8.2 ± 0.2, # 9.2 ± 0.3, # 9.2 ± 0.2, # 9.1 ± 0.4, # 6.7 ± 0.1 LVESD, 4 weeks (mm) 5.8 ± 0.2, # 7.7 ± 0.3, #, ** 8.0 ± 0.1, #, ** 7.3 ± 0.5, #, ** 3.9 ± 0.2 Frational shortening, 4 weeks (%) 28.1 ± ± 1.7, #, ** 13.4 ± 1.3, #, ** 20.2 ± 3.5, #, ** 42 ± 1 Heart rate, 4 weeks (pm) ± 5.8, # ± 6.4, # ± 8.1,#,* ± ± 12 MRI (4 weeks) LVEDV (mm 3 ) 593 ± ± ± 27* 600 ± ± 26 ** LVESV (mm 3 ) 303 ± ± ± 28* 354 ± ± 9 ** LVEF (%) 50.3 ± ± 1.6 ^ 42.0 ± 1.9 ^^ 43.0 ± ± 1 ** Infart wall thikening (%) 27.5 ± ± 1.9 ** 8.8 ± 1.7 ** 9.8 ± 2.0 ** N/A Mid-septal wall thikening (%) 54.5 ± ± ± ± ± 2 Areviations: PSC pro-survival oktail, SFM serum-free medium, MRI magneti resonane imaging, LV left ventrile, LVEDD left ventriular end-diastoli dimension, LVESD left ventriular end-systoli dimension, LVEDV left ventriular end diastoli volume, LVESV left ventriular end systoli volume, LVEF left ventriular ejetion fration. # : signifiant differene versus paired 48 hr time point with p < * : signifiant differene from Cardios + PSC at same time point with p < ** : signifiant differene from Cardios + PSC at same time point with p < ^: orderline signifiane with p = 0.05 y ANOVA. Post ho analysis indiated signifiant differene vs Cardios +PSC (^: p< 0.05; ^^: p < 0.01)

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