Volume 17, Issue 7, Pages 1250-1256 (July 2009) Injection of Bone Marrow Cell Extract Into Infarcted Hearts Results in Functional Improvement Comparable to Intact Cell Therapy  Yerem Yeghiazarians, Yan Zhang, Megha Prasad, Henry Shih, Shereen A Saini, Junya Takagawa, Richard E Sievers, Maelene L Wong, Neel K Kapasi, Rachel Mirsky, Juha Koskenvuo, Petros Minasi, Jianqin Ye, Mohan N Viswanathan, Franca S Angeli, Andrew J Boyle, Matthew L Springer, William Grossman  Molecular Therapy  Volume 17, Issue 7, Pages 1250-1256 (July 2009) DOI: 10.1038/mt.2009.85 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 Limitation of infarct size in BMC and extract groups. Infarct size was determined morphometrically in trichrome-stained sections. Bars show mean ± SD. BMC, bone marrow cells; HBSS, Hank's buffered saline solution; NS, not significant. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 Therapeutic effect of BMCs and extract on left ventricular function measured by echocardiography. (a) LVEF; each line represents the mean of one experimental group. (b) ESV, and (c) EDV at day 28. (d) Wall thickness of the peri-infarct region and the infarct scar. Data are shown as mean ± SD; N = 8/group. BMC, bone marrow cells; EDV, end-diastolic volume; ESV, end-systolic volume; HBSS, Hank's buffered saline solution; LVEF, left ventricular ejection fraction; MI, myocardial infarction. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 Conclusive detection of GFP-expressing cells by colorimetric and multicolor fluorescence approaches. (a) GFP+ cells were detected 3 days after implantation (blue stain). There was no colocalization of GFP with troponin-I (Tn-I; red stain) in any cells. (b) Positive control for GFP fluorescence from implantation of GFP+ BMCs with immediate harvest. Intrinsic GFP fluorescence is shown with the green filter, red filter, and a green/red merged image. Autofluorescence is both green and red, while GFP+ cells are only green; the merged image shows green cells against a yellow background. (c) Adjacent section immunostained for GFP using a far-red secondary antibody and a green tertiary antibody, presented as green/red merged image, red filter, and far-red filter. GFP+ cells are identified as those that are green, are not red, and are far red, with the absence of signal in the red image controlling for wide-spectrum artifactual background fluorescence. (d) Positive control staining for GFP in hearts from GFP transgenic mice, with signal in green and no signal in red. (e) Rare GFP+ cell in border-zone myocardium 25 days after injection, in green/red, red, and far-red colors. (f) A similar presentation of rare GFP+ cells in the infarct scar. (g) Low magnification image of the largest cluster of GFP+ cells that we could detect in the infarct scar, green/red merged image. Clusters of this size were not observed in most hearts injected. (h) High magnification image of a representative GFP+ cell in the infarct scar, with nuclei in blue. Bar = 50 µm for (a–f) at equal magnification; Bar = 100 µm (g) and 10 µm (h). GFP, green fluorescent protein. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 Disappearance of GFP transcripts over time. (a) RT-PCR controls showing GFP PCR product (307 base pairs) and HPRT reference gene with and without reverse transcription (RT), from GFP transgenic heart, wild type heart plus GFP+ BMCs, and wild type heart negative control. (b) Quantitative RT-PCR (TaqMan) of GFP with histone 3.3A reference gene in six hearts per group harvested at 2 hours, 1 day, and 25 days after injection, shown with a negative control lacking GFP+ cells (P = 0.001, 2 hours versus 25 days). GFP transcripts are almost undetectable by day 25. Y-axis shows GFP percent relative expression to the histone reference gene. BMC, bone marrow cells; GFP, green fluorescent protein; HPRT, hypoxanthine phosphoribosyltransferase; neg. ctrol., negative control. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 5 Enhanced vascularity at the infarct BZ. BMC and extract therapies result in both increased vessel density and number of smooth muscle-coated arterioles in the BZ. Bar (upper panel) = 300 µm, lower panel = 100 µm. *P < 0.01 versus HBSS and no injection. †P < 0.01 versus HBSS; P < 0.001 versus no injection. N = 6/group. BMC, bone marrow cells; BZ, border zone; HBSS, Hank's buffered saline solution; HPF, high-power field; SMA, α-smooth muscle actin. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 6 Cardiomyocyte proliferation. BMC therapy causes a significant increase in the number of cycling cardiomyocytes as demonstrated by increased numbers of Ki67+ cardiomyocytes (upper panels) and PCNA+ cardiomyocytes (lower panels). White arrows, apoptotic CMs; yellow arrows, apoptotic cells that are not CMs. Bar = 50 µm. N = 6/group. BMC, bone marrow cell; CM, cardiomyocyte; HPF, high-power field; PCNA, proliferating cell nuclear antigen; Tn-I, troponin-I. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 7 Reduction in CM apoptosis. Extract therapy causes a significant reduction in apoptotic CMs at the infarct BZ on day 6 post-MI. BMC therapy results in a strong trend toward fewer apoptotic CMs. White arrows, apoptotic CMs; yellow arrows, apoptotic cells that are not CMs. Bar = 50 µm. #P = 0.05 versus no injection and 0.08 versus HBSS; *P < 0.01 versus no injection and <0.05 versus HBSS; †P = 0.04 versus no injection and 0.07 versus HBSS; ‡P = 0.011 versus no injection and 0.02 versus HBSS. N = 6/group. BMC, bone marrow cells; BZ, border zone; CM, cardiomyocytes; MI, myocardial infarction; HBSS, Hank's buffered saline solution; HPF, high-power field; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Molecular Therapy 2009 17, 1250-1256DOI: (10.1038/mt.2009.85) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions