1. Dynamics of Human Myocardial Progenitor Cell Populations in the Neonatal Period 
Gabriel Amir, MD, PhD, Xiaoyuan Ma, MD, V. Mohan Reddy, MD, Frank L. Hanley, MD, Olaf Reinhartz, MD, Chandra Ramamoorthy, MD, R. Kirk Riemer, PhD 
The Annals of Thoracic Surgery 
Volume 86, Issue 4, Pages (October 2008)
DOI: /j.athoracsur
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Expression of Ki67 in human myocardium. Ki67 staining (brown) in nuclei of cells is shown in specimens from (a) fetal heart and infant heart at postnatal days (b) 4, (c) 13 (arrowheads), and (d) 22. Note the reduced density of Ki67-positive cells in neonatal myocardium with age and compared with fetal myocardium. Panel e shows a higher magnification of the area indicated by the square in Panel d to better reveal the nuclear localization of Ki67. Panel f indicates the time course of changes in Ki67 expression with postnatal age according to Norwood repair (black circles), aortopulmonary (AP) shunt (clear circles), tetralogy of Fallot (TOF, squares), truncus arteriosis (diamonds), and ventricular septal defect (VSD, plus sign). Panels g and h demonstrate coexpression of Ki67 (green) in a postnatal specimen: consecutive sections of P6 postnatal ([g] troponin T-positive or [h] sarcoplasmic reticulum calcium-regulated ATPase type 2 (SERCA2)-positive stained myocytes in red, nuclei in blue) containing Ki67-positive myocytes, indicated by the white arrowheads is shown. Panel i shows a fetal specimen section stained for the myocardial transcription factor mef2 (red), nuclei in blue, and Ki67 (green), producing a white coloration when all three dyes are colocalized (arrowheads). Figures are representative of results obtained in at least 3 specimens. Magnifications: ×400 (a to d), ×630 (g to i), ×1000 (e). Time course data for Ki67 expression, panel f: Fit of Ki67 expression vs age; linear regression R2 = 0.30; probability > |t| = ; n = 27.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
Expression of stage-specific embryonic antigen 4 (SSEA-4) and islet cell antigen 1 (Isl1) in human myocardium. Panels a to c show SSEA-4 staining. SSEA-4 was localized to cytoplasm and cell membrane (brown diaminobenzidine precipitate). Representative sections from (a) fetal heart near right atrium and postnatal days (b) 5 and (c) 13. Panels d to j show Isl1 staining. A region (panels d to f) of fetal heart where Isl1-positive cells were observed is located between the right ventricular outflow tract bordered by myocardium ([d] troponin T-positive staining) and adjacent to the right atrial wall ([e] atrial natriuretic peptid-positive staining). The area delineated by the rectangle in panels d to f is enlarged in panel g to reveal numerous Isl1-positive cells (some indicated by arrowheads). Panel h depicts another area adjacent to the right atrial wall of the same fetal heart, containing numerous Isl1-positive cells. Images i and j show sparse Isl1 expression (arrowheads) in postnatal day 2 and 6 myocardium. Figures are representative of results obtained in at least 3 specimens. Magnifications: a to c, g to j ×400; and d to f, ×50.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Expression of c-kit in human myocardium. Representative sections from (a) fetal and neonatal myocardium at postnatal days (b) 2 and (c) 10 are shown. The brown diaminobenzidine precipitate indicates the localization of c-kit-positive cells (arrowheads), which are more numerous in fetal than postnatal myocardium but are generally few in number and localized in connective tissue adjacent to blood vessels. Panel d shows the time course for changes in c-kit expression, which declined during the first postnatal month, according to Norwood repair (black circles), aortopulmonary (AP) shunt (clear circles), tetralogy of Fallot (TOF, squares), truncus arteriosis (diamonds), and ventricular septal defect (VSD, + sign). Colocalization of c-kit with Ki67 and sarcoplasmic reticulum calcium-regulated ATPase type 2 (SERCA2) in fetal myocardium: Arrowheads in panels e and f indicate colocalization of c-kit (red) with Ki67 (green) and nucleus (blue) in (e) bright field and (f) rendered laser confocal fluorescence images. Note the presence of cells expressing each antigen alone as well as in combination: Open arrowheads indicate Ki67-positive and c-kit-negative cells; arrows indicate c-kit-positive and Ki67-negative cells. Panel g shows a confocal image of a single cell expressing both SERCA2 (green) and c-kit (red) in a field of SERCA-positive myocardium. Figures are representative of results obtained in at least 3 specimens. Magnifications: a to c, ×400; e to g, ×630. Time course data in panel d: Fit of c-kit expression vs age; linear regression R2 = 0.35; probability > |t| = ; n = 25.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Expression of Nkx2.5 in human myocardium. Specimens of myocardium from postnatal day (a) 2, (b) 6, and (c) 28 hearts are shown. The brown diaminobenzidine precipitate indicates cells expressing Nkx2.5. The relative density of Nkx2.5-positive cells is generally greater in the newborn heart than in the older neonatal heart. Panel d shows the time course for changes in Nkx2.5 expression, which declined nearly threefold during the first postnatal month, according to Norwood repair (black circles), aortopulmonary (AP) shunt (clear circles), tetralogy of Fallot (TOF, squares), truncus arteriosis (diamonds), and ventricular septal defect (VSD, + sign). (Panel e) Double immunostaining analysis shows Nkx2.5 (green) with sarcoplasmic reticulum calcium-regulated ATPase type 2 (SERCA2; red) in fetal specimen. Panels f and g show Nkx2.5 (green) with (f) c-kit (red) or with (g) SERCA2 in the same P6 neonatal heart specimen. Figures are representative of results obtained in at least 3 specimens. Magnifications: a to c, ×400; e to g, ×630. Time course data for Nkx2.5 expression in Panel d: Fit of Nkx2.5 expression vs age; linear regression R2 = 0.56; probability > |t| < ; n = 26.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions