1. Stem Cell–Derived, Tissue-Engineered Pulmonary Artery Augmentation Patches In Vivo 
Bret A. Mettler, MD, Virna L. Sales, MD, Chaz L. Stucken, BA, Vesa Anttila, MD, Karen Mendelson, MA, Joyce Bischoff, PhD, John E. Mayer, MD 
The Annals of Thoracic Surgery 
Volume 86, Issue 1, Pages (July 2008)
DOI: /j.athoracsur
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Immunofluorescent detection of antibodies to ovine endothelial markers CD31 (A) and von Willebrand factor (B). Ovine mesenchymal stem cells (C) are immunoreactive to α-smooth muscle actin similar to blood vessel–derived smooth muscle cells (D). Mesenchymal stem cells were differentiated in osteogenic and adipogenic culture-specific media. Mesenchymal stem cells cultured in osteogenic media (E) stain positive for alkaline phosphatase whereas those cultured in adipogenic media (F) show accumulation of lipid vacuoles when stained with oil-red O. (A–D, ×400; E, F, ×100.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
In vitro culture of labeled endothelial progenitor cells with green fluorescent protein (GFP; A) and labeled mesenchymal stem cells with red fluorescent protein (RFP; B). (C) In vitro tissue-engineered pulmonary artery augmentation patch after 5 days in laminar flow system stained with hematoxylin and eosin. (D) The same section of pulmonary artery augmentation patch under fluorescent microscopy (fluorescein isothiocyanate–Texas red) demonstrating nuclear labeling with 4′,6-diamidino-2-phenylindole (DAPI; blue) colocalized for RFP-labeled mesenchymal stem cells (red arrows) and GFP-labeled endothelial progenitor cells (green arrows) before in vivo implantation. (A, ×100; B, ×200; C, ×40; D, ×400.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Representative patches of progressive tissue formation during a 6-week in vivo maturation period. (A) Thrombus formation is seen after 1 week in vivo with minimal evidence of tissue formation. At 2 (B), 4 (C), and 6 weeks (D), the patches show a progressive smooth endothelial surface with increased tissue formation. After 6 weeks in vivo, the tissue-engineered patch grossly assimilates native pulmonary artery. (E) Light microscopy hematoxylin and eosin section of tissue-engineered pulmonary artery augmentation patch after 7 days in vivo. The region below the line represents the tissue-engineered patch. (PA = native pulmonary artery; + = area of fibrin clot on the luminal surface.) (F) After 6 weeks of in vivo maturation, tissue-engineered patch architecture assimilates architecture of native pulmonary artery (PA). The area to the left of the line represents the native pulmonary artery. (* = anastomotic suture site.) (E, ×100; F, ×40.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Preimplant in vitro seeded tissue-engineered patches compared with the same autologous patch after 1, 2, 4, and 6 weeks of in vivo maturation comparing total tissue cellularity, green fluorescent protein (GFP), and red fluorescent protein (RFP) expression when compared with the number of 4′,6-diamidino-2-phenylindole (DAPI)-expressing cells. (A) Cell seeding is held constant in all autologous in vitro patches used as in vivo controls. As length of time in vivo increases, the number of cells expressing a fluorescent protein marker decreases. (B) Comparison of endothelial progenitor cells (EPC) expressing GFP before implant versus cohort described in vivo maturation. In the preimplant patch, tissue cellularity averages 47% of GFP-positive EPC across all times. After 1 week in vivo, the tissue-engineered patch contains the largest percentage of GFP-positive EPC (58%) and decreases as length in vivo increases. (C) Similar comparison of mesenchymal stem cells (MSC) expressing RFP with 48% of the preimplant tissue-engineered patch composition containing RFP-positive MSC. The maximal number of cells (51%) identified with the RFP marker occurs at 2 weeks of in vivo maturation.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

6. Fig 5
(A) In vivo tissue-engineered patch. Green fluorescent protein (GFP) and red fluorescent protein (RFP) expression in the tissue-engineered patches after 1, 2, 4, and 6 weeks of in vivo maturation. At 1 week, cellularity was predominately GFP-labeled endothelial progenitor cells (EPC) with a more homogeneous distribution as length in vivo increases. (B) Immunofluorescent labeling of the tissue-engineered patch at 1, 2, and 6 weeks. Tissue-engineered patches have maximal endothelial progenitor cell expression at 1 week and maximal mesenchymal stem cell (MSC) expression at 2 weeks. At 6 weeks, the population of nucleated cells has less expression of assayed retroviral markers. (B, ×400.) (green arrows = GFP-positive EPC, red arrows = RFP-positive MSC, blue = 4′,6-diamidino-2-phenylindole (DAPI)-expressing cells.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

7. Fig 6
(A) Representative tissue-engineered patch assayed for proliferation (green) and green fluorescent protein (GFP)–labeled endothelial progenitor cells (EPC) (red). Nucleated cells stained with 4′,6-diamidino-2-phenylindole (DAPl) (blue). Single arrows denote cells expressing both Ki-67 and GFP, indicative of proliferating GFP–labeled EPC (×400). (B) Mesenchymal stem cell (MSC) proliferation evaluated using proliferation (green). Cells were also assayed for red fluorescent protein (RFP) expression of the MSC (red). DAPl was used to stain all cellular nuclei. Single arrows demarcate proliferating MSC. At all time points (data not shown), a greater degree of proliferation is seen near the tissue-engineered construct–native pulmonary artery border (×400). (C) The percentage of EPC and MSC proliferating was determined by counting the number of cells expressing both Ki-67 and either GFP or RFP and dividing by the number of DAPl–positive cells. After 1 week in vivo, 56% of GFP–positive EPC are proliferating, decreasing to 28% at 6 weeks. RFP–positive MSC show maximal proliferation at 2 weeks (51%), decreasing to 28% at 6 weeks. (D) Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end–labeling (TUNEL) staining for apoptosis increased with increased time in vivo. At 1 week, 2.7% of cellular elements were TUNEL-positive, increasing to 10.5% at 6 weeks.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions