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Use of Human Umbilical Cord Blood-Derived Progenitor Cells for Tissue-Engineered Heart Valves  Ralf Sodian, MD, Philipp Schaefermeier, PhD, Sybille Abegg-Zips,

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Presentation on theme: "Use of Human Umbilical Cord Blood-Derived Progenitor Cells for Tissue-Engineered Heart Valves  Ralf Sodian, MD, Philipp Schaefermeier, PhD, Sybille Abegg-Zips,"— Presentation transcript:

1 Use of Human Umbilical Cord Blood-Derived Progenitor Cells for Tissue-Engineered Heart Valves 
Ralf Sodian, MD, Philipp Schaefermeier, PhD, Sybille Abegg-Zips, MD, Wolfgang M. Kuebler, MD, Mehdi Shakibaei, MD, Sabine Daebritz, MD, Johannes Ziegelmueller, MS, Christoph Schmitz, MD, Bruno Reichart, MD  The Annals of Thoracic Surgery  Volume 89, Issue 3, Pages (March 2010) DOI: /j.athoracsur Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

2 Fig 1 (A) Cluster-like colonies of CD133+ cells after 2 days. (B) Myofibroblast-like cells after differentiation, originated from CD133+ cells, stained for fibroblast-specific CD90/AS02, (C) smooth muscle α-actin (αSMA), and (D) cytoskeleton protein desmin. (E) Endothelial-like cells after differentiation, originated from CD133+ cells, stained for CD31, (F) von Willebrand factor, (G) vascular endothelial-cadherin, and (H) factor VIII. (Scale bars are 100 μm.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

3 Fig 2 (A) Polymeric heart valve scaffold seen from the aortic side in a closed position. Direct view of the thin and porous heart valve leaflets. (B) Tissue-engineered heart valve construct explanted from the bioreactor. Pale yellowish surface indicates formation of connective tissue. (C) Bioreactor maturation tissue-engineered heart valve construct in a semiopen position in the “bioreactor” system. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

4 Fig 3 Histologic examinations of tissue-engineered heart valve constructs. (A, B) Toluidin Blue staining shows the myofibroblast-like cells migrate into the deeper areas of the scaffold and are distributed randomly over the entire scaffold (see asterisk [*]). The white areas represent the rest of the polymeric heart valve scaffold (see ×). In A, CD31 staining reveals endothelial cell lining on the surface of the construct (see arrow). (C) Immunohistochemical staining against collagen type I (c), a major extracellular matrix component. (D) Immunohistochemical staining against and β1-integrin, a transmembrane protein important for cell-matrix signaling. (E) Immunohistochemical staining against elastin. This can be observed in all layers of the scaffold, demonstrating diffusion of the myofibroblast-like cells and matrix production over the entire area of the scaffold. (C, D, E) The black areas represent the rest of the polymeric heart valve scaffold (see ×). (Original magnification ×160.) The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

5 Fig 4 Transmission electron microscopy: (A) Longitudinal cut of the scaffold. The cells are arranged in several layers of cells, with endothelial-like cells (*) in the upper layers and myofibroblast-like cells in the lower layers (arrowheads). The endothelial-like cells are in tight cell-to-cell contact and contain a high number of vesicles and cell organelles. The myofibroblast-like cells are large, elongated, and contain large numbers of euchromatin in the nucleus, many mitochondria in the cytoplasm, and are embedded in large amounts of extracellular matrix. (Magnification ×20,000.) (B) Longitudinal cut of the valvular construct. Cells can be observed in layers in the inner area of the scaffold. The cells are embedded in large amounts of extracellular matrix, have established many cell-to-cell contacts, and contain many cell organelles. (Magnification ×20,000.) Scanning electron microscopy: (C) Endothelial-like cells on the inner side of the scaffold form a smooth surface area, as this side is exposed to the flow direction. (D) Endothelial-like cells on the outer side of the scaffold form a rough surface area and are arranged to each other like bricks in a wall. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

6 Fig 5 Extracellular matrix components of tissue-engineered heart valves were compared with native human pulmonary leaflet tissue. The amount of extracellular matrix proteins in explanted human tissue was taken as 100%. Quantification of the extracellular matrix of the in vitro constructs showed an elastin content (hatched bar) of 67% ± 17%, a glycosaminoglycan content (gray bar) of 85% ± 61%, and a collagen content (open bar) of 77% ± 9% compared with native human pulmonary leaflet tissue. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

7 Fig 6 Stress-strain curve of tissue-engineered heart valve constructs. The stress-strain curve of the tissue-engineered heart valve constructs (solid line) shows biomechanical properties almost comparable to those of native human pulmonary arteries (dotted line [control tissue]). The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

8 Fig 7 Fluorescence imaging. Immunofluorescence and nitric oxide (NO) imaging: (A) Representative images show positive staining of the surface cell layer of vascular constructs for the endothelial markers CD31 (platelet/endothelial cell adhesion molecule-1 [PECAM-1]) (top) and vascular endothelial (VE)-cadherin (center). Controls were immunolabeled with secondary FITC-Ab in the absence of primary antisera (bottom). (Replicated in at least n = 3 visual fields each.) (B) Representative images and (C) group data (n = 5 to 7 cells each) of cellular 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescence in the surface layer of vascular constructs. Images were recorded 5 minutes after stimulation with 0, 10−5, 10−4, or 10−3 mol/L acetylcholine (ACh), and DAF-FM fluorescence is expressed relative to its individual baseline (F/F0). Acetylcholine-induced increase in DAF-FM fluorescence demonstrates dose-dependent stimulation of NO synthesis. Intracellular calcium concentration ([Ca2+]i) imaging: (D) Representative tracing of Fluo-3 fluorescence in a single surface cell of a vascular construct. Fluorescence profile was recorded in 5 s intervals over 5 minutes and shows characteristic oscillatory pattern of cellular [Ca2+]i. (E) Representative images and (F) group data (n = 5 to 7 cells each) of cellular Fluo-3 fluorescence in the surface layer of vascular constructs. Images were recorded 5 minutes after stimulation with histamine (10 μM), thapsigargin (2 μM), or an equal volume of buffer (control), and Fluo-3 fluorescence is expressed relative to its individual baseline (F/F0). Increased Fluo-3 fluorescence after stimulation with histamine or thapsigargin demonstrates intactness of basic cellular second messenger responses. The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur ) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

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