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Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall  Alain Mirza, MD,

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Presentation on theme: "Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall  Alain Mirza, MD,"— Presentation transcript:

1 Undifferentiated mesenchymal stem cells seeded on a vascular prosthesis contribute to the restoration of a physiologic vascular wall  Alain Mirza, MD, Jean-Marc Hyvelin, PhD, Gael Y. Rochefort, PhD, Patrick Lermusiaux, MD, PhD, Daniel Antier, PhD, Bonaventure Awede, MD, PhD, Pierre Bonnet, MD, PhD, Jorge Domenech, MD, PhD, Veronique Eder, MD, PhD  Journal of Vascular Surgery  Volume 47, Issue 6, Pages (June 2008) DOI: /j.jvs Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

2 Fig 1 A, Evaluation of endothelialization by scanning electron microscopy: (upper panel) original magnification ×100; (lower panel) original magnification ×1000. Endothelial cells (filled arrow) covered the seeded graft from the seventh day, although the non-seeded graft was essentially covered with collagen fibers (clear arrow), even after 14 days. B, Evaluation of percentage of endothelial surface on seeded graft (S) compared with nonseeded graft (NS) after 1 and 2 weeks in vivo. *Significantly different P < C, Color flow Doppler shows homogenous flow velocity through aorta at the level of prosthesis (arrow). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

3 Fig 2 In vivo smooth muscle differentiation. A, Before implantation, undifferentiated mesenchymal stem cells (MSCs) expressed some smooth muscle proteins, including α-smooth muscle actin (SMA) but not others (desmin, smooth muscle myosin heavy chain [SMMHC], myosin light chain-2 [MLC2]) that are normally expressed by mature smooth muscle cells. The dark blue shows 4′-6-diamidino-2-phenylindole [DAPI] nuclei labeling. B, After in vivo implantation, MSCs seeded on graft were collected and placed in growth medium. They expressed smooth muscle proteins that were not detected before implantation (desmin, SMMHC, MLC2). C, Coexpression of green fluorescent protein (GFP) verified that the collected cells were the MSCs initially seeded on the graft. Secondary antibodies were Alexa (red) or phycoerythrin (green). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

4 Fig 3 Confocal microscopy analysis of grafts after in vivo implantation: smooth muscle protein (α-actin) expression. A, Before implantation, the frontal view of the prosthesis showed smooth muscle actin expression. B, After in vivo implantation, mesenchymal stem cell (MSC) smooth muscle actin appearance was modified in the frontal view on the prosthesis, and the (C) sagittal view showed that cells were (E) organized in a multicellular layer mimicking media in control aortic vascular wall. D, These cells expressed green fluorescent proteins indicating that these cells were the seeded MSCs. F, Nonseeded graft as a negative control. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

5 Fig 4 Messenger RNA expression of smooth muscle cells proteins measured by reverse transcription–polymerase chain reaction. Expression of smooth muscle proteins encoded genes (a-SMA, Desmin, SM-22α, MMHC-11) was still present before in vivo implantation. SM-22α and smooth muscle myosin heavy chain (MMHC)-11 encoding for protein nonexpressed in undifferentiated mesenchymal stem cells enhanced expression after in vivo staying. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; a-SMA, α-smooth muscle actin; SMMHC, smooth muscle myosin heavy chain. *Significantly different P < 0.05. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

6 Fig 5 Functional properties of undifferentiated and in vivo-differentiated mesenchymal stem cells (MSCs) compared with mature aortic and carotid smooth muscle cells (SMCs). A, Comparisons of (left panel) inhibitory effects of iberiotoxin (IBTX) and (right panel) 4-aminopyridine (4-AP) panel at +60 mV of holding potential. Results are expressed in percentage of inhibition of the potassium outward current. Smooth muscle from carotid (1) aorta (2) MSC (3), and MSC (4). *Significantly different P < .05. B, Typical example of differentiated cells in vivo (left image) before and (right image) after perfusion of 80mmol potassium solution. In 30 cells, no contraction was observed. C, left panel, Typical trace showing adenosine triphosphate (ATP)-induced Ca2+ response in MSCs collected from the graft. ATP-induced Ca2+ response in undifferentiated MSCs and MSCs isolated from grafts (in vivo), in the presence (black columns) and in absence (white columns) of extracellular Ca2+. Error bars represent the standard deviation. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

7 Fig 6 Confocal microscopy analysis of a graft seeded with mesenchymal stem cells after in vivo implantation shows expression of endothelial markers. A, Before in vivo implantation, no endothelial marker expression was attested. B, After in vivo implantation, the frontal view showed a CD31-positive area on the prosthesis that looked like the adjacent aortic vascular wall (arrow, anastomosis). C and D, Sagittal view showed endothelium-specific antigen (C) CD31 and (D) von Willebrand factor, which were observed only in the first layer in contact with the artery lumen. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

8 Fig 7 A, Evaluation of wall morphology by transmission electronic microscopy showed that mesenchymal stem cells (MSCs) formed a multilayer coat. B, Cells in contact with artery lumen appeared thin and quite different than initial seeded cells. C, The MSCs kept their initial aspect. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2008 The Society for Vascular Surgery Terms and Conditions

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