Tissue engineering applications to vascular bypass graft development: The use of adipose-derived stem cells Paul DiMuzio, MD, Thomas Tulenko, PhD Journal.

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Tissue engineering applications to vascular bypass graft development: The use of adipose-derived stem cells Paul DiMuzio, MD, Thomas Tulenko, PhD Journal of Vascular Surgery Volume 45, Issue 6, Pages A99-A103 (June 2007) DOI: 10.1016/j.jvs.2007.02.046 Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 1 Morphologic and molecular characterization of adipose-derived stem cells (ASCs). A, Inverted phase light micrograph (original magnification ×40, unstained) of ASCs grown in culture demonstrate spindle-like morphology. After negative selection of cells for CD31 and CD45 using magnetic cell sorting, representative fluorescent activated cell sorting of ASCs grown in culture (passage four) demonstrate a homogeneous population of cells positive for (B) CD29, (C) CD13, and (D) CD90 surface markers. Journal of Vascular Surgery 2007 45, A99-A103DOI: (10.1016/j.jvs.2007.02.046) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 2 Differentiation of adipose-derived stem cells (ASCs) in response to endothelial cell growth supplement (ECGS) and shear stress. A, Morphologic studies. Inverted phase light micrographs (original magnification ×40, unstained) show human endothelial cells (upper two panels) forming cords on Matrigel (left) and aligning in the direction of shear stress (arrow, right). Similar findings are noted in human ASCs cultured in ECGS for a minimum of 3 weeks (lower two panels). B, Molecular studies (CD31 message). Real-time polymerase chain reaction (RT-PCR) detecting CD31 message in human endothelial (left lane), ASCs (middle lane) and smooth muscle (right lane) cells before (upper row) and after (lower row) exposure to 12 dynes shear for 4 days. ASCs grown in ECGS and exposed to shear express CD31 message, an endothelial cell phenotype marker. C, Molecular studies (CD31 protein). In studies analogous to those in B, Western blot for CD31 protein demonstrates that ASCs cultured in ECGS and exposed to shear stress express CD31 at the protein level. D, Immunohistochemical study (CD31). Laser confocal micrograph (original magnification ×40) of ASCs grown in ECGS, exposed to shear stress and stained with human CD31 Mab confirm the presence of CD31 protein within the differentiated ASCs. Table. Summary of molecular studies of ASCs grown in ECGS and exposed to shear stress. Neither von Willebrand factor (vWF) nor endothelial nitric oxide synthase (eNOS) was demonstrated by RT-PCR or Western blot. Journal of Vascular Surgery 2007 45, A99-A103DOI: (10.1016/j.jvs.2007.02.046) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 3 Differentiation of adipose-derived stem cells (ASCs) when cultured in media containing vascular endothelial growth factor (VEGF). Stem cells grown in media containing VEGF (see text) for a minimum of 1 week express message for proteins specific for endothelial cells, suggesting the ability of ASCs to differentiate towards endothelial lineage. Human endothelial (EC) and smooth muscle (SMC) cells served as positive and negative controls, respectively. vWF, Von Willebrand factor; eNOS, endothelial nitric oxide synthase; RT-PCR, reverse transcriptase-polymerase chain reaction; EGM2, endothelial cell growth media. Journal of Vascular Surgery 2007 45, A99-A103DOI: (10.1016/j.jvs.2007.02.046) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 4 Attachment and retention of adipose-derived stem cells to vascular scaffolding. Laser confocal micrograph (original magnification ×100, phalloidin and propidium iodide stain) of human stem cells seeded onto decellularized vein allograft. After 24 hours of static seeding, the graft was exposed to gradually increasing shear force (from 0 to 9 dynes) over 72 hours. The arrow indicates direction of flow. The stem cells aligned with flow and maintained a confluent monolayer of cells under physiological shear force. Journal of Vascular Surgery 2007 45, A99-A103DOI: (10.1016/j.jvs.2007.02.046) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions