This document summarizes a study that investigated co-culturing endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) to generate vascularized tissue engineered grafts. In monolayer cultures, the co-cultures generated networks through endothelial cell aggregation and sprouting, similarly to physiological blood vessel formation. MSC-conditioned media alone did not elicit this response, indicating direct cell contact is needed. The co-cultures were able to form spheroids containing established endothelial networks that were maintained for up to 21 days. When loaded into scaffolds and cultured in a bioreactor, the spheroids showed outgrowth and presence of pre-vascularized networks, unlike direct cell seeding

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Presentation Abstract
Title: CO-CULTURES OF ENDOTHELIAL PROGENITOR CELLS AND
MESENCHYMAL STEM CELLS FOR THE GENERATION OF
VASCULARISED TISSUE ENGINEERED GRAFTS
Category: Stem Cells and Tissue Engineering
Secondary
Category:
Endothelial Cells/Hemangioblasts
Presentation
Start:
6/13/2013 6:00:00 PM
Presentation
End:
6/13/2013 8:00:00 PM
Poster
Board
Number:
T-1193
Author
Block
Mark Chong1, Dedy Sandikin1, Renyi Teo2, Wenhao Leow2, Junwei Goh2,
Toon Tien Foo2, Mahesh Choolani1, Jerry K Y Chan3
1Obstetrics and Gynaecology, National Univ of Singapore, Singapore, Singapore,
2Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore,
Singapore, 3Reproductive Medicine, KK Women's and Children's Hospital,
Singapore, Singapore
Abstract: Engineered bone tissues are currently limited by inadequate vascularisation in
vivo following implantation. Recent research has turned to the use of angiogenic
cell sources, including endothelial progenitor cells (EPCs) to generate pre-
vascularised tissue prior to implantation. In this study, co-cultures of umbilical
cord-blood derived EPCs and fetal bone marrow mesenchymal stem cells (MSCs)
were studied for use in the pre-vascularisation of tissue engineered constructs.
Cells were fluorescently-labelled to facilitate imaging and identification in co-
cultures. Culture conditions were then optimised in monolayer cultures, and
subsequently extended to three-dimensional cultures for applications in tissue
engineering. In monolayer cultures, time-lapsed observations demonstrate the
cocultures to generate networks, following endothelial cell aggregation and
angiogenic sprouting, in a process akin to that of physiological vaculogenesis.

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Cultures of EPC in MSC-conditioned media failed to elicit similar results,
suggesting the need for direct cell contact and the stromal/supportive role of MSC
in the system. A modified image analysis method was then developed to
characterise vasculogenic events, and used in the optimisation of culture
conditions to elicit maximal pre-vascularisation. It was established that culture in
complete endothelial growth medium extensive prevascular networks (1.5-fold
increase in tube length over conventional culture conditions, p<0.01). In addition,
MSC were critical for the provision of stromal support, extending vascular
longevity in a dose-dependent manner. Extending these results to three-
dimensional conditions relevant for tissue engineering, the EPC:MSC co-cultures
were induced to form spheroids by culture on non-adhesive plates. Spheroids
measuring 500 um in diameter were generated with well-established endothelial
networks, which were sustained for up to 21 days of culture. Scaling up to
physiologically relevant conditions, co-cultured spheroids were loaded into tissue
engineering scaffolds and maintained in a bioreactor for 3 weeks. Outgrowth of
cells from spheroids onto the scaffolds was observed and pre-vascularised
networks were found to be present, as opposed to direct cell seeding methods. In
conclusion, a method to generate pre-vascularised tissue engineered constructs
was developed and shown to have several promising features. Work in progress
includes evaluation of the constructs in a murine model.
The information about presentations at the ISSCR 11th Annual Meeting is available for
planning purposes only and is strictly embargoed until presentation.
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