Ex Vivo Reconstitution of Arterial Endothelium by Embryonic Stem Cell-Derived Endothelial Progenitor Cells in Baboons

Abstract

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESCderived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.

Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells

“…The BAB15 baboon ESC lines at passage 39 were obtained from the Pittsburgh Development Center, University of Pittsburgh School of Medicine; they were cultured according to the published method and used within no more than 10 passages. We confirmed the cellular pluripotency by growth behaviour; positive immunostaining for OCT-4, NANOG and SSEA-4; and histochemical staining for ALP [21]. Pooled colonies of high-proliferative-potential endothelial colony-forming cells (HPP-ECFCs), which were considered tissue-resident EPCs, were isolated and cultured according to the method described previously [22].…”

“…As demonstrated previously, our angioblast differentiation protocol can generate angioblasts via mesodermal intermediates and the cells demonstrate dual potential to differentiate into haematopoietic and vascular lineages [21]. To direct angioblast differentiation towards functional EPCs, we tested whether endothelial growth media for mature endothelial cultures could specify and drive the angioblasts into EPCs more efficiently.…”

“…We confirmed the cellular pluripotency by growth behaviour; positive immunostaining for OCT-4, NANOG and SSEA-4; and histochemical staining for ALP [21]. Pooled colonies of high-proliferative-potential endothelial colony-forming cells (HPP-ECFCs), which were considered tissue-resident EPCs, were isolated and cultured according to the method described previously [22].…”

“…Subsequently, we added ADM to EB cultures at gradually increased ratios at the time-points indicated in Figure 1. At the end of day nine, the EBs were transferred onto collagen-coated plates (BD Biosciences, San Jose, CA, USA) for monolayer culture [21]. At this stage, we specified angioblast preparations and generated endothelial progenitors.…”

Tissue Engineered Small Vessel Conduits – The Anti-Thrombotic Effect of Re-Endothelialization of Decellularized Baboon Arteries: A Preliminary Experimental Study

“…Baboon vascular endothelial cell growth characteristics are also thought to be similar to that of humans. Furthermore, they share about 98% homology to human genes, possess similar protein structures to humans, and reflect the anatomical, physiological, and behavioral makeup of humans [ 3 , 12 , 13 ].…”