Abstract
An increasing amount of data has suggested a more dynamic role of vasculogenesis, whereby bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) home and contribute to new blood vessel formation during tumor growth, ischemic injury, and wound healing. EPCs can be obtained by isolating hematopoietic progenitor cells from BM or cord blood. Alternatively, ex vivo expansion of unmobilized human peripheral blood (PB) can generate adherent cells, PB-EPCs, that express endothelial markers and also, upon administration, incorporate into developing neovasculature. The relative ease of obtaining unmobilized human PB has made PB-EPCs an attractive candidate with which to develop cell based therapy to treat ischemia. In parallel with clinical trials designed to understand their therapeutic potential, there is a continued effort to better characterize the PB-EPC and understand its biology. It is currently thought that EPCs are directly derived from a CD34+/lin- faction of hematopoietic stem cells (HSCs). However, in our current study, we have confirmed prior reports that ex vivo expansion of human PB generates similar numbers of EPCs as compared to plating unfractionated human BM, which contains >50-fold higher CD34+/lin- content, suggesting that PB-EPCs may not be derived from the CD34+/lin- population. We used immunofluorescence and FACs analysis to further show that PB-EPCs not only express endothelial markers such as vWF, Vascular Endothelial Growth Factor Receptor 1 and 2 (also known as flt-1 and flk-1, respectively), VE-cadherin, UEA-1 lectin, Tie-1 and Tie-2 but also hematopoietic markers such as CD45 and CD14, a marker enriched on monocytes. To test if PB-derived CD14-positive cells can give rise to PB-EPCs, we isolated them from human PB to >98% purity and plated them on fibronectin-coated coverslips. In vitro culture of CD14-positive cells generated adherent clusters of spindle shaped cells morphologically similar to EPCs. Culture of the CD14-negative fraction failed to yield any adherent cells. After ten days, the coverslips were removed and the cells were stained with various endothelial (flk-1, vWF, uptake of DiI-AcLDL, and UEA-1 lectin) and monocyte/hematopoietic (CD14 and CD45) cell markers. In analysis of these slides, the EPCs derived from the purified CD14 fraction stained positive for all six markers. These observations suggest that PB-EPCs can differentiate from cells of the monocytic lineage in vitro without the necessity of interaction from cells contained in the CD14-negative population. Further experiments will test the possibility that monocytes may be an intermediate in the differentiation of EPCs in vivo.
Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells