An In Vitro Co-Culture Model of Bone Marrow Mesenchymal Stromal Cells and Peripheral Blood Mononuclear Cells Promotes the Differentiation of Myeloid Angiogenic Cells and Pericyte-Like Cells.

Abstract

Mesenchymal stromal cells (MSCs) are known to stimulate the survival and growth of endothelial cells (ECs) by producing paracrine signals, as well as to differentiate into pericytes and thereby support blood vessel formation and stability. On the other hand, cells with an EC-like phenotype have been found within the CD14+ and CD34+ cell populations of peripheral blood (PB) mononuclear cells (MNCs). The aim of this study was to investigate the proangiogenic differentiation potential of human MSC-MNC co-cultures. Bone marrow-derived MSCs (2,500 cells/cm2) were co-cultured with MNCs (50,000 cells/cm2), which were isolated from the PB of healthy donors. MSCs and MNCs cultured alone at same cell densities were used as controls. Cells in MNC fraction and in co-cultures were isolated for CD14, CD34, and CD31 surface markers with magnetic-activated cell sorting. Co-cultures were analyzed for cell proliferation and morphology, as well as for the expression of various hematopoietic, endothelial, and pericyte markers by immunocytochemistry, quantitative PCR (qPCR), and flow cytometry. Vascular endothelial growth factor (VEGF) expression and secretion was measured with qPCR and enzyme-linked immunosorbent assay, respectively. Our results show that in co-cultures with MSCs, CD14+CD45+ MNCs differentiated into spindle-shaped, nonproliferative, EC-like, myeloid angiogenic cells (MACs) expressing CD31, but also into pericyte-like cells expressing neural/glial antigen 2 (NG2) and CD146. Functionality of the isolated MACs was demonstrated in co-cultures with human umbilical vein endothelial cells, where they supported the formation of tube-like structures. NG2+ cells of MNC-origin were found among both CD34-CD14+ and CD34-CD14- cell populations, indicating the existence of different subtypes of pericyte-like cells. In addition, VEGF was shown to be secreted in MSC-MNC co-cultures, mainly by MSCs. In conclusion, MSCs were shown to possess proangiogenic capacity in MSC-MNC co-cultures as they supported the differentiation of functional MACs, as well as the differentiation of pericyte-like cells of MNC origin. This phenomenon was mediated at least partially via secreted VEGF.