Institute of Biomedicine, University of Turku, Turku, Finland.
Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal.
Stem Cells Dev. 2021 Mar;30(6):309-324. doi: 10.1089/scd.2019.0171. Epub 2021 Mar 1.
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/cm) were co-cultured with MNCs (50,000 cells/cm), 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, CD14CD45 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 CD34CD14 and CD34CD14 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.
间充质基质细胞 (MSCs) 通过产生旁分泌信号已知可刺激内皮细胞 (ECs) 的存活和生长，并且分化为周细胞，从而支持血管形成和稳定性。另一方面，在周围血 (PB) 单核细胞 (MNC) 的 CD14 和 CD34 细胞群中已经发现具有 EC 样表型的细胞。本研究的目的是研究人 MSC-MNC 共培养物的促血管生成分化潜能。骨髓来源的 MSCs(2500 个细胞/cm)与从健康供体的 PB 中分离的 MNC(50000 个细胞/cm)共培养。以相同细胞密度单独培养 MSCs 和 MNC 作为对照。使用磁性激活细胞分选从 MNC 部分和共培养物中分离 CD14、CD34 和 CD31 表面标记物。通过免疫细胞化学、定量 PCR(qPCR)和流式细胞术分析共培养物的细胞增殖和形态，以及各种造血、内皮和周细胞标记物的表达。通过 qPCR 和酶联免疫吸附试验分别测量血管内皮生长因子 (VEGF) 的表达和分泌。我们的结果表明，在与 MSCs 的共培养物中，CD14CD45 MNC 分化为具有纺锤形、非增殖性、EC 样、髓样血管生成细胞 (MAC) 的细胞，表达 CD31，但也分化为表达神经/神经胶质抗原 2 (NG2) 和 CD146 的周细胞样细胞。在与人脐静脉内皮细胞的共培养物中证明了分离的 MAC 的功能，其中它们支持管状结构的形成。在 CD34CD14 和 CD34CD14 细胞群中均发现了源自 MNC 的 NG2 细胞，表明存在不同亚型的周细胞样细胞。此外，在 MSC-MNC 共培养物中检测到 VEGF 的分泌，主要由 MSCs 分泌。总之，在 MSC-MNC 共培养物中，MSCs 表现出促血管生成能力，因为它们支持功能性 MAC 的分化，以及源自 MNC 的周细胞样细胞的分化。这种现象至少部分通过分泌的 VEGF 介导。
