Direct cell-cell contact between mesenchymal stem cells and endothelial progenitor cells induces a pericyte-like phenotype in vitro

Abstract

Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs) and endothelial progenitor cells (EPCs). We identified stabilising pericytes (PCs) as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs' differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

Figure 1

Cell culture setups. MSCs or depleted-MSCs were seeded with EPCs in transwell culture (a) or direct coculture (b). MSCs, depleted-MSCs, and EPCs were seeded in single cultures (c) as controls. All experiments were performed in the presence of IMDM-FCS (IMDM supplemented with 10% FCS) or IMDM-PL (IMDM supplemented with 5% FCS, 5% PL).

Figure 2

Baseline gene expression at day 0 of MSCs, depleted-MSCs, and EPCs (ΔCt values relative to GAPDH in logarithmic scale). Pericyte marker genes (CD146, NG2, αSMA, and PDGFR-β) and an endothelial marker gene (PECAM-1) were detected in MSCs (a), depleted-MSCs (b), and EPCs (c) after single culture in culture medium at day 0 in 5 independent experiments.

Figure 3

Gene regulation in MSCs and EPCs after 3 days of culture in IMDM-FCS. Pericyte marker genes CD146 ((a), (f)), NG2 ((b), (g)), αSMA ((c), (h)), and PDGFR-β ((d), (i)) and endothelial marker gene PECAM-1 ((e), (j)) were detected in MSCs and EPCs after 3 days in transwell culture, direct coculture, and single culture in IMDM-FCS (IMDM supplemented with 10% FCS). Results are presented for each gene as relative change in gene expression (2^−ΔΔCt) over time between day 0 and day 3 in 5 independent experiments (donor 1–5) and as average ± error of the mean of all experiments.

Figure 4

Gene regulation in MSCs and EPCs after 3 days of culture in IMDM-PL. Pericyte marker genes CD146 ((a), (f)), NG2 ((b), (g)), αSMA ((c), (h)), and PDGFR-β ((d), (i)) and endothelial marker gene PECAM-1 ((e), (j)) were detected in MSCs and EPCs after 3 days in transwell culture, direct coculture, and single culture in IMDM-PL (IMDM supplemented with 5% FCS and 5% PL). Results are presented for each gene as relative change in gene expression (2^−ΔΔCt) over time between day 0 and day 3 in 5 independent experiments (donor 1–5) and as average ± error of the mean of all experiments.

Figure 5

Coexpression of pericyte markers on MSCs after direct coculture with EPCs for 7 days in IMDM-FCS (top row) or IMDM-PL (bottom row). Immunofluorescence staining for CD146, NG2, and αSMA demonstrated double positive cells for each combination (CD146 and NG2, CD146 and αSMA, and NG2 and αSMA). Negative controls were stained only with the fluorescent labelled secondary antibody. Cell nuclei are stained with DAPI (blue). Scale bars = 50 μm.