Spherical aggregates in HPL-based matrices.

Spherical aggregates consisting of 4500 MSCs and 500 HUVECs were generated by the hanging drop technology and subsequently embedded in 20% HPL matrices. MSCs stained with CellTrackerTM Green and HUVECs stained with CellTrackerTM Red were co-cultured to generate spherical aggregates. These aggregates were embedded into HPL-based matrices for 6 days to see vascular sprouts emanating from the aggregates (A, B). A representative image of two aggregates embedded into the HPL-based matrices in approximately 3 mm showed interconnection of the sprouts after 21 days of cultivation (C, D). Actin filaments of MSCs and HUVECs were stained with phalloidin (green) and HUVECs were stained by the endothelial specific marker vWF (red) (C-F). Branch points and lumen formation are indicated by white arrows.</p

Calculation of extracellular matrix components in spherical aggregates cultivated in MSC-BM with 8% HPL and MSC-GM.

The fluorescence intensity of collagen type I (A), collagen type IV (B), laminin (C) and fibronectin (D) were measured in aggregates grown in MSC-BM+HPL and MSC-GM for 3, 6, 9 and 12 days based on confocal microscopy images using ImageJ. To compare the expression of the ECM components in aggregates grown in MSC-BM+HPL to MSC-GM a two-way ANOVA (n = 2 for each day) was performed using GraphPad Prism 7. * p<0.05.</p

MSCs binding to a layer of HUVECs under flow in a microfluidic system.

MSCs in fluid phase were applied under 2 dyne/mm2 shear rate to bind to a layer of HUVECs in a BioFlux®200 system with a total chamber area is 0.525 mm2 (1500 mm x 350 mm) and one sector indicating 0.1 mm2 (A). A representative area of damaged HUVECs stained with CD144 (VE-cadherin, red) and bound MSCs stained with CD90 (green) (B, C). Some MSCs formed nano-tubular extrusions to interact with the HUVECs (red) (D). Statistical analysis was performed by an unpaired, two-tailed t-test (control n = 34, wound n = 6) using GraphPad Prism 7.</p

Extracellular matrix formation by MSCs in spherical aggregate cultivated in MSC-GM.

Spherical MSC aggregates were cultivated for the time indicated (3d, 6d, 9d and 12d) and cryo-sectioned and stained with mAbs specific form collagen type I (A-C), collagen type IV (D-G), laminin (H-K) and fibronectin (I-L) (red) and counterstained with CD90 (green). For nuclei staining DAPI was applied (blue). (TIF)</p

Extracellular matrix formation by MSCs in spherical aggregate cultivated in MSC-BM with 8% HPL.

Spherical MSC aggregates were cultivated for the indicated time (3d, 6d, 9d and 12d), cryo-sectioned and stained with mAbs specific form collagen type I (A-C), collagen type IV (D-G), laminin (H-K) and fibronectin (I-L) (red) and counterstained with CD90 (green). For nuclei staining DAPI was applied (blue). (TIF)</p

Determination of cross-differentiation of MSCs into an endothelial phenotype.

MSCs were stained with CellTrackerTM Red and co-cultured with HUVECs in HPL-based medium for 21 days. As controls, stained MSCs and unstained HUVECs, respectively, were cultured in HPL-based medium for 21 days. After fixation, cells were stained with a rbAb specific for vWF as endothelial marker, counterstained with anti-rabbit AF488 (green), and DAPI to highlight the nuclei (blue). After 21 days of co-culture with HUVECs in HPL-based medium, no expression of vWF was detected in MSCs. (TIF)</p

Different expression levels of the endothelial marker vWF and focal adhesion proteins in the 3D spheroids compared to 2D cultures.

The expression of the endothelial marker vWF (A) and the focal adhesion proteins vinculin (C) and paxillin (E) are shown in 3D spheroids by confocal microscopy. The fluorescence intensity of vWF (B) in HUVECs, vinculin (D) and paxillin (F) in MSCs were measured based on confocal fluorescent images of 3D aggregates and 2D cell culture using ImageJ. For the analysis of the expression levels of vWF, and the focal adhesion proteins vinculin and paxillin in MSCs in 2D compared to 3D culture, a two-tailed Student’s t-test was performed using GraphPad Prism 7. * p<0.05.</p

Vascular sprout formation in HPL-based matrices.

The capability of two spherical aggregates consisting of 4500 MSCs and 500 HUVECs to bridge distances of up to 5 mm with their vascular sprouts was investigated using phase contrast microscopy (A-E). The closure time of two adjacent spherical aggregates was recorded over a period of 20 days. The joining of vascular sprouts from two adjacent spherical aggregates was recorded at the day indicated with successful joining (filled circles) and failure to close (open circles) (F).</p

Generation of stable spheroids using autologous MSCs and HUVECs.

A) Spherical aggregates consisting of HUVECs only, MSCs only, and autologous MSCs and HUVECs in a ratio of 9:1 in hanging drops observed under a light microscope. Spheroids that contain only HUVECs are instable, whereas the addition of autologous MSCs leads to stable spheroid formation. B) Non-autologous (top) and autologous (bottom) MSCs stained with CellTrackerTM Green and HUVECs stained with CellTrackerTM Red were cultured by hanging drop. Using non-autologous MSCs, the HUVECs showed a round morphology indicating dying cell. In the autologous spheroids the HUVECs are viable, mostly located in the sprouts. (TIF)</p

Phenotypic characterization of MSCs.

MSCs were phenotypically characterized by flow cytometry using the stem cell markers CD73, CD90 and CD105. (TIF)</p