Question:

The general differentiation potential of human adipose tissue-derived mesenchymal stem cells (hASCs) towards many cell types is well known, but conditions to directly differentiate these cells under defined conditions into a high percentage of endothelial cells are not well understood.

Methods:

In this study we isolated hASCs from different donors by collagenase digestion and differential centrifugation. The change of cell surface marker and gene expression was detected by immunofluorescence staining, flow cytometry analysis and real- time PCR.

Results:

Isolated hASCs from excised fat tissue were initially positive for CD29, CD44, CD70, CD90, CD105 and CD166 and negative for CD31, CD34 and CD133 at which the latter three are typical hematopoietic and endothelial cells marker. Interestingly, we found that an exposure of hASCs to hypoxic (3% oxygen) conditions in combination with leptin for ten consecutive days activated the ability of hASCs to directly differentiate into endothelial cells. Furthermore, a co-incubation of VEGF and leptin under these hypoxic conditions significantly increased the number of CD31⁺ cells in hASCs. Real-time PCR analysis demonstrated a significant endothelial differentiation, which was obvious by an up-regulation of major known endothelial cell differentiation markers including CD31, VE-Cadherin, VEGFR2 (Flk-1), VEGFR-1 (Flt-1), Tie2 as well as endothelial cell nitric oxide synthase (eNOS). Moreover, hASCs cultured under hypoxia in combination with VEGF and leptin showed a stable branching network in matrigel cultures, comparably to endothelial cells. To give a further insight into the underlying cell signalling mechanism we demonstrated that the observed CD31 cell differentiation was mainly dependent on the Akt pathway.

Conclusion:

Our data strongly demonstrate that primary isolated hASCs which were negative for VEGFR2 can be differentiate into endothelial in vitro under 3% oxygen in combination with leptin and VEGF.