Creating Endothelial Cells from Adipose-Derived Stem Cells

In conducting experiments to address reviewer comments, we again created endothelial-like cells from adipose derived stem cells (ADSCs). As in the past, the vast majority of ADSCs express some CD31 (PECAM-1) after culture in endothelial media (M200) supplemented with 50ng/mL VEGF (on 0.5LP, 3.0LP and TCP). The expression of this CD31 is relatively low and is uniformly distributed across the cell surface. This is contrast with typical endothelial expression of CD-31 which is typically very strong at cell-cell boundaries. In the two pictures below, image width is 200 microns.

However, when ADSCs are co-cultured with HUVEC (indirect or direct) in M200 supplemented with VEGF, some of the ADSCs begin to show endothelial-like expression of CD31. These EC-like cells are never seen in mono-cultures of ADSCs either on membranes or TCP.

Indirect Co-Culture:
ADSCs were grown on top of a 3LP membrane with HUVEC grown 900 microns below on the TCP substrate in M200 with VEGF. Images were taken 6 days after seeding the ADSC.

Direct Co-Culture:
In this case a nearly confluent monolayer of HUVEC was grown under a 0.5LP membrane and then ADSCs were seeded on top and grown to confluence in M200 supplemented with VEGF. Images were taken 6 days after seeding the ADSC. A subpopulation of the ADSCs have EC-like expression of CD31. The uniform low-level CD31 in the majority of the ADSCs is not easily observed due to the HUVEC background signal in this direct co-culture.

I do not believe the EC-like cells could be due to endothelial contamination in the ADSC isolation. The ADSCs have been through 4-5 passages in the lab with no evidence of strong CD31 labeling in any mono-culture experiments. In each case the “colonies” of EC-like cells have a population of at least 25 cells after just 6 days in co-culture. Additionally, these experiments are from different donor vials.