Limited Endothelial Plasticity of Adipose-derived Mesenchymal Stem Cells Revealed by Quantitative Phenotypic Comparisons to Appropriate Controls

Antonyshyn, Jeremy 1, 2 ; McFadden, Meghan 1, 2 ; Gramolini, Anthony 2, 3 ; Hofer, Stefan 4, 5 ; Santerre, Paul 1, 2, 6

1.  Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2.  Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research; 3.  Department of Physiology, University of Toronto; 4.  Division of Plastic and Reconstructive Surgery, University of Toronto; 5.  Departments of Surgery and Surgical Oncology, University Health Network; 6.  Faculty of Dentistry, University of Toronto;

Introduction. Considerable effort has been directed towards deriving endothelial cells (ECs) from adipose-derived mesenchymal stem cells (ASCs) since it was first suggested in 2004 that ECs and adipocytes share a common progenitor [1]. While many studies have demonstrated the capacity of ASCs to express endothelial markers, most have failed to compare their level of expression to that by ECs [2]. The lack of specificity of these markers to the endothelial lineage undermines their capacity to support an endothelial phenotype [3], and their variable levels of expression between different vascular beds further complicates efforts to define what constitutes the successful endothelial differentiation of ASCs [4]. In this study, quantitative phenotypic comparisons to several EC controls were used to determine the extent of endothelial differentiation achieved with ASCs.

Methods. ASCs were harvested from human adipose tissue, and their endothelial differentiation was induced using biochemical stimuli. Reverse transcription quantitative real-time polymerase chain reaction and parallel reaction monitoring mass spectrometry were used to quantify expression of endothelial genes and proteins, respectively. Flow cytometry was used to quantitatively assess acetylated low density lipoprotein (AcLDL) uptake. Human umbilical vein, coronary artery and dermal microvascular ECs were used as positive controls to reflect the phenotypic heterogeneity between ECs derived from different vascular beds. Their global proteomes were assessed by shotgun proteomics.

Results. Hierarchical clustering of the global proteomes of the EC controls revealed patterns in their protein expression profiles that distinguished macrovascular ECs from microvascular ECs and arterial ECs from venous ECs, supporting their capacity to encompass a broad endothelial phenotype. The abundance of endothelial transcripts and proteins was elevated in biochemically-conditioned ASCs, albeit to levels several orders of magnitude lower than that observed in the EC controls. Similarly, the uptake of AcLDL by conditioned ASCs was 7.2 ± 1.8-fold greater than that by unconditioned controls, but remained significantly lower than levels seen in ECs (71.8 ± 20.1-fold; P < 0.05). Furthermore, conditioned ASCs failed to exhibit a cobblestone-like morphology and contact-mediated growth inhibition, both of which are hallmarks of an EC that were observed in the EC controls.

Implications. This is the first study to emphasize the limited extent of endothelial differentiation that can be achieved with ASCs, suggesting that they may not be suitable EC substitutes for tissue engineering applications. Moreover, this study may serve as a cautionary tale to anyone exploring the plasticity of a cell, where quantitative phenotypic comparisons to appropriate controls may be scarce.

References:

1. Circulation 2004;109:656-663

2. J Vasc Surg 2017;65:1483-1492

3. Circ Res 2007;100:158-173

4. PNAS 2003;100:10623-10628