Application of quantitative proteomic approaches for optimization of the ex-vivo expansion program of human mesenchymal stem cells

Abstract

Human mesenchymal stem cells (MSCs) possess a multilineage differentiation capacity which makes them potentially interesting for therapeutic applications. Expansion protocols for obtaining clinically meaningful cell numbers usually consist of growing MSCs under ambient oxygen conditions. However, hypoxic ex-vivo cultivation has been proven to enhance MSCs proliferation, self-renewal and long-term viability, probably by mimicking the physiological conditions of the low oxygen in-vivo "stem cell niche". To build a better understanding of the molecular mechanisms underlying MSCs behavior under these conditions, a quantitative proteomics approach using two-dimensional gel electrophoresis coupled with mass spectrometry was used to analyse the expression profiles of ex-vivo expanded cultures of bone marrow (BM) MSCs and adipose-derived stromal cells (ASCs) under hypoxia (2% O2) and normoxia (21% O2). Proteins belonging to the functional categories "Structural components and cellular cytoskeleton", "Glycolysis", and "Folding and stress response proteins" are more abundant in hypoxia compared to normoxia in both cell sources, however, there were differences between the BM and adipose tissue (AT) cell proteomes. Proteins in "Cell cycle and regulation" and "Apoptosis" are slightly less abundant in hypoxia compared to normoxia. The high number of multiple size and charge isoforms with an altered content identified in this study also emphasizes the importance of post-translational modifications upon different cell culture conditions. The differential protein expression reported suggests that changes in the actin cytoskeleton structure regulate MSCs cell adhesion, motility and self-renewal, and that a shift towards the glycolytic pathway occurs for energy requirements and protection against oxidative stress. In general, no major variances were observed between BM and AT with respect to the biological categories they were clustered in. However, as revealed by the proteomic expression profiles, differences between protein forms and protein numbers identified for each category were reported along with differences in the expression levels and number of protein isoforms for each tissue type. The global genome-wide expression approach used in this study has helped obtain mechanistic insights into the response to hypoxia, and in the future might contribute to set up a proteome profiling strategy for developing quality controls to assure clinically effective expanded MSCs.