Evaluating the Impact of Oxygen Concentration and Plating Density on Human Wharton's Jelly-Derived Mesenchymal Stromal Cells

Isolates of mesenchymal stromal cells (MSCs) contain a mixed cell population of stem cells, multipotent and unipotent progenitors, and differentiated cells. It is speculated that the useful subpopulation for tissue engineering and cell therapy will be the multipotent progenitor cells or the stem cells. The colony forming unit-fibroblast (CFU-F) assay is an in vitro assay for clonogenicity, which is one property of the stem/progenitor cell population of MSCs. Our goal was to generate standard protocols that would permit the expansion and maintenance of CFU-F. Previous work reported that low plating density and/or exposure to 5% oxygen vs. 21% oxygen increased proliferation rate and enhanced expansion of MSCs. Here, we characterized the effect of both plating density and oxygen concentration on MSCs derived from Wharton's jelly (WJCs). We found that reducing oxygen concentration from 21% (room air) to 5% during expansion increased cell yield and maintained CFU-F, without affecting the expression of surface markers or the differentiation capacity of WJCs. In addition, reducing plating density from 100 cells/cm 2 to 10 cells/cm 2 increased CFU-F frequency. Therefore, plating density and oxygen concentration are two important variables that affect the expansion rate and frequency of CFU-F of WJCs. These results suggest that these two variables might be used to produce different input populations for tissue engineering or cellular therapy.