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Analysis of CD14 expression levels in putative mesenchymal progenitor cells isolated from equine bone marrow.

Stem cells and development (2010-08-21)
Catherine H Hackett, Maria Julia B F Flaminio, Lisa A Fortier

A long-term goal of mesenchymal progenitor cell (MPC) research is to identify cell-surface markers to facilitate MPC isolation. One reported MPC feature in humans and other species is lack of CD14 (lipopolysaccharide receptor) expression. The aim of this study was to evaluate CD14 as an MPC sorting marker. Our hypothesis was that cells negatively selected by CD14 expression would enrich MPC colony formation compared with unsorted and CD14-positive fractions. After validation of reagents, bone marrow aspirate was obtained from 12 horses. Fresh and cultured cells were analyzed by flow cytometry and reverse transcription and quantitative polymerase chain reaction to assess dynamic changes in phenotype. In fresh samples, cells did not consistently express protein markers used for lineage classification. Short-term (2-day) culture allowed distinction between hematopoietic and nonhematopoietic populations. Magnetic activated cell sorting was performed on cells from 6 horses to separate adherent CD14(+) from CD14(-) cells. MPC colony formation was assessed at 7 days. Cells positively selected for CD14 expression were significantly more likely to form MPC colonies than both unsorted and negatively selected cells (P ≤ 0.005). MPCs from all fractions maintained low levels of CD14 expression long term, and upregulated CD14 gene and protein expression when stimulated with lipopolysaccharide. The equine CD14 molecule was trypsin-labile, offering a plausible explanation for the discrepancy with MPC phenotypes reported in other species. By definition, MPCs are considered nonhematopoietic because they lack expression of molecules such as CD14. Our results challenge this assumption, as equine MPCs appear to represent a descendant of a CD14-positive cell.

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Lipopolysaccharides from Escherichia coli O26:B6, purified by gel-filtration chromatography