Analysis of CD14 Expression Levels in Putative Mesenchymal Progenitor Cells Isolated from Equine Bone Marrow

Abstract

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 þ from CD14 À 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.

Equine cellular therapy—from stall to bench to bedside?

“…However, results of a recent study demonstrate that CD14 might not be an appropriate negative MSC marker, as selection of CD14 positive equine bone marrow derived mesenchymal progenitor cells led to higher yields of adherent cells than selection of their CD14 negative counterparts. Furthermore, in this study, high CD14 mean fluorescence intensity was detected in early cell cultures, which decreased over time but then remained constant at a lower level (83). The absence of other negative markers such as CD34 and CD45 seems to be confirmed (75–78).…”

Equine Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: Cytofluorimetric Characterization, In Vitro Differentiation, and Clinical Application

“…The lower expression of CD90 and CD105 than expected, on the basis of ISCT stated [15], could be determined by multiple factors. It has been demonstrated that certain cell surface markers can be trypsin labile, which causes a functional impairment or even their removal [33]. Recently, Paebst et al [18] reported that the CD90 expression on equine cells is very sensitive to the action of enzymes used for detaching cells, including trypsin and accutase.…”

Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood‐derived mononuclear cell fractions

“…Although it has been stated by the International Society of Cellular Therapy that mesenchymal stromal cells should be >95% positive for CD73 and CD105, several studies report a lower or even no expression for human, canine or equine mesenchymal stromal cells . Furthermore, it has been demonstrated that certain cell surface markers can be trypsin‐labile, which causes a functional impairment or even their removal . If this applies to CD73 and CD105, mesenchymal stromal cells would appear negative for markers upon immunophenotyping.…”