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. 2011 Apr;20(4):721-35.
doi: 10.1089/scd.2010.0175. Epub 2010 Oct 12.

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

Catherine H Hackett  1 , Maria Julia B F Flaminio, Lisa A Fortier
Affiliations

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

Catherine H Hackett et al. Stem Cells Dev. 2011 Apr.

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(+) 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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Figures

FIG. 1.
FIG. 1.
Flow cytometric analysis of CD14 cell surface molecule expression of epitope reactivity in freshly isolated peripheral blood leukocytes. Dot plot distribution of uncultured peripheral blood cells isolated using (A1) gradient density centrifugation or (A2) carbonyl iron incubation followed by gradient density centrifugation. The R1 gate corresponds to the size and granularity of neutrophils, R2 lymphocytes, and R3 monocytes. (R1–R3) Histogram analysis of mean fluorescence intensity of CD14 cell surface molecule expression in the gated areas. The shaded curves represent negative isotype control staining in each cell population. Open lines represent the mean fluorescence intensity for CD14. Neutrophils (R1) have low mean fluorescence intensity for CD14 expression, whereas monocytes (R3) have high mean fluorescence intensity. The small population of lymphocytes (R2) with low mean fluorescence intensity likely represents the activated B lymphocyte population (*).
FIG. 2.
FIG. 2.
Immunoprecipitation and Western blot analysis of specificity for CD14 antibody to the equine molecule expressed in peripheral blood cells. (A1) Equine peripheral blood Western blot probed with mouse anti-human CD14 biG10 antibody after immunoprecipitation using either anti-(α)-equine CD14 antibody or biG10 antibody with equine blood cells. WBC lysates immunoprecipitated with both CD14 antibodies retained protein bands, of ∼40 kDa (*). Arrow indicates the heavy chain of IgG; arrowhead indicates the light chain of IgG. (A2) Molecular size standards. WBC, white blood cell. RBC, red blood cell.
FIG. 3.
FIG. 3.
Flow cytometric analysis of cell surface protein expression in freshly isolated bone marrow cells compared with cells cultured for 2 h. (A–C) Dot plot distribution of bone marrow cells isolated using gradient density centrifugation (A) fresh/uncultured, (B) nonadherent cultured, or (C) adherent cultured cell fractions at 2 h of culture. (D–F) Histogram analysis of mean fluorescence intensity of cell surface molecule expression in the gated areas (Regions 1, 2, and 3, respectively). (D) R1 designates cells of the size/granularity equivalent to neutrophils; (E) R2 designates cells of the size/granularity equivalent to lymphocytes; (F) R3 designates cells of the size/granularity equivalent to monocytes. The shaded curves represent negative isotype control staining; open lines represent the labeling for the cell surface markers indicated on the left-hand side. A small fraction (about 15%) of the lymphocyte-like bone marrow cells from both freshly isolated and 2-h nonadherent cultures was positive for CD44 and CD11a/CD18 expression (▾). In the monocyte-like bone marrow cells, about 15% of freshly isolated (†) or nonadherent cells (↓) had detectable expression of some molecules. In contrast, after 2 h of culture, the monocyte-like adherent bone marrow cells (*) had a distinct phenotype.
FIG. 4.
FIG. 4.
Flow cytometric analysis of cell surface molecule expression in cultured bone marrow cells from 2 through 21 days of culture. (A–D) Dot plot distribution of bone marrow cells cultured for (A) 2 days, (B) 7 days, (C) 14 days, or (D) 21 days. (E) Histogram analysis of mean fluorescence intensity of surface molecule expression in Region 3 (R3) cells. The shaded curves represent negative isotype control staining; open lines represent the labeling for the cell surface markers indicated in the left-hand side.
FIG. 5.
FIG. 5.
Gene expression kinetics of CD14 during cell culture. A bone marrow sample cultured for 14 days was used as the calibrator sample for comparison using the 2−ΔΔCt method. Bars represent n = 6 ± SE, letters A and B denote significant differences between groups.
FIG. 6.
FIG. 6.
Flow cytometric dot plot analysis of equine bone marrow cells cultured for 2 days, after MACS sorting using a mouse antiequine CD14 antibody. The percentage of cells in each region are listed.
FIG. 7.
FIG. 7.
Quantification of colony formation after cell sorting using CD14 monoclonal antibody. The number of MPC colonies formed per 106 cells in each fraction were compared between groups. Bars represent n = 6 ± SE, letters A, B, and C denote significant differences between groups. MPC, mesenchymal progenitor cell.
FIG. 8.
FIG. 8.
CD14 gene expression over time between CD14 sorted and unsorted MACS cell fractions on adherent bone marrow cells. A reduced number of samples (n = 3) were available in the negative fractions of samples cultured 7 days or less (*). An unsorted bone marrow sample cultured for 14 days was used as the calibrator sample for comparison using the 2−ΔΔCt method. Bars represent n = 6 ± SE, letters A, B, and C denote significant differences between time period groups.
FIG. 9.
FIG. 9.
Flow cytometric analysis of CD14 cell surface molecule expression in unsorted bone marrow cells in response to LPS stimulation. (A) Dot plot distribution of unsorted bone marrow cells cultured for 21 days. (B) Histogram analysis of CD14 mean fluorescence intensity in Region 3 after overnight incubation of cells with LPS of differing concentrations (0, 1, 5, or 10 ng/mL media). The histograms represent cell surface molecule expression using either a negative isotype control antibody (left column) or the mouse antiequine CD14 antibody (right column). M1 represents the setting used for negative cell percentage calculations based on isotype control labeling. M2 represents the setting for positive cell percentage calculations. (*) A significant difference in percentage of positive cells and mean fluorescence intensity from the untreated sample. LPS, lipopolysaccharide; MFI, mean fluorescence intensity.
FIG. 10.
FIG. 10.
Gene expression kinetics of CD14 in unsorted or CD14-positive selected equine bone marrow cells with or without LPS stimulation. MPCs were cultured 21 days and treated with LPS at dosages of 0, 1, 5, or 10 ng/mL media. (A) 18S gene amplification plot (to verify equal RNA loading). (B) CD14 amplification plot of LPS stimulated and control bone marrow cells. Control MPCs treated with 0 ng/mL of LPS (←) had higher Ct values (lower levels of CD14 expression) than cells that were treated with LPS at any dose (▸).
FIG. 11.
FIG. 11.
Flow cytometric analysis of cell surface molecule expression in sorted and unsorted bone marrow cells treated or not with trypsin. (A, B) Dot plot distribution of bone marrow cells cultured 30 days after separation at 2 days of culture into unsorted, positively, or negatively selected fractions using an antiequine CD14 antibody and MACS. Adherent cells were collected for analysis after 5 min of incubation at 37°C with either Accumax® cell detachment solution (A) or 0.25% trypsin in Hanks balanced salt solution (B). (C–F) Histogram analysis of mean fluorescence intensity of cell surface molecule expression in Region 3. Shaded curves represent negative isotype control staining; open lines represent labeling for the cell surface markers indicated on the left-hand side. When trypsin was used to collect the cells for analysis, a decrease in mean fluorescence intensity was noted for some antibodies (F, *).
FIG. 12.
FIG. 12.
Flow cytometric analysis of cell surface molecule expression in bone marrow cells cultured for 30 days and treated with either Accumax or 0.25% trypsin in Hanks balanced salt solution. The percentage of positive cells using the respective antibody was compared between solutions (n = 4 ± SE). There were significant reductions (P ≤ 0.005) in the percentage of positive cells for some molecules (*) when trypsin was used instead of Accumax for cell preparation. The 0.25% trypsin solution reduces the detection of some, but not all cell surface markers in flow cytometric analysis.
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