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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(+) 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.

SUBMITTER: Hackett CH

PROVIDER: S-EPMC3128771 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Hackett Catherine H CH Flaminio Maria Julia B F MJ Fortier Lisa A LA

Stem cells and development 20101012 4

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 ma ...[more]

PMID: 20722500

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Project description:BackgroundThe use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA.ResultsRelative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P < 0.001) and 5-bromo-2'-deoxyuridine (BrdU) incorporation (167 ± 6 and 120 ± 6%, respectively; P < 0.001). Treatment with DEX increased ALP activity compared with control (1,638 ± 38%; P < 0.001). In the absence and presence of Dex, BMP-2 did not alter ALP activity (P > 0.8). Runt-related transcription factor2 expression increased 3-fold (P < 0.001) by d 6 of culture. Osterix expression increased 9-fold (P < 0.05) by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 (P < 0.01); however expression was reduced 4-fold at d 18 (P < 0.01).ConclusionsDexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

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

Publications

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

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