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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.
Publication Date: 2010-10-12 PubMed ID: 20722500PubMed Central: PMC3128771DOI: 10.1089/scd.2010.0175Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- N.I.H.
- Extramural
- Research Support
- Non-U.S. Gov't
Summary
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The research explores the role of CD14, a receptor protein, in identifying and isolating Mesenchymal Progenitor Cells (MPCs) from horse bone marrow. Whilst CD14 is commonly absent in MPCs in humans, the study found that in equine MPCs, those cells that were selected based on the presence of CD14 were more likely to form colonies, challenging previous biological assumptions.
Research Purpose and Hypothesis
- The study aimed to assess the potential of CD14, a lipopolysaccharide receptor, as a marker for sorting Mesenchymal Progenitor Cells (MPCs). MPCs are a type of stem cell with huge potential for tissue repair and regeneration.
- In humans and other species, CD14 is not typically expressed on MPCs. The researchers hypothesized that by selecting cells that did not express CD14, they would enrich the formation of MPC colonies when compared with unsorted or CD14-positive cells.
Methodology
- Bone marrow aspirate was obtained from 12 horses and both fresh and cultured cells were analysed using flow cytometry and reverse transcription and quantitative polymerase chain reaction. These techniques allowed researchers to track changes in cell phenotype, or observable characteristics.
- Cells were sorted using magnetic activated cell sorting. This process involved isolating CD14(+) cells from CD14(-) cells in order to assess MPC colony formation at seven days.
Results and Findings
- The results showed that cells which were positively selected for CD14 expression were significantly more likely to form MPC colonies than those that were unsorted or negatively selected.
- In the resulting MPCs, low levels of CD14 expression were maintained over a long period of time. Moreover, CD14 gene and protein expression were upregulated when the cells were stimulated with lipopolysaccharide.
- The researchers found that the equine CD14 molecule was trypsin-labile, meaning that it could be broken down by trypsin, a digestive enzyme. This could explain why the findings of this study differ from the typical MPC phenotypes found in other species.
Conclusions
- According to the standard definition, MPCs do not express CD14 and are considered nonhematopoietic. However, this study suggests that equine MPCs could potentially originate from a CD14-positive cell. This discovery challenges previous assumptions and increases our understanding of the complex nature of progenitor cells.
Cite This Article
APA
Hackett CH, Flaminio MJ, Fortier LA.
(2010).
Analysis of CD14 expression levels in putative mesenchymal progenitor cells isolated from equine bone marrow.
Stem Cells Dev, 20(4), 721-735.
https://doi.org/10.1089/scd.2010.0175 Publication
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
MeSH Terms
- Animals
- Antibody Specificity
- Antigens, Differentiation / genetics
- Antigens, Differentiation / metabolism
- Blotting, Western
- Bone Marrow Cells / cytology
- Bone Marrow Cells / metabolism
- Cell Separation / methods
- Colony-Forming Units Assay
- Flow Cytometry
- Horses
- Lipopolysaccharide Receptors / metabolism
- Lipopolysaccharides / pharmacology
- Membrane Proteins / genetics
- Membrane Proteins / metabolism
- Mesenchymal Stem Cells / metabolism
- Protein Biosynthesis / drug effects
- Transcription, Genetic
- Trypsin / pharmacology
Grant Funding
- T32RR07059 / NCRR NIH HHS
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Citations
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