Comparison of Surface Markers between Human and Rabbit Mesenchymal Stem Cells

<div><p>This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.</p></div

Rabbit MSCs observed using an inverted microscope after differentiation and staining.

<p>A. After osteogenic differentiation of rabbit MSCs and Alizarin red staining, calcium deposition is observed (arrows; original magnification, 100×; scale bar, 10 µm). B. After chondrogenic differentiation of rabbit MSCs and Alcian blue staining, sulfated cartilage GAGs are observed (arrows; original magnification, 100×; scale bar, 100 µm). C. After adipogenic differentiation of rabbit MSCs and Oil Red O staining, accumulation of intracellular neutral lipid vacuoles is observed (arrows; original magnification, 100×; scale bar, 10 µm).</p

Comparison of data from the current study with data from four previous studies that examined the phenotypes of human and rabbit MSCs.

<p>The studies of Pittenger et al. and Lapi et al. did not specify how MSCs were defined as positive or negative for a given marker. Only Dominici's study strictly defined the positive or negative reactivity of MSCs for a given marker according to the percentage of marker-labeled cells^§; however, only a small number of markers was studied. The study by Martínez-Lorenzo et al. reported the percentage of MSCs that were labeled with a marker; however, many of their results did not fulfill Dominici's criteria^§.</p>§<p>Dominici's criteria: a cell sample is positive for a given marker if more than 95% of cells express the marker, whereas it is negative if less than 2% of cells express the marker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111390#pone.0111390-Dominici1" target="_blank">[1]</a>.</p><p>*: ≥95% of cells labeled by the marker (+), ≤2% of cells labeled by the marker (-).</p><p>Comparison of data from the current study with data from four previous studies that examined the phenotypes of human and rabbit MSCs.</p

Mean percentages of rabbit adipose derived stem cells that were labeled with each of the 25 markers, as determined by flow cytometry.

<p>Mean percentages of rabbit adipose derived stem cells that were labeled with each of the 25 markers, as determined by flow cytometry.</p

RT-PCR analysis of CD44 and CD105 mRNA expression in rabbit and human MSCs.

<p>The RT-PCR products from rabbit and human MSCs were normalized to the expression of β-actin. The electrophoresis of the rabbit MSCs showed a normalized expression of CD44 mRNA of 0.8, and CD105 mRNA of 0.59. The electrophoresis of the human MSCs showed a normalized expression of CD44 mRNA of 0.71, and CD105 mRNA of 2.4. The results indicated that the ratio between rabbit and human CD44 expression was 1∶0.89, while the ratio between rabbit and human CD105 expression was 1∶4.08. These results indicated that rabbit and human MSCs had similar expression of CD44 at the mRNA level. However, human MSCs, compared with rabbit MSCs, had apparent more expression of CD105 at the mRNA level.</p

Morphology of human MSCs observed using an inverted microscope.

<p>A. At 1 day after seeding, only a few fibroblast-like cells are adhered to the culture surface (original magnification, 100×; scale bar, 10 µm). B. At 3–4 days after seeding, a large number of fibroblast-like cells are adhered to the culture surface (original magnification, 100×; scale bar, 10 µm).</p

Mean percentages of rabbit MSCs that were labeled with each of the 25 markers, as determined by flow cytometry.

<p>Among the 25 mean percentages, 23 fulfilled or were close to fulfilling Dominici's criteria to define rabbit MSCs as being positive or negative for the given marker*. Only two mean values (12.99% for CD51 and 77.91% for desmin) had slight deviation from Dominici's criteria. If CD51 was considered to be absent, while desmin present, our result show that CD13, CD14, CD29, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II and cytokeratin are absent from rabbit MSCs, whereas CD44, α-SMA, vimentin and desmin are present on rabbit MSCs.</p><p>*Dominici's criteria: a cell sample is positive for a given marker if more than 95% of cells express the marker, whereas it is negative if less than 2% of cells express the marker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111390#pone.0111390-Dominici1" target="_blank">[1]</a>.</p><p>Mean percentages of rabbit MSCs that were labeled with each of the 25 markers, as determined by flow cytometry.</p

Morphology of rabbit MSCs observed using an inverted microscope.

<p>A. At 1 week after seeding, only a few fibroblast-like cells (arrows) adhere to the culture surface (original magnification, 100×; scale bar, 10 µm). B. At 2 weeks after seeding, a large number of fibroblast-like cells adhere to the culture surface (original magnification, 100×; scale bar, 10 µm).</p

Human MSCs observed using an inverted microscope after differentiation and staining.

<p>A. After osteogenic differentiation of human MSCs and Alizarin red staining, calcium deposition is observed (arrow, original magnification, 100×; scale bar, 10 µm). B. After chondrogenic differentiation of human MSCs and Alcian blue staining, sulfated cartilage GAGs are observed (arrows; original magnification, 100×; scale bar, 100 µm). C. After adipogenic differentiation of human MSCs and Oil Red O staining, accumulation of intracellular neutral lipid vacuoles is observed (arrows; original magnification, 100×; scale bar, 10 µm).</p