Expression of neural cell adhesion molecule and polysialic acid in human bone marrow-derived mesenchymal stromal cells

Abstract

Background: In order to develop novel clinical applications and to gain insights into possible therapeutic mechanisms, detailed molecular characterization of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) is needed. Neural cell adhesion molecule (NCAM, CD56) is a transmembrane glycoprotein modulating cell-cell and cell-matrix interactions. An additional post-translational modification of NCAM is the α2,8-linked polysialic acid (polySia). Because of its background, NCAM is often considered a marker of neural lineage commitment. Generally, hBM-MSCs are considered to be devoid of NCAM expression, but more rigorous characterization is needed.

Methods: We have studied NCAM and polySia expression in five hBM-MSC lines at mRNA and protein levels. Cell surface localization was confirmed by immunofluorescence staining and expression frequency in the donor-specific lines by flow cytometry. For the detection of poorly immunogenic polySia, a fluorochrome-tagged catalytically defective enzyme was employed.

Results: All five known NCAM isoforms are expressed in these cells at mRNA level and the three main isoforms are present at protein level. Both polysialyltransferases, generally responsible for NCAM polysialylation, are expressed at mRNA level, but only very few cells express polySia at the cell surface.

Conclusions: Our results underline the need for a careful control of methods and conditions in the characterization of MSCs. This study shows that, against the generally held view, clinical-grade hBM-MSCs do express NCAM. In contrast, although both polysialyltransferase genes are transcribed in these cells, very few express polySia at the cell surface. NCAM and polySia represent new candidate molecules for influencing MSC interactions.

Keywords: Bone marrow; Clinical grade; Mesenchymal stromal cell; Neural cell adhesion molecule; Polysialic acid.

Fig. 1

Multilineage differentiation of hBM-MSCs. Representative light microscopic photographs of control and differentiated hBM-MSCs. Adipogenic differentiation was confirmed by Sudan III staining, osteogenic differentiation by von Kossa staining, and chondrogenic differentiation by Safranin O staining. All hBM-MSC lines displayed typical differentiation capacity. Adipogenic and chondrogenic differentiation images were taken at 10× lens objective magnification, osteogenic differentiation images at 4× lens objective magnification. MSC mesenchymal stromal cell

Fig. 2

NCAM transcription in hBM-MSCs. NCAM is transcribed from a single gene, located in chromosome 11q23.1, consisting of at least 25 exons. As a result of alternative splicing, the protein exists in multiple isoforms. Qualitative reverse transcription PCR analysis was performed to gain an overview of NCAM mRNA expression. a Five distinct isoforms are known and can be detected with a common primer pair (NCAM-All) or isoform-specific primers as indicated. b All known NCAM isoforms are expressed in hBM-MSCs at the mRNA level. NCAM-expressing neuroblastoma cell line kSK-N-SH served as a control. bp base pairs, GAPDH glyceraldehyde 3-phosphate dehydrogenase, MSC mesenchymal stromal cell, NCAM neural cell adhesion molecule, RT reverse transcriptase

Fig. 3

Polysialyltransferase transcription in hBM-MSCs. NCAM polysialylation is catalyzed by two Golgi resident enzymes, polysialyltransferases ST8SIA2 and ST8SIA4. Qualitative reverse transcription PCR analysis was performed to indicate the presence of polysialyltransferases. a ST8SIA2 gene is located in chromosome 15q26 and consists of six exons, whereas ST8SIA4 gene is located in chromosome 5q21 and is comprised of five exons. b Both polysialyltransferases are transcribed in hBM-MSCs. The polySia-expressing neuroblastoma cell line kSK-N-SH was used as a control. bp base pairs, GAPDH glyceraldehyde 3-phosphate dehydrogenase, MSC mesenchymal stromal cell, RT reverse transcriptase

Fig. 4

Flow cytometric analysis of marker expression in hBM-MSCs. Flow cytometric analyses were performed to determine the prevalence of marker expression in hBM-MSC lines. a Plots of NCAM antibody and endosialidase-GFP fusion protein-labeled cells shown in comparison with plots of secondary antibody controls. NCAM was detected in all hBM-MSC lines, but the proportion of positive cells varied. In contrast, the proportion of polySia positive cells was very low. The NCAM and polySia-expressing cell line kSK-N-SH served as a control. b For comparison, hBM-MSC lines were analyzed for the expression of CD44, a common MSC marker, and tissue nonspecific alkaline phosphatase (TNAP), an early osteogenic marker. Standard deviation is indicated. No correlation between marker expressions was detected. MSC mesenchymal stromal cell, NCAM neural cell adhesion molecule, polySia polysialic acid

Fig. 5

NCAM protein expression in hBM-MSCs. NCAM is expressed as transmembrane (NCAM-180 and NCAM-140), membrane-anchored (NCAM-125 and NCAM-120), or secreted (any isoform) protein. Western blotting was performed to identify which NCAM isoforms are expressed at the protein level in hBM-MSCs. In gel electrophoresis, polySia may affect the mobility of NCAM. Parallel samples were thus treated with endosialidase (+Endo) to remove polySia. NCAM was detected with a mixture of two primary antibodies. All of the main NCAM isoforms—NCAM-180, NCAM-140, and NCAM-120—were detected from the whole cell lysates of hBM-MSCs. The NCAM-120 and NCAM-125 isoforms could not be distinguished from one another. The NCAM-expressing neuroblastoma cell line kSK-N-SH served as a positive control. Secondary antibody control confirmed the specificity of the labeling and α-Tubulin served as a loading control. MSC mesenchymal stromal cell, NCAM neural cell adhesion molecule

Fig. 6

NCAM localization in hBM-MSCs. Immunofluorescence staining was performed to study NCAM localization. Exemplified fluorescence microscopic photographs of antibody-labeled nonpermeabilized cells. On hBM-MSCs, NCAM is expressed in a clustered manner around the cell surface. In contrast, NCAM expression on kSK-N-SH cells appears to be even and smooth, and concentrating towards cell–cell contact sites. Secondary antibody control confirmed the specificity of the staining. Scale bar = 25 μm. DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stromal cell, NCAM neural cell adhesion molecule

Fig. 7

PolySia localization in hBM-MSCs. Immunofluorescence staining was performed to study polySia localization. Exemplified fluorescence microscopic photographs of antibody and fusion protein-labeled nonpermeabilized cells. Only very little polySia was detected on hBM-MSCs, mostly on cell extensions (white arrows). In contrast, polySia expression on kSK-N-SH cells is fairly strong and smooth throughout the cell surface. Secondary antibody control confirmed the specificity of the staining. Scale bar = 25 μm. DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stromal cell, NCAM neural cell adhesion molecule, polySia polysialic acid