Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells

Abstract

Background: Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.

Results: Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.

Conclusion: Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Figure 1

In vitro differentiation of BM-MSCs and AT-MSCs. Both types of cell were maintained in control medium (A, B). Osteogenic differentiation was identified by von Kossa staining (C, D) and adipogenic differentiation by Oil Red O staining (E, F). Scale bar, 200 μm.

Figure 2

Flow cytometry. Comparison of cell surface proteins CD29, CD44, CD90, CD34, CD45, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 on primary cultures of BM-MSCs (A, C) and AT-MSCs (B, D). Solid histograms show nonspecific staining and open histograms show specific staining for the indicated marker. Three different donor MSC populations from each tissue type were analyzed and representative samples are shown.

Figure 3

Quantitative RT-PCR. Expression levels of mRNAs for stem cell markers Oct3/4, Sox2, and Nanog in BM-MSCs and AT-MSCs. Each value was normalized to beta-actin expression. Statistical comparisons were made using Student’s t test (**p < 0.01).

Figure 4

Immunocytochemistry. Expression and localization of Oct3/4 and Sox2 in BM-MSCs and AT-MSCs. Immunofluorescent localization of Oct3/4 in BM-MSCs with DAPI counterstaining (C, D). Immunofluorescent localization of Sox2 in BM-MSCs with DAPI counterstaining (E, F). Immunofluorescent localization of Oct3/4 in AT-MSCs with DAPI counterstaining (I, J). Immunofluorescent localization of Oct3/4 in BM-MSCs with DAPI counterstaining (K, L). Scale bar, 40 μm.