A comparative study of mouse bone marrow mesenchymal stem cells isolated using three easy-to-perform approaches

Abstract

Mouse bone marrow mesenchymal stem cells (mBM-MSCs) are important for preclinical tissue regeneration and repair studies. In the present study, we isolated mBM-MSCs using three easy-to-perform methods (whole bone marrow-adherent culture, density-gradient centrifugation, and bone digestion), and then compared the morphology, proliferation, differentiation, and paracrine factor profiles of the isolated mBM-MSCs. Of these three isolation methods, the bone digestion method resulted in the highest quantity of mBM-MSCs with high growth potential and moderate differentiation. Conversely, the mBM-MSCs isolated through the whole bone marrow-adherent method exhibited the lowest potency for proliferation and differentiation. The differentially expressed factors between mBM-MSCs were primarily those involved in immune responses. The highly expressed secreted factors included cytokines/members of the chemokine family, growth factors, and protein binding/proteinase activity. These findings provide a fundamental reference for development of MSC isolation methods.

Keywords: bone digestion; density-gradient centrifugation; mouse bone marrow mesenchymal stem cells; secretome; whole bone marrow-adherent culture.

Fig. 1

Flow chart of isolation of mBM‐MSCs using various isolation methods; BM‐MSC‐A, mBM‐MSCs from whole bone marrow adherent culture; BM‐MSC‐G, mBM‐MSCs from density‐gradient centrifugation; BM‐MSC‐D, mBM‐MSCs from bone digestion method.

Fig. 2

Morphology and proliferation of mBM‐MSCs obtained using three isolation approaches. (A) The morphology was viewed using an inverted microscope; scale bar = 50 μm. (B) Growth curves were measured by a CCK‐8 assay. (C, D) Colonies were stained with crystal violet (C) and CFU‐F was counted (D) (n = 3). Data are analyzed by one‐way ANOVA and expressed as the mean ± SEM, ***P < 0.001 and ****P < 0.0001.

Fig. 3

Cell markers of MSCs were analyzed by flow cytometry. (A–D) Surface antigen markers CD29 (A), Sca I (B), CD34 (C), and CD45 (D) were detected and quantified from the three groups (n = 3). Red markers represent positive surface antigens; blue markers represent negative surface antigens. Data are expressed as the mean ± SEM; ns, not significant.

Fig. 4

Differentiation potency of mBM‐MSCs. (A) Representative alizarin red S staining following osteogenetic induction medium culture. The histogram (right) shows the quantification of mineralization levels (n = 5). (B) Representative oil red O staining and quantification of adipocyte number per field for each of the three mBM‐MSC groups (n = 6–9). (C) Representative Alcian blue staining and quantification of chondrogenic differentiation from three isolations of mBM‐MSCs (n = 5). Scale bar = 200 μm; data are analyzed by one‐way ANOVA and expressed as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5

Secretome released by mBM‐MSCs. (A) Principal component analysis of expression profiles of mBM‐MSCs from three experimental groups. PC1 and PC2 refer to two different principal components. (B) In total, 139 significantly differentially expressed genes were selected and hierarchically clustered in a heatmap.

Fig. 6

Functional analysis of differentially expressed mBM‐MSC secretive factors. (A–C) GO terms [biological progress (A), cellular component (B), and molecular function (C)] enrichment analysis among the three experimental groups were measured using david. (D) Bubble diagram of significantly enriched KEGG signaling pathways of the differentially expressed mBM‐MSC secretive factors.

Fig. 7

Quantification of highly expressed cytokines of mBM‐MSCs. (A–C) Histogram representing the highly expressed cytokines/chemokines (A), growth factors (B), and protein and proteinase (C) (n = 3). Data are expressed as the mean ± SEM; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.