Identification of mesenchymal stem cell populations with high osteogenic potential using difference in cell division rate

Abstract

Introduction: In bone regenerative medicine, mesenchymal stem cells (MSCs) have been widely investigated for their potential in bone regeneration. However, MSCs are a heterogeneous cell population containing a variety of cell types, making it difficult to obtain a homogeneous MSC population sufficient for tissue regeneration. Our group previously reported that by selecting rapidly dividing human auricular chondrocytes, it was possible to enrich for more chondrogenic cells. In this study, we aimed to identify a highly osteogenic MSC population by using a similar approach for mouse bone marrow MSCs.

Methods: Mouse bone marrow MSCs were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) and sorted according to the fluorescence intensity using flow cytometry on day 3 after labeling. To compare the ability to produce bone matrix in vitro, osteogenic differentiation cultures were performed and mineral deposition was confirmed by alizarin red staining. Real-time qPCR was also performed to examine the differences in gene expression between the fast- and slow-dividing cell groups immediately after aliquoting and after osteogenic differentiation.

Results: Differences in the growth rate of the fractionated cells were maintained after culture. Results of osteogenic differentiation culture and alizarin red staining showed more extensive mineral deposition in the slow cell group than in the fast cell group. Calcium quantification also showed higher absorbance in the slow cell group compared to the fast cell group, indicating higher osteogenic differentiation potential in the slow cell group. Furthermore, real-time qPCR analysis showed that osteocalcin expression was higher in the slow cell group in cells immediately after preparative differentiation. In addition, the expression of osteocalcin and sclerostin were higher in the slow cells after osteogenic differentiation.

Conclusion: The slow cell population contains many highly differentiated cells that are already more deeply committed to the bone lineage, suggesting that they have higher osteogenic differentiation potential than the fast cell population. This study will contribute to the realization of better bone regenerative medicine by utilizing the high osteogenic differentiation potential of the slow cell population.

Keywords: Cell division rate; Flow cytometry; Mesenchymal stem cells; Osteogenic differentiation; Regenerative medicine.

Fig. 1

Flow cytometric analysis of cell surface markers and CFSE fluorescence. Positive markers: CD29, CD44, CD73, and CD90; Negative markers: CD31, CD34, CD45.

Fig. 2

Cell labeling with CFSE. (A) Flow cytometric analysis of mouse MSCs on the day of CFSE staining and 3 and 7 days after staining. The concentrations of CFSE and incubation times were set to 5 μM 15 min, 10 μM 20 min, 15 μM 10 min, and 20 μM 5 min, respectively. Unstained cells were also analyzed on the same day as negative controls. Horizontal lines indicate the intensity of CFSE fluorescence and vertical lines indicate the percentage of cell count. (B) Phase contrast and fluorescence images of cells stained or unstained with CFSE for 20 μM 5 min. Images were taken immediately before and after staining; Scale bars = 75 μm.

Fig. 3

Sorting of rapid cell groups and slow cell groups. (A) Sorting strategy for rapid and slow cell groups by fluorescence intensity. (B) FSC/SSC plots of total cells and rapid and slow cell groups. (C) Histograms of CFSE fluorescence during sorting of all cells and rapid and slow cell groups. Areas of rapid cell group (50 %) and slow cell group (50 %) are shown. (D) Cell viability of rapid and slow cell groups. Values are means ± SD.

Fig. 4

Proliferation of rapid cell groups and slow cell groups. (A) Phase contrast image of sorted rapid cell group/slow cell group. Photographs were taken after 1, 3, 5, and 7 days of culture. Scale bars = 100 μm. (B) CCK-8 assay of rapid cells/slow cells. Cell proliferation of sorted rapid and slow cell groups was examined by CCK-8 assay on days 1, 3, 5, and 7 (n = 3, ∗P < 0.05).

Fig. 5

Osteogenic activity of rapid cell groups and slow cell groups. (A) Alizarin red staining at 28 days after osteogenic differentiation. Left: Photograph of each well (3 wells per group), Right: Micrograph (3 fields of view per group). Scale bars = 500 μm. (B) Quantification of calcium deposition. (n = 3, ∗P < 0.01).

Fig. 6

Gene expression analysis by real-time qPCR. Gene expression of rapid cell group/slow cell group immediately after sorting and on day 28 of bone differentiation culture is shown. (n = 4, ∗P < 0.05, ∗∗P < 0.01).