Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow-derived mesenchymal stem cells

Abstract

In vitro expansion of mesenchymal stem cell (MSCs) into large number is necessary for their application in cell-based treatment of articular cartilage defects. On the other hand, some studies have indicated that BIO (6-Bromoindirubin-3-Oxime) possesses mitogenic effects on cell culture. The objective of the present study was to examine the effect of BIO on in vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. The culture was established using bone marrow tissue obtained from 10 NMRI mice. MSC nature of the isolated cells was verified according to the minimal criteria proposed for MSC. Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and 1.5 µM BIO for seven days. The culture without BIO was taken as the control. At the end of cultivation period, the cultures were examined for viable cell number which was then used to calculate population doubling time (PDT). The BIO with higher proliferation-promoting effect was investigated for its chondrogenic effect on MSC culture. There was significantly more viable cells at the cultures treated by 0.1 µM BIO. At this culture the cells tended to double their population in rapid rate (each 43.07 hr) than the cells treated with the other BIO concentrations (p < 0.05). Interestingly treatment of MSC chondrogenic culture with 0.1 µM BIO led to the up-regulation of cartilage specific genes including aggrecan, collagen II and Sox9. In conclusion BIO at 0.1 µM could enhance mouse MSC in vitro proliferation as well as their chondrogenic differentiation. These findings would be of great importance for the field of regenerative medicine.

Keywords: BIO; Cartilage differentiation; Mesenchymal stem cells; Mouse; Proliferation.

Fig.1

Mouse marrow-derived mesenchymal stem cells. A) The primary culture contained a variety of cell morphology (bar = 100 µm); B) Confluent culture was composed of fibroblastic cells (bar = 200 µm); C) Majority of the isolated cells expressed mesenchymal markers including CD44 and CD73. Hematopoietic (CD11b and CD34) as well as endothelial (CD31) markers were expressed on a very low percentage of the isolated cells.

Fig. 2

Multi-lineage differentiation of the isolated cells from mouse bone marrow. A) Osteogenic culture stained by alizarin red; B) Bone-specific genes expressed in the osteogenic culture; C) Adipogenic culture stained by Oil red; D) Adipose-related genes expressed in the adipogenic culture; E) Section prepared from chondrogenic pellets stained by toluidin blue; F) Cartilage-specific genes expressed in chondrogenic culture. Bar = 200 µm.

Fig. 3

Cell viability and population doubling number (PDT) at cultures treated by BIO. A) The number of viable cells was significantly higher at culture treated by 0.1 and 0.05 µM BIO than the cultures with 0.01, 1.0 and 1.5 µM BIO and the control. * indicates a difference, (p < 0.5). There were no significant difference between the cultures with 0.1 and 0.05 BIO concentrations; B) The same relationships were observed among the studied cultures regarding PDN values

Fig. 4

Chondrogenic culture of mouse marrow-derived MSCs treated by BIO. There was apparently more metachromasia (i.e. the intensity of purple stain) at cultures with BIO treatment (A) than without BIO (B). This was in accordance with the real-time PCR findings indicating that cartilage-specific genes were significantly up-regulated at BIO-treated cultures, (Bar = 200 µm). C) Comparative analysis of fold change of cartilage-specific genes using quantitative PCR: The level of aggreacan, collagen and Sox9 expression in cultures with BIO was significantly higher than the culture without BIO (the control). * indicates a significant difference, (p < 0.05).