Gene expression profiles of early chondrogenic markers in dedifferentiated fat cells stimulated by bone morphogenetic protein 4 under monolayer and spheroid culture conditions in vitro

Abstract

Purpose

Human dedifferentiated fat (hDFAT) cells are thought to be a promising cell source for cartilage regeneration therapy. Nevertheless, the responses of hDFAT cells to bone morphogenetic proteins (BMPs) are still unclear. Here, we elucidated the effects of BMP-4 on the mRNA expression of early chondrogenic markers in hDFAT cells under monolayer or pellet cell culture conditions.

Materials and methods

Monolayer and pellet cell cultures of hDFAT cells were grown with control medium or chondrogenic medium (CM) with or without BMP-2, BMP-4, or BMP-7. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of nine genes: chondrogenic markers, i.e., SOX9, SOX5, SOX6, aggrecan, type 2 collagen, type 10 collagen, and matrix metalloproteinase (MMP) 13; type 1 collagen; and MMP3. The BMP signaling inhibitor dorsomorphin was used to verify the mechanisms of BMP-4-induced chondrogenesis.

Results

Recombinant BMP-4 (100 ng/mL) increased the expression of SOX9, SOX6, and aggrecan mRNAs in monolayer cells compared with that in cells treated with BMP-2 or BMP-7 on day 3. Chondrogenically differentiated hDFAT cells induced by CM containing BMP-4 showed higher expression of eight genes (excluding SOX5) in monolayer cultures and nine genes (including SOX5) in pellet cultures compared with those in control medium on day 14. Dorsomorphin attenuated the effects of BMP-4.

Conclusion

These results showed that BMP-4 had the potential to modulate the early chondrogenesis of hDFAT cells under both monolayer and pellet cell culture conditions.

Keywords:

• Dedifferentiated fat cell
• Adipocyte
• Chondrocyte
• BMP-4
• Differentiation

Acknowledgments

This study was partially supported by a Grant-in-aid (25861904, 26463082, and 26463033) from the Japan Science and Technology Agency. We thank N. Okita (Department of Orthodontics, Osaka Dental University) for technical advice for cell culturing.