Suppression of cbfa1 expression in C3H10T1/2 mesenchymal stem cells by anti-cbfa1 siRNA blocks osteoblastic develoment, but does not affect chondrogenesis

Introduction

Mesenchymal stem cells (MSC) may differentiate into adipocytes, osteoblasts or chondrocytes, the latter leading to hypertrophic chondrocytes responsible for endochondral ossification. The presence of the transcription factor cbfa1 is a prerequisite for osteoblast development, but is also of major importance in chondrocyte transition to the hypertrophic state. For cartilage tissue engineering, we need to prevent hypertrophic phenotype of MSC differentiated to chondrocytes. For this, we transfected mesenchymal stem cells transiently with anti-cbfa1 siRNA and investigated its effect on bone morphogenic protein (BMP)-2-induced osteogenesis in monolayer culture, or chondrogenesis in cultured micropellets.

Methods

We used the murine MSC C3H10T1/2 to obtain chondrogenic differentiation in micropellets. In order to assess impact of cbfa1 inhibition, we used a plasmid encoding the osteocalcin promoter coupled to the luciferase gene. Cells were transfected using 1–30 pmoles siRNA designed to inhibit cbfa1 per 10,000 cells and oligofectamine according to standard protocols, and thereafter incubated for up to 28 days.

Results

It appeared that both the cbfa1 expression (RT-PCR) and nuclear protein levels (immunohistochemistry) were diminished up to 75% at day 5. Furthermore, cells transfected with a plasmid containing the osteocalcin promoter coupled to the luciferase gene displayed an approximate 65% reduction in luciferase activity subsequent to transient exposure to anti-cbfa1 siRNA. cbfa1 suppression also markedly reduced the expression of several osteoblast-related genes (i.e. osterix; collagen-I; alkaline phosphatase; osteocalcin; secreted protein acidic, cysteine-rich or osteonectin; and osteopontin).

Functional parameters such as enzymatic activity of alkaline phosphatase and mineralization surface profiles over a period of 28 days were both delayed by a mean of 8 days. In contrast, cells differentiated to chondrocytes in micropellets and transfected with the siRNA duplex did not display altered expression of aggrecan or collagen II, as compared with nontransfected cells.

Conclusion

In summary, these experiments indicate that a transient and substantial suppression of cbfa1 in C3H10T1/2 cells, grown in the presence of BMP-2, is sufficient to delay mineralization, while not reducing their ability to differentiate towards functional chondrocytes. Vectors to express anti-cbfa1 siRNA and to stably transfect stem cells are in progress.