Abstract
Our bone, to an extent, is an organ whose maintenance and rebuilding process is facilitated by bone marrow resident mesenchymal stem cells (MSCs). MSCs can differentiate into a variety of lineages including but not limited to osteoblasts, adipocytes and chondrocytes. One of the many functions that MSCs are responsible for is the healing of bone fractures through differentiation and paracrine activity, such as promoting angiogenesis and modulating the immune response. However, due to several factors such as poor overall health or aging, regeneration of bone caused by a fracture may be prolonged or may not occur at all. There is a need to develop therapeutics that safely promote bone fracture healing, but more research is needed. Although MSCs have shown a good safety profile, they have often not met the expected therapeutic outcomes. One approach to increase the efficacy of MSCs is by genetically modifying the cells, to exacerbate the secretion of therapeutic (paracrine) factors.
The WNT family of proteins plays an important role in the differentiation of MSCs into osteoblasts. It has also been observed that WNT4 and WNT5a expression are significantly increased in fibrous dysplasia, a condition where bone mass is significantly increased. Thus, we over-expressed these two genes individually in MSCs in vitro and analyzed effects that led us to believe that these findings can lead to a therapeutic that can promote and expedite bone fracture healing. MSCs need to be first tested in vitro to analyze what the over-expression of these Wnt genes does to the MSCs themselves. We hypothesized that osteoblast precursor differentiation would increase, however mature osteoblast differentiation may not. Each experiment was done with at least two biological replicates and three technical replicates. To over-express WNT, lentiviruses to express the previously stated Wnt genes were cloned. To show successful functionality of the lentivirus, MSCs transduced with the respective lentiviruses were tested for over-expression of WNT by means of PCR (mRNA level). Once over- expression was confirmed, cells were tested for any changes in proliferation by measuring metabolic activity using an MTT assay and by cell counting with a hemocytometer. To characterize MSC differentiation, cells were tested for differentiation into two cell types, adipocytes, and osteoblasts. To measure osteoblastic differentiation, MSCs were tested using two different methods. The alkaline phosphatase assay tested for the activity of alkaline phosphatase, and Alizarin Red S staining showed the presence of mineral deposits in cells. To test adipocytic differentiation, we used the Oil Red O assay to stain for droplets. Results showed that proliferation was not affected by the overexpression of WNT4 or WNT5a. However, as expected, early osteoblast formation was upregulated while mature osteoblast formation was also increased in both conditions, as compared to controls. Because results are looking promising and suggesting that there may in fact be an induction of osteogenesis, future experiments will test these cells in fracture models.