Abstract
Fibrous Dysplasia (FD) is a rare bone disorder that causes abnormalities characterized by replacing healthy bone tissue with abnormal fibrous connective tissue. The severity of the symptoms varies from a mild form of the disease in which one bone is affected termed monostotic, to a more severe form in which several bones are affected termed polyostotic. FD is not inherited but rather is due to post-zygotic somatic mutations in the GNAS gene complex locus (Guanine Nucleotide binding protein, Alpha Stimulating activity polypeptide). The GNAS gene makes a specific transcript leading to production of the Gsα polypeptide which is a regulatory subunit for a G protein signal transduction complex. Specifically, mutations in Gsα subunit at amino acid position 201 which is normally an arginine is substituted with cysteine (R201C) or histidine (R201H). The goal of this study was to investigate the paracrine effect of FD-like Mesenchymal stem cells / multipotent stromal cells (MSCs) expressing mutant forms of Gsα on healthy MSCs. MSCs show potent osteogenic differentiation potential and are used in this project to replicate the FD model. We hypothesized that the healthy MSCs exposed to MSCs expressing Gsα-R201C or Gsα-R201H genes would have increased osteogenic differentiation, meaning the paracrine effect is causing MSCs to become more osteoblastic than usual, mimicking the dysregulation we see in FD patients. Lentiviral transduction was used to introduce GFP along with overexpressed Gsα mutations (Gsα-R201C or Gsα-R201H) into MSCs and are compared to MSCs transduced with control constructs with only GFP or another copy of the normal gene for overexpression of Gsα wild-type (Gsα-WT). To evaluate proliferation, we performed cell counts at specific day time points, which showed that Gsα-WT and control had the most proliferation overall, however statistically not significant. To test differentiation, we used an Alkaline phosphatase assay (ALP) which has the highest level of activity in early-stage osteoblast cell types. ALP assays revealed reduced pre-osteoblastic differentiation in MSCs exposed to the supernatant of Gsα-R201C or Gsα-R201H transduced MSCs as compared to controls. Conversely, Alizarin Red Staining (ARS) was also performed to compare the amount of hydroxyapatite deposits indicating enhanced mineralization. The ARS results showed that healthy MSCs exposed to the supernatant of Gsα-R201C or Gsα-R201H transduced MSCs had enhanced osteoblastic differentiation. In addition, flow cytometry and a Phospho-Kinase Array analysis was performed to evaluate which signaling pathway had been activated in MSCs when exposed to the supernatant of mutant MSCs. These studies revealed an increase in the activation of bone formation-related pathways, most notably the Wnt signaling pathway, in cells exposed to supernatant from mutated MSCs as compared to controls. In conclusion, our studies indicate that there are possible paracrine effects from MSCs harboring mutant Gsα genes which is a promising first step in exploring potential therapeutics to help treat or cure Fibrous Dysplasia.