CELLULAR DETERMINANTS OF ECTOPIC BONE FORMATION IN ALBRIGHT HEREDITARY OSTEODYSTROPHY, PSEUDOHYPOPARATHYROIDISM AND GNAS-RELATED DISEASES

Pseudohypoparathyroidism is a term used to identify a heterogeneous group of rare diseases associated with genetic and/or epigenetic defects at the imprinted GNAS locus, that encodes also for the α subunit of the stimulatory G protein (Gsα). Most GNAS-based disorders share the common feature of episodic de novo formation of heterotopic ossifications (HO), a pathological formation of bone in extra-skeletal tissues. The mosaic tissue distribution of HOs suggested that their pathogenesis involved the abnormal differentiation of precursor cells located in subcutaneous tissues, mesenchymal stem cells, and that GNAS defects provided a sensitized background promoting osteoblastogenesis. This study attempted to clarify the molecular mechanisms underlying HO formation in GNASrelated diseases, as, although the growing knowledge, they are not completely understood. To this aim, we developed an in vitro human model of adipose-derived mesenchymal stem cells (ADMSCs) from surgically removed samples of subcutaneous fat of healthy donors. Firstly, we confirmed the ability of our ADMSCs to differentiate into osteoblasts and adipocytes by specific biochemical assays, stainings and lineage-specific markers expression. We determined that in vitro manipulation did not affect the maintenance of GNAS imprinting status. Finally, In order to investigate the effect of GNAS haploinsufficiency, we transfected ADMSCs with siRNAs designed to specifically silence different GNAS transcripts, obtaining an efficiency of about 75% at day 4, which decreased at about 50% at day 21. Then, we analyzed the expression of different osteogenic markers in GNAS-silenced ADMSCs and we observed that Gs alpha haploinsufficiency promoted their commitment towards the osteoblastic differentiation. In conclusion, our study showed that ADMSCs are a good model to study the osteogenic differentiation and the formation of HOs in GNAS-related diseases. In particular, we observed that GNAS silencing promotes ADMSCs commitment towards the osteoblast lineage, even in the absence of additional osteogenic-inducing stimuli. Moreover, no significant differences due to use of different GNAS siRNAs were observed, further supporting the hypothesis that the formation of ectopic bone in GNAS-related diseases is predominantly Gsα-mediated.