Endocrine Abstracts

Epi/genetic defects at the imprinted GNAS locus, that encodes the α subunit of the stimulatory G protein (Gsα), have been associated with a heterogeneous group of rare diseases, termed as Pseudohypoparathyroidism. Most GNAS-based disorders have the common feature of episodic de novo formation of heterotopic ossifications (HO) in subcutaneous tissues. The mosaic tissue distribution of HO suggests that pathogenesis involves an abnormal differentiation of precursor cells, and that GNAS defects provide a sensitized background, but the molecular mechanism is still under investigation. To date, investigations have been mainly conducted in mouse models, thus we have developed an in vitro human model from surgically removed samples of subcutaneous fat (adipose-derived mesenchymal stem cells, ADMSCs) to study the determinants underlying HO formation in GNAS-related diseases. The multilineage differentiation ability of ADMSCs was confirmed via biochemical assays, specific stainings and expression analysis of specific markers. We also determined the preservation of GNAS imprinting profile during cell culture and handling. Subsequently, cells were treated with siRNAs against Gsα-specific or non-specific GNAS transcripts, the efficiency of transfection (ranging from about 75% at day 4, up to about 50% at day 21) was evaluated, as well as the effect on cell differentiation toward osteoblasts. No significant differences due to different siRNA target sequences were observed. The increase of alkaline phosphatase activity reflected cell maturation and non-induced silenced cells displayed an intermediate phenotype between cells grown in control and inducing medium (Abs 405 nm at day 14: silenced=0.507±0.16, control=0.076±0.01, induced=0.861±0.13, P<0.05). Moreover, Gsα-deficient cells, although not able to completely differentiate and depose mineralized matrix, began to lay down few, interspersed and very small calcium deposits. Future experiments will expand our knowledge in molecular mechanisms underlying HO formation in GNAS-related disorders, in order to test pharmacological treatments against HO formation and progression.