In Vitro And In Vivo Disease Modeling Using Patient-Derived iPSCs To Characterize The Calcification Phenotype In Arterial Calcification Due To Deficiency of CD73 (ACDC)

ACDC is a recessive disease resulting from mutations in CD73 and manifests with tortuosity and vascular calcification of the medial layer. We previously modeled this disease in vitro; in vivo studies of the vasculature are... [ view full abstract ]

ACDC is a recessive disease resulting from mutations in CD73 and manifests with tortuosity and vascular calcification of the medial layer. We previously modeled this disease in vitro; in vivo studies of the vasculature are limited, as CD73 knockout murine models do not recapitulate the human disease. For further mechanistic studies we created ACDC and Control patient iPSCs and using a teratoma assay discovered that ACDC iPSC teratomas exhibit extensive bone-like calcifications, while Control iPSC tereatomas do not. To study osteogenesis Control and ACDC iPSCs were differentiated down the mesenchymal stromal cell (MSC) lineage. There was no difference in chondrogenesis and adipogenesis, however ACDC iMSCs underwent osteogenesis sooner than Control iPSC, showed higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP ablated this increase in adenosine suggesting that TNAP is upregulated in attempt to compensate for CD73-deficiency. Further, pyrophosphate, the TNAP substrate and endogenous inhibitor of ectopic calcification, was significantly reduced. Treatment with an A2b adenosine receptor agonist or rapamycin drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas in vivo. These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP attempts to compensate for CD73 deficiency, but subsequently induces calcification, which can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.