ECTS2016 Poster Presentations Other diseases of bone and mineral metabolism (52 abstracts)
Rankl−/− mesenchymal stromal cells have an unexpected osteogenic differentiation defect which is improved by a RANKL-expressing lentiviral vector
Ciro Menale 1, , Francesca Schena 3 , Lorenzo Diomede 1, , Lucia Sergi Sergi 4 , Lucia Susani 1, , Eleonora Palagano 5, , Elisabetta Traggiai 6 , Cristina Sobacchi 1, & Anna Villa 1,
1CNR-IRGB, Milan Unit, Milan, Italy; 2Humanitas Clinical and Research Institute, Rozzano, Milan, Italy; 3Istituto Gaslini, Genoa, Italy; 4San Raffaele TIGET, Milan, Italy; 5University of Milan, Milan, Italy; 6Novartis Institute for Research in Biomedicine, Basel, Switzerland.
Osteoclast-poor RANKL-dependent autosomal recessive osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Haematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Whether also these cells, besides the osteoclast, are in some way affected by RANKL deficiency is not known. To verify this, we established and characterized bone marrow derived Mesenchymal Stromal Cells (BM-MSC) from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from WT mice. No differences in morphology, immunophenotype and proliferation capacity were found between KO and WT MSC. However, KO MSC displayed a reduced clonogenic potential with a decrease of stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while displayed a significantly impaired osteogenic differentiation capacity compared to the WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we corrected the genetic defect by transducing KO MSC with a third generation lentiviral vector expressing human soluble RANKL (hsRL). In corrected MSC hsRL production and secretion was measurable, stable over time and comparable to sRL levels in WT MSC. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to the KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. The expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Overall, the gene therapy approach to restore hsRL expression ameliorated KO MSC functionality and could be considered for an MSC-based therapy to treat RANKL-dependent ARO.
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Bone Abstracts
ISSN 2052-1219 (online)
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