Murine Rankl−/− Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL-Expressing Lentiviral Vector

Abstract

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl mice, and found that Rankl BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.

Mesenchymal Stromal Cell-Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl-Deficient Osteopetrosis

“…We already reported evidence showing that a similar cell‐scaffold construct was well tolerated and osteoinductive in immunodeficient mice . Here, we verified the in vivo biocompatibility of the cell‐seeded MgHA/Col scaffold and the effect of lentivirally mediated RANKL production in the Rankl −/− mouse.…”

“…Rankl −/− MSCs were transduced with either LVhsRL or mock lentiviral vector at 20 MOI, as previously described, and maintained in MSC complete Mesencult medium (Stemcell Technologies, Vancouver, BC) .…”

Soluble Factors on Stage to Direct Mesenchymal Stem Cells Fate

“…In line with these observations, our group has found that BM-MSCs derived from RANKL-deficient mice display a partial osteogenic differentiation defect, which is improved by restoring the production of the soluble form of the cytokine. Our data suggest that RANKL might contribute to direct MSCs fate in an autocrine/paracrine manner, likely through the interaction with either its receptor RANK (Schena et al, 2017) or the recently identified RANKL receptor LGR4 (Luo et al, 2016) (an R-spondin receptor, suggested to regulate bone formation in synergy with Wnt3a), which are both expressed in MSCs (Schena et al, 2017). On this basis, we might speculate that fine tuning, rather than completely blocking, RANKL could be relevant to regulate bone physiology.…”

“…MSCs, identified in bone tissue as precursor cells of osteoblasts\osteocytes, chondrocytes, and marrow adipocytes, are defined as multipotent cells that can be easily isolated from the stromal fraction. MSCs exhibit in vitro plastic adherence, fibroblast spindle-like shaped morphology, and expression of a panel of surface molecules that is continuously refined to identify unique markers for bona fide MSCs definition (Bourin et al, 2013; Schena et al, 2017). MSCs possess self-renewal and clonogenic capacity and highly proliferate and differentiate at least toward the osteogenic, adipogenic, and chondrogenic lineages both in vitro , by means of specific differentiation media, and in vivo in an ectopic bone formation assay (Schena et al, 2017).…”

“…MSCs exhibit in vitro plastic adherence, fibroblast spindle-like shaped morphology, and expression of a panel of surface molecules that is continuously refined to identify unique markers for bona fide MSCs definition (Bourin et al, 2013; Schena et al, 2017). MSCs possess self-renewal and clonogenic capacity and highly proliferate and differentiate at least toward the osteogenic, adipogenic, and chondrogenic lineages both in vitro , by means of specific differentiation media, and in vivo in an ectopic bone formation assay (Schena et al, 2017). In bone, MSCs are located around sinusoids and along the perivascular network in the stroma (Sacchetti et al, 2007; Mendez-Ferrer et al, 2010), where they take part in the generation of the complex and heterogeneous system of the bone marrow microenviroment (BM-ME).…”