Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation

Abstract

Background Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. Methods To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl−/−) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). Results We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl−/− mice as compared to wild-type. Rankl−/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl−/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. Conclusions We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

1. Introduction

Skeletal stem cells (SSCs) are a bone-specific subtype of somatic stem cells crucial for bone physiology [1]. They are endowed with self-renewal and multipotency capacity, and give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes in different proportions, depending on the compartment of origin (i.e., bone marrow, periosteum, growth plate) [2–6]. Gene expression analysis of SSCs and downstream progenitor populations identified ligands and receptors of signaling pathways that may modulate the activity of the SSCs and their progeny, and suggested a control based also on paracrine and/or autocrine molecular cues [7, 8]. Intrinsic regulatory factors may represent a means to tune the regenerative potential of mesenchymal progenitors, which is a highly pursued (but poorly achieved) goal due to the expected broad application in the field of regenerative medicine [9].

The Receptor Activator of NF-kB Ligand (RANKL) cytokine is recognized as a pleiotropic factor since its discovery; in fact, it was originally cloned by three independent groups and classified as a dendritic cell survival factor [10], a regulator of T cell function [11] and an essential osteoclast differentiation factor [12]. In line with this key function in the bone microenvironment, Rankl deficient (Rankl−/−) mice as well as patients bearing mutations in the TNFSF11 gene (encoding RANKL) display severe osteopetrosis owing to lack of osteoclast formation [13, 14]. Several additional roles have been recognized for RANKL in pathophysiological conditions, making this cytokine an interesting target for therapy [15–19]. Of note, we previously reported that murine bone marrow-derived mesenchymal stromal cells (BMSCs) lacking Rankl (Rankl−/−) displayed a partial osteogenic defect that was corrected