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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.
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.
摘要：
背景：骨骼干细胞（SSC）是骨骼发育所必需的，稳态，和修复。它们在再生医学方法中广泛应用的观点支持了这一领域的研究，尽管到目前为止诊所的结果还没有达到预期，可能也是由于内在的部分知识，潜在可行的SSC调节因素。其中,多效性细胞因子RANKL，在骨生物学中也有重要作用，是值得深入研究的候选人。
方法：为了剖析RANKL细胞因子在SSC生物学中的作用，我们通过细胞荧光分选和分析来自不同骨骼区室的SSC群体，在缺乏Rankl（Rankl-/-）的小鼠中进行了SSC和下游祖细胞（SSPC）的离体表征。基因表达分析,和体外成骨分化。此外,我们评估了抗RANKL阻断抗体Denosumab(已批准用于病理性骨丢失患者的治疗)药物治疗对人健康受试者骨髓来源基质细胞(hBMSCs)成骨潜能的影响.
结果：我们发现，不管骨化类型的骨，与野生型相比,Rankl-/-小鼠中的骨软骨SSC具有更高的频率和沿着骨软骨谱系的分化受损。Rankl-/-小鼠也具有来自血管周围SSC的定型骨软骨形成和脂肪形成祖细胞的频率增加。这些变化不是由于缺乏破骨细胞吸收（明确的骨硬化）引起的密度增加的特殊骨表型;它们没有在另一个石骨鼠模型中发现，即，oc/oc鼠标，因此不是由于石骨症本身。此外,Rankl-/-SSC和原代成骨细胞显示出降低的矿化能力。值得注意的是，与对照培养物相比,用Denosumab体外处理的hBMSCs具有降低的成骨能力。
结论：我们首次提供了来自严重隐性石斑病小鼠模型的SSPCs特征。我们证明了鼠SSC中的Rankl遗传缺陷和hBMSCs中的功能阻断降低了其成骨潜力。因此,我们认为RANKL是具有翻译相关性的SSC特征的重要调控因子。
方法：为了剖析RANKL细胞因子在SSC生物学中的作用，我们通过细胞荧光分选和分析来自不同骨骼区室的SSC群体，在缺乏Rankl（Rankl-/-）的小鼠中进行了SSC和下游祖细胞（SSPC）的离体表征。基因表达分析,和体外成骨分化。此外,我们评估了抗RANKL阻断抗体Denosumab(已批准用于病理性骨丢失患者的治疗)药物治疗对人健康受试者骨髓来源基质细胞(hBMSCs)成骨潜能的影响.
结果：我们发现，不管骨化类型的骨，与野生型相比,Rankl-/-小鼠中的骨软骨SSC具有更高的频率和沿着骨软骨谱系的分化受损。Rankl-/-小鼠也具有来自血管周围SSC的定型骨软骨形成和脂肪形成祖细胞的频率增加。这些变化不是由于缺乏破骨细胞吸收（明确的骨硬化）引起的密度增加的特殊骨表型;它们没有在另一个石骨鼠模型中发现，即，oc/oc鼠标，因此不是由于石骨症本身。此外,Rankl-/-SSC和原代成骨细胞显示出降低的矿化能力。值得注意的是，与对照培养物相比,用Denosumab体外处理的hBMSCs具有降低的成骨能力。
结论：我们首次提供了来自严重隐性石斑病小鼠模型的SSPCs特征。我们证明了鼠SSC中的Rankl遗传缺陷和hBMSCs中的功能阻断降低了其成骨潜力。因此,我们认为RANKL是具有翻译相关性的SSC特征的重要调控因子。
