A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as bone morphogenic protein-2 (BMP-2) for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs (hMSCs) and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and induced pluripotent stem cell (iPS)-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase (ALP) activity, Alizarin Red staining of osteogenic monolayers, and quantitative polymerase chain reaction. Data were reported as mean ± standard deviation and compared using one- or two-way analysis of variance and Tukey or Sidak post hoc tests. Significance was established at P < 0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.

中文翻译：

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用地塞米松和 BMP-2 优化犬自体和诱导多能干细胞衍生的间充质基质细胞的体外成骨

越来越多的研究表明，犬间充质基质细胞 (cMSCs) 需要额外的激动剂，例如骨形态发生蛋白 2 (BMP-2)，以实现一致的体外成骨分化。BMP-2 成本高，可能会挑战犬模型的转化相关性。地塞米松增强人 MSCs (hMSCs) 的成骨分化，并广泛用于成骨方案。本研究的目的是确定 BMP-2 和地塞米松对自体和诱导多能干细胞 (iPS) 衍生的 cMSCs 早期和晚期成骨的影响。选择两种骨髓来源的 cMSCs 制剂来代表异常或边缘成骨的自体 cMSCs。iPS 衍生的 cMSCs 由犬成纤维细胞产生。使用碱性磷酸酶 (ALP) 活性评估所有制剂，成骨单层的茜素红染色和定量聚合酶链反应。数据报告为平均值±标准差，并使用单向或双向方差分析和 Tukey 或 Sidak 事后检验进行比较。意义成立于磷 < 0.05。在早期试验中，地塞米松在 BMP-2 存在的情况下降低了所有 cMSCs 的 ALP 活性。在后期检测中，在培养的第 1 天加入地塞米松和 BMP-2 为自体 cMSCs 产生了强大的单层矿化。在第 1 天提供 100 nM 地塞米松改善了矿化并降低了实现边缘 cMSC 矿化所需的 BMP-2 浓度。对于 iPS-cMSCs，地塞米松对 ALP 活性和单层矿化均有抑制作用。自体 cMSCs 中骨钙素和 osterix 与 BMP-2 的表达增加，但 iPS cMSCs 中的表达更温和。虽然自体和 iPS 衍生的 cMSC 在早期成骨试验中的反应相似，但它们在晚期矿化试验中对地塞米松和 BMP-2 表现出独特的反应。该研究表明，地塞米松和 BMP-2 可以以时间和浓度依赖性方式滴定，以增强自体 cMSC 制剂的成骨。这些结果将证明对使用 cMSCs 进行转化研究的研究人员有用，同时提供对 iPS 衍生的 cMSC 成骨的洞察力。