Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.
Department of Clinical Sciences, Center for Immune and Regenerative Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.
Stem Cells Dev. 2021 Feb;30(4):214-226. doi: 10.1089/scd.2020.0144. Epub 2021 Feb 8.
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 < 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.
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，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 成本高昂，可能会影响犬模型的转化相关性。地塞米松增强了人骨髓间充质干细胞（hMSCs）的成骨分化，并且广泛用于成骨方案中。本研究旨在确定 BMP-2 和地塞米松对自体和诱导多能干细胞（iPS）衍生的 cMSCs 的早期和晚期成骨的影响。选择两种骨髓来源的 cMSC 制剂来代表异常或边缘成骨的自体 cMSCs。iPS 衍生的 cMSCs 是从犬成纤维细胞中生成的。所有制剂均通过碱性磷酸酶（ALP）活性、成骨单层的茜素红染色和定量聚合酶链反应进行评估。数据以平均值±标准差表示，并使用单因素或双因素方差分析以及 Tukey 或 Sidak 事后检验进行比较。设定显著性水平为 < 0.05。在早期测定中，地塞米松在存在 BMP-2 的情况下降低了所有 cMSCs 的 ALP 活性。在晚期测定中，在培养的第 1 天添加地塞米松和 BMP-2 可使自体 cMSCs 的单层矿化非常强烈。在第 1 天提供 100 nM 地塞米松可改善矿化并减少达到边缘 cMSCs 矿化所需的 BMP-2 浓度。对于 iPS-cMSCs，地塞米松对 ALP 活性和单层矿化均具有抑制作用。在自体 cMSCs 中，BMP-2 可增加骨钙素和骨桥蛋白的表达，但在 iPS cMSCs 中则表达更为温和。虽然自体和 iPS 衍生的 cMSCs 在早期成骨测定中反应相似，但它们在晚期矿化测定中对地塞米松和 BMP-2 的反应独特。本研究表明，地塞米松和 BMP-2 可以在时间和浓度依赖性方式中进行滴定，以增强自体 cMSC 制剂的成骨作用。这些结果将有助于从事 cMSCs 转化研究的研究人员，并提供对 iPS 衍生的 cMSC 成骨作用的深入了解。
