Optimizing In Vitro Osteogenesis in Canine Autologous and Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells with Dexamethasone and BMP-2

Abstract

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.

Keywords: MSCs; bone morphogenic protein-2; canine; induced pluripotent cells; mesenchymal stromal cells; osteogenic differentiation.

FIG. 1.

Overall experimental approach and design. On Day 0, passage 2 (autologous) and passage 21 (iPS-derived) cMSCs were seeded at 1 × 10⁴ cells/cm² and allowed to attach overnight. On Day 1, osteogenic media were supplemented. For ALP and qPCR assays, dexamethasone was supplemented beginning at this Day 1 timepoint. For ALZ assays, dexamethasone was supplemented beginning at this Day 1 timepoint only for the Early Dex treatment groups. On Day 7, dexamethasone was introduced for the first time to the Late Dex ALZ treatment groups. Media exchange for all treatment groups was performed twice weekly. RNA was isolated for qPCR at Days 3, 7, 14, and 21. All ALP assays were performed at Day 7. All ALZ assays were performed at Day 21. ALP, alkaline phosphatase; ALZ, Alizarin red stain; cMSCs, canine mesenchymal stromal cells; iPS, induced pluripotent stem cell; qPCR, quantitative polymerase chain reaction. Color images are available online.

FIG. 2.

Early-stage osteogenesis of marrow-derived and iPS-derived cMSCs. Osteogenesis was evaluated using the ALP activity assay. Passage 2 (autologous) and passage 21 (iPS-derived) cMSCs were cultured in CCM or osteogenic media supplemented with varying concentrations of dexamethasone and rhBMP-2 (n = 3 wells/condition). At Day 7, ALP activity was determined by measuring the colorimetric conversion of PNPP to PNP and normalizing to cell number through DNA quantification. Statistical analysis was performed using two-way ANOVA and Tukey's post hoc test. Letters denote significant differences in ALP activity between different concentrations of dexamethasone within individual rhBMP-2 treatment groups (P < 0.05). Symbols denote significant differences in ALP activity between different concentrations of rhBMP-2 within individual dexamethasone treatment groups (P < 0.05). (a) IC018 ALP activity. Increasing concentrations of dexamethasone significantly reduced ALP activity in the presence of rhBMP-2. (b) H012 ALP activity. Increasing concentrations of dexamethasone reduced ALP activity in the presence of rhBMP-2, but this finding was only significant at higher concentrations of rhBMP-2. (c) iPS cMSC ALP activity. ALP activity was significantly reduced at high concentrations of dexamethasone within all concentrations of rhBMP-2. ANOVA, analysis of variance; rhBMP-2, recombinant human bone morphogenic protein-2; CCM, complete culture medium; PNPP, p-nitrophenylphosphate. Color images are available online.

FIG. 3.

Late-stage osteogenesis of IC018 marrow-derived cMSCs. Passage 2 cMSCs were cultured in CCM or osteogenic media supplemented with varying concentrations of dexamethasone and rhBMP-2 (n = 3 wells/condition). At Day 21, monolayers were formalin fixed, stained with ALZ, and photographed. Statistical analysis was performed using two-way ANOVA and Tukey's post hoc test with P < 0.05. (a) Representative photos demonstrate ALZ staining of monolayers from IC018 cells. (b) ALZ extraction demonstrates that early supplementation of dexamethasone at 10 or 100 nM resulted in the greatest degrees of monolayer mineralization, especially at high concentrations of rhBMP-2. Letters denote significant differences in ALZ recovery between different concentrations of dexamethasone administered beginning at Day 1, while symbols denote significant differences between dexamethasone administered beginning at Day 7 within individual rhBMP-2 treatment groups. cMSCs from this preparation have historical excellent osteogenic performance in our laboratory, and these data demonstrate that the osteogenic performance can be further improved by supplementing dexamethasone at high doses early in the assay. Color images are available online.

FIG. 4.

Late-stage osteogenesis of H012 marrow-derived cMSCs. Late-stage osteogenesis was performed as described. (a) Representative photos demonstrate ALZ staining of monolayers from H012 cells. (b) ALZ extraction demonstrates that early supplementation of dexamethasone at 100 nM resulted in the greatest degree of monolayer mineralization, especially at high concentrations of rhBMP-2. Letters denote significant differences in ALZ recovery between different concentrations of dexamethasone administered beginning at Day 1, while symbols denote significant differences between dexamethasone administered beginning at Day 7 within individual rhBMP-2 treatment groups (P < 0.05). cMSCs from this preparation have historical fair to poor osteogenic performance in our laboratory. These data demonstrate that the osteogenic performance of poor-performing cells may be enhanced by providing early supplementation of high dexamethasone concentrations. Color images are available online.

FIG. 5.

Late-stage osteogenesis of iPS-derived cMSCs. Late-stage osteogenesis was performed as described in Fig. 4. (a) Representative photos demonstrate ALZ staining of monolayers from iPS-derived cMSCs. (b) ALZ extraction demonstrates that the highest degree of ALZ recovery was achieved when cells were cultured in the absence of dexamethasone, but at high concentrations of rhBMP-2. In contrast, inclusion of dexamethasone, especially at early time points and high concentrations (10 and 100 nM), reduced ALZ recovery. Letters denote significant differences in ALZ recovery between different concentrations of dexamethasone administered beginning at Day 1, while symbols denote significant differences between dexamethasone administered beginning at Day 7 within individual rhBMP-2 treatment groups (P < 0.05). These data demonstrate that the osteogenic performance of iPS-derived cMSCs is markedly different from tissue-derived cMSCs in response to varying concentrations of dexamethasone and rhBMP-2. Color images are available online.

FIG. 6.

Quantitative assessment of gene transcription in bone marrow-derived and iPS-derived cMSCs. cMSCs were cultured as described in Fig. 1 for 3, 7, 14, and 21 days. At each time point total RNA was isolated and normalized to 125 ng to generate cDNA. Real-time qPCR was performed for three canine osteogenic genes of interest. All genes were normalized to a housekeeping gene (RPL32). Relative gene expression is provided for (a) osteocalcin, (b) osterix, and (c) Runx2. Statistical analysis was performed using two-way ANOVA and Tukey's post hoc test with P < 0.05. Letters denote significant differences in relative gene expression for each cell preparation within individual rhBMP-2 treatment groups. cDNA, complimentary DNA. Color images are available online.