Comparison of osteogenic differentiation of embryonic stem cells and primary osteoblasts revealed by responses to IL-1β, TNF-α, and IFN-γ

Abstract

There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.

FIG. 1.

Osteogenic differentiation of mouse embryonic stem cells (osteo-mESCs) and mouse primary calvarial osteoblasts. (A) Representative images of alizarin red S staining of bone nodule formation by osteo-mESCs (a, c, e) and primary osteoblasts (b, d, f) in the osteogenic (a, b, e, f) and nonosteogenic medium (c, d) at 21 days of culture. Whole-well images of bone nodule formation (scale bar=2 mm) (a–d). Higher magnification image of osteo-mESC (e) and primary osteoblast (f) bone nodules in the osteogenic medium (scale bar=20 μm). (B) Immunocytochemistry showing expression of collagen-I (col-I) (green), cadherin-11 (cad-11) (red), and nuclei (blue) in bone nodules formed by osteo-mESCs (a) and primary osteoblasts (b) at day 18 of the osteogenic culture (representative images, scale bar=48 μm). (C) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis performed on primary osteoblasts and osteo-mESCs at day 18 of the culture for osteogenic markers Runx2, Col1a1, Opn, and Ocn. Gene expression of each target gene normalized to Rpl32, and expression of osteo-mESC genes expressed relative to primary osteoblasts. Data represent mean±SD of three independent experiments (n=3). *Statistical significance of primary osteoblasts versus osteo-mESCs, P≤0.05. (D) Immunocytochemistry showing expression of osteopontin (OPN) (red) and osteocalcin (OCN) (green) by osteo-mESCs (a–c, g–i) and primary osteoblasts (d–f, j–l) cultured in the osteogenic (a–f) and nonosteogenic (g–l) medium. Merge image shows OPN and OCN with Hoechst nuclear staining (representative images, scale bar=90 μm). Color images available online at www.liebertpub.com/scd

FIG. 2.

Response of osteo-mESCs to proinflammatory cytokine stimulation compared to primary osteoblasts. (A, B) Nitrite and prostaglandin E₂ (PGE₂) concentration released into the culture medium, respectively. Osteo-mESCs were treated with interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) for 48 h at day 0, 7, 14, or 21 of osteogenic culture. Primary osteoblasts were treated on day 7 of culture as a comparator. Nitrite and PGE₂ concentrations corrected for DNA content. Values are mean±SD for three separate experiments each with n=6. Statistical significance versus control of same cell type, *P≤0.01. (C) Immunocytochemistry showing inducible enzyme expression (inducible nitric oxide synthase [iNOS]: a–e, scale bar=90 μm; cyclooxygenase-2 [COX-2]: f–k, scale bar=46 μm; prostaglandin E synthase [PGES]: l–p, scale bar=46 μm) of primary osteoblasts at day 14 and osteo-mESCs at day 0, 7, 14, or 21 of osteogenic culture, in response to 48 h stimulation with IL-1β, TNF-α, and IFN-γ, before fixation. (D, E) Dose response effect of proinflammatory cytokines on cell viability of primary osteoblasts (D) and osteo-mESCs (E). Cells were treated with increasing concentrations of a combination of IL-1β, TNF-α, and IFN-γ over a 24-day time period. Cytokine doses: A (IL-1β 0.03125 ng/mL, TNF-α 0.3125 ng/mL, IFN-γ 3.125 ng/mL); B (IL-1β 0.0625 ng/mL, TNF-α 0.625 ng/mL, IFN-γ 6.35 ng/mL); C (IL-1β 0.125 ng/mL, TNF-α 1.25 ng/mL, IFN-γ 12.5 ng/mL); D (IL-1β 0.25 ng/mL, TNF-α 2.5 ng/mL, IFN-γ 25 ng/mL); E (IL-1β 0.5 ng/mL, TNF-α 5 ng/mL, IFN-γ 50 ng/mL); F (IL-1β 1 ng/mL, TNF-α 10 ng/mL, IFN-γ 100 ng/mL). Viability of cells at certain time points was measured by MTS assay. Data shown as optical absorbance proportional to cell viability. Values are mean±SD (n=6, experiment repeated in triplicate). Statistical significance against control *P≤0.05. Color images available online at www.liebertpub.com/scd

FIG. 3.

Effect of IL-1β, TNF-α, and IFN-γ on bone nodule formation of osteo-mESCs and primary osteoblasts. Cells were stimulated at day 0, 3, 7, or 14 with IL-1β, TNF-α, and IFN-γ for 48 h, before being returned to the osteogenic medium and cultured continuously until either day 14 (alkaline phosphatase) or day 21. (A) Representative images of alizarin red S staining of calcium deposits in bone nodules formed by osteo-mESCs (a–e) and primary osteoblasts (f–j). Scale bar=2 mm. (B) Representative phase-contrast images of osteo-mESCs (a–e) and primary osteoblasts (f–j) at day 21. (C) Immunocytochemistry showing OPN (red) and OCN (green) staining at day 21. Hoechst nuclear counterstain. (D) Immunocytochemistry showing staining of cad-11 (red) and col-I (green) at day 21. Hoechst nuclear counterstain. Scale bars for (B–D) at 90 μm. (E, F) Image quantification of alizarin red staining of osteo-mESCs and primary osteoblasts, respectively. Values corrected to proportion of osteogenic control. Mean±SD (n=6). (G, H) Alkaline phosphatase activity of osteo-mESCs and primary osteoblasts, respectively. Values are mean±SD, experiment repeated three times, each with n=6. Statistical significance compared to control; **P≤0.01, ***P≤0.001. Color images available online at www.liebertpub.com/scd

FIG. 4.

Osteogenic differentiation of cad-11 sorted osteo-mESCs and effect of proinflammatory cytokines on bone nodule formation. Cells were sorted for cad-11 at day 16 of osteogenic culture and positive and negative fractions cultured separately in osteogenic medium. Cells were stimulated with IL-1β, TNF-α, and IFN-γ for 48 h, at day 7 or 14 post-sorting before being returned to the osteogenic medium and cultured until day 21. (A) Alizarin red staining of calcium deposits in bone nodules formed by cad-11⁺ mESCs (a–c) and cad-11⁻ mESCs (d–f). Scale bar=2 mm, representative images. (B) Phase-contrast images of cad-11⁺ mESCs (a–c) and cad-11⁻ mESCs (d–f). (C) Immunocytochemistry showing OPN (red) and OCN (green) staining of cad-11⁺ mESCs (a–c) and cad-11⁻ mESCs (d–f). (D) Immunocytochemistry showing col-I staining of cad-11⁺ mESCs (a–c) and cad-11⁻ mESCs (d–f). (C, D) Hoechst nuclear counterstaining, scale bars=46 μm, representative images. Color images available online at www.liebertpub.com/scd

FIG. 5.

Response of cad-11 sorted cells to proinflammatory cytokine stimulation. (A, B) Nitrite concentration in the culture medium at day 7 and 14, respectively, after 48 h of IL-1β, TNF-α, and IFN-γ treatment. Values are mean±SD, n=9. Statistical significance of cytokine treated versus control, ***P≤0.001. Statistical significance of cad-11⁻ versus cad-11⁺, ^###P≤0.001. (C, D) PGE₂ concentration in the culture medium at day 7 and 14, respectively, after 48 h of IL-1β, TNF-α, and IFN-γ treatment. Values are mean±SD, n=9. Statistical significance of cytokine treated versus control, ***P≤0.001, **P≤0.01. Statistical significance of cad-11⁻ versus cad-11⁺, ^#P≤0.05. (E) Phase-contrast images and immunocytochemistry of iNOS expression, in both control cultures (a–d, upper images) and cytokine stimulated (e–h, lower images) of cad-11⁺ (a, e, c, g) and cad-11⁻ (b, d, f, h) cultures, at day 7 (a, b, e, f) and day 14 (c, d, g, h) post-sorting. Representative images, scale bar=46 μm. Color images available online at www.liebertpub.com/scd