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. Author manuscript; available in PMC: 2015 Mar 10.
Published in final edited form as: Stem Cells. 2011 Aug;29(8):1241–1255. doi: 10.1002/stem.670

Figure 5.

Figure 5

Bone morphogenetic protein (BMP) signaling underlies the pro-osteogenic effect of the dura mater (DM)-human adipose-derived stro-mal cell (hASC) paracrine relationship. (A): Analysis of relative BMP-2 secretion, as assessed by BMP-2 enzyme-linked immunosorbent assay. Conditioned media (CM) was collected under serum-free conditions from mouse DM cells, hASCs, and mouse osteoblasts as a positive control. BMP-2 quantification (in picomoles) is based on known quantities of recombinant BMP-2, n = 3; *, p < .05. A two-tailed Student’s t test was performed to assess significance. (B): Western blot image and quantification of SMAD-5 and phosphorylated SMAD (pSMAD)-1/5 as well as alpha tubulin control for ASCs after exposure to osteogenic differentiation medium (ODM) alone, ODM with DM CM, or ODM with DM CM and a BMP-2/4 inhibitory antibody (BMPi) at 3, 6, and 12 hours. (C): Quantification of SMAD-5 protein at 0, 3, 6, and 12 hours after exposure to ODM, ODM plus DM CM, or CM or ODM with DM CM and a BMP-2/4 inhibitory antibody. (D): Quantification of active or pSMAD-1/5 protein at 0, 3, 6, and 12 hours after exposure to ODM, ODM plus DM CM, or CM or ODM with DM CM and a BMP-2/4 inhibitory antibody. (E) Expression of Runt-related protein-2 after 3, 6, and 12 hours of coculture of hASCs with DM cells by quantitative real-time polymerase chain reaction (qRT-PCR); *, p < .05. A two-tailed Student’s t test was performed to assess significance, n = 3. (F): Expression of BMP-associated genes among hASCs with or without coculture with DM cells, by qRT-PCR; *, p < .05. A two-tailed Student’s t test was performed to assess significance, n = 3. (G): Expression of BMP-associated genes among hASCs with or without supplementation with DM derived CM, by qRT-PCR; *, p < .05. A two-tailed Student’s t test was performed to assess significance, n = 3. (H–K): Osteogenic differentiation assays of hASCs with or without DM CM and with or without neutralization by a anti-BMP-2/4 inhibitory antibody. (H, J): Alkaline phosphatase stain and quantification of hASCs under control conditions (ODM alone), ODM with BMPi, ODM with DM-derived CM, or ODM with DM CM and BMPi. (I, K): Alizarin Red stain and quantification of hASCs under control conditions (ODM alone), ODM with BMPi, ODM with DM-derived CM, or ODM with DM CM and BMPi, n = 3 for all assays; *, p < .05. A one-factor analysis of variance was used to compare different treatment groups, followed by a post hoc student’s t test to assess significance. Abbreviations: ASC, adipose-derived stromal cell; BMP-2, bone morphogenetic protein-2; CM, conditioned media; ODM, osteogenic differentiation medium; pSMAD, phosphorylated SMAD; RUNX-2, Runt-related protein-2.