Rhtgf β3

ELISA was used to quantify TGF-β3 levels in conditioned cell culture media from primary endometrial stromal cells and leiomyoma cells. Culture media, consisting of DMEM containing 1% penicillin, 1% streptomycin, 1% Amphotericin B, and 10% FBS was also analyzed. TGF-β ELISA was performed using a commercially available kit per the manufacturer’s protocol (R&D Systems, Minneapolis, MN). Briefly, the microplate was coated with capture antibody overnight. Aliquots of conditioned culture media were obtained from cell culture flasks 24 hours after addition of fresh media, when cells were at 60–70% confluence. Latent TGF-β3 in samples was activated to the immunoreactive form by addition of 1N HCl, followed by 1.2N NaOH/0.5M HEPES prior to assay. After blocking to prevent non-specific binding, diluted samples and standards (rhTGF-β3, R&D Systems, Minneapolis, MN) were plated, followed by detection antibody, Streptavidin-HRP, substrate solution, and a stop solution of 2N H₂SO₄. Color intensity was quantified by spectrophotometry at 450 nm using an iMark microplate absorbance reader (BioRad, Hercules, CA. A standard curve was generated and the TGF-β3 concentration of each sample was calculated by regression analysis. Each sample was assayed in duplicate. Mean TGF-β3 concentration was calculated for un-conditioned media, ESC-conditioned media, and Leiomyoma cell conditioned media (LCM).

Flag-Smad3ΔC (aa 1–381, lacks the C-terminal 43 amino acids and functions as DN-Smad3) vector and wild type and null Smad3 MEFs were provided by Dr. Rik Derynck, University of California at San Francisco. Smad7 (catalog #11733) and Smad3 (#11742) plasmids were obtained from Addgene. Galectin-3 promoter and its 5′ deletion constructs have been described earlier [20 (link)]. The vector pRL-TK (Promega) containing Renilla luciferase gene was used as an internal transfection control. The amount of transfected plasmid, the pre-transfection period after seeding, and the post-transfection period before harvesting, have been optimized for NP cells using pSV β-galactosidase plasmid (Promega) [43 (link)]. rhTGFβ3 was from R&D systems. rTNF-α and Galectin-3 was from Peprotech.

To evaluate transduction efficiency during scaffold-mediated transduction, MSCs were seeded and transduced as above with either constitutive or inducible eGFP LV (n=3) or remained non-transduced (n=2). Two days following transduction, all constructs were given 1 μg/mL dox. Six days after transduction, constructs were digested in Pronase (Calbiochem, San Diego, CA) and Collagenase type II (Worthington, Lakewood, NJ) and minced to isolate MSCs for flow cytometry, as previously described [37 ].
To assess the extent to which IL-1Ra production with the dox-inducible vector was tunable and repeatable during development of engineered cartilage constructs, MSCs were seeded and transduced as described above with inducible IL-1Ra LV or inducible eGFP LV. After 11 days of pre-culture in expansion medium to allow the seeded cells to proliferate and fill the pores within the scaffold, constructs were switched to chondrogenic medium for 36 days and subjected to different treatment courses of 1 μg/mL dox (n=4/group). Chondrogenic medium consisted of DMEM-HG (Gibco), 1% pen/strep (Gibco), 1% ITS⁺, 100 nM dexamethasone (Sigma-Aldrich), 50 μg/ml L-ascorbic acid (Sigma-Aldrich), 40 μg/ml L-proline (Sigma-Aldrich), and 10 ng/mL rhTGF-β3 (R&D Systems, Minneapolis, MN). Medium was collected and replaced every 3 days.