Adipose-derived Stromal Cell Implantation

Donor adipose-derived stromal cells (ASCs) were isolated from 4-week-old R26-CreERT2; Gnas^fl/fl; and Ai9^fl/+ and R26-CreERT2; Gnas^+/+; and Ai9^fl/+ control mice (Pignolo et al., 2011 (link)). Briefly, inguinal white adipose was dissected, minced using the two-scalpel method, treated with collagenase (Sigma) for 1 h at 37°C, filtered through 100 μm mesh, recovered by centrifugation, and plated in growth medium (DMEM/F12 with 20% FBS and 2% Antibiotic-Antimycotic). Media was changed after 24 h, and adherent cells were expanded and maintained in DMEM/F12 with 10% FBS and 1% Antibiotic-Antimycotic. Recombination was induced in vitro with 1 μM 4-hydroxytamoxifen (4-HT, Sigma) 48 h prior to implantation; control cells were also treated with 4-HT. ASCs were genotyped and checked for Ai9 expression via fluorescent imaging prior to implant. 4-week-old host animals (R26-CreERT2;Gnas^fl/fl) were treated with 3 consecutive daily subcutaneous injections of 4-HT, as described above, to both hindlimbs; 6 weeks after 4-HT treatment, 400,000 cells were suspended in 100 μL of Matrigel and implanted subcutaneously into hindlimbs (Culbert et al., 2014 (link)). Implanted mice were imaged via in vivo μCT at the indicated timepoints as described above.