Pam3Cys promotes MSC proliferation, inhibits in vitro “wound healing,” and does not affect the MSC's ability to inhibit T-cell response. MSCs were plated in MSC medium and starved with 2% serum-containing media for 24 hours following 10% FCS-containing media with Pam3Cys. MSC proliferation was measured after 48 hours by 3H thymidine incorporation (Ai) and by cell count (Aii). Time response (Aiii) of an experiment similar to that in panel Aii, where MSCs were treated with medium alone (dashed line), 1 μg/mL Pam3Cys (dotted line), or 10 μg/mL Pam3Cys (solid line) were counted every 24 hours for 3 days. For migration assay, a round 5-mm diameter space was made in confluent MSC cultures and DMEM containing 10% FCS without (Bi,iii) or with (Bii,iv) 20 μg/mL Pam3Cys was added. Five days later, cells were fixed and stained. The diameters of the circles were measured and quantified in panel Bv. Original magnifications: × 10 (Biii-iv); × 6.3 (Bi-ii). The results represent the means ± SD of a total of 5 “wounds” in duplicate wells. For the immunosuppression assay, MSCs were incubated for 48 hours with 1 μg/mL Pam3Cys (□), 10 μg/mL Pam3Cys (yellow bars), 20 μg/mL Pam3Cys (⊡), or without Pam3Cys (▪), washed, and added to a T-cell line activated by its cognate antigen in different ratios. After 72 hours, cells were pulsed with 0.037 MBq (1 μCi) 3H thymidine and measured for 3H thymidine incorporation (C). The results represent the means ± SD of triplicate wells. *P < .05, Pam3Cys treatment versus medium alone, by 2-tailed Welch t test.
