Pam3Cys modulates MSC differentation. (A) Pam3Cys inhibits spontaneous adipogenesis, induces calcium deposition, and increases ALP activity in noninduced MSCs. MSCs were plated in MSC medium. Later (48 hours), the medium was replaced with DMEM containing 10% FCS with 20 μg/mL (Aii), 10 μg/mL (Aiv), and 5 μg/mL (Avi) or without (Ai,iii,v) Pam3Cys. Later (2 to 3 weeks), the cells were stained with Oil red O (Ai-ii), Alizarin red (Aiii-iv), or ALP substrate (Av-vi) to examine possible spontaneous differentiation. Original magnifications: × 10 for all micrographs. Alizarin red stain from experiment shown in panels Aii and Aiii was extracted from the cell culture and quantified by light absorbance in 405 nm (B). Pam3Cys reduces MSC-induced differentation into adipogenic, osteogenic, and chondrogenic pathways. MSCs were induced to differentiate into osteocytes (Cii-iii, v-vi), adipocytes (Cviii-ix), or chondrocytes (Cxi-xii) with 5 μg/mL (Ciii, vi, xii) and 10 μg/mL (Aviii) or without (Cii, v, viii, and xi) Pam3Cys. After 1 week (adipogenic differentiation) or 3 weeks (osteogenic and chondrogenic differentiation), cell cultures were fixed and stained with Alizarin red (Ci-iii), ALP substrate (Civ-vi), Oil red O (Cvii-ix) or Alcian blue (Cx-xii). Original magnifications: × 10 (Ci-ix); × 20 (Cx-xii). MSCs incubated with osteogenic induction media with or without 1 μg/mL Pam3Cys for 3 weeks were stained with Alizarin red, which was then extracted and measured for light absorbance at 405 nm (D). MSCs incubated with adipogenic induction media with or without 10 μg/mL Pam3Cys for 1 week were fixed and stained with Oil red O, which was then extracted and measured for light absorbance at 492 nm (E). The results represent the means ± SE of duplicate wells. *P < .04, Pam3Cys versus untreated control by the 2-tailed Welch t test.