Fig. 1.

iPSC-derived MSCs phenotypically resemble native MSCs, respond to IFNγ licensing, and dampen PBMC activation potential. iPSC-MSCs were plated as described; IFNγ was added to some cells. 24 or 48 h later, cells were harvested and stained with antibodies specific for (A) PD-L1 or (B) IDO. Cells were permeabilized prior to IDO staining. Data were acquired on a BD LSR Fortessa Flow Cytometer and analyzed using FlowJo software. For (C) IDO gene expression, cells were harvested after 24 or 48 h of culture with or without IFNγ.

Total RNA was reverse transcribed, and IDO expression determined by quantitative real-time PCR using specific forward and reverse primers. For co-culture experiments, stimulated PBMCs were added to iPSC-MSCs and cultured an additional 24, 48, or 72 h. PBMCs were harvested and stained with antibodies specific for (D) T-BET. Percent positive cells and amount of protein expressed, indicated by median fluorescence intensity (MFI), was determined using flow cytometry. For some cultures, golgi plug was added during the last 6 h and (E) IFNγ levels were determined by intracellular staining and flow cytometric analysis. iPSC-MSCs were cultured without or with INFγ for 48 h. Stimulated PBMCs were added to iPSC-MSCs and cultured an additional 96 h. Expression of (F) pPKCθ was determined by immunoblotting. Loading control was β-actin. Data are the mean + SEM of three independent experiments or, for immunoblotting, are representative of two independent replicates that showed similar results. *P < .05; unpaired Student’s t-test.