hESC- and fetal liver–derived monocytic cells have a pattern of cytokine expression characteristic of an anti-inflammatory state. (A) Gene expression profile of sorted CD14⁺CD16⁺ from hESC-derived (n = 6) and fetal liver–derived (n = 4) CD34⁺ cell cultures. quantitative RT-PCR analysis shows the expression level of relevant genes of the M1 and M2 polarization paradigm. Results are expressed as 1/ΔCT values (mean ± SEM) over the endogenous controls 18S and Gadph. (B-C) Cytokine production was measured in the supernatant from 3 × 10⁵ hESCs and fetal-sorted CD14⁺CD16⁺ cells, and comparatively assessed with CD14⁺CD16⁺ peripheral blood monocytes (n = 4 for each population). Tested cells were either left untreated (basal; B) or were stimulated with LPS and IFN-γ for 24 hours (C); the resulting supernatants were subsequently analyzed using a Luminex assay. Results are expressed in picograms per milliliter. Note that the response to LPS and IFN-γ in terms of inflammatory cytokine production is decreased from embryonic and fetal cells compared with the adult. (D) Comparative analysis of TGF-β secretion from hESC-derived, fetal liver–derived, and adult peripheral blood CD14⁺CD16⁺ cells. Note that in contrast to other cytokines, TGF-β was oversecreted by embryonic and fetal cells compared with adult cells. Results are expressed in picograms per milliliter. †Cytokine level was over the maximal range of the assay. Statistical differences between groups were calculated by pairwise t test. *P < .05 between the expression and production of some proinflammatory cytokines after stimulation with IFN-γ and LPS by adult versus hESC- and fetal liver–derived cells.