FIG. 2.

In vitro effects of human ESC-MSC-CM and BM-MSC-CM on hepatocyte viability and MNC immunomodulation. (A) Schematic diagram of the experimental procedure to study the trophic potential of MSC-CM on mouse primary hepatocytes. Two-step collagenase perfusion was used for mouse hepatocyte isolation. Isolated cells were initially cultured on plates that contained Williams' medium for 3 h, after which the medium was replaced by hepatocyte medium (HepatoZYME + supplements) for 1 day and then incubated in Williams' medium + CMs for 2 days. (B) Immunostaining for ALB. (i) Cultured hepatocytes represented epithelial morphology and formed a stable monolayer such as polygonal cells with one or two round, prominent nuclei. (ii) Albumin-expressing hepatocytes visualized by immunostaining and fluorescent microscopy. Nuclei stained with DAPI. Scale bar: 100 μm. (C) We used the MTS assay to assess for primary cultured hepatocyte viability in the presence of MSC-CM or NCM. Human BM-MSC-CM and human ESC-derived MSC-CM (RH6-MSC-CM and RH5-MSC-CM) increased hepatocyte survival compared with NCM. (D) Schematic diagram of an in vitro immunomodulation assay. Primary human peripheral blood MNCs were first incubated in the presence of MSC-CM and NCM for 18 h, then stimulated with LPS for 5 h. Finally, we measured the level of IL-10 secreted from MNCs as an indicator for anti-inflammatory activity. (E) ELISA was used to assess the level of IL-10 in MNC culture supernatant. Incubation of MNCs with MSC-CM led to increased secretion of IL-10 from MNCs. Results have been reported as a mean of experiments performed in triplicate. ***P < 0.001 for MSC-CM compared with NCM. ^#P < 0.05 for ESC-MSC-CM compared with BM-MSC-CM. CM, conditioned medium; DAPI, 4, 6-diamidino-2-phenylindole; IL-10, interleukin-10; LPS, lipopolysaccharide; MNCs, mononuclear cells; NCM, nonconditioned medium. Color images available online at www.liebertpub.com/scd