This proinflammatory pleiotropic cytokine is produced primarily by the host T lymphocytes in response to antigen recognition and has also been shown to induce the expression of immune-relevant molecules in several stem cell populations, including neural stem cells  and MSCs [24, 28]. associated with indoleamine 2,3-dioxygenase (IDO) enzyme-mediated tryptophan metabolism. Such findings constitute a step forward in better understanding the mechanisms of action of transplanted hCSCs in allogeneic settings. Electronic supplementary material The online version of this article (10.1186/s13287-018-1010-2) contains supplementary material, which is available to authorized users. values are shown hCSCs immunomodulatory capacity can occur in the absence of cell-cell contact To evaluate the importance of IDO enzyme and Trp metabolism in the immunosuppressive capacity of hCSCs, we carried out T lymphocyte proliferation assays in which hCSCs were not in direct contact (DC) with hPBMCs and therefore cannot exert their immunomodulatory activity through the PDL-1/PD1 axis. We carried out hCSC-hPBMC coculture under transwell conditions (TW), allowing paracrine interaction between the cell types. At 72 h of incubation, although slightly Regadenoson lower when compared with DC, hCSCs do exert a significant suppressive effect on T lymphocyte proliferation under TW conditions. Moreover, such a difference between TW and DC conditions was lost after 96 h of incubation (Fig. ?(Fig.4a4a). Open in a separate windows Fig. 4 Human cardiac/stem progenitor cells (hCSCs) inhibit T lymphocyte proliferation via a paracrine mechanism. a CFSE-labeled hPBMCs were stimulated with PHA and cultured alone, in direct contact (DC), or in a transwell setting (TW) with hCSCs (ratio 1:10 hCSCs:hPBMCs). b Concentrations of tryptophan (Trp) and kynurenine (Kyn) were determined by HPLC in the supernatants. c CFSE-labeled hPBMCs were stimulated with PHA Mouse monoclonal to HDAC4 and cultured alone or in conditioned medium (Cond.M.) from hCSCs cultures activated or not with interferon (IFN)-. Conditioned media were generated for 24 h (white bars), 36 h (grey bars), and 48h (black bars). d Concentrations of Trp and Kyn were determined by HPLC in the conditioned media. Proliferation of the viable population of CD3 T lymphocytes (CD3+/7AADC) was assayed by loss of CFSE staining after 72 h (white bars) and 96 h (black bars) for TW and DC experiments (a) and after 96 h for Cond.M. experiments (c). Percentage of cells per generation and percentage of inhibition of Regadenoson proliferation was decided using FSC Express software against Regadenoson proliferation of activated hPBMCs alone. Human adipose-derived mesenchymal stem cells (hASCs) were used as a positive control for T cell proliferation inhibition (ratio 1:25 hASCs:hPBMCs). Adjusted values are shown Trp metabolism was also assessed by measuring Trp and kynurenine (Kyn; a Trp metabolite described as cytotoxic for T lymphocytes ) concentrations in the conditioned medium. As shown in Fig. ?Fig.4b,4b, Trp is fully depleted at 72 h under the DC condition, and in the TW setting it is also significantly diminished when compared with stimulated hPBMCs alone. Furthermore, the accumulation of Kyn occurred in both experimental setups (Fig. ?(Fig.4b4b). Besides the TW experiments, hCSC conditioned medium was generated for 24 h, 36 h, and 48 h using control and IFN–stimulated hCSCs. Similar to the hASC control, hCSC-derived conditioned medium significantly inhibited T lymphocyte proliferation, with a significant increase in IFN–stimulated cells (51.79??11.67 % versus 15.49??8.10% with 36-h conditioned medium; 100??0.00 % versus 19.01??7.22% with 48-h conditioned medium; Fig. ?Fig.4c).4c). Moreover, conditioned medium from longer IFN–stimulated hCSC cultures prompted higher inhibition of T lymphocyte proliferation (Fig. ?(Fig.4c).4c). Such findings are also in accordance with the Trp and Kyn measurements, where Trp Regadenoson is usually gradually depleted and Kyn gradually accumulates in the supernatant of hCSC cultures (Fig. ?(Fig.4d4d). Discussion Allogeneic hCSC-based therapies continue to be explored as an alternative for AMI patients. However, hCSC regenerative medicine.