ESC-derived factors modulate T helper responses during an allogeneic immune response.

<p>A one way mixed lymphocyte reaction was performed, C57BL/6 splenocytes were used as responders and CD1 splenocytes as stimulators in the presence of ESC-derived factors or vehicle control. Cells were harvested at the indicated time points and total RNA was isolated. Subsequently, cDNA was synthesized and used to carry out QPCR to examine the expression of cytokines and master regulator transcription factors of T helper cells. <i>a)</i> IL-2, <i>b)</i> IFN-γ, <i>c)</i> TGF-β, <i>d)</i> T-bet, <i>e)</i> Foxp3. Responders alone were used as baseline. Results are representative of 3 separate experiments. Data points represent mean ± SD. * indicates p value≤0.05. White bars represent results obtained from vehicle treated MLRs and black bars indicate results obtained from ESC-derived factor treated MLRs.</p

ESC-derived factors in combination with cyclosporin A enhance inhibition of allogeneic immune activation.

<p>A one way mixed lymphocyte reaction was performed, C57BL/6 splenocytes were used as responders and CD1 splenocytes as stimulators in the presence of vehicle control, ESC-derived factors and CsA. Moreover, ESC-derived factors were used in combination with CsA to determine whether they can complement one another in preventing allo-immune activation.</p

ESC-derived factors inhibit PMA mediated PKC-θ activation in T cells.

<p><i>a)</i> C57BL/6 splenocytes were stained with CellTrace Violet Cell Proliferation dye and activated with 50 ng/ml of PMA for 24 hours in the presence of 0.23 mg/ml of ESC- derived factors (without pre-treatment). Cells were harvested, washed with PBS and examined for proliferation by flow cytometry. <i>b)</i> C57BL/6 splenocytes were pre-treated over night with 0.23 mg/ml of ESC-derived factors and stimulated with 50 ng/ml of PMA for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for PKC-θ phosphorylation (Thre 538), and total PKC-θ. <i>c)</i> C57BL/6 CD3+ T cells were pre-treated overnight with 0.23 mg/ml ESC-derived factors and stimulated with 50 ng/ml of PMA for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for IκB-α degradation. Results are representative of 4 separate experiments.</p

ESC-derived factors skew T cell helper responses towards T regulatory cells.

<p><i>a)</i> C57BL/6 splenocytes were pre-treated over night with ESC-derived factors and stimulated with PMA and Ionomycin or anti-CD3/anti-CD28 for 6 hours. Protein transport inhibitor cocktail was added to the cells 1 hour following stimulation. Cells were harvested and stained for surface CD8. After washing, the cells were fixed, permeabilized and stained for intracellular IFN-γ. <i>b)</i> C57BL/6 splenocytes were treated with ESC-derived factors and stimulated with anti-CD3 and anti-CD28 for 3 days. The cells were harvested and stained for CD3, CD4 and CD25. Subsequently, cells were fixed, permeabilized and stained for Foxp3. Gates were set on CD3+ followed by CD4+ cells. Results are representative of at least 3 separate experiments.</p

ESC-derived factors specifically inhibit PKC-θ activation without affecting upstream signaling molecules.

<p>C57BL/6 splenocytes were pre-treated with ESC-derived factors overnight and stimulated with anti-CD3/anti-CD28 for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for PLC-γ, AKT and PKC-θ phosphorylation. Results are representative of 3 separate experiments.</p

ESC-conditioned media and cellular factors from ESC-extracts inhibit T cell proliferation in response to anti-CD3/anti-CD28 stimulation.

<p><i>a)</i> C57BL/6 splenocytes were labeled with CFSE and activated with anti-CD3/anti-CD28 in RPMI media (unstimulated cells presented in panel 1 and stimulated cells in panel 2). Cells were also activated in 50% RPMI along with 50% or 100% fresh unconditioned media (panels 3 and 4), 50% RPMI along with 50% or 100% mouse embryonic fibroblast-conditioned media (MEF-CM, panels 5 and 6), and 50% RPMI along with 50% or 100% mouse ESC-conditioned medium (ESC-CM, panels 7 and 8). After 48 hours the cells were analyzed by flow cytometry for proliferation. <i>b)</i> ESCs were grown in feeder free cultures, harvested and lysed by sonication. Cell membrane, mitochondria and nucleus were removed by centrifuging the sonicate at 15000 g for 15 minutes. Proliferation of B6 splenocytes stimulated with anti-CD3/anti-CD28 in RPMI media was assessed using extraction buffer alone (vehicle, panel 1), lysates from C2C12 cells (Control-Factors) or increasing concentration of ESC-derived factors (ESC-Factors). Results are representative of 4 separate experiments.</p