The low rate of HLA class I molecules on the human embryonic stem cell line HS293 is associated with the APM components' expression level.

Human embryonic stem cells (hESCs) represent a promise for future strategies of tissue replacement. However, there are different issues that should be resolved before these cells can be used in cellular therapies; among others, the rejection of transplantable hESCs as a result of HLA incompatibility between donor cells and recipients. The hESCs exhibit a weak HLA class I expression on the cell surface, but today the responsible mechanisms are unknown. We have analyzed the level expression of HLA class I heavy chain, beta2-microglobulin (beta2-m), and antigen-processing machinery (APM) components (TAP1, TAP2, LMP2, LMP7, and Tapasin) using the HS293 hESC line by real-time quantitative RT-PCR. This analysis has revealed a low expression of beta2-m, HLA-B, and Tapasin, and an absence of expression of: TAP1, TAP2, LMP2, and LMP7 genes in the HS293 hESC line respect to the embryoid bodies (EBs) and the induced stem cells with IFNgamma (with significant differences, p<0.05). The lack or loss of HLA class I molecules due to the down-regulation of the APM components has been frequently found in tumors of different histology as specific mechanisms of immune-evasion. We described for the first time in this report that the hESCs shared similar mechanisms with respect to tumor cells responsible for the weak HLA class I expression on the cell surface.

Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embryonic stem cells: immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry.

Mitomycin C (MMC) treatment has been used to arrest cell proliferation but not much is known about the effect of MMC on human foreskin fibroblasts (HFF) used as feeders for human embryonic stem cells (hESC). We tested the ability of MMC to stop the proliferation of HFF and to induce apoptosis. MMC inhibited the proliferation of HFF at 10 microg/ml over 2.5h of MMC treatment showing a decrease in the proliferation index measured by Ki-67 and S and G2/M phases related to active HFF. A low percentage of cells showed necrotic or apoptotic features using different lengths of incubation. Over time, the majority of cells remained in a mitotically inactive state. The percentage of apoptotic cells increased from day 2 to day 10, at the same time as the necrotic ones increased. The HS181 hESC line grew in an undifferentiated state on inactive HFF throughout the study.