Differential expression of nitric oxide signaling components in undifferentiated and differentiated human embryonic stem cells

Nitric oxide (NO) is an uncharged free-radical gas that is involved in a number of physiological and pathological events. We have examined the expression of various subunits of soluble guanylyl cyclase (sGC α₁, α₂, β₁, β₂), nitric oxide synthase (s) (NOS-1, -2, -3), MLC2 (cardiac marker) and a cardiac-specific transcription factor (Nkx2.5) in human embryonic stem (hES) cells (H-9 cells) and differentiated cells subjected to differentiation in cell suspension using embryoid body (EB) formation. Our results demonstrate a time-dependent increase in the expression of sGC α₁ and β₁ the mRNA and protein levels in differentiated cells compared to undifferentiated H-9 cells as examined by real-time PCR and western blot analysis. mRNA for sGC α₂ also showed a time-dependent increase compared to undifferentiated cells. In contrast, there was a time-dependent decrease in sGC β₂ mRNA expression in differentiated cells compared to undifferentiated H-9 cells. In contrast to undifferentiated H-9 cells, the maximum mRNA expression of cardiac marker MLC2 and the cardiac-specific transcription factor Nkx2.5 was observed at day 14 of the differentiated H-9 cells. The protein levels of MLC2 were stable up to day 25 compared to mRNA levels, which showed a sharp decline after day 15. Using immunofluorescence, we also demonstrate positive staining of cardiac markers such as troponin I, α-actinin, atrial natriuretic peptide, and SGC Ot1 at days 8-37 post-differentiation. These results clearly demonstrate the role of NO signaling components in differentiation events or physiological processes of human ES or ES cell-derived cardiomyocytes.