Human embryonic stem cells (hESC) have the capacity to differentiate into a number of specialised cell types including cardiomyocytes and may have utility in the treatment of cardiac disease (Kehat et al., 2004). Calcium signalling is central to cell differentiation, however at this time we do not fully understand when or how this signalling capacity is acquired during differentiation. TRPC channels play an important role in modulating the calcium response to multiple stimuli and have been implicated as candidates for store operated and receptor operated calcium release (Clapham et al., 2003).

The aim of this study was to determine which components of TRPC calcium signalling machinery are present in undifferentiated hESCs in order to investigate how signalling capacity is developed.

RT-PCR was used to determine the presence or absence of TRPC1 and TRPC3-7 at the mRNA level in undifferentiated BG01 hESCs of two different passage numbers (pB+29 and pB+36). As positive controls we used Human brain RNA (Ambion) and primary cultured Human Airway Smooth Muscle (HASM) (Corteling et al., 2004). TRPC2 was excluded from these studies as a suitable positive control had not been obtained. RNA from Human heart tissue (Ambion), known to have fully developed, functional calcium signalling was also evaluated. RT-PCR conditions were optimised for each of the TRPC reactions. All RT-PCRs were performed in duplicate with appropriate RT negative controls. Sequencing was used to confirm the identity of the generated TRPC PCR fragments.

TRPC1 and TRPC3-7 were shown to be expressed in undifferentiated hESC BG01 for both passages (pB+29 and pB+36). In contrast, expression of TRPC1, TRPC3, TRPC4 and TRPC6 but not TRPC5 and TRPC7 was shown in the human heart under these experimental conditions. Assays for TRPC1-7 were validated using a combination of the control templates. Expression of TRPC1, 3, 4 and 6 was shown in HASM in agreement with the literature. TRPC5 was detected in HASM in contrast to previous studies (Corteling et al 2004). TRPC7 was not detected in HASM and TRPC1 together with TRPC3-7 were detected in the human brain.

TRPC channels appear to be ubiquitously expressed in undifferentiated hESCs. Moreover, selective expression of TRPC channels appears to be acquired in the fully differentiated human heart. The expression of TRPC1, TRPC3-7 in BG01hESC and the absence of TRPC5 and TRPC7 in the human heart suggests active regulation of TRPC during cardiomyocyte differentiation.

Clapham D. E. (2003) Nature 426(6966): 517-24.
Corteling R., et al., (2004) Am J Respir Cell Biol 30:145-154.
Kehat I., et al., (2004) Nature Biotech advance online publication.