Abstract
This study investigated the effects of exposing human embryonic stem cells (hESC) to 4 degrees C and 25 degrees C for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25 degrees C and 4 degrees C for 24h was 77.3 +/- 4.8 % and 64.4 +/- 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25 degrees C and 4 degrees C was 71.0 +/- 0.5 % and 69.0 +/- 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37 degrees C. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure.