Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture

Abstract

Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3β (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage

“…Immunocytochemistry was performed as described in previous report (Park et al, 2008). The cells were incubated with the following primary antibodies: rabbit anti-Oct 4 antibody (1:400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), mouse anti-Tra-1-60 (1:400; Chemicon, Temecula, CA, http://www.millipore.com), rabbit anti-brachyury ( …”

Telomerase and Oxidative Stress in Embryonic Stem Cells

“…Both groups consistently reported an increased proliferation of ESCs at higher passages, no changes in telomerase activity as well as a reduced differentiation ability. However, while Park et al (2008) found no karyotypic changes Xie et al (2010) described some karyotypic abnormalities (translocation) in one of the two hES cell lines analysed while Maitra and co-workers (2005) and found karyotypic anomalies in 8 out of 9 analysed human ESC lines at high passage numbers. These included copy number changes (amplifications and deletions of chromosomes, 45%) and gene promoter methylation (90%).…”

“…This might depend on the species (mouse or human) as well as the (often sub-optimal) culture conditions. Various groups had cultivated human embryonic stem cell lines for around 100 passages (Maitra et al, 2005, Park et al, 2008, Xie et al, 2010. Park et al (2008) and Xie et al (2010) did not find any gross morphological changes or different expression levels for stem cell markers between early and late passages.…”

“…Various groups had cultivated human embryonic stem cell lines for around 100 passages (Maitra et al, 2005, Park et al, 2008, Xie et al, 2010. Park et al (2008) and Xie et al (2010) did not find any gross morphological changes or different expression levels for stem cell markers between early and late passages. Both groups consistently reported an increased proliferation of ESCs at higher passages, no changes in telomerase activity as well as a reduced differentiation ability.…”

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

“…19,20 Thus, for a given cell line, when considering an equal inoculum concentration and an equal elapsed time in culture, it is expected that colonies with similar areas be formed.…”