Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells

Abstract

Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression.

Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells

“…Many EC cell lines show reduced tendency to undergo spontaneous differentiation when compared with hESCs, suggesting that they have been subjected to strong selections for culture adaptations/ mutations that lead to an increase in self-renewal compared with differentiation (Andrews et al, 2005). Higher expression, therefore, of some cell cycle regulatory components (for example, c-MYC, CDK2, CYCLIN D1) might be responsible for tuning the fine balance between self-renewal and differentiation, and needs to be investigated in detail in hESCs and during their culture adaptation (Enver et al, 2005).…”

Two Novel Splice Variants of SOX2OT, SOX2OT-S1, and SOX2OT-S2 are Coupregulated with SOX2 and OCT4 in Esophageal Squamous Cell Carcinoma

“…NTERA2 (NT2), a human embryonal carcinoma (EC) stem cell line, shares many features with hESCs, including similar patterns of gene expression [30][31][32][33]. Thus, this cell type is an excellent prototype model of pluripotent stem cells to study gene expression and differentiation associated to human embryonic development [34].…”

Human Embryonic Stem Cells Fail to Activate CHK1 and Commit to Apoptosis in Response to DNA Replication Stress

“…Accumulating evidence now points to the frequent occurrence of genetic alterations in many commonly used human ESC lines [8,9]. Similar chromosomal abnormalities, notably gains of chromosome 12 and 17, are commonly found in both culture-adapted human ESCs [10,11] and in embryonal carcinoma (EC) cells, their malignant counterparts, and the stem cells of teratocarcinomas, raising concerns that ESCs, or their derivatives, may be susceptible to develop malignant phenotypes in vivo after transplantation [8,9,12,13]. Such changes may reflect errors in DNA processing following disruptions of DNA synthesis or repair.…”