Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
1. Isolation and characterization of human embryonic stem cells and future applications in tissue engineering therapies
1.2. Basic characterization of hESCs
Besides the typical cell/colony morphology, which is a routine check during cell culture, hESCs are characterized mainly by their expression of a variety of specific cell-surface and intracellular protein markers using antibodies (usually monoclonal), often in combination with flow cytometry or high-content image analysis. These cell-surface markers were first identified in the preimplantation mouse embryo or in embryonal carcinoma cells (ECCs; pluripotent cancer cell lines). The phenotypic morphology of a hESC may alter as spontaneous differentiation occurs during cell culture, with cells gradually losing expression of markers associated with pluripotency and upregulating those associated with differentiation; therefore, a panel of markers can rapidly identify subpopulations of cells. If quantitative analysis is used, the stability of a hESC culture can be monitored accurately over time. Surface markers indicative of an undifferentiated hESC state include SSEA-3, SSEA-4 and the high-molecular-weight glycoproteins TRA-1-60 and TRA-1-81 (Thomson et al., 1998). HESCs also express the intracellular markers OCT4, Nanog and REX1 and stain positive for alkaline phosphatase activity (Figure 1.3).
Figure 1.3. (A) Main intracellular and extracellular markers used to identify hESCs. (B) A colony of Shef1 hESCs plated on ECM (Matrigel). Immunofluorescent localization of cell-surface markers Tra-1-60 (green), SSEA3 (blue) and SSEA4 (red). Although all three markers identify pluripotent cells, the expression patterns in the colony differ.
Significantly, mESCs differ in their surface-antigen profile, failing to express SSEA-3 or SSEA-4 but expressing SSEA-1, a cell-surface marker characteristic of differentiated hESCs. The markers display differences in sensitivity to shifts in the differentiation status of the cell, which can be exploited to some extent to forecast developmental changes. For example, SSEA-3 expression is the first to downregulate upon early differentiation while markers such as SSEA-4 and TRA1-60 lag behind (Henderson et al., 2002).