Table 1.
Main assays for pluripotency characterization according to their priority
| Pluripotency characterization | Significance | Importance | Drawbacks |
|---|---|---|---|
| Alkaline phosphatase activity | Placental AP is upregulated in PSCs | – AP staining is used for early screening (initial indicator of successful reprogramming) and distinguishing PSCs from feeder and parental cells – May serve as a reporter of transfected cells after thermal treatment |
– Extra pluripotent tests needed – Some reagents toxic for PCSs result in loss of cellular morphology and preclude further propagation – Level of AP expression depends on cell line – Background noise (signals) |
| Stem cell surface marker detection (immunostaining) | The keratan sulfate antigens TRA-1-60, TRA-1-81 and the glycolipid antigens SSEA3 and SSEA4 determine pluripotent cells | – Phenotypic assessment of the pluripotent status and homogeneity in cell culture – Valuable time– saving and easy– to– handle method to distinguish, select and isolate reprogrammed colonies |
– required “negative control” to suppress a background signal – Incomplete qualitative confirmation of pluripotency at the protein level |
| Pluripotent marker expression (RT-PCR and protein assays) | Detection of the set of pluripotent genes and proteins | – Quantitative analysis of undifferentiated marker gene expression and efficacy confirmation of transgene silencing by RT-PCR – Complementary detection of pluripotent nuclear protein markers |
– Assays do not fully confirm the complete reprogramming process – Positive results do not prejudge the pluripotent abilities in in vivo conditions |
| Epigenetic status of promoter | Indicator of gene expression related to pluripotency | – Evaluation of the methylation status of CpG in pluripotency-associated gene promoters (bisulphite sequencing) and epigenetic status of H3 histones (e.g. by chromatin immunoprecipitation) in comparison to PSCs and parental cells verifies pluripotent status of cells and allows to check whether epigenetic memory remained from somatic cells and PSCs is safe and effective for clinical application – Level of transcriptional activity may be detected via luciferase reporter assay – Global DNA methylations ensures to assess the reprogramming progress |
– The analysis alone is not sufficient to conclude pluripotency – Multistep and complex test to conduct – The role of epigenetic regulation of gene expression not fully understood |
| Gene expression profiling (DNA microarray) | Global gene expression patterns as a relative for ES and parental cell expression profile | – Straightforward and relatively inexpensive method for verification of differences between pluripotency status of iPSCs, ESCs and descendant cell lines – Convincing prove for complete reprogramming process |
– Positive results do not prejudge the pluripotent abilities in in vivo conditions |
| Differentiation tests in vitro (EBs) | Ability of PSCs for forming tissues derived from the three primordial germ layers | – Quick and easy-to-handle for demonstration of pluripotent abilities of PSC lines – Immunohistochemistry assays detect three germ layer markers |
– Positive results do not prejudge the pluripotent abilities in in vivo conditions – Still recommended direct in vitro differentiation |
| Direct in vitro differentiation | Assessment of pluripotency towards specific cellular lineage | – Ability to differentiate into specific tissue confirmed by immunostaining and RT-PCR analysis | – Does not prove full pluripotency of particular iPS cell line |
| Differentiation tests in vivo (teratoma) | The most rigorous and definite landmark test for pluripotency | – Easy to obtain, no problematic procedure of cell administration into animal – Immunohistochemistry and histopathological analysis detects three derivation tissues of germ layers – Spontaneous teratoma formation may help in understanding potential hazards of stem cell therapies |
– Poor reproducibility and high degree of variability – Time-consuming, laborious procedure – Teratoma assay requires a threshold level of cells to prove successful reprogramming – Not standardized in terms of graft size, number of implanted cells, mice age, PSC preparation |
The tests presented in the table are directly related to assessing pluripotency of PSC lines—gene expression profiling, epigenetic status (promoter), teratoma, formation and in vitro cell differentiation. However, additional assays connected with utility in further application studies are highly recommended, namely telomerase activity (TERT expression is responsible for self-renewal abilities of PSCs), doubling time (for comparing further rate of cell divisions between iPSCs, ESCs and descendant cell lines), cross-contamination test (DNA fingerprinting like STR analysis confirms the origin from parental cells), karyotyping (chromosomal G-band analysis enables detection of chromosomal abnormalities but does not detect minor genetic variations; FISH analysis indicates chromosomal translocation or deletion), genome-wide single-nucleotide polymorphism array analysis (detects the DNA mutation ratio)
AP alkaline phosphatase; EB embryoid body; PSC pluripotent stem cell; SSEA3 stage specific embryonic antigen-3; SSEA4 stage specific embryonic antigen-4; TRA-1-60 tissue rejection antigen 1–60; TRA-1-81 tissue rejection antigen 1–81