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Table 1 Parameters of interest for hPSC characterisation and methods for their assessment

From: Defining synthetic surfaces for human pluripotent stem cell culture

Parameters Method Strength of evidence of pluripotency
Physical characteristics (daily/weekly) Daily visual assessment of cell/colony morphology Weak, subjective
  calculate adhesion efficiency, population doubling time  
Expression of molecular markers eg. OCT4, NANOG, SOX2, REX1 (following passages 1, 5 and >10) Immunocytochemical staining, flow cytometry, RT-PCR, microarray assays Moderate-strong. Depending on marker(s) assessed.
Epigenetic profiling Bisulfite sequencing, ChIP, microarray assays Moderate-strong. Depending on marker(s) assessed.
Differentiation potential (following >10 passages) Embryoid body differentiation (in vitro) with RT-PCR analysis for molecular markers of differentiation Very strong
  Teratoma formation assay (in vivo) with histological determination of cells from the three embryonic germ layers Gold standard
Genetic stability (following >10 passages) G-banding, FISH, SNP analysis Not applicable. Important to identify genetically transformed cultures, not indicative of differentiation potential
  1. Physical characteristics, molecular markers, epigenetic profiling, differentiation potential and genetic stability can be assessed by the range of methods listed (not comprehensive). We recommend the methods highlighted in bold performed at frequencies indicated in the first column as the minimum requirements for validating novel culture systems. Unbolded methods should also be considered for more thorough characterisation of hPSCs.