Defining synthetic surfaces for human pluripotent stem cell culture

Cell Regeneration

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

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

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.