Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges

Table 1 Methods for evaluating human pluripotent stem cell lines for pluripotency, differentiation potential, and malignant potential

Aim	Techniques	Targets	Cell treatment	Timepoint detection	Ref.
Pluripotency	DNA methylation sequencing and microarray	Deviation scorecard: The cell line-specific number of outliers relative to the human ES cell reference	N/A	N/A	[1]
	Microarray	PluriTest: Pluripotency score (refers to gene expression profiles of a large collection of human PSCs) and novelty score (refers to gene expression patterns not typically associated with human PSCs)	N/A	N/A	[21-23]
	qPCR	Level of CHD7	N/A	N/A	[43]
Differentiation potential	Microarray	Differentiation scale: a subset of the 1000 most informative genes	W/O	At any time of differentiation	[27]
	Microarray	Lineage scorecard: 500 lineage marker genes to monitor cell state, pluripotency, and differentiation	Nondirected EB differentiation	At 16 d of differentiation	[1]
	qPCR	Lineage scorecard: 15 selected marker genes per lineage	Nondirected and directed differentiation into three germ lineages		[22]
	qPCR	hPSC ScoreCard: 9 self-renewal genes and 70 genes representing specific lineages	Monolayer or EB protocol differentiation into three germ lineages	At 5 or 9 d	[23]
	Single-cell qPCR	96 developmental genes; a transcriptional circuit (HAND1-SOX17) and phenotypic readout (cKIT distribution)	Cardiomyocyte differentiation	On day 2	[31]
	qPCR	Improved scorecard: 96 specific gene markers	Directed EB differentiation into three germ lineages	At 12 d of differentiation.	[28]
	Microarray or RNA-sequencing	TeratoScore: 100 tissue-specific genes representing the three embryonic germ layers and extra- embryonic membranes	Xenograft teratoma formation	A suitable growth period	[22,23,26]
	Morphology	Definitive endoderm morphology production	Treated with small molecules	48 h after induction	[44]
	qPCR	SALL3 for ectodermal differentiation	N/A	N/A	[32]
	PCR array	Prediction scores for hepatic differentiation based on the expression of the three genes FGF-1, RHOU, and TYMP	N/A	N/A	[33]
Malignant potential	Histology, qPCR, and microarray	TeratoScore: 10 undifferentiated hPSC markers; embryonal carcinoma-like cells with yolk sac elements; undifferentiated hPSC marker and malignancy marker expression	Xenograft teratoma formation	Suitable growth period	[22]
		TeratoScore: 100 tissue-specific genes; embryonal carcinoma-like cells; undifferentiated hPSC marker and malignancy marker expression			[23]
	Histology, microarray, karyotype analysis, and whole exome sequencing	Formation of immature teratomas, carcinoma and sarcoma; mutation of cancer-related genes; chromosomal abnormalities	Xenograft teratoma formation; N/A	Suitable growth period; N/A	[40]

hPSCs: Human pluripotent stem cells; N/A: Not available.