Table 1.

Primary Refinements of the Embryonic Stem Cell Test

Chemicals	Cell line	Exposure duration	Differences from Spielmann's original EST	Findings	Reference
15 pharmaceuticals	DBA/1lacJ murine derived ESCs, 3T3 A31	Cytotoxicity assay: 7 days	Different ESC line	Other murine ESC lines can replace D3 in the EST; false positives and negatives can still be significant	7
		Differentiation assay: 10 days
Penicillin G, 5-FU, RA, diphenylhydantoin, valproic acid, thalidomide	Balb/c 3T3, D3	Cell viability: 10 days	Involvement of multiple marker genes of other lineages (neurogenesis, osteogenesis, and chondrogenesis markers)	First time to include multiple gene expressions in the EST; results are promising and open to further improvement and optimization	8
		Differentiation assays (contraction assay and collection of RNA samples): 10 days
6-Aminonicotinamide, 5-FU, methylmercury chloride, hydroxyl urea, valproic acid, boric acid, methoxyacetic acid, lithium chloride, penicillin G, saccharin, diphenhydramine, acrylamide	D3	Cell viability assay: 6 days	Instead of being seeded in 24-well plates, mESCs are seeded and cultured in low attachment 96-well plate to facilitate high-throughput tests. Introduce relative embryotoxicity potency values that are derived from the ranking of the embryotoxic potential of test compounds relative to positive controls	No need for 3T3 to provide reference toxicity information. Toxicity ranking seems more reliable than the absolute classification of toxicity	9
		Differentiation assay: 10 days
Monobutyl phthalate, methoxyacetic acid, valproic acid, RA, 5-FU, penicillin G	D3	Cell viability assay: 3 days	Use resazurin proliferation assay; involve whole-genome gene expression profiling	Several genes are very sensitive and significantly dysregulated after compound exposure, which are useful as additional endpoints in the EST	10
		Differentiation assay: 10 days
Triazoles, flusilazole, hexaconazole, cyproconazole, myclobutanil, triadimefon, triticonazole, 5-FU	D3	Cell viability assay: 5 days	Compare the EST with in vivo tests, the rat postimplantation WEC, and zebrafish embryotoxicity test	The EST is able to represent developmental toxicity, and partially mimic the processes in utero	11
		Differentiation assay: 10 days
9-cis-RA, 13-cis-RA, acitretin, ATRA, TTNPB, etretinate, retinol	D3	Cell viability: 10 days	Compare the EST with in vivo tests, the rat limb bud micromass test, and the postimplantation rat WEC	The EST, although it could not completely imitate toxicological kinetics in vivo, was overall a promising in vitro alternative in toxicology	12
		Cell differentiation: 10 days
Methylmercury, valproic acid, AsV, AsIII, saccharin, isoniazid, ascorbic acid	D3, 3T3	Cytotoxicity: 10 days	Neural differentiation instead of cardiac induction with exposure duration of 12 days. Use flow cytometry and detection of neural gene expression as endpoints	Neural differentiation could serve as an alternative process in the EST. The molecular endpoints introduced in the neural differentiation-based EST were Tuj1 mRNA and protein levels	13
		Neural differentiation assay: 12 days
Mono-ethlhexyl phthalate, valproic acid, methotrexate, 6-aminonicotinamde, methoxyacetic acid, penicillin G	D3	Cell viability: 5 days	Involvement of osteoblast differentiation and molecular endpoints to evaluate it. Comparison between osteoblast and cardiomyocyte differentiations on exposure to same chemicals	Osteoblast differentiation can be an alternative to cardiogenesis in the EST, and may give different results	14
		Osteoblast differentiation assay: 21 days. Cardiac differentiation assay: 10 days
Phenol, p-fluorophenol, p-heptyloxyphenol, p-mercaptophenol, p-methylketophenol	D3	Cell differentiation assay: 10 days	Compare the EST with in vivo tests and the WEC assay	The EST gives toxicity rankings of tested phenols that are different from the rankings given by in vivo tests and the WEC assay; exposure doses in the EST have to consider the kinetics of in vivo absorption, metabolism, elimination, and excretion	15
Acealdehyde, carbamazepine, flusilazole, monoethylhexylaphthalate, penicillin G sodium salt, phenytoin	D3	Cell viability test: 48 h	Neural differentiation	The neural differentiation-modified EST is valid; transcriptomics provides mechanistic information	16
		Morphological scoring: 72 h	Different exposure durations
		Whole-genome expression profiling: 24 h	Resazurin cell viability assay
			Include genome profiling
MeHgCl, monosodium l-glutamate, penicillin G, poly-l-ornithine, sodium arsenite, sodium valproate, chlorpyrifoe-ethyl, parathion-ethyl	D3	Cell viability: 4 or 5 days Differentiation: 2 or 3 days	Differentiation to neural cells	This method is suitable for high-throughput screening but does not necessarily represent relevant concentrations in vivo and is not applicable for acute and chronic toxicities	17
			Cell proliferation tests are based on ELISA. Cell viability tests are based on CellTiter-Blue Cell Viability Assay. Involvement of βIII-Tubulin enzyme-linked immunosorbent assay
Bisphenol A, genistein, as well as combined with bisphenol A and 5-FU	D3, 3T3	Cell viability test: 10 days	Cell Titer 96 Aqueous One Solution Cell Proliferation Assay for cell viability test; cells are exposed to two chemicals	Bisphenol A and genistein, to which we are exposed daily unintentionally, have combined embryotoxic effects that become synergistic at low concentrations	18
		Differentiation assay: 10 days
38 teratogens	D3	Cell viability test: 72 h	Shorter exposure times; include gene expression analysis for 12 potential molecular endpoints	The Molecular Embryonic Stem Cell Developmental Toxicity Assay facilitates high-throughput screenings of potential teratogens with good predictivity and concordance with in vivo data	19
39 nonteratogens		Cell differentiation assay: 96 h
Monobutyl phthalate, monobenzyl phthalate, mono-(2-ethylhexyl) phthalate, monomethyl phthalate	D3	Cell viability: 5 days Differentiation assay: 10 days	Incorporate RNA microarray analyses as additional endpoints	A total of 668 commonly expressed genes are altered after exposure, proving the validity of transcriptomics in the EST	20
5-FU, hydroxyurea, saccharin; silver nanomaterial, coated and uncoated zinc oxide, titanium and silica nanomaterials	D3, 3T3	Cell viability: 10 days	Skip the step of EB formation in petri dishes and transfer EBs directly to 24-well plates. Add nanomaterial once to avoid continuous accumulation in cells	This simplified protocol shows to be more suitable to facilitate nanotoxicity research for medical or therapeutic nanomaterial uses	21
		Cell differentiation: 10 days
6-aminonicotinamide, all-trans RA, 5-bromo-2′-deoxyuridine, dexamethasone, methoxyacetic acid, salicylic acid sodium salt, ascorbic acid, acrylamide, d-(+)-camphor, 5-FU	Linearized Hand1-promoter-Luc plasmid transfected C57BL/6 mice derived ESCs	Cell viability: 5 days	Monitor Hand1 expression via Luciferase reporter assay, which at the same time indicates both proliferation and differentiation	The expression of Hand1 by Luciferase reporter gene assay is reproducible and relatively accurate	22
		Differentiation assay: 5 days
Simvastatin	D3, 3T3	Cytotoxicity: 10 days.	Include both EB hanging drop method and monolayer differentiation. Molecular endpoints are maker genes for each germ layer	Genes of the mesodermal lineage are most sensitive to the two drugs; the hanging drop method and monolayer differentiation give rise to consistent results	23
		Differentiation assay (both hanging drop method and monolayer differentiation): 10 days
Chinese herbal extracts from Atractylodes macrocephala, Coptis chinensis, Radix isatidis, Flos genkwa	OG2 mESCs, BALB/c 3T3	Cell viability: 10 days	Cell viability assay: CCK8 assay	Atractylodes macrocephala and Radix isatidis are non-embryotoxic, Coptis chinensis is weakly embryotoxic whereas Flos genkwa is strongly embryotoxic	24
		Differentiation assay: 10 days	Differentiation assay based on myosin heavy chain gene expression
5-FU, RA, valproic acid, diphenhydramine, LiCl, saccharin, penicillin G	D3, 3T3	Cell viability: 5 days	Based on only monolayer culture with 5-day exposure. Check 16 genes for the three germ layers as endpoints for differentiation	Monolayer culture is applicable in the EST with gene expression detection for three germ layers	25
		Differentiation assay: 5 days

ATRA, all trans retinoic acid; EB, embryoid body; ESC, embryonic stem cell; EST, embryonic stem cell test; 5-FU, 5-fluorouracil; mESCs, mouse embryonic stem cells; RA, retinoic acid; TTNPB, (E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; WEC, whole embryo culture.