Evaluation of the Embryotoxic Potency of Compounds in a Newly Revised High Throughput Embryonic Stem Cell Test

Abstract

The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals.

In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing

“…The embryonic stem cell test was performed as described previously [29] with some minor modifications. For the differentiation process, hanging drops of 20 µl cell suspension (3.75.10 4 cells/mL) were prepared at day 0 on the cover of 96-well plates.…”

Mouse and Human Embryonic Stem Cells: Can They Improve Human Health by Preventing Disease?

“…Progress toward this goal has been made recently by adapting the EST to 96-well low attachment dishes [22]. Twelve chemicals with known embryotoxicity were compared using the EST endpoints.…”

Embryonic stem cell application in drug discovery

“…Mul- [13,16,17] . For example [1] , high-throughput identification of small molecules revealed that the orphan ligand phosphoserine (P-Ser) was an enhancer of neurogenesis.…”

“…Mouse ES (mES) cells have been in use for several years in drug discovery to develop genetically modified mice for target validation, target selectivity, model development and toxicity evaluation [12][13][14][15][16][17] . Combined with advances in hES cell technology, the drug discovery community may soon have a physiologically relevant screening tool (with limitless availability) that shows normal growth and genetic structure.…”