embryotoxicity tests are currently conducted with organization for economic cooperation and development ( oecd)guidelines . in vivo tests used now days are more time consuming , expensive , and require lot of skill and expertise . moreover these experiments require a number of laboratory animals to be sacrificed which raises ethical concerns . therefore , to lower down the animal experimentation , many in vitro methods have been developed . these include whole embryos from whole embryo culture ( wec ) , xenopus ( fetax ) test , or chicken ( chest ) ; however , all these assays have been used rarely because their predictive valve is only 7080%.[36 ] according to stringent testing measures 30 000 chemicals that are currently on the market will have to be re - evaluated over the next 10 years within the european union with an estimated use of 10 million animals for in vivo teratogenicity testing . therefore , in vitro developmental toxicity tests need to be established in order to reduce the number of test animals and expenses , without compromising the safety of consumers and patients . furthermore , such in vitro methods should be better suited to test a larger number of chemicals than those employed in vivo tests . more than 30 in vitro assays using invertebrates or vertebrates to predict the embryotoxic potential of test compounds have been developed . for the prediction of reproductive effects in humans , mammalian in vitro assays are the first choice . three assays based on ontogenesis have been validated by an international study , namely , the micromass ( mm ) test systems which use dissociated cells from the limb buds and brains of rat embryos , whole frog embryos ( the frog embryo teratogenesis assay ) and whole rat embryos the whole embryo culture ( wec ) test.[4912 ] animal - free , cell - line - based assays include , the european centre for the validation of alternative methods ( ecvam ) validated embryonic stem cell test ( est ) which is probably the most extensively studied test in its class as no pregnant animals have to be sacrificed , since two permanent mouse cell lines ( d3 and 3t3 ) are used . embryonic stem ( es ) cell lines are established from the inner cell mass of the 3.5 day mouse blastocyst . they can be expanded under the control conditions apparently without limit in culture , while remaining pluripotent . this means they can give rise to more specialized cells of the ectodermal , mesodermal , and endodermal lineages , such as neuronal cells , heart , liver , blood vessel , and pancreatic islet on the addition or removal of certain growth factors . they are the only known truly immortal stem cells and most importantly maintain a normal diploid karyotype . due to their fundamental attributes of unlimited expansion and pluripotency , embryonic stem cells have gained considerable interest from the biopharmaceutical sector for their use in drug discovery and developmental toxicity testing . within the toxicological field es cells have been utilized in two approaches ; established mouse lines are used in the embryonic stem cell test ( est ) for developmental toxicology and mouse and human es - like derivatives of particular lineages are being used for early stage assessment of drug adsorption , metabolism and toxicity . the est was developed by horst spielmann and his group in 1997 as an in vitro model for the screening of embryotoxicity , based on a blastocyst - derived permanent embryonic mouse esc ( mesc ) d3 cell line derived from mouse 129 strains . the mouse embryonic stem cell test ( mest ) is based on the assessment of three toxicological endpoints which are : inhibition of growth ( cytotoxicity ) of ( i ) 3t3 cells , which represent differentiated cells and ( ii ) undifferentiated es cells after 10 days of treatment . this cytotoxicity is determined by a test which utilizes the dehydrogenase enzymes present in the intact mitochondria of living cells to convert yellow soluble substrate 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide ( mtt ) , into a dark blue insoluble formazan product , which gets sequestered within the cells and is detected quantitatively using a microplate elisa reader , after solubilizing the cell membrane . the third endpoint is the inhibition of differentiation of es cells into myoblasts which are cardiac muscle cell precursors after 10 days of treatment . contracting myocardiocytes were chosen as an endpoint since it not only detects the mere differentiation into a certain cell type , but also resembles the intact functional interplay between several related cell types such as sinusnode , atrial , and ventricular cells . the est categorizes 20 potentially embryotoxic test chemicals with known embryotoxic potential , selected from a published list recommended by the us teratology society , for the validation of in vitro embryotoxicity tests into three classes of in vivo embryo toxicity , strong , weak and nonembryo toxic . the selection criterion for the chemicals was based upon sufficient high quality in vivo data from both testing in animals and human pregnancies . chemicals selected for testing during the pre - validation study mainly consisted of drugs which had been used in human pregnancy , for example in the case of bacterial infections or the therapy of cancer . the classification of embryotoxicity potential of chemicals after 10-day exposure is based on statistical model using half - maximal inhibition ( id50 ) value of inhibited differentiation of es cells and half - maximal inhibition ( ic50 ) values of decreased viability of 3t3 and es cells assessed from the concentration response curves as endpoints . the prevalidation study showed that the sop of the est was successfully transferred to other laboratories in europe and the usa . the reproducibility of results in each laboratory was very high ; as a result the est was a suitable test for embroyotoxicity testing . the assay was endorsed by the ecvam scientific advisory committee ( esac ) as scientifically validated and ready to be considered for regulatory acceptance and application . however the opinion of reproductive toxicologist was that the database of 20 validated chemicals was not sufficient and insisting that the database should be expanded . owing to the requirement of skilled personnel , and the fact that this test is labor intensive many alternative strategies including those suitable for a high - throughput screening of chemicals for embryotoxicity test systems have been developed . in addition , it was suggested that additional molecular endpoints should be included in the mest and the development of reporter gene assays based on the mest.[1315 ] one approach was the assessment of marker genes ( molecular endpoints ) which get expressed when the es cell differentiated along a specific lineage . when stem cells differentiate into an embryoid body ( a three - dimensional mass of cells of the various lineages resembling an early embryo ) , many different cell types arise . therefore , it would be possible to assess this differentiation by simultaneously measuring the expression of genes characteristic of many different cell types . although there are several common features in both embryonic stem cell lines , differences are also present , e.g. , expression of differentiation markers and the cell cycle . this type of data was used as a valuable marker for choosing new end points and evaluating the similarities and differences between different cell lines and species , thus improving the toxicological risk assessment . specifically , the analysis of cardiomyocyte differentiation alone as an endpoint does not suffice for the determination of embryotoxic effects . methods have been developed for further improvement of est to include molecular endpoints for bone , cartilage , as well as tissues of neuronal and endodermal origin . further investigations are on the way to replace subjective assessment by objective measurements . as such , genetic modifications and molecular markers such as the ones detected by facs and quantitative gene expression analyses offer significant advantages in developing a new test approach . the gene expression can be quantitated by real - time quantitative polymerase chain reaction ( pcr ) in the presence or absence of test chemicals.[1520 ] a genetically engineered mouse es cell line expressing green fluorescent protein ( gfp ) under the control of cardiac -actinin was developed which could be easily analyzed using fluorescence - activated cell sorting ( facs ) method . tissue - specific proteins in es cell cultures could also be studied using immune fluorescent antibody technique and facs analysis . the expression of tissue - specific marker proteins , -actinin and myosin heavy chain was quantified by intracellular flow cytometry assay . based on kinetic analysis , the strongest signals were observed at day 7 of differentiation indicating that a reduction in protein expression induced by embryotoxic compounds could be best monitored at day 7 of culture . to determine whether facs with cardiac - specific marker proteins could be used as a new toxicological endpoint in the est , selected compounds with known teratogenic potential were tested and the results were compared to those obtained with the existing est . facs analysis therefore can replace the microscopic evaluation of beating cardiomyocytes in the est and -actinin and mhc can be used as marker proteins for cardiac development . tissue- or organ - specific antibodies labeled with immune fluorescent dyes may be useful in screening a high number of test chemicals in the est.[2024 ] although the toxicological endpoint used in the classical est , holds true for toxicological interpretations in humans , it has been demonstrated that high interspecies variations are a serious problem for assessing developmental toxicity in vivo . the most famous examples are that of thalidomide and isotretinoin which both demonstrated no obvious effects in mice but led to severe malformations in human embryos or selected laboratory species . therefore , the use of human embryonic stem cells provides an added value for reliable safety assessments of drugs and chemicals in comparison to the current practice of in vivo testing since the data are directly applicable for hazard assessment . human es cell lines may prove clinically more relevant in developing safer and more effective drugs for human diseases . the establishment of human embryonic stem cells raises hopes to increase the safety of patients and consumers due to humanized and sophisticated pharmacological and toxicological in vitro tests . human es cells show many advantages in the field of toxicology testing , due to their unlimited proliferation ability , their pluripotency and their potential to generate derivatives of all three germ layers that makes them a readily available source of human cells . since the initial report of the derivation of he s cell lines , a variety of studies have established in vitro spontaneous and directed differentiation systems to several lineages , including cardiac tissue , neuronal tissue , etc . however , there are some morphological as well as functional differences between mes and he s cells . the he s cells form flat colonies that can be dissociated into single cells with common mechanical and enzymatic techniques . however , they do not form new colonies out of single cells but form cell aggregates . a very important difference between the mouse and human es cells in contrast to mouse model , in which the addition of leukemia inhibitory factor ( lif ) to the culture medium is sufficient to maintain the mes cells in an undifferentiated state , the he s cells require the presence of feeder layer consisting of mouse embryonic fibroblast ( mefs ) as well as culture medium supplemented human basic fibroblast growth factor ( bfgf ) . further , there are also important variations in the in vitro cardiomyocyte differentiation properties between the he s and mes cell lines . human es cells differentiate in vitro into cardiomyocyte at a slower rate than mes cells . moreover , it is not necessary that only cardiomyocyte will be affected by potential drugs and other sensitive cells and organs may be more prone to these drugs.[2232 ] well - chosen endpoints may contribute to examining the mechanism of teratogens and deciding the dose concentrations . in vitro testing must include homogenous cell interactions so that a response for particular tissue or cell can be evaluated . the embryonic stem cell could be used directly for those toxicants which could alter the events in the development of embryo and show us how stem cell regulate their fate in response to drugs or teratogens . understanding the basics of embryonic stem cell biology and factors related to them , can lead us to develop new approaches in toxicity testing from the stages of embryo development to fetus and finally to the mature and fully developed organ . stem cells can provide sufficient quantities of tissue - specific cells for screening or toxicity testing . an ideal test system in toxicology the key characteristics for adoption of stem cells are , they exhibit key cellular characteristics , stem cell can recapitulate normal human biology ; these can be reproducible and provide us with known and relevant genome . basically stem cells , give us quality , quantity and purity for testing toxicology pharmacological industry and academia need to develop in vitro assays based on the human es cell lines . this would provide an opportunity to assess the species - specific toxicity endpoints early in drug development and research . not only would this lead to lesser use of animals but also would dramatically cut down the cost of drug development . es - derived cells would be a great approach which could improve drug research and development . es derived differentiated cells could also be used to learn more about diseases of interest . well - explored in vitro method of human escs will throw some light on effect of new drugs on fetal development which mimics in vivo fetal development .
stem cells in the body have a unique ability to renew themselves and give rise to more specialized cell types having functional commitments . under specified growth conditions , 
 these cell types remain unspecialized but can be triggered to become specific cell type of the body such as heart , nerve , or skin cells . 
 this ability of embryonic stem cells for directed differentiation makes it a prominent candidate as a screening tool in revealing safer and better drugs . 
 in addition , genetic variations and birth defects caused by mutations and teratogens affecting early human development could also be studied on this basis . 
 moreover , replacement of animal testing is needed because it involves ethical , legal , and cost issues . 
 thus , there is a strong requirement for validated and reliable , if achievable , human stem cell - based developmental assays for pharmacological and toxicological screening .