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Development of Stem Cell-based Assay for High-Throughput Screening of Chemicals of Toxicological Concern

Adversehumanhealthoutcomes ? a.k.a., ?toxicity? ? caused by pharmaceutical or environmental compounds are a major cause of drug development failure and public health concern. Methods to evaluate the potential of chemical compounds to induce toxicity are based largely on animal testing, are low-throughput and expensive while giving little insight into mechanisms of compound toxicity, and have not changed appreciably in the last 50 years despite enormous advances in science. Multiple efforts, including Tox21 program in the U.S., REACH Program in the E.U., and multiple industrial collaborations, are attempting to develop in vitro methods using induced pluripotent stem cell (iPSC)-derived cells to assess chemical toxicity. Stem cells or iPSC-derived cell types have great potential to provide more physiological relevance than immortalized/transformed cell lines and to provide larger quantities and higher assay reproducibility than primary cells. These programs must assess toxicity potential in every organ system and identify pathways and/or targets affected.
Given the protean nature of these effects, it is likely that hundreds of in vitro assays will need to be developed and evaluated for their ability to profile chemical effects on particular cell types and pathways. Progress in the field is currently limited by the relatively small number of pathways and cell types that have been developed into high- throughput screening (HTS)-ready assays, and the artificial nature of many of the assays that have been developed (e.g., immortalized/transformed cell lines, heterologous expression with lack of physiologically accurate regulation).
The development of HTS-ready assays using stem cells or iPSC-derived cells, which can report on particular pathways and cellular phenotypes across the full spectrum of pathway space and toxicological outcomes, is needed. Such assays would need to meet strict performance criteria of robustness, reproducibility, and physiological relevance. The assays developed would need to be capable of being run in 384-well or (ideally) 1536-well format and must allow the testing of >100,000 samples per week.
Mainrequirements
Theoutcomeofthiscontractisexpectedtobeoneormorenovelassays that can be performed in stem cells or iPSC-derived cells fortargets,pathways,andcellularphenotypesrelatedtoanytypeofxenobiotictoxicity.Theseassayswouldutilizehumancells,includingprimarycellsandstemcellderivedcells,andmustbefunctionalinmulti-wellformatwithcharacteristicssuitableforautomatedhigh-throughputscreening.Suchassaysshouldbenovel, having metabolic capability, reflecting new pathways or cellular endpoints than are currently available, and be clearlyconnectedtosometypeofhumantoxicologicalresponse.Suchassayscouldfindutilityasinchemicalassessmentandriskmanagementafter validation.
DeliverablesPhase1
Anassaythatmeetstherequirementslistedaboveandalsomeetsthefollowing:
?          Developaworkingassayin96-wellordenser(384,1536)micro-wellformat
?          Characterizethesensitivity,specificity,variability,reproducibility,signal:background,dynamicrange,and accuracyoftheassay,utilizingstandardpositiveandnegativecontrols,Z?values>0.5
?          Demonstratetheutilityoftheassaybycharacterizingitsabilitytodetecttheeffectsofcompoundsknowntoaffectthepathway/cellularphenotype,withathroughputofatleast10,000samples/daywithworkstationautomation
?          Arenotduplicativeofassaysalreadyavailablecommercially
?          Delivertheassay/SOPtoNCATSforevaluation
 
DeliverablesPhase2
?          Demonstrateminiaturizationofassaytoworkinatleast384-well(preferably1536-well)formatwithsametechnicalspecificationsaslistedabove
?          DemonstrateamenabilityforHTSbysuccessfultestingof>100,000samples/dayinfullyautomatedroboticformatwithmaintenanceofassayperformance
 
?          Deliverfinalassay/SOPtoNCATSforevaluation.

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