Embryonic stem cell high throughput screen accurately reports toxic stress

State of the art techniques in Developmental and Reproductive Technology use slowexpensive testing of pregnant female rodentsor of embryonic stem cellsESCsthat are cultured to lose stem cell qualities and differentiate into organ tissuesThis only produces toxicological outcomes similar to those in adult versions of these organsReproductive StressM has established high throughput screensHTSsthat test toxicants on transgenic reporter ESCs cultured as stem cells that accurately report stunted ESC quality and growthDecreased ESC quality and growth predict drugs and commercial compounds that are embryotoxic and unsafe for development and useBy pre differentiating ESCs forweekscurrent methods limit pluripotent ESCsusefulnessOur uniquepatented ESC quality reporters reveal adaptive responses that force differentiation that compensates for fewer stem cellsCompensatory differentiative ESC stress responses occur at lower doses and are more sensitive than ESC death assaysReproductive StressMs ESC HTS accurately identifies nonembryotoxic drugs such as Penicillinweak or strong embryotoxic drugs such as Salicylate or MethotrexaterespectivelyStressed ESCs increase their first differentiated lineage and suppress later onesThis should eliminate false negatives of other assays as toxicants may stress ESCs and decrease later differentiated organs although the toxicants don t directly affect these organsOur stemness reporting ESC HTS identifies doses of toxicants that stunt ESC growth and stemness which would lead to miscarriageBut since we test pluripotent ESCthese may survive stress and transmit errors into the next generationA final improvement in our ESC HTS is to switch in Aimour current fluorescent reporterwhich has cellular background for some toxicantsfor alactamase reporter without backgroundIn Aimwe do global mRNA marker analysis to support the interpretation that stress forces differentiation unique to first lineage and decreases later lineagesthus leading to miscarriage or later deficient organogenesisIn Aimwe increase the validating drug setssize to show the HTSsreproducibility and predictive nature for in vivo toxicological outcomes occurring to embryos after gestational exposures of the same compounds tested here in vitroThese three aims will lead toin a Phasefollow up granta multilab validation and increased numbers of pharma from thek FDA approved set and thek NIEHS environmental toxicant set to gain greater approval of pharma and compound manufacturers in need of highly predictiverapid and inexpensive toxicology testing In this STTRReproStress M will revise a previously patented embryonic stem cell line that reports toxicant stress forced loss of quality and predicts embryo miscarriage at high stress and suppressed later embryo function at lower stress levelsReproStress M company labs will bioengineer the revised reporter stem cells and the academic lab will validate and prove that strongweak and nonembryotoxic pharma have proportional effects as we have shown on the unrevisedpatented stem cellsBoth labs will use global mRNA markersthrough NextGen sequencing that shows that toxic stress causes potency loss to match proliferation lossinduction of first lineage differentiationand suppression of later lineagesTogether these data will show that our assay is precise with low false positive and negative rates and predicts miscarriage as well as later organogenic defects missed by the assays used by other toxicology labs