State-of-the-art techniques in Developmental and Reproductive Technology use slow, expensive testing of pregnant female rodents, or of embryonic stem cells (ESCs) that are cultured to lose stem cell qualities and differentiate into organ tissues. This only produces toxicological outcomes similar to those in adult versions of these organs. Reproductive Stress 3M has established high throughput screens (HTSs) that test toxicants on transgenic reporter ESCs cultured as stem cells that accurately report stunted ESC quality and growth. Decreased ESC quality and growth predict drugs and commercial compounds that are embryotoxic and unsafe for development and use. By pre-differentiating ESCs for 1-3 weeks, current methods limit pluripotent ESCs? usefulness. Our unique, patented ESC quality reporters reveal adaptive responses that force differentiation that compensates for fewer stem cells. Compensatory differentiative ESC stress responses occur at lower doses and are more sensitive than ESC death assays. Reproductive Stress 3Ms ESC HTS accurately identifies nonembryotoxic drugs such as Penicillin, weak or strong embryotoxic drugs such as Salicylate or Methotrexate, respectively. Stressed ESCs increase their first differentiated lineage and suppress later ones. This 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 organs. Our stemness-reporting ESC HTS identifies doses of toxicants that stunt ESC growth and stemness which would lead to miscarriage. But since we test pluripotent ESC, these may survive stress and transmit errors into the next generation. A final improvement in our ESC HTS is to switch in Aim 1 our current fluorescent reporter, which has cellular background for some toxicants, for a ?- lactamase reporter without background.
In Aim 2, we do global mRNA marker analysis to support the interpretation that stress forces differentiation unique to first lineage and decreases later lineages; thus leading to miscarriage or later deficient organogenesis.
In Aim 3, we increase the validating drug sets? size to show the HTSs? reproducibility and predictive nature for in vivo toxicological outcomes occurring to embryos after gestational exposures of the same compounds tested here in vitro. These three aims will lead to, in a Phase 2 follow up grant, a multilab validation and increased numbers of pharma from the 2.4k FDA approved set and the 10k NIEHS environmental toxicant set to gain greater approval of pharma and compound manufacturers in need of highly predictive, rapid and inexpensive toxicology testing.
In this STTR, ReproStress3M 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 levels. ReproStress3M company labs will bioengineer the revised reporter stem cells and the academic lab will validate and prove that strong, weak and nonembryotoxic pharma have proportional effects as we have shown on the unrevised, patented stem cells. Both labs will use global mRNA markers, through NextGen sequencing that shows that toxic stress causes potency loss to match proliferation loss, induction of first lineage differentiation, and suppression of later lineages. Together 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.
