Stress forces huESC differentiation; confirmation as an accurate DevTox HTS

EXECUTIVE SUMMARY - huESC HTS2 - human
Toxic stress effects in 1st trimester pregnancy leads to $10s of billions of US health care costs/year and is a
significant part of the $2.5-4B/year US Developmental and Reproductive Technology (DART industry. Current
DART techniques use slow, expensive pregnant rodent tests, or tests of embryonic stem cells (ESCs) that are
cultured to lose “stemness” and differentiate into organ tissues. As differentiated organ tissues, our competitors
DART high throughput screens (HTSs) test only metabolic or cytotoxic effects of compounds on cell survival and
function. ReproStress Inc. exposes undifferentiated ESCs to stress and test for organismal survival outcomes of
developing embryos. We have shown, and developed ESC HTSs to test, stress-forced changes in stemness
loss and abnormal differentiation to 1st lineage while suppressing later lineages. These outcomes should assess
organismal risks for stunting, teratogenesis and miscarriage. Our HTSs should be able to reduce, refine, and
replace many organismal DART assays now done in pregnant females. Reproductive Stress 3M has invented
HTSs that test toxicants on transgenic reporter ESCs cultured as stem cells that accurately report stunted ESC
quality and growth. These HTSs show stress-forced stem cell stunting, differentiation and depletion that predicts
drug exposures that are embryotoxic and unsafe for development. ReproStress’ patented stemness loss ESC
HTS(1) identifies nonembryotoxic drugs such as Penicillin, weak or strong embryotoxic drugs such as Salicylate
or Methotrexate, respectively. In 2019, we published a paper and converted the provisional patent for mouse
(mu)ESC HTS2 which reports 1st lineage increase. Commercial customers and government regulators want
conversion of mouse to human (hu)ESC HTS. Although rare in huESC, we found 2 huESC lines in the GEO
transcriptomic databases, NIH-approved H1 and H9 lines, that do differentiate 1st lineage. In Aim 1 we will confirm
that H1 and/or H9 huESCs produce 1st lineage during normal differentiation, and more 1st lineage marker/cell
during stress-forced differentiation. In Aim 1 we will make transgenic huESC with H1 or H9 with two
improvements over muESC HTS2; 1) we will change high background fluorescent reporters to low background,
rapid turnover luminescent reporters. To drive luminescence reporters, our current R41 transcriptomic data
suggested stronger promoters for 1st lineage exist by two criteria. Stronger promoters should enable a higher
resolution, more reproducible assay and we will test patented Pdgfra promoter and a second, stronger 1st lineage
reporter for both criteria. In Aim 1, we increase the validating drug set size to show the in vitro HTSs’
reproducibility and predictive nature for in vivo toxicological outcomes after gestational exposures of the same
compounds. In Aim 2, we analyze single cell global transcriptomic marker to corroborate the luminescence
marker itself and support the theory that stress forces differentiation unique to 1st lineage and decreases later
ones; leads to teratogenesis. These aims will lead to a Phase 2 grant to test compounds from the large 10k
NIEHS toxicant set, leading to regulatory approval and sales to DevTox testing for commercial customers.Narrative Grant R41 STTR huESC DevTox HTS
Miscarriage, or some degree of teratogenesis and epigenetic disease affect most human embryos in the first half
of the first trimester of pregnancy and cause tens of billions of dollars of yearly, US health care and other costs.
The market for cell-based toxicology is the fastest growing one in all toxicology and in vitro Developmental toxi-
cology is the fastest growing in all DART assays, where all DART is 2.6B/year and in vitro DART is 118M/year.
We will transfer a high-throughput screens in mouse ESC to human ESCs and show that these predict exposures
of 6 categories of stressful toxicants from the National Research Council’s 21st century toxicology report, and 22
chemicals from the Multilab European ESC DevTox validation and thus develop a new HTS to help predict
miscarriage and other development diseases causing billions of dollars of health costs yearly.