Periodic Reporting for period 1 - ReproTox (Developing a human-based stem cell model for reproductive toxicity)
Periodo di rendicontazione: 2024-08-01 al 2026-01-31
Environmental factors and chemicals are thought to be one cause of declining male fertility.
Reproductive toxicity is becoming a major public health problem. Currently, new pharmacological or
chemical compounds are tested in animals for their effects on reproduction before entering the market.
This risk assessment is essential but time-consuming, expensive and is not always predictive in humans
since animals have different reproductive characteristics and physiology. A validated and qualified
human system to assess reproductive toxicity would be invaluable in addressing this conundrum between
regulatory requirements and true relevance.
Through my ERC-CoG, I have built a large repository of knowledge and developed robust technology
on human in vitro gametogenesis, more specifically on how to generate male gamete progenitors from
human pluripotent stem cells. This, together with my bioinformatics knowhow, makes me exceptionally
well-placed to optimise a human-based high-throughput screening assay for male reproductive toxicity
for use by the private sector in filing reproductive safety data to the regulatory authorities. This
ReproTox assay will be further optimised (increase throughput, standardise measurements) and
integrated with a software innovation (machine learning algorithm to automate quantification and
webtool), before we carry out a pilot screen using FDA-approved spermatotoxic compounds and market
research to define the commercialisation route.
Reducing the number of compounds that show male reproductive toxicity at very early stages of drug
development will contribute to an effective first-tier toxicity screening, not only providing standard
protocols for quantitative, rapid, cost-effective, high-throughput, scalable and reproducible toxicity
testing, but also reducing, perhaps eventually replacing animal use. ReproTox will ultimately lead to
economic, societal, environmental, health and ethical benefits.
Reproductive toxicity is becoming a major public health problem. Currently, new pharmacological or
chemical compounds are tested in animals for their effects on reproduction before entering the market.
This risk assessment is essential but time-consuming, expensive and is not always predictive in humans
since animals have different reproductive characteristics and physiology. A validated and qualified
human system to assess reproductive toxicity would be invaluable in addressing this conundrum between
regulatory requirements and true relevance.
Through my ERC-CoG, I have built a large repository of knowledge and developed robust technology
on human in vitro gametogenesis, more specifically on how to generate male gamete progenitors from
human pluripotent stem cells. This, together with my bioinformatics knowhow, makes me exceptionally
well-placed to optimise a human-based high-throughput screening assay for male reproductive toxicity
for use by the private sector in filing reproductive safety data to the regulatory authorities. This
ReproTox assay will be further optimised (increase throughput, standardise measurements) and
integrated with a software innovation (machine learning algorithm to automate quantification and
webtool), before we carry out a pilot screen using FDA-approved spermatotoxic compounds and market
research to define the commercialisation route.
Reducing the number of compounds that show male reproductive toxicity at very early stages of drug
development will contribute to an effective first-tier toxicity screening, not only providing standard
protocols for quantitative, rapid, cost-effective, high-throughput, scalable and reproducible toxicity
testing, but also reducing, perhaps eventually replacing animal use. ReproTox will ultimately lead to
economic, societal, environmental, health and ethical benefits.
During ReproTox, we have increased robustness, using different hiPSC lines to validate our assay and optimizing the protocol further. Importantly, we adapted the assay successfully to a 384-plate format, generated single cell transcriptomics data and generated IC50 for spermatotoxic compounds of interest, showing toxicity in germ cells, but not in non-germ cells in our differentiated assay.
In addition, we have generated a machine-learning algorithm/AI to predict germ cell identity based on DAPI staining alone. This bioinformatics tool allows to rapidly screen for germline toxicity, without the need to perform immunofluorescence for additional markers. Furthermore, we developed an interactive software module or webtool platform DASHBOARD for quantification of the total number of germ cells, to allow fast and accurate quantification of toxicity on a 384-well format. This is an easy-to-use webtool for direct visualization of results.
Finally, an independent market research was performed to define a route for commercialization towards regulatory acceptance as well IP protection of ReproTox.
In addition, we have generated a machine-learning algorithm/AI to predict germ cell identity based on DAPI staining alone. This bioinformatics tool allows to rapidly screen for germline toxicity, without the need to perform immunofluorescence for additional markers. Furthermore, we developed an interactive software module or webtool platform DASHBOARD for quantification of the total number of germ cells, to allow fast and accurate quantification of toxicity on a 384-well format. This is an easy-to-use webtool for direct visualization of results.
Finally, an independent market research was performed to define a route for commercialization towards regulatory acceptance as well IP protection of ReproTox.
During ReproTox, we have optimized, miniaturized, automized and used machine learning into our high-throughput screening assay for human male reproductive toxicity. We are now moving towards validation following OECD-toxicology guidelines and we will apply for Dutch/EU funding for this to continue on this path.
We are still in the process of extending the list toxic compounds tested in ReproTox to collect and broaden evidence of the efficacy of the assay.
We are also in the process of contacting relevant stakeholders to further validate ReproTox, to ultimately use our assay for DART testing, while reducing the costs associated with animals used, the reducing the number of animals used, while increasing efficacy and speeding up results.
We are still in the process of extending the list toxic compounds tested in ReproTox to collect and broaden evidence of the efficacy of the assay.
We are also in the process of contacting relevant stakeholders to further validate ReproTox, to ultimately use our assay for DART testing, while reducing the costs associated with animals used, the reducing the number of animals used, while increasing efficacy and speeding up results.