Periodic Reporting for period 1 - hiPSCmore (Instrumented human stem cells to see more during high throughput screening in drugInstrumented human stem cells to see more during high throughput screening in drug discovery and regenerative medicine)
Periodo di rendicontazione: 2024-05-01 al 2025-10-31
Early decision-making in drug discovery is limited by models that do not adequately recapitulate human cardiac biology and by workflows dependent on single-endpoint dyes with poor scalability. This project addresses those constraints by engineering human induced pluripotent stem cells (hiPSCs) with multiplex, endogenous reporters, enabling the same living cells to provide structural, functional, and cell-cycle information in real time. A technology we called TEMPO: Timing Early and Mature Phenotype, Optically. In parallel, we target a bioprocess that yields mature hiPSC-derived cardiomyocytes (hiPSC-CMs) in high quantity by scaling up expansion and differentiation in stirred systems. Together, these elements are intended to deliver human-relevant readouts earlier and at lower marginal cost and complexity.
Objectives.
OBJ1 – Technology feasibility: scale hiPSC manufacturing in stirred bioreactors; validate TEMPO hiPSC-CMs in image-based high-throughput screening for drug discovery and regeneration readouts.
OBJ2 – Business feasibility: quantify cost of goods (COGS) and cost-down levers; conduct market and customer research; develop a cost–benefit model for initial launch segments.
OBJ3 – Executive management: secure IP/FTO; select go-to-market pathway (cells vs contract research service vs co-development); prepare the spin-off plan.
Definition of success (public, general terms).
• Bioprocess: materially higher yields than conventional 2D approaches in a workflow compatible with stirred bioreactors, specifically geared to produce mature hiPSC-CMs at scale.
• Assay validity: reagent-free, multi-signal screening demonstrated using compounds of known pharmacology (e.g. electrophysiology modulators and regenerative cues), evidencing both safety and regeneration readouts.
• Business readiness: a credible cost model with clear levers, an executable IP/licensing pathway, and a spin-off-ready plan.
Expected impact. The platform aims to enable earlier and more reliable decisions, reduce false calls through cell-cycle-aware analysis, decrease dependence on single-use dyes, and offer a practical route from academic innovation to deployable industrial screening—aligned with EU priorities in human-relevant testing and advanced manufacturing.
Objectives.
OBJ1 – Technology feasibility: scale hiPSC manufacturing in stirred bioreactors; validate TEMPO hiPSC-CMs in image-based high-throughput screening for drug discovery and regeneration readouts.
OBJ2 – Business feasibility: quantify cost of goods (COGS) and cost-down levers; conduct market and customer research; develop a cost–benefit model for initial launch segments.
OBJ3 – Executive management: secure IP/FTO; select go-to-market pathway (cells vs contract research service vs co-development); prepare the spin-off plan.
Definition of success (public, general terms).
• Bioprocess: materially higher yields than conventional 2D approaches in a workflow compatible with stirred bioreactors, specifically geared to produce mature hiPSC-CMs at scale.
• Assay validity: reagent-free, multi-signal screening demonstrated using compounds of known pharmacology (e.g. electrophysiology modulators and regenerative cues), evidencing both safety and regeneration readouts.
• Business readiness: a credible cost model with clear levers, an executable IP/licensing pathway, and a spin-off-ready plan.
Expected impact. The platform aims to enable earlier and more reliable decisions, reduce false calls through cell-cycle-aware analysis, decrease dependence on single-use dyes, and offer a practical route from academic innovation to deployable industrial screening—aligned with EU priorities in human-relevant testing and advanced manufacturing.
Progress is reported by work package, with emphasis on risks foreseen at proposal stage and the corresponding mitigations.
WP1: Technological Feasibility.
In mature cardiomyocytes, the original multi-sensor cassette in TEMPO exhibited broad epigenetic silencing—an anticipated risk. As planned, we executed the backup: genome editing into a safe-harbor locus with a revised promoter/architecture. We called this new technology CALIPERS for Cell-cycle Aware Live-Imaging Platform for Enhanced Readouts & Screening. CALIPERS maintains expression in mature hiPSC-CMs and supports phase-aware analysis of structure and function. Given the effort required to re-engineer the line, the scope of the remaining WP1 tasks was rescaled preserving their intent.
• Bioprocessing. Transitioning to a 3D stirred bioreactor increased hiPSC expansion but did not improve maturation to hiPSC-CMs—consistent with common field observations when 2D differentiation is directly ported to 3D vessels. We therefore implemented an alternative route: 3D cardiac organoid differentiation followed by dissociation and re-seeding of hiPSC-CMs. This workflow yielded structurally and functionally mature cardiomyocytes suitable for live high-content imaging. Its direct deployment in bioreactors is planned for the next phase; time invested in CALIPERS limited in-grant bioreactor re-testing of the organoid protocol.
• Validation. Using CALIPERS, we performed proof-of-concept assays to confirm intended behavior. Regenerative cues (e.g. agrin) induced cell-cycle re-entry with concordant structural/functional changes captured by the multiplex reporters. A focused subset of compounds with known electrophysiology mechanisms (e.g. nifedipine) produced the expected direction and pattern in calcium handling and contractility. Full-scale validation panels will be executed in future work.
WP2: Business Feasibility. A parametric COGS tool was assembled to compare workflows and identify principal levers (automation, plate strategy, media/QC bundling). Market mapping and stakeholder interviews indicate interest in mature-cardiomyocyte, multi-signal screening with manageable switching costs when provided through a simple workflow and clear analytics. A cost–benefit framework links fewer false calls and faster time-to-decision to downstream savings.
WP3: Executive Management. IP/FTO and licensing pathways with the originating institute were clarified; an academic material-sharing framework is active. Two go-to-market options remain viable in principle: a contract research service for innovative cardiac screening and/or a biotech model using the platform as a proprietary discovery asset. To strengthen long-term defensibility, the spin-off strategy emphasizes establishing a proprietary, diverse hiPSC panel (sex/ancestry) beyond prototyping lines.
WP1: Technological Feasibility.
In mature cardiomyocytes, the original multi-sensor cassette in TEMPO exhibited broad epigenetic silencing—an anticipated risk. As planned, we executed the backup: genome editing into a safe-harbor locus with a revised promoter/architecture. We called this new technology CALIPERS for Cell-cycle Aware Live-Imaging Platform for Enhanced Readouts & Screening. CALIPERS maintains expression in mature hiPSC-CMs and supports phase-aware analysis of structure and function. Given the effort required to re-engineer the line, the scope of the remaining WP1 tasks was rescaled preserving their intent.
• Bioprocessing. Transitioning to a 3D stirred bioreactor increased hiPSC expansion but did not improve maturation to hiPSC-CMs—consistent with common field observations when 2D differentiation is directly ported to 3D vessels. We therefore implemented an alternative route: 3D cardiac organoid differentiation followed by dissociation and re-seeding of hiPSC-CMs. This workflow yielded structurally and functionally mature cardiomyocytes suitable for live high-content imaging. Its direct deployment in bioreactors is planned for the next phase; time invested in CALIPERS limited in-grant bioreactor re-testing of the organoid protocol.
• Validation. Using CALIPERS, we performed proof-of-concept assays to confirm intended behavior. Regenerative cues (e.g. agrin) induced cell-cycle re-entry with concordant structural/functional changes captured by the multiplex reporters. A focused subset of compounds with known electrophysiology mechanisms (e.g. nifedipine) produced the expected direction and pattern in calcium handling and contractility. Full-scale validation panels will be executed in future work.
WP2: Business Feasibility. A parametric COGS tool was assembled to compare workflows and identify principal levers (automation, plate strategy, media/QC bundling). Market mapping and stakeholder interviews indicate interest in mature-cardiomyocyte, multi-signal screening with manageable switching costs when provided through a simple workflow and clear analytics. A cost–benefit framework links fewer false calls and faster time-to-decision to downstream savings.
WP3: Executive Management. IP/FTO and licensing pathways with the originating institute were clarified; an academic material-sharing framework is active. Two go-to-market options remain viable in principle: a contract research service for innovative cardiac screening and/or a biotech model using the platform as a proprietary discovery asset. To strengthen long-term defensibility, the spin-off strategy emphasizes establishing a proprietary, diverse hiPSC panel (sex/ancestry) beyond prototyping lines.
The anticipated silencing risk in TEMPO hiPSC materialized and was resolved by implementing the planned backup, yielding CALIPERS hiPSCs. The bioprocess strategy was adapted to a 3D-first organoid route, yielding promising maturation outcomes. Initial validation with representative regenerative and safety compounds confirmed the functionality of CALIPERS hiPSC-CM in advanced imaging-based early drug screening experiments, an advantage that we will exploit with the creation of a spin-off CRO company.