A strong evidence base for the effective and safe use of iPSC-based strategies for regenerative treatment of IDD, restoring the IVD to a functional unit.
Building on the standard operating procedures developed by the consortium, we further improved the protocols to drive robust differentiation into iPS-notochordal-like cells (iPS-NLCs), the disc cells that reside within the core of the juvenile IVD. The best performing biomaterials were selected, optimized, and further characterized in their capacity to support the native notochordal cells (NCs). Initial studies confirmed the viability of the iPS-NLC-progenitors within the six selected biomaterials and ongoing efforts focus on selecting the two best-performing biomaterials for further translation into the ex vivo and in vivo models of IVD degeneration. Upon ethical approval a pilot study was conducted to assess the initial biodistribution and viability of the iPS-NLC-progenitors in a sheep disc degeneration model. Newly designed equipment has been constructed and proof-of-concept studies have demonstrated the usability of the portable spine biomechanics tester and the dynamic loading bioreactor for the human disc tissue. Furthermore, the deep machine learning algorithm developed for staging disc degeneration/regeneration is now readily available. The external scientific advisory board embraced the activities performed. The consortium is now ready for the first discussion with a national regulatory authority. Furthermore, we have consolidated the available knowledge, expertise, and experiences to create guidelines for specimen processing as well as functional and architectural evaluations for an optimal study design that utilizes large animal models in disc regeneration studies.
Enabling technologies for the development, characterisation, and validation of the iPSCbased strategy.
The consortium has focused its efforts on improving the purification methods by employing multiple surface markers to exclude unwanted cell-linages and by determining new NC-specific markers to improve the iPS-NLCs differentiation. Within this context, an Aggrecan iPS reporter cell line has been developed and functionally characterized, while a new cell line is under development to monitor NOTO expression.
The developed complementary methods and tools developed (flow cytometry, immunostainings, DigiWest proteomics technology and transcriptomic analyses) have been successfully implemented to demonstrate iPS-NLC differentiation efficiency, phenotypic identity, and purity. The consortium successfully generated cloaked/FailSafe cells with a dual fluorescence reporter system to facilitate the in vitro and in vivo tracing of iPS-NLCs. The intracellular delivery (iTOP) technology has been shown to be feasible for iPS cells and the consortium has demonstrated that CRISPR activation can be used to target multiple transcription factors essential in the iPS-NLC differentiation process.
Much effort was placed on completing the development and validation of all novel and advanced platforms, which were used to initially start evaluating the performance of biomaterials combined with cells for the regeneration of the IVD. Finally, a unique open digital platform prototype to guide the design of preclinical studies for advanced therapies was completed. Together with the evaluation platforms, comprehensive pre-in-vivo testing system for the evaluation of IVD advanced therapeutics complying with the 3Rs philosophy for animal testing has been developed.
A recommendation on ethical and regulatory affairs to enhance and accelerate further development and implementation of iPSC-based therapeutic strategies.
To broaden the understanding of ethical aspects in stem cell research, hard and soft impacts were mapped and published in an open access journal. We have spoken with Dutch patients with chronic LBP to identify and understand what they perceive to be the ethical dimensions of the iPSpine research and the potential iPSC-based treatment. To explore the interplay of ethical, policy, and regulatory aspects we have produced a comprehensive compendium of European laws (46 countries), international norms, and policies (hard and soft norms) regulating stem cell research. We completed a preliminary analysis of this compendium by focusing on central ethical and policy issues regarding contentious applications, including criteria for permissibility, oversight, and enforcement mechanisms.
The Partners received further training from regulatory experts and in the iPSpine research activities, regulatory advice is continuously being embedded to guide the consortium through the regulatory field of ATMP. Importantly, we prepared together with key researchers from the different work packages for the first National Scientific Advice which will take place in March 2022. The Advice will be focusing on the design of safety and efficacy studies in large animal models, as well as feedback to iPS cell line development and product and biomaterial classification. Hereby, iPSpine envisions to set an example on long-term, fast-forwarding, preclinical research that is closer to the patient in need.
Progress beyond the state of the art, expected results until the end of the project and potential impacts.
Upon successful implementation of the iPSpine therapy, we envision improved outcome and QoL for patients with IVD degeneration-related LBP, through effective regeneration of the degenerated tissue with restored biomechanical function of the disc, and long-lasting reduction of LBP. Altogether, this will result in reduced LBP-related premature retirement and improved socio-economic contribution.
The novel and extended knowledge, tools and technologies that will be the outcome of the iPSpine project, will provide unique platforms of knowledge, testing, and exploitation with broad applicability. These platforms will contribute to acceleration, innovation, and implementation of novel technologies and ATMPs for treatment of large patient groups. Hereby, iPSpine realizes its ambition to make a significant contribution by reducing translational bottlenecks through open innovation and taking European leadership in the development of ATMPs.