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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - LSFM4LIFE (Production and characterization of endocrine cells derived from human pancreas organoids for the cell-based therapy of type 1 diabetes)

Teaser

Summary

The worldwide incidence of type 1 diabetes (T1D) is increasing at a rate of 4% yearly. This alarming epidemiological data strongly motivates the development of a cellular treatment of T1D, in which the missing beta-cells are replenished with exogenous cells. Despite many advancements, the allogeneic transplantation of Langerhans islets is only recommended for a small group of patients. The suitability of islets transplantation is limited both by donor availability and by the need of immunosuppressive treatment.
In contrast, the cellular therapy with beta-cells obtained by differentiating adult stem cells can drastically revolutionize this picture and realize the dream of a curative therapy of T1D. The stem cell approach aims to establish a virtually endless source of beta-cells, cultured accordingly to the highest safety standards defined by EMA (European Medicine Agency) and FDA (Federal Drug Administration). Moreover, Stemcell-derived beta-cells are suitable for autologous transplantation, which allows to avoid immunosuppressive treatment. A fundamental issue concerns the type of stem cells of choice. While embryonic stem cells (ESC) pose significant ethical challenges, inducible pluripotent stem (iPSC) cells could have a safety issue due to the activation of oncogenes during the reprogramming of somatic cells.
A novel strategy, consisting in the isolation and culture of pancreas progenitor cells (human pancreas organoids, hPOs), allows overcoming the drawbacks of both ESC and iPSC. Academic and industrial partners from France, Germany, Great Britain, Italy, Netherlands, and Switzerland joined in the European consortium LSFM4LIFE to develop the hPO technology. LSFM4LIFE is supported for the period 2016-2020 by a Horizon 2020 grant. The main goal of LSFM4LIFE is to achieve the culture and differentiation of therapeutic batches of hPO accordingly to Good Manufacturing Practice (GMP) quality control criteria, qualifying them for clinical testing in patients.

Work performed

At month 36 of the project, the group led by Meritxell Huch has completed the development of an improved culture media formulation for the growth of the hPO in vitro. A patent for this new formulation has been submitted. Kourosh Saeb-Parsy and collaborators have successfully transplanted early- and late-passage hPO in mouse and verified their integration and long-term survival in the host. Moreover, immunogenicity and genomic stability tests on the transplanted hPO have been performed by the UCAM groups. In this period, the UCAM laboratories have also established a method that enables freezing the original biopsy (prior to cell isolation) and its subsequent recovery for organoid formation with a similar efficiency of fresh isolated biopsies. That will facilitate the potential future bio-banking of tissues for organoid generation purposes. A GMP freezing/thawing protocol of both cultured hPO and raw pancreas tissues allowing bio-banking has been established by Cell Factory in Milan, led by Lorenza Lazzari. Moreover, Cell Factory has successfully achieved a major goal of LSFM4LIFE: the SOPs for the GMP manufacture of hPO have been accomplished. These are essential for the translation in the clinics and the start of clinical trials. The group at Goethe University Frankfurt (GUF), led by Francesco Pampaloni has successfully established the specimen preparation and assay/imaging procedures for the characterization of the hPO. The GUF group has first developed all the protocols for the imaging with all the planned microscopies, including light sheet-based fluorescence microscopy, confocal microscopy, wide-field fluorescence microscopy and bright-field microscopy. Procedures for both immunofluorescence and long-term live imaging have been elaborated. Immunostaining protocols for the detection of the growth and differentiation markers defined in close collaboration with UCAM have been optimized and are routinely and continuously applied. The growth of the hPO in the synthetic hydrogel has been thoroughly characterized. As planned in the project, the Frankfurt group has elaborated image processing and data storage/sharing pipelines. P01 has submitted several patents in the field of optical technology applied to hPO and organoid analysis supported by LSFM4LIFE. A major goal of LSFM4LIFE has been achieved by Cellendes, led by Brigitte Angres and Helmut Wurst. In close cooperation with GUF and UCAM, a new hydrogel efficiently supporting the growth of hPO has been designed, synthesized and tested. The new hydrogel, fully defined and GMP compliant, allows the expansion of hPO in the new medium developed by Meritxell Huch group at UCAM, both from already established frozen stocks as well as from tissue biopsies. Moreover, the first hydrogel specified for the culture of mouse organoids has been brought on the market in 2018. The partner Lonza (Bart van Dijk, Nasser Sadr) has performed a detailed and comprehensive gap analysis for the commercial production of hPO and their use as a cellular medicinal product, which together with the GMP procedures established by Cell Factory, represents a milestone for the translation of the hPO technology to the clinical trials. Considerable and consistent effort has been devoted to the dissemination activities of LSFM4LIFE. Partner P08 (Sparks&Co) has performed an excellent job in ensuring a constant and well-structured coverage of the advancements of LSFM4LIFE on the website and social media, and has efficiently supported the dissemination activities of the individual LSFM4LIFE partners producing flyers, posters, and multi-language roll-ups.

Final results

The ambition of LSFM4LIFE is advancing the state-of-the-art in several biotechnological areas on the way towards the production of human pancreas organoids at GMP-level. The biotechnological areas of innovation pursued by LSFM4LIFE cluster into the following main fields:

â€¢ Cell-based therapy.
â€¢ Biomimetic hydrogel scaffolds.
â€¢ Therapeutic-scale production of cells at GMP standard.
â€¢ Optical technology for 3D tissues.
â€¢ Cell-based assay.

hPOs produced at GMP-level as a long-term source of pancreas stem cells, represent a breakthrough in the cellular therapy of Type 1 Diabetes. A further groundbreaking and ambitious goal is the production of a cost-effective surrogate for Matrigel, which is a hydrogel of animal origin unsuitable for clinical application. LSFM4LIFE also advances the field of cell-based assay by developing standardized assays on pancreas organoids in a multi-well format for drug and toxicity screening. Moreover, with the high-throughput light sheet microscope (HT-LSFM) LSFM4LIFE realizes an innovative optical technology for the imaging of 3D tissues, which shows much better performance than state-of-the-art confocal fluorescence microscopes in terms of high speed, high resolution and low photo-bleaching.