LSFM4LIFE Progress

Context and objectives

The cellular therapy with beta-cells obtained by differentiating adult stem cells can realize the dream of a curative therapy of T1D. The stem cell approach aims to establish a virtually endless source of beta-cells, cultured according to the highest safety standards defined by EMA (European Medicines Agency) and FDA (Food and Drug Administration). Moreover, stem cell-derived beta-cells are suitable for autologous transplantation, which avoids immunosuppressive treatment.

A fundamental issue concerns the type of stem cells of choice. While embryonic stem cells (ESCs) pose significant ethical challenges, inducible pluripotent stem cells (iPSCs) could have a safety issue due to the activation of oncogenes during the reprogramming of somatic cells.

A novel strategy that consists in the isolation and culture of pancreas progenitor cells (human pancreas organoids, hPOs) allows overcoming the drawbacks of both ESCs and iPSCs.

The main goal of LSFM4LIFE is to achieve the culture and differentiation of therapeutic batches of hPOs accordingly to Good Manufacturing Practice (GMP) quality control criteria, qualifying them for clinical testing in patients.

Work performed so far

Reporting Period 1

The first phase of the project ran from January 2016 to June 2017.

Meritxell Huch’s and Kourosh Saeb-Parsy’s labs in Cambridge established 25 lines of hPOs from healthy donors, with a success rate of 81%. These were then made available to the other project partners for performing the different tasks.

Meritxell Huch’s group improved the formulation of the media for the culture of pancreas organoids in vitro. Moreover, their long-term genetic stability and safety in vivo was analyzed by Kourosh Saeb-Parsy’s group by transplanting the organoids under the kidney capsule of immunodeficient mice.

The close collaboration between the groups in Cambridge, Frankfurt and Switzerland (InSphero) produced robust assays to test the proliferation and differentiation of the pancreas organoids, establishing an initial panel of quality assurance criteria.

Moreover, the group at the University of Frankfurt developed novel optical technology for the imaging of organoids in three-dimension with Light Sheet Fluorescence Microscopy (LSFM), as well as a novel laboratory tool for the recovery of organoids from the culture wells. Two patents concerning these innovations were submitted.

A remarkable amount of work was performed by Cellendes (Reutlingen), which successfully generated a biomimetic synthetic hydrogel that supports the growth of the organoids. Therefore, the goal of exchanging Matrigel (which is a hydrogel of animal origin unsuitable for clinical application) with a GMP-level hydrogel that is completely safe for human and 3R-compatible is getting closer.

LSFM4LIFE’s GMP experts at the Cell Factory in Milan and Lonza Netherlands made great progress for converting the laboratory-scale culture of the organoids to a GMP-level process, pursuing the final goal of translating hPO culture into a commercial Advanced Therapeutic Medicinal Product (ATMP).

The LSFM4LIFE consortium is also strongly committed to informing T1D patients and their families, as well as medical doctors, about the ongoing research and the therapeutic potential of hPOs. Several presentations to patients and medical doctors have been given by LSFM4LIFE’s scientific coordinator Francesco Pampaloni.

Reporting Period 2

The second phase of the project ran from July 2017 to December 2018.

The group led by Meritxell Huch has completed the development of an improved culture media formulation for the growth of the hPO in vitro and a patent for it has been submitted.

Kourosh Saeb-Parsy and his collaborators have successfully transplanted early- and late-passage hPOs in mouse and verified their integration and long-term survival in the host. Moreover, immunogenicity and genomic stability tests have been performed by the University of Cambridge groups on the transplanted hPOs. In this period, the Cambridge laboratories have also established a method that enables freezing the original biopsy (prior to cell isolation) and its subsequent recovery for organoid formation. This will facilitate the potential future bio-banking of tissues for organoid generation purposes.

A GMP freezing/thawing protocol of both cultured hPOs and raw pancreas tissues has been established by Cell Factory in Milan, led by Lorenza Lazzari. Moreover, Cell Factory has successfully achieved a major goal of LSFM4LIFE: the Standard Operating Procedures (SOPs) for the GMP manufacture of hPOs have been accomplished. These are essential for the translation in the clinics and the start of clinical trials.

The group at Goethe University Frankfurt, led by Francesco Pampaloni, has successfully established the specimen preparation and assay/imaging procedures for the characterization of hPOs. The Frankfurt group has first developed all the protocols for the imaging with all the planned microscopies, including LSFM, 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 Cambridge have been optimized and are routinely and continuously applied. The growth of hPOs 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.

A major goal of LSFM4LIFE has been achieved by Cellendes, led by Brigitte Angres and Helmut Wurst. In close cooperation with the Goethe University Frankfurt and the University of Cambridge, a new hydrogel efficiently supporting the growth of hPOs has been designed, synthesized and tested. The new hydrogel, fully defined and GMP compliant, allows the expansion of hPOs in the new medium developed by Meritxell Huch’s group. 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 and Nasser Sadr) has performed a detailed and comprehensive gap analysis for the commercial production of hPOs 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. 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 multilanguage roll-ups.

Reporting Period 3

The last phase of the project runs from January 2019 to December 2019.

Progress and impact

hPOs produced at GMP-level as a long-term source of pancreas stem cells represent a breakthrough in the cellular therapy of T1D. A further groundbreaking and ambitious goal is the production of a cost-effective surrogate for Matrigel.

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 speed, resolution and photo-bleaching.