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- Cheung, Pierre, et al.
(författare)
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Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells
- 2023
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Ingår i: EJNMMI Research. - : Springer Nature. - 2191-219X. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.
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- Thorngren, Julia, et al.
(författare)
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Differentiation of human pluripotent stem cells into insulin-producing islet-like clusters using nanofiltered cell culture medium
- 2024
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Ingår i: Frontiers in Membrane Science and Technology. - : Frontiers Media S.A.. - 2813-1010. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- The challenge of using patient-specific, autologous stem cell therapies in clinical settings is the need for advanced cell processing and expansion technologies. These include decentralized, small-scale manufacturing at the point of care in hospitals. The highest risk for contamination in cell-based therapy products comes from animal- and human-derived components such as serum, blood components, and growth factors. To mitigate the risk of adventitious microorganism contamination, preventive measures like size-exclusion virus removal filtration of cell media components can be employed. This article examines the impact of nanofiltration using nanocellulose-based virus clearance filter paper on the differentiation of human pluripotent stem cells into insulin-producing pancreatic islets (SC-islets). The cells were monitored for biomarkers using flow cytometry and immunohistochemistry along the 7-stage differentiation protocol. The produced SC-islets were evaluated functionally using low and high glucose stimulation under dynamic perifusion conditions. Pluripotent stem cells grown in culture media filtered through 20 nm cut-off nanocellulose filters showed similar expression of desired biomarkers at each stage compared to the control group. At the end of stage 7, SC-islets exhibited a rounded shape and strong expression of insulin, glucagon, and somatostatin in both the control and filtered media groups. The present study demonstrates that SC-islets differentiated with nanofiltered media were functional.
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- Thorngren, Julia, 1993-
(författare)
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Transplantation of stem cell-derived islets as a treatment for type 1 diabetes
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 1 diabetes (T1D) is an autoimmune disease that leads to an immune attack on insulin-producing beta cells, necessitating lifelong insulin therapy. For individuals with brittle diabetes and poor metabolic control, the option of pancreatic human islet transplantation exists. However, the shortage of organ donors and the need for life-long immune suppressive agents pose significant challenges. Stem cell-derived islets (SC-islets) present a promising alternative for diabetes treatment. This thesis explores the differentiation and transplantation of SC-islets as a treatment for diabetes. In paper I, three months post-transplantation, the ingrowth of recipient blood vessels and the neural density was higher in SC-islet grafts compared to human islet grafts. Furthermore, there was a higher tendency of blood flow, whereas the oxygenation was twice as high in SC-islet grafts. Both transplanted SC-islets and human islets had formation of amyloid depositions, which can affect the long-term survival and function of transplanted cells. In paper II, a humanized mouse model transplanted with SC-islets or human islets was validated. Transplanted SC-islets or human islets were not completely rejected 11 days after injection with human peripheral blood mononuclear cells (PBMCs). In vivo imaging and flow cytometry confirmed the presence of injected human immune cells, demonstrating an effective model for studying the human immune responses of allogeneically transplanted islets or SC-islets. In paper III, positron emission tomography (PET) imaging, using the DGCR2 affibody, for monitoring transplanted beta cells revealed successful binding to SC-islets and human islets in vitro. PET imaging in vivo demonstrated successful detection of the affibody in transplanted SC-islets. Although, the affibody could be optimized since the signal vanished 30 min after administration. However, DGCR2 remains a promising marker for SC-islet imaging. In paper IV, nanofiltration with a virus clearance filter paper during SC-islet differentiation was evaluated. Filter SC-islets expressed essential markers for beta cells during differentiation in comparable amounts as the control. The filtered SC-islets demonstrated physiological insulin-releasing function similar to that of the control. Nanofiltration did not seem to affect the differentiation of SC-islets. In conclusion, transplantation of SC-islets is a promising future treatment for diabetes, however, long-term effects need to be evaluated.
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