Periodic Reporting for period 4 - EYELETS (A regenerative medicine approach in diabetes.)
Periodo di rendicontazione: 2024-07-01 al 2025-06-30
Diabetes mellitus has reached pandemic proportions in Europe. Besides the personal suffering the disease represents a substantial economic burden to society. Diabetes develops when the supply of insulin by pancreatic islets is insufficient to maintain blood glucose homeostasis. In insulin-dependent diabetes islet transplantation is considered a promising therapy. However, the outcome varies due to transplantation site, quality of islets and the fact that transplanted islets are affected by the same challenges as in situ islets. Tailor-making islets for transplantation by tissue engineering combined with a more favorable transplantation site that allows for both monitoring and local modulation of islet cells is thus instrumental. We have established the anterior chamber of the eye (ACE) as a favorable environment for long term survival of islet grafts and the cornea as a natural body window for non-invasive, longitudinal optical monitoring of islet function. ACE engrafted islets are able to maintain blood glucose homeostasis in diabetic animals. Tissue engineering of native islets is technically difficult. We will therefore apply genetically engineered islet organoids. This allows us to generate i) standardized material optimized for transplantation, function and survival, as well as ii) islet organoids suitable for monitoring (sensor islet organoids) and treating (metabolic islet organoids) insulin-dependent diabetes. Our overall aim is to create a platform allowing monitoring and treatment of insulin-dependent diabetes in mice that can be transferred to large animals for validation.
The overall objective of the present proposal is to design a novel strategy to settle the so far unmet scientific/clinical problems currently associated with islet transplantation. This will be achieved by taking a regenerative medicine approach involving tailor-making islet organoids by tissue engineering, transplanting these organoids to the ACE, a site that allows for both monitoring and local modulation of the graft and combine this approach with synthetic biology and the development of novel microelectronic/micro optical readout systems for islet cells.
The overall objective of the present proposal is to design a novel strategy to settle the so far unmet scientific/clinical problems currently associated with islet transplantation. This will be achieved by taking a regenerative medicine approach involving tailor-making islet organoids by tissue engineering, transplanting these organoids to the ACE, a site that allows for both monitoring and local modulation of the graft and combine this approach with synthetic biology and the development of novel microelectronic/micro optical readout systems for islet cells.
Aim 1. Sensor islet organoids for monitoring function and survival of metabolic islet organoids.. We equipped islet organoids with biosensors that report on changes in cytoplasmic free Ca2+ concentration, stimulus-induced gene transcription and insulin resistance. Their combinatorial use allowed us to monitor changes in functional β-cell mass during diabetes development (Paschen et al., FASEB J 2019; Mir-Coll et a., IJMS 2021). Employing Ca2+-biosensor GCaMP3 allows Ca2+-imaging in real-time online at the single-cell level in vivo (Jacob et al., FASEB J, 2020; Visa et al,. Diabetologia 2024; Xiong et al., JCI 2025). We tested adenoviruses and adeno-associated viruses as gene delivery systems (Voznesenskaya et al., Front Bioeng Biotechnol. 2023). We established an inducible expression system allowing to switch on/off gene expression (Leibiger et al., Metabolites 2021). Furthermore, we established protocols tomanipulate gene expression in islet organoids by siRNA (Barker et al., Diabetes 2020) or by saRNA.
Aim 2. Islets organoids for treatment of insulin-dependent diabetes. We verified the beta-3 subunit of voltage-dependent L-type calcium channels and apolipoprotein CIII as targets for genetic downregulation in islet cells to generate islet organoids that are resilient to a diabetic mileu. We evaluated the number of islet organoids needed to treat insulin-dependent diabetes in a streptozotocine (STZ)-diabetes mouse model. Our data also show that transplantation of approx. 600 stem cell-derived human islets into the ACE of STZ-diabetic mice normalizes glycemia.
Aim 3. Local intraocular treatment strategies for the modulation of islet organoid function. We showed that local intravitreal administration of dexamethasone implants improves survival of islet allografts transplanted to the ACE of non-human primates (Tun et al., Cell Transplant 2022). An eye-drop application of doxycycline allowed us to induce gene expression in intraocular islet organoids equipped with Tet-ON gene cassettes. The intravitreal infusion of NNC55-0396 demonstrated that the inhibition of Cav3 channels facilitates the maturation of human pluripotent stem cell-derived islets (Zhao et al., Biomedicines 2024).
We summarized the potential of the anterior chamber of the eye as a transplantation site to monitor and manipulate islet grafts longitudinally in a current review article (Yang, Shi and Berggren, Physiol Rev 2024).
Aim 2. Islets organoids for treatment of insulin-dependent diabetes. We verified the beta-3 subunit of voltage-dependent L-type calcium channels and apolipoprotein CIII as targets for genetic downregulation in islet cells to generate islet organoids that are resilient to a diabetic mileu. We evaluated the number of islet organoids needed to treat insulin-dependent diabetes in a streptozotocine (STZ)-diabetes mouse model. Our data also show that transplantation of approx. 600 stem cell-derived human islets into the ACE of STZ-diabetic mice normalizes glycemia.
Aim 3. Local intraocular treatment strategies for the modulation of islet organoid function. We showed that local intravitreal administration of dexamethasone implants improves survival of islet allografts transplanted to the ACE of non-human primates (Tun et al., Cell Transplant 2022). An eye-drop application of doxycycline allowed us to induce gene expression in intraocular islet organoids equipped with Tet-ON gene cassettes. The intravitreal infusion of NNC55-0396 demonstrated that the inhibition of Cav3 channels facilitates the maturation of human pluripotent stem cell-derived islets (Zhao et al., Biomedicines 2024).
We summarized the potential of the anterior chamber of the eye as a transplantation site to monitor and manipulate islet grafts longitudinally in a current review article (Yang, Shi and Berggren, Physiol Rev 2024).
Real-time imaging of pancreatic islet/organoid function in awake non-anesthetized mice. We explored the possibility to transplant reporter islets onto the dura mater of the brain and monitor islet function through a cranial body window by fluorescence microscopy in awake mice utilizing the Mobile HomeCage technology (Neurotar). In addition to avoiding negative effects resulting from anesthesia, this set-up, due to its high stability, will allow analysis of subcellular compartments in engrafted islets/organoids in real-time in awake mice (Tröster et al., Nat Commun, accepted).
Non-invasive in vivo imaging of intraocular liver spheroids. We tested the possibility to monitor liver spheroids engrafted in the anterior chamber of mice as metabolic sensors for in situ liver function. Our published data show that liver spheroids engraft, become vascularized and innervated and can be readily imaged by fluorescence microscopy in living animals. In combination with intraocular sensor islet organoids this approach will broaden the spectrum to monitor both liver and islet function in diabetes and MAFLD (Lazzeri-Barcelo et al., Nat Commun 2024; Lazzeri-Barcelo et al., J Vis Exp. 2024).
Diet-induced obesity results in endothelial cell desensitization to VEGF-A and permanent islet vascular dysfunction (Xiong et al., JCI 2025). While we hypothesized that islet endothelial cells might become desensitized to VEGF-A and affect glucose homeostasis by impairing insulin release into the blood circulation, we unexpectedly found that these impairments were of irreversible nature. In contrast to the resilience of islet endocrine cells to western diet intervention, islet vessels exhibited remarkable memory of the previously increased metabolic stress and continued to undermine glucose homeostasis even after the removal of the diet stressor.
Structural changes within the pancreatic islet result in enhanced Ca2+ dynamics and prediabetes-induced beta-cell adaptation. (Visa et al., Diabetologia 2024). In this study we show increased β-cell cytoplasmic free Ca2+ concentration dynamics linked to enhanced insulin secretion as a compensatory mechanism after short-term exposure to western diet. The unexpected finding was that this response is likely to be explained by western diet-induced structural changes within the islet and thereby enhanced paracrine input from adjacent α-cells, adjusting the glucose set-point and amplifying the insulin secretion pathway.
Non-invasive in vivo imaging of intraocular liver spheroids. We tested the possibility to monitor liver spheroids engrafted in the anterior chamber of mice as metabolic sensors for in situ liver function. Our published data show that liver spheroids engraft, become vascularized and innervated and can be readily imaged by fluorescence microscopy in living animals. In combination with intraocular sensor islet organoids this approach will broaden the spectrum to monitor both liver and islet function in diabetes and MAFLD (Lazzeri-Barcelo et al., Nat Commun 2024; Lazzeri-Barcelo et al., J Vis Exp. 2024).
Diet-induced obesity results in endothelial cell desensitization to VEGF-A and permanent islet vascular dysfunction (Xiong et al., JCI 2025). While we hypothesized that islet endothelial cells might become desensitized to VEGF-A and affect glucose homeostasis by impairing insulin release into the blood circulation, we unexpectedly found that these impairments were of irreversible nature. In contrast to the resilience of islet endocrine cells to western diet intervention, islet vessels exhibited remarkable memory of the previously increased metabolic stress and continued to undermine glucose homeostasis even after the removal of the diet stressor.
Structural changes within the pancreatic islet result in enhanced Ca2+ dynamics and prediabetes-induced beta-cell adaptation. (Visa et al., Diabetologia 2024). In this study we show increased β-cell cytoplasmic free Ca2+ concentration dynamics linked to enhanced insulin secretion as a compensatory mechanism after short-term exposure to western diet. The unexpected finding was that this response is likely to be explained by western diet-induced structural changes within the islet and thereby enhanced paracrine input from adjacent α-cells, adjusting the glucose set-point and amplifying the insulin secretion pathway.