Objective
The objective of this research is to assess the potential of a novel platform designed for subcutaneous islet transplantation. The subcutaneous site, while the most accessible in a clinical setting, presents challenges such as limited vascularization and low oxygen concentrations, making it less favorable for densely packed islet cell grafts. The research aims to overcome these limitations by investigating a groundbreaking approach that combines vascular guiding hydrogels with temporally proangiogenic and pro-lymphangiogenic engineered cells that lead to a robust vascular and lymphatic environment in close proximity to transplanted islets.
The proposed platform seeks to promote vascular network formation within the hydrogel, facilitated by the use of engineered cells that secrete pro-angiogenic and pro-lymphangiogenic growth factors. Vascular and lymphatic growth factors will help generate new blood vessels which will provide large amounts of oxygen and nutrients to transplanted cells while newly formed lymphatic vessels will improve the bioavailability and uptake of insulin into the system. In addition, using FDA-approved small molecules will allow us to temporally control the secretion of the growth factors, which means the growth factors will only be secreted when they are more needed to generate vasculature and lymphatic vessels. The vascular guiding hydrogel loaded with the engineered cells as well as islet cells will be implanted in the subcutaneous space of rats. The final goal of this 3-year project is to evaluate the capabilities of this innovative technology specifically in supporting the long-term survival of syngeneic rat islets in the subcutaneous site. The project aims to achieve physiologically necessary glucose-stimulated insulin secretion kinetics addressing issues such as early graft failure due to delayed vascularization. The utilization of STZ-treated Sprague Dawley rats in this study will provide valuable insights into the success and viability of the proposed platform for subcutaneous islet transplantation.
Background Rationale
The prevailing treatment for T1D involves a meticulous routine of blood glucose monitoring, exogenous insulin administration, and dietary modifications. Despite the remarkable progress in T1D treatment technologies, the ability to attain target glycemic levels for patients suffering from T1D remains low, underscoring the need for a more effective approach. Beta cell replacement therapy emerges as the potential cure for T1D, capitalizing on recent strides in stem cell differentiation technologies. These advancements enable the development of a replenishable source of clinical beta cells. While studies have explored various transplantation sites (hepatic portal vein, omentum, intramuscular, and subcutaneous), optimization is imperative to enhance islet graft survival. Additionally, the success of islet transplantation is hampered by oxygen and nutrient deficiencies at the transplantation site, inhibiting islet transplantation. Oxygen, a critical factor for cell viability, faces constraints such as insufficient tension, high consumption, and barriers to diffusion. In addition, low insulin uptake from transplanted islets as well as the lack of drainage of waste products generated by transplanted islets are other challenges that reduce the efficacy of some of these technologies.
Vascularization, essential for nutrient and oxygen access, is often delayed in current implantable platforms, leading to initial transplantation failure. The subcutaneous space, despite its accessibility, poses challenges due to low basal oxygen concentration and inferior diffusivity. Early oxygenation and complementary vascularization strategies are deemed promising for transplanted pancreatic islets. Vascularization remains an unmet need in islet transplantation. The natural pancreas relies on a dense vascular network to supply nutrients and oxygen to nearby islets, ensuring effective insulin secretion for blood glucose regulation. The presence of lymphatic vessels and the lymphatic system around the pancreas plays an important role in the uptake of insulin and also in the removal of waste products from the robust vascular network surrounding the pancreas and the islet cells. Current efforts, including decellularization, face challenges in maintaining the desired vascular network for islet integration, emphasizing the critical gap in scaffold fabrication for vascularized and lymphatic islet constructs in clinical translation.
Our previous publication in Science Advances, showcasing the 3D printing of gelatin-based hydrogels with subsequent implantation in rodent models, resulted in the generation of mature vasculature within the hydrogels. Therefore, the proposed research aims to address the identified challenges by investigating a novel platform that combines both our vascular guiding hydrogels with pro-angiogenic and pro-lymphangiogenic engineered cells that will be temporally controlled to generate fast and mature blood and lymphatic vessels around transplanted islets. By promoting vascular network formation within the hydrogel and improving the long-term viability of transplanted islets, this innovative 3D-printed cell carrier is designed for subcutaneous islet transplantation. The three-year project seeks to evaluate the platform’s capabilities in supporting the long-term survival of syngeneic rat islets and achieving physiologically necessary glucose-stimulated secretion kinetics, thereby advancing the field of islet transplantation for T1D.
Description of Project
Type 1 Diabetes (T1D) is a chronic condition characterized by the immune system's destruction of insulin-producing beta cells in the pancreas, necessitating a lifelong reliance on exogenous insulin along with strict blood glucose monitoring, insulin administration, and dietary modifications. While there have been significant advancements in treating T1D, challenges persist with only 17% of children and adolescents meeting target glycemic levels. Therefore, the goal in diabetes treatment is achieving insulin dependency, often pursued through islet transplantation.
Islet transplantation, aiming to replace damaged pancreatic cells, faces obstacles such as the need for immunosuppression and challenges in providing adequate oxygen and nutrients. Oxygen plays a crucial role in supporting cell viability and function, and transplanting islets requires the formation of new blood vessels or supplemental oxygenation. Unfortunately, current approaches face difficulties due to hypoxic stress to transplanted cells, caused by factors like insufficient oxygen tension, high cell density, and barriers to oxygen diffusion. Various strategies have been explored to enhance oxygen delivery to transplanted cells, however, they have yet to succeed.
Vascularization, the formation of new blood vessels, is crucial for ensuring proper access to nutrients and oxygen and is therefore a potential strategy to increase oxygen levels in transplanted cells. However, current implantable platforms often experience delays in vascularization, leading to transplantation failure. Dynamic Light 3D Printing (DLP) has shown significant promise in the field of tissue engineering due to advances in resolution and biocompatible materials. In fact, we have previously developed a 3D-printed cell carrier containing printed blood vessel conduits that demonstrate angiogenesis from nearby vasculature when implanted in vivo. However, the use of vascular guiding devices and biomaterials is not enough to generate fast and mature blood vessel networks to keep transplanted cells and islets alive and functional for long periods of time.
The lymphatic system helps with the uptake of different molecules such as proteins as well as the removal of waste products and it is a potential route for systemic absorption following the subcutaneous administration of therapeutics. In fact, recent studies have shown faster transfer of insulin via the lymphatic system. Therefore, having a robust lymphatic vessel network in close proximity to transplanted islets will help with insulin bioavailability and uptake into the system for faster and more effective blood glucose correction. In addition, the presence of lymphatic vessels will aid with the removal of waste products from the blood system.
The proposed solution is to incorporate cells that have been engineered to secrete growth factors that promote blood vessel growth and lymphatic vessel growth within our vascular guiding devices. A newly generated blood vessel network will increase oxygen and nutrient levels in transplanted cells while newly generated lymphatic vessels will increase the uptake and bioavailability of insulin in the system. In addition, the engineered cells will respond to FDA-approved small molecules that will regulate when each of the growth factors gets secreted independently. This will help activate the necessary growth factors only when they are needed, leading to improved vascular and lymphatic vessel growth. Therefore, the combination of syngeneic rat islets with temporally regulated pro-angiogenic and pro-lymphangiogenic engineered cells within our vascular guiding platform will allow for generating an extrahepatic site for the transplantation of islets.
Anticipated Outcome
In the pursuit of Aim 1, our objective is to engineer ARPE-19 cells to secrete both proangiogenic growth factors VEGF-A and PDGF-A and pro-lymphangiogenic growth factor VEGF-C and test their pro-angiogenic and pro-lymphangiogenic potential first in vitro using a tubule assay and then in vivo in a rat model. Monoclonal highly stable and producing cell lines will be generated for these studies where multiple dosages of 10, 30, and 50 e6 cells/ml will be tested by introducing our engineered cells inside vascular guiding hydrogels previously optimized in the lab made of 10%w/v GelMA. The platform will be tested for vascular and lymphatic growth in the subcutaneous space of Sprague Dawley rats (7-8 weeks old). Once the platform has been validated in terms of pharmacokinetics and pro-angiogenic and pro-lymphangiogenic potential we will implement a safety switch within our engineered cells to terminate the secretion of the growth factors upon administration of a small molecule. In Aim 2, our goal will be to introduce a temporally regulated system within our engineered constructs. FDA-approved small molecules will be administered to the engineered cells to activate the transcription process and therefore the secretion of the growth factors of interest. Activating each growth factor at a different time point will enhance vascular and lymphatic growth. The implementation of these temporally regulated systems will be tested first in vitro where different doses of each small molecule will be studied to lead to a 10x increase in productivity and then cells will be placed within our vascularizing hydrogels, and we will test each growth factor independently first and then in combination by implanting the vascular guiding hydrogels in the subcutaneous space of Sprague Dawley rats (7-8 weeks old). In Aim 3, we will use the optimized platform from Aims 1 and 2 to correct blood glucose levels in STZ diabetic rat models. First, we will assess biocompatibility in vitro and then we will run two glycemic correction studies, a short one for 30 days and if successful a long-term study for 100 days.
If successful, the combination of 3D-printed gelatin-based hydrogels, with our temporally regulated engineered cells secreting pro-angiogenic and pro-lymphangiogenic growth factors holds an innovative opportunity to develop an extrahepatic site for islet transplantation. This innovative approach offers short-term islet support within a vascularizing environment, allowing for the long-term survival of islets. The culmination of these efforts has the capacity to revolutionize therapeutic solutions for patients suffering from T1D. As we progress, we remain poised to contribute significant advancements to the field of islet transplantation, paving the way for improved outcomes and potential cures for T1D patients.
Relevance to T1D
The proposed work directly addresses the limitations and challenges that have hindered the widespread success of islet transplantation for T1D such as the lack of a mature vascular network or lymphatic network in close proximity to transplanted islets. By enhancing oxygenation during the critical early stages and promoting prolonged viability in vivo through improved blood vessel growth, and by improving insulin bioavailability, uptake, and removal of waste products by the lymphatic vessels, the research seeks to significantly improve the outcomes of islet transplantation. If successful, this innovative approach has the potential to provide a transformative therapeutic solution for patients afflicted with T1D. It aims to offer not only short-term islet support within a vascularizing and lymphangiogenic environment but also the long-term survival and functionality of transplanted islets, marking a significant leap forward in the pursuit of a cure for T1D.
In conclusion, the research's relevance for T1D lies in its potential to overcome existing challenges, enhance the success of islet transplantation by generating an engineered extrahepatic site for islet transplantation, and contribute to the development of a more effective and sustainable therapeutic solution for T1D patients. As the research progresses, it holds the promise of advancing the field, bringing us closer to realizing a cure for T1D through innovative and transformative approaches.