Objective
The objective of the proposal is to generate optimized islet vascularized extracellular matrix organoids, made entirely from human pluripotent stem cells (hPSCs), that are suitable for transplantation. By first forming hPSC-derived vascular networks (VNs) in a human pancreas matrix scaffolding in culture together with hPSC-derived islet like clusters we predict these organoids can serve as an excellent culture and transplant platform. We expect to demonstrate that these organoid structures provide an enhanced microenvironment that more closely recapitulates important cell-matrix and cell-cell interactions which are missing in currently lab produced hPSC-derived islets that do not possess these vascular cell types or a normal complement of matrix scaffolding proteins. Thus, through this project we will determine the importance of these critical interactions and potentially produce a more potent stem cell-islet therapy.
Background Rationale
T1D patient’s beta cells are destroyed by autoimmune mechanisms leading to near complete insulin and beta cell deficiency associated with erratic blood glucose control. Though insulin injections, advanced insulin delivery systems and glucose monitoring technologies may be effective in some patients, many patients unfortunately still fail to achieve ADA glucose targets and suffer from the consequences of acute hypoglycemia or chronic hyperglycemia leading to premature death and/or accelerated kidney, eye, nerve and cardiovascular disease. Islet and pancreas transplantation offer proof-of-principle that a beta cell replacement therapy can be effective in normalizing blood glucose long-term without the need for insulin; however, beta cell replacement therapies are not available for most patients due to the shortage of cadaver donor sourced beta cells, among other reasons. Moreover, islet transplantation is associated with poor cell engraftment and early cell death after the transplant procedure, especially in the inflammation-prone liver transplant site, and alternate less invasive, retrievable transplant sites are being investigated.
Through recent achievements in many labs and several companies, human pluripotent stem cells can be turned into islet-like cell clusters in the lab, have been shown to be able to cure diabetes in rodents and have entered clinical trials. However, stem cell-derived islet-like clusters lack typical matrix scaffolding and blood vessels that are present in normal human islets in the body. As a result, some critical signals for complete maturation and survival after transplantation are missing. Therefore, we propose to overcome these barriers through generating a stem cell-derived islet organoid by embedding stem cell-derived islet clusters in a relevant biological scaffolding and generating vessels in this construct prior to transplantation. Thus, it is possible that organoids are made entirely from the same human pluripotent stem cell lines.
Description of Project
Human pluripotent stem cell (hPSC)-derived islets have the potential to provide an unlimited supply of insulin-producing cells for treating patients with T1D. It is now well established that hPSCs can be turned into islet-like cell clusters in culture, can cure diabetes in rodents and have entered clinical trials. However, stem cell-derived islet-like clusters (SCILCs) lack typical tissue matrix scaffolding and blood vessels that are present in normal human islets in the pancreas. As a result, some critical signals for maturation and survival after transplantation are missing.
We propose to combine our expertise in generating stem cell-islets and producing a well-characterized scaffolding from the human pancreas with the expertise of Drs. Murphy and Palecek in generating hPSC-derived blood vessels to build a more complete stem cell-derived islet organoid. Our objective is to produce a 3D bioengineered islet-like cluster entirely from hPSCs. We predict that combining SCILCs with hPSC-derived vascular networks (VNs) in human pancreas scaffolding will provide an enhanced environment more closely recapitulating important cell-scaffolding and cell-cell interactions. We anticipate that this enhanced niche will promote more complete achievement of mature islet-like insulin secretion and/or more rapid revascularization post-transplant, thereby enhancing survival and transplant success, especially at the more retrievable subcutaneous implant site.
We propose to address our overall objective in a stepwise fashion, by first confirming our promising preliminary data suggesting that co-transplantation of human islets and human endothelial cell-derived vascular networks (VNs) embedded in pancreas scaffolding improves transplant success in a diabetic mouse model. We expect to show better survival of beta cells post-transplant and intend to use this transplant model to validate a novel beta cell imaging modality in collaboration with Dr. Engle in our Small Animal Imaging and Radiotherapy Facility. The possibility of a serial, noninvasive beta cell mass imaging method would be of great value to monitoring beta cell transplant therapies in patients, yet despite extensive investigation in the field over decades, none are in clinical use today. We also intend to establish optimal methods for forming a vascular plexus in hydrogels using hPSC-derived endothelial cells and determine if vessel mural cells, called pericytes, can improve the quality and stability of vascular networks. Finally, we will combine what we learn in the prior experiments, and test whether combining SCILCs with hPSC-derived vascular networks (VNs) in a pancreas matrix scaffolding or synthetic scaffolding promotes more complete acquisition of mature islet function from the stem cells in culture, and then test whether these 3D bioengineered SCILC organoids containing VN and scaffolding can reverse diabetes in mice more effectively and rapidly than the SCILC alone. These experiments are designed to demonstrate the importance of critical vascular and matrix niche signals for proper stem cell-islet development and function. By overcoming existing challenges of beta cell replacement therapies, we believe that generating an abundant supply of 3D bioengineered human stem cell-derived islet organoids would have significant implications for treating T1D.
Anticipated Outcome
We expect to demonstrate that these organoid structures provide an enhanced microenvironment that more closely recapitulates important cell-matrix and cell-cell interactions which are absent in currently lab produced hPSC-derived islets. Thus, we anticipate positive effects on culture-based pre-transplant maturation of glucose-regulatory function in these organoids compared to stem cell-derived islets alone. Further, we predict that these organoids, by virtue of providing key niche survival signals, will survive better post-transplantation, especially in the poorly vascularized subcutaneous transplant site, and therefore will be able to reverse diabetes in standard animal models more effectively (i.e. at a lower dose) and more rapidly that stem cell-derived islet clusters do that lack these key biological components. These experiments will also provide the first evidence supporting the use of a novel, non-invasive clinically-translatable beta cell mass imaging modality to estimate functional beta cell mass after islet or stem cell-islet transplantation into non-liver sites.
Relevance to T1D
Given the shortage of beta cells for transplantation, as well as biological, and transplant site challenges of current beta cell replacement therapies, human pluripotent stem cell- derived islet-like clusters offer great promise to overcome the limitations of current therapies. Islet vascularized extracellular matrix organoids, made entirely from human pluripotent stem cells (hPSCs) and suitable for transplantation, may improve on the stem cell-derived islets currently grown in labs and being used in clinical trials. By forming hPSC-derived vascular networks (VNs) in a human pancreas matrix scaffolding in culture and combining these preformed VNs together with hPSC-derived islet like clusters we predict these organoids can be transplanted with enhanced potency and effectiveness. We will first test them in routine preclinical animal models of diabetes, and if successful, then test the effectiveness of these organoids in large animal models and ultimately human trials, as the materials proposed to be used have potential for clinical translation.
In addition to being a potentially more potent stem cell-derived islet transplant therapy, these organoids may also serve as a model system that can then be used in the future to gain insights into the nature of these cell-cell and cell-matrix interactions in islet biology by intentionally perturbing the system. Islet organoids may also be integrated into microphysiological systems that could be developed and used for a variety of purposes. Some examples include gaining a better understanding of islet-immune cell interactions related to the pathogenesis of T1D, screening compound libraries for discovering drugs offering beta cell protection or enhanced secretion, or studying the liver- islet axis in diabetes pathogenesis, among others.