Objective
Replacing lost islets is a major goal for the treatment of T1D to reduce or eliminate the need for daily insulin injection. Transplantation of islets, however, requires islets from 2-4 cadaveric donors, reflecting poor survival of the grafts. In addition, life-long immunosuppression is required to prevent immune rejection of the transplanted tissue. Generating insulin-secreting β cells from human stem cells (hSCs) is a renewable source and others are working on modification to reduce or abolish immune recognition. Recent trials with hSCs are promising but, similar to donor islets, survival of these cells is low and functionally closer to fetal than adult human β cells.
We hypothesize that mixing of donor islets with hSC-derived β-like cells into spherical cell clusters, called pseudoislets, leads to maturation of the hSC-derived β-like cells. In Aim 1, I will generate endothelial blood vessel cells (ECs) from hSCs and test whether inclusion of these cells can similarly induce maturation of the hSC-derived β-like cells. In mice, blood vessel cells are important to maintain function and gene expression of adult mouse β cells. This aim will test if similar effects are present in pseudoislets assembled from hSC-ECs with islets or hSC- β-like cells.
In Aim 2, I will test whether pseudoislets from ECs and donor islet cells can increase survival and function after transplantation into mice. After transplantation, islets need to establish access to nutrients and oxygen supplied by the recipients’ blood stream. This process often takes several weeks, during which most islets do not survive. Rapid revascularization of the transplanted islets is therefore critical to improve transplantation outcome and reduce the required amount of already scarce donor islets. I hypothesize that addition of hSC-ECs will speed up revascularization, increasing islet viability and improving diabetes reversal in diabetic mice.
Prevascularized islets have the potential to generate fully functional islets for transplantation. Combined with hSC or patient-derived stem cells, this would be an important step towards personalized, regenerative medicine.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disease targeting and destroying the insulin-producing islet β cells with an onset typically in childhood. Transplantation of donor islets or replacement islets is FDA-endorsed, and potentially curative for type 1 diabetes (T1D). However, transplantation is limited by poor islet survival, reflecting poor vascularization and other stressors, and necessitates islets from 2-4 cadaveric donors, impeding widespread adoption. Thus, T1D research is challenged to improve transplanted islet survival and function.
Islet blood vessels, comprised of arterial, capillary, and venous endothelial cells (ECs), and other cell types, are crucial for nutrient transport, oxygen supply, and distribution of endocrine hormones within and outside islets. EC-associated cells, such as pericytes likely also have important roles in regulating β cell function and insulin secretion. Experimental deletion of pericytes in mice, for example, leads to reduced insulin content and secretion; effects correlated to reduced β cell function, highlighting the importance of other cell types in maintaining a mature β cell phenotype.
Islet β cells, in turn, are thought to provide signals that help maintain islet vascular structure. For example, vascular endothelial growth factor A (VEGFA) is secreted from β cells under conditions of low oxygen and sensed by endothelial cells. In mice, interference with β cell VEGFA expression reduces islet vascularization and insulin secretion, resulting in reduced glucose tolerance. However, we and others postulate that the cell interactions between endothelial cells and endocrine islets cells - to maintain health, or during disease development - are far richer and more complex than currently known. Addition of primary umbilical cord endothelial cells or microvascular fragments to islets prior to transplantation can improve islet survival and function, but neither source is suitable for widespread adoption. These data underscore the importance of vascular elements in the survival, function, and maturation of β cells.
Stem cell-derived β-like cells are considered a viable and renewable source of β cells for clinical transplantation. Despite progress in this area, β-like cells are not functionally mature with recent work identifying molecular features of this incomplete development. Given the importance of the vasculature in maintenance of a mature β cell phenotype in mice, we hypothesize that addition of ECs from renewable sources will foster maturation of stem cell-derived β-like cells and potentially speed up ‘revascularization’ of transplanted islets in the recipient, thereby providing access to oxygen and nutrients. We will test if addition of vascular elements will shorten the vascularization time of grafts and enhance survivability and function.
Description of Project
Diabetes mellitus is a disease characterized by defects in hormone production in the pancreatic islets of Langerhans that affects hundreds of millions of people worldwide. In healthy individuals, islets maintain normal circulating blood glucose levels by secreting hormones, such as insulin and glucagon, into the blood stream. Insulin replacement therapy and/or drugs targeting insulin or glucagon secretion are the most common treatments for diabetes. Type 1 diabetes (T1D) is an autoimmune disease that most commonly develops in childhood where the body’s own immune system erroneously targets and destroys the insulin producing islet β cells. Standard care for T1D involves insulin replacement therapy and is life-saving, but this treatment is not a cure. Indeed, average life expectancy remains shorter by 10 years and can be accompanied by lifelong co-morbidities, including organ failure. Replacement of lost islet cells and insulin independence is the ultimate goal for curing T1D.
Transplantation of cadaveric donor islets or replacement islets derived from human stem cells represent a viable approach for T1D, and clinical studies show promising results with both approaches. However, islet cell survival after transplantation is poor and limits broader application of transplant-based therapies. Islet cells need to reconnect to the blood vessels of the recipient to survive and function, and prolonged oxygen and nutrient deprivation during this 'revascularization' period are thought to reduce islet cell survival. Our work with human islet cell transplantation suggests that re-establishment of blood vessels in islets is favored by simple physical manipulations, including cell dispersion and reaggregation. To further enhance this process, I propose to mix blood vessel cells with cadaveric human islets or replacement islets and investigate if this can enhance islet cell survival and function after transplantation. If successful, the integration of blood vessel cells will increase the survival and function of transplanted islets and enhance islet transplantation approaches to cure T1D.
Anticipated Outcome
Type 1 diabetes (T1D) is the result of an autoimmune reaction destroying the insulin-secreting pancreatic β cells. Replacement of these islet cells either by cadaveric donor islets or replacement islets derived from human stem cells (hSCs) is considered a viable long-term therapeutic approach. Recent trials showed some promise in utilizing hSC-derived β-like cells to treat T1D patients, however, two factors, amongst others, limit their wider use in T1D treatment: First, donor and replacement islets may not be fully functional after transplantation. This may be attributed to a lack of interacting cell types, such as blood vessel cells, which have been shown to positively regulate β cell function. Second, survival rates of transplanted donor or replacement islets are very low (<10%), likely due to slow connection to the recipients’ vascular system. I anticipate addressing these limitations by the incorporation of stem cell-derived ECs in cell clusters with islets or hSC-derived β-like cells, enhancing the revascularization process and initiating β cell-EC interactions.
Our preliminary data suggests that hSC-derived ECs can be stably aggregated into cell clusters with donor cells or hSC-derived β-like cells. I expect to see improved insulin secretion from these clusters compared to their controls. If so, this would suggest that β cell-ECs interaction improves function and I might then observe corresponding changes in their gene expression profiles. If not, prolonged interaction or additional vascular elements may be required to induce maturation of hSC-derived β-like and endothelial cells.
The addition of hSC-derived ECs could improve access to the recipients’ vascular system leading to increased survival of transplanted vascularized islet cell clusters, likely due to faster access to oxygen and nutrients. This may translate into increased insulin secretion, improved islet cell survival, and a reduction in the required islet clusters for the reversal of diabetes in animal models. If so, I will analyze the gene expression profiles of these cells and compare them to control clusters. Lack of changes in insulin secretion after addition of hSC-derived ECs may indicate the need for other vascular elements.
Choice of transplantation site might be important for the survival and function of transplanted islets, since every tissue has a distinct composition of cell types, blood vessels, access to nutrients and hormones. I will explore other sites than the traditionally used mouse kidney to investigate clinically relevant transplantation sites, such as the omentum. I anticipate seeing differences in survival, function and gene expression of the transplanted clusters compared to transplantation into the kidney. If not, I will explore additional transplantation sites to increase survival of the grafts.
The outcomes of this study will determine if addition of hSC-derived ECs to islets or islet-like cells can enhance the function and survival of transplants. If so, this could reduce the amount of required tissue to reverse T1D, and enhance clinical outcomes.
Relevance to T1D
The clinical focus of our translational work is type 1 diabetes (T1D), an autoimmune disease that classically presents early in childhood, specifically targeting insulin-producing β cells located in the pancreatic islets of Langerhans. Once β cell mass is reduced to critical levels, patients require daily insulin injections for life. Although exogenous insulin supply via multiple daily injections or insulin pumps helps maintain healthy blood glucose levels, long-term co-morbidities and increased mortality in patients with T1D, such as damage to eyes, kidneys, blood vessels, and end-organ failure, remain common. In addition, insulin injections may be accompanied by a economic, social, physical and mental burden for patients and caregivers. Thus, exogenous insulin replacement, while life-saving, is not considered curative, and alternative cell-based therapies are intensively sought.
To meet these challenges, we and others are investigating islet cell replacement therapy, whereby donor islets or stem cell-derived islet-like cells are transplanted into patients with diabetes to replace lost β cells. Despite encouraging progress, survival of primary donor islets and stem cell-derived islet β cells is poor (generally thought to be <10%) after transplantation, reflecting a period of hypoxia and nutrient deprivation when islets are not yet incorporated into the blood supply, or “re-vascularized.” Here I propose to build on recent advances in stem cell, islet cell, genetic and molecular medicine to test the hypothesis that mixing vascular endothelial cells with human islets prior to transplantation will enhance islet cell vascularization, integration, survival and function. I will test the hypothesis that engineering pre-vascularized islet organoids (pseudoislets) - with primary donor islets, or with stem cell-derived β-like cells - will enhance and improve the survival and function of islets after transplantation, including in diabetic animal models. If so, this could transform the research and clinical landscape of islet replacement in diabetes. Ultimately, these studies could enhance the outcomes of islet transplantation for the treatment of T1D and greatly improve the lives of children and adults living with T1D.