Objective
The overall goal of this proposal is to assess the efficacy of reversing high blood glucose levels with subcutaneous functional engraftment of islets, which will be enabled by R-VECs to blood vessel and PEG-4MAL to deliver active peptides. Our first objective is to determine the best transplantation route. In the real practice, we will first mix islets with endothelial cells in the PEG-4MAL hydrogel. One way to proceed is to let the mixture solidify in the culture dish, pick the solid gel droplets, and insert the droplets into the subcutaneous space by inserting through a skin incision. Or, we could promptly inject the liquid mixture under the skin and let it solidify within the subcutaneous tissue of the transplant recipient. In the objective 1, we will also test different endothelial cell concentrations. It is actually one of the advantages of PEG-4MAL, which is capable of encapsulating a higher cell density than the mostly used matrix collagen type I. The 2nd objective of this proposal is designed to pick 2 of the best active peptides to support islet vascularization in the subcutaneous space. Using single-cell RNA-sequencing, we have identified several extra cellular matrix proteins that are highly expressed in islets, including collagen type IV, laminin α1, laminin α4, and laminin α5. From these proteins, we have extracted 7 active peptides. All the 7 active peptides, along with 1 generic peptide as the control, will be firstly tested in dishes to see which 2 best support the long-term function sustaining of in vitro cultured islets. Next, 2 of the best active peptides will be further tested in vivo by transplanting them into the subcutaneous space to see which one will stimulate the best subcutaneous islet vascularization. Lastly, our objective 3 will determine if the islets transplanted in this manner will eventually reverse the hyperglycemia in diabetic mice. We will also use different dosages of islets, full dose and reduced dose, to evaluate if our approach can reduce the required quantity of transplanted islet to achieve insulin independence. In objective 3, we will also transplant stem cell-derived islet to evaluate if our technology can also facility the functional engraftment of stem cell-derived islets. The current differentiation protocol can achieve islet that secrete insulin in response to glucose stimulation, but the derived islets are still not fully functionally comparable to native islets in multiple aspects such as the dynamic regulation of insulin secretion. Hence, we will try if the combination of induced islet-specific endothelial cells and the PEG-4MAL containing islet-specific peptides will further improve the functional maturation of the stem cell-derived islets. Taken together, we hope that we will develop a novel technology to treat patients with Type 1 Diabetes (T1D) and achieve long-term glucose control independent of exogenous insulin injection. Completion of these objectives will set stage for filing an IND application to Food and Drug Administration (FDA).
Background Rationale
The success of therapeutic transplantation of replacement organs depends on the speed by which these tissues are able to recruit the nurturing blood supplies to survive and maintain proper metabolic function. Blood vessels, lined by single layer of cells known as endothelial cells, also supply numerous nurturing factors that are essential for the survival and function of the islet cells. Acquiring blood supply upon transplantation is particularly necessary for islets, which in their native location in the pancreas, are richly vascularized with extensive network of nourishing blood vessels. Although islets only make up 2% of the pancreas, they consume 20% of the blood flow through the pancreas. It has also been shown that every single insulin, and glucagon secreting islet cell is in direct contact with the blood vessel endothelial cells. The re-establishment of blood supply is less a problem in the case of whole organ transplant, such as pancreas transplantation, when the organ’s own arteries and veins can be directly connected to the recipient’s blood vessels through surgical procedure. However, in the case of islet cell transplantation, since all the vascular structure has been destroyed during the islet isolation, the re-establishment of blood supply in the transplanted islets relies on the self-formation of blood vessels contributed by the blood vessel endothelial cells from the host transplantation site and the residual endothelial cells from the implanted islets. Nonetheless, the degree of vascularization in the transplanted islets is much less than that of the native pancreatic islets. Therefore, here we have engineered both the extracellular matrix and the co-transplanted blood vessel cells to facilitate the in-growth of blood vessels into the transplanted islets. For better engineering the extra cellular matrix, we carried out gene profiling of the islet cells and discovered the specific matrix proteins in native islets, which are essential to support islet cell function and survival. The matrix will be designed accordingly so that the matrix will not only support blood vessel in-growth but also deliver peptides providing essential signaling to sustain islet cell survival and function. The endothelial cells that will be used are adaptive endothelial cells. We have shown that the adaptive endothelial cells actively form connective blood vessels beneath the skin. In addition, the adaptive endothelial cells will also adapt to islet cells and acquire the specific features of native islet endothelial cells to meet the special requirement of islet endocrine cells. Hence, the blood supply for islets is only half of the story. Our labs and others have pioneered the concept that each tissue has its own specialized niche, which consists of endothelial cells, mesenchymal cells, and extra cellular matrix. Such special niche makes critical contributions to sustain the long term function and survival of islet cells. Using the active peptides that are delivered by PEG-4MAL and the islet-specific ECs self-adapted from R-VECs, we expect to establish a highly vascularized and islet-specific niche to support the long-term functional mass of transplanted islets to treat diabetes.
Description of Project
Current treatments for type 1 diabetes (T1D) involve the administration of insulin, either by daily self-injections or insulin pumps, which effectively reduce blood glucose. However, without second-to-second fine tuning of insulin secretion, these approaches still result in intermittent elevations in blood glucose levels and as such may not always prevent complications of diabetes. Furthermore, those with “brittle diabetes” can have difficulty in managing their diabetes, with large swings in blood glucose that can result in severe hypoglycemia or hyperglycemia. To date, pancreas and islet cell transplantation are the only approaches that achieve physiologic regulation of blood glucose. Given the invasive surgical approach required and potential complications associated with pancreas transplantation, islet cell transplantation represents the most promising therapy to endow T1D patients with normal life. The overarching objective of our proposal is to expedite and translate the potential of islet transplantation to a broader population of patients with T1D. Our approach provides the essential blood supply to the donor-derived islets through a technology developed in our laboratory, which will augment the safety, efficacy and durability of islet cell transplantation. The current clinical practice of islet cell transplantation often requires collection of islets from 2-3 donors and infusion of these islets into the recipient’s liver. Because the liver is not an ideal site for the engraftment of islets, the majority of the transplanted islets do not survive. Therefore, many of these patients will need a 2nd or 3rd transplantation to achieve normal blood glucose. Also, about 80% have to resume insulin therapy 10 years after the first transplantation due to gradual loss of functional islet mass. The main bottleneck of delivering islets to the liver is that the transplanted islets are not fully functional within the liver because they do not provide the specific niche for long-term sustaining islet function, which is largely contributed by the specific endothelial cells and the extra cellular matrix. To circumvent this hurdle, we will transplant islets to the subcutaneous space, which enables the manipulation possibility to re-construct a islet-specific niche beneath the skin. To establish the islet-specific niche, we will recruit 2 approaches synchronically, the adaptable blood vessel cells to connect the islet graft to the host circulatory system and the synthetic encapsulation matrix to deliver peptides to build the islet-specific extracellular matrix. We hope to develop the above strategy to achieve long-term and physiologic glucose control in T1D patients. Th Rafii lab recently published a paper (Palikuqi B et al., Nature, 2020) reporting engineering adaptable human blood vessels, known as Reset-Vascular-Endothelial-Cells (R-VECs), that have the capacity to form blood vessels and to self-shape to meet the metabolic requirements of islets. The Garcia lab has developed a fully synthetic matrix, called PEG-4MAL (Weaver et al., Science Advance, 2017), which has the capacity of delivering peptides of interest along with the transplanted islets. Combining these 2 advanced technologies, we will transplant either donor-derived or stem cell-derived islets to the subcutaneous space, together with adaptive blood vessel endothelial cells. and encapsulated in PEG-4MAL that delivers the islet-specific peptides. We expect that the transplanted islets will functionally engraft beneath the skin, and normal blood glucose levels will be achieved using a low number of islets for a long term. We hope to develop the above strategy to achieve long-term and physiologic glucose control in T1D patients.
Anticipated Outcome
The overall goal of this study is to reduce the blood glucose levels in the diabetic transplantation recipients with subcutaneous islet transplantation along with adaptive endothelial cells and customized matrix. To achieve that goal, we proposed 3 objectives to gradually optimize our technology.
The objective 1 is aimed at optimizing the transplantation route, either inserting solid cell mixture through the skin incision or inject liquid cell mixture directly under the skin. Based on our previous results, we expect that the injection of liquid cell mixture would have a better outcome in term of islet vascularization. Clinically, subcutaneous injection will be more convenient without the cutting and suturing. In addition, we also expect that the islet vascularization will be higher when we use higher concentration of endothelial cells.
In the objective 2, we will test various matrix protein-derived active peptides. Totally 7 peptides will be tested, which are derived collagen type IV, laminin 111, laminin 411, and laminin 511 that are expressed in the islets. Firstly, we expect at least 2 peptides will improve islet function during in vitro culture, compared to the ubiquitous fibronectin-derived RGD. Secondly, among different peptides, we have the expectation on those derived from laminin 411, which is the most specifically expressed in islet endothelial cells. Lastly, we expect at least 1 of the peptides will further increase the islet vascularization after subcutaneous transplantation.
The efficacy of treating diabetes will be tested in the objective 3. We will use the transplantation route and endothelial cell concentration that are optimized in objective 1, the best peptides that are screened out in objective 2, and use either donor-derived human islets or stem cell-derived islets. Using donor-derived islets, we expect to achieve normal blood glucose levels in that all recipients using the full dosage as 3,000 islet equivalents, and a high ratio of euglycemia using the reduced dosage as 1,000 islet equivalents. By generating stem cell-derived islets using islet-specific endothelial cells in matrix containing islet-specific matrix peptides, we first expect that the islet maturation will be further improved with more dynamic insulin secretion and gene profile more similar to native islet cells. Transplanting stem cell-derived islet together with adaptive R-VECs and customized PEG-4MAL, we expect to reverse the hyperglycemia and to prove that our approach is compatible with the stem cell-derived islets, which may be the future of islet transplantation.
Relevance to T1D
Type 1 diabetes (T1D) is caused by insufficient insulin secretion by the pancreatic beta cells, resulting in high levels of blood sugar. Managing diabetes can be challenging, and the most common treatment is daily self-injections of insulin. An alternative approach is the insulin pump, which is a device implanted under the skin and programed to inject insulin subcutaneously. A step forward that offers more consistent diabetic management is the use of an insulin pump in combination with a continuous glucose monitoring system. However, besides the inconvenience and cost of daily insulin administration and of these devices, they are not capable of the fine regulation of insulin secretion that is optimal. Under normal conditions, the body adjusts insulin secretion every millisecond to ensure constant control of blood glucose. In contrast, devices such as insulin pumps and glucose monitors sense blood glucose every 5 to 15 minutes, which inevitably results in peaks and drops in glucose that can lead to diabetic complications, such as cardiovascular disease, kidney damage, nerve damage, stroke, accelerated aging and eyesight impairments. Transplanting healthy islets with adequate blood supply into an individual with Type 1 diabetes offers the opportunity for a cure that can achieve physiologic regulation of blood glucose. In particular, islet transplantation offers great promise for treating type 1 diabetes in those young or old patients that suffer from “brittle type 1 diabetes,” in which patients can have severe hypoglycemic events and great difficulty in controlling their glucose with insulin injections. This approach could also potentially mitigate complications associated with Type 1 diabetes, including impaired kidney, heart, eye and brain functions. Both types of islet cell replacement therapy, however, whole pancreas transplantation and islet cell transplantation, have major hurdles that hamper wide clinical application (discussed above). Most importantly, in these approaches the cadaveric islets have lost their blood vessels and as such will not be able to connect to the host blood vessels that will lead to the poor function of islets and death of the islets. To circumvent this formidable obstacle in this proposal, we have engineered adaptable endothelial cells with peptide-delivering PEG-4MAL hydrogels that will rapidly vascularize human donor-derived islets or stem cell-derived islets. The combination of these resilient endothelial cells and customized hydrogel is expected to quickly establish islet-specific niches and extensive nurturing blood supply after transplantation. This will enable timely transplantation of these vascularized islets under the skin to ensure blood supply and durable engraftment. In this regard, our approach obviates the need for commonly practiced procedure of islet cell transplantation directly into the unfavorable liver portal circulation, which is often associated with major complications, including clotting and gradually loss of the islet function. The advantage of our approach to re-construct the islet-specific niche over the liver approach is that the co-transplanted adaptive endothelial cells with islet-specific peptides will expedite permanent blood vessel connection to the host circulation and support long-lasting function of transplanted islets to modulate finely insulin secretion for glucose control and provide the essential signaling to sustain the long-term reserve of the islet functional mass. We envision the application of islet/R-VECs/PEG-4MAL as a subcutaneous insertion of gel or semi-liquid mixture of islets with adaptable endothelial cells, to provide long-term, constant control of blood glucose without the need for insulin and serve as a major game changing approach for treatment of Type I Diabetes at low cost and large margin of safety. The capacity of the vascularized islets to survive under the skin will allow for close monitoring, potential retrieval and a drastic decrease in vascular complication associated with diabetes.