Objective
The objective of this study is to establish novel methods to generate insulin-producing human islet-like mini organs (HILOs) derived from human induced pluripotent stem cells (hiPSCs) that can avoid rejection by host immune cells in a fully scalable way. For this purpose, I will investigate the molecular mechanisms by which HILOs acquire the ability to avoid attack from immune cells. In addition, I have preliminary methods that generate scalable functional HILOs in a dish using a three-dimensional gel culture system. Since the shortage of cadaveric human islets and necessary use of immune suppressive drugs to prevent rejection of the transplanted cells limits the successful cure of type 1 diabetes by islet transplantation, finding a way to generate alternative human islets from a limitless source such as human pluripotent stem cells and removing immune cell recognition would be a major goal for therapeutics in the disease. Understanding the novel molecular mechanisms by which insulin-producing cells acquire the ability to avoid immune cells would be a ground-breaking step in the field. My preliminary findings reveal a unique function of insulin-producing cells, suggesting that they memorize previous signals from immune cells to enhance their tolerance to immune rejection from the host body (I term these cells as MPS-HILOs). This phenomenon is constructed from two unique features: (1) MPS-HILOs have tolerance to immune cell signals that reduce insulin secretion function, and (2) MPS-HILOs highly and sustainably express an immune cell suppressive molecule called PD-L1. Unveiling these mechanisms may lead to novel therapeutics for type 1 diabetes that do not require immune suppressive drug treatment. Investigation into a safety switch system to eliminate transplanted cells when they become dysfunctional will be another objective for this study.
Background Rationale
Both Type 1 diabetes (T1D) and Type 2 diabetes (T2D) are characterized by progressive failure of insulin producing β-cell function. Since insulin is the only hormone to reduce blood sugar in our body, loss of insulin-producing β-cells leads to high blood sugar and causes many complications such as blindness, kidney failure and cardiovascular disease. T2D is often accompanied with environmental stresses such as obesity and aging, while T1D is often found in young children due to autoimmune rejection of pancreatic β-cells. These pathological differences distinguish T1D and T2D. However, both diseases have common features such as morphological and functional defects in the gut and insulin-producing pancreatic β-cells. Thus, gut to pancreas is a main therapeutic target for treating both T1D and T2D. In the past decade, the number of juvenile Type1 diabetic patients has dramatically increased worldwide. In the U.S, the number of patients has increased 60% from 2003 to 2013. Lifestyle changes such as increased consumption of cow milk and processed foods rather than breast milk and natural foods are believed to affect the microbiome and, along with viral infections, are suspected to contribute to the pandemic of T1D by triggering the activation of immune cells responsible for attacking insulin-producing β-cells.
Human islet transplantation confers significant improvement in glycemic control and prevents life-threatening severe hypoglycemia in T1D patients. However, the shortage of human islets limits the therapeutic opportunities. In addition, chronic immunosuppression, which is required to avoid rejection of transplanted islets, is associated with severe complications such as increased risk of cancer and infections. Thus, there is a significant need for novel approaches for large-scale generation of functional human islets that have immune tolerance to ensure durable graft acceptance without immunosuppression or its complications. An important step in addressing this need is increasing our understanding of transplant immune tolerance mechanisms for both graft rejection and autoimmune rejection. Using this system, I found that the protective mechanism used by cancer cells to escape from T-cell recognition and adaptation, characterized by PD-L1 expression, can be induced by IFNγ in human islets and HILOs. Since forced expression of PD-L1 in HILOs significantly reduced the possibility of xenograft rejection in immune-competent mice, understanding how to achieve sustainable induction of PD-L1 in HILOs may lead to the generation of immune-tolerant human islets from hiPSCs. My objective is to progress this technology towards preclinical evaluation by demonstrating the efficacy and safety of immuno-protected HILOs. The expected findings will provide novel mechanistic insight into induced and naturally occurring islet immune tolerance, as well as a conceptional breakthrough in the field.
Description of Project
Type 1 diabetes (T1D) is a major form of diabetes, which often begins in young children. T1D is a lifelong condition that causes the patient’s blood sugar levels to become too high due to lack of insulin, a hormone that reduces blood sugar levels. Insulin is produced from pancreatic islet β cells. Therefore, human islet transplantation, coupled with immune-suppressive, drugs has been acknowledged as a functional cure for T1D, despite the shortage of human islets and the side effects of immune-suppressive drug treatment, including increased risk of infection and cancer.
Generation of functional human pancreatic islets, which can avoid attack from host immune cells, would provide an infinite source for transplantation therapy. Human induced pluripotent stem cells (hiPSCs) offer an infinite supply of cells because of their function of self-renewal and pluripotency. Despite recent progress in the generation of insulin-producing β-like cells from pluripotent stem cells, further improvements in functional maturation, a process which is activated after birth in humans, are required to achieve generation of fully functional mature β cells. Moreover, although using hiPSCs deliver autologous, and thus immune-matched, insulin-producing β cells, life-long immune suppression may still be required to protect transplanted insulin-producing β cells because of the hyperactive immune reaction in type 1 diabetic patients. Therefore universal hiPSCs, which are tolerant to immune rejection, offer an alternative way to reduce the risk for autoimmune rejection and costly personalized therapy.
I will seek to understand how human islet β cells transform into immune-tolerant and -evasive β cells, which are characterized by the sustainable expression of a deactivator of T-cells (PD-L1) and T-cell-derived small molecules. Since T-cells directly and indirectly contribute to the immune rejection of transplanted cells from the host body, understanding the mechanisms of how β cells express PD-L1 and acquire the function for tolerance from autoimmune rejection may be the key for successful stem cell-based cell therapy for T1D.
My recent findings revealed that constitutive expression of PD-L1 (T-cell deactivator) in hiPSC-derived mature functional islet-like mini organs (HILOs) rapidly ameliorates diabetes in autoimmunity without the need for immune-suppressive drugs. I found that HILOs can acquire PD-L1 expression and tolerance for T-cell-derived small molecules by repeated stimulation of IFNγ, which causes genomic memory to adopt an immune rejection condition. I refer to this novel phenomenon as “genomic memory of β cells”. I will dissect the mechanisms underlying the genomic memory of cells to induce sustainable PD-L1 expression in HILOs by using a combination of molecular, genomic and physiological approaches as well as a unique differentiation technology of HILOs from human pluripotent stem cells and humanized diabetic model mice. By establishing the genomic signature using state-of-the-art genome wide analysis and bioinformatic techniques, as well as the immune-tolerant function in immune competent mice, the proposed study should provide new insight into immune-tolerance induction in human β cells.
To achieve this goal, I will address the above questions by using humanized T1D mouse models as well as human insulin-producing islet-like organoids created from pluripotent stem cells in a dish. I will also engineer a kill switch system for HILOs as safety backup to enable removal of all transplanted HILOs if or when they become dysfunctional after transplantation.
Anticipated Outcome
Unveiling the mechanism by which human β-cells acquire immune tolerance and evasive function will lead to a strategy to generate an alternative source of human islets for the functional cure of type 1 diabetes that does not require immune suppressive drugs. I would anticipate that this study will elucidate novel mechanisms for the induction of immune tolerance in human β-cells as well as a platform for generating scalable, highly reproducible human islet-like mini organs from human iPSCs (HILOs). The generated HILOs will not only be a valuable resource for transplantation into diabetic animals (and eventually human patients) but also a resource for the disease modeling of type 1 diabetes and drug screening. A valuable “what if” safety feature for transplanted cells is the ability to be eliminated should the need arise. I also anticipate we will know the efficacy of “safety switch system” by using induced caspase 9 from this study. Anticipated outcomes from this study are listed below.
1. Transcriptomic (genomic) information on how human β-cells acquire the immune tolerance and evasive function.
2. Elucidating the novel phenomenon named “adaptive transcriptional memory for human β-cells,” which will be defined by repeated stimulation by small molecules secreted from immune cells that induce dynamic but stable transcriptional changes, including expression of PD-L1 and tolerance for toxic inflammatory signals in T1D conditions.
3. Long-term efficacy of immune tolerant HILOs for T1D mice model in immune competent conditions.
4. Single cell level of genomic information on how long-term survived HILOs and immune tolerance induction.
Relevance to T1D
For type 1 diabetes, insulin injection is currently the major therapy. Human islet transplantation confers significant improvement in glycemic control and prevents severe life-threatening hypoglycemia in type 1 diabetes (T1D) patients. However, the shortage of human islets limits the potential of this therapy. In addition, chronic immunosuppression, which is required to avoid rejection of transplanted islets, is associated with severe complications such as increased risk of cancer and infections. Thus, there is a significant need for novel approaches for large-scale generation of functional mature human islets with immune tolerance to ensure durable graft acceptance in the absence of immunosuppression or its complications. An important step in addressing this need is increasing our understanding of transplant immune tolerance mechanisms for both graft rejection and autoimmune rejection. Generation of functional human pancreatic islets that can avoid attacks from host immune cells would provide an alternative safe resource for transplantation therapy. Human induced pluripotent stem cells (hiPSCs) offer a potentially limitless supply of cells because of their ability of self-renewal and pluripotency. Therefore studying immune tolerance induction in hiPSC derived human pancreatic islet-like organoids (HILOs) will directly contribute towards the goal for functional cure of T1D.