Objective
In this proposed study, we plan to explore the role of scaffold microarchitecture on the differentiation and maintenance of stem cell derived beta cells in vitro and in vivo. In order to enhance the efficiency of insulin-producing cells, we propose to culture these cells on 3D microporous and microwell scaffolds with controlled architecture. The in vitro differentiation of the pancreatic progenitors to beta-cells on the scaffolds will be compared to traditional approaches, using previously reported techniques such as FACS, immunostaining, and qRT-PCR. We will assess the effect of the scaffold on cell morphology, organization, glucose responsiveness, and ECM secretion. Then, we will assess whether beta-cells grown in a 3D environment will lead to improved efficiency and survival in vivo in a clinically translatable site.
We will develop PCL based scaffolds which have been shown to promote vascularization with minimal fibrosis. Survival and function of stem cell-derived beta cells will be analyzed for scaffolds transplanted subcutaneously. We will also assess the rate of neovascularization at the site. In addition, we believe the scaffold benefits provided in vitro can extend to the in vivo environment, and that this environment can provide the proper cues to maintain beta-cell maturation long term. Importantly, the scaffold/cell constructs can be directly transplanted, which is anticipated to enhance engraftment and function by avoiding the disruption of cell-matrix and cell-cell contacts that occurs during the manipulation involved with preparing suspension cultured cells for transplant. With these studies, we aim to investigate whether stem cell derived beta cells placed in a 3D environment that mimics in vivo pancreatic extracellular matrix have greater survival and are more functional, and whether these cells can reverse hyperglycemia upon transplantation.
Background Rationale
Type I diabetes (T1D), which affects an estimated 3 million Americans, is caused by autoimmune destruction of pancreatic beta-cells that results in the need for life-long insulin therapy. Although insulin therapy has been successful, hypoglycemic events and vascular complications persist. One current treatment for T1D is allogeneic islet transplantation where donor islets are infused intrahepatically leading to a transient reversal of diabetes. Yet, islet allogeneic transplantation has several therapeutic limitations including a shortage of donor islets, long-term immunosuppression, and high risk of tissue rejection. These limitations have led to the investigation of stem cells as an unlimited source of functional beta-cells. However, there remain several challenges in the efficiency of differentiation and the maintenance of beta-cell viability and function once transplanted. While stem cell derived beta-cells have been previously shown to reverse diabetes, new approaches to ensure long term maturation and function of the beta cells are required for clinical translation to treat T1D.
An engineered scaffold can recapitulate pancreatic architecture found in vivo by providing spatial control over cell-cell and cell-extracellular matrix (ECM) interactions. The ECM forms a three-dimensional (3D) environment and offers niches for cell adhesion, migration, proliferation, and differentiation. 3D microporous scaffolds can provide a more suitable microenvironment for stem cell derived beta cells to undergo proliferation, differentiation, and secretion of specific ECM molecules, which can form additional scaffold and promote cell adhesion and proliferation in vivo. Microporous and microwell scaffolds allow for both cell-cell and cell-matrix signaling that supports the self-organization of the cells into functional tissue structures. In vivo, beta cells naturally congregate into islet structures that are surrounded by a supportive extracellular matrix. Thus, it is important to mimic these aspects for the long-term maintenance of glucose-responsive, insulin-secreting cells for cell-based diabetes therapy.
An ideal material for beta-cell delivery should support rapid and robust vascularization without provoking foreign body reaction and fibrosis that limit vascular ingrowth to the grafts from adjacent tissues. Our extensive experience with PCL for islet encapsulation has shown that it is able to induce neovascularization with minimal fibrosis. Moreover, PCL can be easily modified both chemically and topographically using surface modification and photolithography allowing for maximal control of scaffold surface chemistry and pore size/shape, respectively. Lastly, PCL is already used in FDA-approved implanted scaffolds, thus the regulatory path for its clinical development is relatively more straightforward than completely new materials.
Description of Project
Type I diabetes (T1D) is a chronic metabolic disorder characterized by autoimmune destruction of the pancreatic beta-cells that results in the need for life-long insulin therapy. This disease represents 5–10% of the diagnosed cases of diabetes, corresponding to more than 3 million individuals in the United States. Several secondary metabolic disorders can arise from this disease as well, including retinopathy, neuropathy, nephropathy, stroke and heart failure. Although exogenous insulin injections have been successful, hypoglycemic events and vascular complications persist. Thus, recent research has turned to cell-based therapies focused on replacing lost insulin-producing cells. Initial enthusiasm in cell replacement therapies for diabetes was primarily driven by the dramatic progress in allogeneic islet transplantation using the Edmonton protocol for islet harvesting and rejection prevention. However, the widespread application of islet transplantation has been tempered by the lack of availability of islets, the need for life-long immunosuppression, and the increasing reversal of insulin independence.
All of these limitations have led to the investigation of human pluripotent stem cells (hPSCs), as an unlimited source of functional beta-cells. Multiple investigators have demonstrated the feasibility of differentiating stem cells to immature beta-cells in vitro, and transplanting these cells to support their maturation into glucose-responsive insulin-producing beta-cells. Despite these advancements, many transplants are not capable of producing a therapeutically functional population of glucose-responsive insulin-producing cells and transplants are typically performed at non-translatable sites.
Our research proposes to culture stem cell derived beta-cells on microporous and microwell scaffolds with controlled architecture in order to recapitulate cell-cell and cell-matrix interactions of the pancreatic niche and, thus, promote the maturation of beta-cells. The scaffolds will help elucidate what interactions within the niche environment are important during β-cell development. The scaffolds can be transplanted to a clinically relevant site, such as the subcutaneous space, for the β-cells to continue to mature. The subcutaneous site has been a desirable clinical site for transplantation as it provides easy accessibility however, there is a lack of vascularization that can lead to cell death due to low oxygen supply. Despite the diversity of the transplant sites that have been investigated to date, as outlined above, their ability to successfully promote islet graft survival is linked to a common ability to foster revascularization. Using this rationale, we plan to investigate the effects of scaffold architecture on beta cell function and vascularization.
Anticipated Outcome
We anticipate that the scaffolds seeded with the stem cell derived beta-cells will display increased maturation, survival, and function. The scaffold design is expected to facilitate the organization of cells into 3D structures with a more physiological extracellular matrix, which we anticipate will increase the expression of genes associated with differentiation, maturation, and secretion. ECM proteins produced on the scaffold by the cells are anticipated to aid in cell maturation by mimicking the basement membrane found in the pancreas, thereby facilitating cell-matrix interactions necessary for enhanced cell maturation. These findings will establish the role of scaffold architecture on stem cell derived beta-cell function in vitro and in vivo.
Relevance to T1D
Islet transplantation requires the procurement of large numbers of islets, highlighting the need for an alternative source of donor cells. Stem cell derived beta-cells have the potential to provide that cell source, but the consistency and efficiency of maturation must be significantly enhanced. 3D environments can be employed to enhance maturation and promote more functional tissues. During pancreatic development, progenitor cells congregate into structures called islets that are surrounded by a supportive extracellular matrix. We are developing engineered scaffolds with controlled microarchitecture that can provide a 3D environment where stem cell derived beta cells can be surrounded by a supportive extracellular matrix that better mimics the architecture of the native islet environment.
The goal of the proposed research is to develop engineered scaffolds that support and maintain differentiation and function of stem cell derived beta-cells for transplantation into a clinically relevant site. We hypothesize that the scaffolds can provide matrix-based signals that enable a more physiologic niche and allow for stem cell derived β-cells to have better long term survival and function. Furthermore, our scaffolds will provide an environment that supports vascularization, cell engraftment, maturation, and function in the post-transplantation time period. Ultimately, this strategy can be used in the clinic as an implantable device for the treatment of T1D.