Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis.

In type 1 diabetes (T1D), autoimmune destruction of pancreatic ß cells leads to insulin deficiency and loss of glycemic control. However, knowledge about human pancreas pathophysiology in T1D remains incomplete. To address this limitation, we established a pancreas tissue slice platform of donor organs with and without diabetes, facilitating the first live cell studies of human pancreas in T1D pathogenesis to our knowledge. We show that pancreas tissue slices from organ donors allow thorough assessment of processes critical for disease development, including insulin secretion, ß cell physiology, endocrine cell morphology, and immune infiltration within the same donor organ. Using this approach, we compared detailed pathophysiological profiles for 4 pancreata from donors with T1D with 19 nondiabetic control donors. We demonstrate that ß cell loss, ß cell dysfunction, alterations of ß cell physiology, and islet infiltration contributed differently to individual cases of T1D, allowing insight into pathophysiology and heterogeneity of T1D pathogenesis. Thus, our study demonstrates that organ donor pancreas tissue slices represent a promising and potentially novel approach in the search for successful prevention and reversal strategies of T1D.