ABSTRACT
Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.
Subject(s)
Genomics , Humans , Gene Expression , Transgenes , Cell DifferentiationABSTRACT
Diabetes mellitus is a global disease requiring long-term treatment and monitoring. At present, pancreas or islet transplantation is the only reliable treatment for achieving stable euglycemia in Type I diabetes patients. However, the shortage of viable pancreata for transplantation limits the use of this therapy for the majority of patients. Organ decellularization and recellularization is emerging as a promising solution to overcome the shortage of viable organs for transplantation by providing a potential alternative source of donor organs. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have been performed, and show promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we provide an overview of the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with an outlook on the remaining work that needs to be done in order to realize the goal of using this technology to create bioengineered pancreata for transplantation in diabetes patients. STATEMENT OF SIGNIFICANCE: Pancreas or islet transplantation is a means of providing insulin-independence in diabetes patients. However, due to the shortage of viable pancreata, whole-organ decellularization and recellularization is emerging as a promising solution to overcome organ shortage for transplantation. Several studies on decellularization and recellularization of rodent, porcine, and human pancreata have shown promise for generating usable decellularized pancreas scaffolds for subsequent recellularization and transplantation. In this state-of-the-art review, we highlight the latest advances in pancreas decellularization, recellularization, and revascularization. We also discuss clinical considerations such as potential transplantation sites, donor source, and immune considerations. We conclude with future work that needs to be done in order to realize clinical translation of bioengineered pancreata for transplantation in diabetes patients.
Subject(s)
Diabetes Mellitus, Type 1 , Tissue Engineering , Humans , Animals , Swine , Regenerative Medicine , Tissue Scaffolds , Pancreas , Extracellular MatrixABSTRACT
Heterozygous HNF1A gene mutations can cause maturity onset diabetes of the young 3 (MODY3), characterized by insulin secretion defects. However, specific mechanisms of MODY3 in humans remain unclear due to lack of access to diseased human pancreatic cells. Here, we utilize MODY3 patient-derived human induced pluripotent stem cells (hiPSCs) to study the effect(s) of a causal HNF1A+/H126D mutation on pancreatic function. Molecular dynamics simulations predict that the H126D mutation could compromise DNA binding and gene target transcription. Genome-wide RNA-Seq and ChIP-Seq analyses on MODY3 hiPSC-derived endocrine progenitors reveal numerous HNF1A gene targets affected by the mutation. We find decreased glucose transporter GLUT2 expression, which is associated with reduced glucose uptake and ATP production in the MODY3 hiPSC-derived ß-like cells. Overall, our findings reveal the importance of HNF1A in regulating GLUT2 and several genes involved in insulin secretion that can account for the insulin secretory defect clinically observed in MODY3 patients.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucose Transporter Type 2/genetics , Glucose/metabolism , Hepatocyte Nuclear Factor 1-alpha/genetics , Insulin Secretion/genetics , Insulin-Secreting Cells/metabolism , Mutation , Cells, Cultured , Chromatin Immunoprecipitation Sequencing/methods , Diabetes Mellitus, Type 2/metabolism , Female , Glucose Transporter Type 2/metabolism , Hepatocyte Nuclear Factor 1-alpha/chemistry , Hepatocyte Nuclear Factor 1-alpha/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Insulin-Secreting Cells/cytology , Male , Molecular Dynamics Simulation , Pedigree , Protein DomainsABSTRACT
Metformin is becoming a popular treatment before and during pregnancy, but current literature on in utero exposure to metformin lacks long-term clinical trials and mechanistic studies. Current literature on the effects of metformin on mature pancreatic ß-cells highlights its dual, opposing, protective, or inhibitory effects, depending on metabolic environment. However, the impact of metformin on developing human pancreatic ß-cells remains unknown. In this study, we investigated the potential effects of metformin exposure on human pancreatic ß-cell development and function in vitro. In the absence of metabolic challenges such as high levels of glucose and fatty acids, metformin exposure impaired the development and function of pancreatic ß-cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic ß-cells. These findings call for further in-depth evaluation of the exposure of human embryonic and fetal tissue during pregnancy to metformin and its implications for long-term offspring health.
Subject(s)
Cell Differentiation/drug effects , Human Embryonic Stem Cells/drug effects , Hypoglycemic Agents/pharmacology , Insulin-Secreting Cells/drug effects , Metformin/pharmacology , Pancreas/drug effects , Cell Survival/drug effects , Human Embryonic Stem Cells/cytology , Humans , Insulin Secretion/drug effects , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Pancreas/cytology , Pancreas/metabolismABSTRACT
There is now a sizeable number of single cell transcriptomics studies performed on human and rodent pancreatic islets that have shed light on the unique gene signatures and level of heterogeneity within each individual islet cell type. Following closely from these studies, there is also rapidly-growing activity on characterizing islet-like cells derived from in vitro differentiation of human pluripotent stem cells (hPSCs) at the single cell level. The overall consensus across the studies so far suggests that the first few stages of differentiation are largely uniform, whereas during pancreatic endocrine commitment, cell trajectories start to diverge, resulting in multiple end-stage pancreatic cells that include progenitor-like, endocrine and non-endocrine cells. Comprehensive transcriptional profiling is important for understanding how and why islet cells, especially the insulin-secreting beta cells, exist in subpopulations that differ in maturity, proliferation rate, sensitivity to stress, and insulin secretion function. For hPSC-derived beta cells to be used confidently for cell therapy, optimal differentiation and thorough characterization is required. The key questions to address are-What is the trajectory of differentiation? Is heterogeneity a natural occurrence or is it a consequence of imperfect differentiation protocols? Can lessons be drawn from the extensive single cell transcriptomic data to help guide maturation of beta cells in vitro? This book chapter seeks to address some of these questions, and facilitate ongoing efforts in improving the beta cell differentiation pipeline or enriching for desired beta cell populations following differentiation, to make way for better mechanistic studies and future clinical translation.
Subject(s)
Insulin-Secreting Cells , Islets of Langerhans , Neuroendocrine Cells , Pluripotent Stem Cells , Cell Differentiation , Humans , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolismABSTRACT
Multipotent mesenchymal stem cells (MSCs) have been employed in numerous pre-clinical and clinical settings for various diseases. MSCs have been used in treating degenerative disorders pertaining to the eye, for example, age-related macular degeneration, glaucoma, retinitis pigmentosa, diabetic retinopathy, and optic neuritis. Despite the known therapeutic role and mechanisms of MSCs, low cell precision towards the targeted area and cell survivability at tissue needing repair often resulted in a disparity in therapeutic outcomes. In this review, we will discuss the current and feasible strategy options to enhance treatment outcomes with MSC therapy. We will review the application of various types of biomaterials and advances in nanotechnology, which have been employed on MSCs to augment cellular function and differentiation for improving treatment of visual functions. In addition, several modes of gene delivery into MSCs and the types of associated therapeutic genes that are important for modulation of ocular tissue function and repair will be highlighted.