Stem Cell-derived Islet Cells Research Articles

Optimizing Generation of Stem Cell-Derived Islet Cells.

Islet transplantation is a highly effective treatment for select patients with type 1 diabetes. Unfortunately, current use is limited to those with brittle disease due to donor limitations and immunosuppression requirements. Discovery of factors for induction of pluripotent stem cells from adult somatic cells into a malleable state has reinvigorated the possibility of autologous-based regenerative cell therapies. Similarly, recent progress in allogeneic human embryonic stem cell islet products is showing early success in clinical trials. Describing safe and standardized differentiation protocols with clear pathways to optimize yield and minimize off-target growth is needed to efficiently move the field forward. This review discusses current islet differentiation protocols with a detailed break-down of differentiation stages to guide step-wise controlled generation of functional islet products.

Development of a scalable method to isolate subsets of stem cell-derived pancreatic islet cells.

SummaryCell replacement therapy using β cells derived from stem cells is a promising alternative to conventional diabetes treatment options. Although current differentiation methods produce glucose-responsive β cells, they can also yield populations of undesired endocrine progenitors and other proliferating cell types that might interfere with long-term islet function and safety of transplanted cells. Here, we describe the generation of an array of monoclonal antibodies against cell surface markers that selectively label stem cell-derived islet cells. A high-throughput screen identified promising candidates, including three clones that mark a high proportion of endocrine cells in differentiated cultures. A scalable magnetic sorting method was developed to enrich for human pluripotent stem cell (hPSC)-derived islet cells using these three antibodies, leading to the formation of islet-like clusters with improved glucose-stimulated insulin secretion and reduced growth upon transplantation. This strategy should facilitate large-scale production of functional islet clusters from stem cells for disease modeling and cell replacement therapy.

209.2: Engineering Immune-evasive Human Stem Cell-derived Islet Cells

Introduction: Protection against the human immune system is required to realize the therapeutic potential of stem cell (SC)-islet cells for the treatment of type 1 diabetes. Various groups have attempted to address this challenge by engineering immune-evasive human embryonic stem cells (hESCs) that can be differentiated into various cell types. In many cases, over-expression of the tolerogenic molecules at safe harbor loci is lost due to transgene silencing. We recently addressed this issue by targeting Firefly luciferase (Luc2) to the constitutively expressed GAPDH locus of hESCs (GAPluc), followed by successful differentiation into SC-islet cells that maintain Luc2 expression. Thus, in this study we used a similar engineering strategy to generate hypoimmunogenic SC-islet cells. Methods: Using CRISPR/Cas9 we introduced Luc2 and the B2M::HLA-E fusion to the GAPDH locus of hESCs, followed by CRISPR knockout of endogenous B2M to eliminate class I HLA expression. Using sequential antibiotic selection of GAPDH-targeted and FACS sorting of HLA-ABC-/- hESCs, we generated a hypoimmunogenic hESC line (BEC) that was differentiated into SC-islet cells. SC-islet cell immunogenicity was assessed in in vitro PBMC and NK cell co-culture assays using cells isolated from allogeneic human peripheral blood (n=5 donors), with Luc2 expression serving as a reporter of cell viability. Wild-type (WT) and B2M-/- SC-islet cells served as controls. To assess SC-islet cell survival in vivo, 5x106 WT and BEC SC-islet cells were transplanted under the kidney capsule of HLA-A2 mismatched humanized NSG-(Kb Db)null (IAnull) mice (n=4/group). Graft survival was assessed via bioluminescence imaging using the IVIS Spectrum. Results: Differentiation of WT, B2M-/- and BEC hESCs into SC-islet cells resulted in a similar yield (~20%) of Nkx6.1+/C-peptide+ SC-beta cells. HLA-E and Luc2 expression correlated throughout the course of differentiation, with a ~10-fold decrease in expression in hESCs vs CD49a-enriched SC-beta cells. No significant difference in Luc2 expression was observed in CD49a-enriched SC-beta cells derived from all gene-modified hESC lines. In in vitro PBMC co-culture assays, B2M-/- and BEC SC-islet cells were significantly protected from PBMC cytotoxicity by comparison to WT. In in vitro NK cell co-culture assays, BEC SC-islet cells were significantly protected against NK cell cytotoxicity by comparison to B2M-/-. Additionally, following transplant into the kidney capsule of humanized NSG-(Kb Db)null (IAnull) mice, BEC SC-islet cells showed a significant survival advantage by comparison to WT at 35 days post transplantation. Conclusion: In this study we show that targeting transgenes to constitutively expressed loci results in persistent transgene expression throughout the course of islet cell differentiation. Furthermore, our engineering strategy resulted in the generation of hypoimmunogenic SC-islet cells that evade immune rejection both in vitro and in vivo.

The Feasibility and Applicability of Stem Cell Therapy for the Cure of Type 1 Diabetes.

Stem cell therapy using islet-like insulin-producing cells derived from human pluripotent stem cells has the potential to allow patients with type 1 diabetes to withdraw from insulin therapy. However, several issues exist regarding the use of stem cell therapy to treat type 1 diabetes. In this review, we will focus on the following topics: (1) autoimmune responses during the autologous transplantation of stem cell-derived islet cells, (2) a comparison of stem cell therapy with insulin injection therapy, (3) the impact of the islet microenvironment on stem cell-derived islet cells, and (4) the cost-effectiveness of stem cell-derived islet cell transplantation. Based on these various viewpoints, we will discuss what is required to perform stem cell therapy for patients with type 1 diabetes.

Insights from single cell studies of human pancreatic islets and stem cell-derived islet cells to guide functional beta cell maturation in vitro.

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.

YIPF5 mutations cause neonatal diabetes and microcephaly through endoplasmic reticulum stress.

Neonatal diabetes is caused by single gene mutations reducing pancreatic β cell number or impairing β cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in β cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human β cell models (YIPF5 silencing in EndoC-βH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects β cells. Loss of YIPF5 function in stem cell–derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and β cell failure. Partial YIPF5 silencing in EndoC-βH1 cells and a patient mutation in stem cells increased the β cell sensitivity to ER stress–induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in β cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes.

A hydrogel platform for in vitro three dimensional assembly of human stem cell-derived islet cells and endothelial cells

Differentiation of stem cells into functional replacement cells and tissues is a major goal of the regenerative medicine field. However, one limitation has been organization of differentiated cells into multi-cellular, three-dimensional assemblies. The islets of Langerhans contain many endocrine and non-endocrine cell types, such as insulin-producing β cells and endothelial cells. Despite the potential importance of endothelial cells to islet function, facilitating interactions between endothelial cells and islet endocrine cell types already differentiated from human embryonic stem cells has been difficult in vitro. We have developed a strategy of assembling human embryonic stem cell-derived islet cells with endothelial cells into three-dimensional aggregates on a hydrogel. The resulting islet organoids express β cell and other endocrine markers and are functional, capable of undergoing glucose-stimulated insulin secretion. This assembly was not observed on traditional tissue culture plastic and in aggregates generated in suspension culture, highlighting how physical culture conditions greatly influence the interactions among these cell types. These results provide a platform for evaluating the effects of the islet tissue microenvironment on human embryonic stem cell-derived β cells and other islet endocrine cells to develop tissue engineered islets. Statement of SignificanceDifferentiation of insulin-producing cells and tissues from human pluripotent stem cells is being investigated for diabetes cell replacement therapies. Despite successes generating β cells, the cell type responsible for glucose-stimulated insulin secretion within the islets of Langerhans found in the pancreas, successful assembly with other non-endocrine cell types, particularly endothelial cells, has been technically challenging. The present study provides a platform for the assembly of endothelial cells with SC-β and other endocrine cells, producing islet organoids that are functional and express β cell markers, that can be used to study the islet microenvironment and islet tissue engineering.

Diabetes and stem cells: Endogenous effects and reparative mechanisms

Stem cells offer a novel approach to diabetes care based on regeneration, which can potentially shift treatment paradigms towards curation; therapeutic approaches have honed in on generating functional stem cell-derived islet cells for transplantation. Other approaches include stimulating endogenous stem cell replication followed by differentiation into functional islet cells in situ. This review critically considers the impact of diabetes on endogenous stem cells and discusses the potential utility of stem cell therapies for the treatment of diabetes.

Stem cell-derived islet cells for transplantation

The promise of islet transplantation for type 1 diabetes has been hampered by the lack of a renewable source of insulin-producing cells. However, steadfast advances in the field have set the stage for stem cell-based approaches to take over in the near future. This review focuses on the most intriguing findings reported in recent years, which include not only progress in adult and embryonic stem cell differentiation, but also the direct reprogramming of nonendocrine tissues into insulin-producing beta cells. In spite of their potential for tumorigenesis, human embryonic stem (hES) cells are poised to be in clinical trials within the next decade. This situation is mainly due to the preclinical success of a differentiation method that recapitulates beta cell development. In contrast, adult stem cells still need one such gold standard of differentiation, and progress is somewhat impeded by the lack of consensus on the best source. A concerted effort is necessary to bring their potential to clinical fruition. In the meantime, reported success in reprogramming might offer a 'third way' towards the rescue of pancreatic endocrine function. Here we discuss the important strategic decisions that need to be made in order to maximize the therapeutic chances of each of the presented approaches.