Generation of functional human pancreatic ß cells in vitro.

The generation of insulin-producing pancreatic ß cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide ß cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive ß cells from hPSC in vitro. These stem-cell-derived ß cells (SC-ß) express markers found in mature ß cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult ß cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.

Charting cellular identity during human in vitro ß-cell differentiation.

In vitro differentiation of human stem cells can produce pancreatic ß-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro ß-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to ß-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo ß-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the ß-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro ß-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine.

How to make a functional ß-cell.

Insulin-secreting pancreatic ß-cells are essential regulators of mammalian metabolism. The absence of functional ß-cells leads to hyperglycemia and diabetes, making patients dependent on exogenously supplied insulin. Recent insights into ß-cell development, combined with the discovery of pluripotent stem cells, have led to an unprecedented opportunity to generate new ß-cells for transplantation therapy and drug screening. Progress has also been made in converting terminally differentiated cell types into ß-cells using transcriptional regulators identified as key players in normal development, and in identifying conditions that induce ß-cell replication in vivo and in vitro. Here, we summarize what is currently known about how these strategies could be utilized to generate new ß-cells and highlight how further study into the mechanisms governing later stages of differentiation and the acquisition of functional capabilities could inform this effort.

Conformal Coating of Stem Cell-Derived Islets for ß Cell Replacement in Type 1 Diabetes.

The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species.

Autologous Pluripotent Stem Cell-Derived ß-Like Cells for Diabetes Cellular Therapy.

Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing ß-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional ß-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into ß-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous ß-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach.

Generation of stem cell-derived ß-cells from patients with type 1 diabetes.

We recently reported the scalable in vitro production of functional stem cell-derived ß-cells (SC-ß cells). Here we extend this approach to generate the first SC-ß cells from type 1 diabetic patients (T1D). ß-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-ß cells express ß-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of ß-cell stress. Using these assays, we find no major differences in T1D SC-ß cells compared with SC-ß cells derived from non-diabetic patients. These results show that T1D SC-ß cells could potentially be used for the treatment of diabetes, drug screening and the study of ß-cell biology.

Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-ß cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-ß cells. SC-ß cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.