Generation of pancreatic β cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin ...cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived β (SC-β) cells with improved in vitro and in vivo function. SC-β cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.
The generation of insulin-producing pancreatic β 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 β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β 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.
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•Stem-cell-derived β (SC-β) cells secrete insulin upon glucose stimulation in vitro•SC-β cells resemble human islet β cells by gene expression and ultrastructure•SC-β cell transplantation ameliorates hyperglycemia in mice•SC-β cells provide a platform for therapeutic development and disease modeling
Functional pancreatic β cells are generated from human pluripotent stem cells using a scalable, in vitro differentiation protocol. This Resource provides a valuable platform for therapeutic development and disease modeling in diabetes.
Human pluripotent stem cells differentiated to insulin-secreting β cells (SC-β cells) in islet organoids could provide an unlimited cell source for diabetes cell replacement therapy. However, current ...SC-β cells generated in vitro are transcriptionally and functionally immature compared to native adult β cells. Here, we use single-cell transcriptomic profiling to catalog changes that occur in transplanted SC-β cells. We find that transplanted SC-β cells exhibit drastic transcriptional changes and mature to more closely resemble adult β cells. Insulin and IAPP protein secretions increase upon transplantation, along with expression of maturation genes lacking with differentiation in vitro, including INS, MAFA, CHGB, and G6PC2. Other differentiated cell types, such as SC-α and SC-enterochromaffin (SC-EC) cells, also exhibit large transcriptional changes. This study provides a comprehensive resource for understanding human islet cell maturation and provides important insights into maturation of SC-β cells and other SC-islet cell types to enable future differentiation strategy improvements.
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•SC-islets show improved insulin and IAPP release with time in transplanted diabetic mice•SC-islet cells undergo drastic transcriptome changes after 6 months in vivo•Grafted SC-β cells mature and express MAFA, CHGB, G6PC2, and FAM159B in vivo•Differentially expressed genes upon transplantation resemble native human islet cells
Augsornworawat et al. demonstrate that in vitro human stem-cell-derived islets have immature transcriptome profiles when compared to native human islets. After transplantation, immature SC-islet cells undergo drastic changes in transcriptome and acquire mature gene expression. Grafted SC-β cells express mature β cell genes, including MAFA and G6PC2, resembling native human islets.
The generation of islet-like endocrine clusters from human pluripotent stem cells (hPSCs) has the potential to provide an unlimited source of insulin-producing β cells for the treatment of diabetes. ...In order for this cell therapy to become widely adopted, highly functional and well-characterized stem cell-derived islets (SC-islets) need to be manufactured at scale. Furthermore, successful SC-islet replacement strategies should prevent significant cell loss immediately following transplantation and avoid long-term immune rejection. This review highlights the most recent advances in the generation and characterization of highly functional SC-islets as well as strategies to ensure graft viability and safety after transplantation.
Stem cell-derived islets (SC-islets) have the potential to provide an unlimited source of insulin-producing β cells for the treatment of diabetes. Here, we review the most recent developments associated with generating highly functional SC-islets, solving the immune rejection problem, and overcoming practical challenges associated with SC-islet transplantation.
We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients ...(T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-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 β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology.
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-β 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-β cells. SC-β 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.
Recent advances in human pluripotent stem cell (hPSC) differentiation protocols have generated insulin-producing cells resembling pancreatic β cells. While these stem cell-derived β (SC-β) cells are ...capable of undergoing glucose-stimulated insulin secretion (GSIS), insulin secretion per cell remains low compared with islets and cells lack dynamic insulin release. Herein, we report a differentiation strategy focused on modulating transforming growth factor β (TGF-β) signaling, controlling cellular cluster size, and using an enriched serum-free media to generate SC-β cells that express β cell markers and undergo GSIS with first- and second-phase dynamic insulin secretion. Transplantation of these cells into mice greatly improves glucose tolerance. These results reveal that specific time frames for inhibiting and permitting TGF-β signaling are required during SC-β cell differentiation to achieve dynamic function. The capacity of these cells to undergo GSIS with dynamic insulin release makes them a promising cell source for diabetes cellular therapy.
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•Development of differentiation protocol to β-like cells with enhanced function•TGF-β signaling promotes acquisition of dynamic function in maturing β-like cells•Transplanted cells rapidly restore glucose tolerance in mice
In this study, Millman and colleagues report a differentiation strategy to generate β-like cells from human pluripotent stem cells with islet-like dynamic insulin release that rapidly reverses diabetes in mice. The authors elucidate that stage-specific control of TGF-β signaling during endocrine induction and maturation to be critical for robust function.
Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated ...cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS ⁺) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS ⁺ cells and (ii) hPSC-derived INS ⁺ (hPSC-INS ⁺) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS ⁺ cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS ⁺ cells into functional β cells.
We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell-derived pancreatic β cells (SC-β cells) that secrete high amounts of insulin in response to glucose ...stimulation. This protocol first induces definitive endoderm by treatment with Activin A and CHIR99021, then generates PDX1+/NKX6-1+ pancreatic progenitors through the timed application of keratinocyte growth factor, SANT1, TPPB, LDN193189 and retinoic acid. Endocrine induction and subsequent SC-β-cell specification is achieved with a cocktail consisting of the cytoskeletal depolymerizing compound latrunculin A combined with XXI, T3, ALK5 inhibitor II, SANT1 and retinoic acid. The resulting SC-β cells and other endocrine cell types can then be aggregated into islet-like clusters for analysis and transplantation. This differentiation methodology takes ~34 d to generate functional SC-β cells, plus an additional 1-2 weeks for initial stem cell expansion and final cell assessment. This protocol builds upon a large body of previous work for generating β-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-β cells to be done entirely on tissue culture polystyrene. This change simplifies the differentiation methodology, requiring only basic stem cell culture experience as well as familiarity with assessment techniques common in biology laboratories. In addition to expanding protocol accessibility and simplifying SC-β-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-β cells from a wide variety of cell lines from various sources, broadening its applicability.
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 β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-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 β-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 β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach.
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