Explore the vast range of titles available.

Identifying gene expression programs underlying both cell-type identity and cellular activities (e.g. life-cycle processes, responses to environmental cues) is crucial for understanding the organization of cells and tissues. Although single-cell RNA-Seq (scRNA-Seq) can quantify transcripts in individual cells, each cell’s expression profile may be a mixture of both types of programs, making them difficult to disentangle. Here, we benchmark and enhance the use of matrix factorization to solve this problem. We show with simulations that a method we call consensus non-negative matrix factorization (cNMF) accurately infers identity and activity programs, including their relative contributions in each cell. To illustrate the insights this approach enables, we apply it to published brain organoid and visual cortex scRNA-Seq datasets; cNMF refines cell types and identifies both expected (e.g. cell cycle and hypoxia) and novel activity programs, including programs that may underlie a neurosecretory phenotype and synaptogenesis.

Stem cell-derived insulin-producing beta cells (SC-β) offer an inexhaustible supply of functional β cells for cell replacement therapies and disease modeling for diabetes. While successful directed differentiation protocols for this cell type have been described, the mechanisms controlling its differentiation and function are not fully understood. Here we report that the Hippo pathway controls the proliferation and specification of pancreatic progenitors into the endocrine lineage. Downregulation of YAP, an effector of the pathway, enhances endocrine progenitor differentiation and the generation of SC-β cells with improved insulin secretion. A chemical inhibitor of YAP acts as an inducer of endocrine differentiation and reduces the presence of proliferative progenitor cells. Conversely, sustained activation of YAP results in impaired differentiation, blunted glucose-stimulated insulin secretion, and increased proliferation of SC-β cells. Together these results support a role for YAP in controlling the self-renewal and differentiation balance of pancreatic progenitors and limiting endocrine differentiation in vitro. Pluripotent stem cells can be directed into insulin-producing beta cells in vitro. Here, the authors show that downregulation of YAP, an effector of the Hippo pathway, enhances endocrine progenitor differentiation and the generation of beta-cells with improved insulin secretion.

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. Pancreatic β cells can be generated from pluripotent stem cells. Here, the authors show that human induced pluripotent stem cells from patients with type 1 diabetes can be differentiated into β-like cells that have no detectable differences compared with cells from non-diabetic individuals.

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

When encapsulated with alginate derivatives that resist the foreign-body response, human embryonic stem cell–derived beta cells restore long-term normoglycemia in immunocompetent mice without the need for immunosuppression. The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes 1 . Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically 2 , 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 3 . 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 4 . Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier 5 , 6 . However, clinical implementation has been challenging because of host immune responses to the implant materials 7 . 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.

The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet β-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of β-cell mass is through the generation and transplantation of new β-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous β-cell proliferation, reprogramming non-β-cells to β-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration.

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes, but its efficacy is hindered by low oxygen supply post-transplantation, particularly in subcutaneous spaces and encapsulation devices, leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here, we show that β cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes ( EGR1 , FOS , and JUN ), which downregulates key β cell transcription factors. We further identified genes important for maintaining β cell fitness in hypoxia, with EDN3 as a potent player. Elevated EDN3 expression preserves β cell identity and function in hypoxia by modulating genes involved in β cell maturation, glucose sensing and regulation. These insights improve the understanding of hypoxia’s impact on stem cell-derived islets, offering a potential intervention for clinical applications. Hypoxia impairs the efficacy of stem cell-derived islet cell therapy, making it a potential barrier for treatment of type 1 diabetes. Wang et al. identify EDN3 as a key factor that preserves β cell identity and function in hypoxia, offering possible strategies to improve therapeutic outcomes.

Cultured human pluripotent stem cells can be differentiated to immature pancreatic beta cells, but no one has yet succeeded in maturing these cells in vitro . Blum et al . define markers of beta-cell maturation that can be used to screen conditions for generating fully functional beta cells. Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro . We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.

Existing methods for reprogramming somatic cells to 'induced pluripotent stem cells' are inefficient, with only a small fraction of the starting cell population becoming pluripotent. Working with mouse embryonic fibroblasts, Hunagfu et al . increase reprogramming efficiency by treatment with DNA methyltransferase and histone deacetylase inhibitors. Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc .

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.