Hypoimmune induced pluripotent stem cells survive long term in fully immunocompetent, allogeneic rhesus macaques.

Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M-/-CIITA-/-CD47+). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.

Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.

Insulin-producing ß cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between ß cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes.

The generation of islet-like endocrine clusters from human pluripotent stem cells (hPSCs) has the potential to provide an unlimited source of insulin-producing ß 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.

Generation of insulin-producing pancreatic ß cells from multiple human stem cell lines.

We detail a six-stage planar differentiation methodology for generating human pluripotent stem cell-derived pancreatic ß cells (SC-ß 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-ß-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-ß 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-ß 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 ß-like cells. In this iteration, we have eliminated the need for 3D culture during endocrine induction, allowing for the generation of highly functional SC-ß 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-ß-cell generation, we demonstrate that this planar methodology is amenable for differentiating SC-ß cells from a wide variety of cell lines from various sources, broadening its applicability.

SIX2 Regulates Human ß Cell Differentiation from Stem Cells and Functional Maturation In Vitro.

Generation of insulin-secreting ß cells in vitro is a promising approach for diabetes cell therapy. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are differentiated to ß cells (SC-ß cells) and mature to undergo glucose-stimulated insulin secretion, but molecular regulation of this defining ß cell phenotype is unknown. Here, we show that maturation of SC-ß cells is regulated by the transcription factor SIX2. Knockdown (KD) or knockout (KO) of SIX2 in SC-ß cells drastically limits glucose-stimulated insulin secretion in both static and dynamic assays, along with the upstream processes of cytoplasmic calcium flux and mitochondrial respiration. Furthermore, SIX2 regulates the expression of genes associated with these key ß cell processes, and its expression is restricted to endocrine cells. Our results demonstrate that expression of SIX2 influences the generation of human SC-ß cells in vitro.

Gene-edited human stem cell-derived ß cells from a patient with monogenic diabetes reverse preexisting diabetes in mice.

Differentiation of insulin-producing pancreatic ß cells from induced pluripotent stem cells (iPSCs) derived from patients with diabetes promises to provide autologous cells for diabetes cell replacement therapy. However, current approaches produce patient iPSC-derived ß (SC-ß) cells with poor function in vitro and in vivo. Here, we used CRISPR-Cas9 to correct a diabetes-causing pathogenic variant in Wolfram syndrome 1 (WFS1) in iPSCs derived from a patient with Wolfram syndrome (WS). After differentiation to ß cells with our recent six-stage differentiation strategy, corrected WS SC-ß cells performed robust dynamic insulin secretion in vitro in response to glucose and reversed preexisting streptozocin-induced diabetes after transplantation into mice. Single-cell transcriptomics showed that corrected SC-ß cells displayed increased insulin and decreased expression of genes associated with endoplasmic reticulum stress. CRISPR-Cas9 correction of a diabetes-inducing gene variant thus allows for robust differentiation of autologous SC-ß cells that can reverse severe diabetes in an animal model.

Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells.

Generation of pancreatic ß 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 ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß 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.

Acquisition of Dynamic Function in Human Stem Cell-Derived ß Cells.

Recent advances in human pluripotent stem cell (hPSC) differentiation protocols have generated insulin-producing cells resembling pancreatic ß cells. While these stem cell-derived ß (SC-ß) 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 ß (TGF-ß) signaling, controlling cellular cluster size, and using an enriched serum-free media to generate SC-ß cells that express ß 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-ß signaling are required during SC-ß 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.

Independent control of matrix adhesiveness and stiffness within a 3D self-assembling peptide hydrogel.

A cell's insoluble microenvironment has increasingly been shown to exert influence on its function. In particular, matrix stiffness and adhesiveness strongly impact behaviors such as cell spreading and differentiation, but materials that allow for independent control of these parameters within a fibrous, stromal-like microenvironment are very limited. In the current work, we devise a self-assembling peptide (SAP) system that facilitates user-friendly control of matrix stiffness and RGD (Arg-Gly-Asp) concentration within a hydrogel possessing a microarchitecture similar to stromal extracellular matrix. In this system, the RGD-modified SAP sequence KFE-RGD and the scrambled sequence KFE-RDG can be directly swapped for one another to change RGD concentration at a given matrix stiffness and total peptide concentration. Stiffness is controlled by altering total peptide concentration, and the unmodified base peptide KFE-8 can be included to further increase this stiffness range due to its higher modulus. With this tunable system, we demonstrate that human mesenchymal stem cell morphology and differentiation are influenced by both gel stiffness and the presence of functional cell binding sites in 3D culture. Specifically, cells 24 hours after encapsulation were only able to spread out in stiffer matrices containing KFE-RGD. Upon addition of soluble adipogenic factors, soft gels facilitated the greatest adipogenesis as determined by the presence of lipid vacuoles and PPARγ-2 expression, while increasing KFE-RGD concentration at a given stiffness had a negative effect on adipogenesis. This three-component hydrogel system thus allows for systematic investigation of matrix stiffness and RGD concentration on cell behavior within a fibrous, three-dimensional matrix. STATEMENT OF SIGNIFICANCE: Physical cues from a cell's surrounding environment-such as the density of cell binding sites and the stiffness of the surrounding material-are increasingly being recognized as key regulators of cell function. Currently, most synthetic biomaterials used to independently tune these parameters lack the fibrous structure characteristic of stromal extracellular matrix, which can be important to cells naturally residing within stromal tissues. In this manuscript, we describe a 3D hydrogel encapsulation system that provides user-friendly control over matrix stiffness and binding site concentration within the context of a stromal-like microarchitecture. Binding site concentration and gel stiffness both influenced cell spreading and differentiation, highlighting the utility of this system to study the independent effects of these material properties on cell function.

Biomaterial microarchitecture: a potent regulator of individual cell behavior and multicellular organization.

Insoluble cues from a cell's surrounding microenvironment have increasingly been shown to be important regulators of cell behavior. The microarchitecture of biomaterials used for 3D cell encapsulation, however, is often underappreciated as an important insoluble factor guiding cell activity. In this review, we illustrate that the subcellular physical features of a scaffold influence a range of cell behaviors, including morphology, cytoskeletal organization, migration, matrix remodeling, and long-range force transmission. We emphasize that the microarchitecture of stromal extracellular matrix (ECM)-specifically the fact that it consists of a network of long interconnecting fibers with micron and nanometer-sized diameters-is an important determinant of how cells naturally interact with their surrounding matrix and each other. Synthetic biomaterials with a microarchitecture similar to stromal ECM can support analogous cellular responses, suggesting that this fibrous microarchitecture is a key regulator of these cell behaviors. Drawing upon examples from in vitro, in silico, and in vivo studies, we compare these behaviors in fibrous matrices to those of cells cultured within nanoporous matrices (e.g., alginate and PEG gels) as well as macroporous scaffolds to highlight key differences in the cellular response to each type of microarchitecture. Understanding how microarchitecture affects cell behavior can lead to more efficient biomaterial selection when designing tissue engineered scaffolds for therapeutic applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 640-661, 2017.

The effect of RGD peptide on 2D and miniaturized 3D culture of HEPM cells, MSCs, and ADSCs with alginate hydrogel.

Advancements in tissue engineering require the development of new technologies to study cell behavior in vitro. This study focuses on stem cell behavior within various miniaturized three-dimensional (3D) culture conditions of alginate biomaterials modified with the Arg-Gly-Asp (RGD) peptide known for its role in cell adhesion/attachment. Human embryonic palatal mesenchyme (HEPM) cells, bone marrow derived mesenchymal stem cells (MSCs), and human adipose derived stem cells (ADSCs) were cultured on a flat hydrogel of different concentrations of alginate-RGD, and in the miniaturized 3D core of microcapsules with either a 2% alginate or 2% alginate-RGD shell. The core was made of 0%, 0.5%, or 2% alginate-RGD. Cell spreading was observed in all systems containing the RGD peptide, and the cell morphology was quantified by measuring the cell surface area and circularity. In all types of stem cells, there was a significant increase in the cell surface area (p < 0.05) and a significant decrease in cell circularity (p < 0.01) in alginate-RGD conditions, indicating that cells spread much more readily in environments containing the peptide. This control over the cell spreading within a 3D microenvironment can help to create the ideal biomimetic condition in which to conduct further studies on cell behavior.

Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.

Much is unknown about the effects of culture dimensionality on cell behavior due to the lack of biomimetic substrates that are suitable for directly comparing cells grown on two-dimensional (2D) and encapsulated within three-dimensional (3D) matrices of the same stiffness and biochemistry. To overcome this limitation, we used a self-assembling peptide hydrogel system that has tunable stiffness and cell-binding site density as well as a fibrous microarchitecture resembling the structure of collagen. We investigated the effect of culture dimensionality on human mesenchymal stem cell differentiation at different values of matrix stiffness (G' = 0.25, 1.25, 5, and 10 kPa) and a constant RGD (Arg-Gly-Asp) binding site concentration. In the presence of the same soluble induction factors, culture on top of stiff gels facilitated the most efficient osteogenesis, while encapsulation within the same stiff gels resulted in a switch to predominantly terminal chondrogenesis. Adipogenesis dominated at soft conditions, and 3D culture induced better adipogenic differentiation than 2D culture at a given stiffness. Interestingly, initial matrix-induced cell morphology was predictive of these end phenotypes. Furthermore, optimal culture conditions corresponded to each cell type's natural niche within the body, highlighting the importance of incorporating native matrix dimensionality and stiffness into tissue engineering strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2356-2368, 2016.