Enrichment of stem cell-derived pancreatic beta-like cells and controlled graft size through pharmacological removal of proliferating cells.

Transplantation of limited human cadaveric islets into type 1 diabetic patients results in ∼35 months of insulin independence. Direct differentiation of stem cell-derived insulin-producing beta-like cells (sBCs) that can reverse diabetes in animal models effectively removes this shortage constraint, but uncontrolled graft growth remains a concern. Current protocols do not generate pure sBCs, but consist of only 20%-50% insulin-expressing cells with additional cell types present, some of which are proliferative. Here, we show the selective ablation of proliferative cells marked by SOX9 by simple pharmacological treatment in vitro. This treatment concomitantly enriches for sBCs by ∼1.7-fold. Treated sBC clusters show improved function in vitro and in vivo transplantation controls graft size. Overall, our study provides a convenient and effective approach to enrich for sBCs while minimizing the presence of unwanted proliferative cells and thus has important implications for current cell therapy approaches.

Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

Generation of functional thymic organoids from human pluripotent stem cells.

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system but involutes with age, resulting in reduced naive T cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) only form months after transplantation of TEPs in vivo. We show the generation of TECs in vitro in isogenic stem cell-derived thymic organoids (sTOs) consisting of TEPs, hematopoietic progenitor cells, and mesenchymal cells, differentiated from the same hPSC line. sTOs support T cell development, express key markers of negative selection, including the autoimmune regulator (AIRE) protein, and facilitate regulatory T cell development. sTOs provide the basis for functional patient-specific thymic organoid models, allowing for the study of human thymus function, T cell development, and transplant immunity.

Cell Replacement Therapy for Type 1 Diabetes Patients: Potential Mechanisms Leading to Stem-Cell-Derived Pancreatic ß-Cell Loss upon Transplant.

Cell replacement therapy using stem-cell-derived insulin-producing ß-like cells (sBCs) has been proposed as a practical cure for patients with type one diabetes (T1D). sBCs can correct diabetes in preclinical animal models, demonstrating the promise of this stem cell-based approach. However, in vivo studies have demonstrated that most sBCs, similarly to cadaveric human islets, are lost upon transplantation due to ischemia and other unknown mechanisms. Hence, there is a critical knowledge gap in the current field concerning the fate of sBCs upon engraftment. Here we review, discuss effects, and propose additional potential mechanisms that could contribute toward ß-cell loss in vivo. We summarize and highlight some of the literature on phenotypic loss in ß-cells under both steady, stressed, and diseased diabetic conditions. Specifically, we focus on ß-cell death, dedifferentiation into progenitors, trans-differentiation into other hormone-expressing cells, and/or interconversion into less functional ß-cell subtypes as potential mechanisms. While current cell replacement therapy efforts employing sBCs carry great promise as an abundant cell source, addressing the somewhat neglected aspect of ß-cell loss in vivo will further accelerate sBC transplantation as a promising therapeutic modality that could significantly enhance the life quality of T1D patients.

Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

High-throughput identification of RNA localization elements in neuronal cells.

Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.

From the dish to humans: A stem cell recipe for success.

Islet transplantation has proven to be an effective treatment for type 1 diabetes (T1D) yet is hampered by the shortage of available tissue. Recently, two reports from a Viacyte multicenter clinical trial demonstrate the feasibility, safety, and potential efficacy of transplanting macro-encapsulated human stem cell-derived pancreatic endoderm cells into patients with T1D, highlighting the promise of a stem cell-based therapeutic approach.

Generation of functional human thymic cells from induced pluripotent stem cells.

BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition.

Type 1 diabetes results from an autoimmune attack directed at pancreatic beta cells predominantly mediated by T cells. Transplantation of stem cell derived beta-like cells (sBC) have been shown to rescue diabetes in preclinical animal models. However, how sBC will respond to an inflammatory environment with diabetogenic T cells in a strict human setting has not been determined. This is due to the lack of model systems that closely recapitulates human T1D. Here, we present a reliable in vitro assay to measure autologous CD8 T cell stimulation against sBC in a human setting. Our data shows that upon pro-inflammatory cytokine exposure, sBC upregulate Human Leukocyte Antigen (HLA) class I molecules which allows for their recognition by diabetogenic CD8 T cells. To protect sBC from this immune recognition, we utilized genome engineering to delete surface expression of HLA class I molecules and to integrate an inducible overexpression system for the immune checkpoint inhibitor Programmed Death Ligand 1 (PD-L1). Genetically engineered sBC that lack HLA surface expression or overexpress PD-L1 showed reduced stimulation of diabetogenic CD8 T cells when compared to unmodified cells. Here, we present evidence that manipulation of HLA class I and PD-L1 receptors on sBC can provide protection from diabetes-specific immune recognition in a human setting.

Strategies for durable ß cell replacement in type 1 diabetes.

Technological advancements in blood glucose monitoring and therapeutic insulin administration have improved the quality of life for people with type 1 diabetes. However, these efforts fall short of replicating the exquisite metabolic control provided by native islets. We examine the integrated advancements in islet cell replacement and immunomodulatory therapies that are coalescing to enable the restoration of endogenous glucose regulation. We highlight advances in stem cell biology and graft site design, which offer innovative sources of cellular material and improved engraftment. We also cover cutting-edge approaches for preventing allograft rejection and recurrent autoimmunity. These insights reflect a growing understanding of type 1 diabetes etiology, ß cell biology, and biomaterial design, together highlighting therapeutic opportunities to durably replace the ß cells destroyed in type 1 diabetes.

In vitro generation of peri-islet basement membrane-like structures.

The peri-islet extracellular matrix (ECM) is a key component of the microenvironmental niche surrounding pancreatic islets of Langerhans. The cell anchorage and signaling provided by the peri-islet ECM is critical for optimum beta cell glucose responsiveness, but islets lose this important native ECM when isolated for transplantation or in vitro studies. Here, we established a method to construct a peri-islet ECM on the surfaces of isolated rat and human islets by the co-assembly from solution of laminin, nidogen and collagen IV proteins. Successful deposition of contiguous peri-islet ECM networks was confirmed by immunofluorescence, western blot, and transmission electron microscopy. The ECM coatings were disrupted when assembly occurred in Ca2+/Mg2+-free conditions. As laminin network polymerization is divalent cation dependent, our data are consistent with receptor-driven ordered ECM network formation rather than passive protein adsorption. To further illustrate the utility of ECM coatings, we employed stem cell derived beta-like cell clusters (sBCs) as a renewable source of functional beta cells for cell replacement therapy. We observe that sBC pseudo-islets lack an endogenous peri-islet ECM, but successfully applied our approach to construct a de novo ECM coating on the surfaces of sBCs.

Amylin, Aß42, and Amyloid in Varicella Zoster Virus Vasculopathy Cerebrospinal Fluid and Infected Vascular Cells.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid. METHODS: Aß peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin's function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated. RESULTS: Compared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aß40 was reduced and Aß42 unchanged. Intracellular amylin, Aß42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA. CONCLUSIONS: VZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin.

Loss of the transcription factor MAFB limits ß-cell derivation from human PSCs.

Next generation sequencing studies have highlighted discrepancies in ß-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human ß-cells postnatally, while its expression is restricted to embryonic and neo-natal ß-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to ß-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.

Varicella-Zoster Virus Infection of Primary Human Spinal Astrocytes Produces Intracellular Amylin, Amyloid-ß, and an Amyloidogenic Extracellular Environment.

BACKGROUND: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid. METHODS: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-ß [Aß]) and amyloid, as well as extracellular amylin, Aß, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aß42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined. RESULTS: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aß, and amyloid. No differences in extracellular amylin, Aß40, or Aß42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aß42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aß42 aggregation. CONCLUSIONS: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.

Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived ß cells.

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional ß cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and ß cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature ß-like cells to form islet-sized enriched ß-clusters (eBCs). eBCs display physiological properties analogous to primary human ß cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell clustering induces metabolic maturation by driving mitochondrial oxidative respiration, a process central to stimulus-secretion coupling in mature ß cells. eBCs display glucose-stimulated insulin secretion as early as three days after transplantation in mice. In summary, replicating aspects of endocrine cell clustering permits the generation of stem-cell-derived ß cells that resemble their endogenous counterparts.

ß Cell replacement: improving on the design.

PURPOSE OF REVIEW: Here we summarize recent advancements in ß cell replacement as a therapy for type 1 diabetes. RECENT FINDINGS: ß cell replacement therapy has been proposed as a cure for type 1 diabetes with the introduction of the Edmonton protocol for cadaveric islet transplantation. To allow widespread use of this approach, efforts have focused on establishing an abundant source of insulin-producing ß cells, protecting transplanted cells from ischemia-mediated death, immune rejection, and re-occurring autoimmunity. Recent developments addressing these issues include generation of insulin-producing cells from human pluripotent stem cells, different encapsulation strategies and prevention of ischemia upon transplant. SUMMARY: Despite significant advances in generating functional ß cells from human pluripotent stem cells, several key challenges remain in regard to the survival of ß cell grafts, protection from (auto-) immune destruction and implementation of additional safety mechanisms before a stem cell-based cell replacement therapy approach can be widely applied. Taking current findings into consideration, we outline a multilayered approach to design immune-privileged ß cells from stem cells using state of the art genome editing technologies that if successfully incorporated could result in great benefit for diabetic patients and improve clinical results for cell replacement therapy.

Replication confers ß cell immaturity.

Pancreatic ß cells are highly specialized to regulate systemic glucose levels by secreting insulin. In adults, increase in ß-cell mass is limited due to brakes on cell replication. In contrast, proliferation is robust in neonatal ß cells that are functionally immature as defined by a lower set point for glucose-stimulated insulin secretion. Here we show that ß-cell proliferation and immaturity are linked by tuning expression of physiologically relevant, non-oncogenic levels of c-Myc. Adult ß cells induced to replicate adopt gene expression and metabolic profiles resembling those of immature neonatal ß that proliferate readily. We directly demonstrate that priming insulin-producing cells to enter the cell cycle promotes a functionally immature phenotype. We suggest that there exists a balance between mature functionality and the ability to expand, as the phenotypic state of the ß cell reverts to a less functional one in response to proliferative cues.

Nanoporous Immunoprotective Device for Stem-Cell-Derived ß-Cell Replacement Therapy.

Encapsulation of human embryonic stem-cell-differentiated beta cell clusters (hES-ßC) holds great promise for cell replacement therapy for the treatment of diabetics without the need for chronic systemic immune suppression. Here, we demonstrate a nanoporous immunoprotective polymer thin film cell encapsulation device that can exclude immune molecules while allowing exchange of oxygen and nutrients necessary for in vitro and in vivo stem cell viability and function. Biocompatibility studies show the device promotes neovascular formation with limited foreign body response in vivo. The device also successfully prevented teratoma escape into the peritoneal cavity of mice. Long-term animal studies demonstrate evidence of engraftment, viability, and function of cells encapsulated in the device after 6 months. Finally, in vivo study confirms that the device was able to effectively immuno-isolate cells from the host immune system.

Mitigating Ischemic Injury of Stem Cell-Derived Insulin-Producing Cells after Transplant.

The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC) has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant.