Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus.

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.

Lung viral infection modelling in a bioengineered whole-organ.

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

An In Vitro Whole-Organ Liver Engineering for Testing of Genetic Therapies.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells.

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.

Loss of prohibitin induces mitochondrial damages altering ß-cell function and survival and is responsible for gradual diabetes development.

Prohibitins are highly conserved proteins mainly implicated in the maintenance of mitochondrial function and architecture. Their dysfunctions are associated with aging, cancer, obesity, and inflammation. However, their possible role in pancreatic ß-cells remains unknown. The current study documents the expression of prohibitins in human and rodent islets and their key role for ß-cell function and survival. Ablation of Phb2 in mouse ß-cells sequentially resulted in impairment of mitochondrial function and insulin secretion, loss of ß-cells, progressive alteration of glucose homeostasis, and, ultimately, severe diabetes. Remarkably, these events progressed over a 3-week period of time after weaning. Defective insulin supply in ß-Phb2(-/-) mice was contributed by both ß-cell dysfunction and apoptosis, temporarily compensated by increased ß-cell proliferation. At the molecular level, we observed that deletion of Phb2 caused mitochondrial abnormalities, including reduction of mitochondrial DNA copy number and respiratory chain complex IV levels, altered mitochondrial activity, cleavage of L-optic atrophy 1, and mitochondrial fragmentation. Overall, our data demonstrate that Phb2 is essential for metabolic activation of mitochondria and, as a consequence, for function and survival of ß-cells.

Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

In vivo conditional Pax4 overexpression in mature islet ß-cells prevents stress-induced hyperglycemia in mice.

OBJECTIVE: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in ß-cells. RESEARCH DESIGN AND METHODS: Glucose homeostasis and ß-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and ß-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated ß-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1ß transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of ß-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

The Lou/C rat: a model of spontaneous food restriction associated with improved insulin sensitivity and decreased lipid storage in adipose tissue.

The inbred Lou/C rat, originating from the Wistar strain, has been described as a model of resistance to diet-induced obesity, but little is known about its metabolism. Since this knowledge could provide some clues about the etiology of obesity/insulin resistance, this study aimed at characterizing glucose and lipid metabolism in Lou/C vs. Wistar rats. This was achieved by performing glucose and insulin tolerance tests, euglycemic hyperinsulinemic clamps, and characterization of intracellular insulin signaling in skeletal muscle. Substrate-induced insulin secretion was evaluated using perfused pancreas and isolated islets. Finally, body fat composition and the expression of various factors involved in lipid metabolism were determined. Body weight and caloric intake were lower in Lou/C than in Wistar rats, whereas food efficiency was similar. Improved glucose tolerance of Lou/C rats was not related to increased insulin output but was related to improved insulin sensitivity/responsiveness in the liver and in skeletal muscles. In the latter tissue, this was accompanied by improved insulin signaling, as suggested by higher activation of the insulin receptor and of the Akt/protein kinase B pathway. Fat deposition was markedly lower in Lou/C than in Wistar rats, especially in visceral adipose tissue. In the inguinal adipose depot, expression of uncoupling protein-1 was detected in Lou/C but not in Wistar rats, in keeping with a higher expression of peroxisome proliferator-activated receptor-gamma coactivator-1 in these animals. The Lou/C rat is a valuable model of spontaneous food restriction with associated improved insulin sensitivity. Independently from its reduced caloric intake, it also exhibits a preferential channeling of nutrients toward utilization rather than storage.

Deletion of glutamate dehydrogenase in beta-cells abolishes part of the insulin secretory response not required for glucose homeostasis.

Insulin exocytosis is regulated in pancreatic ss-cells by a cascade of intracellular signals translating glucose levels into corresponding secretory responses. The mitochondrial enzyme glutamate dehydrogenase (GDH) is regarded as a major player in this process, although its abrogation has not been tested yet in animal models. Here, we generated transgenic mice, named betaGlud1(-/-), with ss-cell-specific GDH deletion. Our results show that GDH plays an essential role in the full development of the insulin secretory response. In situ pancreatic perfusion revealed that glucose-stimulated insulin secretion was reduced by 37% in betaGlud1(-/-). Furthermore, isolated islets with either constitutive or acute adenovirus-mediated knock-out of GDH showed a 49 and 38% reduction in glucose-induced insulin release, respectively. Adenovirus-mediated re-expression of GDH in betaGlud1(-/-) islets fully restored glucose-induced insulin release. Thus, GDH appears to account for about 40% of glucose-stimulated insulin secretion and to lack redundant mechanisms. In betaGlud1(-/-) mice, the reduced secretory capacity resulted in lower plasma insulin levels in response to both feeding and glucose load, while body weight gain was preserved. The results demonstrate that GDH is essential for the full development of the secretory response in beta-cells. However, maximal secretory capacity is not required for maintenance of glucose homeostasis in normo-caloric conditions.

The Fas pathway is involved in pancreatic beta cell secretory function.

Pancreatic beta cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in beta cell apoptosis or proliferation, depending on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates beta cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a beta cell-specific transcription factor regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient beta cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased beta cell mass. Up-regulation of FLIP enhanced NF-kappaB activity via NF-kappaB-inducing kinase and RelB. This led to increased PDX-1 and insulin production independent of changes in cell turnover. The results support a previously undescribed role for the Fas pathway in regulating insulin production and release.

Beta-cell secretory products activate alpha-cell ATP-dependent potassium channels to inhibit glucagon release.

Glucagon, secreted from islet alpha-cells, mobilizes liver glucose. During hyperglycemia, glucagon secretion is inhibited by paracrine factors from other islet cells, but in type 1 and type 2 diabetic patients, this suppression is lost. We investigated the effects of beta-cell secretory products zinc and insulin on isolated rat alpha-cells, intact islets, and perfused pancreata. Islet glucagon secretion was markedly zinc sensitive (IC(50) = 2.7 micromol/l) more than insulin release (IC(50) = 10.7 micromol/l). Glucose, the mitochondrial substrate pyruvate, and the ATP-sensitive K(+) channel (K(ATP) channel) inhibitor tolbutamide stimulated isolated alpha-cell electrical activity and glucagon secretion. Zinc opened K(ATP) channels and inhibited both electrical activity and pyruvate (but not arginine)-stimulated glucagon secretion in alpha-cells. Insulin transiently increased K(ATP) channel activity, inhibited electrical activity and glucagon secretion in alpha-cells, and inhibited pancreatic glucagon output. Insulin receptor and K(ATP) channel subunit transcripts were more abundant in alpha- than beta-cells. Transcript for the glucagon-like peptide 1 (GLP-1) receptor was not detected in alpha-cells nor did GLP-1 stimulate alpha-cell glucagon release. beta-Cell secretory products zinc and insulin therefore inhibit glucagon secretion most probably by direct activation of K(ATP) channels, thereby masking an alpha-cell metabolism secretion coupling pathway similar to beta-cells.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from beta-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from alpha-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet alpha- and beta-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although alpha-cells, like beta-cells, possess the inherent capacity to respond to nutrients, secretion from alpha-cells is normally suppressed by the simultaneous activation of beta-cells. Zinc released from beta-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.

Discrimination between signaling pathways in regulation of specific gene expression by insulin and growth hormone in hepatocytes.

Insulin and GH can activate common signaling elements in many tissues and cell lines. We investigated the possibility of overlap in signaling pathways activated by insulin and GH in a key target cell, the hepatocyte. In primary cultures of rat hepatocytes, GH caused a dose- and time-dependent increase in tyrosine phosphorylation of signal transducer and activator of transcription 5. This was accompanied by the induction of the mRNA encoding suppressor of cytokine signaling 2. Neither of these effects took place in companion hepatocytes challenged with insulin. By contrast, insulin caused a rapid and sustained phosphorylation of protein kinase B, accompanied by a massive induction of the mRNA encoding glucokinase. GH had no detectable effect on phosphorylation of protein kinase B or level of glucokinase mRNA. Insulin also elicited brief hyperphosphorylation of ERK1 and 2, an effect not seen in GH-stimulated hepatocytes. Thus, there was a clear demarcation of signaling events triggered in hepatocytes by insulin and GH, and this was accompanied by hormone-specific responses with respect to the induction of gene expression. Additionally, the current results show that signal transducer and activator of transcription 5 activation is neither necessary nor sufficient for the insulin-dependent induction of hepatic glucokinase.