RESUMEN
Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting ß-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-ß-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.
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Diabetes Mellitus/patología , Diabetes Mellitus/terapia , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/patología , Animales , Biomarcadores/análisis , Linaje de la Célula/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Diabetes Mellitus/inmunología , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Femenino , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/trasplante , Glucosa/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Masculino , Ratones , Especificidad de Órganos/efectos de los fármacos , Polipéptido Pancreático/metabolismo , Células Secretoras de Polipéptido Pancreático/citología , Células Secretoras de Polipéptido Pancreático/efectos de los fármacos , Células Secretoras de Polipéptido Pancreático/metabolismo , Proteómica , Análisis de Secuencia de ARN , Transactivadores/genética , Transactivadores/metabolismo , Transcriptoma , Transducción GenéticaRESUMEN
The generation of insulin-producing cells from human-induced pluripotent stem cells holds great potential for diabetes modeling and treatment. However, existing protocols typically involve incubating cells with un-physiologically high concentrations of glucose, which often fail to generate fully functional IPCs. Here, we investigated the influence of high (20 mM) versus low (5.5 mM) glucose concentrations on IPCs differentiation in three hiPSC lines. In two hiPSC lines that were unable to differentiate to IPCs sufficiently, we found that high glucose during differentiation leads to a shortage of NKX6.1+ cells that have co-expression with PDX1 due to insufficient NKX6.1 gene activation, thus further reducing differentiation efficiency. Furthermore, high glucose during differentiation weakened mitochondrial respiration ability. In the third iPSC line, which is IPC differentiation amenable, glucose concentrations did not affect the PDX1/NKX6.1 expression and differentiation efficiency. In addition, glucose-stimulated insulin secretion was only seen in the differentiation under a high glucose condition. These IPCs have higher KATP channel activity and were linked to sufficient ABCC8 gene expression under a high glucose condition. These data suggest high glucose concentration during IPC differentiation is necessary to generate functional IPCs. However, in cell lines that were IPC differentiation unamenable, high glucose could worsen the situation.
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Células Madre Pluripotentes Inducidas , Células Secretoras de Insulina , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Insulina/metabolismo , Diferenciación Celular , Glucosa/farmacología , Glucosa/metabolismoRESUMEN
Diabetes is a metabolic disease that currently affects nearly half a billion people worldwide. ß-cells dysfunction is one of the main causes of diabetes. Exposure to endocrine-disrupting chemicals is correlated with increased diabetes incidence. We hypothesized that treatment with bisphenol A (BPA) induces endoplasmic reticulum (ER) stress that activates the unfolded protein response (UPR), leading to impaired function of the ß-cells, which over time, can cause diabetes. In this study, we aimed to evaluate UPR pathways activation under BPA treatment in ß-cells and possible recovery of ER homeostasis. MIN6 cells (mouse insulinoma cell line) and isolated pancreatic islets from NOR (non-obese diabetes resistant) mice were treated with BPA. We analyzed the impact of BPA on ß-cell viability, the architecture of the early secretory pathway, the synthesis and processing of insulin and the activation of UPR sensors and effectors. We found that the addition of the chemical chaperone TUDCA rescues the deleterious effects of BPA, resulting in improved viability, morphology and function of the ß-cells. In conclusion, we propose that modulators of UPR can be used as therapeutic interventions targeted towards regaining ß-cells homeostasis.
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Diabetes Mellitus , Disruptores Endocrinos , Células Secretoras de Insulina , Animales , Ratones , Disruptores Endocrinos/farmacología , Respuesta de Proteína Desplegada , Estrés del Retículo Endoplásmico , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos NODRESUMEN
To date, most attention on tissue regeneration has focused on the exploration of positive cues promoting or allowing the engagement of natural cellular restoration upon injury. In contrast, the signals fostering cell identity maintenance in the vertebrate body have been poorly investigated; yet they are crucial, for their counteraction could become a powerful method to induce and modulate regeneration. Here we review the mechanisms inhibiting pro-regenerative spontaneous adaptive cell responses in different model organisms and organs. The pharmacological or genetic/epigenetic modulation of such regenerative brakes could release a dormant but innate adaptive competence of certain cell types and therefore boost tissue regeneration in different situations.
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Medicina Regenerativa/métodos , Cicatrización de Heridas/fisiología , HumanosRESUMEN
The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing ß-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature.
Asunto(s)
Diferenciación Celular , Islotes Pancreáticos/metabolismo , Proteoma , Metabolismo Energético , Glucosa , Humanos , Células Madre Pluripotentes Inducidas , Islotes Pancreáticos/citología , Proteómica , Vía de Señalización WntRESUMEN
Total or near-total loss of insulin-producing ß-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which ß-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-ß-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on ß-cell reconstitution from heterologous islet cells after near-total ß-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after ß-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although ß-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-ß-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.
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Envejecimiento/fisiología , Transdiferenciación Celular , Diabetes Mellitus Experimental/patología , Células Secretoras de Insulina/citología , Insulina/biosíntesis , Regeneración , Células Secretoras de Somatostatina/citología , Animales , Desdiferenciación Celular , Proliferación Celular , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Maduración Sexual , Somatostatina/biosíntesis , Somatostatina/metabolismo , Células Secretoras de Somatostatina/metabolismoRESUMEN
Differentiation of human induced pluripotent stem cells towards pancreatic islet endocrine cells is a complex process, involving the stepwise modulation of key developmental pathways, such as the Hedgehog signaling inhibition during early differentiation stages. In tandem with this active inhibition, key transcription factors for the islet endocrine cell fate, such as HNF1A, show specific changes in their expression patterns. Here we designed a pilot study aimed at investigating the potential interconnection between HH-signaling inhibition and the increase in the HNF1A expression during early regeneration, by inducing changes in the GLI code. This unveiled a link between the two, where GLI3-R mediated Hedgehog target genes inhibition is apparently required for HNF1A efficient expression.
RESUMEN
Animal longevity is a function of global vital organ functionality and, consequently, a complex polygenic trait. Yet, monogenic regulators controlling overall or organ-specific ageing exist, owing their conservation to their function in growth and development. Here, by using pathway analysis combined with wet-biology methods on several dynamic timelines, we identified Hnf1a as a novel master regulator of the maturation and ageing in the adult pancreatic islet during the first year of life. Conditional transgenic mice bearing suboptimal levels of this transcription factor in the pancreatic islets displayed age-dependent changes, with a profile echoing precocious maturation. Additionally, the comparative pathway analysis revealed a link between Hnf1a age-dependent regulation and immune signaling, which was confirmed in the ageing timeline of an overly immunodeficient mouse model. Last, the global proteome analysis of human islets spanning three decades of life largely backed the age-specific regulation observed in mice. Collectively, our results suggest a novel role of Hnf1a as a monogenic regulator of the maturation and ageing process in the pancreatic islet via a direct or indirect regulatory loop with immune signaling.
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Envejecimiento , Factor Nuclear 1-alfa del Hepatocito , Islotes Pancreáticos , Transducción de Señal , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Animales , Islotes Pancreáticos/metabolismo , Ratones , Humanos , Transducción de Señal/fisiología , Envejecimiento/metabolismo , Envejecimiento/fisiología , Ratones TransgénicosRESUMEN
Regeneration, the ability to replace injured tissues and organs, is a phenomenon commonly associated with lower vertebrates but is also observed in mammals, in specific tissues. In this study, we investigated the regenerative potential of pancreatic islets following moderate beta-cell loss in mice. Using a rapid model of moderate ablation, we observed a compensatory response characterized by transient inflammation and proliferation signatures, ultimately leading to the recovery of beta-cell identity and function. Interestingly, this proliferative response occurred independently of inflammation, as demonstrated in ablated immunodeficient mice. Furthermore, exposure to high-fat diet stimulated beta-cell proliferation but negatively impacted beta-cell function. In contrast, an equivalent slower ablation model revealed a delayed but similar proliferative response, suggesting proliferation as a common regenerative response. However, high-fat diet failed to promote proliferation in this model, indicating a differential response to metabolic stressors. Overall, our findings shed light on the complex interplay between beta-cell loss, inflammation, and stress in modulating pancreatic islet regeneration. Understanding these mechanisms could pave the way for novel therapeutic strategies based on beta-cell proliferation.
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Proliferación Celular , Dieta Alta en Grasa , Células Secretoras de Insulina , Regeneración , Animales , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Ratones , Dieta Alta en Grasa/efectos adversos , Masculino , Ratones Endogámicos C57BL , Inflamación/metabolismo , Inflamación/patologíaRESUMEN
Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.
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Factor de Transcripción Activador 3 , Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Ratones Endogámicos NOD , Transducción de Señal , Animales , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 3/genética , Ratones , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Perfilación de la Expresión Génica , Modelos Animales de EnfermedadRESUMEN
Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.
RESUMEN
AIM: The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples. METHODS: By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis. RESULTS: In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity. CONCLUSIONS: Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.
Asunto(s)
Insulina , Islotes Pancreáticos , Humanos , Insulina/metabolismo , Proteómica , Proteoma/metabolismo , Proyectos Piloto , Islotes Pancreáticos/metabolismo , HomeostasisRESUMEN
Pancreatic islet endocrine cells generated from patient-derived induced pluripotent stem cells represent a great strategy for both disease modeling and regenerative medicine. Nevertheless, these cells inherently miss the effects of the intricate network of systemic signals characterizing the living organisms. Xenotransplantation of in vitro differentiating cells into murine hosts substantially compensates for this drawback.Here we describe our transplantation strategy of encapsulated differentiating pancreatic progenitors into diabetic immunosuppressed (NSG) overtly diabetic mice generated by the total ablation of insulin-producing cells following diphtheria toxin administration. We will detail the differentiation protocol employed, the alginate encapsulation procedure, and the xenotransplantation steps required for a successful and reproducible experiment.
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Diabetes Mellitus Experimental , Células Madre Pluripotentes Inducidas , Células Secretoras de Insulina , Animales , Diferenciación Celular , Diabetes Mellitus Experimental/terapia , Humanos , Insulina , Ratones , PáncreasRESUMEN
Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet ß-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.
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Diabetes Mellitus Experimental , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Diabetes Mellitus Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Inmunología del TrasplanteRESUMEN
Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic ß-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic ß-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.
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Diabetes Mellitus Tipo 2 , Células Madre Pluripotentes Inducidas , Alginatos/metabolismo , Cápsulas/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Mutación , ProteomaRESUMEN
After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.
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Apoptosis/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hydra/metabolismo , Hydra/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Hydra/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/genéticaRESUMEN
New perspectives on the origin of neurogenesis emerged with the identification of genes encoding post-synaptic proteins as well as many "neurogenic" regulators as the NK, Six, Pax, bHLH proteins in the Demosponge genome, a species that might differentiate sensory cells but no neurons. However, poriferans seem to miss some key regulators of the neurogenic circuitry as the Hox/paraHox and Otx-like gene families. Moreover as a general feature, many gene families encoding evolutionarily-conserved signaling proteins and transcription factors were submitted to a wave of gene duplication in the last common eumetazoan ancestor, after Porifera divergence. In contrast gene duplications in the last common bilaterian ancestor, Urbilateria, are limited, except for the bHLH Atonal-class. Hence Cnidaria share with Bilateria a large number of genetic tools. The expression and functional analyses currently available suggest a neurogenic function for numerous orthologs in developing or adult cnidarians where neurogenesis takes place continuously. As an example, in the Hydra polyp, the Clytia medusa and the Acropora coral, the Gsx/cnox2/Anthox-2 ParaHox gene likely supports neurogenesis. Also neurons and nematocytes (mechanosensory cells) share in hydrozoans a common stem cell and several regulatory genes indicating that they can be considered as sister cells. Performed in anthozoan and medusozoan species, these studies should tell us more about the way(s) evolution hazards achieved the transition from epithelial to neuronal cell fate, and about the robustness of the genetic circuitry that allowed neuromuscular transmission to arise and be maintained across evolution.
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Evolución Biológica , Cnidarios/crecimiento & desarrollo , Cnidarios/genética , Neurogénesis , Animales , Cnidarios/anatomía & histología , Sistema Nervioso/anatomía & histología , Sistema Nervioso/crecimiento & desarrollo , Neurogénesis/genéticaRESUMEN
The Hydra polyp provides a powerful model system to investigate the regulation of cell survival and cell death in homeostasis and regeneration as Hydra survive weeks without feeding and regenerates any missing part after bisection. Induction of autophagy during starvation is the main surviving strategy in Hydra as autophagic vacuoles form in most myoepithelial cells after several days. When the autophagic process is inhibited, animal survival is actually rapidly jeopardized. An appropriate regulation of autophagy is also essential during regeneration as Hydra RNAi knocked-down for the serine protease inhibitor Kazal-type (SPINK) gene Kazal1, exhibit a massive autophagy after amputation that rapidly compromises cell and animal survival. This excessive autophagy phenotype actually mimics that observed in the mammalian pancreas when SPINK genes are mutated, highlighting the paradigmatic value of the Hydra model system for deciphering pathological processes. Interestingly autophagy during starvation predominantly affects ectodermal epithelial cells and lead to cell survival whereas Kazal1(RNAi)-induced autophagy is restricted to endodermal digestive cells that rapidly undergo cell death. This indicates that distinct regulations that remain to be identified, are at work in these two contexts. Cnidarian express orthologs for most components of the autophagy and TOR pathways suggesting evolutionarily-conserved roles during starvation.
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Autofagia , Evolución Biológica , Hydra/citología , Hydra/fisiología , Estrés Fisiológico , Animales , Supervivencia Celular , Hydra/genética , Transducción de SeñalRESUMEN
Over the past decades, genetic analyses performed in vertebrate and invertebrate organisms deciphered numerous cellular and molecular mechanisms deployed during sexual development and identified genetic circuitries largely shared among bilaterians. In contrast, the functional analysis of the mechanisms that support regenerative processes in species randomly scattered among the animal kingdom, were limited by the lack of genetic tools. Consequently, unifying principles explaining how stress and injury can lead to the reactivation of a complete developmental program with restoration of original shape and function remained beyond reach of understanding. Recent data on cell plasticity suggest that beside the classical developmental approach, the analysis of homeostasis and asexual reproduction in adult organisms provides novel entry points to dissect the regenerative potential of a given species, a given organ or a given tissue. As a clue, both tissue homeostasis and regeneration dynamics rely on the availability of stem cells and/or on the plasticity of differentiated cells to replenish the missing structure. The freshwater Hydra polyp provides us with a unique model system to study the intricate relationships between the mechanisms that regulate the maintenance of homeostasis, even in extreme conditions (starvation and overfeeding) and the reactivation of developmental programs after bisection or during budding. Interestingly head regeneration in Hydra can follow several routes according to the level of amputation, suggesting that indeed the homeostatic background dramatically influences the route taken to bridge injury and regeneration.
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Fenómenos Fisiológicos Celulares , Biología Evolutiva , Homeostasis , Modelos Biológicos , Regeneración , Animales , Hydra , Estadios del Ciclo de VidaRESUMEN
AIM: The loss of insulin-secreting ß-cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. METHODS: To rigorously study the effect of hyperglycaemia, in vitro differentiated human-induced pluripotent stem cells (hiPSC)-derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)-diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1-week or 1-month exposure to overt hyperglycaemia and analysed by large-scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP-DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute ß-cell ablation property in the absence of inflammation or other organ toxicity. RESULTS: Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. CONCLUSION: Hyperglycaemia exposure induced distinct, period-dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival.