Étude du rôle du glucose dans l'ingénierie tissulaire médiée par les cellules souches mésenchymateuses

Les cellules souches mésenchymateuses (CSMs), aussi appelé cellules stromales multipotentes, ont été présenté comme des candidates prometteuses pour leurs applications possibles en médecine régénérative. Cependant, leur efficacité thérapeutique est remise en question étant donné leur faible taux de survie post implantation. Le microenvironnement ischémique, principalement caractérisé par une déprivation d'oxygène et de nutriments, est responsable de la mort massive et rapide des CSMs suite à leur implantation. Des études préalables au laboratoire B3OA ont mis en évidence que l'absence de glucose (et non le manque d'oxygène) était responsable de la mort des CSMs. L'hypothèse centrale de cette étude est que le glucose, un régulateur clé de l'activité des cellules souches, est essentiel pour que les CSM puissent survivre à leur implantation, être fonctionnelles in vivo et exprimer pleinement leur potentiel thérapeutique. L'objectif de cette thèse est double : (i) étudier, si et comment, le glucose affecte les fonctions paracrines pro-angiogènes des CSM ; et (ii) développer un hydrogel nutritif à base d'amidon, qui par dégradation enzymatique libère du glucose promouvant ainsi la survie et la fonctionnalité des CSM humaines (hCSM) après implantation. Pour notre premier objectif, nous avons constaté que le milieu conditionné obtenu à partir de hCSM cultivées en présence de glucose en quasi-anoxie (0,1 % d'O2) présentait un potentiel angiogénique significativement plus élevé que celui collecté à partir de hCSM témoins cultivées sans glucose. De façon intéressante, les CSMh cultivées en quasi-anoxie en l'absence de glucose ont présenté un niveau plus élevé de stress du réticulum endoplasmique (RE), par rapport aux hCSM cultivées dans les mêmes conditions en présence de glucose. Enfin, il s'est avéré que des hydrogels cellularisés contenant du glucose implantés en site ectopique murin induisaient une augmentation du volume des vaisseaux sanguins néoformés autour de l'implant plus importante que des hydrogels cellularisés ne contenant pas de glucose. Pour notre second objectif, nous avons développé un hydrogel à base de fibrine, d'amidon (un polymère de glucose) et d'amyloglucosidase (AMG, une enzyme permettant l'hydrolyse du glucose à partir d'amidon) afin de fournir du glucose aux hCSMs à une concentration physiologiquement pertinente. Nous avons observé une augmentation de la survie et des fonctions paracrines des hCSM contenues dans ces hydrogels fibrine/amidon/AMG par rapport aux hydrogels témoins à la fois in vitro (modèle de culture en quasi-anoxie sans glucose) et in vivo (modèle ectopique murin) . Cependant, la cinétique de délivrance à long terme du glucose et la dégradation incomplète de l'amidon pouvant créer un encombrement stérique empêchant la formation de néo-tissus sont des limitations potentielles de cet hydrogel qui nécessitent des recherches supplémentaires afin d'obtenir une formulation optimale de l'hydrogel pour son application en reconstruction tissulaire. En conclusion, ce travail contribue à démontrer l'effet positif d'un apport exogène de glucose sur la survie, les fonctions pro-angiogènes et la régulation du stress du réticulum endoplasmique des hCSM dans un site ischémié. Ce travail permet aussi d'établir la preuve de concept d'un hydrogel, à base de fibrine, d'amidon et d'AMG comme système de délivrance de glucose. Ces découvertes encouragent à employer des stratégies permettant l'apport de glucose afin d'augmenter l'efficacité thérapeutique des produits à base de hCSM utilisé en médecine régénérative.Mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, are promising candidates for tissue engineering and regenerative medicine applications, however, increasing evidence shows that the therapeutic efficacy of MSCs has fallen short of their initial promise and hype due to the observed poor MSC survival and engraftment postimplantation. The hostile ischemic microenvironment, mainly characterized by deprivation of oxygen and nutrients, is responsible for the massive and rapid MSC death upon implantation. Previous studies from the B3OA lab established that the absence of glucose (but not the lack of oxygen), is the culprit for this MSC death. The central hypothesis of this work is that glucose metabolism, a central regulator of stem cell activity, is instrumental for MSCs to enhance their survival and functionalities upon transplantation, and ultimately improve their therapeutic efficacy. As a step towards the engineering of a glucose supplying construct, we hereby propose to investigate whether and how glucose affects MSC paracrine functions pertinent to angiogenesis, and then to develop an enzyme-controlled, nutritive hydrogel with an inbuilt system of glucose delivery to improve MSC survival and functionalities postimplantation. For our first aim, we found that conditioned media (CM) collected from hMSCs cultured in the presence of glucose under near-anoxia (0.1% O2) exhibited significantly higher angiogenic potential when compared to the one collected from hMSCs cultured without glucose. In addition, hMSCs cultured in the absence of glucose under near-anoxia exhibited a higher level of endoplasmic reticulum (ER) stress evidenced by less nascent protein biosynthesis, blockage of protein secretion, and higher activity of PERK pathway when compared to hMSCs cultured in the presence of glucose under near-anoxia. Most importantly, implanted hMSC-containing hydrogels loaded with glucose exhibited an increase in the volume of newly-formed blood vessels when compared to hMSC-containing hydrogels without glucose. For our second aim, we developed a novel fibrin hydrogel, which consists of starch (a polymer of glucose) and amyloglucosidase (AMG, an enzyme that release glucose from starch), to provide physiological levels of glucose to fuel MSCs via glycolysis. hMSCs loaded in this novel starch/AMG hydrogel showed improved cell survival and paracrine functions in vitro up to 14 days. Moreover, this novel nutritive hydrogel enhanced MSC survival and paracrine function pertinent to angiogenesis when implanted subcutaneously in nude mice. However, long-term sustainable production of glucose inside hydrogel and complete resorption of this novel nutritive hydrogel need further investigation. Taken together, our findings demonstrate the positive effects of exogenous glucose on improving hMSCs paracrine functions pertinent to angiogenesis and regulating hMSCs ER stress under an ischemic environment, and establish the proof of concept that a novel inbuilt system of glucose delivery improves MSC survival and angiogenesis post-implantation. These findings inspire the field to employ a glucose-supply strategy in order to improve the therapeutic efficiency of MSC-based cell constructs

Étude du rôle du glucose dans l'ingénierie tissulaire médiée par les cellules souches mésenchymateuses

Mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, are promising candidates for tissue engineering and regenerative medicine applications, however, increasing evidence shows that the therapeutic efficacy of MSCs has fallen short of their initial promise and hype due to the observed poor MSC survival and engraftment postimplantation. The hostile ischemic microenvironment, mainly characterized by deprivation of oxygen and nutrients, is responsible for the massive and rapid MSC death upon implantation. Previous studies from the B3OA lab established that the absence of glucose (but not the lack of oxygen), is the culprit for this MSC death. The central hypothesis of this work is that glucose metabolism, a central regulator of stem cell activity, is instrumental for MSCs to enhance their survival and functionalities upon transplantation, and ultimately improve their therapeutic efficacy. As a step towards the engineering of a glucose supplying construct, we hereby propose to investigate whether and how glucose affects MSC paracrine functions pertinent to angiogenesis, and then to develop an enzyme-controlled, nutritive hydrogel with an inbuilt system of glucose delivery to improve MSC survival and functionalities postimplantation. For our first aim, we found that conditioned media (CM) collected from hMSCs cultured in the presence of glucose under near-anoxia (0.1% O2) exhibited significantly higher angiogenic potential when compared to the one collected from hMSCs cultured without glucose. In addition, hMSCs cultured in the absence of glucose under near-anoxia exhibited a higher level of endoplasmic reticulum (ER) stress evidenced by less nascent protein biosynthesis, blockage of protein secretion, and higher activity of PERK pathway when compared to hMSCs cultured in the presence of glucose under near-anoxia. Most importantly, implanted hMSC-containing hydrogels loaded with glucose exhibited an increase in the volume of newly-formed blood vessels when compared to hMSC-containing hydrogels without glucose. For our second aim, we developed a novel fibrin hydrogel, which consists of starch (a polymer of glucose) and amyloglucosidase (AMG, an enzyme that release glucose from starch), to provide physiological levels of glucose to fuel MSCs via glycolysis. hMSCs loaded in this novel starch/AMG hydrogel showed improved cell survival and paracrine functions in vitro up to 14 days. Moreover, this novel nutritive hydrogel enhanced MSC survival and paracrine function pertinent to angiogenesis when implanted subcutaneously in nude mice. However, long-term sustainable production of glucose inside hydrogel and complete resorption of this novel nutritive hydrogel need further investigation. Taken together, our findings demonstrate the positive effects of exogenous glucose on improving hMSCs paracrine functions pertinent to angiogenesis and regulating hMSCs ER stress under an ischemic environment, and establish the proof of concept that a novel inbuilt system of glucose delivery improves MSC survival and angiogenesis post-implantation. These findings inspire the field to employ a glucose-supply strategy in order to improve the therapeutic efficiency of MSC-based cell constructs.Les cellules souches mésenchymateuses (CSMs), aussi appelé cellules stromales multipotentes, ont été présenté comme des candidates prometteuses pour leurs applications possibles en médecine régénérative. Cependant, leur efficacité thérapeutique est remise en question étant donné leur faible taux de survie post implantation. Le microenvironnement ischémique, principalement caractérisé par une déprivation d'oxygène et de nutriments, est responsable de la mort massive et rapide des CSMs suite à leur implantation. Des études préalables au laboratoire B3OA ont mis en évidence que l'absence de glucose (et non le manque d'oxygène) était responsable de la mort des CSMs. L'hypothèse centrale de cette étude est que le glucose, un régulateur clé de l'activité des cellules souches, est essentiel pour que les CSM puissent survivre à leur implantation, être fonctionnelles in vivo et exprimer pleinement leur potentiel thérapeutique. L'objectif de cette thèse est double : (i) étudier, si et comment, le glucose affecte les fonctions paracrines pro-angiogènes des CSM ; et (ii) développer un hydrogel nutritif à base d'amidon, qui par dégradation enzymatique libère du glucose promouvant ainsi la survie et la fonctionnalité des CSM humaines (hCSM) après implantation. Pour notre premier objectif, nous avons constaté que le milieu conditionné obtenu à partir de hCSM cultivées en présence de glucose en quasi-anoxie (0,1 % d'O2) présentait un potentiel angiogénique significativement plus élevé que celui collecté à partir de hCSM témoins cultivées sans glucose. De façon intéressante, les CSMh cultivées en quasi-anoxie en l'absence de glucose ont présenté un niveau plus élevé de stress du réticulum endoplasmique (RE), par rapport aux hCSM cultivées dans les mêmes conditions en présence de glucose. Enfin, il s'est avéré que des hydrogels cellularisés contenant du glucose implantés en site ectopique murin induisaient une augmentation du volume des vaisseaux sanguins néoformés autour de l'implant plus importante que des hydrogels cellularisés ne contenant pas de glucose. Pour notre second objectif, nous avons développé un hydrogel à base de fibrine, d'amidon (un polymère de glucose) et d'amyloglucosidase (AMG, une enzyme permettant l'hydrolyse du glucose à partir d'amidon) afin de fournir du glucose aux hCSMs à une concentration physiologiquement pertinente. Nous avons observé une augmentation de la survie et des fonctions paracrines des hCSM contenues dans ces hydrogels fibrine/amidon/AMG par rapport aux hydrogels témoins à la fois in vitro (modèle de culture en quasi-anoxie sans glucose) et in vivo (modèle ectopique murin) . Cependant, la cinétique de délivrance à long terme du glucose et la dégradation incomplète de l'amidon pouvant créer un encombrement stérique empêchant la formation de néo-tissus sont des limitations potentielles de cet hydrogel qui nécessitent des recherches supplémentaires afin d'obtenir une formulation optimale de l'hydrogel pour son application en reconstruction tissulaire. En conclusion, ce travail contribue à démontrer l'effet positif d'un apport exogène de glucose sur la survie, les fonctions pro-angiogènes et la régulation du stress du réticulum endoplasmique des hCSM dans un site ischémié. Ce travail permet aussi d'établir la preuve de concept d'un hydrogel, à base de fibrine, d'amidon et d'AMG comme système de délivrance de glucose. Ces découvertes encouragent à employer des stratégies permettant l'apport de glucose afin d'augmenter l'efficacité thérapeutique des produits à base de hCSM utilisé en médecine régénérative

A study on atmospheric environmental resource accounting: A case of SO2 capacity resources in Chinese provinces

Ecological civilization construction in China is in its critical period and the natural resources assets are audited to the administration. However, the natural resources accounting is still in its infancy, especially the atmospheric environmental resources accounting, which refers to the ability of the atmospheric environment to accommodate and purify certain pollutants. This paper established a methodology to calculate the atmospheric resources assets with the index of SO2, a convenient method to calculate the physical accounts for atmospheric environmental resources based on the capacity of SO2 and an accessible way to estimate the monetary accounts with market-based price. Based on the methodology, a calculation was conducted on the assets and liabilities of SO2 capacity resources for 31 provinces of China in 2015. Empirical results showed that the physical accounts for SO(2 )capacity resources quantify the environmental pollution status in each region, and the monetary accounts reflect whether the industry and energy structures in one region are sound and sustainable. The findings provide specific profit or loss in terms of physical and monetary accounts for each region, which enable to quantify the government's occupancy and affordability of SO2 capacity resources, and contribute to the establishment of natural resource balance sheet and ecological compensation mechanism

Friend or foe? Essential roles of osteoclast in maintaining skeletal health

Heightened activity of osteoclast is considered to be the culprit in breaking the balance during bone remodeling in pathological conditions, such as osteoporosis. As a “foe” of skeletal health, many antiosteoporosis therapies aim to inhibit osteoclastogenesis. However, bone remodeling is a dynamic process that requires the subtle coordination of osteoclasts and osteoblasts. Severe suppression of osteoclast differentiation will impair bone formation because of the coupling effect. Thus, understanding the complex roles of osteoclast in maintaining proper bone remodeling is highly warranted to develop better management of osteoporosis. This review aimed to determine the varied roles of osteoclasts in maintaining skeletal health and to highlight the positive roles of osteoclasts in maintaining normal bone remodeling. Generally, osteoclasts interact with osteocytes to initiate targeted bone remodeling and have crosstalk with mesenchymal stem cells and osteoblasts via secreted factors or cell-cell contact to promote bone formation. We believe that a better outcome of bone remodeling disorders will be achieved when proper strategies are made to coordinate osteoclasts and osteoblasts in managing such disorders

Cu₂O-Catalyzed C(sp³)-H/C(sp³)-H Cross-Coupling Using TEMPO: Synthesis of 3-(2-Oxoalkyl)-3-hydroxyoxindoles

<div><p></p><p>A simple, convenient and efficient oxidative cross-coupling reaction of oxindoles with ketones toward a variety of 3-(2-oxoalkyl)-3-hydroxyoxindoles in moderate to excellent yields has been developed. This transformation proceeds<i>via</i> a tandem oxidative cross-coupling by using TEMPO in air as an environmentally benign oxidant. This methodology provides an alternative approach for the direct generation of all-carbon quaternary centers at the C3 position of oxindoles.</p></div

Effects of Postharvest Time, Heat Treatment, pH and Filtration on the Limonin Content in Newhall Navel Orange (Citrus sinensis Osbeck cv. Newhall) Juice

Delayed bitterness causes severe economic loss in citrus juice industry worldwide, which is mostly due to the formation of limonoid compounds, especially limonin, in juice. In this study, effects of postharvest time of fruits, heat treatment, pH and filtration of juice on limonin content in Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) juice were investigated. Our research indicated for the first time that: (1) limonin content in juice would gradually increase to a maximal level and then remained almost constant thereafter as storage time going on, whereas the maximum constant value (MCV) of limonin content in juice significantly (p &lt; 0.05) decreased with the increment of postharvest time of fruits being juiced; (2) heat treatment and acidification of juice only speeded up the formation of limonin to the maximal level while without changing the MCV of limonin content; (3) the juice after filtration exhibited much lower MCV of limonin content compared with the unfiltered one. These experimental observations might not only provide useful information for the development of new debitterness method for navel orange juice, but also strongly support the acid-promoted delayed bitterness mechanism, suggesting the formation of delayed bitterness might primary due to the acid-promoted rather than the enzyme-catalyzed lactonization of limonoate A-ring lactone (LARL) to produce limonin in juice of navel orange

A novel transcription factor UvCGBP1 regulates development and virulence of rice false smut fungus Ustilaginoidea virens

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1

Understanding and leveraging cell metabolism to enhance mesenchymal stem cell transplantation survival in tissue engineering and regenerative medicine applications

International audienceIn tissue engineering and regenerative medicine, stem cell-specifically, mesenchymal stromal/stem cells (MSCs)-therapies have fallen short of their initial promise and hype. The observed marginal, to no benefit, success in several applications has been attributed primarily to poor cell survival and engraftment at transplantation sites. MSCs have a metabolism that is flexible enough to enable them to fulfill their various cellular functions and remarkably sensitive to different cellular and environmental cues. At the transplantation sites, MSCs experience hostile environments devoid or, at the very least, severely depleted of oxygen and nutrients. The impact of this particular setting on MSC metabolism ultimately affects their survival and function. In order to develop the next generation of cell-delivery materials and methods, scientists must have a better understanding of the metabolic switches MSCs experience upon transplantation. By designing treatment strategies with cell metabolism in mind, scientists may improve survival and the overall therapeutic potential of MSCs. Here, we provide a comprehensive review of plausible metabolic switches in response to implantation and of the various strategies currently used to leverage MSC metabolism to improve stem cell-based therapeutics. Significance statement: Lack of success of stem cell-based therapies has been largely attributed to the massive cell death observed post-transplantation, which is caused by the metabolic shock these cells experience as they transition from in vitro to a hostile, injured site in vivo. The metabolism in mesenchymal stem cells (MSCs), specifically, is highly sensitive to cellular and environmental cues. In order to improve cell survival rate posttransplantation, it is important that scientists understand, and take into account, the needs and demands of MSC metabolism as they design the next generation of MSC-based therapies

Wear Particles Promote Reactive Oxygen Species-Mediated Inflammation via the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Pathway in Macrophages Surrounding Loosened Implants

Background/Aims: Prosthesis loosening is closely associated with chronic inflammatory cytokine secretion by macrophages, which are activated by wear particles or inflammatory stimulants such as lipopolysaccharide (LPS). Reactive oxygen species (ROS) are critical regulators of inflammation, but their enzymatic sources in response to wear particles and their effects on peri-implant LPS-tolerance remain unclear. Methods: Three ROS-related enzymes—nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1 and -2 and catalase—were investigated in interface membrane tissues and in titanium (Ti) particle-stimulated macrophages in vitro. The generation of ROS and downstream inflammatory effects were measured with or without pre-incubation with apocynin, an NOX inhibitor. Results: Pre-exposure to Ti particles attenuated NF-&#954;B activation in LPS-stimulated macrophages, indicating that wear particles suppress immune response, which may lead to chronic inflammation. NOX-1 and -2 were highly expressed in aseptically loosened interface membranes and in macrophages stimulated with Ti particles; the particles induced a moderate amount of ROS generation, NF-&#954;B activation, and TNF-a secretion in macrophages, and these effects were suppressed by apocynin. Conclusion: Wear particles induce ROS generation through the NOX signaling pathway, resulting in persistent inflammation and delayed loosening. Thus, the suppression of NOX activity may be a useful strategy for preventing prosthesis loosening