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In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.
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Substância Própria , Células Estromais , Adulto , Humanos , Diferenciação Celular/fisiologia , Linhagem Celular , Células-TroncoRESUMO
BACKGROUND: In France, information collected during postdonation interviews showed that a majority of human immunodeficiency virus (HIV)-infected donors were not eligible to donate as per donor selection criteria. In the interest of blood safety, this study aimed to explore the mechanisms of noncompliance with blood donor selection criteria, notably the permanent deferral of men who have sex with men (MSM). STUDY DESIGN AND METHODS: Semistructured individual interviews were conducted with 32 blood donors found positive for HIV between mid-2011 and 2014. Topics such as the experience and motivations for donating blood, understanding of selection criteria, sexual risk management, and opinions on donor selection were discussed. Transcripts were analyzed inductively. RESULTS: More than 50% of study participants were noncompliant with donor selection criteria. Reasons for nondisclosure of risk factors in the predonation questionnaire or the predonation interview included stigma, test-seeking motivations, symbolic attachment to blood donation, and context of donation. Compliance to donor criteria was seen as secondary by donors who reaped personal benefits from the symbolism of their donation. Donors lacked self-reflexivity in their assessment of risky sexual behavior. The "window period" and the underlying epidemiologic arguments for donor selection criteria were poorly understood. Nearly all participants disapproved of the permanent ban on blood donations from MSM. CONCLUSION: This study demonstrated the need for more communication on the epidemiologic basis for donor selection criteria and on the window period to facilitate donor compliance. These findings have already advanced improvements to predonation documents, in a larger context of 2016 donor selection criteria revision.
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Doadores de Sangue/psicologia , Segurança do Sangue/normas , Seleção do Doador/métodos , Percepção Social , Adulto , Feminino , França , Infecções por HIV/sangue , Humanos , Masculino , Inquéritos e QuestionáriosRESUMO
Initial conditions disclosed for the Passerini-Smiles reaction are associated with a lack of efficiency that has prevented chemists from using it since its discovery. We wish to report herein our thorough study in the development of new experimental conditions for this coupling between electron-poor phenols, isocyanides, and carbonyl derivatives. These new conditions have been applied to several synthetic strategies towards benzoxazinones.
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The salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 belong to the adenosine monophosphate-activated protein kinase (AMPK) family of serine/threonine kinases. SIK inhibition represents a new therapeutic approach modulating pro-inflammatory and immunoregulatory pathways that holds potential for the treatment of inflammatory diseases. Here, we describe the identification of GLPG3970 (32), a first-in-class dual SIK2/SIK3 inhibitor with selectivity against SIK1 (IC50 of 282.8 nM on SIK1, 7.8 nM on SIK2 and 3.8 nM on SIK3). We outline efforts made to increase selectivity against SIK1 and improve CYP time-dependent inhibition properties through the structure-activity relationship. The dual activity of 32 in modulating the pro-inflammatory cytokine TNFα and the immunoregulatory cytokine IL-10 is demonstrated in vitro in human primary myeloid cells and human whole blood, and in vivo in mice stimulated with lipopolysaccharide. Compound 32 shows dose-dependent activity in disease-relevant mouse pharmacological models.
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Proteínas Quinases , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Proteínas Quinases/metabolismo , Citocinas , Fator de Necrose Tumoral alfaRESUMO
2-Benzyl benzothiazoles and benzimidazoles are easily oxidized under air and basic conditions to give the corresponding ketones in good yields. The use of palladium acetate as a catalyst has little effect and even gives, in some cases, much lower yields.
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Benzimidazóis/química , Benzotiazóis/química , Benzimidazóis/síntese química , Benzotiazóis/síntese química , Estrutura Molecular , OxirreduçãoRESUMO
With significant advancement and development of extracellular vesicle (EV)-based therapies, there is a growing need to understand how their storage affects their physical and functional characteristics. EVs were isolated from the conditioned medium of a corneal stromal stem cell line (imCSSC) using Total Exosome isolation kit (TEI) and ultracentrifugation (UC) combined protocol. Purified EVs were stored at 4°C, - 80°C, room temperature (RT) after lyophilization with or without trehalose for 4 weeks. EVs stored at - 80°C and RT (lyophilization with trehalose) demonstrated a comparable morphology, while the freeze-dried samples without trehalose showed aggregation and degradation under a transmission electron microscope (TEM). Lyophilized samples without trehalose demonstrated a decreased particle concentration, recovery rate and protein concentration, which was remediated by the addition of trehalose. EVs stored at - 80â showed no change in the protein expression of CD9, CD63, and CD81. Regardless of the storage condition, all EV samples investigated reduced inflammation, as well as inhibited expression of fibrotic markers in vitro. Lyophilization of EVs with trehalose was a feasible storage method that retained the physical property and in vitro biological activities of EVs after 4 weeks of storage, while - 80°C offered the best retention of imCSSC-derived EV physical properties. For the first time, this data demonstrated a practical and translatable method for the storage of CSSC-derived EVs for clinical use.
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Vesículas Extracelulares , Células-Tronco Mesenquimais , Humanos , Trealose/farmacologia , Trealose/metabolismo , Estudo de Prova de Conceito , Vesículas Extracelulares/metabolismo , UltracentrifugaçãoRESUMO
Pyrazolidinones were prepared in a two-step sequence starting from α-hydrazonocarboxylic acids. After a four-component Ugi coupling, the resulting hydrazone was engaged in a copper triggered [3 + 2] cycloaddition/aerobic oxidation cascade.
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Limbal stem cell deficiency and corneal disorders are among the top global threats for human vision. Emerging therapies that integrate stem cell transplantation with engineered hydrogel scaffolds for biological and mechanical support are becoming a rising trend in the field. However, methods for high-throughput fabrication of hydrogel scaffolds, as well as knowledge of the interaction between limbal stem/progenitor cells (LSCs) and the surrounding extracellular matrix (ECM) are still much needed. Here, we employed digital light processing (DLP)-based bioprinting to fabricate hydrogel scaffolds encapsulating primary LSCs and studied the ECM-dependent LSC phenotypes. The DLP-based bioprinting with gelatin methacrylate (GelMA) or hyaluronic acid glycidyl methacrylate (HAGM) generated microscale hydrogel scaffolds that could support the viability of the encapsulated primary rabbit LSCs (rbLSCs) in culture. Immunocytochemistry and transcriptional analysis showed that the encapsulated rbLSCs remained active in GelMA-based scaffolds while exhibited quiescence in the HAGM-based scaffolds. The primary human LSCs encapsulated within bioprinted scaffolds showed consistent ECM-dependent active/quiescent statuses. Based on these results, we have developed a novel bioprinted dual ECM 'Yin-Yang' model encapsulating LSCs to support both active and quiescent statues. Our findings provide valuable insights towards stem cell therapies and regenerative medicine for corneal reconstruction.
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Bioimpressão , Animais , Matriz Extracelular , Gelatina , Coelhos , Células-Tronco , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Infection, trauma, and chemical exposure of the ocular surface can severely damage the cornea, resulting in visually significant stromal scars. Current medical treatments are ineffective in mitigating corneal scarring, and corneal transplantation is the only therapy able to restore vision in these eyes. However, because of a severe shortage of corneal tissues, risks of blinding complications associated with corneal transplants, and a higher rate of graft failure in these eyes, an effective and deliverable alternative therapy for the prevention and treatment of corneal scarring remains a significant unmet medical need globally. In recent years, the therapeutic potential of extracellular vesicles (EVs) secreted by cells to mediate cell-cell communication has been a topic of increasing interest. EVs derived from mesenchymal stem cells, in particular human corneal stromal stem cells, have antifibrotic, anti-inflammatory, and regenerative effects in injured corneas. The exact mechanism of action of these functional EVs are largely unknown. Therapeutic development of EVs is at an early stage and warrants further preclinical studies.
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Lesões da Córnea , Vesículas Extracelulares , Células-Tronco Mesenquimais , Cicatriz/terapia , Córnea/patologia , Lesões da Córnea/terapia , HumanosRESUMO
Introduction: Human corneal stromal stem cells (CSSCs) have gained increasing attention in the treatment of corneal stromal scars. In view of this, the preparation and storage of CSSCs are critical to maintaining the regenerative potential of CSSCs. The goal of the study was to investigate the human serum (HS) concentration in the cryomedia that could best preserve CSSCs. Materials and Methods: Three different cryopreservation media, varying in HS concentration were evaluated in their ability to preserve the viability and phenotype of CSSCs: 2% HS (FS1), 4% HS (FS2), and 90% HS (FS3). After thawing, CSSCs morphology, recovery rate, cell proliferation, relative gene expression of CSSC markers (ABCG2, SOX2, NANOG, PAX6, and SIX3), and their anti-inflammatory response (level of TNFAIP6) were compared with those of unfrozen CSSCs (control). Results: Cryopreserved CSSCs had similar cell morphology as the control. Cell viability was significantly higher using FS2 (92.7 ± 1.3%) compared with FS1 (88 ± 0.8%, p = 0.018). Doubling times of CSSCs were maintained in all cryopreserved conditions, as in the control (p > 0.05), which were 0.9 ± 0.1 days and 1.8 ± 0.0 days at passages 3 and 4, then increased to 18.2 ± 1.9 days at passage 6 (p > 0.05). The expression level of stem cell/progenitor cell markers investigated was not affected by the cryopreservation with any of the three media. In addition, cryopreserved CSSCs have a similar expression level of TNFAIP6 after stimulation with proinflammatory cytokines as the control (p > 0.05). Conclusion: Our results indicated that all three cryopreservation media maintained CSSCs phenotype after undergoing one freezing/thawing cycle. Impact Statement Corneal stromal stem cells (CSSCs) offer an alternative for the treatment of corneal stromal scars. Cryopreservation of CSSCs is necessary as it enables feasibility of using CSSCs as a cell therapy candidate. The current study shows that media used to cryopreserve CSSCs could be optimized to maintain cell viability, phenotype, and potency of CSSCs after thawing.
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Diferenciação Celular , Proliferação de Células , Substância Própria/citologia , Criopreservação/métodos , Meios de Cultivo Condicionados/química , Células Estromais/citologia , Sobrevivência Celular , Células Cultivadas , HumanosRESUMO
The coupling of an acyl chloride with an isocyanide affords 2,5-disubstituted oxazoles under mild basic conditions instead of 4,5-disubstituted derivatives when using Schöllkopf conditions (butyllithium); this reaction constitutes a remarkable example of a base-induced chemoselective process in isocyanide chemistry.
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Cianetos/metabolismo , Oxazóis/síntese química , Oxazóis/metabolismo , Estrutura Molecular , Oxazóis/químicaRESUMO
Purpose: Mesenchymal stem cells (MSCs) have been extensively studied for their capacity to enhance wound healing and represent a promising research field for generating cell therapies for corneal scars. In the present study, we investigated MSCs from different tissues and their potential to differentiate toward corneal keratocytes. Methods: Adipose-derived stem cells, bone marrow MSCs, umbilical cord stem cells, and corneal stromal stem cells (CSSCs) were characterized by their expression of surface markers CD105, CD90, and CD73, and their multilineage differentiation capacity into adipocytes, osteoblasts, and chondrocytes. MSCs were also evaluated for their potential to differentiate toward keratocytes, and for upregulation of the anti-inflammatory protein TNFα-stimulated gene-6 (TNFAIP6) after simulation by IFN-γ and TNF-α. Results: Keratocyte lineage induction was achieved in all MSCs as indicated by the upregulated expression of keratocyte markers, including keratocan, lumican, and carbohydrate sulfotransferase. TNFAIP6 response to inflammatory stimulation was observed only in CSSCs; increasing by 3-fold compared with the control (P < 0.05). Conclusions: Based on our findings, CSSCs appeared to have the greatest differentiation potential toward the keratocyte lineage and the greatest anti-inflammatory properties in vitro.
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Moléculas de Adesão Celular/genética , Ceratócitos da Córnea/citologia , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular , Células Cultivadas , Ceratócitos da Córnea/metabolismo , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , RNA/genética , Fator de Necrose Tumoral alfaRESUMO
Mesenchymal stem cells from corneal stromal stem cells (CSSC) prevent fibrotic scarring and stimulate regeneration of transparent stromal tissue after corneal wounding in mice. These effects rely on the ability of CSSC to block neutrophil infiltration into the damaged cornea. The current study investigated the hypothesis that tissue regeneration by CSSC is mediated by secreted extracellular vesicles (EVs). CSSC produced EVs 130-150 nm in diameter with surface proteins that include CD63, CD81, and CD9. EVs from CSSC reduced visual scarring in murine corneal wounds as effectively as did live cells, but EVs from human embryonic kidney (HEK)293T cells had no regenerative properties. CSSC EV treatment of wounds decreased expression of fibrotic genes Col3a1 and Acta2, blocked neutrophil infiltration, and restored normal tissue morphology. CSSC EVs labeled with carboxyfluorescein succinimidyl ester dye, rapidly fused with corneal epithelial and stromal cells in culture, transferring microRNA (miRNA) to the target cells. Knockdown of mRNA for Alix, a component of the endosomal sorting complex required for transport, using siRNA, resulted in an 85% reduction of miRNA in the secreted EVs. The EVs with reduced miRNA were ineffective at blocking corneal scarring. Furthermore, CSSC with reduced Alix expression also lost their regenerative function, suggesting EVs as an obligate component in the delivery of miRNA. The results of these studies support an essential role for extracellular vesicles in the process by which CSSC cells block scarring and initiate regeneration of transparent corneal tissue after wounding. EVs appear to serve as a delivery vehicle for miRNA, which affects the regenerative action. Stem Cells Translational Medicine 2019;8:1192-1201.
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Doenças da Córnea/terapia , Vesículas Extracelulares/transplante , Fibrose/terapia , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , MicroRNAs/administração & dosagem , Animais , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , CicatrizaçãoRESUMO
INTRODUCTION AND AIMS: People who inject drugs (PWID) are exposed to associated viral, bacterial and fungal risks. These risks can be reduced by filtration. Large disparities in the quality of filtration exist between the various available filters. This paper compares both performance and user acceptability of three filters for drug injection (cotton filters, Sterifilt® and wheel filters) by combining epidemiological and bacteriological analyses. DESIGN AND METHODS: A cross-sectional epidemiological study (ANRS-Coquelicot) using time-location sampling combined with the generalised weight sampling method was conducted among 985 PWID in France. Two filtration-based bacteriological studies of 0.20- and 0.45-µm wheel filters, Sterifilt filters and cotton filters were also conducted. RESULTS: The bacteriological study highlighted the value of using wheel filters with a porosity of less than 0.5 µm, as they limit the risk of bacterial and fungal infection. The results of this study clearly highlight a distinction between the efficiency of Sterifilt and wheel filters, the latter being more effective. Our epidemiological study highlighted that the use of cotton filters is widespread and routine, but is the subject of much criticism among PWID. Sterifilt is not widely used, and its adoption is slow. Finally, the wheel filter remains a largely untested tool. DISCUSSION AND CONCLUSIONS: Low product retention and ease of use are the two most important factors for filters for PWID. Bacterial and fungal risk filtration is less important. It is essential to educate PWID about the benefits of wheel filters. [Jauffret-Roustide M, Chollet A, Santos A, Benoit T, Péchiné S, Duplessy C, Bara J-L, Lévi Y, Karolak S, Néfau T. Theory versus practice, bacteriological efficiency versus personal habits: A bacteriological and user acceptability evaluation of filtering tools for people who inject drugs. Drug Alcohol Rev 2018;37:106-115].
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Usuários de Drogas , Redução do Dano , Controle de Infecções/métodos , Infecções/etiologia , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Feminino , Filtração , Humanos , Masculino , Pessoa de Meia-Idade , Uso Comum de Agulhas e SeringasRESUMO
The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16ink4a in vitro. Viral vector-mediated overexpression of p16ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres in vitro. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16ink4a.
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Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Órgão Espiral/citologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Orelha Interna/metabolismo , Orelha Interna/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Complexo Repressor Polycomb 1/deficiência , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Células-Tronco/citologia , Transcriptoma , Regulação para CimaRESUMO
The Passerini reaction of α,ß-unsaturated aldehydes affords suitable substrates for the Tsuji-Trost reaction with various carbon based-nucleophiles. The resulting α,ß-unsaturated amides may be cyclized to lactams or converted into cyclopentane derivatives if bis-nucleophiles are used in the Tsuji-Trost step.
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Correction for 'Formal [3+2] cycloaddition of Ugi adducts towards pyrrolines' by Abdelbari Ben Abdessalem et al., Chem. Commun., 2015, 51, 1116-1119.
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Ugi adducts derived from aromatic aldehydes may be converted to pyrrolines via addition of Michael acceptors under microwave irradiation. The reaction may proceed via unusual formation of azomethine ylides followed by a [3+2] cycloaddition using Michael acceptors.