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1.
Methods Mol Biol ; 2848: 187-196, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39240524

RESUMO

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas , Células Ganglionares da Retina , Humanos , Animais , Camundongos , Células-Tronco Embrionárias Humanas/citologia , Células Ganglionares da Retina/citologia , Transplante de Células-Tronco/métodos , Organoides/citologia , Organoides/transplante , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Retina/citologia , Células-Tronco Embrionárias/citologia
2.
Methods Mol Biol ; 2848: 37-58, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39240515

RESUMO

Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).


Assuntos
Cristalino , Organoides , Regeneração , Cristalino/citologia , Cristalino/metabolismo , Organoides/metabolismo , Organoides/citologia , Animais , Camundongos , Regeneração/genética , Perfilação da Expressão Gênica/métodos , Biologia Computacional/métodos , Simulação por Computador , Humanos , Catarata/genética , Catarata/patologia , Catarata/metabolismo , Transcriptoma , Bases de Dados Genéticas
3.
Methods Mol Biol ; 2848: 197-214, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39240525

RESUMO

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Organoides , Epitélio Pigmentado da Retina , Humanos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Organoides/citologia , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Técnicas de Cultura de Células/métodos , Células Cultivadas
4.
Methods Mol Biol ; 2854: 199-212, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39192131

RESUMO

Antiviral innate immunity plays a critical role in the defense against viral infections, yet its complex interactions with viruses have been challenging to study using traditional models. Organoids, three-dimensional (3D) tissue-like structures derived from stem cells, have emerged as powerful tools for modeling human tissues and studying the complex interactions between viruses and the host innate immune system. This chapter summarizes relevant applications of organoids in antiviral innate immunity studies and provides detailed information and experimental procedures for using organoids to study antiviral innate immunity.


Assuntos
Imunidade Inata , Organoides , Viroses , Organoides/imunologia , Organoides/virologia , Humanos , Viroses/imunologia , Viroses/virologia , Animais , Interações Hospedeiro-Patógeno/imunologia , Vírus/imunologia
5.
Anal Chim Acta ; 1325: 342989, 2024 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-39244298

RESUMO

BACKGROUND: Patient-derived organoids (PDOs) are multi-cellular cultures with specific three-dimensional (3D) structures. Tumor organoids (TOs) offer a personalized perspective for assessing treatment response. However, the presence of normal organoid (NO) residuals poses a potential threat to their utility for personalized medicine. There is a crucial need for an effective platform capable of distinguishing between TO and NO in cancer organoid cultures. RESULTS: We introduced a whole-mount (WM) preparation protocol for in-situ visualization of the lipidomic distribution of organoids. To assess the efficacy of this method, nine breast cancer organoids (BCOs) and six normal breast organoids (NBOs) were analyzed. Poly-l-lysine (PLL) coated slides, equipped with 12 well chambers, were utilized as a carrier for the high-throughput analysis of PDOs. Optimizing the fixation time to 30 min, preserved the integrity of organoids and the fidelity of lipid compounds. The PDOs derived from the same organoid lines exhibited similar lipidomic profiles. BCOs and NBOs were obviously distinguished based on their lipidomic signatures detected by WM autofocusing (AF) scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging (MSI). SIGNIFICANCE: A whole-mount (WM) preparation protocol was developed to visualize lipidomic distributions of the organoids' surface. Using poly-l-lysine coated slides for high-throughput analysis, the method preserved organoid integrity and distinguished breast cancer organoids (BCOs) from normal breast organoids (NBOs) based on their unique lipidomic profiles using autofocusing scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging.


Assuntos
Neoplasias da Mama , Lipidômica , Organoides , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Humanos , Organoides/metabolismo , Organoides/citologia , Lipidômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Lipídeos/análise , Lipídeos/química
6.
AMA J Ethics ; 26(9): E709-715, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39250943

RESUMO

A transition from nonhuman animal models to engineered microphysiological systems (MPS), such as organoids and organ-on-a-chip technologies, would signal a paradigm shift in biomedical research. Despite MPS' potential to more accurately model human physiology, reduce high failure rates of drugs in clinical trials, and limit unnecessary animal use, widespread adoption is hampered by public opinion and lack of scalability, standardization, and current regulatory uptake. This article suggests how 5 key concepts (awareness, access, education, application, and rewards) could help address these barriers. These concepts are part of a framework that underscores a need to integrate MPS into mainstream biomedical research and to better promote ethical responsibility for the means of biomedical innovation.


Assuntos
Pesquisa Biomédica , Modelos Animais , Pesquisa Biomédica/ética , Humanos , Animais , Organoides , Dispositivos Lab-On-A-Chip , Conscientização , Recompensa , Experimentação Animal/ética , Opinião Pública , Sistemas Microfisiológicos
7.
Biochemistry (Mosc) ; 89(8): 1474-1489, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39245456

RESUMO

Expansion of CAG repeats in certain genes is a known cause of several neurodegenerative diseases, but exact mechanism behind this is not yet fully understood. It is believed that the double-stranded RNA regions formed by CAG repeats could be harmful to the cell. This study aimed to test the hypothesis that these RNA regions might potentially interfere with ADAR RNA editing enzymes, leading to the reduced A-to-I editing of RNA and activation of the interferon response. We studied induced pluripotent stem cells (iPSCs) derived from the patients with Huntington's disease or ataxia type 17, as well as midbrain organoids developed from these cells. A targeted panel for next-generation sequencing was used to assess editing in the specific RNA regions. Differentiation of iPSCs into brain organoids led to increase in the ADAR2 gene expression and decrease in the expression of protein inhibitors of RNA editing. As a result, there was increase in the editing of specific ADAR2 substrates, which allowed identification of differential substrates of ADAR isoforms. However, comparison of the pathology and control groups did not show differences in the editing levels among the iPSCs. Additionally, brain organoids with 42-46 CAG repeats did not exhibit global changes. On the other hand, brain organoids with the highest number of CAG repeats in the huntingtin gene (76) showed significant decrease in the level of RNA editing of specific transcripts, potentially involving ADAR1. Notably, editing of the long non-coding RNA PWAR5 was nearly absent in this sample. It could be stated in conclusion that in most cultures with repeat expansion, the hypothesized effect on RNA editing was not confirmed.


Assuntos
Adenosina Desaminase , Encéfalo , Diferenciação Celular , Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Organoides , Edição de RNA , Proteínas de Ligação a RNA , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Humanos , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Encéfalo/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Expansão das Repetições de Trinucleotídeos
8.
J Nanobiotechnology ; 22(1): 550, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39243057

RESUMO

Non-healing skin wounds pose significant clinical challenges, with biologic products like exosomes showing promise for wound healing. Saliva and saliva-derived exosomes, known to accelerate wound repair, yet their extraction is difficult due to the complex environment of oral cavity. In this study, as a viable alternative, we established human minor salivary gland organoids (hMSG-ORG) to produce exosomes (MsOrg-Exo). In vitro, MsOrg-Exo significantly enhanced cell proliferation, migration, and angiogenesis. When incorporated into a GelMA-based controlled-release system, MsOrg-Exo demonstrated controlled release, effectively improving wound closure, collagen synthesis, angiogenesis, and cellular proliferation in a murine skin wound model. Further molecular analyses revealed that MsOrg-Exo promotes proliferation, angiogenesis and the secretion of growth factors in wound sites. Proteomic profiling showed that MsOrg-Exo's protein composition is similar to human saliva and enriched in proteins essential for wound repair, immune modulation, and coagulation. Additionally, MsOrg-Exo was found to modulate macrophage polarization, inducing a shift towards M1 and M2 phenotypes in vitro within 48 h and predominantly towards the M2 phenotype in vivo after 15 days. In conclusion, our study successfully extracted MsOrg-Exo from hMSG-ORGs, confirmed the effectiveness of the controlled-release system combining MsOrg-Exo with GelMA in promoting skin wound healing, and explored the potential role of macrophages in this action.


Assuntos
Exossomos , Macrófagos , Organoides , Cicatrização , Exossomos/metabolismo , Cicatrização/efeitos dos fármacos , Humanos , Animais , Macrófagos/metabolismo , Organoides/metabolismo , Camundongos , Proliferação de Células , Hidrogéis/química , Hidrogéis/farmacologia , Glândulas Salivares/metabolismo , Saliva/química , Saliva/metabolismo , Movimento Celular , Pele/metabolismo , Pele/lesões
9.
Commun Biol ; 7(1): 1089, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39237833

RESUMO

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here, we systematically investigated the neutral lipid-synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein, indicating that protein interaction might be required for ZIKV replication. Importantly, inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions, indicating that ZIKV morphogenesis is compromised, likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target.


Assuntos
Organoides , Esterol O-Aciltransferase , Replicação Viral , Infecção por Zika virus , Zika virus , Humanos , Infecção por Zika virus/virologia , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Organoides/virologia , Organoides/metabolismo , Replicação Viral/efeitos dos fármacos , Esterol O-Aciltransferase/metabolismo , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Antivirais/farmacologia
10.
Stem Cell Res Ther ; 15(1): 274, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39218930

RESUMO

BACKGROUND: Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule. METHODS: To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses. RESULTS: We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations. CONCLUSIONS: This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.


Assuntos
Diferenciação Celular , Linhagem da Célula , Proteínas de Homeodomínio , Células-Tronco Embrionárias Humanas , Próstata , Fatores de Transcrição , Humanos , Próstata/citologia , Próstata/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Masculino , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Organoides/metabolismo , Organoides/citologia , Camundongos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Animais , Linhagem Celular
11.
Stem Cell Res Ther ; 15(1): 273, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39218985

RESUMO

BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.


Assuntos
Diferenciação Celular , Pulmão , Organoides , Humanos , Organoides/citologia , Organoides/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Técnicas de Cocultura/métodos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo
12.
J Vis Exp ; (210)2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39221930

RESUMO

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.


Assuntos
Organoides , Ovário , Regeneração , Humanos , Organoides/citologia , Feminino , Ovário/citologia , Ovário/fisiologia , Regeneração/fisiologia , Epitélio/fisiologia
13.
Sci Adv ; 10(37): eadk3700, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39259788

RESUMO

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doença por Corpos de Lewy , Organoides , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Organoides/metabolismo , Organoides/efeitos dos fármacos , Organoides/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Doença por Corpos de Lewy/patologia , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/tratamento farmacológico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos
14.
J Exp Clin Cancer Res ; 43(1): 259, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261955

RESUMO

Patient-derived organoids (PDOs) established from tissues from various tumor types gave the foundation of ex vivo models to screen and/or validate the activity of many cancer drug candidates. Due to their phenotypic and genotypic similarity to the tumor of which they were derived, PDOs offer results that effectively complement those obtained from more complex models. Yet, their potential for predicting sensitivity to combination therapy remains underexplored. In this review, we discuss the use of PDOs in both validation and optimization of multi-drug combinations for personalized treatment strategies in CRC. Moreover, we present recent advancements in enriching PDOs with diverse cell types, enhancing their ability to mimic the complexity of in vivo environments. Finally, we debate how such sophisticated models are narrowing the gap in personalized medicine, particularly through immunotherapy strategies and discuss the challenges and future direction in this promising field.


Assuntos
Neoplasias Colorretais , Organoides , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/terapia , Medicina de Precisão/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia
15.
Nat Biotechnol ; 42(9): 1357, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39271831
16.
Orphanet J Rare Dis ; 19(1): 343, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39272186

RESUMO

BACKGROUND: Predictions based on patient-derived materials of CFTR modulators efficacy have been performed lately in patient-derived cells, extending FDA-approved drugs for CF patients harboring rare variants. Here we developed intestinal organoids from subjects carrying S737F- and T465N-CFTR in trans with null alleles to evaluate their functional impact on CFTR protein function and their restoration upon CFTR modulator treatment. The characterization of S737F-CFTR was performed in two subjects recently assessed in nasal epithelial cells but not in colonoids. RESULTS: Our functional analysis (Ussing chamber) confirmed that S737F-CFTR is a mild variant with residual function as investigated in colonoids of patients with S737F/Dele22-24 and S737F/W1282X genotypes. An increase of current upon Elexacaftor/Tezacaftor/Ivacaftor (ETI) treatment was recorded for the former genotype. T465N is a poorly characterized missense variant that strongly impacts CFTR function, as almost no CFTR-mediated anion secretion was registered for T465N/Q39X colonoids. ETI treatment substantially improved CFTR-mediated anion secretion and increased the rescue of mature CFTR expression compared to either untreated colonoids or to dual CFTR modulator therapies. CONCLUSIONS: Our study confirms the presence of a residual function of the S737F variant and its limited response to CFTR modulators while predicting for the first time the potential clinical benefit of Trikafta® for patients carrying the rare T465N variant.


Assuntos
Aminofenóis , Benzodioxóis , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Organoides , Quinolonas , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Organoides/metabolismo , Organoides/efeitos dos fármacos , Benzodioxóis/farmacologia , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Quinolonas/farmacologia , Aminofenóis/farmacologia , Indóis/farmacologia , Combinação de Medicamentos , Pirazóis/farmacologia , Masculino , Feminino , Quinolinas/farmacologia , Piridinas , Pirrolidinas
17.
Cells ; 13(17)2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39273033

RESUMO

Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling.


Assuntos
Sobrevivência Celular , Fator de Transcrição STAT3 , Esferoides Celulares , Neoplasias da Bexiga Urinária , Fator de Transcrição STAT3/metabolismo , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Humanos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia
18.
Parasit Vectors ; 17(1): 393, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39285481

RESUMO

BACKGROUND: Anisakis spp. are zoonotic nematodes causing mild to severe acute and chronic gastrointestinal infections. Chronic anisakiasis can lead to erosive mucosal ulcers, granulomas and inflammation, potential tumorigenic triggers. How Anisakis exerts its pathogenic potential through extracellular vesicles (EVs) and whether third-stage infective larvae may favor a tumorigenic microenvironment remain unclear. METHODS: Here, we investigated the parasite's tumorigenic and immunomodulatory capabilities using comparative transcriptomics, qRT-PCR and protein analysis with multiplex ELISA on human intestinal organoids exposed to Anisakis EVs. Moreover, EVs were characterized in terms of shape, size and concentration using classic TEM, SEM and NTA analyses and advanced interferometric NTA. RESULTS: Anisakis EVs showed classic shape features and a median average diameter of around 100 nm, according to NTA and iNTA. Moreover, a refractive index of 5-20% of non-water content suggested their effective biological cargo. After treatment of human intestinal organoids with Anisakis EVs, an overall parasitic strategy based on mitigation of the immune and inflammatory response was observed. Anisakis EVs impacted gene expression of main cytokines, cell cycle regulation and protein products. Seven key genes related to cell cycle regulation and apoptosis were differentially expressed in organoids exposed to EVs. In particular, the downregulation of EPHB2 and LEFTY1 and upregulation of NUPR1 genes known to be associated with colorectal cancer were observed, suggesting their involvement in tumorigenic microenvironment. A statistically significant reduction in specific mediators of inflammation and cell-cycle regulation from the polarized epithelium as IL-33R, CD40 and CEACAM1 from the apical chambers and IL-1B, GM-CSF, IL-15 and IL-23 from both chambers were observed. CONCLUSIONS: The results here obtained unravel intestinal epithelium response to Anisakis EVs, impacting host's anthelminthic strategies and revealing for the first time to our knowledge the host-parasite interactions in the niche environment of an emerging accidental zoonosis. Use of an innovative EV characterization approach may also be useful for study of other helminth EVs, since the knowledge in this field is very limited.


Assuntos
Anisakis , Vesículas Extracelulares , Organoides , Humanos , Organoides/parasitologia , Organoides/imunologia , Anisakis/imunologia , Anisakis/genética , Animais , Vesículas Extracelulares/imunologia , Anisaquíase/parasitologia , Anisaquíase/imunologia , Citocinas/metabolismo , Citocinas/genética , Intestinos/parasitologia , Intestinos/imunologia , Carcinogênese , Imunomodulação
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