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1.
Cell Mol Life Sci ; 81(1): 267, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38884678

RESUMO

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.


Assuntos
Anticorpos Neutralizantes , Receptor 1 de Quimiocina CX3C , Testes de Neutralização , Organoides , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Humanos , Vírus Sincicial Respiratório Humano/imunologia , Anticorpos Neutralizantes/imunologia , Organoides/metabolismo , Organoides/imunologia , Organoides/virologia , Organoides/citologia , Animais , Testes de Neutralização/métodos , Chlorocebus aethiops , Células Vero , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Receptor 1 de Quimiocina CX3C/metabolismo , Receptor 1 de Quimiocina CX3C/imunologia , Anticorpos Antivirais/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Lactente , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/virologia , Anticorpos Monoclonais/imunologia
2.
Adv Exp Med Biol ; 1441: 239-252, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38884715

RESUMO

Congenital heart disease (CHD) is a leading cause of birth defect-related death. Despite significant advances, the mechanisms underlying the development of CHD are complex and remain elusive due to a lack of efficient, reproducible, and translational model systems. Investigations relied on animal models have inherent limitations due to interspecies differences. Human induced pluripotent stem cells (iPSCs) have emerged as an effective platform for disease modeling. iPSCs allow for the production of a limitless supply of patient-specific somatic cells that enable advancement in cardiovascular precision medicine. Over the past decade, researchers have developed protocols to differentiate iPSCs to multiple cardiovascular lineages, as well as to enhance the maturity and functionality of these cells. With the development of physiologic three-dimensional cardiac organoids, iPSCs represent a powerful platform to mechanistically dissect CHD and serve as a foundation for future translational research.


Assuntos
Diferenciação Celular , Cardiopatias Congênitas , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/terapia , Organoides/citologia , Organoides/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Animais
3.
PLoS One ; 19(6): e0304526, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38857221

RESUMO

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.


Assuntos
L-Lactato Desidrogenase , Organoides , Humanos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , L-Lactato Desidrogenase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Intestinos/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo
4.
Sci Rep ; 14(1): 13525, 2024 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-38866945

RESUMO

The traditional nomenclature of enteroendocrine cells (EECs), established in 1977, applied the "one cell - one hormone" dogma, which distinguishes subpopulations based on the secretion of a specific hormone. These hormone-specific subpopulations included S cells for secretin (SCT), K cells for glucose-dependent insulinotropic polypeptide (GIP), N cells producing neurotensin (NTS), I cells producing cholecystokinin (CCK), D cells producing somatostatin (SST), and others. In the past 15 years, reinvestigations into murine and human organoid-derived EECs, however, strongly questioned this dogma and established that certain EECs coexpress multiple hormones. Using the Gut Cell Atlas, the largest available single-cell transcriptome dataset of human intestinal cells, this study consolidates that the original dogma is outdated not only for murine and human organoid-derived EECs, but also for primary human EECs, showing that the expression of certain hormones is not restricted to their designated cell type. Moreover, specific analyses into SCT-expressing cells reject the presence of any cell population that exhibits significantly elevated secretin expression compared to other cell populations, previously referred to as S cells. Instead, this investigation indicates that secretin production is realized jointly by other enteroendocrine subpopulations, validating corresponding observations in murine EECs also for human EECs. Furthermore, our findings corroborate that SCT expression peaks in mature EECs, in contrast, progenitor EECs exhibit markedly lower expression levels, supporting the hypothesis that SCT expression is a hallmark of EEC maturation.


Assuntos
Células Enteroendócrinas , Perfilação da Expressão Gênica , Secretina , Análise de Célula Única , Humanos , Células Enteroendócrinas/metabolismo , Secretina/metabolismo , Secretina/genética , Análise de Célula Única/métodos , Camundongos , Animais , Transcriptoma , Diferenciação Celular , Organoides/metabolismo , Organoides/citologia , Colecistocinina/metabolismo , Colecistocinina/genética , Somatostatina/metabolismo , Somatostatina/genética , Análise da Expressão Gênica de Célula Única
5.
Proc Natl Acad Sci U S A ; 121(25): e2317285121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38870053

RESUMO

Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.


Assuntos
Diferenciação Celular , Organoides , Células-Tronco Pluripotentes , Retina , Humanos , Organoides/citologia , Organoides/metabolismo , Retina/citologia , Retina/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Reprodutibilidade dos Testes , Proteínas Morfogenéticas Ósseas/metabolismo
6.
EBioMedicine ; 104: 105181, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38838469

RESUMO

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).


Assuntos
COVID-19 , Quirópteros , SARS-CoV-2 , Animais , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Humanos , COVID-19/virologia , Quirópteros/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Organoides/virologia , Organoides/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/virologia , Cricetinae , Furina/metabolismo , Células Epiteliais/virologia , Células Vero , Chlorocebus aethiops
7.
J Neurosci Methods ; 408: 110181, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38823594

RESUMO

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.


Assuntos
Exossomos , Organoides , Retina , Animais , Organoides/metabolismo , Retina/citologia , Retina/metabolismo , Exossomos/metabolismo , Perfusão/métodos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Tecidos/métodos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos
8.
Stem Cell Res Ther ; 15(1): 183, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38902814

RESUMO

In the realm of studying joint-related diseases, there is a continuous quest for more accurate and representative models. Recently, regenerative medicine and tissue engineering have seen a growing interest in utilizing organoids as powerful tools for studying complex biological systems in vitro. Organoids, three-dimensional structures replicating the architecture and function of organs, provide a unique platform for investigating disease mechanisms, drug responses, and tissue regeneration. The surge in organoid research is fueled by the need for physiologically relevant models to bridge the gap between traditional cell cultures and in vivo studies. Osteochondral organoids have emerged as a promising avenue in this pursuit, offering a better platform to mimic the intricate biological interactions within bone and cartilage. This review explores the significance of osteochondral organoids and the need for their development in advancing our understanding and treatment of bone and cartilage-related diseases. It summarizes osteochondral organoids' insights and research progress, focusing on their composition, materials, cell sources, and cultivation methods, as well as the concept of organoids on chips and application scenarios. Additionally, we address the limitations and challenges these organoids face, emphasizing the necessity for further research to overcome these obstacles and facilitate orthopedic regeneration.


Assuntos
Organoides , Engenharia Tecidual , Organoides/citologia , Organoides/metabolismo , Humanos , Engenharia Tecidual/métodos , Animais , Cartilagem/citologia , Medicina Regenerativa/métodos , Osso e Ossos/citologia , Osso e Ossos/fisiologia
9.
Molecules ; 29(11)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38893345

RESUMO

Among brain tumors, glioblastoma (GBM) is very challenging to treat as chemotherapeutic drugs can only penetrate the brain to a limited extent due to the blood-brain barrier (BBB). Nanoparticles can be an attractive solution for the treatment of GBM as they can transport drugs across the BBB into the tumor. In this study, normal and GBM organoids comprising six brain cell types were developed and applied to study the uptake, BBB penetration, distribution, and efficacy of fluorescent, ultrasmall gold nanoparticles (AuTio-Dox-AF647s) conjugated with doxorubicin (Dox) and AlexaFluor-647-cadaverine (AF647) by confocal laser scanning microscopy (CLSM), using a mixture of dissolved doxorubicin and fluorescent AF647 molecules as a control. It was shown that the nanoparticles could easily penetrate the BBB and were found in normal and GBM organoids, while the dissolved Dox and AF647 molecules alone were unable to penetrate the BBB. Flow cytometry showed a reduction in glioblastoma cells after treatment with AuTio-Dox nanoparticles, as well as a higher uptake of these nanoparticles by GBM cells in the GBM model compared to astrocytes in the normal cell organoids. In summary, our results show that ultrasmall gold nanoparticles can serve as suitable carriers for the delivery of drugs into organoids to study BBB function.


Assuntos
Barreira Hematoencefálica , Doxorrubicina , Glioblastoma , Ouro , Nanopartículas Metálicas , Organoides , Doxorrubicina/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Nanopartículas Metálicas/química , Ouro/química , Humanos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral
10.
Cells ; 13(11)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38891075

RESUMO

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others' previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and ß-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal-epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level.


Assuntos
Adipócitos , Transdiferenciação Celular , Células Epiteliais , Humanos , Feminino , Adipócitos/citologia , Adipócitos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Organoides/citologia , Organoides/metabolismo , Técnicas de Cocultura , Camundongos , Modelos Biológicos
11.
Cell Genom ; 4(6): 100581, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38823397

RESUMO

Cell atlases serve as vital references for automating cell labeling in new samples, yet existing classification algorithms struggle with accuracy. Here we introduce SIMS (scalable, interpretable machine learning for single cell), a low-code data-efficient pipeline for single-cell RNA classification. We benchmark SIMS against datasets from different tissues and species. We demonstrate SIMS's efficacy in classifying cells in the brain, achieving high accuracy even with small training sets (<3,500 cells) and across different samples. SIMS accurately predicts neuronal subtypes in the developing brain, shedding light on genetic changes during neuronal differentiation and postmitotic fate refinement. Finally, we apply SIMS to single-cell RNA datasets of cortical organoids to predict cell identities and uncover genetic variations between cell lines. SIMS identifies cell-line differences and misannotated cell lineages in human cortical organoids derived from different pluripotent stem cell lines. Altogether, we show that SIMS is a versatile and robust tool for cell-type classification from single-cell datasets.


Assuntos
Aprendizado Profundo , Análise de Sequência de RNA , Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Análise de Sequência de RNA/métodos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Neurônios/metabolismo , Neurônios/citologia , Organoides/metabolismo , Organoides/citologia , Diferenciação Celular/genética , Camundongos
12.
Nat Commun ; 15(1): 4976, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862520

RESUMO

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.


Assuntos
Proteínas Morfogenéticas Ósseas , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Camundongos , Humanos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/química , Glicoproteínas/metabolismo , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Sítios de Ligação , Domínios Proteicos , Ligação Proteica , Organoides/metabolismo , Organoides/embriologia , Células HEK293 , Gastrulação/genética , Mutação , Cristalografia por Raios X , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas
13.
Stem Cell Res Ther ; 15(1): 171, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38886860

RESUMO

BACKGROUND: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). METHODS: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. RESULTS: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. CONCLUSIONS: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.


Assuntos
Reatores Biológicos , Células-Tronco Pluripotentes Induzidas , Macrófagos , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Organoides/citologia , Organoides/metabolismo , Diferenciação Celular , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/instrumentação , Hematopoese
14.
Biomed Pharmacother ; 176: 116871, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38861856

RESUMO

Breast cancer (BC) is the most prevalent cancer among women around the world. Finding new and efficient drugs has become a crucial aspect of BC treatment. Liensinine diperchlorate (LIN) and artemisitene (ATT) are natural compounds with potential anti-cancer activities extracted from lotus (Nelumbo nucifera Gaertn) seeds and Artemisia annua, respectively. However, the synergistic anti-breast cancer effectiveness and mechanism of LIN and ATT remain unknown. This study intended to reveal the biological functions and underlying mechanism of combined LIN and ATT treatment in BC. Herein, we first reported that LIN and ATT synergistically mitigated the proliferation, migration as well as invasion of BC cells. Besides, LIN boosted the stimulatory effect of ATT on reactive oxygen species (ROS)-mediated apoptosis in BC cells. Interestingly, LIN and ATT synergistically attenuated the growth of BC patient-derived organoids. Moreover, LIN augmented the inhibitory efficacy of ATT on BC growth in vivo without obvious side effects. Furthermore, the inactivation of PI3K-AKT pathway and its regulated proteins contributed to the therapeutic role of LIN and ATT treatment in BC. Intriguingly, a prediction model constructed as per RNA sequencing data indicated that the combination of LIN and ATT treatment might ameliorate the prognosis of BC patients. In conclusion, our present investigation demonstrated that LIN and ATT synergistically inhibited BC cell proliferation, migration as well as invasion and enhanced ROS-mediated apoptosis via suppressing the PI3K-AKT signaling, and suggested that combining LIN and ATT treatment might be a promising choice for BC therapy.


Assuntos
Apoptose , Neoplasias da Mama , Proliferação de Células , Sinergismo Farmacológico , Camundongos Nus , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Camundongos , Progressão da Doença , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto , Células MCF-7 , Isoquinolinas , Fenóis
15.
Anal Chem ; 96(24): 10092-10101, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38833634

RESUMO

Tumor patients-derived organoids, as a promising preclinical prediction model, have been utilized to evaluate ex vivo drug responses for formulating optimal therapeutic strategies. Detecting adenosine triphosphate (ATP) has been widely used in existing organoid-based drug response tests. However, all commercial ATP detection kits containing the cell lysis procedure can only be applied for single time point ATP detection, resulting in the neglect of dynamic ATP variations in living cells. Meanwhile, due to the limited number of viable organoids from a single patient, it is impractical to exhaustively test all potential time points in search of optimal ones. In this work, a multifunctional microfluidic chip was developed to perform all procedures of organoid-based drug response tests, including establishment, culturing, drug treatment, and ATP monitoring of organoids. An ATP sensor was developed to facilitate the first successful attempt on whole-course monitoring the growth status of fragile organoids. To realize a clinically applicable automatic system for the drug testing of lung cancer, a microfluidic chip based automated system was developed to perform entire organoid-based drug response test, bridging the gap between laboratorial manipulation and clinical practices, as it outperformed previous methods by improving data repeatability, eliminating human error/sample loss, and more importantly, providing a more accurate and comprehensive evaluation of drug effects.


Assuntos
Trifosfato de Adenosina , Dispositivos Lab-On-A-Chip , Organoides , Humanos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Automação
16.
Nat Commun ; 15(1): 4866, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849373

RESUMO

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neoplasias da Mama , Colágeno Tipo I , Mecanotransdução Celular , Invasividade Neoplásica , Fatores de Transcrição , Proteínas de Sinalização YAP , Animais , Feminino , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Regulação Neoplásica da Expressão Gênica , Organoides/metabolismo , Organoides/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismo
17.
Mol Biol Rep ; 51(1): 704, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824233

RESUMO

BACKGROUND: Tumor modeling using organoids holds potential in studies of cancer development, enlightening both the intracellular and extracellular molecular mechanisms behind different cancer types, biobanking, and drug screening. Intestinal organoids can be generated in vitro using a unique type of adult stem cells which are found at the base of crypts and are characterized by their high Lgr5 expression levels. METHODS AND RESULTS: In this study, we successfully established intestinal cancer organoid models by using both the BALB/c derived and mouse embryonic stem cells (mESCs)-derived intestinal organoids. In both cases, carcinogenesis-like model was developed by using azoxymethane (AOM) treatment. Carcinogenesis-like model was verified by H&E staining, immunostaining, relative mRNA expression analysis, and LC/MS analysis. The morphologic analysis demonstrated that the number of generated organoids, the number of crypts, and the intensity of the organoids were significantly augmented in AOM-treated intestinal organoids compared to non-AOM-treated ones. Relative mRNA expression data revealed that there was a significant increase in both Wnt signaling pathway-related genes and pluripotency transcription factors in the AOM-induced intestinal organoids. CONCLUSION: We successfully developed simple carcinogenesis-like models using mESC-based and Lgr5 + stem cell-based intestinal organoids. Intestinal organoid based carcinogenesi models might be used for personalized cancer therapy in the future.


Assuntos
Azoximetano , Carcinogênese , Células-Tronco Embrionárias Murinas , Organoides , Via de Sinalização Wnt , Animais , Organoides/metabolismo , Organoides/patologia , Camundongos , Azoximetano/toxicidade , Carcinogênese/patologia , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Células-Tronco Embrionárias Murinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Camundongos Endogâmicos BALB C , Intestinos/patologia , Neoplasias Intestinais/patologia , Neoplasias Intestinais/induzido quimicamente , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Modelos Animais de Doenças , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia
18.
Physiol Rep ; 12(11): e16057, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38825580

RESUMO

The bronchoalveolar organoid (BAO) model is increasingly acknowledged as an ex-vivo platform that accurately emulates the structural and functional attributes of proximal airway tissue. The transition from bronchoalveolar progenitor cells to alveolar organoids is a common event during the generation of BAOs. However, there is a pressing need for comprehensive analysis to elucidate the molecular distinctions characterizing the pre-differentiated and post-differentiated states within BAO models. This study established a murine BAO model and subsequently triggered its differentiation. Thereafter, a suite of multidimensional analytical procedures was employed, including the morphological recognition and examination of organoids utilizing an established artificial intelligence (AI) image tracking system, quantification of cellular composition, proteomic profiling and immunoblots of selected proteins. Our investigation yielded a detailed evaluation of the morphologic, cellular, and molecular variances demarcating the pre- and post-differentiation phases of the BAO model. We also identified of a potential molecular signature reflective of the observed morphological transformations. The integration of cutting-edge AI-driven image analysis with traditional cellular and molecular investigative methods has illuminated key features of this nascent model.


Assuntos
Diferenciação Celular , Organoides , Organoides/metabolismo , Organoides/citologia , Animais , Camundongos , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Inteligência Artificial , Proteômica/métodos , Camundongos Endogâmicos C57BL
19.
Sci Rep ; 14(1): 12879, 2024 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-38839896

RESUMO

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn's disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.


Assuntos
Azatioprina , Diferenciação Celular , Doença de Crohn , Mucosa Intestinal , Celulas de Paneth , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Azatioprina/farmacologia , Celulas de Paneth/metabolismo , Celulas de Paneth/efeitos dos fármacos , Celulas de Paneth/patologia , Humanos , Diferenciação Celular/efeitos dos fármacos , Animais , Camundongos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Feminino , Masculino , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Adulto , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proliferação de Células/efeitos dos fármacos , Pessoa de Meia-Idade , Linhagem Celular , Índice de Gravidade de Doença
20.
Sci Adv ; 10(23): eadj4735, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38838150

RESUMO

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.


Assuntos
Encéfalo , COVID-19 , Plexo Corióideo , Síndrome de Down , Organoides , SARS-CoV-2 , Serina Endopeptidases , Plexo Corióideo/virologia , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Organoides/virologia , Organoides/metabolismo , Organoides/patologia , Humanos , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/patologia , COVID-19/metabolismo , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Síndrome de Down/genética , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Neurônios/patologia , Replicação Viral , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/virologia , Furina/metabolismo , Furina/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Tropismo Viral
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