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1.
Artigo em Inglês | MEDLINE | ID: mdl-38713614

RESUMO

Although impaired regeneration is important in many gastrointestinal diseases including ulcerative colitis (UC), the dynamics of mucosal regeneration in humans are poorly investigated. We have developed a model to study these processes in vivo in humans. Epithelial restitution (ER) and extracellular matrix (ECM) regulation after an experimental injury of the sigmoid colonic mucosa was assessed by repeated high resolution endoscopic imaging, histologic assessment, RNA sequencing, deconvolution analysis, and 16S rDNA sequencing of the injury niche microbiome of 19 UC patients in remission and 20 control subjects. Human ER had a 48-hour lag before induction of regenerative epithelial cells (WAE and TA cells) along with increase of fibroblast derived stem cell growth factor Gremlin 1 mRNA (GREM1). However, in UC deconvolution data showed aby rapid induction of inflammatory fibroblasts, and upregulation of major structural ECM collagen mRNAs and along with tissue inhibitor of metalloproteinase 1 (TIMP1) suggesting increased profibrotic ECM deposition. No change was seen in transforming growth factor ß (TGFß) mRNA whereas and the profibrotic cytokines interleukin 13 (IL13) and IL11 were upregulated in UC suggesting that human post injury responses could be TGFß-independent. In conclusion, we found distinct regulatory layers of regeneration in the normal human colon and a potential targetable profibrotic dysregulation in UC that could lead to long-term end organ failure - i.e., intestinal damage.

2.
bioRxiv ; 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38077056

RESUMO

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi -omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi -omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.

3.
bioRxiv ; 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36747859

RESUMO

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

4.
Proc Natl Acad Sci U S A ; 119(15): e2121720119, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35377806

RESUMO

Human breast milk (hBM) is a dynamic fluid that contains millions of cells, but their identities and phenotypic properties are poorly understood. We generated and analyzed single-cell RNA-sequencing (scRNA-seq) data to characterize the transcriptomes of cells from hBM across lactational time from 3 to 632 d postpartum in 15 donors. We found that the majority of cells in hBM are lactocytes, a specialized epithelial subset, and that cell-type frequencies shift over the course of lactation, yielding greater epithelial diversity at later points. Analysis of lactocytes reveals a continuum of cell states characterized by transcriptional changes in hormone-, growth factor-, and milk production-related pathways. Generalized additive models suggest that one subcluster, LC1 epithelial cells, increases as a function of time postpartum, daycare attendance, and the use of hormonal birth control. We identify several subclusters of macrophages in hBM that are enriched for tolerogenic functions, possibly playing a role in protecting the mammary gland during lactation. Our description of the cellular components of breast milk, their association with maternal­infant dyad metadata, and our quantification of alterations at the gene and pathway levels provide a detailed longitudinal picture of hBM cells across lactational time. This work paves the way for future investigations of how a potential division of cellular labor and differential hormone regulation might be leveraged therapeutically to support healthy lactation and potentially aid in milk production.


Assuntos
Lactação , Leite Humano , Aleitamento Materno , Feminino , Perfilação da Expressão Gênica , Humanos , Lactação/genética , Leite Humano/citologia , Leite Humano/metabolismo , RNA-Seq , Transcriptoma
5.
Nat Biomed Eng ; 6(4): 476-494, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35314801

RESUMO

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.


Assuntos
Organoides , Celulas de Paneth , Animais , Diferenciação Celular , Intestinos , Camundongos , Celulas de Paneth/fisiologia , Células-Tronco
7.
Nat Commun ; 12(1): 4995, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404785

RESUMO

A cell's phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.


Assuntos
Rastreamento de Células/psicologia , Corantes , Transcriptoma , Animais , Bioensaio , Citocinas , Feminino , Fluoresceínas , Corantes Fluorescentes , Células HEK293 , Nível de Saúde , Humanos , Neoplasias Pulmonares , Masculino , Camundongos , Células Mieloides , Organoides , Fenótipo , Células-Tronco , Microambiente Tumoral , Raios Ultravioleta
8.
Cell Mol Gastroenterol Hepatol ; 12(4): 1281-1296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118489

RESUMO

BACKGROUND AND AIMS: The trigger hypothesis opens the possibility of anti-flare initiation therapies by stating that ulcerative colitis (UC) flares originate from inadequate responses to acute mucosal injuries. However, experimental evidence is restricted by a limited use of suitable human models. We thus aimed to investigate the acute mucosal barrier injury responses in humans with and without UC using an experimental injury model. METHODS: A standardized mucosal break was inflicted in the sigmoid colon of 19 patients with UC in endoscopic and histological remission and 20 control subjects. Postinjury responses were assessed repeatedly by high-resolution imaging and sampling to perform Geboes scoring, RNA sequencing, and injury niche microbiota 16S ribosomal RNA gene sequencing. RESULTS: UC patients had more severe endoscopic postinjury inflammation than did control subjects (P < .01), an elevated modified Geboes score (P < .05), a rapid induction of innate response gene sets (P < .05) and antimicrobial peptides (P < .01), and engagement of neutrophils (P < .01). Innate lymphoid cell type 3 (ILC3) markers were increased preinjury (P < .01), and ILC3 activating cytokines were highly induced postinjury, resulting in an increase in ILC3-type cytokine interleukin-17A. Across groups, the postinjury mucosal microbiome had higher bacterial load (P < .0001) and lower α-diversity (P < .05). CONCLUSIONS: UC patients in remission respond to mucosal breaks by an innate hyperresponse engaging resident regulatory ILC3s and a subsequent adaptive activation. The postinjury inflammatory bowel disease-like microbiota diversity decrease is irrespective of diagnosis, suggesting that the dysbiosis is secondary to host injury responses. We provide a model for the study of flare initiation in the search for antitrigger-directed therapies.


Assuntos
Colite Ulcerativa/etiologia , Colite Ulcerativa/patologia , Microbioma Gastrointestinal , Imunidade Inata , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Adulto , Idoso , Biomarcadores , Estudos de Casos e Controles , Colite Ulcerativa/diagnóstico por imagem , Colite Ulcerativa/metabolismo , Citocinas/metabolismo , Progressão da Doença , Suscetibilidade a Doenças , Disbiose , Endoscopia , Feminino , Interações entre Hospedeiro e Microrganismos , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade
9.
Stem Cells ; 39(3): 296-305, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33438789

RESUMO

Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self-renewing tissues. Here we investigated the role of mTert-expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert-GFP+ cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert-expressing cells are a distinct SSC population with enriched colony-forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage-tracing studies identified mTert+ cells as osteochondral progenitors and contribute to the bone-forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance.


Assuntos
Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Telomerase/metabolismo , Animais , Medula Óssea/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Camundongos
10.
Cell ; 181(5): 1016-1035.e19, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32413319

RESUMO

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.


Assuntos
Células Epiteliais Alveolares/metabolismo , Enterócitos/metabolismo , Células Caliciformes/metabolismo , Interferon Tipo I/metabolismo , Mucosa Nasal/citologia , Peptidil Dipeptidase A/genética , Adolescente , Células Epiteliais Alveolares/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/fisiologia , COVID-19 , Linhagem Celular , Células Cultivadas , Criança , Infecções por Coronavirus/virologia , Enterócitos/imunologia , Células Caliciformes/imunologia , Infecções por HIV/imunologia , Humanos , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/citologia , Pulmão/patologia , Macaca mulatta , Camundongos , Mycobacterium tuberculosis , Mucosa Nasal/imunologia , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/genética , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Análise de Célula Única , Tuberculose/imunologia , Regulação para Cima
11.
Development ; 146(19)2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515224

RESUMO

Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data provide a high-resolution view into the consequences of depleting the three catalytically active DNMTs in human pluripotent stem cells.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Ciclo Celular/genética , Diferenciação Celular/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Elementos Facilitadores Genéticos/genética , Entropia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , DNA Metiltransferase 3B
12.
Nat Biomed Eng ; 3(8): 621-631, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391590

RESUMO

The precision of the delivery of therapeutics to the desired injection site by syringes and hollow needles typically depends on the operator. Here, we introduce a highly sensitive, completely mechanical and cost-effective injector for targeting tissue reliably and precisely. As the operator pushes the syringe plunger, the injector senses the loss-of-resistance on encountering a softer tissue or a cavity, stops advancing the needle and delivers the payload. We demonstrate that the injector can reliably deliver liquids to the suprachoroidal space-a challenging injection site that provides access to the back of the eye-for a wide range of eye sizes, scleral thicknesses and intraocular pressures, and target sites relevant for epidural injections, subcutaneous injections and intraperitoneal access. The design of this simple and effective injector can be adapted for a broad variety of clinical applications.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Injeções/instrumentação , Injeções/métodos , Animais , Sistemas de Liberação de Medicamentos/efeitos adversos , Desenho de Equipamento/instrumentação , Desenho de Equipamento/métodos , Olho/patologia , Humanos , Bombas de Infusão/efeitos adversos , Injeções/efeitos adversos , Injeções Epidurais/instrumentação , Injeções Epidurais/métodos , Injeções Intraperitoneais/instrumentação , Injeções Intraperitoneais/métodos , Injeções Subcutâneas/instrumentação , Injeções Subcutâneas/métodos , Agulhas , Coelhos , Seringas , Ferimentos e Lesões
13.
Stem Cell Res Ther ; 10(1): 148, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133056

RESUMO

BACKGROUND: Intestinal stem cell transplantation has been shown to promote mucosal healing and to engender fully functional epithelium in experimental colitis. Hence, stem cell therapies may provide an innovative approach to accomplish mucosal healing in patients with debilitating conditions such as inflammatory bowel disease. However, an approach to label and trace transplanted cells, in order to assess engraftment efficiency and to monitor wound healing, is a key hurdle to overcome prior to initiating human studies. Genetic engineering is commonly employed in animal studies, but may be problematic in humans due to potential off-target and long-term adverse effects. METHODS: We investigated the applicability of a panel of fluorescent dyes and nanoparticles to label intestinal organoids for visualization using the clinically approved imaging modality, confocal laser endomicroscopy (CLE). Staining homogeneity, durability, cell viability, differentiation capacity, and organoid forming efficiency were evaluated, together with visualization of labeled organoids in vitro and ex vivo using CLE. RESULTS: 5-Chloromethylfluorescein diacetate (CMFDA) proved to be suitable as it efficiently stained all organoids without transfer to unstained organoids in co-cultures. No noticeable adverse effects on viability, organoid growth, or stem cell differentiation capacity were observed, although single-cell reseeding revealed a dose-dependent reduction in organoid forming efficiency. Labeled organoids were easily identified in vitro using CLE for a duration of at least 3 days and could additionally be detected ex vivo following transplantation into murine experimental colitis. CONCLUSIONS: It is highly feasible to use fluorescent dye-based labeling in combination with CLE to trace intestinal organoids following transplantation to confirm implantation at the intestinal target site.


Assuntos
Células Epiteliais/metabolismo , Fluorescência , Mucosa Intestinal/metabolismo , Microscopia Confocal/métodos , Animais , Humanos , Masculino , Camundongos
14.
15.
Biotechnol Bioeng ; 116(2): 307-319, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30063243

RESUMO

Newly recognized as natural nanocarriers that deliver biological information between cells, extracellular vesicles (EVs), including exosomes and microvesicles, provide unprecedented therapeutic opportunities. Large-scale and cost-effective manufacturing is imperative for EV products to meet commercial and clinical demands; successful translation requires careful decisions that minimize financial and technological risks. Here, we develop a decision support tool (DST) that computes the most cost-effective technologies for manufacturing EVs at different scales, by examining the costs of goods associated with using published protocols. The DST identifies costs of labor and consumables during EV harvest as key cost drivers, substantiating a need for larger-scale, higher-throughput, and automated technologies for harvesting EVs. Importantly, we highlight a lack of appropriate technologies for meeting clinical demands, and propose a potentially cost-effective solution. This DST can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes when commercializing EV products.


Assuntos
Biotecnologia/métodos , Técnicas de Apoio para a Decisão , Vesículas Extracelulares/metabolismo , Biotecnologia/economia
16.
BMC Biol ; 16(1): 62, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29871632

RESUMO

BACKGROUND: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. RESULTS: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. CONCLUSIONS: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.


Assuntos
Genômica/métodos , Organoides/citologia , Celulas de Paneth/citologia , Análise de Célula Única/métodos , Humanos , Modelos Biológicos , Proteômica , Análise de Sequência de RNA , Nicho de Células-Tronco
17.
Biomaterials ; 154: 60-73, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29120819

RESUMO

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2ß1, integrin ß4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy).


Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/metabolismo , Intestinos/citologia , Células-Tronco/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Colágeno/farmacologia , Colágeno Tipo IV/farmacologia , Combinação de Medicamentos , Células Epiteliais/citologia , Matriz Extracelular/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Laminina/farmacologia , Camundongos Endogâmicos C57BL , Proteoglicanas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/efeitos dos fármacos
18.
EMBO Mol Med ; 9(5): 558-570, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28283650

RESUMO

Both the incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally; in the industrialized world up to 0.5% of the population are affected and around 4.2 million individuals suffer from IBD in Europe and North America combined. Successful engraftment in experimental colitis models suggests that intestinal stem cell transplantation could constitute a novel treatment strategy to re-establish mucosal barrier function in patients with severe disease. Intestinal stem cells can be grown in vitro in organoid structures, though only a fraction of the cells contained are stem cells with regenerative capabilities. Hence, techniques to enrich stem cell populations are being pursued through the development of multiple two-dimensional and three-dimensional culture protocols, as well as co-culture techniques and multiple growth medium compositions. Moreover, research in support matrices allowing for efficient clinical application is in progress. In vitro culture is accomplished by modulating the signaling pathways fundamental for the stem cell niche with a suitable culture matrix to provide additional contact-dependent stimuli and structural support. The aim of this review was to discuss medium compositions and support matrices for optimal intestinal stem cell culture, as well as potential modifications to advance clinical use in IBD.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Doenças Inflamatórias Intestinais/terapia , Intestinos/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Alicerces Teciduais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Meios de Cultura/química , Dinoprostona/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Intestinos/fisiologia , Receptores Notch/metabolismo , Regeneração , Medicina Regenerativa/métodos , Transdução de Sinais , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo , Alicerces Teciduais/química , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
19.
Cell Stem Cell ; 18(1): 25-38, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26748754

RESUMO

Organoid systems leverage the self-organizing properties of stem cells to create diverse multi-cellular tissue proxies. Most organoid models only represent single or partial components of a tissue, and it is often difficult to control the cell type, organization, and cell-cell/cell-matrix interactions within these systems. Herein, we discuss basic approaches to generate stem cell-based organoids, their advantages and limitations, and how bioengineering strategies can be used to steer the cell composition and their 3D organization within organoids to further enhance their utility in research and therapies.


Assuntos
Técnicas de Cultura de Células , Organoides/citologia , Células-Tronco/citologia , Animais , Encéfalo/metabolismo , Comunicação Celular , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Microfluídica , Organogênese , Regeneração , Engenharia Tecidual/métodos
20.
Cell Stem Cell ; 16(1): 13-7, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25575079

RESUMO

Induced pluripotent stem cells (iPSCs) have the potential to transform drug discovery and healthcare in the 21(st) century. However, successful commercialization will require standardized manufacturing platforms. Here we highlight the need to define standardized practices for iPSC generation and processing and discuss current challenges to the robust manufacture of iPSC products.


Assuntos
Técnicas de Cultura de Células/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Humanos , Reprodutibilidade dos Testes , Transplante de Células-Tronco
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